c-MYC and reactive oxygen species play roles in tetrandrine-induced leukemia differentiation

Guixian Wu; Ting Liu; Han Li; Yafang Li; Dengju Li; Wenhua Li

doi:10.1038/s41419-018-0498-9

c-MYC and reactive oxygen species play roles in tetrandrine-induced leukemia differentiation

Wu, Guixian; Liu, Ting; Li, Han; Li, Yafang; Li, Dengju; Li, Wenhua 2018-04-27 00:00:00 Tetrandrine is a broadly used bisbenzylisoquinoline alkaloid component of traditional Chinese medicine that has antitumor effects in some cancer types. In this study, we investigated the effects of tetrandrine on leukemia in vitro and in vivo. The results showed that tetrandrine effectively induced differentiation and autophagy in leukemia cells. In addition, tetrandrine treatment activated the accumulation of reactive oxygen species (ROS) and inhibited c-MYC protein expression. Further, we found that treatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and Tiron as well as overexpression of c-MYC reduced tetrandrine-induced autophagy and differentiation. Moreover, a small molecular c-MYC inhibitor, 10058-F4, enhanced the tetrandrine-induced differentiation of leukemia cells. These results suggest that ROS generation and c-MYC suppression play important roles in tetrandrine-induced autophagy and differentiation, and the results from in vivo experiments were consistent with those from in vitro studies. Therefore, our data suggest that tetrandrine may be a promising agent for the treatment of leukemia. Introduction drugs have prompted the search for new therapeutic agents. Leukemia is a disease caused by malignant proliferation of hematopoietic stem cells. The most important char- Tetrandrine is a bisbenzylisoquinoline alkaloid isolated acteristic of leukemia is that cells are blocked at an early from the roots of the traditional Chinese medicine plant stage of development and fail to differentiate into func- Stephaniae tetrandrae. Tetrandrine has been broadly used tional mature cells . In the 1970s and 1980s, studies for anti-allergic, anti-inﬂammatory and anti-silicosis 2,8,9 showing the capabilities of certain chemicals to induce the treatments . Some studies have shown that tetran- differentiation of leukemia cell lines fostered the concept drine can inhibit proliferation and induce apoptosis in 10–12 of treating leukemia by forcing malignant cells to undergo lung carcinoma, bladder cancer and colon cancer . terminal differentiation instead of killing them through We have reported that relatively high concentrations of 2,3 cytotoxicity . The best proof of principle for differ- tetrandrine induce apoptosis through the reactive oxygen entiation therapy has been the treatment of acute pro- species (ROS)/Akt pathway and that low doses of tet- myelocytic leukemia (APL) with all-trans retinoic acid randrine trigger autophagy via ATG7 and the ROS/ERK 4–7 13,14 (ATRA) . Although various chemicals are used to treat pathway in human hepatocellular carcinoma . These leukemia, tumor resistance and the cytotoxicity of many studies suggest that tetrandrine can exhibit strong anti- tumor effects and has potential as a cancer chemother- apeutic agent. Autophagy, which is a dynamic process induced by Correspondence: Wenhua Li (whli@whu.edu.cn) Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan starvation or cellular stress, is essential for cell differ- 15–17 University, Wuhan 430072, P. R. China entiation, cell survival, aging and the cell cycle . Department of Hematology, Tongji Hospital of Tongji Medical College, Although autophagy is a self-protecting mechanism Huazhong University of Science and Technology, Wuhan, P. R. China Edited by M Diederich © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Ofﬁcial journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Wu et al. Cell Death and Disease (2018) 9:473 Page 2 of 16 24h 80000 b K562 K562 48h 60000 100 72h 24h 48h 72h Te trandrine (µM) Te trandrine (µM) 24h THP-1 THP-1 48h 72h 24h 40000 60 48h 72h Te trandrine (µM) Te trandrine (µM ) 24h U937 U937 48h 45000 100 72h 24h 15000 48h 72h Te trandrine (µM) Te trandrine (µM) 24h HL60 HL60 48h 240000 100 72h 24h 48h 40000 20 72h Te trandrine (µM) Te trandrine (µM) Con Tet d Con T et 2 μM T et 3 μM G0/G1:39.61% G0/G1:50.12% G2/M:24.31% G2/M:18.74% S:36.22% S:33.64% 0.365% 1.9% 7.35% 0.941% 9.26% 25.2% G0/G1:33.82% G0/G1:50.92% G2/M:20.69% G2/M:14.35% S:46.04% S:36.63% 1.48% 2.87% 2.85% 0.468% 0.788% 0.76% G0/G1:51.17% G0/G1:35.42% G2/M:21.75% G2/M:32.5% S:28.65% S:32.8% 13.7% 9.87% 43.3% 2.77% 4.02% 13.4% DNA Conte nt K562 THP-1 0.711% 2.24% 45.3% Tet μM 0 2 3 0 2 3 0.294% 4.38% 13.6% 116KD Anne xin-V-FITC PARP 89KD 47KD 80 K562 THP-1 *** U937 HL60 *** Caspase9 *** 35KD NS 36KD GAPDH Con Tet 2 Tet 3 Fig. 1 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Ce ll numbe r ce ll numbe r cell number cell number cell numbe r HL60 THP-1 K562 Apoptotic cells (%) PI cell viability(%) cell viability (%) cell viability (%) cell viability (%) HL60 U937 THP-1 K562 Wu et al. Cell Death and Disease (2018) 9:473 Page 3 of 16 (see ﬁgure on previous page) Fig. 1 Tetrandrine at 2 µM inhibited leukemia cell proliferation but did not induce apoptosis. DMSO was used as a negative control (Con). The data are presented as the mean ± S.D. (a) Cells were treated with tetrandrine (0, 1, 2 or 3 μM) for 24 h, 48 h and 72 h and then cell proliferation was assessed using a cell counting method. (b) Cell viability was determined by the trypan blue dye-exclusion assay. n = 3. (c) After 48 h of treatment with DMSO or 2 μM tetrandrine, cells were stained with PI and the cell cycle stage was determined by ﬂow cytometry. (d) The cells were treated with tetrandrine (0, 2, or 3 μM) for 48 h. Apoptotic cells were detected by ﬂow cytometry. n = 3. ***p < 0.001, NS, not signiﬁcant. (e) Western blot analysis of the PARP and caspase-9 expression in the cells after tetrandrine (0, 2 or 3 μM) treatment for 48 h. GAPDH was used as a loading control regulated by nutritional deﬁciencies, excessive autophagy nucleus/cytoplasm ratio and the cells with horseshoe leads to cell death . In recent years, autophagy was found shape nuclei (Fig. 2a). Then, we found that tetrandrine- to be closely related to cancer , and ATG7 or ATG4B treated cells exhibited increased nitroblue tetrazolium knockdown has been reported to alter the viability of (NBT) reduction in a time-dependent manner (Fig. 2b). primary chronic myeloid leukemia CD34+ progenitor Moreover, ﬂow cytometry showed that tetrandrine cells. Many studies have shown that autophagy is remarkably increased CD14 and CD11b expression 20–24 important for myeloid cell differentiation . Hence, levels in a time- and dose-dependent manner (Fig. 2c, d). enhanced autophagy may be a promising treatment to Next, ﬂow cytometry analysis showed that tetrandrine- promote differentiation in leukemia patients. treated cells expressed higher levels of CD14 and In our study, we investigated the mechanism of CD11b than DMSO-treated cells when cells were co- tetrandrine-induced leukemia differentiation in vitro and stained with CD14 and CD11b (Fig. 2e). Finally, western in vivo. Our results demonstrated that tetrandrine trig- blot analysis also showed that CD14 expression was gered autophagy, induced ROS generation, and inhibited remarkably higher in tetrandrine-treated cells than in c-MYC expression, which can regulate differentiation. DMSO-treated control cells (Fig. 2f). Altogether, these These ﬁndings suggest that tetrandrine may be a pro- ﬁndings strongly suggest that tetrandrine induced leuke- mising agent for leukemia treatment. mia differentiation. Results Tetrandrine-facilitated differentiation was related to Tetrandrine inhibited cell proliferation in leukemia cells autophagy First, leukemia cells were counted to examine In recent years, several agents that induce cell differ- the effects of tetrandrine on leukemia cell proliferation, entiation and cell death have been shown to activate 26,27 and the results suggested that 2 μM and 3 μM tetrandrine autophagy . We examined whether tetrandrine could dramatically inhibited cell proliferation (Fig. 1a). induce autophagy in leukemia cells. Western blot analysis However, cell viability analysis demonstrated that showed that tetrandrine remarkably increased LC3-II 0–2 μM tetrandrine did not increase cell death (Fig. 1b). protein levels in a dose- and time-dependent manner To further investigate proliferation inhibition, cell (Fig. 3a, b). And acridine orange staining assay demon- cycle analysis was performed and showed signiﬁcant strated that the intensity of acridine orange red staining cell cycle arrest at G0/G1 phase (Fig. 1c), the statistic was signiﬁcantly enhanced in the tetrandrine-treated cells analysis was shown in Figure S1. Moreover, cell (Figure S2A and B). To conﬁrm tetrandrine-induced apoptosis analysis by ﬂow cytometry indicated that 2 μM autophagy, K562 cells were transfected with the GFP-LC3 tetrandrine did not kill cells (Fig. 1d), and western blot plasmid and treated with tetrandrine, followed by visua- analysis of PARP and caspase-9 expression revealed lization of the punctate ﬂuorescent pattern of GFP-LC3 similar results (Fig. 1e). In conclusion, 2 μM tetrandrine by ﬂuorescence microscopy (Fig. 3c). Further, tetrandrine- inhibited proliferation but did not induce apoptosis in induced autophagy ﬂux was investigated by expression leukemia cells. ptfLC3 plasmid and with or without CQ. After 48 h treatment, tetrandrine-treated cells showed only mRFP- Tetrandrine treatment induced differentiation in leukemia LC3 dots, and tetrandrine combine CQ-treated cells cells showed both mRFP and GFP-LC3 dots, which indicated Differentiation therapy can improve the cure rates of that tetrandrine induced autophagy ﬂux been blocked by patients with leukemia . To investigate whether tetran- CQ (Fig. 3d). To examine the relationship between drine induces differentiation in leukemia cells, we used autophagy and differentiation in tetrandrine-induced Wright-Giemsa staining to detect the morphologic fea- cells, 3-methyladenine (3-MA) was used to inhibit tures of tetrandrine-treated cells. The 1.5% DMSO- autophagy, revealing that the tetrandrine-mediated treated K562 cells was used as a positive control . The autophagy process and CD14 expression were strongly results revealed that tetrandrine-treated cells showed a prevented (Fig. 3e, S2C-E and 3F). Next, we also found highly differentiated cellular morphology, such as a lower that tetrandrine-treated cell differentiated morphology Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 4 of 16 a b 0d 4d 5d 6d K562 THP-1 K562 + 0.45 1.5% DMSO *** Con Tet Con Tet 0.4 *** *** 0.35 *** ** 0.3 0.25 60 Con Tet 0.2 *** *** 0.15 0.1 0.05 K562 THP-1 K562 THP-1 K562 THP-1 1d 2d 3d 4d Tet Tet 3 μM Con Tet 2 μM Tet 1 μM Con Tet CD14-FITC Con Tet 3 μM CD14-FITC Tet 2 μM Tet 1 μM Tet Con Con CD11b-PE (fluore s cence intensity) Tet *** ** *** NS Con ** Con 1 μM 50 NS 2 μM 3 μM CD11b-PE (fluore s cence intensity) K562 THP-1 4d 4d Con Tet Con Tet 100 120 ** *** ** ** ** ** ** ** Con 80 NS NS 50 1 μM 2 μM 0 0 3 μM K562 THP-1 K562 THP-1 K562 THP-1 Con Tet 1 μM Tet 2 μM Tet 3 μM K562 THP-1 Tet -+ -+ 82.5% 82.3% CD14 55KD 93.6% 91% GAPDH 36KD 91.6% 82.3% 62.6% 70.6% CD11b-PE ** 30 * Con NS 20 ** 1 μM ** NS 2 μM 3 μM K562 THP-1 Fig. 2 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Re lative cell number CD14 positive Wright-Gie ms a CD14-FLTC cells (%) CD14 and CD11b % differentiation positive cells (%) CD11b positive cells (%) THP-1 K562 THP-1 K562 THP-1 K562 Re lative cell number NBT reducon OD 570 nm CD11b positive CD14 positive cells (%) cells (%) Wu et al. Cell Death and Disease (2018) 9:473 Page 5 of 16 (see ﬁgure on previous page) Fig. 2 Tetrandrine at 2 μM induced differentiation in leukemia cells. The bars indicate the S.D. *p < 0.05, **p < 0.01, ***p < 0.001, NS, not signiﬁcant. (a) The morphology of the Wright-Giemsa-stained cells was observed under a microscope after the cells were treated with DMSO or 2 μM tetrandrine for 4 days. The 1.5% DMSO-treated K562 cells was used as positive control. The arrows indicate lower nucleus/cytoplasm ratio and the cells with horseshoe shape nuclei. n = 3. (b) NBT reduction assay detected cell differentiation after the cells were treated with 2 μM tetrandrine for 0, 4, 5 and 6 days. (c) CD14 or CD11b antigen expression were measured after the cells were treated with DMSO or 2 μM tetrandrine treatment at the indicated times (d) or were measured by ﬂow cytometry after tetrandrine treatment at the indicated concentrations for 4 days. (e) CD14 and CD11b expression was co-detected by ﬂow cytometry after 4 days of 0, 1, 2 or 3 μM tetrandrine treatment. (f) CD14 and GAPDH levels were measured by western blot after the cells were treated with DMSO or 2 μM tetrandrine for 4 days features were restored by 3-MA (Figure S2F). To further activation of c-MYC can inhibit terminal differentiation . determine autophagy was involved in tetrandrine-induced In our study, we treated cells with tetrandrine for various differentiation, ATG7 knockout K562 cells were used treatment durations. Western blot analysis indicated that (Fig. 3g). Flow cytometry detection showed that tetrandrine dramatically suppressed c-MYC expression tetrandrine-induced CD14 expression was effectively (Fig. 5a). Moreover, tetrandrine also decreased c-MYC inhibited in ATG7 knockout K562 cells (Fig. 3h). These mRNA expression (Fig. 5b). To further establish the role data suggest that tetrandrine induces autophagy of leu- of c-MYC in tetrandrine-induced differentiation, c-MYC kemia cells and that autophagy plays a critical role in the was stably expressed in K562 and THP-1 cells (Fig. 5c). As tetrandrine-induced differentiation. shown in Fig. 5d, the tetrandrine-induced cell morphology features were restored by overexpression of c-MYC. Flow Tetrandrine promoted intracellular ROS accumulation, an cytometry analysis showed that tetrandrine-induced important event in tetrandrine-induced autophagy and CD14 expression was prevented in cells overexpressing differentiation c-MYC (Fig. 5e), and western blot analysis for CD14 Some reports have suggested that ROS may be involved revealed similar results (Fig. 5f). To examine whether in cell differentiation . Therefore, we aimed to determine reduction of c-MYC activity can enhance tetradrine- whether tetrandrine-induced differentiation was asso- induced differentiation, c-MYC inhibitor 10058-F4 was ciated with intracellular ROS activation. As shown in used to co-treatment with tetrandrine. As shown in Fig. 4a, we found that tetrandrine-treated cells had higher Fig. 5g, h, though 40 μM 10058-F4 can’t induce cell intracellular ROS levels than DMSO-treated cells. To differentiation, tetrandrine treatment combined with 40 further investigate whether ROS accumulation was μM 10058-F4 can inhibit c-MYC expression and involved in tetrandrine-induced differentiation, CD14 promote CD14 expression, revealing that further expression was assessed after tetrandrine treatment in the inhibition of c-MYC expression can promote cell presence or absence of N-acetyl-L-cysteine (NAC) and differentiation. However, tetrandrine-induced ROS Tiron. The results indicated that tetrandrine-induced accumulation was still activated in the overexpression ROS generation and differentiation were markedly c-MYC cells (Fig. 5i). Further, western blot analysis restrained by NAC and Tiron (Fig. 4b, c), and western blot indicated that c-MYC expression inhibited the LC3-II analysis of CD14 expression revealed similar results accumulation induced by tetrandrine (Fig. 5j). Detection (Fig. 4d). Recent studies have shown that ROS can initiate of tetrandrine-induced acridine orange-positive cells autophagosome formation. Therefore, LC3-II levels and further conﬁrmed that expression of c-MYC may be acridine orange-positive cells were examined with or associated with autophagy (Figure S4). The above results without the addition of NAC. Notably, the results shown suggest that tetrandrine-induced differentiation was that NAC remarkably inhibited the increased LC3-II associated with c-MYC expression. levels and the intensity of acridine orange red staining in the tetrandrine-treated cells (Fig. 4e, f and S3). Impor- Tetrandrine induced differentiation in an in vivo xenograft tantly, ROS accumulation was detected in the cells in model which autophagy was inhibited (Fig. 4g). Thus, these data To investigate the effects of tetrandrine on differentia- suggest that ROS generation may mediate autophagy and tion in vivo, we established a subcutaneous tumor xeno- differentiation in response to tetrandrine-treated leuke- graft model in athymic nude mice using the THP-1 cells mia cells. overexpressing c-MYC or a control vector. A week later, mice were intragastrically administered vehicle or tet- Inhibition of c-MYC expression facilitated tetrandrine- randrine (25 or 50 mg/kg body weight) for 13 days, and induced differentiation their body weight and tumor size were measured daily. Recently, deregulated or elevated expression of c-MYC Importantly, tetrandrine treatment reduced tumor has been reported in many cancers, and sustained growth, as evidenced by the reduction in the tumor Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 6 of 16 Tet 2 μM 24h b Tet Time 0 3 6 9 12 24 48h 0 0.5 1 1.5 2 2.5 µM 18KD LC3-I LC3-I 18KD 16KD 16KD LC3-II LC3-II 36KD GAPDH GAPDH 36KD LC3-I 18KD LC3-I 18KD 16KD LC3-II LC3-II 16KD GAPDH 36KD GAPDH 36KD K562 THP-1 Tet+3-MA 3-MA Tet Con K562 WT ATG7 KO K562 CD14-FITC - + - + Tet (fluore s cence intensity) WT ATG7 KO LC3-I 18KD Con Tet 3-MA Tet+3-MA ATG7 55KD 16KD 100 LC3-II GAPDH GAPDH 36KD 36KD K562 THP-1 K562 Con Tet Con+ATG7 KO Tet+ATG7 KO Tet+ATG7 KO ** Con+ATG7 KO 50 Tet CD14-FITC (fluore s cence intensity) Fig. 3 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Re lative cell number CD14 positive cells (%) THP-1 K562 CD14 positive Re lative cell number cells (%) THP-1 K562 Wu et al. Cell Death and Disease (2018) 9:473 Page 7 of 16 (see ﬁgure on previous page) Fig. 3 Autophagy played an important role in tetrandrine-induced differentiation. The bars indicate the S.D. (a) K562 and THP-1 cells were treated with the indicated concentration of tetrandrine for 24 h or (b) with 2 μM tetrandrine for the indicated durations. Western blot analysis of LC3 levels. GAPDH was used as a loading control. (c) K562 cells were transfected with the GFP-LC3 plasmid for 24 h, subsequently treated with DMSO or 2 μM tetrandrine for 24 h and observed by ﬂuorescence microscopy. Representative experiments are shown to indicate the cellular localization patterns of the GFP-LC3 fusion protein (magniﬁcation ×400), and percentage of cells with GFP-LC3 puncta were used to quantify the percentage of autophagic cells. n = 3, ***p < 0.001. (d) After a 1 h pretreatment with 10 μM CQ and 48 h of subsequent treatment with DMSO or 2 μM tetrandrine, ﬂuorescence microscopy detected green and red ﬂuorescent spots in K562 cells transfected with the ptfLC3 plasmid and percentage of cells with red dots were used to quantify the autophagic ﬂux, while yellow dots (the overlay of green and red ﬂuorescence) were increased if the process of autophagosomes fusing with lysosomes was inhibited. n = 3, *p < 0.05, **p < 0.01. (e) GFP-LC3-transfected cells were pretreated with or without 1.5 mM 3-MA for 1 h. The cells were then exposed to DMSO or 2 μM tetrandrine for 24 h, and the localization of GFP-LC3 was observed using a ﬂuorescent microscope (magniﬁcation ×400). n = 3, **p < 0.01. (f) CD14 antigen expression was analyzed by ﬂow cytometry after treatment for 4 days with or without pretreatment with 1.5 mM 3-MA for 1 h, and a statistic analysis for three experiments in the bottom. *p < 0.05. (g) The expression of ATG7 and LC3-II proteins in K562 ATG7 KO cells were assessed via western blot, and K562 WT cells were used as a positive control. (h) CD14 expression was determined by ﬂow cytometry in K562 ATG7 and K562 WT cells after treatment for 4 days, and a statistic analysis for three experiments in the right. **p < 0.01. WT wild type volume and lower tumor weight in mice treated with the tetrandrine, trypan blue dye-exclusion assay and THP-1 cells overexpressing the control vector compared Annexin-V/PI staining showed that tetrandrine had sig- to those in the mice treated with vehicle, but had a weak niﬁcant anti-leukemia effects (Fig. 7a, b). Next, we found effect on tumors wherein c-MYC was overexpressed tetrandrine facilitated increase CD14 and CD11b expres- (Fig. 6a, b). Notably, we found that tetrandrine treatment sion on the surface of patient cells compared to vehicle was well tolerated by all mice as the animals did not treatment (Fig. 7c, d). As shown in Fig. 7f, western blot display weight loss (Figure S5A). Immunohistochemistry results also showed that tetrandrine treatment promoted results showed that tetrandrine effectively promoted CD14 expression and inhibited c-MYC levels. In addition, CD14 expression and decreased c-MYC expression in the tetrandrine-treated cells dramatically increased the per- mice with THP-1 cells overexpressing the control vector centage of acridine orange-positive cells and up-regulated but had minimal effects on tumors overexpressing c-MYC LC3-II levels (Fig. 7e, f). And we also found that the (Fig. 6c). Moreover, the levels of the lipid peroxidation intracellular ROS levels were signiﬁcantly increased in the product malondialdehyde (MDA), which was used as a tetrandrine-treated patient cells (Fig. 7g). Tetrandrine- presumptive measure of the ROS levels, were higher in induced CD14 expression was also prevented by NAC and both the THP-1 vector and THP-1 c-MYC tumor tissues 3-MA in the patient cells (Figure S6B and C). These following tetrandrine treatment than in those treated with results revealed that tetrandrine may effectively regulated vehicle (Fig. 6d). Finally, decreased c-MYC and increased proliferation, apoptosis, differentiation, autophagy and LC3-II protein levels were detected by western blot in the ROS accumulation in primary leukemia cells. Moreover, tetrandrine-treated THP-1 vector tumors (Fig. 6e). ROS activation and autophagy may be associated with Immunohistochemistry also showed more LC3 protein in tetrandrine-induced differentiation. the tetrandrine-treated THP-1 vector tumors than THP-1 c-MYC tumors (Figure S5B). These results demonstrate Tetrandrine at 2 μM had no cytotoxicity or effects on that tetrandrine exhibited good antitumor activity in vivo, differentiation in human CD34+ progenitor cells and the potential mechanism was associated with the In this study, the effect of 2 μM tetrandrine on human induction of tumor cells autophagy and differentiation by CD34+ progenitor cells has been studied. As shown in inhibiting c-MYC expression. Fig. 8, the results showed that 2 μΜ terandrine has no effect on human CD34+ progenitor cells viability and Tetrandrine affected differentiation in AML patient cells differentiation but may be trigger autophagy. In addition, To determine the effects of tetrandrine on human pri- we also found that tetrandrine has no signiﬁcant effects mary leukemia cells, here, cells were obtained from four on cell viability and CD14 expression but triggered AML patients who received no chemotherapy. Because of autophagy of healthy mouse haematopoietic stem cells the small number of cells isolated from the patient 2# and (Figure S7). 3# blood samples, apoptosis, acridine orange staining, western blot and ROS levels analysis were just validated in Discussion patient 1# and 4#. As shown in Figs. S6A, 2–3 μM tet- Studies have demonstrated that induced cell differ- 30–34 randrine can inhibit cell proliferation. When the cells entiation can effectively treat leukemia . However, the were treated with the indicated concentrations of side effects of drugs are of primary consideration. Based Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 8 of 16 a b 24h 12h K562 THP-1 Tet Tet+NAC Con NAC Tet Con Tet Con Tet+Tiron Tiron Tet Con Intracellular ROS Intracellular ROS Con Tet 24h Con Tet Tiron Tet+Tiron Con Tet NAC Tet+NAC ** * ** 100 ** 100 *** *** 50 50 0 0 K562 THP-1 K562 THP-1 K562 THP-1 K562 THP-1 THP-1 K562 Tet+NAC Tet - + - + - + - + NAC - - + + - - + + NAC Tet CD14 55KD Con 36KD GAPDH Tet+Tiron Tiron Tet Con K562 THP-1 CD14-FITC (fluorescence intensity) Tet - + - + - + - + Con Tet - - + + - - + + NAC Con Tet Tiron Tet+Tiron NAC Tet+NAC LC3-I 18KD ** ** * 16KD * LC3-II 36KD GAPDH K562 THP-1 K562 THP-1 f g K562 Con Tet NAC Tet+NAC Tet+3-MA 1.09% 52.7% 1.92% 10.7% 3-MA Tet Con 0.127% 52.4% 0.041% 1.7% Tet+ATG7 KO Con+ATG7 KO Tet Con FL1-Gre e n Intracellular ROS Fig. 4 Tetrandrine induced accumulation of intracellular ROS, leading to cell autophagy and differentiation. The bars indicate the S.D. *p < 0.05, **p < 0.01, ***p < 0.001. (a) Intracellular ROS levels were assessed by ﬂow cytometry after treatment with DMSO or 2 μM tetrandrine for 12 h or 24 h. (b) The cells were pretreated with NAC (K562 with 15 mM and THP-1 with 10 mM) and Tiron (K562 with 0.5 mM and THP-1 with 0.2 mM) for 1 h, and then treated with DMSO or 2 μM tetrandrine for 24 h. The intracellular ROS levels were measured by ﬂow cytometry (c) or CD14 expression was measured by ﬂow cytometry after 4 days of treatment. (d) CD14 and GAPDH levels were analysis by western blot after the indicated treatments for 4 days. (e) After 24 h of treatment, the LC3 and GAPDH levels were measured by western blots and (f) cells were stained with acridine orange and then analyzed by ﬂow cytometry. (g) After 24 h of DMSO or 2 μM tetrandrine treatment, ﬂow cytometry was used to detect the generation of ROS in K562 ATG7 KO cells and K562 cells pretreated with 1.5 mM 3-MA Ofﬁcial journal of the Cell Death Differentiation Association CD14 positive cells (%) Relative cell number Intracellular Relative cell number FL3-Orange ROS (%) CD14 positive cells (%) THP-1 K562 Intracellular ROS (%) THP-1 K562 Re lative ce ll numbe r Re lative cell numbe r Intracellular ROS (%) Wu et al. Cell Death and Disease (2018) 9:473 Page 9 of 16 T et 2μM ac b K562 THP-1 0 3 6 9 12 24 48 72h 65KD c-MYC Con Tet 1.5 GAPDH 36KD * * c-MYC 65KD 65KD 0.5 c-MYC * * GAPDH 36KD GAPDH 36KD K562 THP-1 THP-1 d K562 e K562 THP-1 Con Tet Con Tet c-MYC-Tet c-MYC-Con Vector-Tet Vector-Con CD14-FITC (fluore s cence intensity) Con Tet *** *** NS NS Con Tet 60 ** ** 40 NS NS Vector c-MYC Vector c-MYC K562 THP-1 Vector c-MYC Vector c-MYC K562 THP-1 g K562 THP-1 - + - + -+ - + Tet - - + + - - + + K562 10058-F4 f THP-1 Vector c-MYC Vector c-MYC 65KD c-MYC - + - + - + - + Tet GAPDH 36KD CD14 55KD C-MYC 65KD i K562 THP-1 GAPDH 36KD c-MYC-Tet c-MYC-Con Vector-Tet Vector-Con K562 THP-1 Intracellular ROS Tet+10058-F4 10058-F4 K562 THP-1 Tet Ve ctor c-M YC Ve ctor c-M YC Con -+ - + - + - + Tet CD14-FITC 18KD (fluore s cence intensity) LC3-I long exposition 16KD Con Tet LC3-II 10058-F4 Tet+10058 -F4 ** short exposition LC3-II 16KD GAPDH 36KD K562 THP-1 LC3-II / GAPDH 0.61 3.02 0.4 1.25 1.29 3.57 0.73 1.63 Fig. 5 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Re lative cell c-MYC Vector CD14 positive numbe r cells (%) % differentiation THP-1 K562 Relative mRNA expression Re lative cell Re lative cell numbe r numbe r CD14 positive cells (%) Wu et al. Cell Death and Disease (2018) 9:473 Page 10 of 16 (see ﬁgure on previous page) Fig. 5 Tetrandrine induced differentiation by inhibiting c-MYC expression. The bars indicate the S.D. (a) Western blot analysis of c-MYC and GAPDH levels in cells treated with 2 μM tetrandrine for different durations. (b) qRT–PCR analysis of c-MYC expression in cells after 24 h of treatment. n = 3. **p < 0.01. (c) Western blot analysis of c-MYC and GAPDH levels in cells overexpressing either vector or c-MYC. (d) Wright-Giemsa staining was used to assess cell morphology after 4 days of DMSO or 2 μM tetrandrine treatment in cells overexpressing either vector or c-MYC. The arrows indicate lower nucleus/cytoplasm ratio and the cells with horseshoe shape nuclei. **p < 0.01, NS, not signiﬁcant. (e) After 4 days treatment, CD14 expression was detected by ﬂow cytometry and (f) the CD14 and c-MYC levels were measured by western blot. n = 3. ***p < 0.001, NS, not signiﬁcant. (g) The cells were pretreated with 40 μM 10058-F4 and then treated with DMSO or 2 μM tetrandrine for 24 h. Western blot analysis of c- MYC and GAPDH levels. (h) Flow cytometry was used to analyze CD14 expression after the indicated treatments for 4 days. (i) Intracellular ROS was assessed via FACS analysis in cells overexpressing either vector or c-MYC after treatment with DMSO or 2 μM tetrandrine for 24 h. (j) Western blot analysis of LC3 and GAPDH levels on a long history of clinical applications of traditional MYC protein can regulate terminal differentiation of 43–45 Chinese medicine, tetrandrine is considered to be a safe hematopoietic cells . In our study, tetrandrine was agent. Tetrandrine has been used for cancer chemopre- shown to reduce c-MYC protein expression. Then, over- 14,35,36 vention and therapy . In this study, we demonstrated expression of c-MYC in K562 and THP-1 cells inhibited that tetrandrine can inhibit leukemia cell proliferation and tetrandrine-induced cell differentiation, and animal induce differentiation. Mechanistically, tetrandrine- experiments also validated these results. Moreover, we induced differentiation was mainly associated with the found that 40 μM 10058-F4 can enhance tetrandrine- stimulation of ROS and autophagy. Moreover, inhibited c- induced differentiation (Fig. 5h) but 80 μM 10058-F4 MYC protein expression played a critical role in alone induce K562 cells differentiation (date not shown). tetrandrine-induced leukemia cell differentiation. However, the signaling pathways involved in tetrandrine- Most normal cell growth and development requires a induced differentiation that are modulated by c-MYC well-controlled balance of protein synthesis and organelle remain unclear and will require further investigation. biogenesis to protein degradation and organelle renewal. In summary, we demonstrated that tetrandrine has the The major pathways for the degradation of cellular con- potential to treat leukemia. Above all, we discovered that stituents are autophagy and cytosolic turnover by the tetrandrine can induce autophagy and differentiation both proteasome . Differentiation of cells is usually associated in vitro and in vivo. The potential molecular mechanisms involve activation of ROS accumulation with slowed cell growth, which is caused by an altered rate of macromolecule synthesis and degradation. Research and inhibition of c-MYC expression. These studies has shown that autophagy can regulate myeloid cell dif- provide the rationale for application of tetrandrine in 22,38 ferentiation . In this study, we observed that early clinical therapies and in therapeutic regimens for autophagy mediated the tetrandrine-induced differentia- leukemia patients. tion. Further inhibition of autophagy by knockout of ATG7 gene decreased tetrandrine-induced differentiation Materials and methods in K562 cells. Moreover, others have reported that Cell lines and cell culture autophagy is required for differentiation of non- HL60 cells were purchased from CCTCC (China Center hematologic cells and tissues. Together, these results for Type Culture Collection; Wuhan, China). K562, U937 show that autophagy is important for cell differentiation. and THP-1 cells were kindly provided by Dr. Zan Huang ROS have emerged in recent years as important reg- (Wuhan University). All cell lines were grown in RPMI ulators of cell division and differentiation . There are 1640 medium supplemented with 10% FBS (fetal bovine reports that ROS are produced in the early stage of serum, Hyclone), 1% streptomycin and 1% penicillin. monocyte–macrophage differentiation. ROS generation AML patient cells were provided by Dengju Li (Tongji blockade induced by BHA, TEMPO, NAC and apocynin Hospital of Tongji Medical College; Wuhan, China). The 40,41 speciﬁcally inhibits M2 macrophage differentiation .In informed consent was obtained from all of the examined the present study, we observed that tetrandrine treatment subjects, and the related studies were approved by the signiﬁcantly stimulated ROS generation. We also ethics committees of the participating hospitals and observed that the ROS scavengers NAC and Tiron can institute. AML patient cells were cultured in expansion inhibit tetrandrine-induced leukemia cell autophagy and media (RPMI 1640 with 10% FBS, 10 ng/ml recombinant differentiation. These results suggest that tetrandrine human IL-3, 10 ng/ml rhIL-6, and 50 ng/ml stem cell accelerated intracellular ROS generation that is important factor). All cells were cultured in a humidiﬁed atmosphere for tetrandrine-induced autophagy and differentiation. containing 5% CO at 37 °C. Cell culture dishes and plates The MYC oncogene contributes to the genesis of many were purchased from Wuxi NEST Biotechnology Co. Ltd. human cancers . Many reports have indicated that c- (Wuxi, China). Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 11 of 16 THP-1 Vector THP-1 Vector P=0.1546 0 mg/kg 2000 25 mg/kg P=0.000276 50 mg/kg P=0.0121 P=0.000846 Vehicle 1d 3d 5d 7d 9d 11d 13d Tet 25mg/kg Tet 50mg/kg THP-1 c-MYC THP-1 c-MYC P=0.1317 0 mg/kg 3000 P=0.1048 P=0.073 25 mg/kg P=0.02 50 mg/kg 1d 3d 5d 7d 9d 11d 13d Vehicle Te t 25mg/kg Te t 50mg/kg THP-1 Vector THP-1 c-MYC P=0.0116 P=0.0469 P=0.4806 P<0.0001 Vehicle Tet 25 mg/kg Tet 50 mg/kg Vehicle Tet 25 mg/kg Tet 50 mg/kg Vehicle T et 25 mg/kg T et 50 mg/kg Vehicle T et 25 mg/kg T et 50 mg/kg 1# 2# 3# 4# 1# 2# 3# 4# 1# 2# 3# 4# 1# 2# 3# 4# 1# 2# 3# 4# 1# 2# 3# 4# LC3-I 18KD LC3-II 16KD 65KD c-MYC 16KD GAPDH THP-1 Vector THP-1 c-MYC Fig. 6 Tetrandrine induced differentiation in an in vivo xenograft model. The bars represent the mean ± S.D. (a), (b) Tumor volume was measured daily. The tumors were removed by dissection and weighed after 13 days of treatment. (c) CD14 and c-MYC expression were evaluated by immunohistochemistry analysis in tumor tissues. Magniﬁcation: ×400. (d) Tumor tissue proteins were extracted from the THP-1 vector and c-MYC xenografts and were subjected to MDA assay to analyze tissue ROS levels. (e) Western blot analysis of c-MYC, LC3 and GAPDH levels in tumor tissues Ofﬁcial journal of the Cell Death Differentiation Association Tumor Volume (cm ) 3 Tumor Volume (cm ) MDA (μmol/mg tumor tissue protein) MDA (μmol/mg tumor Tumor weight (g) Tumor weight (g) tissue protein) Wu et al. Cell Death and Disease (2018) 9:473 Page 12 of 16 Con T et 2 μM T et 3 μM 24h 48h patient 1# patient 4# Te trandrine (µM ) Anne xin-V-FITC patient 1# patient 2# patient 3# patient 4# patient 1# patient 2# patient 3# patient 4# Tet Con 77.1% 87.4% 96.4% 95.1% CD14-FITC (fluore s cence intensity) 62.6% 69.5% 64.3% 57.3% CD11b-PE Tet Con 8.19% 31.4% patient 1# patient 1# patient 4# -+ -+ Tet LC3-I 18KD 2.5% 21.9% LC3-II 16KD patient 4# 55KD CD14 FL1-Gre e n c-MYC 65KD 36KD GAPDH patient 4# patient 1# Tet Con Intracellular ROS Fig. 7 Tetrandrine induced differentiation and autophagy in AML patient cells. (a) Cell viability was analyzed by trypan blue dye-exclusion assays. The bars indicate the S.D. n = 3. (b) Patient #1 and #4 apoptotic cells were analyzed by ﬂow cytometry after treatment with tetrandrine at the indicated concentrations. (c) Analysis of CD14 or (d) CD14 and CD11b expression by ﬂow cytometry after DMSO or 2 μM tetrandrine treatment in all patient cells for 3 days. (e) The patient #1 and #4 cells were treated with DMSO or 2 μM tetrandrine for 24 h, stained with acridine orange, and then analyzed by ﬂow cytometry. (f) Western blot analysis of LC3, CD14, c-MYC and GAPDH in tetrandrine-treated #1 and #4 patient cells. (g) After 24 h treatment, the intracellular ROS levels were assessed via ﬂow cytometry analysis in #1 and #4 patient cells Ofﬁcial journal of the Cell Death Differentiation Association Re lative cell Re lative ce ll FL3-Orange numbe r cell viability (%) numbe r CD14-FLTC PI Tet Con Wu et al. Cell Death and Disease (2018) 9:473 Page 13 of 16 a b Con Tet 24h 48h 72h NS NS NS NS NS NS NS NS Anne xin-V-FITC NS 0 10 Te trandrine (µM) Con Tet Tet Con 0.073% 43.6% Tet Con CD14-FITC (fluore s cence intensity) FL1-Green NS Con Tet Fig. 8 Tetrandrine at 2 μM had no toxicity on human CD34 + progenitor cells. The bars indicate the S.D. *p < 0.05. (a) Cell viability of human CD34+ progenitor cells was assessed by MTS assay. (b) Apoptotic cells were detected by ﬂow cytometry in CD34+ cells following treatment with DMSO or 2 μM tetrandrine for 48 h. (c) After 24 h of treatment with DMSO or 2 μM tetrandrine, cells were stained with acridine orange and analyzed by ﬂow cytometry. (d) The expression of CD14 in the CD34+ cells was measured by ﬂow cytometry after treated with DMSO or 2 μM tetrandrine for 4 days. NS not signiﬁcant Chemical reagents and antibodies (goat–anti-rabbit and goat–anti-mouse) were acquired Tetrandrine was acquired from Shanghai Ronghe from Beyotime (Nantong, China). Medical (Shanghai, China). Wright-Giemsa stain was obtained from Baso (Zhuhai, China). DCFH-DA was from Cell proliferation and viability assays Invitrogen (Carlsbad, CA). Acridine orange, 3-MA, Tetrandrine was dissolved in DMSO to a ﬁnal con- DMSO, NAC, and Tiron were purchased from Sigma- centration of 10 mM and then stored at −80 °C. For Aldrich (St. Louis, MO). NBT was from Beyotime (Nan- proliferation and viability assays, 3000 cells (K562 and tong, China). The FITC Annexin V Apoptosis Detection HL60) or 5000 cells (THP-1 and U937) per well were Kit I, CD14-FITC and CD11b-PE were obtained from BD seeded in a 96-well plate with 100 μl medium, cultured for Biosciences. Red blood cell lysis buffer, IL-3, IL6 and stem 24, 48 and 72 h in the presence of varying concentrations cell factor were kindly provided by Dr. Zan Huang of tetrandrine or DMSO (con). The total cell count was (Wuhan University). MTS was acquired from Promega then detected by a hemocytometer, and cell viability was (Madison, USA). The antibody against LC3 was from measured as the percentage of living cells demonstrated Sigma-Aldrich (St. Louis, MO). The caspase-3, PARP, and by the trypan blue dye-exclusion assay according to ATG7 antibodies were obtained from Cell Signaling established protocols. Technology (Beverly, MA). Antibodies against CD14 and c-MYC were purchased from Proteintech Group Inc. Cell cycle analysis (Chicago, IL). The anti-GAPDH antibody and the horse- Cells were cultured for 48 h in the presence of 2 μM radish peroxidase (HRP)-conjugated secondary antibodies tetrandrine or DMSO (con). The cells were collected and Ofﬁcial journal of the Cell Death Differentiation Association cell viability (%) FL3-Orange PI CD14 positive Apoptotic cells % Re lative cell numbe r cells (%) Wu et al. Cell Death and Disease (2018) 9:473 Page 14 of 16 washed with PBS and then ﬁxed with 70% ethanol for at cells were plated in 12-well plates and treated with 2 μM least 4 h at 4 °C. The ﬁxed cells were collected and washed tetrandrine for 24 h. Cells were then stained with acridine twice with PBS, suspended in cold PBS containing 50 μg/ orange (1 μg/ml) at 37 °C for 25 min before observation. ml of PI and 100 μg/ml of Rnase A and then kept in the The red acidic vesicular organelles (AVOs) in autophagy dark for 30 min. The samples were transferred to the ﬂow cells were measured by ﬂow cytometry or visualized by cytometer (Beckman, Indianapolis, CA, USA) and cell ﬂuorescence microscopy. ﬂuorescence were measured. The data were analyzed using FlowJo software (TreeStar, San Carlos, CA, USA). Measurement of intracellular ROS levels The cell-permeant probe, DCFH-DA, which ﬂuoresces Cell apoptosis analysis by ﬂow cytometry when it is oxidized, was used to measure intracellular ROS Cells were treated with 2 μM tetrandrine or DMSO levels. Cells were treated with DMSO or the indicated (con) for 48 h, collected, washed twice with PBS, resus- concentrations of tetrandrine in a 12-well plate for 12 h pended in binding buffer and then dyed with Annexin V- and 24 h. Then, cells were collected and washed with PBS FITC and PI for 15 min in the dark according to the and resuspended in 500 μl of serum-free RPMI 1640 manufacturer’s instructions. Annexin V ﬂuorescence was medium containing 0.5 μl DCFH-DA at 37 °C for measured by ﬂow cytometry, and the membrane integrity 30 min. The prepared cells were evaluated using ﬂow of the cells was simultaneously assessed by the PI exclu- cytometry. sion method. Western blot analysis Morphological observation After treatment, cells were collected and washed with Cell morphology was determined by a Wright-Giemsa PBS and then lysed in 1% sodium dodecyl sulfate (SDS) on staining assay following 4 days of 2 μM tetrandrine ice. Cell lysates were heated to 98 °C for 15 min and then treatment. Cells were collected and smeared on micro- centrifuged at 12,000×g for 15 min. The supernatant was scope slides from Citoglas (Jiangsu, China). After Wright- collected, and protein concentrations were assessed using Giemsa staining, the slides were cleaned gently, observed the Bicinchoninic Acid Protein Assay Kit (Thermo sci- under a light microscope (CKX41, Olympus Optical Co., entiﬁc). Equal amounts of protein were separated by Ltd.) and photographed. SDS–PAGE and transferred to a PVDF membrane (Mil- lipore), which was then immunoblotted with the indicated antibodies. NBT reduction assay For the NBT reduction analysis, cells were treated with 2 μM tetrandrine for 0, 4, 5 and 6 days. Then, the cell Quantitative real-time PCR suspensions were incubated with 1 mg/ml of NBT and 20 Cells were treated with 2 μM tetrandrine or DMSO for ng/ml of TPA in an equal volume of RPMI1640 for 30 24 h. Total RNA was isolated using the Total RNA Kit I min at 37 °C. After 30 min, the cells were washed with (Omega Bio-Tek, Inc., GA). Then, RNA was transcribed PBS and resuspended in 100 μl of PBS. NBT was evaluated into cDNA using the Transcriptor First Strand cDNA both as the percentage of positive cells and the intensity of Synthesis Kit (Roche Life Science, USA) according to the reduction when measured at a wavelength of 570 nm with manufacturer’s instructions. qRT–PCR was performed microplate readers (SpectraMax M5). using the FastStart Universal SYBR Green Master kit (Rox) (Roche Life Science, USA) on the Applied Biosys- Detection of CD14 and CD11b expression by ﬂow tems 7500 Fast Real-Time PCR System (PerkinElmer, cytometry Torrance, CA). The following primer pairs were used for Cells were plated in 12-well plates and mixed with 2 μM qRT–PCR: c-MYC: forward, 5′-CACCGAGTCGTAGTC tetrandrine for indicated time. To measure the expression GAGGT-3′ and reverse, 5′-TTTCGGGTAGTGGAAAA of differentiation markers CD14 or CD11b, cells were CCA-3′. GAPDH: forward, 5′-TCCACCACCCTGTTG washed twice in cold PBS after dilution to a density of 1 × CTGTA-3′ and reverse 5′-ACCACAGTCCATGCCA 10 cells per well, and then incubated with the CD14- TCAC-3′. All reactions were performed in triplicate in a FITC and/or CD11b-PE antibody for 20 min in the dark. 20-μl reaction volume. Fold changes in gene expression −ΔΔCt Samples were washed with cold PBS and measured by were determined using the 2 method with GAPDH ﬂow cytometry. as an endogenous control. Acridine orange staining assay Plasmids and transient transfection Acridine orange staining can detect intracellular acidic The GFP-LC3 plasmid was kindly provided by Dr. autophagic vacuoles by ﬂow cytometry or ﬂuorescence Tamotsu Yoshimori (National Institute of Genetics, microscopy. For the acridine orange staining assay, the Mishima, Japan). The pEGFP-mRFP tandem ﬂuorescent- Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 15 of 16 tagged LC3 (ptfLC3) plasmid was purchased from Immunohistochemistry Addgene (Cambridge, MA). To detect autophagy and The tumor tissue sections were ﬁxed by 4% paraf- autophagic ﬂux, the cells were seeded in a 12-well plate, ormaldehyde, embedded in parafﬁn and sliced at a 5 μm then transfected with the GFP-LC3 or ptfLC3 plasmids thickness for immunohistochemical analysis. After for 24 h, and then treatment with indicated drugs to deparafﬁnization and the appropriate epitope retrieval, present autophagy and autophagic ﬂux. After treatment the sections were stained with c-MYC, CD14 and LC3 the cells were collected and observed with a ﬂuorescence antibodies and further incubated with biotinylated microscope (Olympus BX51). goat–anti-rabbit antibodies. The speciﬁc signals were then detected with streptavidin-conjugated HRP and Plasmids and lentiviral transfection diaminobenzidine as the chromogen. The pHAGE.puro-c-MYC plasmid and the empty vec- tor plasmid were kindly provided by Dr. Li Y (College of Healthy progenitor cell study Life Sciences, Wuhan University, China). Transfection Mouse hematopoietic stem cells and human CD34+ reagents were kindly provided by Dr. Xiaodong Zhang progenitor cells were used to measure tetrandrine drug (College of Life Sciences, Wuhan University, China). In toxicity. ICR mice were obtained from the Disease Pre- our study, HEK-293T cells were transfected with either vention Center of Hubei Province. In the experiment, the c-MYC plasmid or an empty vector together with the mice embryos were collected after 12.5 days of conception pMD2.G and psPAX2 plasmids using transfection and the fetal livers were titrated with a pipette needle to reagents. At 48 h post-transfection, the supernatants were obtain a single-cell suspension. Human CD34+ pro- collected and ﬁltered with 0.45-μm ﬁlters. Then, K562 genitor cells were provided by Dr. Zan Huang (Wuhan and THP-1 cells were cultured with the supernatants University). Then, cells were cultured with complete containing 5 μg/ml polybrene. After 24 h, the virus- RPMI 1640 media containing IL-3 and IL6 at 10 ng/ml containing medium was replaced by fresh medium with and stem cell factor at 100 ng/ml. To induce the cells to 2 μg/ml puromycin. Stable cells were selected with differentiate, cells were cultured in differentiation media puromycin. (RPMI 1640 with 10% FBS, 10 ng/ml stem cell factor and 50 ng/ml GM-CSF). In this study, mouse cells used mouse Tumor xenograft model cytokines and human cells with human cytokines. The xenograft model in athymic nude mice was per- formed to evaluate the in vivo efﬁcacy of tetrandrine. Statistical analysis Animal experimental protocols and care were approved Data are expressed as the mean ± S.D. A two-tailed by the Experimental Animal Center of Wuhan University. unpaired Student’s t-test was used to analyze data con- Male nude mice of 4 to 5 weeks of age were obtained from taining two groups unless otherwise speciﬁed. Statistical the Model Animal Research Center (Changsha, China). signiﬁcance was denoted as follows: NS, not signiﬁcant; THP-1 cells (~1 × 10 cells) and the same number of *P < 0.05, **P < 0.01 and ***P < 0.001 were deemed statis- THP-1 cells overexpressing c-MYC in a total volume of tically signiﬁcant. 0.2 mL of PBS were inoculated subcutaneously over the right ﬂank of each mouse. Tumor diameter and body Acknowledgements weights were measured every day. Tumor volume was This study was supported by the National Basic Research Program of China (2014CB910600), the National Natural Science Foundation of China (81273540, calculated by the following formula: 0.52 × length × 81472684 and 81770168) and Fundamental Research Funds for the Central width . Mice were randomized into three groups Universities (2042017KF0242). (ﬁve per group) when the tumor volume reached ~50 mm . There were two treatment groups of mice that Author details received tetrandrine at 25 or 50 mg/kg, while the other Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan mouse group was given a vehicle treatment of 0.5% University, Wuhan 430072, P. R. China. Department of Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and methylcellulose. Technology, Wuhan, P. R. China Malondialdehyde (MDA) assay Author contributions Mice were killed after 13 days of tetrandrine treatment, G.W. performed the research, analyzed the data and wrote the manuscript; T.L. and tumor tissues were removed. For the MDA assay, provided the ATG7 knockout K562 cells; H.L. and Y.L. analyzed and interpreted tissue proteins were prepared according to the description the data; D.L. provided the AML patient cells and W.L. designed the research and wrote the manuscript. in the Lipid Peroxidation MDA assay kit (Beyotime, Nantong, China). The MDA levels were evaluated by Multi-Mode Microplate Readers (SpectraMax M5) at 532 Conﬂict of interest nm using 490 nm as a control. The authors declare that they have no conﬂict of interest. Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 16 of 16 Publisher's note 21. Karvela, M. et al. ATG7 regulates energy metabolism, differentiation and sur- Springer Nature remains neutral with regard to jurisdictional claims in vival of Philadelphia-chromosome-positive cells. Autophagy 12,936–948 published maps and institutional afﬁliations. (2016). 22. Isakson, P., Bjoras, M., Boe, S. O. & Simonsen, A. 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Publisher: Springer Journals
Copyright: Copyright © 2018 by The Author(s)
Subject: Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Immunology; Cell Culture; Antibodies
eISSN: 2041-4889
DOI: 10.1038/s41419-018-0498-9
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Abstract

Tetrandrine is a broadly used bisbenzylisoquinoline alkaloid component of traditional Chinese medicine that has antitumor effects in some cancer types. In this study, we investigated the effects of tetrandrine on leukemia in vitro and in vivo. The results showed that tetrandrine effectively induced differentiation and autophagy in leukemia cells. In addition, tetrandrine treatment activated the accumulation of reactive oxygen species (ROS) and inhibited c-MYC protein expression. Further, we found that treatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and Tiron as well as overexpression of c-MYC reduced tetrandrine-induced autophagy and differentiation. Moreover, a small molecular c-MYC inhibitor, 10058-F4, enhanced the tetrandrine-induced differentiation of leukemia cells. These results suggest that ROS generation and c-MYC suppression play important roles in tetrandrine-induced autophagy and differentiation, and the results from in vivo experiments were consistent with those from in vitro studies. Therefore, our data suggest that tetrandrine may be a promising agent for the treatment of leukemia. Introduction drugs have prompted the search for new therapeutic agents. Leukemia is a disease caused by malignant proliferation of hematopoietic stem cells. The most important char- Tetrandrine is a bisbenzylisoquinoline alkaloid isolated acteristic of leukemia is that cells are blocked at an early from the roots of the traditional Chinese medicine plant stage of development and fail to differentiate into func- Stephaniae tetrandrae. Tetrandrine has been broadly used tional mature cells . In the 1970s and 1980s, studies for anti-allergic, anti-inﬂammatory and anti-silicosis 2,8,9 showing the capabilities of certain chemicals to induce the treatments . Some studies have shown that tetran- differentiation of leukemia cell lines fostered the concept drine can inhibit proliferation and induce apoptosis in 10–12 of treating leukemia by forcing malignant cells to undergo lung carcinoma, bladder cancer and colon cancer . terminal differentiation instead of killing them through We have reported that relatively high concentrations of 2,3 cytotoxicity . The best proof of principle for differ- tetrandrine induce apoptosis through the reactive oxygen entiation therapy has been the treatment of acute pro- species (ROS)/Akt pathway and that low doses of tet- myelocytic leukemia (APL) with all-trans retinoic acid randrine trigger autophagy via ATG7 and the ROS/ERK 4–7 13,14 (ATRA) . Although various chemicals are used to treat pathway in human hepatocellular carcinoma . These leukemia, tumor resistance and the cytotoxicity of many studies suggest that tetrandrine can exhibit strong anti- tumor effects and has potential as a cancer chemother- apeutic agent. Autophagy, which is a dynamic process induced by Correspondence: Wenhua Li (whli@whu.edu.cn) Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan starvation or cellular stress, is essential for cell differ- 15–17 University, Wuhan 430072, P. R. China entiation, cell survival, aging and the cell cycle . Department of Hematology, Tongji Hospital of Tongji Medical College, Although autophagy is a self-protecting mechanism Huazhong University of Science and Technology, Wuhan, P. R. China Edited by M Diederich © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Ofﬁcial journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Wu et al. Cell Death and Disease (2018) 9:473 Page 2 of 16 24h 80000 b K562 K562 48h 60000 100 72h 24h 48h 72h Te trandrine (µM) Te trandrine (µM) 24h THP-1 THP-1 48h 72h 24h 40000 60 48h 72h Te trandrine (µM) Te trandrine (µM ) 24h U937 U937 48h 45000 100 72h 24h 15000 48h 72h Te trandrine (µM) Te trandrine (µM) 24h HL60 HL60 48h 240000 100 72h 24h 48h 40000 20 72h Te trandrine (µM) Te trandrine (µM) Con Tet d Con T et 2 μM T et 3 μM G0/G1:39.61% G0/G1:50.12% G2/M:24.31% G2/M:18.74% S:36.22% S:33.64% 0.365% 1.9% 7.35% 0.941% 9.26% 25.2% G0/G1:33.82% G0/G1:50.92% G2/M:20.69% G2/M:14.35% S:46.04% S:36.63% 1.48% 2.87% 2.85% 0.468% 0.788% 0.76% G0/G1:51.17% G0/G1:35.42% G2/M:21.75% G2/M:32.5% S:28.65% S:32.8% 13.7% 9.87% 43.3% 2.77% 4.02% 13.4% DNA Conte nt K562 THP-1 0.711% 2.24% 45.3% Tet μM 0 2 3 0 2 3 0.294% 4.38% 13.6% 116KD Anne xin-V-FITC PARP 89KD 47KD 80 K562 THP-1 *** U937 HL60 *** Caspase9 *** 35KD NS 36KD GAPDH Con Tet 2 Tet 3 Fig. 1 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Ce ll numbe r ce ll numbe r cell number cell number cell numbe r HL60 THP-1 K562 Apoptotic cells (%) PI cell viability(%) cell viability (%) cell viability (%) cell viability (%) HL60 U937 THP-1 K562 Wu et al. Cell Death and Disease (2018) 9:473 Page 3 of 16 (see ﬁgure on previous page) Fig. 1 Tetrandrine at 2 µM inhibited leukemia cell proliferation but did not induce apoptosis. DMSO was used as a negative control (Con). The data are presented as the mean ± S.D. (a) Cells were treated with tetrandrine (0, 1, 2 or 3 μM) for 24 h, 48 h and 72 h and then cell proliferation was assessed using a cell counting method. (b) Cell viability was determined by the trypan blue dye-exclusion assay. n = 3. (c) After 48 h of treatment with DMSO or 2 μM tetrandrine, cells were stained with PI and the cell cycle stage was determined by ﬂow cytometry. (d) The cells were treated with tetrandrine (0, 2, or 3 μM) for 48 h. Apoptotic cells were detected by ﬂow cytometry. n = 3. ***p < 0.001, NS, not signiﬁcant. (e) Western blot analysis of the PARP and caspase-9 expression in the cells after tetrandrine (0, 2 or 3 μM) treatment for 48 h. GAPDH was used as a loading control regulated by nutritional deﬁciencies, excessive autophagy nucleus/cytoplasm ratio and the cells with horseshoe leads to cell death . In recent years, autophagy was found shape nuclei (Fig. 2a). Then, we found that tetrandrine- to be closely related to cancer , and ATG7 or ATG4B treated cells exhibited increased nitroblue tetrazolium knockdown has been reported to alter the viability of (NBT) reduction in a time-dependent manner (Fig. 2b). primary chronic myeloid leukemia CD34+ progenitor Moreover, ﬂow cytometry showed that tetrandrine cells. Many studies have shown that autophagy is remarkably increased CD14 and CD11b expression 20–24 important for myeloid cell differentiation . Hence, levels in a time- and dose-dependent manner (Fig. 2c, d). enhanced autophagy may be a promising treatment to Next, ﬂow cytometry analysis showed that tetrandrine- promote differentiation in leukemia patients. treated cells expressed higher levels of CD14 and In our study, we investigated the mechanism of CD11b than DMSO-treated cells when cells were co- tetrandrine-induced leukemia differentiation in vitro and stained with CD14 and CD11b (Fig. 2e). Finally, western in vivo. Our results demonstrated that tetrandrine trig- blot analysis also showed that CD14 expression was gered autophagy, induced ROS generation, and inhibited remarkably higher in tetrandrine-treated cells than in c-MYC expression, which can regulate differentiation. DMSO-treated control cells (Fig. 2f). Altogether, these These ﬁndings suggest that tetrandrine may be a pro- ﬁndings strongly suggest that tetrandrine induced leuke- mising agent for leukemia treatment. mia differentiation. Results Tetrandrine-facilitated differentiation was related to Tetrandrine inhibited cell proliferation in leukemia cells autophagy First, leukemia cells were counted to examine In recent years, several agents that induce cell differ- the effects of tetrandrine on leukemia cell proliferation, entiation and cell death have been shown to activate 26,27 and the results suggested that 2 μM and 3 μM tetrandrine autophagy . We examined whether tetrandrine could dramatically inhibited cell proliferation (Fig. 1a). induce autophagy in leukemia cells. Western blot analysis However, cell viability analysis demonstrated that showed that tetrandrine remarkably increased LC3-II 0–2 μM tetrandrine did not increase cell death (Fig. 1b). protein levels in a dose- and time-dependent manner To further investigate proliferation inhibition, cell (Fig. 3a, b). And acridine orange staining assay demon- cycle analysis was performed and showed signiﬁcant strated that the intensity of acridine orange red staining cell cycle arrest at G0/G1 phase (Fig. 1c), the statistic was signiﬁcantly enhanced in the tetrandrine-treated cells analysis was shown in Figure S1. Moreover, cell (Figure S2A and B). To conﬁrm tetrandrine-induced apoptosis analysis by ﬂow cytometry indicated that 2 μM autophagy, K562 cells were transfected with the GFP-LC3 tetrandrine did not kill cells (Fig. 1d), and western blot plasmid and treated with tetrandrine, followed by visua- analysis of PARP and caspase-9 expression revealed lization of the punctate ﬂuorescent pattern of GFP-LC3 similar results (Fig. 1e). In conclusion, 2 μM tetrandrine by ﬂuorescence microscopy (Fig. 3c). Further, tetrandrine- inhibited proliferation but did not induce apoptosis in induced autophagy ﬂux was investigated by expression leukemia cells. ptfLC3 plasmid and with or without CQ. After 48 h treatment, tetrandrine-treated cells showed only mRFP- Tetrandrine treatment induced differentiation in leukemia LC3 dots, and tetrandrine combine CQ-treated cells cells showed both mRFP and GFP-LC3 dots, which indicated Differentiation therapy can improve the cure rates of that tetrandrine induced autophagy ﬂux been blocked by patients with leukemia . To investigate whether tetran- CQ (Fig. 3d). To examine the relationship between drine induces differentiation in leukemia cells, we used autophagy and differentiation in tetrandrine-induced Wright-Giemsa staining to detect the morphologic fea- cells, 3-methyladenine (3-MA) was used to inhibit tures of tetrandrine-treated cells. The 1.5% DMSO- autophagy, revealing that the tetrandrine-mediated treated K562 cells was used as a positive control . The autophagy process and CD14 expression were strongly results revealed that tetrandrine-treated cells showed a prevented (Fig. 3e, S2C-E and 3F). Next, we also found highly differentiated cellular morphology, such as a lower that tetrandrine-treated cell differentiated morphology Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 4 of 16 a b 0d 4d 5d 6d K562 THP-1 K562 + 0.45 1.5% DMSO *** Con Tet Con Tet 0.4 *** *** 0.35 *** ** 0.3 0.25 60 Con Tet 0.2 *** *** 0.15 0.1 0.05 K562 THP-1 K562 THP-1 K562 THP-1 1d 2d 3d 4d Tet Tet 3 μM Con Tet 2 μM Tet 1 μM Con Tet CD14-FITC Con Tet 3 μM CD14-FITC Tet 2 μM Tet 1 μM Tet Con Con CD11b-PE (fluore s cence intensity) Tet *** ** *** NS Con ** Con 1 μM 50 NS 2 μM 3 μM CD11b-PE (fluore s cence intensity) K562 THP-1 4d 4d Con Tet Con Tet 100 120 ** *** ** ** ** ** ** ** Con 80 NS NS 50 1 μM 2 μM 0 0 3 μM K562 THP-1 K562 THP-1 K562 THP-1 Con Tet 1 μM Tet 2 μM Tet 3 μM K562 THP-1 Tet -+ -+ 82.5% 82.3% CD14 55KD 93.6% 91% GAPDH 36KD 91.6% 82.3% 62.6% 70.6% CD11b-PE ** 30 * Con NS 20 ** 1 μM ** NS 2 μM 3 μM K562 THP-1 Fig. 2 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Re lative cell number CD14 positive Wright-Gie ms a CD14-FLTC cells (%) CD14 and CD11b % differentiation positive cells (%) CD11b positive cells (%) THP-1 K562 THP-1 K562 THP-1 K562 Re lative cell number NBT reducon OD 570 nm CD11b positive CD14 positive cells (%) cells (%) Wu et al. Cell Death and Disease (2018) 9:473 Page 5 of 16 (see ﬁgure on previous page) Fig. 2 Tetrandrine at 2 μM induced differentiation in leukemia cells. The bars indicate the S.D. *p < 0.05, **p < 0.01, ***p < 0.001, NS, not signiﬁcant. (a) The morphology of the Wright-Giemsa-stained cells was observed under a microscope after the cells were treated with DMSO or 2 μM tetrandrine for 4 days. The 1.5% DMSO-treated K562 cells was used as positive control. The arrows indicate lower nucleus/cytoplasm ratio and the cells with horseshoe shape nuclei. n = 3. (b) NBT reduction assay detected cell differentiation after the cells were treated with 2 μM tetrandrine for 0, 4, 5 and 6 days. (c) CD14 or CD11b antigen expression were measured after the cells were treated with DMSO or 2 μM tetrandrine treatment at the indicated times (d) or were measured by ﬂow cytometry after tetrandrine treatment at the indicated concentrations for 4 days. (e) CD14 and CD11b expression was co-detected by ﬂow cytometry after 4 days of 0, 1, 2 or 3 μM tetrandrine treatment. (f) CD14 and GAPDH levels were measured by western blot after the cells were treated with DMSO or 2 μM tetrandrine for 4 days features were restored by 3-MA (Figure S2F). To further activation of c-MYC can inhibit terminal differentiation . determine autophagy was involved in tetrandrine-induced In our study, we treated cells with tetrandrine for various differentiation, ATG7 knockout K562 cells were used treatment durations. Western blot analysis indicated that (Fig. 3g). Flow cytometry detection showed that tetrandrine dramatically suppressed c-MYC expression tetrandrine-induced CD14 expression was effectively (Fig. 5a). Moreover, tetrandrine also decreased c-MYC inhibited in ATG7 knockout K562 cells (Fig. 3h). These mRNA expression (Fig. 5b). To further establish the role data suggest that tetrandrine induces autophagy of leu- of c-MYC in tetrandrine-induced differentiation, c-MYC kemia cells and that autophagy plays a critical role in the was stably expressed in K562 and THP-1 cells (Fig. 5c). As tetrandrine-induced differentiation. shown in Fig. 5d, the tetrandrine-induced cell morphology features were restored by overexpression of c-MYC. Flow Tetrandrine promoted intracellular ROS accumulation, an cytometry analysis showed that tetrandrine-induced important event in tetrandrine-induced autophagy and CD14 expression was prevented in cells overexpressing differentiation c-MYC (Fig. 5e), and western blot analysis for CD14 Some reports have suggested that ROS may be involved revealed similar results (Fig. 5f). To examine whether in cell differentiation . Therefore, we aimed to determine reduction of c-MYC activity can enhance tetradrine- whether tetrandrine-induced differentiation was asso- induced differentiation, c-MYC inhibitor 10058-F4 was ciated with intracellular ROS activation. As shown in used to co-treatment with tetrandrine. As shown in Fig. 4a, we found that tetrandrine-treated cells had higher Fig. 5g, h, though 40 μM 10058-F4 can’t induce cell intracellular ROS levels than DMSO-treated cells. To differentiation, tetrandrine treatment combined with 40 further investigate whether ROS accumulation was μM 10058-F4 can inhibit c-MYC expression and involved in tetrandrine-induced differentiation, CD14 promote CD14 expression, revealing that further expression was assessed after tetrandrine treatment in the inhibition of c-MYC expression can promote cell presence or absence of N-acetyl-L-cysteine (NAC) and differentiation. However, tetrandrine-induced ROS Tiron. The results indicated that tetrandrine-induced accumulation was still activated in the overexpression ROS generation and differentiation were markedly c-MYC cells (Fig. 5i). Further, western blot analysis restrained by NAC and Tiron (Fig. 4b, c), and western blot indicated that c-MYC expression inhibited the LC3-II analysis of CD14 expression revealed similar results accumulation induced by tetrandrine (Fig. 5j). Detection (Fig. 4d). Recent studies have shown that ROS can initiate of tetrandrine-induced acridine orange-positive cells autophagosome formation. Therefore, LC3-II levels and further conﬁrmed that expression of c-MYC may be acridine orange-positive cells were examined with or associated with autophagy (Figure S4). The above results without the addition of NAC. Notably, the results shown suggest that tetrandrine-induced differentiation was that NAC remarkably inhibited the increased LC3-II associated with c-MYC expression. levels and the intensity of acridine orange red staining in the tetrandrine-treated cells (Fig. 4e, f and S3). Impor- Tetrandrine induced differentiation in an in vivo xenograft tantly, ROS accumulation was detected in the cells in model which autophagy was inhibited (Fig. 4g). Thus, these data To investigate the effects of tetrandrine on differentia- suggest that ROS generation may mediate autophagy and tion in vivo, we established a subcutaneous tumor xeno- differentiation in response to tetrandrine-treated leuke- graft model in athymic nude mice using the THP-1 cells mia cells. overexpressing c-MYC or a control vector. A week later, mice were intragastrically administered vehicle or tet- Inhibition of c-MYC expression facilitated tetrandrine- randrine (25 or 50 mg/kg body weight) for 13 days, and induced differentiation their body weight and tumor size were measured daily. Recently, deregulated or elevated expression of c-MYC Importantly, tetrandrine treatment reduced tumor has been reported in many cancers, and sustained growth, as evidenced by the reduction in the tumor Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 6 of 16 Tet 2 μM 24h b Tet Time 0 3 6 9 12 24 48h 0 0.5 1 1.5 2 2.5 µM 18KD LC3-I LC3-I 18KD 16KD 16KD LC3-II LC3-II 36KD GAPDH GAPDH 36KD LC3-I 18KD LC3-I 18KD 16KD LC3-II LC3-II 16KD GAPDH 36KD GAPDH 36KD K562 THP-1 Tet+3-MA 3-MA Tet Con K562 WT ATG7 KO K562 CD14-FITC - + - + Tet (fluore s cence intensity) WT ATG7 KO LC3-I 18KD Con Tet 3-MA Tet+3-MA ATG7 55KD 16KD 100 LC3-II GAPDH GAPDH 36KD 36KD K562 THP-1 K562 Con Tet Con+ATG7 KO Tet+ATG7 KO Tet+ATG7 KO ** Con+ATG7 KO 50 Tet CD14-FITC (fluore s cence intensity) Fig. 3 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Re lative cell number CD14 positive cells (%) THP-1 K562 CD14 positive Re lative cell number cells (%) THP-1 K562 Wu et al. Cell Death and Disease (2018) 9:473 Page 7 of 16 (see ﬁgure on previous page) Fig. 3 Autophagy played an important role in tetrandrine-induced differentiation. The bars indicate the S.D. (a) K562 and THP-1 cells were treated with the indicated concentration of tetrandrine for 24 h or (b) with 2 μM tetrandrine for the indicated durations. Western blot analysis of LC3 levels. GAPDH was used as a loading control. (c) K562 cells were transfected with the GFP-LC3 plasmid for 24 h, subsequently treated with DMSO or 2 μM tetrandrine for 24 h and observed by ﬂuorescence microscopy. Representative experiments are shown to indicate the cellular localization patterns of the GFP-LC3 fusion protein (magniﬁcation ×400), and percentage of cells with GFP-LC3 puncta were used to quantify the percentage of autophagic cells. n = 3, ***p < 0.001. (d) After a 1 h pretreatment with 10 μM CQ and 48 h of subsequent treatment with DMSO or 2 μM tetrandrine, ﬂuorescence microscopy detected green and red ﬂuorescent spots in K562 cells transfected with the ptfLC3 plasmid and percentage of cells with red dots were used to quantify the autophagic ﬂux, while yellow dots (the overlay of green and red ﬂuorescence) were increased if the process of autophagosomes fusing with lysosomes was inhibited. n = 3, *p < 0.05, **p < 0.01. (e) GFP-LC3-transfected cells were pretreated with or without 1.5 mM 3-MA for 1 h. The cells were then exposed to DMSO or 2 μM tetrandrine for 24 h, and the localization of GFP-LC3 was observed using a ﬂuorescent microscope (magniﬁcation ×400). n = 3, **p < 0.01. (f) CD14 antigen expression was analyzed by ﬂow cytometry after treatment for 4 days with or without pretreatment with 1.5 mM 3-MA for 1 h, and a statistic analysis for three experiments in the bottom. *p < 0.05. (g) The expression of ATG7 and LC3-II proteins in K562 ATG7 KO cells were assessed via western blot, and K562 WT cells were used as a positive control. (h) CD14 expression was determined by ﬂow cytometry in K562 ATG7 and K562 WT cells after treatment for 4 days, and a statistic analysis for three experiments in the right. **p < 0.01. WT wild type volume and lower tumor weight in mice treated with the tetrandrine, trypan blue dye-exclusion assay and THP-1 cells overexpressing the control vector compared Annexin-V/PI staining showed that tetrandrine had sig- to those in the mice treated with vehicle, but had a weak niﬁcant anti-leukemia effects (Fig. 7a, b). Next, we found effect on tumors wherein c-MYC was overexpressed tetrandrine facilitated increase CD14 and CD11b expres- (Fig. 6a, b). Notably, we found that tetrandrine treatment sion on the surface of patient cells compared to vehicle was well tolerated by all mice as the animals did not treatment (Fig. 7c, d). As shown in Fig. 7f, western blot display weight loss (Figure S5A). Immunohistochemistry results also showed that tetrandrine treatment promoted results showed that tetrandrine effectively promoted CD14 expression and inhibited c-MYC levels. In addition, CD14 expression and decreased c-MYC expression in the tetrandrine-treated cells dramatically increased the per- mice with THP-1 cells overexpressing the control vector centage of acridine orange-positive cells and up-regulated but had minimal effects on tumors overexpressing c-MYC LC3-II levels (Fig. 7e, f). And we also found that the (Fig. 6c). Moreover, the levels of the lipid peroxidation intracellular ROS levels were signiﬁcantly increased in the product malondialdehyde (MDA), which was used as a tetrandrine-treated patient cells (Fig. 7g). Tetrandrine- presumptive measure of the ROS levels, were higher in induced CD14 expression was also prevented by NAC and both the THP-1 vector and THP-1 c-MYC tumor tissues 3-MA in the patient cells (Figure S6B and C). These following tetrandrine treatment than in those treated with results revealed that tetrandrine may effectively regulated vehicle (Fig. 6d). Finally, decreased c-MYC and increased proliferation, apoptosis, differentiation, autophagy and LC3-II protein levels were detected by western blot in the ROS accumulation in primary leukemia cells. Moreover, tetrandrine-treated THP-1 vector tumors (Fig. 6e). ROS activation and autophagy may be associated with Immunohistochemistry also showed more LC3 protein in tetrandrine-induced differentiation. the tetrandrine-treated THP-1 vector tumors than THP-1 c-MYC tumors (Figure S5B). These results demonstrate Tetrandrine at 2 μM had no cytotoxicity or effects on that tetrandrine exhibited good antitumor activity in vivo, differentiation in human CD34+ progenitor cells and the potential mechanism was associated with the In this study, the effect of 2 μM tetrandrine on human induction of tumor cells autophagy and differentiation by CD34+ progenitor cells has been studied. As shown in inhibiting c-MYC expression. Fig. 8, the results showed that 2 μΜ terandrine has no effect on human CD34+ progenitor cells viability and Tetrandrine affected differentiation in AML patient cells differentiation but may be trigger autophagy. In addition, To determine the effects of tetrandrine on human pri- we also found that tetrandrine has no signiﬁcant effects mary leukemia cells, here, cells were obtained from four on cell viability and CD14 expression but triggered AML patients who received no chemotherapy. Because of autophagy of healthy mouse haematopoietic stem cells the small number of cells isolated from the patient 2# and (Figure S7). 3# blood samples, apoptosis, acridine orange staining, western blot and ROS levels analysis were just validated in Discussion patient 1# and 4#. As shown in Figs. S6A, 2–3 μM tet- Studies have demonstrated that induced cell differ- 30–34 randrine can inhibit cell proliferation. When the cells entiation can effectively treat leukemia . However, the were treated with the indicated concentrations of side effects of drugs are of primary consideration. Based Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 8 of 16 a b 24h 12h K562 THP-1 Tet Tet+NAC Con NAC Tet Con Tet Con Tet+Tiron Tiron Tet Con Intracellular ROS Intracellular ROS Con Tet 24h Con Tet Tiron Tet+Tiron Con Tet NAC Tet+NAC ** * ** 100 ** 100 *** *** 50 50 0 0 K562 THP-1 K562 THP-1 K562 THP-1 K562 THP-1 THP-1 K562 Tet+NAC Tet - + - + - + - + NAC - - + + - - + + NAC Tet CD14 55KD Con 36KD GAPDH Tet+Tiron Tiron Tet Con K562 THP-1 CD14-FITC (fluorescence intensity) Tet - + - + - + - + Con Tet - - + + - - + + NAC Con Tet Tiron Tet+Tiron NAC Tet+NAC LC3-I 18KD ** ** * 16KD * LC3-II 36KD GAPDH K562 THP-1 K562 THP-1 f g K562 Con Tet NAC Tet+NAC Tet+3-MA 1.09% 52.7% 1.92% 10.7% 3-MA Tet Con 0.127% 52.4% 0.041% 1.7% Tet+ATG7 KO Con+ATG7 KO Tet Con FL1-Gre e n Intracellular ROS Fig. 4 Tetrandrine induced accumulation of intracellular ROS, leading to cell autophagy and differentiation. The bars indicate the S.D. *p < 0.05, **p < 0.01, ***p < 0.001. (a) Intracellular ROS levels were assessed by ﬂow cytometry after treatment with DMSO or 2 μM tetrandrine for 12 h or 24 h. (b) The cells were pretreated with NAC (K562 with 15 mM and THP-1 with 10 mM) and Tiron (K562 with 0.5 mM and THP-1 with 0.2 mM) for 1 h, and then treated with DMSO or 2 μM tetrandrine for 24 h. The intracellular ROS levels were measured by ﬂow cytometry (c) or CD14 expression was measured by ﬂow cytometry after 4 days of treatment. (d) CD14 and GAPDH levels were analysis by western blot after the indicated treatments for 4 days. (e) After 24 h of treatment, the LC3 and GAPDH levels were measured by western blots and (f) cells were stained with acridine orange and then analyzed by ﬂow cytometry. (g) After 24 h of DMSO or 2 μM tetrandrine treatment, ﬂow cytometry was used to detect the generation of ROS in K562 ATG7 KO cells and K562 cells pretreated with 1.5 mM 3-MA Ofﬁcial journal of the Cell Death Differentiation Association CD14 positive cells (%) Relative cell number Intracellular Relative cell number FL3-Orange ROS (%) CD14 positive cells (%) THP-1 K562 Intracellular ROS (%) THP-1 K562 Re lative ce ll numbe r Re lative cell numbe r Intracellular ROS (%) Wu et al. Cell Death and Disease (2018) 9:473 Page 9 of 16 T et 2μM ac b K562 THP-1 0 3 6 9 12 24 48 72h 65KD c-MYC Con Tet 1.5 GAPDH 36KD * * c-MYC 65KD 65KD 0.5 c-MYC * * GAPDH 36KD GAPDH 36KD K562 THP-1 THP-1 d K562 e K562 THP-1 Con Tet Con Tet c-MYC-Tet c-MYC-Con Vector-Tet Vector-Con CD14-FITC (fluore s cence intensity) Con Tet *** *** NS NS Con Tet 60 ** ** 40 NS NS Vector c-MYC Vector c-MYC K562 THP-1 Vector c-MYC Vector c-MYC K562 THP-1 g K562 THP-1 - + - + -+ - + Tet - - + + - - + + K562 10058-F4 f THP-1 Vector c-MYC Vector c-MYC 65KD c-MYC - + - + - + - + Tet GAPDH 36KD CD14 55KD C-MYC 65KD i K562 THP-1 GAPDH 36KD c-MYC-Tet c-MYC-Con Vector-Tet Vector-Con K562 THP-1 Intracellular ROS Tet+10058-F4 10058-F4 K562 THP-1 Tet Ve ctor c-M YC Ve ctor c-M YC Con -+ - + - + - + Tet CD14-FITC 18KD (fluore s cence intensity) LC3-I long exposition 16KD Con Tet LC3-II 10058-F4 Tet+10058 -F4 ** short exposition LC3-II 16KD GAPDH 36KD K562 THP-1 LC3-II / GAPDH 0.61 3.02 0.4 1.25 1.29 3.57 0.73 1.63 Fig. 5 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Re lative cell c-MYC Vector CD14 positive numbe r cells (%) % differentiation THP-1 K562 Relative mRNA expression Re lative cell Re lative cell numbe r numbe r CD14 positive cells (%) Wu et al. Cell Death and Disease (2018) 9:473 Page 10 of 16 (see ﬁgure on previous page) Fig. 5 Tetrandrine induced differentiation by inhibiting c-MYC expression. The bars indicate the S.D. (a) Western blot analysis of c-MYC and GAPDH levels in cells treated with 2 μM tetrandrine for different durations. (b) qRT–PCR analysis of c-MYC expression in cells after 24 h of treatment. n = 3. **p < 0.01. (c) Western blot analysis of c-MYC and GAPDH levels in cells overexpressing either vector or c-MYC. (d) Wright-Giemsa staining was used to assess cell morphology after 4 days of DMSO or 2 μM tetrandrine treatment in cells overexpressing either vector or c-MYC. The arrows indicate lower nucleus/cytoplasm ratio and the cells with horseshoe shape nuclei. **p < 0.01, NS, not signiﬁcant. (e) After 4 days treatment, CD14 expression was detected by ﬂow cytometry and (f) the CD14 and c-MYC levels were measured by western blot. n = 3. ***p < 0.001, NS, not signiﬁcant. (g) The cells were pretreated with 40 μM 10058-F4 and then treated with DMSO or 2 μM tetrandrine for 24 h. Western blot analysis of c- MYC and GAPDH levels. (h) Flow cytometry was used to analyze CD14 expression after the indicated treatments for 4 days. (i) Intracellular ROS was assessed via FACS analysis in cells overexpressing either vector or c-MYC after treatment with DMSO or 2 μM tetrandrine for 24 h. (j) Western blot analysis of LC3 and GAPDH levels on a long history of clinical applications of traditional MYC protein can regulate terminal differentiation of 43–45 Chinese medicine, tetrandrine is considered to be a safe hematopoietic cells . In our study, tetrandrine was agent. Tetrandrine has been used for cancer chemopre- shown to reduce c-MYC protein expression. Then, over- 14,35,36 vention and therapy . In this study, we demonstrated expression of c-MYC in K562 and THP-1 cells inhibited that tetrandrine can inhibit leukemia cell proliferation and tetrandrine-induced cell differentiation, and animal induce differentiation. Mechanistically, tetrandrine- experiments also validated these results. Moreover, we induced differentiation was mainly associated with the found that 40 μM 10058-F4 can enhance tetrandrine- stimulation of ROS and autophagy. Moreover, inhibited c- induced differentiation (Fig. 5h) but 80 μM 10058-F4 MYC protein expression played a critical role in alone induce K562 cells differentiation (date not shown). tetrandrine-induced leukemia cell differentiation. However, the signaling pathways involved in tetrandrine- Most normal cell growth and development requires a induced differentiation that are modulated by c-MYC well-controlled balance of protein synthesis and organelle remain unclear and will require further investigation. biogenesis to protein degradation and organelle renewal. In summary, we demonstrated that tetrandrine has the The major pathways for the degradation of cellular con- potential to treat leukemia. Above all, we discovered that stituents are autophagy and cytosolic turnover by the tetrandrine can induce autophagy and differentiation both proteasome . Differentiation of cells is usually associated in vitro and in vivo. The potential molecular mechanisms involve activation of ROS accumulation with slowed cell growth, which is caused by an altered rate of macromolecule synthesis and degradation. Research and inhibition of c-MYC expression. These studies has shown that autophagy can regulate myeloid cell dif- provide the rationale for application of tetrandrine in 22,38 ferentiation . In this study, we observed that early clinical therapies and in therapeutic regimens for autophagy mediated the tetrandrine-induced differentia- leukemia patients. tion. Further inhibition of autophagy by knockout of ATG7 gene decreased tetrandrine-induced differentiation Materials and methods in K562 cells. Moreover, others have reported that Cell lines and cell culture autophagy is required for differentiation of non- HL60 cells were purchased from CCTCC (China Center hematologic cells and tissues. Together, these results for Type Culture Collection; Wuhan, China). K562, U937 show that autophagy is important for cell differentiation. and THP-1 cells were kindly provided by Dr. Zan Huang ROS have emerged in recent years as important reg- (Wuhan University). All cell lines were grown in RPMI ulators of cell division and differentiation . There are 1640 medium supplemented with 10% FBS (fetal bovine reports that ROS are produced in the early stage of serum, Hyclone), 1% streptomycin and 1% penicillin. monocyte–macrophage differentiation. ROS generation AML patient cells were provided by Dengju Li (Tongji blockade induced by BHA, TEMPO, NAC and apocynin Hospital of Tongji Medical College; Wuhan, China). The 40,41 speciﬁcally inhibits M2 macrophage differentiation .In informed consent was obtained from all of the examined the present study, we observed that tetrandrine treatment subjects, and the related studies were approved by the signiﬁcantly stimulated ROS generation. We also ethics committees of the participating hospitals and observed that the ROS scavengers NAC and Tiron can institute. AML patient cells were cultured in expansion inhibit tetrandrine-induced leukemia cell autophagy and media (RPMI 1640 with 10% FBS, 10 ng/ml recombinant differentiation. These results suggest that tetrandrine human IL-3, 10 ng/ml rhIL-6, and 50 ng/ml stem cell accelerated intracellular ROS generation that is important factor). All cells were cultured in a humidiﬁed atmosphere for tetrandrine-induced autophagy and differentiation. containing 5% CO at 37 °C. Cell culture dishes and plates The MYC oncogene contributes to the genesis of many were purchased from Wuxi NEST Biotechnology Co. Ltd. human cancers . Many reports have indicated that c- (Wuxi, China). Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 11 of 16 THP-1 Vector THP-1 Vector P=0.1546 0 mg/kg 2000 25 mg/kg P=0.000276 50 mg/kg P=0.0121 P=0.000846 Vehicle 1d 3d 5d 7d 9d 11d 13d Tet 25mg/kg Tet 50mg/kg THP-1 c-MYC THP-1 c-MYC P=0.1317 0 mg/kg 3000 P=0.1048 P=0.073 25 mg/kg P=0.02 50 mg/kg 1d 3d 5d 7d 9d 11d 13d Vehicle Te t 25mg/kg Te t 50mg/kg THP-1 Vector THP-1 c-MYC P=0.0116 P=0.0469 P=0.4806 P<0.0001 Vehicle Tet 25 mg/kg Tet 50 mg/kg Vehicle Tet 25 mg/kg Tet 50 mg/kg Vehicle T et 25 mg/kg T et 50 mg/kg Vehicle T et 25 mg/kg T et 50 mg/kg 1# 2# 3# 4# 1# 2# 3# 4# 1# 2# 3# 4# 1# 2# 3# 4# 1# 2# 3# 4# 1# 2# 3# 4# LC3-I 18KD LC3-II 16KD 65KD c-MYC 16KD GAPDH THP-1 Vector THP-1 c-MYC Fig. 6 Tetrandrine induced differentiation in an in vivo xenograft model. The bars represent the mean ± S.D. (a), (b) Tumor volume was measured daily. The tumors were removed by dissection and weighed after 13 days of treatment. (c) CD14 and c-MYC expression were evaluated by immunohistochemistry analysis in tumor tissues. Magniﬁcation: ×400. (d) Tumor tissue proteins were extracted from the THP-1 vector and c-MYC xenografts and were subjected to MDA assay to analyze tissue ROS levels. (e) Western blot analysis of c-MYC, LC3 and GAPDH levels in tumor tissues Ofﬁcial journal of the Cell Death Differentiation Association Tumor Volume (cm ) 3 Tumor Volume (cm ) MDA (μmol/mg tumor tissue protein) MDA (μmol/mg tumor Tumor weight (g) Tumor weight (g) tissue protein) Wu et al. Cell Death and Disease (2018) 9:473 Page 12 of 16 Con T et 2 μM T et 3 μM 24h 48h patient 1# patient 4# Te trandrine (µM ) Anne xin-V-FITC patient 1# patient 2# patient 3# patient 4# patient 1# patient 2# patient 3# patient 4# Tet Con 77.1% 87.4% 96.4% 95.1% CD14-FITC (fluore s cence intensity) 62.6% 69.5% 64.3% 57.3% CD11b-PE Tet Con 8.19% 31.4% patient 1# patient 1# patient 4# -+ -+ Tet LC3-I 18KD 2.5% 21.9% LC3-II 16KD patient 4# 55KD CD14 FL1-Gre e n c-MYC 65KD 36KD GAPDH patient 4# patient 1# Tet Con Intracellular ROS Fig. 7 Tetrandrine induced differentiation and autophagy in AML patient cells. (a) Cell viability was analyzed by trypan blue dye-exclusion assays. The bars indicate the S.D. n = 3. (b) Patient #1 and #4 apoptotic cells were analyzed by ﬂow cytometry after treatment with tetrandrine at the indicated concentrations. (c) Analysis of CD14 or (d) CD14 and CD11b expression by ﬂow cytometry after DMSO or 2 μM tetrandrine treatment in all patient cells for 3 days. (e) The patient #1 and #4 cells were treated with DMSO or 2 μM tetrandrine for 24 h, stained with acridine orange, and then analyzed by ﬂow cytometry. (f) Western blot analysis of LC3, CD14, c-MYC and GAPDH in tetrandrine-treated #1 and #4 patient cells. (g) After 24 h treatment, the intracellular ROS levels were assessed via ﬂow cytometry analysis in #1 and #4 patient cells Ofﬁcial journal of the Cell Death Differentiation Association Re lative cell Re lative ce ll FL3-Orange numbe r cell viability (%) numbe r CD14-FLTC PI Tet Con Wu et al. Cell Death and Disease (2018) 9:473 Page 13 of 16 a b Con Tet 24h 48h 72h NS NS NS NS NS NS NS NS Anne xin-V-FITC NS 0 10 Te trandrine (µM) Con Tet Tet Con 0.073% 43.6% Tet Con CD14-FITC (fluore s cence intensity) FL1-Green NS Con Tet Fig. 8 Tetrandrine at 2 μM had no toxicity on human CD34 + progenitor cells. The bars indicate the S.D. *p < 0.05. (a) Cell viability of human CD34+ progenitor cells was assessed by MTS assay. (b) Apoptotic cells were detected by ﬂow cytometry in CD34+ cells following treatment with DMSO or 2 μM tetrandrine for 48 h. (c) After 24 h of treatment with DMSO or 2 μM tetrandrine, cells were stained with acridine orange and analyzed by ﬂow cytometry. (d) The expression of CD14 in the CD34+ cells was measured by ﬂow cytometry after treated with DMSO or 2 μM tetrandrine for 4 days. NS not signiﬁcant Chemical reagents and antibodies (goat–anti-rabbit and goat–anti-mouse) were acquired Tetrandrine was acquired from Shanghai Ronghe from Beyotime (Nantong, China). Medical (Shanghai, China). Wright-Giemsa stain was obtained from Baso (Zhuhai, China). DCFH-DA was from Cell proliferation and viability assays Invitrogen (Carlsbad, CA). Acridine orange, 3-MA, Tetrandrine was dissolved in DMSO to a ﬁnal con- DMSO, NAC, and Tiron were purchased from Sigma- centration of 10 mM and then stored at −80 °C. For Aldrich (St. Louis, MO). NBT was from Beyotime (Nan- proliferation and viability assays, 3000 cells (K562 and tong, China). The FITC Annexin V Apoptosis Detection HL60) or 5000 cells (THP-1 and U937) per well were Kit I, CD14-FITC and CD11b-PE were obtained from BD seeded in a 96-well plate with 100 μl medium, cultured for Biosciences. Red blood cell lysis buffer, IL-3, IL6 and stem 24, 48 and 72 h in the presence of varying concentrations cell factor were kindly provided by Dr. Zan Huang of tetrandrine or DMSO (con). The total cell count was (Wuhan University). MTS was acquired from Promega then detected by a hemocytometer, and cell viability was (Madison, USA). The antibody against LC3 was from measured as the percentage of living cells demonstrated Sigma-Aldrich (St. Louis, MO). The caspase-3, PARP, and by the trypan blue dye-exclusion assay according to ATG7 antibodies were obtained from Cell Signaling established protocols. Technology (Beverly, MA). Antibodies against CD14 and c-MYC were purchased from Proteintech Group Inc. Cell cycle analysis (Chicago, IL). The anti-GAPDH antibody and the horse- Cells were cultured for 48 h in the presence of 2 μM radish peroxidase (HRP)-conjugated secondary antibodies tetrandrine or DMSO (con). The cells were collected and Ofﬁcial journal of the Cell Death Differentiation Association cell viability (%) FL3-Orange PI CD14 positive Apoptotic cells % Re lative cell numbe r cells (%) Wu et al. Cell Death and Disease (2018) 9:473 Page 14 of 16 washed with PBS and then ﬁxed with 70% ethanol for at cells were plated in 12-well plates and treated with 2 μM least 4 h at 4 °C. The ﬁxed cells were collected and washed tetrandrine for 24 h. Cells were then stained with acridine twice with PBS, suspended in cold PBS containing 50 μg/ orange (1 μg/ml) at 37 °C for 25 min before observation. ml of PI and 100 μg/ml of Rnase A and then kept in the The red acidic vesicular organelles (AVOs) in autophagy dark for 30 min. The samples were transferred to the ﬂow cells were measured by ﬂow cytometry or visualized by cytometer (Beckman, Indianapolis, CA, USA) and cell ﬂuorescence microscopy. ﬂuorescence were measured. The data were analyzed using FlowJo software (TreeStar, San Carlos, CA, USA). Measurement of intracellular ROS levels The cell-permeant probe, DCFH-DA, which ﬂuoresces Cell apoptosis analysis by ﬂow cytometry when it is oxidized, was used to measure intracellular ROS Cells were treated with 2 μM tetrandrine or DMSO levels. Cells were treated with DMSO or the indicated (con) for 48 h, collected, washed twice with PBS, resus- concentrations of tetrandrine in a 12-well plate for 12 h pended in binding buffer and then dyed with Annexin V- and 24 h. Then, cells were collected and washed with PBS FITC and PI for 15 min in the dark according to the and resuspended in 500 μl of serum-free RPMI 1640 manufacturer’s instructions. Annexin V ﬂuorescence was medium containing 0.5 μl DCFH-DA at 37 °C for measured by ﬂow cytometry, and the membrane integrity 30 min. The prepared cells were evaluated using ﬂow of the cells was simultaneously assessed by the PI exclu- cytometry. sion method. Western blot analysis Morphological observation After treatment, cells were collected and washed with Cell morphology was determined by a Wright-Giemsa PBS and then lysed in 1% sodium dodecyl sulfate (SDS) on staining assay following 4 days of 2 μM tetrandrine ice. Cell lysates were heated to 98 °C for 15 min and then treatment. Cells were collected and smeared on micro- centrifuged at 12,000×g for 15 min. The supernatant was scope slides from Citoglas (Jiangsu, China). After Wright- collected, and protein concentrations were assessed using Giemsa staining, the slides were cleaned gently, observed the Bicinchoninic Acid Protein Assay Kit (Thermo sci- under a light microscope (CKX41, Olympus Optical Co., entiﬁc). Equal amounts of protein were separated by Ltd.) and photographed. SDS–PAGE and transferred to a PVDF membrane (Mil- lipore), which was then immunoblotted with the indicated antibodies. NBT reduction assay For the NBT reduction analysis, cells were treated with 2 μM tetrandrine for 0, 4, 5 and 6 days. Then, the cell Quantitative real-time PCR suspensions were incubated with 1 mg/ml of NBT and 20 Cells were treated with 2 μM tetrandrine or DMSO for ng/ml of TPA in an equal volume of RPMI1640 for 30 24 h. Total RNA was isolated using the Total RNA Kit I min at 37 °C. After 30 min, the cells were washed with (Omega Bio-Tek, Inc., GA). Then, RNA was transcribed PBS and resuspended in 100 μl of PBS. NBT was evaluated into cDNA using the Transcriptor First Strand cDNA both as the percentage of positive cells and the intensity of Synthesis Kit (Roche Life Science, USA) according to the reduction when measured at a wavelength of 570 nm with manufacturer’s instructions. qRT–PCR was performed microplate readers (SpectraMax M5). using the FastStart Universal SYBR Green Master kit (Rox) (Roche Life Science, USA) on the Applied Biosys- Detection of CD14 and CD11b expression by ﬂow tems 7500 Fast Real-Time PCR System (PerkinElmer, cytometry Torrance, CA). The following primer pairs were used for Cells were plated in 12-well plates and mixed with 2 μM qRT–PCR: c-MYC: forward, 5′-CACCGAGTCGTAGTC tetrandrine for indicated time. To measure the expression GAGGT-3′ and reverse, 5′-TTTCGGGTAGTGGAAAA of differentiation markers CD14 or CD11b, cells were CCA-3′. GAPDH: forward, 5′-TCCACCACCCTGTTG washed twice in cold PBS after dilution to a density of 1 × CTGTA-3′ and reverse 5′-ACCACAGTCCATGCCA 10 cells per well, and then incubated with the CD14- TCAC-3′. All reactions were performed in triplicate in a FITC and/or CD11b-PE antibody for 20 min in the dark. 20-μl reaction volume. Fold changes in gene expression −ΔΔCt Samples were washed with cold PBS and measured by were determined using the 2 method with GAPDH ﬂow cytometry. as an endogenous control. Acridine orange staining assay Plasmids and transient transfection Acridine orange staining can detect intracellular acidic The GFP-LC3 plasmid was kindly provided by Dr. autophagic vacuoles by ﬂow cytometry or ﬂuorescence Tamotsu Yoshimori (National Institute of Genetics, microscopy. For the acridine orange staining assay, the Mishima, Japan). The pEGFP-mRFP tandem ﬂuorescent- Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 15 of 16 tagged LC3 (ptfLC3) plasmid was purchased from Immunohistochemistry Addgene (Cambridge, MA). To detect autophagy and The tumor tissue sections were ﬁxed by 4% paraf- autophagic ﬂux, the cells were seeded in a 12-well plate, ormaldehyde, embedded in parafﬁn and sliced at a 5 μm then transfected with the GFP-LC3 or ptfLC3 plasmids thickness for immunohistochemical analysis. After for 24 h, and then treatment with indicated drugs to deparafﬁnization and the appropriate epitope retrieval, present autophagy and autophagic ﬂux. After treatment the sections were stained with c-MYC, CD14 and LC3 the cells were collected and observed with a ﬂuorescence antibodies and further incubated with biotinylated microscope (Olympus BX51). goat–anti-rabbit antibodies. The speciﬁc signals were then detected with streptavidin-conjugated HRP and Plasmids and lentiviral transfection diaminobenzidine as the chromogen. The pHAGE.puro-c-MYC plasmid and the empty vec- tor plasmid were kindly provided by Dr. Li Y (College of Healthy progenitor cell study Life Sciences, Wuhan University, China). Transfection Mouse hematopoietic stem cells and human CD34+ reagents were kindly provided by Dr. Xiaodong Zhang progenitor cells were used to measure tetrandrine drug (College of Life Sciences, Wuhan University, China). In toxicity. ICR mice were obtained from the Disease Pre- our study, HEK-293T cells were transfected with either vention Center of Hubei Province. In the experiment, the c-MYC plasmid or an empty vector together with the mice embryos were collected after 12.5 days of conception pMD2.G and psPAX2 plasmids using transfection and the fetal livers were titrated with a pipette needle to reagents. At 48 h post-transfection, the supernatants were obtain a single-cell suspension. Human CD34+ pro- collected and ﬁltered with 0.45-μm ﬁlters. Then, K562 genitor cells were provided by Dr. Zan Huang (Wuhan and THP-1 cells were cultured with the supernatants University). Then, cells were cultured with complete containing 5 μg/ml polybrene. After 24 h, the virus- RPMI 1640 media containing IL-3 and IL6 at 10 ng/ml containing medium was replaced by fresh medium with and stem cell factor at 100 ng/ml. To induce the cells to 2 μg/ml puromycin. Stable cells were selected with differentiate, cells were cultured in differentiation media puromycin. (RPMI 1640 with 10% FBS, 10 ng/ml stem cell factor and 50 ng/ml GM-CSF). In this study, mouse cells used mouse Tumor xenograft model cytokines and human cells with human cytokines. The xenograft model in athymic nude mice was per- formed to evaluate the in vivo efﬁcacy of tetrandrine. Statistical analysis Animal experimental protocols and care were approved Data are expressed as the mean ± S.D. A two-tailed by the Experimental Animal Center of Wuhan University. unpaired Student’s t-test was used to analyze data con- Male nude mice of 4 to 5 weeks of age were obtained from taining two groups unless otherwise speciﬁed. Statistical the Model Animal Research Center (Changsha, China). signiﬁcance was denoted as follows: NS, not signiﬁcant; THP-1 cells (~1 × 10 cells) and the same number of *P < 0.05, **P < 0.01 and ***P < 0.001 were deemed statis- THP-1 cells overexpressing c-MYC in a total volume of tically signiﬁcant. 0.2 mL of PBS were inoculated subcutaneously over the right ﬂank of each mouse. Tumor diameter and body Acknowledgements weights were measured every day. Tumor volume was This study was supported by the National Basic Research Program of China (2014CB910600), the National Natural Science Foundation of China (81273540, calculated by the following formula: 0.52 × length × 81472684 and 81770168) and Fundamental Research Funds for the Central width . Mice were randomized into three groups Universities (2042017KF0242). (ﬁve per group) when the tumor volume reached ~50 mm . There were two treatment groups of mice that Author details received tetrandrine at 25 or 50 mg/kg, while the other Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan mouse group was given a vehicle treatment of 0.5% University, Wuhan 430072, P. R. China. Department of Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and methylcellulose. Technology, Wuhan, P. R. China Malondialdehyde (MDA) assay Author contributions Mice were killed after 13 days of tetrandrine treatment, G.W. performed the research, analyzed the data and wrote the manuscript; T.L. and tumor tissues were removed. For the MDA assay, provided the ATG7 knockout K562 cells; H.L. and Y.L. analyzed and interpreted tissue proteins were prepared according to the description the data; D.L. provided the AML patient cells and W.L. designed the research and wrote the manuscript. in the Lipid Peroxidation MDA assay kit (Beyotime, Nantong, China). The MDA levels were evaluated by Multi-Mode Microplate Readers (SpectraMax M5) at 532 Conﬂict of interest nm using 490 nm as a control. The authors declare that they have no conﬂict of interest. Ofﬁcial journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2018) 9:473 Page 16 of 16 Publisher's note 21. Karvela, M. et al. ATG7 regulates energy metabolism, differentiation and sur- Springer Nature remains neutral with regard to jurisdictional claims in vival of Philadelphia-chromosome-positive cells. Autophagy 12,936–948 published maps and institutional afﬁliations. (2016). 22. Isakson, P., Bjoras, M., Boe, S. O. & Simonsen, A. 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Journal

Cell Death & Disease – Springer Journals

Published: Apr 27, 2018

References

Differentiation therapy of leukemia: 3 decades of development

Nowak, D; Stewart, D; Koeffler, HP
Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide

Friend, C; Scher, W; Holland, J; Sato, T
Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid

Breitman, TR; Selonick, SE; Collins, SJ
15/17 translocation, a consistent chromosomal change in acute promyelocytic leukaemia

Rowley, JD; Golomb, HM; Dougherty, C
Probable in vivo induction of differentiation by retinoic acid of promyelocytes in acute promyelocytic leukaemia

Nilsson, B
Retinoic acid as antileukemic therapy in a patient with acute promyelocytic leukemia and Aspergillus pneumonia

Daenen, S; Vellenga, E; Dobbenburgh, OA; Halie, MR
Anti-inflammatory and immunosuppressive properties of the bis-benzylisoquinolines: in vitro comparisons of tetrandrine and berbamine

Li, SY; Lh, Ling; Teh, BS; Seow, WK; Thong, YH
Immunomodulatory effects and mechanisms of plant alkaloid tetrandrine in autoimmune diseases

Lai, JH
Tetrandrine induces apoptosis and growth suppression of colon cancer cells in mice

Wu, JM; Chen, Y; Chen, JC; Lin, TY; Tseng, SH
Tetrandrine induces apoptosis and triggers caspase cascade in human bladder cancer cells

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c-MYC and reactive oxygen species play roles in tetrandrine-induced leukemia differentiation

c-MYC and reactive oxygen species play roles in tetrandrine-induced leukemia differentiation

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c-MYC and reactive oxygen species play roles in tetrandrine-induced leukemia differentiation

c-MYC and reactive oxygen species play roles in tetrandrine-induced leukemia differentiation

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