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Trans-vaccenic acid inhibits proliferation and induces apoptosis of human nasopharyngeal carcinoma cells via a mitochondrial-mediated apoptosis pathway

Trans-vaccenic acid inhibits proliferation and induces apoptosis of human nasopharyngeal... Background: Intake of trans fatty acids (TFAs) from partially hydrogenated vegetable oil is associated with a variety of adverse outcomes, but little is known about the health effects of ruminant trans fats. Trans-vaccenic acid (TVA) is a naturally occurring TFA found in the fat of ruminants and in human dairy products. The present study was conducted to investigate the anticancer activity and underlying mechanisms of TVA on human nasopharyngeal carcinoma (NPC) 5-8F and CNE-2 cells. Methods: A CCK8 assay was used to determine the effect of TVA and the Mcl-1 inhibitor S63845 on the proliferation of NPC cells. Apoptosis was measured using flow cytometry. Western blotting was used to detect the protein expression levels of factors associated with Bcl-2-family protein signaling and Akt signaling. Results: TVA significantly inhibited cell proliferation in a dose-dependent manner. Mechanistic investigation demonstrated that TVA significantly decreased p-Akt levels and Bad phosphorylation on Ser-136 and Ser-112. More importantly, we discovered that the Mcl-1 inhibitor S63845 synergistically sensitized NPC cells to apoptosis induction by TVA. Conclusion: TVA can inhibit NPC cell growth and induced apoptosis through the inhibition of Bad/Akt phosphorylation. The combined use of TVA and Mcl-1 inhibitors offers a potential advantage for nasopharyngeal cancer treatment. Keywords: Trans-vaccenic acid, Apoptosis, Nasopharyngeal carcinoma, Akt, Bad, Mcl-1 Introduction different sources cause various biological effects on hu- Trans fatty acids (TFAs) is a general term for unsatur- man health that may be beneficial or unfavorable [2, 3]. ated fatty acids with at least one double bond in the The impact of TFAs on the cardiovascular system has trans configuration [1]. TFAs within the human diet are been extensively studied, and many epidemiological mainly derived from industrial partial hydrogenation of investigations and experiments have shown that TFAs vegetable oils and from natural sources, such as rumin- from partially hydrogenated oils have adverse effects on ant animal products. Evidence suggests that TFAs from the cardiovascular system [4]. TFAs formed via indus- trial hydrogenation could significantly accelerate the development of atherosclerosis by increasing the ratio of low-density lipoprotein (LDL) to high-density lipopro- * Correspondence: nghui@21cn.com tein (HDL) [5]. In addition, many other studies have Department of Otolaryngology, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen 518035, People’s shown that these types of TFAs also have adverse effects Republic of China on blood lipids [6], inflammation [7], oxidative stress [8], Institute of Translational Medicine, Shenzhen Second People’s Hospital, The endothelial health [9], body weight [10], insulin sensitiv- First Affiliated Hospital of Shenzhen University, Shenzhen 518035, People’s Republic of China ity [11] and cancer [12]. However, emerging evidence Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Song et al. Lipids in Health and Disease (2019) 18:46 Page 2 of 9 indicates that trans fats derived from milk or ruminant For treatments, the cells were grown in 6-well plates to body fats are beneficial for reducing the incidence of 60–70% confluence and were then treated with different cardiovascular disease, cancer and obesity [13]. concentrations of TFAs (EA, LA, and TVA; Nu-Chek Trans-vaccenic acid (TVA) is ubiquitous in ruminant- Prep) (0, 25, 50, 100, 200 μM) for 24 h. Equal derived fats and human dairy products such as milk and concentrations of DMSO were used as the controls. butter. It is worth noting that TVA is also the predomin- ant TFA in human milk. Supplementation with milk Cell viability assay lipids that contain TVA triggers a pronounced cytotoxic The effect of TFAs on the viability of NPC cells was effect on HT29 cell due to conversion to c9,t11-conju- measured by CCK8 assay. 5-8F and CNE-2 cells were gated linoleic acid (CLA) [14]. Suppression of tumor cell plated (1.5 × 10 cells) in 96-well culture plates and growth by TVA treatment of the MCF7 and SW480 cell incubated in serum-containing medium for 24 h. The lines can be attributed to the induction of apoptosis medium was then replaced with serum-free media con- though increased DNA fragmentation and reduced cyto- taining different concentrations of TFA and/or S63845 solic glutathione levels [15]. Preclinical studies have (HY-100741, MedChem Express) for 24 h. After incuba- shown that the use of various types of fatty acids alone tion, cell proliferation was analyzed with a CCK8 assay or combined with other anticancer drugs has promising kit following the manufacturer’s instructions (CK04, therapeutic application prospects [16]. Dojindo Laboratories), and the absorbance at 450 nm Nasopharyngeal carcinoma (NPC), the most common was measured using a microplate reader (BioTek). cancer originating in the nasopharynx, has a high inci- dence in Southern China and Southeast Asia [17]. Annexin V and propidium iodide (PI) staining Radiotherapy is currently the preferred method of treat- The effects of the TFAs on apoptosis were determined ment for early-stage NPC because most NPCs are poorly by dual staining with annexin V-FITC and PI using an differentiated cancers with high sensitivity to radiation apoptosis detection kit from BD Bioscience. After treat- and because the primary and neck lymphatic drainage ment with the indicated TFA concentrations for 24 h, areas are easily included in the radiotherapy field [18]. the cells were harvested and washed with PBS. The cells Clinical treatment of recurrent or metastatic NPC is were then incubated with 5 μL of annexin V-FITC and more difficult than primary NPC treatment. The routine PI for 15 min. The fluorescence of the cells was analyzed treatment for these groups of patients is platinum-based by flow cytometry (Beckman Coulter). chemotherapy, which confers a median progression-free survival time of 7 months [19]. Therefore, it is urgent to Western blot assay identify a more effective treatment option for patients After treatment, lysates from the cultured cells were pre- with recurrent or metastatic NPC. pared with RIPA buffer, and the protein concentrations In the present study, we demonstrate that TVA effect- were determined with a BCA Protein Assay Reagent Kit ively induces NPC apoptosis in 5-8F and CNE-2 cells. (23227, Thermo Scientific). The lysates were separated Mechanism studies indicate that TVA significantly by electrophoresis on a 12% SDS-polyacrylamide gel and inhibits Akt/Bad phosphorylation. More importantly, we then transferred to PVDF Western Blotting Membranes found that TVA treatment also led to the upregulation (IPVH00010, Millipore). The membranes were blocked of Mcl-1 as a novel mechanism involved in TVA resist- with 5% milk and incubated at 4 °C with primary anti- ance, which could be overcome by treatment with the bodies against Bad (9292, Cell Signaling Technology, Mcl-1 inhibitor S63845. These results suggest that the 1:1000), phosphor-Bad (Ser-112) (5284, Cell Signaling combination of TVA and Mcl-1 inhibitors is a promising Technology, 1:1000), phosphor-Bad (Ser-136) (4366, Cell approach for NPC treatment strategies. Signaling Technology, 1:1000), Mcl-1 (94,296, Cell Signaling Technology, 1:1000), Bcl-xL (2764, Cell Signal- Materials and methods ing Technology, 1:1000), Bcl-2 (12789–1-AP, Protein- Cell culture and treatments tech, 1:1000), pan-Akt (4685, Cell Signaling Technology, The human NPC cell lines 5-8F and CNE-2 were a gen- 1:1000), p-Akt (Ser-473) (4060, Cell Signaling Technol- erous gift from Prof. Chao-Nan Qian at the State Key ogy, 1:1000), p-Akt (Thr-308) (13,038, Cell Signaling Laboratory of Oncology in South China and the Collab- Technology, 1:1000), and β-tubulin (10068–1-AP, orative Innovation Center for Cancer Medicine, Sun Proteintech, 1:5000). After overnight incubation with the Yat-Sen University Cancer Center. All NPC cell lines primary antibodies, the membranes were incubated with were maintained in RPMI-1640 medium (SH30809.01, secondary anti-mouse (115–035-003, Jackson, 1:5000) or HyClone) supplemented with 10% FBS (10099–141, anti-rabbit (111–035-003, Jackson, 1:5000) antibodies. Gibco) and 1% penicillin-streptomycin (15070–063, Then, the signals were detected with an Amersham Gibco) at 37 °C with 5% CO . Imager 600. 2 Song et al. Lipids in Health and Disease (2019) 18:46 Page 3 of 9 Calculation of the combination index flow cytometry analysis of annexin V/PI-stained cells. Whether the synergistic inhibitory effect between TVA We found that TVA significantly induced apoptosis in a and S63845 can be determined by the combination dose-dependent manner. Incubation of 5-8F cells with index (CI) using the Chou-Talalay equation [20]. 5-8F 25, 50 or 100 μM TVA for 24 h caused 7.67, 12.9% or and CNE-2cells were treated with various concentrations 35% increases in total apoptosis, respectively. Similarly, of TVA and S63845 separately or in combination. The after incubation with 25 50 or 100 μM CNE-2, the per- total inhibitory effect was assessed by CCK8 assay as de- centage of apoptotic cells was increased by 12.9, 15.1% scribed above. The CI value was determined as follow: or 22.3%, respectively (Fig. 2a). Cleaved poly (ADP-ri- bose) polymerase (PARP) and cleaved caspase-3 are DA; x DB; x widely used to detect apoptosis in cells. Therefore, we CI ¼ þ assessed the protein levels of cleaved PARP and cleaved ICx; A ICx; B caspase-3 in the presence or absence of TVA treatment. A, B represents two different agent, ICX, A and ICX, Immunoblot analysis revealed that TVA treatment sig- B are the concentration when the two agent are used nificantly increased the levels of cleaved PARP and alone to achieve a growth inhibition rate of X%, and cleaved caspase-3 in a time-dependent manner (Fig. 2c). DA,x and DB,x are the concentration of two agent com- bined to achieve a growth inhibition rate of X%. CI < 1 TVA induces apoptosis in NPC cells through Akt and bad indicate synergism, CI = 1indicates additive effects, and inactivation CI > 1 indicates antagonism. The Bcl-2-associated death promoter (Bad) is a pro- apoptotic member of the Bcl-2 family that can form a Statistical analysis heterodimer with the antiapoptotic proteins Bcl-2 and Statistical analysis was performed using the standard Bcl-XL and prevent them from inhibiting apoptosis [21]. Student t test for pair comparisons and ANOVA analysis We were thus interested in determining whether TVA for multiple factors. The statistical values of *P < 0.05, could affect endogenous Bad activity. The role of Bad in **P < 0.01 and ***P < 0.001 were considered statistically promoting apoptosis is mainly involves phosphorylation significant. Values of mean determinants are presented of Ser-136 and Ser-112. Bad is rapidly dephosphorylated as ± s.e.m. and transferred to the mitochondria to induce apoptosis in response to external stimuli. As shown in Fig. 3a and Results b, TVA treatment induced a decrease in Bad phosphor- TVA inhibits NPC cell viability in a dose-dependent ylation on Ser-136 and Ser-112 in a concentration- manner dependent manner. Protein kinases such as Akt To determine the effect of TVA on NPC cells viability, phosphorylate Bad at Ser136, thereby blocking Bad-in- we treated two human NPC cell line subtypes, 5-8F and duced apoptosis. We were further interested in deter- CNE-2 (highly metastatic strain) with TFAs using a mining whether the Akt pathway is involved in range of doses. After 24 h of treatment, cell viability was TVA-induced apoptosis. The expression levels of Akt in evaluated using a CCK8 assay. We found that all 3 TFAs the TVA groups were not significantly different from exerted inhibitory effects on 5-8F and CNE-2 cell viabil- those in the control group; however, the expression ity in a dose-dependent manner (Fig. 1a). The number levels of p-Akt were significantly reduced in a of cells was significantly lower in the low-dose TVA dose-dependent manner compared to those in the (50 μM) group than in the control group, whereas control group, correlating closely with the findings re- 100 μM LA and 200 μM EA supplementation suppressed garding Bad Ser-136 phosphorylation. To confirm the NPC cells viability to a significant degree. The growth role of Akt activation in apoptosis induction by TVA, we inhibitory ratios of 5-8F cells treated with 25, 50, 100 determined the effects of IGF-1, an activator of Akt. As and 200 μM TVA were 10.8, 18.9, 49.3 and 80.2%, shown in Fig. 3c, the combination of IGF-1 and TVA respectively (P < 0.05), and those of CNE-2 cells were could neutralize the inhibitory effect of TVA alone. The 7.9, 15.2, 45.3 and 70.5%, respectively (P < 0.05) (Fig. 1b). above results indicate that the Akt/Bad pathway is The cytotoxic effect of TVA was much greater in the involved in TVA-induced apoptosis. 5-8F NPC cells (EC50 = 81.5 μM) than in CNE-2 cells (EC50 = 124 μM). Synergistic inhibitory effect of combining TVA with S63845 in NPC cells TVA induces apoptosis in NPC cells in a dose-dependent Mcl-1 is an anti-apoptotic member of the Bcl-2 family manner that inhibits apoptosis in response to a number of cyto- To investigate whether apoptosis causes inhibition of cell toxic stimuli [22]. Mcl-1 is widely expressed in normal growth, we analyzed apoptosis after TVA treatment by human tissues and is abnormally highly expressed in Song et al. Lipids in Health and Disease (2019) 18:46 Page 4 of 9 Fig. 1 Effects of TVA on cell growth in human NPC cells. a. Optical density at 450 nm (OD450) values of NPC cells after TFA treatment for 24 h. b. Cell viability was assessed by CCK8 assays to calculate the survival rates. c. Cell morphology shrinkage was consistent with apoptotic cell death. *P<0.05, **P<0.01 and ***P<0.001 versus the control group Song et al. Lipids in Health and Disease (2019) 18:46 Page 5 of 9 Fig. 2 TVA treatment induces apoptosis in NPC cells. a. Apoptosis was analyzed by flow cytometry with PI and annexin V-FITC staining after 5-8F and CNE-2 cells were treated with TVA for 24 h at the indicated concentrations. b. The percentage of apoptotic cells was calculated as the apoptosis rate. c. Cleaved PARP (c-PARP) and cleaved caspase-3 (c-Caspase-3) protein levels after TVA treatment for 24 h. β-tubulin was used as an internal control. *P<0.05 and **P<0.01 versus the control group many malignant tumor tissues. As shown in Fig. 3a and alone. The CI value was less than 1 at all doses, suggest- b, TVA caused a significant increase in Mcl-1 expression ing that these two compounds have synergistic inhibitory in 5-8F and CNE-2 cells, which indicates that tumor effects on 5-8F and CNE-2 cells. cells are resistant to TVA. To verify our hypothesis, we treated cells with the Mcl-1 inhibitor S63845 combined Discussion with TVA and measured the overall inhibitory effects of TFAs from different sources have unique biological the agents individually and in combination at a fixed effects. Many clinical and experimental studies have ratio of 25:1 (TVA:S63845) using a CCK8 assay. As shown that the consumption of TFA from partially shown in Fig. 4a, S63845 treatment alone caused the in- hydrogenated oils not only adversely affects the cardio- hibition of NPC cell growth, while the combination of vascular system but also accelerates the occurrence of TVA and S63845 was much more effectively than either diseases such as obesity and tumors [23, 24]. However, Song et al. Lipids in Health and Disease (2019) 18:46 Page 6 of 9 Fig. 3 TVA induces apoptosis in NPC cells through Akt and Bad inactivation. a. The expression of Akt, p-Akt, Bad, p-Bad and Mcl-1 in 5-8F cells was analyzed by western blot assay after the cells were treated with TVA. b. The expression of Akt, p-Akt, Bad, p-Bad and Mcl-1 in CNE-2 cells was analyzed by western blot assay after the cells were treated with TVA. Protein expression was quantified by normalization to the level of β-tubulin. c. Effects of IGF-1 on TVA-induced apoptosis as detected by an annexin V-FITC/PI staining assay. The number of apoptotic cells was determined after treatment with TVA (100 μM) in the presence or absence of IGF-1 (50 ng/mL) for 24 h. The values represent the means±standard deviation of three independent experiments. *P<0.05 and **P<0.01 versus the control group in recent years, increasing evidences has demonstrated Apoptosis is an autonomous, ordered death of cells that TFAs from ruminant trans fats have beneficial controlled by gene regulation. Biochemical events occur effects on human health [25]. In the present study, during this process, which is characterized by blebbing, we examined the anticancer effects of TVA on human cell shrinkage and nuclear fragmentation [26]. Apoptosis NPC 5-8F and CNE-2 cells and explored the related of cancer cells is an important mechanism to inhibit cell molecular mechanisms. Furthermore, we found that growth, and inducing tumor cell apoptosis has become TVA treatment can promote a significant increase in the first choice for clinical anticancer therapy [27]. Mcl-1 leading to drug resistance in NPC cells, which Therefore, we examined whether TVA could induce couldbeovercomeby treatment with theMcl-1 apoptosis in NPC cells in the present study. The CCK8, inhibitor S63845. annexin V-FITC/PI staining, and western blot assay Song et al. Lipids in Health and Disease (2019) 18:46 Page 7 of 9 Fig. 4 Synergistic inhibitory effect of the combination of TVA and S63845 on NPC cells. a. Cell viability of NPC cells after treatment with TVA (25, 50, 75, 100 and 125 μM), S683845 (1, 2, 3, 4 and 5 μM) or a combination of TVA and S63845 in a fixed ratio of 25:1 (TVA:S63845) for 24 h. b.CI value of the TVA/S63845 combination at each fixed ratio concentration. c. Signaling pathways underlying TVA and/or S63845-induced apoptosis in NPC cells results revealed that the viability of 5-8F and CNE-2 Phosphorylation of Ser-112 and/or Ser-136 of Bad leads cells was inhibited by TVA in the range of 25–100 μM to the loss of proapoptotic activity [30]. Our results in a dose-dependent manner (P < 0.05). show that TVA can induce a concentration-dependent Bad is a pro-apoptotic member of the Bcl-2 family. Re- decrease in Bad phosphorylation at both Ser-136 and markably, dephosphorylated Bad, but not phosphory- Ser-112. However, as a common protein in multiple lated Bad, forms a heterodimer with the antiapoptotic signaling pathway, Bad is phosphorylated by several pro- proteins Bcl-2 and Bcl-XL, inactivating them and thus tein kinases such as those induced by survival signals. allowing apoptosis [28]. Phosphorylated Bad is located in The Akt signaling pathway plays an important role in the cytoplasm and has no pro-apoptotic activity [29]. controlling tumor cell proliferation, the cell cycle and Song et al. Lipids in Health and Disease (2019) 18:46 Page 8 of 9 metastasis [31]. Furthermore, Akt, have been reported to Ethics approval and consent to participate The study protocol was approved by the Ethics Committee of Shenzhen phosphorylate Bad in response to survival signals [32]. Second People’s Hospital. IOur study, we found that the decrease in Bad phos- phorylation was consistent with Akt-mediated dephos- Consent for publication All authors have approved the final manuscript for publication. phorylation. Specific activation of Akt (by IGF-1) inhibited apoptosis and significantly attenuated the in- Competing interests hibitory effect of TVA. TVA promotes NPC apoptosis by The authors declare that they have no competing interests. targeting the Akt/Bad signaling pathway. Tumor cells develop drug resistance for many reasons, Publisher’sNote among which apoptotic regulation of protein expression Springer Nature remains neutral with regard to jurisdictional claims in is an important factor [33]. In various types of tumor published maps and institutional affiliations. cells, anti-apoptotic genes of the Bcl-2 family are highly Author details expressed, thereby preventing apoptosis [34]. Mcl-1 is a 1 Department of Otolaryngology, Shenzhen Second People’s Hospital, The member of the antiapoptotic Bcl-2 family of proteins. First Affiliated Hospital of Shenzhen University, Shenzhen 518035, People’s Republic of China. Institute of Translational Medicine, Shenzhen Second Abundant evidence to suggests that Mcl-1 is an import- People’s Hospital, The First Affiliated Hospital of Shenzhen University, ant cancer target [35]. The root cause of resistance to 3 Shenzhen 518035, People’s Republic of China. Department of widely used anticancer drugs, including Bcl-2 inhibitors Otolaryngology, Peking University Shenzhen Hospital, Shenzhen 518036, People’s Republic of China. Department of Otolaryngology, Shenzhen [36], paclitaxel [37], vincristine [37], and gemcitabine People’s Hospital, The second Affiliated Hospital of Jinan University, [38] is upregulation of the Mcl-1 level. In the present Shenzhen 518000, People’s Republic of China. study, TVA treatment induced Mcl-1 expression. To Received: 7 November 2018 Accepted: 4 February 2019 inhibit drug resistance resulting from Mcl-1 upregula- tion, we used the newly developed Mcl-1 inhibitor S63845 combinated with TVA and showed that the two References compounds have a synergistic effect. Therefore, targeting 1. Lichtenstein AH. Dietary trans fatty acids and cardiovascular disease risk: past and present. Curr Atheroscler Rep. 2014;16:433. Mcl-1 is a rational strategy to improve the efficacy of TVA. 2. Gebauer SK, Psota TL, Kris-Etherton PM. 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Trans-vaccenic acid inhibits proliferation and induces apoptosis of human nasopharyngeal carcinoma cells via a mitochondrial-mediated apoptosis pathway

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Life Sciences; Lipidology; Medical Biochemistry; Clinical Nutrition
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Abstract

Background: Intake of trans fatty acids (TFAs) from partially hydrogenated vegetable oil is associated with a variety of adverse outcomes, but little is known about the health effects of ruminant trans fats. Trans-vaccenic acid (TVA) is a naturally occurring TFA found in the fat of ruminants and in human dairy products. The present study was conducted to investigate the anticancer activity and underlying mechanisms of TVA on human nasopharyngeal carcinoma (NPC) 5-8F and CNE-2 cells. Methods: A CCK8 assay was used to determine the effect of TVA and the Mcl-1 inhibitor S63845 on the proliferation of NPC cells. Apoptosis was measured using flow cytometry. Western blotting was used to detect the protein expression levels of factors associated with Bcl-2-family protein signaling and Akt signaling. Results: TVA significantly inhibited cell proliferation in a dose-dependent manner. Mechanistic investigation demonstrated that TVA significantly decreased p-Akt levels and Bad phosphorylation on Ser-136 and Ser-112. More importantly, we discovered that the Mcl-1 inhibitor S63845 synergistically sensitized NPC cells to apoptosis induction by TVA. Conclusion: TVA can inhibit NPC cell growth and induced apoptosis through the inhibition of Bad/Akt phosphorylation. The combined use of TVA and Mcl-1 inhibitors offers a potential advantage for nasopharyngeal cancer treatment. Keywords: Trans-vaccenic acid, Apoptosis, Nasopharyngeal carcinoma, Akt, Bad, Mcl-1 Introduction different sources cause various biological effects on hu- Trans fatty acids (TFAs) is a general term for unsatur- man health that may be beneficial or unfavorable [2, 3]. ated fatty acids with at least one double bond in the The impact of TFAs on the cardiovascular system has trans configuration [1]. TFAs within the human diet are been extensively studied, and many epidemiological mainly derived from industrial partial hydrogenation of investigations and experiments have shown that TFAs vegetable oils and from natural sources, such as rumin- from partially hydrogenated oils have adverse effects on ant animal products. Evidence suggests that TFAs from the cardiovascular system [4]. TFAs formed via indus- trial hydrogenation could significantly accelerate the development of atherosclerosis by increasing the ratio of low-density lipoprotein (LDL) to high-density lipopro- * Correspondence: nghui@21cn.com tein (HDL) [5]. In addition, many other studies have Department of Otolaryngology, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen 518035, People’s shown that these types of TFAs also have adverse effects Republic of China on blood lipids [6], inflammation [7], oxidative stress [8], Institute of Translational Medicine, Shenzhen Second People’s Hospital, The endothelial health [9], body weight [10], insulin sensitiv- First Affiliated Hospital of Shenzhen University, Shenzhen 518035, People’s Republic of China ity [11] and cancer [12]. However, emerging evidence Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Song et al. Lipids in Health and Disease (2019) 18:46 Page 2 of 9 indicates that trans fats derived from milk or ruminant For treatments, the cells were grown in 6-well plates to body fats are beneficial for reducing the incidence of 60–70% confluence and were then treated with different cardiovascular disease, cancer and obesity [13]. concentrations of TFAs (EA, LA, and TVA; Nu-Chek Trans-vaccenic acid (TVA) is ubiquitous in ruminant- Prep) (0, 25, 50, 100, 200 μM) for 24 h. Equal derived fats and human dairy products such as milk and concentrations of DMSO were used as the controls. butter. It is worth noting that TVA is also the predomin- ant TFA in human milk. Supplementation with milk Cell viability assay lipids that contain TVA triggers a pronounced cytotoxic The effect of TFAs on the viability of NPC cells was effect on HT29 cell due to conversion to c9,t11-conju- measured by CCK8 assay. 5-8F and CNE-2 cells were gated linoleic acid (CLA) [14]. Suppression of tumor cell plated (1.5 × 10 cells) in 96-well culture plates and growth by TVA treatment of the MCF7 and SW480 cell incubated in serum-containing medium for 24 h. The lines can be attributed to the induction of apoptosis medium was then replaced with serum-free media con- though increased DNA fragmentation and reduced cyto- taining different concentrations of TFA and/or S63845 solic glutathione levels [15]. Preclinical studies have (HY-100741, MedChem Express) for 24 h. After incuba- shown that the use of various types of fatty acids alone tion, cell proliferation was analyzed with a CCK8 assay or combined with other anticancer drugs has promising kit following the manufacturer’s instructions (CK04, therapeutic application prospects [16]. Dojindo Laboratories), and the absorbance at 450 nm Nasopharyngeal carcinoma (NPC), the most common was measured using a microplate reader (BioTek). cancer originating in the nasopharynx, has a high inci- dence in Southern China and Southeast Asia [17]. Annexin V and propidium iodide (PI) staining Radiotherapy is currently the preferred method of treat- The effects of the TFAs on apoptosis were determined ment for early-stage NPC because most NPCs are poorly by dual staining with annexin V-FITC and PI using an differentiated cancers with high sensitivity to radiation apoptosis detection kit from BD Bioscience. After treat- and because the primary and neck lymphatic drainage ment with the indicated TFA concentrations for 24 h, areas are easily included in the radiotherapy field [18]. the cells were harvested and washed with PBS. The cells Clinical treatment of recurrent or metastatic NPC is were then incubated with 5 μL of annexin V-FITC and more difficult than primary NPC treatment. The routine PI for 15 min. The fluorescence of the cells was analyzed treatment for these groups of patients is platinum-based by flow cytometry (Beckman Coulter). chemotherapy, which confers a median progression-free survival time of 7 months [19]. Therefore, it is urgent to Western blot assay identify a more effective treatment option for patients After treatment, lysates from the cultured cells were pre- with recurrent or metastatic NPC. pared with RIPA buffer, and the protein concentrations In the present study, we demonstrate that TVA effect- were determined with a BCA Protein Assay Reagent Kit ively induces NPC apoptosis in 5-8F and CNE-2 cells. (23227, Thermo Scientific). The lysates were separated Mechanism studies indicate that TVA significantly by electrophoresis on a 12% SDS-polyacrylamide gel and inhibits Akt/Bad phosphorylation. More importantly, we then transferred to PVDF Western Blotting Membranes found that TVA treatment also led to the upregulation (IPVH00010, Millipore). The membranes were blocked of Mcl-1 as a novel mechanism involved in TVA resist- with 5% milk and incubated at 4 °C with primary anti- ance, which could be overcome by treatment with the bodies against Bad (9292, Cell Signaling Technology, Mcl-1 inhibitor S63845. These results suggest that the 1:1000), phosphor-Bad (Ser-112) (5284, Cell Signaling combination of TVA and Mcl-1 inhibitors is a promising Technology, 1:1000), phosphor-Bad (Ser-136) (4366, Cell approach for NPC treatment strategies. Signaling Technology, 1:1000), Mcl-1 (94,296, Cell Signaling Technology, 1:1000), Bcl-xL (2764, Cell Signal- Materials and methods ing Technology, 1:1000), Bcl-2 (12789–1-AP, Protein- Cell culture and treatments tech, 1:1000), pan-Akt (4685, Cell Signaling Technology, The human NPC cell lines 5-8F and CNE-2 were a gen- 1:1000), p-Akt (Ser-473) (4060, Cell Signaling Technol- erous gift from Prof. Chao-Nan Qian at the State Key ogy, 1:1000), p-Akt (Thr-308) (13,038, Cell Signaling Laboratory of Oncology in South China and the Collab- Technology, 1:1000), and β-tubulin (10068–1-AP, orative Innovation Center for Cancer Medicine, Sun Proteintech, 1:5000). After overnight incubation with the Yat-Sen University Cancer Center. All NPC cell lines primary antibodies, the membranes were incubated with were maintained in RPMI-1640 medium (SH30809.01, secondary anti-mouse (115–035-003, Jackson, 1:5000) or HyClone) supplemented with 10% FBS (10099–141, anti-rabbit (111–035-003, Jackson, 1:5000) antibodies. Gibco) and 1% penicillin-streptomycin (15070–063, Then, the signals were detected with an Amersham Gibco) at 37 °C with 5% CO . Imager 600. 2 Song et al. Lipids in Health and Disease (2019) 18:46 Page 3 of 9 Calculation of the combination index flow cytometry analysis of annexin V/PI-stained cells. Whether the synergistic inhibitory effect between TVA We found that TVA significantly induced apoptosis in a and S63845 can be determined by the combination dose-dependent manner. Incubation of 5-8F cells with index (CI) using the Chou-Talalay equation [20]. 5-8F 25, 50 or 100 μM TVA for 24 h caused 7.67, 12.9% or and CNE-2cells were treated with various concentrations 35% increases in total apoptosis, respectively. Similarly, of TVA and S63845 separately or in combination. The after incubation with 25 50 or 100 μM CNE-2, the per- total inhibitory effect was assessed by CCK8 assay as de- centage of apoptotic cells was increased by 12.9, 15.1% scribed above. The CI value was determined as follow: or 22.3%, respectively (Fig. 2a). Cleaved poly (ADP-ri- bose) polymerase (PARP) and cleaved caspase-3 are DA; x DB; x widely used to detect apoptosis in cells. Therefore, we CI ¼ þ assessed the protein levels of cleaved PARP and cleaved ICx; A ICx; B caspase-3 in the presence or absence of TVA treatment. A, B represents two different agent, ICX, A and ICX, Immunoblot analysis revealed that TVA treatment sig- B are the concentration when the two agent are used nificantly increased the levels of cleaved PARP and alone to achieve a growth inhibition rate of X%, and cleaved caspase-3 in a time-dependent manner (Fig. 2c). DA,x and DB,x are the concentration of two agent com- bined to achieve a growth inhibition rate of X%. CI < 1 TVA induces apoptosis in NPC cells through Akt and bad indicate synergism, CI = 1indicates additive effects, and inactivation CI > 1 indicates antagonism. The Bcl-2-associated death promoter (Bad) is a pro- apoptotic member of the Bcl-2 family that can form a Statistical analysis heterodimer with the antiapoptotic proteins Bcl-2 and Statistical analysis was performed using the standard Bcl-XL and prevent them from inhibiting apoptosis [21]. Student t test for pair comparisons and ANOVA analysis We were thus interested in determining whether TVA for multiple factors. The statistical values of *P < 0.05, could affect endogenous Bad activity. The role of Bad in **P < 0.01 and ***P < 0.001 were considered statistically promoting apoptosis is mainly involves phosphorylation significant. Values of mean determinants are presented of Ser-136 and Ser-112. Bad is rapidly dephosphorylated as ± s.e.m. and transferred to the mitochondria to induce apoptosis in response to external stimuli. As shown in Fig. 3a and Results b, TVA treatment induced a decrease in Bad phosphor- TVA inhibits NPC cell viability in a dose-dependent ylation on Ser-136 and Ser-112 in a concentration- manner dependent manner. Protein kinases such as Akt To determine the effect of TVA on NPC cells viability, phosphorylate Bad at Ser136, thereby blocking Bad-in- we treated two human NPC cell line subtypes, 5-8F and duced apoptosis. We were further interested in deter- CNE-2 (highly metastatic strain) with TFAs using a mining whether the Akt pathway is involved in range of doses. After 24 h of treatment, cell viability was TVA-induced apoptosis. The expression levels of Akt in evaluated using a CCK8 assay. We found that all 3 TFAs the TVA groups were not significantly different from exerted inhibitory effects on 5-8F and CNE-2 cell viabil- those in the control group; however, the expression ity in a dose-dependent manner (Fig. 1a). The number levels of p-Akt were significantly reduced in a of cells was significantly lower in the low-dose TVA dose-dependent manner compared to those in the (50 μM) group than in the control group, whereas control group, correlating closely with the findings re- 100 μM LA and 200 μM EA supplementation suppressed garding Bad Ser-136 phosphorylation. To confirm the NPC cells viability to a significant degree. The growth role of Akt activation in apoptosis induction by TVA, we inhibitory ratios of 5-8F cells treated with 25, 50, 100 determined the effects of IGF-1, an activator of Akt. As and 200 μM TVA were 10.8, 18.9, 49.3 and 80.2%, shown in Fig. 3c, the combination of IGF-1 and TVA respectively (P < 0.05), and those of CNE-2 cells were could neutralize the inhibitory effect of TVA alone. The 7.9, 15.2, 45.3 and 70.5%, respectively (P < 0.05) (Fig. 1b). above results indicate that the Akt/Bad pathway is The cytotoxic effect of TVA was much greater in the involved in TVA-induced apoptosis. 5-8F NPC cells (EC50 = 81.5 μM) than in CNE-2 cells (EC50 = 124 μM). Synergistic inhibitory effect of combining TVA with S63845 in NPC cells TVA induces apoptosis in NPC cells in a dose-dependent Mcl-1 is an anti-apoptotic member of the Bcl-2 family manner that inhibits apoptosis in response to a number of cyto- To investigate whether apoptosis causes inhibition of cell toxic stimuli [22]. Mcl-1 is widely expressed in normal growth, we analyzed apoptosis after TVA treatment by human tissues and is abnormally highly expressed in Song et al. Lipids in Health and Disease (2019) 18:46 Page 4 of 9 Fig. 1 Effects of TVA on cell growth in human NPC cells. a. Optical density at 450 nm (OD450) values of NPC cells after TFA treatment for 24 h. b. Cell viability was assessed by CCK8 assays to calculate the survival rates. c. Cell morphology shrinkage was consistent with apoptotic cell death. *P<0.05, **P<0.01 and ***P<0.001 versus the control group Song et al. Lipids in Health and Disease (2019) 18:46 Page 5 of 9 Fig. 2 TVA treatment induces apoptosis in NPC cells. a. Apoptosis was analyzed by flow cytometry with PI and annexin V-FITC staining after 5-8F and CNE-2 cells were treated with TVA for 24 h at the indicated concentrations. b. The percentage of apoptotic cells was calculated as the apoptosis rate. c. Cleaved PARP (c-PARP) and cleaved caspase-3 (c-Caspase-3) protein levels after TVA treatment for 24 h. β-tubulin was used as an internal control. *P<0.05 and **P<0.01 versus the control group many malignant tumor tissues. As shown in Fig. 3a and alone. The CI value was less than 1 at all doses, suggest- b, TVA caused a significant increase in Mcl-1 expression ing that these two compounds have synergistic inhibitory in 5-8F and CNE-2 cells, which indicates that tumor effects on 5-8F and CNE-2 cells. cells are resistant to TVA. To verify our hypothesis, we treated cells with the Mcl-1 inhibitor S63845 combined Discussion with TVA and measured the overall inhibitory effects of TFAs from different sources have unique biological the agents individually and in combination at a fixed effects. Many clinical and experimental studies have ratio of 25:1 (TVA:S63845) using a CCK8 assay. As shown that the consumption of TFA from partially shown in Fig. 4a, S63845 treatment alone caused the in- hydrogenated oils not only adversely affects the cardio- hibition of NPC cell growth, while the combination of vascular system but also accelerates the occurrence of TVA and S63845 was much more effectively than either diseases such as obesity and tumors [23, 24]. However, Song et al. Lipids in Health and Disease (2019) 18:46 Page 6 of 9 Fig. 3 TVA induces apoptosis in NPC cells through Akt and Bad inactivation. a. The expression of Akt, p-Akt, Bad, p-Bad and Mcl-1 in 5-8F cells was analyzed by western blot assay after the cells were treated with TVA. b. The expression of Akt, p-Akt, Bad, p-Bad and Mcl-1 in CNE-2 cells was analyzed by western blot assay after the cells were treated with TVA. Protein expression was quantified by normalization to the level of β-tubulin. c. Effects of IGF-1 on TVA-induced apoptosis as detected by an annexin V-FITC/PI staining assay. The number of apoptotic cells was determined after treatment with TVA (100 μM) in the presence or absence of IGF-1 (50 ng/mL) for 24 h. The values represent the means±standard deviation of three independent experiments. *P<0.05 and **P<0.01 versus the control group in recent years, increasing evidences has demonstrated Apoptosis is an autonomous, ordered death of cells that TFAs from ruminant trans fats have beneficial controlled by gene regulation. Biochemical events occur effects on human health [25]. In the present study, during this process, which is characterized by blebbing, we examined the anticancer effects of TVA on human cell shrinkage and nuclear fragmentation [26]. Apoptosis NPC 5-8F and CNE-2 cells and explored the related of cancer cells is an important mechanism to inhibit cell molecular mechanisms. Furthermore, we found that growth, and inducing tumor cell apoptosis has become TVA treatment can promote a significant increase in the first choice for clinical anticancer therapy [27]. Mcl-1 leading to drug resistance in NPC cells, which Therefore, we examined whether TVA could induce couldbeovercomeby treatment with theMcl-1 apoptosis in NPC cells in the present study. The CCK8, inhibitor S63845. annexin V-FITC/PI staining, and western blot assay Song et al. Lipids in Health and Disease (2019) 18:46 Page 7 of 9 Fig. 4 Synergistic inhibitory effect of the combination of TVA and S63845 on NPC cells. a. Cell viability of NPC cells after treatment with TVA (25, 50, 75, 100 and 125 μM), S683845 (1, 2, 3, 4 and 5 μM) or a combination of TVA and S63845 in a fixed ratio of 25:1 (TVA:S63845) for 24 h. b.CI value of the TVA/S63845 combination at each fixed ratio concentration. c. Signaling pathways underlying TVA and/or S63845-induced apoptosis in NPC cells results revealed that the viability of 5-8F and CNE-2 Phosphorylation of Ser-112 and/or Ser-136 of Bad leads cells was inhibited by TVA in the range of 25–100 μM to the loss of proapoptotic activity [30]. Our results in a dose-dependent manner (P < 0.05). show that TVA can induce a concentration-dependent Bad is a pro-apoptotic member of the Bcl-2 family. Re- decrease in Bad phosphorylation at both Ser-136 and markably, dephosphorylated Bad, but not phosphory- Ser-112. However, as a common protein in multiple lated Bad, forms a heterodimer with the antiapoptotic signaling pathway, Bad is phosphorylated by several pro- proteins Bcl-2 and Bcl-XL, inactivating them and thus tein kinases such as those induced by survival signals. allowing apoptosis [28]. Phosphorylated Bad is located in The Akt signaling pathway plays an important role in the cytoplasm and has no pro-apoptotic activity [29]. controlling tumor cell proliferation, the cell cycle and Song et al. Lipids in Health and Disease (2019) 18:46 Page 8 of 9 metastasis [31]. Furthermore, Akt, have been reported to Ethics approval and consent to participate The study protocol was approved by the Ethics Committee of Shenzhen phosphorylate Bad in response to survival signals [32]. Second People’s Hospital. IOur study, we found that the decrease in Bad phos- phorylation was consistent with Akt-mediated dephos- Consent for publication All authors have approved the final manuscript for publication. phorylation. Specific activation of Akt (by IGF-1) inhibited apoptosis and significantly attenuated the in- Competing interests hibitory effect of TVA. TVA promotes NPC apoptosis by The authors declare that they have no competing interests. targeting the Akt/Bad signaling pathway. Tumor cells develop drug resistance for many reasons, Publisher’sNote among which apoptotic regulation of protein expression Springer Nature remains neutral with regard to jurisdictional claims in is an important factor [33]. In various types of tumor published maps and institutional affiliations. cells, anti-apoptotic genes of the Bcl-2 family are highly Author details expressed, thereby preventing apoptosis [34]. Mcl-1 is a 1 Department of Otolaryngology, Shenzhen Second People’s Hospital, The member of the antiapoptotic Bcl-2 family of proteins. First Affiliated Hospital of Shenzhen University, Shenzhen 518035, People’s Republic of China. Institute of Translational Medicine, Shenzhen Second Abundant evidence to suggests that Mcl-1 is an import- People’s Hospital, The First Affiliated Hospital of Shenzhen University, ant cancer target [35]. The root cause of resistance to 3 Shenzhen 518035, People’s Republic of China. Department of widely used anticancer drugs, including Bcl-2 inhibitors Otolaryngology, Peking University Shenzhen Hospital, Shenzhen 518036, People’s Republic of China. Department of Otolaryngology, Shenzhen [36], paclitaxel [37], vincristine [37], and gemcitabine People’s Hospital, The second Affiliated Hospital of Jinan University, [38] is upregulation of the Mcl-1 level. In the present Shenzhen 518000, People’s Republic of China. study, TVA treatment induced Mcl-1 expression. To Received: 7 November 2018 Accepted: 4 February 2019 inhibit drug resistance resulting from Mcl-1 upregula- tion, we used the newly developed Mcl-1 inhibitor S63845 combinated with TVA and showed that the two References compounds have a synergistic effect. Therefore, targeting 1. Lichtenstein AH. Dietary trans fatty acids and cardiovascular disease risk: past and present. Curr Atheroscler Rep. 2014;16:433. Mcl-1 is a rational strategy to improve the efficacy of TVA. 2. Gebauer SK, Psota TL, Kris-Etherton PM. 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Lipids in Health and Disease – Springer Journals

Published: Feb 9, 2019

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