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Attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving ERK activation in renal cell carcinoma cells

Attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving ERK... Journal name: Drug Design, Development and Therapy Article Designation: Original Research Year: 2018 Volume: 12 Running head verso: Zeng et al Drug Design, Development and Therapy Dovepress Running head recto: Attenuation of everolimus-induced cytotoxicity by involving ERK activation open access to scientific and medical research DOI: 160557 Open access Full Text article O rig inal r esearch attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving erK activation in renal cell carcinoma cells Yizhou Zeng Aim: The mammalian target of rapamycin (mTOR) pathway is a critical target for cancer treatment and the mTOR inhibitor everolimus (RAD001) has been approved for treatment of Xiaofang Tian renal cell carcinoma (RCC). However, the limited efficacy of RAD001 has led to the develop - Quan Wang ment of drug resistance. Autophagy is closely related to cell survival and death, which may be Weiyang he activated under RAD001 stimulation. The aim of the present study was to identify the underlying Jing Fan mechanisms of RAD001 resistance in RCC cells through cytoprotective autophagy involving Xin gou activation of the extracellular signal-regulated kinase (ERK) pathway. Department of Urinary surgery, The Methods and results: RAD001 strongly induced autophagy of RCC cells in a dose- and time- First a ffiliated h ospital of c hongqing dependent manner, as conr fi med by Western blot analysis. Importantly, suppression of autophagy Medical University, Yuzhong District, chongqing, china by the pharmacological inhibitor chloroquine effectively enhanced RAD001-induced apoptotic cytotoxicity, as demonstrated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western blot analysis, indicating a cytoprotective role for RAD001- induced autophagy. In addition, as was shown by the MTT assay, flow cytometry, and Western blot analysis, RAD001 robustly activated ERK, but not c-Jun N-terminal kinase and p38. Activation of ERK was inhibited by the pharmacological inhibitor selumetinib (AZD6244), which effectively promoted RAD001-induced cell death. Moreover, employing AZD6244 markedly attenuated RAD001-induced autophagy and enhanced RAD001-induced apoptosis, which play a central role in RAD001-induced cell death. Furthermore, RAD001-induced autophagy is regulated by ERK-mediated phosphorylation of Beclin-1 and B-cell lymphoma 2, as confirmed by Western blot analysis. Conclusion: These results suggest that RAD001-induced autophagy involves activation of the ERK, which may impair cytotoxicity of RAD001 in RCC cells. Thus, inhibition of the activation of ERK pathway-mediated autophagy may be useful to overcome chemoresistance to RAD001. Keywords: apoptosis, autophagy, everolimus, ERK, renal cancer, selumetinib Introduction Renal cell carcinoma (RCC) is the most common form of kidney cancer, with ~338,000 new diagnoses and 144,000 deaths occurring annually worldwide. Surgical resection is generally performed to treat this disease; however, nephrectomy is not a feasible option for about 30% of patients with metastatic disease. Therefore, to further improve correspondence: Jing Fan; Xin gou the quality of life and survival of patients, systemic treatment may be a more effec- Department of Urinary surgery, The First tive choice. Affiliated Hospital of Chongqing Medical University, no 1 Youyi road, Yuzhong Everolimus (RAD001), a mammalian target of rapamycin (mTOR) inhibitor, has District, chongqing 400016, china been demonstrated to exert cytotoxicity against human cancers of the breast, stomach, email fjfj8003@163.com; 4–6 omegamanman@163.com and prostate, and is currently used as a sequential or second-line therapy for RCC submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 911–920 Dovepress © 2018 Zeng et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you http://dx.doi.org/10.2147/DDDT.S160557 hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Zeng et al Dovepress refractory to sunitinib or sorafenib. However, the efficacy phospho-Bcl-2, Beclin-1, ERK, phospho-ERK, p38, phospho- of RAD001 is thought to be limited by feedback loops and p38, c-Jun N-terminal kinase (JNK), and phospho-JNK were cross talk with other pathways, resulting in drug resistance. purchased from Cell Signaling Technology (Danvers, MA, Karam et al established and characterized a panel of mouse USA). Anti-cleaved poly ADP ribose polymerase (PARP) models of RCC derived from patients undergoing radical and p62 were obtained from BD Biosciences (San Jose, CA, nephrectomy. Using these models, resistance to the mTOR USA). Anti-LC3 and chloroquine (CQ) were purchased from inhibitor RAD001 was identified. François et al stated that Sigma-Aldrich Co. (St Louis, MO, USA). Anti-β-actin anti- RAD001 rarely induces regression of pancreatic neuroen- body was purchased from Zoonbio Biotechnology Co., Ltd docrine tumors. In addition, as compared with RAD001 (Nanjing, China). All secondary antibodies were obtained alone, co-treatment seems to be potentially more effective from Abgent (San Diego, CA, USA). Roswell Park Memorial 9,10 11 in controlling cell signaling. Motzer et al declared that Institute (RPMI)-1640 medium and trypsin were purchased a combination therapy of RAD001 and lenvatinib showed from HyClone (Logan, UT, USA). Fetal bovine serum was a favorably synergistic effect in patients with advanced or purchased from Gibco (Thermo Fisher Scientific, Waltham, metastatic RCC, which was the first successful combination MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- therapy approved by the US Food and Drug Administration. zolium bromide (MTT) cell kit was purchased from Beyotime Thus, based on these findings, it is necessary to explore the Institute of Biotechnology (Shanghai, China). underlying mechanism of the drug resistance of RAD001. Autophagy is a highly conserved intracellular catabolic cell culture process that degrades and recycles cellular components for The human RCC cell lines 786-O and A498 were pur- cell survival under certain conditions, and is closely related to chased from American Type Culture Collection (Manassas, cell survival or death. For cancer cells resistant to chemother- VA, USA) and grown in RPMI-1640 medium supplemented apy, autophagy presents either protective or injurious effects. with 10% fetal bovine serum, 1 mmol/L glutamine, 10 U/L For example, RAD001 is able to induce autophagy in human penicillin, and 100 g/L streptomycin at 37°C, under a humidi- renal cancer cells, which then promotes tumor cell survival, fied atmosphere containing 5% CO . resulting in a limited anticancer effect. The mTOR complex is now regarded as an autophagy switch to promote prolifera- MTT assay tion and inhibit autophagy, although the potential mechanisms Cell viability was assessed with the MTT assay. Briefly, involved in the cell signal pathways for this process are still 786-O and A498 cells were seeded into 96-well plates at not fully understood. Interestingly, Butler et al found that 5,000 cells/well and cultivated for 24 hours. After adher- inhibition of the phosphatidylinositol 3 kinase (PI3K)/protein ence, the 786-O and A498 cells were treated with various kinase B (AKT)/mTOR pathway activates autophagy and concentrations of RAD001 for 24 hours. MTT (5 mg/mL, compensatory Ras/Raf/MEK/extracellular signal-regulated 20 μL/well) was added to each well and the plate was incu- kinase (ERK) signaling in prostate cancer. Besides, activation bated at 37°C for 4 hours. Dimethyl sulfoxide was then added of the PI3K/AKT/mTOR and Ras/MEK/ERK cell signaling (150 μL/well) to each well to dissolve any crystals and the pathways is also critical for autophagy. Particularly, cross plates were agitated for 15 minutes. Absorbance at 570 nm talk between these two pathways has been well illustrated in was measured using an Infinite F200/M200 type multifunc - 15–17 previous studies. Moreover, several studies have shown tion microplate reader (Tecan Trading AG, Männedorf, that inhibition of the ERK pathway enhanced the antitumor Switzerland). Cell viability was calculated by OD values of 18 19 activity of RAD001 in pediatric gliomas, neuroblastoma, treated groups/OD values of control group) ×100%. Each and acute myelogenous leukemia. Thus, the aim of this study experiment was repeated three times. was to identify the underlying mechanisms and biochemical pathways involved in RAD001 resistance in RCC. Apoptosis analysis by flow cytometry After treatment with RAD001 and AZD6244, the cells Materials and methods were trypsinized, washed with phosphate-buffered saline, Materials and suspended in 195 μL of Annexin V-fluorescein isothio - The small molecular inhibitors RAD001 and AZD6244 were cyanate (FITC) binding buffer containing 5 μL of Annexin obtained from MedChem Express (Monmouth Junction, V-FITC and 10 μL of propidium iodide (Beyotime Institute NJ, USA). Antibodies against B-cell lymphoma 2 (Bcl-2), of Biotechnology). After incubation for 10–20 minutes at submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Dovepress attenuation of everolimus-induced cytotoxicity by involving erK activation room temperature in the dark, the cells were subjected to conversion of LC3-I to LC3-II was in line with a gradual flow cytometry analysis using a FACSCanto 6-color flow decrease in autophagic protein p62 in both cell lines after cytometer (BD Biosciences). RAD001 treatment in a time-dependent manner (Figure 1C and D). Taken together, these n fi dings further emphasize that Western blot analysis RAD001 is able to induce autophagy in RCC cells. To extract total protein lysates, the cells were lysed in cold autophagy protected rcc cells against radioimmunoprecipitation assay lysis buffer (Beyotime raD001-induced cell death, which Institute of Biotechnology) containing phenylmethanesul- fonyl fluoride (Beyotime Institute of Biotechnology), and involved activation of the erK pathway then centrifuged at 12,000 rpm for 15 minutes at 4°C to To further determine whether RAD001 could induce death remove debris. Protein concentrations were measured with of RCC cells, the MTT assay was performed with 786-O the bicinchoninic acid protein assay kit (Beyotime Institute and A498 cells following exposure to RAD001. Pretreat- of Biotechnology). The protein extracts were heat denatured ment of both cell lines with RAD001 (0.01–100 μmol/L) in sodium dodecyl sulfate polyacrylamide gel electrophoresis for 24 hours revealed a dose-dependent reduction in cell Sample Loading Buffer (Beyotime Institute of Biotech- survival. The half IC (half maximal inhibitor concentra- nology). Equal amounts of protein from each cell lysate tion) of RAD001 at 24 hours for 786-O and A498 cells was (40 μg/lane) were separated in 12% polyacrylamide gels 10 and 1 μmol/L, respectively. Thus, these values were cho- and transferred on to a membrane. After blocking with 5% sen for subsequent experiments (Figure 2A). Based on the nonfat dried milk for 2 hours, the membrane was incubated observation that RAD001 also induced autophagy, we were with the primary antibodies overnight at 4°C. Afterward, the particularly interested in investigating whether autophagy proteins were visualized by enhanced chemiluminescence was involved in RAD001-induced cytotoxicity. For this using horseradish peroxidase-conjugated immunoglobulin G purpose, the lysosome inhibitor CQ was used in this study, secondary antibodies. as it inhibits autophagy via blocking autophagolysosome- mediated degradation of the p62 and LC3-II proteins. statistical analysis Expectedly, the RAD001-induced downregulation of p62 Data are expressed as mean ± SD. Statistical analyses were expression was effectively inhibited by CQ, although a performed with GraphPad Prism 6 software (GraphPad Soft- higher LC3-II level was observed in 786-O and A498 cells ware, Inc, San Diego, CA, USA) and IBM SPSS Statistics after exposure to co-treatment with both RAD001and CQ, for Windows, version 20.0 (IBM Corporation, Armonk, suggesting that RAD001 induces a continuous autophagic NY, USA). Based on the completely random design of this flux in both cell lines (Figure 2B and C). These results experiment, the results were analyzed by one-way analysis indicate that autophagy was indeed induced by RAD001. of variance or t-test. A probability value of ,0.05 was con- More importantly, in line with these findings, the results of sidered statistically significant. the MTT assay showed that the effect of RAD001-induced cell cytotoxicity was significantly enhanced by inhibition Results of autophagy in 786-O and A498 cells, suggesting that raD001-induced autophagy in rcc cells autophagy may play a protective role in RAD001-induced It has been well demonstrated that the mTOR pathway inhibi- cytotoxicity of RCC (Figure 2D). Because cell cytotoxicity tor RAD001 can induce autophagy in human RCC cells. The is closely associated with apoptosis, to further determine aim of the present study was to determine whether RAD001 whether the protective effects of autophagy for RAD001- can induce autophagy in RCC cells. For this purpose, dif- induced cell death were due to the activation of apoptotic ferent concentrations of RAD001 were added to cultures of signaling, Western blot analysis was conducted to measure 786-O and A498 cells. The results showed that RAD001- expression levels of cleaved PARP protein. Our results induced autophagy of 786-O and A498 cells in a dose- demonstrated that the apoptosis biomarker cleaved-PARP dependent manner, which was indicated by a typical pheno- was significantly upregulated by RAD001 and this effect type of autophagy, including upregulation of LC3-II/I and was enhanced by inhibition of autophagy with CQ (Figure 2E downregulation of p62 (Figure 1A and B). Next, to further and F). Altogether, these results suggest that autophagy pro- validate this effect, 786-O and A498 cells were treated with tects RCC cells against RAD001-induced cytotoxicity in part RAD001 for 0, 1, 2, 4, or 8 hours. As expected, the increased by suppressing the apoptosis biomarker-cleaved PARP. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Zeng et al Dovepress A 786-O A498 RAD (µmol/L) 00.010.1 11020 00.010.1 11020 LC3-I LC3-II p62 β-Actin B 786-O A498 1.5 5 1.0 0.5 0.0 0 LC3-II/I p62 LC3-II/I p62 0 0.01 0.1 1 10 20 C 786-O A498 RAD 0 h1 h2 h4 h8 h 0 h1 h2 h4 h8 h LC3-I LC3-II p62 β-Actin D 786-O A498 2.0 4 1.5 1.0 2 0.5 1 0.0 0 LC3-II/I p62 LC3-II/I p62 0 h 1 h 2 h 4 h 8 h Figure 1 everolimus (raD001) induces autophagy in rccs. Notes: (A, B) 786-O and a498 cell lines were treated by raD001 with different concentrations (0, 0.01, 0.1, 1, 10, and 20 μmol/l) for 24 hours. ( A) immunoblotting showed that raD001 induced autophagy indicators including upregulation of lc3-ii/i and downregulation of p62 in a dose-dependent manner. ( B) Quantification of the immunoblots. *P,0.05 versus 0 μmol/l group. n =3. (C, D) 786-O and a498 cell lines were treated by raD001 for 0, 1, 2, 4, and 8 hours. ( C) immunoblotting showed that raD001 induced autophagy in 786-O and a498 cells as time prolonged, which peaked at 8 hours. ( D) Quantification of the immunoblots. * P,0.05 versus 0 hour group. n=3. Abbreviation: rccs, renal cell carcinoma cells. We next sought to explore the molecular mechanism by cell death on the activity of the JNK, ERK, and p38 cell sig- which RAD001 induces death of RCC cells. As it has been naling pathways. Briey fl , 786-O and A498 cells were treated recognized that mitogen-activated protein kinases play a with RAD001 for the indicated times, and the activities of critical regulative role in cancer cell death and survival, the the involved signaling pathways were assessed by Western focus of the present study was the effect of RAD001-induced blot analysis. The results showed that following RAD001 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Protein relative levels Protein relative levels Protein relative levels Protein relative levels Dovepress attenuation of everolimus-induced cytotoxicity by involving erK activation $ ) 2 $ IROG IROG $53 $53 &HOOYLDELOLW\ &OHDYH3 $FWLQ &OHDYH3 $FWLQ FKDQJHRYHU FKDQJHRYHU &4 &4 5$' —PRO/ &RQWURO &RQWURO 5$' 5$' 5$'&4 5$'&4 % 2 $ &4 ±± ±± * 2 $ 5$' ±  ±  ±  ± 5$' K K K K K K K K K K /&, /&,, S-1. -1. β$FWLQ SS & 2 $ S(5. (5. 3URWHLQ 3URWHLQ β$FWLQ UHODWLYHOHYHOV UHODWLYHOHYHOV /&,,, S /&,,, S &RQ 5$' &4 5$'&4 ' + 2 &RQ 5$' &4 5$'&4 3URWHLQ UHODWLYHOHYHOV &HOOYLDELOLW\ S-1.-1. SSS S(5.(5. 2 $ ( 2 $ &4 ±± ±± 5$' ±  ±  ±  ± 3URWHLQ 3 $53 &OHDYHG3 $53 UHODWLYHOHYHOV β$FWLQ S-1.-1. SSS S(5.(5. K K K K K Figure 2 inhibition of autophagy enhances raD001-induced cytotoxicity, raD001-induced activation of erK signaling pathway in rccs. Notes: 786-O and a498 cells were pretreated with cQ (autophagy inhibitor) (10 mmol/l) for 30 minutes and then treated with raD001 for 8 hours. ( A) MTT assay showed that raD001 impaired cell viability of 786-O and a498 cells in a dose-dependent manner. n=3. (B) immunoblotting of lc3-ii/i and p62 showed that cQ could restore the downregulation of p62, but would not impair the upregulation of lc3-ii/i because cQ blocks autophagy at the lysosomal degradation step. (C) Densitometric analysis was performed to quantify the immunoblots. *P,0.05 versus control. P,0.05 versus raD001 group. n =3. (D) MTT assay displayed that autophagy inhibition by cQ promoted raD001-induced cytotoxicity in 786-O and a498 cells. * P,0.05 versus control. P,0.05 versus raD001 group. n =3. (E) The apoptosis indicator cleave-ParP detected by immunoblot indicated that cells apoptosis was increased in 786-O and a498 cells after cQ intervention, compared to the raD001 group. ( F) Densitometric analysis was performed to quantify the immunoblots. *P,0.05 versus control group. P,0.05 versus raD001 group. n =3. 786-O and a498 cells were treated by raD001 for 0, 1, 2, 4, and 8 hours. ( G) immunoblotting analyzed the JnK, p38, and erK signaling molecules in 786-O and a498 cells after raD001 treatment. (H) Quantification of the immunoblots. * P,0.05 versus 0 hour group. n=3. Abbreviations: cQ, chloroquine; con, control; rccs, renal cell carcinoma cells; erK, extracellular signal-regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; ParP, poly aDP ribose polymerase; JnK, c-Jun n-terminal kinase. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress β Zeng et al Dovepress pretreatment, p-ERK expression was significantly upregu - enhanced in both cell lines (Figure 3H and I). In summary, lated in both cell lines. However, the expression levels of these data suggest that inhibition of ERK activation not only p-JNK and p-p38 were largely unchanged following RAD001 suppressed RAD001-induced autophagy, but also enhanced exposure (Figure 2G and H). RAD001-induced apoptosis, which plays a central role in RAD001-induced cell death. inhibition of erK activation suppressed raD001-induced autophagy and erK mediated autophagy by upregulating enhanced raD001-induced apoptosis expression of Beclin-1 and p-Bcl-2 The pharmacological inhibitor AZD6244 was employed It is reported that ERK activation mediates phosphorylation to block ERK signaling to investigate the role of ERK of Bcl-2, resulting in dissociation of the Bcl-2/Beclin-1 in RAD001-induced cell death. The results showed that complex. This process releases Beclin-1 and leads to AZD6244 markedly inhibited activation of the ERK pathway autophagy induction. Next, we further explored the mecha- induced by RAD001 (Figure 3A and B). More importantly, nism whereby RAD001 induced ERK activation and led to consistent with the effective suppressive effects of AZD6244 the induction of autophagy. Beclin-1 is critical for the ini- on ERK, co-treatment of both RAD001 and AZD6244 tiation of autophagy. The downstream factors of the ERK induced a greater extent of cell death than pretreatment pathway, including Beclin-1 and p-Bcl-2, were upregulated of RAD001 alone, which indicated that inhibition of ERK as early as 1 hour after RAD001 treatment in both cell lines effectively synergized with RAD001 to impair cell viability (Figure 4A and B). Moreover, inhibition of ERK activation (Figure 3C). Overall, these results suggest that RAD001- with AZD6244 significantly decreased the expression levels induced cytotoxicity involves ERK signaling. of Beclin-1 and p-Bcl-2 (Figure 4C and D). These results A series of studies, including those by our group, have suggest that RAD001-induced ERK-activated autophagy is demonstrated that ERK signaling is closely related to at least in part due to upregulating the expression levels of autophagy in various cancers. To further confirm whether Beclin-1 and p-Bcl-2. this signaling is involved in RAD001-induced autophagy, the specific inhibitor AZD6244 for ERK was used to Discussion interfere with autophagy induction. Indeed, employing The results of the present study showed that RAD001- AZD6244 markedly attenuated the autophagy effect induced induced autophagy may play an important role in RAD001- by RAD001 in both 786-O and A498 cells, as indicated by induced cytotoxicity of RCC cells, although the underlying the decreased expression of LC3-II/I and increased expres- mechanism remains unknown. Furthermore, cytoprotective sion of p62 (Figure 3D and E). It is well established that autophagy involving the ERK pathway was induced by cell death after exposure to cytotoxic chemotherapeutics is RAD001, thereby presenting a novel combination strategy partially mediated by apoptosis. Moreover, as suggested by to target ERK signaling. AZD6244 is a highly selective ERK the above observation that autophagy protected RCC cells inhibitor. Interestingly, AZD6244 significantly inhibited against RAD001-induced cytotoxicity in part by suppressing RAD001-induced autophagy and enhanced RAD001-induced the apoptotic biomarker-cleaved PARP, we were interested apoptosis of 786-O and A498 cells. These findings sug - in investigating whether ERK signaling was involved in the gested that RCC cell resistance to RAD001 may be closely apoptosis process that caused RAD001-induced cytotoxicity. related to cytoprotective autophagy linked to the activation To test this hypothesis, we evaluated cell apoptosis by flow of the ERK pathway. cytometry and the protein expression of cleaved PARP Evidence has confirmed that RAD001 can substantially by Western blot analysis. Immunoblotting showed that induce autophagy, as well as promote the apoptosis of 22 23,24 AZD6244 exaggerated RAD001-induced cleavage of PARP breast cancer cells and hepatocellular carcinoma cells. (Figure 3F and G), indicating that the RAD001-induced Similarly, the results of the present experiment showed that apoptosis was enhanced by AZD6244. Furthermore, the RAD001 treatment of RCC cells resulted in the upregulation flow cytometry results showed that pretreatment with of LC3-II/I and downregulation of p62, which are typical RAD001 resulted in a higher rate of apoptosis in both cell features of autophagy induction, suggesting that autophagy lines, compared with no RAD001 exposure, suggesting by 786-O and A498 cells was induced by RAD001 treat- that RAD001 did indeed induce cell apoptosis. Moreover, ment. So, high concentrations of RAD001 were used in this when these cell lines were treated with both AZD6244 and experiment due to the grade of the RCC cell lines 786-O RAD001, this RAD001-induced apoptosis effect was further and A498. In addition, it is reported that RAD001 did submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Dovepress attenuation of everolimus-induced cytotoxicity by involving erK activation $% 2 & $=' ± ±  ± ± ±  ±  ± ± 5$' S(5. FKDQJH (5. S(5.(5.IROG &HOOYLDELOLW\ β$FWLQ 2 $ 2 $ &RQ 5$' $=' 5$' $=' $ 2 $ '( 2 ± ±  ± ± $=' ±  ±  ± ± 5$' /&, /&,, OHYHOV OHYHOV 3URWHLQUHODWLYH 3URWHLQUHODWLYH β$FWLQ /&,,, S /&,,, S &RQ 5$' $=' 5$' $=' 2 $ 2 $ IROG  IROG $=' ± ±  ± ± 5$' ±  ±  ±  ± $53 $53 3 $53 &OHDYHG3 $53 β$FWLQ &OHDYH3 DFWLQ &OHDYH3 DFWLQ FKDQJHRYHU FKDQJHRYHU &RQWURO &RQWURO 5$' 5$' $=' $=' 5$' 5$' $=' $=' $=' ± ± 5$' ±  ± 34 34 34 34 34 34 34 34 34 34  34 34  34 34  34 34 3, $SRSWRVLV 34 34 34 34 34 34 34 34 SURSRUWLRQ &RQ 5$' $=' 34  34 34  34 34  34 34  34 $='5$' $QQH[LQ9 ),7& Figure 3 Selumetinib (AZD6244) significantly enhances RAD001-induced apoptosis of RCCs. Notes: 786-O and a498 cells were pretreated with aZD6244 (1 μmol/l) for 30 minutes and then treated with raD001 for 8 hours. ( A) immunoblotting showed that AZD6244 significantly inhibited the activation of ERK pathway in 786-O and A498 cells. ( B) Quantification of the immunoblots. ( C) MTT assay detected the effect of aZD6244 on cell viability of 786-O and a498 cells, which suggested that aZD6244 effectively blunts raD001-induced cell viability. ( D) immunoblotting demonstrates that aZD6244 could effectively restore upregulation of lc3-ii/i and downregulation of p62 induced by raD001 in 786-O and a498 cells. ( E) Densitometric analysis was performed to quantify the immunoblots. (F) immunoblot analysis indicated that combination treatment of raD001 with aZD6244 could increase cleave-ParP level in 786-O and a498 cells, compared to the raD001 group. ( G) Densitometric analysis was performed to quantify the cleave-ParP immunoblots. ( H) The difference of apoptosis determined by flow cytometry showed that AZD6244 significantly increased RAD001-induced cells apoptosis in 786-O and A498 cell lines. ( I) Quantification of the apoptosis proportion. *P,0.05 versus control group. P,0.05 versus raD001 group. n =3. Abbreviations: rccs, renal cell carcinoma cells; erK, extracellular signal-regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ParP, poly ADP ribose polymerase; FITC, fluorescein isothiocyanate; PI, propidium iodide; RAD001, everolimus; AZD6244, selumetinib; Con, control. not induce autophagy in RCC cells at low concentrations to the ability of CQ to effectively block autophagy at the 25 26 such as 0.1 μM. Our data showed that the combination lysosomal degradation step. However, currently available of RAD001 with CQ restored downregulation of p62, but autophagy blockers are highly unspecific and lack potency, 27–30 further promoted the upregulation of LC3-II/I, which is due and are limited by the severe toxicity of these compounds. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress β Zeng et al Dovepress $ 2 $ 5$' —PRO/ K K K K K K K K K K %HFOLQ S%FO %FO β$FWLQ % 2 $ OHYHOV OHYHOV 3URWHLQUHODWLYH 3URWHLQUHODWLYH %HFOLQ S%FO%FO %HFOLQ S%FO%FO K K K K K & 2 $ $=' ± ±   ± ± 5$' ±  ±  ±  ± %HFOLQ S%FO %FO β$FWLQ ' 2 $ OHYHOV OHYHOV 3URWHLQUHODWLYH 3URWHLQUHODWLYH %HFOLQ S%FO%FO %HFOLQ S%FO%FO &RQ 5$' $=' 5$' $=' Figure 4 erK mediated autophagy through upregulating Beclin-1 and p-Bcl-2 expression. Notes: (A, B) 786-O and a498 cells were treated by raD001 for 0, 1, 2, 4, and 8 hours. ( A) immunoblotting showed that raD001 induced upregulation of downstream signaling molecules of the erK pathway including Beclin-1 and Bcl-2 in 786-O and a498 cells in a time-dependent manner. ( B) Densitometric analysis was performed to quantify the immunoblots. *P,0.05 versus 0 hour group. n=3. (C, D) 786-O and a498 cells were pretreated with aZD6244 (1 μmol/l) for 30 minutes and then treated with raD001 for 8 hours. ( C) Immunoblotting showed that AZD6244 significantly decreased protein levels of Beclin-1 and Bcl-2 in 786-O and A498 cells. ( D) Densitometric analysis was performed to quantify the immunoblots. *P,0.05 versus control group. P,0.05 versus raD001 group. n =3. Abbreviations: erK, extracellular signal-regulated kinase; raD001, everolimus. Collectively, RAD001-induced LC3 upregulation was due necessary to further explore the effectiveness of this novel to the excessive production and accumulation of LC3-II. combination therapy with RAD001. In addition, autophagy inhibition enhanced RAD001-induced Emerging evidence shows that the JNK, p38, and ERK sig- 31–33 apoptosis of RCC cells. However, due to the clinical naling pathways are involved in cell death and autophagy. limitations of the autophagy inhibitor CQ in RCC cells, it is Previous studies have indicated that ERK activation mediates submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Dovepress attenuation of everolimus-induced cytotoxicity by involving erK activation phosphorylation of Bcl-2, leading to dissociation of the Bcl-2/ References Beclin-1 complex, which releases Beclin-1 and triggers 1. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mor- 34,35 autophagy induction. In the present study, ERK, but not tality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359–E386. the p38 and JNK pathways, was activated in RCC cells after 2. Fisher R, Gore M, Larkin J. 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BRAF status in personalizing treat- ment approaches for pediatric gliomas. Clin Cancer Res. 2016;22: Chongqing Health Bureau (2013-1-006). We sincerely thank 5312–5321. Lixue Chen, Wenhui Yan, Xiaojuan Deng, and Guangchen 19. Kiessling MK, Curioni-Fontecedro A, Samaras P, et al. Targeting Qin of the Central Laboratory of the First Afl fi iated Hospital the mTOR complex by everolimus in NRAS mutant neuroblastoma. PLoS One. 2016;11:e0147682. of Chongqing Medical University for their assistance with 20. Nishioka C, Ikezoe T, Yang J, Yokoyama A. Inhibition of MEK/ERK these experiments. signaling induces apoptosis of acute myelogenous leukemia cells via inhibition of eukaryotic initiation factor 4E-binding protein 1 and down-regulation of Mcl-1. Apoptosis. 2010;15:795–804. Disclosure 21. Sahni S, Merlot AM, Krishan S, et al. Gene of the month: BECN1. The authors report no conflicts of interest in this work. 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The mTOR inhibitor RAD001 32. Hung AC, Tsai CH, Hou MF, et al. The synthetic β-nitrostyrene deriva- augments radiation induced growth inhibition in a hepatocellular tive CYT-Rx20 induces breast cancer cell death and autophagy via carcinoma cell line by increasing autophagy. Int J Oncol. 2012;41: ROS-mediated MEK/ERK pathway. Cancer Lett. 2016;371:251–261. 1381–1386. 33. Huang XL, Zhang H, Yang XY, et al. Activation of a C-Jun N-terminal 25. Zou Y, Wang J, Leng X, et al. The selective ERK inhibitor selumetinib kinase-mediated autophagy pathway attenuates the anticancer activity enhances the antitumor activity of everolimus against renal cell carci- of gemcitabine in human bladder cancer cells. Anticancer Drugs. noma in vitro and in vivo. Oncotarget. 2017;8:20825–20833. 2017;28:596–602. 26. Wang A, Zhang H, Liang Z, et al. Antagonistic effects of chloroquine 34. Dai JP, Zhao XF, Zeng J, et al. Drug screening for autophagy inhibi- on autophagy occurrence potentiate the anticancer effects of everolimus tors based on the dissociation of Beclin1-Bcl2 complex using BiFC on renal cancer cells. Cancer Biol Ther. 2015;16:567–579. technique and mechanism of eugenol on anti-infl uenza A virus activity. 27. Yang Y, Hu L, Zheng H, et al. Application and interpretation of current PLoS One. 2013;8:e61026. autophagy inhibitors and activators. Acta Pharmacol Sin. 2013;34: 35. He W, Wang Q, Srinivasan B, et al. A JNK-mediated autophagy path- 625–635. way that triggers c-IAP degradation and necroptosis for anticancer 28. Shi TT, Yu XX, Yan LJ, et al. Research progress of hydroxychloroquine chemotherapy. Oncogene. 2014;33:3004–3013. and autophagy inhibitors on cancer. Cancer Chemother Pharmacol. 36. Yuen JS, Sim MY, Sim HG, et al. Combination of the ERK inhibitor 2017;79:287–294. AZD6244 and low-dose sorafenib in a xenograft model of human renal 29. Rosenfeld MR, Ye X, Supko JG, et al. A phase I/II trial of hydroxy- cell carcinoma. Int J Oncol. 2012;41:712–720. chloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme. Autophagy. 2014;10:1359–1368. Drug Design, Development and Therapy Dovepress Publish your work in this journal Drug Design, Development and Therapy is an international, peer- has also been accepted for indexing on PubMed Central. The manu- reviewed open-access journal that spans the spectrum of drug design script management system is completely online and includes a very and development through to clinical applications. Clinical outcomes, quick and fair peer-review system, which is all easy to use. Visit patient safety, and programs for the development and effective, safe, http://www.dovepress.com/testimonials.php to read real quotes from and sustained use of medicines are the features of the journal, which published authors. 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Attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving ERK activation in renal cell carcinoma cells

Zeng, Yizhou; Tian, Xiaofang; Wang, Quan; He, Weiyang; Fan, Jing; Gou, Xin
Drug Design, Development and Therapy , Volume 12 – Apr 19, 2018
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Abstract

Journal name: Drug Design, Development and Therapy Article Designation: Original Research Year: 2018 Volume: 12 Running head verso: Zeng et al Drug Design, Development and Therapy Dovepress Running head recto: Attenuation of everolimus-induced cytotoxicity by involving ERK activation open access to scientific and medical research DOI: 160557 Open access Full Text article O rig inal r esearch attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving erK activation in renal cell carcinoma cells Yizhou Zeng Aim: The mammalian target of rapamycin (mTOR) pathway is a critical target for cancer treatment and the mTOR inhibitor everolimus (RAD001) has been approved for treatment of Xiaofang Tian renal cell carcinoma (RCC). However, the limited efficacy of RAD001 has led to the develop - Quan Wang ment of drug resistance. Autophagy is closely related to cell survival and death, which may be Weiyang he activated under RAD001 stimulation. The aim of the present study was to identify the underlying Jing Fan mechanisms of RAD001 resistance in RCC cells through cytoprotective autophagy involving Xin gou activation of the extracellular signal-regulated kinase (ERK) pathway. Department of Urinary surgery, The Methods and results: RAD001 strongly induced autophagy of RCC cells in a dose- and time- First a ffiliated h ospital of c hongqing dependent manner, as conr fi med by Western blot analysis. Importantly, suppression of autophagy Medical University, Yuzhong District, chongqing, china by the pharmacological inhibitor chloroquine effectively enhanced RAD001-induced apoptotic cytotoxicity, as demonstrated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western blot analysis, indicating a cytoprotective role for RAD001- induced autophagy. In addition, as was shown by the MTT assay, flow cytometry, and Western blot analysis, RAD001 robustly activated ERK, but not c-Jun N-terminal kinase and p38. Activation of ERK was inhibited by the pharmacological inhibitor selumetinib (AZD6244), which effectively promoted RAD001-induced cell death. Moreover, employing AZD6244 markedly attenuated RAD001-induced autophagy and enhanced RAD001-induced apoptosis, which play a central role in RAD001-induced cell death. Furthermore, RAD001-induced autophagy is regulated by ERK-mediated phosphorylation of Beclin-1 and B-cell lymphoma 2, as confirmed by Western blot analysis. Conclusion: These results suggest that RAD001-induced autophagy involves activation of the ERK, which may impair cytotoxicity of RAD001 in RCC cells. Thus, inhibition of the activation of ERK pathway-mediated autophagy may be useful to overcome chemoresistance to RAD001. Keywords: apoptosis, autophagy, everolimus, ERK, renal cancer, selumetinib Introduction Renal cell carcinoma (RCC) is the most common form of kidney cancer, with ~338,000 new diagnoses and 144,000 deaths occurring annually worldwide. Surgical resection is generally performed to treat this disease; however, nephrectomy is not a feasible option for about 30% of patients with metastatic disease. Therefore, to further improve correspondence: Jing Fan; Xin gou the quality of life and survival of patients, systemic treatment may be a more effec- Department of Urinary surgery, The First tive choice. Affiliated Hospital of Chongqing Medical University, no 1 Youyi road, Yuzhong Everolimus (RAD001), a mammalian target of rapamycin (mTOR) inhibitor, has District, chongqing 400016, china been demonstrated to exert cytotoxicity against human cancers of the breast, stomach, email fjfj8003@163.com; 4–6 omegamanman@163.com and prostate, and is currently used as a sequential or second-line therapy for RCC submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 911–920 Dovepress © 2018 Zeng et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you http://dx.doi.org/10.2147/DDDT.S160557 hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Zeng et al Dovepress refractory to sunitinib or sorafenib. However, the efficacy phospho-Bcl-2, Beclin-1, ERK, phospho-ERK, p38, phospho- of RAD001 is thought to be limited by feedback loops and p38, c-Jun N-terminal kinase (JNK), and phospho-JNK were cross talk with other pathways, resulting in drug resistance. purchased from Cell Signaling Technology (Danvers, MA, Karam et al established and characterized a panel of mouse USA). Anti-cleaved poly ADP ribose polymerase (PARP) models of RCC derived from patients undergoing radical and p62 were obtained from BD Biosciences (San Jose, CA, nephrectomy. Using these models, resistance to the mTOR USA). Anti-LC3 and chloroquine (CQ) were purchased from inhibitor RAD001 was identified. François et al stated that Sigma-Aldrich Co. (St Louis, MO, USA). Anti-β-actin anti- RAD001 rarely induces regression of pancreatic neuroen- body was purchased from Zoonbio Biotechnology Co., Ltd docrine tumors. In addition, as compared with RAD001 (Nanjing, China). All secondary antibodies were obtained alone, co-treatment seems to be potentially more effective from Abgent (San Diego, CA, USA). Roswell Park Memorial 9,10 11 in controlling cell signaling. Motzer et al declared that Institute (RPMI)-1640 medium and trypsin were purchased a combination therapy of RAD001 and lenvatinib showed from HyClone (Logan, UT, USA). Fetal bovine serum was a favorably synergistic effect in patients with advanced or purchased from Gibco (Thermo Fisher Scientific, Waltham, metastatic RCC, which was the first successful combination MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- therapy approved by the US Food and Drug Administration. zolium bromide (MTT) cell kit was purchased from Beyotime Thus, based on these findings, it is necessary to explore the Institute of Biotechnology (Shanghai, China). underlying mechanism of the drug resistance of RAD001. Autophagy is a highly conserved intracellular catabolic cell culture process that degrades and recycles cellular components for The human RCC cell lines 786-O and A498 were pur- cell survival under certain conditions, and is closely related to chased from American Type Culture Collection (Manassas, cell survival or death. For cancer cells resistant to chemother- VA, USA) and grown in RPMI-1640 medium supplemented apy, autophagy presents either protective or injurious effects. with 10% fetal bovine serum, 1 mmol/L glutamine, 10 U/L For example, RAD001 is able to induce autophagy in human penicillin, and 100 g/L streptomycin at 37°C, under a humidi- renal cancer cells, which then promotes tumor cell survival, fied atmosphere containing 5% CO . resulting in a limited anticancer effect. The mTOR complex is now regarded as an autophagy switch to promote prolifera- MTT assay tion and inhibit autophagy, although the potential mechanisms Cell viability was assessed with the MTT assay. Briefly, involved in the cell signal pathways for this process are still 786-O and A498 cells were seeded into 96-well plates at not fully understood. Interestingly, Butler et al found that 5,000 cells/well and cultivated for 24 hours. After adher- inhibition of the phosphatidylinositol 3 kinase (PI3K)/protein ence, the 786-O and A498 cells were treated with various kinase B (AKT)/mTOR pathway activates autophagy and concentrations of RAD001 for 24 hours. MTT (5 mg/mL, compensatory Ras/Raf/MEK/extracellular signal-regulated 20 μL/well) was added to each well and the plate was incu- kinase (ERK) signaling in prostate cancer. Besides, activation bated at 37°C for 4 hours. Dimethyl sulfoxide was then added of the PI3K/AKT/mTOR and Ras/MEK/ERK cell signaling (150 μL/well) to each well to dissolve any crystals and the pathways is also critical for autophagy. Particularly, cross plates were agitated for 15 minutes. Absorbance at 570 nm talk between these two pathways has been well illustrated in was measured using an Infinite F200/M200 type multifunc - 15–17 previous studies. Moreover, several studies have shown tion microplate reader (Tecan Trading AG, Männedorf, that inhibition of the ERK pathway enhanced the antitumor Switzerland). Cell viability was calculated by OD values of 18 19 activity of RAD001 in pediatric gliomas, neuroblastoma, treated groups/OD values of control group) ×100%. Each and acute myelogenous leukemia. Thus, the aim of this study experiment was repeated three times. was to identify the underlying mechanisms and biochemical pathways involved in RAD001 resistance in RCC. Apoptosis analysis by flow cytometry After treatment with RAD001 and AZD6244, the cells Materials and methods were trypsinized, washed with phosphate-buffered saline, Materials and suspended in 195 μL of Annexin V-fluorescein isothio - The small molecular inhibitors RAD001 and AZD6244 were cyanate (FITC) binding buffer containing 5 μL of Annexin obtained from MedChem Express (Monmouth Junction, V-FITC and 10 μL of propidium iodide (Beyotime Institute NJ, USA). Antibodies against B-cell lymphoma 2 (Bcl-2), of Biotechnology). After incubation for 10–20 minutes at submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Dovepress attenuation of everolimus-induced cytotoxicity by involving erK activation room temperature in the dark, the cells were subjected to conversion of LC3-I to LC3-II was in line with a gradual flow cytometry analysis using a FACSCanto 6-color flow decrease in autophagic protein p62 in both cell lines after cytometer (BD Biosciences). RAD001 treatment in a time-dependent manner (Figure 1C and D). Taken together, these n fi dings further emphasize that Western blot analysis RAD001 is able to induce autophagy in RCC cells. To extract total protein lysates, the cells were lysed in cold autophagy protected rcc cells against radioimmunoprecipitation assay lysis buffer (Beyotime raD001-induced cell death, which Institute of Biotechnology) containing phenylmethanesul- fonyl fluoride (Beyotime Institute of Biotechnology), and involved activation of the erK pathway then centrifuged at 12,000 rpm for 15 minutes at 4°C to To further determine whether RAD001 could induce death remove debris. Protein concentrations were measured with of RCC cells, the MTT assay was performed with 786-O the bicinchoninic acid protein assay kit (Beyotime Institute and A498 cells following exposure to RAD001. Pretreat- of Biotechnology). The protein extracts were heat denatured ment of both cell lines with RAD001 (0.01–100 μmol/L) in sodium dodecyl sulfate polyacrylamide gel electrophoresis for 24 hours revealed a dose-dependent reduction in cell Sample Loading Buffer (Beyotime Institute of Biotech- survival. The half IC (half maximal inhibitor concentra- nology). Equal amounts of protein from each cell lysate tion) of RAD001 at 24 hours for 786-O and A498 cells was (40 μg/lane) were separated in 12% polyacrylamide gels 10 and 1 μmol/L, respectively. Thus, these values were cho- and transferred on to a membrane. After blocking with 5% sen for subsequent experiments (Figure 2A). Based on the nonfat dried milk for 2 hours, the membrane was incubated observation that RAD001 also induced autophagy, we were with the primary antibodies overnight at 4°C. Afterward, the particularly interested in investigating whether autophagy proteins were visualized by enhanced chemiluminescence was involved in RAD001-induced cytotoxicity. For this using horseradish peroxidase-conjugated immunoglobulin G purpose, the lysosome inhibitor CQ was used in this study, secondary antibodies. as it inhibits autophagy via blocking autophagolysosome- mediated degradation of the p62 and LC3-II proteins. statistical analysis Expectedly, the RAD001-induced downregulation of p62 Data are expressed as mean ± SD. Statistical analyses were expression was effectively inhibited by CQ, although a performed with GraphPad Prism 6 software (GraphPad Soft- higher LC3-II level was observed in 786-O and A498 cells ware, Inc, San Diego, CA, USA) and IBM SPSS Statistics after exposure to co-treatment with both RAD001and CQ, for Windows, version 20.0 (IBM Corporation, Armonk, suggesting that RAD001 induces a continuous autophagic NY, USA). Based on the completely random design of this flux in both cell lines (Figure 2B and C). These results experiment, the results were analyzed by one-way analysis indicate that autophagy was indeed induced by RAD001. of variance or t-test. A probability value of ,0.05 was con- More importantly, in line with these findings, the results of sidered statistically significant. the MTT assay showed that the effect of RAD001-induced cell cytotoxicity was significantly enhanced by inhibition Results of autophagy in 786-O and A498 cells, suggesting that raD001-induced autophagy in rcc cells autophagy may play a protective role in RAD001-induced It has been well demonstrated that the mTOR pathway inhibi- cytotoxicity of RCC (Figure 2D). Because cell cytotoxicity tor RAD001 can induce autophagy in human RCC cells. The is closely associated with apoptosis, to further determine aim of the present study was to determine whether RAD001 whether the protective effects of autophagy for RAD001- can induce autophagy in RCC cells. For this purpose, dif- induced cell death were due to the activation of apoptotic ferent concentrations of RAD001 were added to cultures of signaling, Western blot analysis was conducted to measure 786-O and A498 cells. The results showed that RAD001- expression levels of cleaved PARP protein. Our results induced autophagy of 786-O and A498 cells in a dose- demonstrated that the apoptosis biomarker cleaved-PARP dependent manner, which was indicated by a typical pheno- was significantly upregulated by RAD001 and this effect type of autophagy, including upregulation of LC3-II/I and was enhanced by inhibition of autophagy with CQ (Figure 2E downregulation of p62 (Figure 1A and B). Next, to further and F). Altogether, these results suggest that autophagy pro- validate this effect, 786-O and A498 cells were treated with tects RCC cells against RAD001-induced cytotoxicity in part RAD001 for 0, 1, 2, 4, or 8 hours. As expected, the increased by suppressing the apoptosis biomarker-cleaved PARP. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Zeng et al Dovepress A 786-O A498 RAD (µmol/L) 00.010.1 11020 00.010.1 11020 LC3-I LC3-II p62 β-Actin B 786-O A498 1.5 5 1.0 0.5 0.0 0 LC3-II/I p62 LC3-II/I p62 0 0.01 0.1 1 10 20 C 786-O A498 RAD 0 h1 h2 h4 h8 h 0 h1 h2 h4 h8 h LC3-I LC3-II p62 β-Actin D 786-O A498 2.0 4 1.5 1.0 2 0.5 1 0.0 0 LC3-II/I p62 LC3-II/I p62 0 h 1 h 2 h 4 h 8 h Figure 1 everolimus (raD001) induces autophagy in rccs. Notes: (A, B) 786-O and a498 cell lines were treated by raD001 with different concentrations (0, 0.01, 0.1, 1, 10, and 20 μmol/l) for 24 hours. ( A) immunoblotting showed that raD001 induced autophagy indicators including upregulation of lc3-ii/i and downregulation of p62 in a dose-dependent manner. ( B) Quantification of the immunoblots. *P,0.05 versus 0 μmol/l group. n =3. (C, D) 786-O and a498 cell lines were treated by raD001 for 0, 1, 2, 4, and 8 hours. ( C) immunoblotting showed that raD001 induced autophagy in 786-O and a498 cells as time prolonged, which peaked at 8 hours. ( D) Quantification of the immunoblots. * P,0.05 versus 0 hour group. n=3. Abbreviation: rccs, renal cell carcinoma cells. We next sought to explore the molecular mechanism by cell death on the activity of the JNK, ERK, and p38 cell sig- which RAD001 induces death of RCC cells. As it has been naling pathways. Briey fl , 786-O and A498 cells were treated recognized that mitogen-activated protein kinases play a with RAD001 for the indicated times, and the activities of critical regulative role in cancer cell death and survival, the the involved signaling pathways were assessed by Western focus of the present study was the effect of RAD001-induced blot analysis. The results showed that following RAD001 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Protein relative levels Protein relative levels Protein relative levels Protein relative levels Dovepress attenuation of everolimus-induced cytotoxicity by involving erK activation $ ) 2 $ IROG IROG $53 $53 &HOOYLDELOLW\ &OHDYH3 $FWLQ &OHDYH3 $FWLQ FKDQJHRYHU FKDQJHRYHU &4 &4 5$' —PRO/ &RQWURO &RQWURO 5$' 5$' 5$'&4 5$'&4 % 2 $ &4 ±± ±± * 2 $ 5$' ±  ±  ±  ± 5$' K K K K K K K K K K /&, /&,, S-1. -1. β$FWLQ SS & 2 $ S(5. (5. 3URWHLQ 3URWHLQ β$FWLQ UHODWLYHOHYHOV UHODWLYHOHYHOV /&,,, S /&,,, S &RQ 5$' &4 5$'&4 ' + 2 &RQ 5$' &4 5$'&4 3URWHLQ UHODWLYHOHYHOV &HOOYLDELOLW\ S-1.-1. SSS S(5.(5. 2 $ ( 2 $ &4 ±± ±± 5$' ±  ±  ±  ± 3URWHLQ 3 $53 &OHDYHG3 $53 UHODWLYHOHYHOV β$FWLQ S-1.-1. SSS S(5.(5. K K K K K Figure 2 inhibition of autophagy enhances raD001-induced cytotoxicity, raD001-induced activation of erK signaling pathway in rccs. Notes: 786-O and a498 cells were pretreated with cQ (autophagy inhibitor) (10 mmol/l) for 30 minutes and then treated with raD001 for 8 hours. ( A) MTT assay showed that raD001 impaired cell viability of 786-O and a498 cells in a dose-dependent manner. n=3. (B) immunoblotting of lc3-ii/i and p62 showed that cQ could restore the downregulation of p62, but would not impair the upregulation of lc3-ii/i because cQ blocks autophagy at the lysosomal degradation step. (C) Densitometric analysis was performed to quantify the immunoblots. *P,0.05 versus control. P,0.05 versus raD001 group. n =3. (D) MTT assay displayed that autophagy inhibition by cQ promoted raD001-induced cytotoxicity in 786-O and a498 cells. * P,0.05 versus control. P,0.05 versus raD001 group. n =3. (E) The apoptosis indicator cleave-ParP detected by immunoblot indicated that cells apoptosis was increased in 786-O and a498 cells after cQ intervention, compared to the raD001 group. ( F) Densitometric analysis was performed to quantify the immunoblots. *P,0.05 versus control group. P,0.05 versus raD001 group. n =3. 786-O and a498 cells were treated by raD001 for 0, 1, 2, 4, and 8 hours. ( G) immunoblotting analyzed the JnK, p38, and erK signaling molecules in 786-O and a498 cells after raD001 treatment. (H) Quantification of the immunoblots. * P,0.05 versus 0 hour group. n=3. Abbreviations: cQ, chloroquine; con, control; rccs, renal cell carcinoma cells; erK, extracellular signal-regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; ParP, poly aDP ribose polymerase; JnK, c-Jun n-terminal kinase. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress β Zeng et al Dovepress pretreatment, p-ERK expression was significantly upregu - enhanced in both cell lines (Figure 3H and I). In summary, lated in both cell lines. However, the expression levels of these data suggest that inhibition of ERK activation not only p-JNK and p-p38 were largely unchanged following RAD001 suppressed RAD001-induced autophagy, but also enhanced exposure (Figure 2G and H). RAD001-induced apoptosis, which plays a central role in RAD001-induced cell death. inhibition of erK activation suppressed raD001-induced autophagy and erK mediated autophagy by upregulating enhanced raD001-induced apoptosis expression of Beclin-1 and p-Bcl-2 The pharmacological inhibitor AZD6244 was employed It is reported that ERK activation mediates phosphorylation to block ERK signaling to investigate the role of ERK of Bcl-2, resulting in dissociation of the Bcl-2/Beclin-1 in RAD001-induced cell death. The results showed that complex. This process releases Beclin-1 and leads to AZD6244 markedly inhibited activation of the ERK pathway autophagy induction. Next, we further explored the mecha- induced by RAD001 (Figure 3A and B). More importantly, nism whereby RAD001 induced ERK activation and led to consistent with the effective suppressive effects of AZD6244 the induction of autophagy. Beclin-1 is critical for the ini- on ERK, co-treatment of both RAD001 and AZD6244 tiation of autophagy. The downstream factors of the ERK induced a greater extent of cell death than pretreatment pathway, including Beclin-1 and p-Bcl-2, were upregulated of RAD001 alone, which indicated that inhibition of ERK as early as 1 hour after RAD001 treatment in both cell lines effectively synergized with RAD001 to impair cell viability (Figure 4A and B). Moreover, inhibition of ERK activation (Figure 3C). Overall, these results suggest that RAD001- with AZD6244 significantly decreased the expression levels induced cytotoxicity involves ERK signaling. of Beclin-1 and p-Bcl-2 (Figure 4C and D). These results A series of studies, including those by our group, have suggest that RAD001-induced ERK-activated autophagy is demonstrated that ERK signaling is closely related to at least in part due to upregulating the expression levels of autophagy in various cancers. To further confirm whether Beclin-1 and p-Bcl-2. this signaling is involved in RAD001-induced autophagy, the specific inhibitor AZD6244 for ERK was used to Discussion interfere with autophagy induction. Indeed, employing The results of the present study showed that RAD001- AZD6244 markedly attenuated the autophagy effect induced induced autophagy may play an important role in RAD001- by RAD001 in both 786-O and A498 cells, as indicated by induced cytotoxicity of RCC cells, although the underlying the decreased expression of LC3-II/I and increased expres- mechanism remains unknown. Furthermore, cytoprotective sion of p62 (Figure 3D and E). It is well established that autophagy involving the ERK pathway was induced by cell death after exposure to cytotoxic chemotherapeutics is RAD001, thereby presenting a novel combination strategy partially mediated by apoptosis. Moreover, as suggested by to target ERK signaling. AZD6244 is a highly selective ERK the above observation that autophagy protected RCC cells inhibitor. Interestingly, AZD6244 significantly inhibited against RAD001-induced cytotoxicity in part by suppressing RAD001-induced autophagy and enhanced RAD001-induced the apoptotic biomarker-cleaved PARP, we were interested apoptosis of 786-O and A498 cells. These findings sug - in investigating whether ERK signaling was involved in the gested that RCC cell resistance to RAD001 may be closely apoptosis process that caused RAD001-induced cytotoxicity. related to cytoprotective autophagy linked to the activation To test this hypothesis, we evaluated cell apoptosis by flow of the ERK pathway. cytometry and the protein expression of cleaved PARP Evidence has confirmed that RAD001 can substantially by Western blot analysis. Immunoblotting showed that induce autophagy, as well as promote the apoptosis of 22 23,24 AZD6244 exaggerated RAD001-induced cleavage of PARP breast cancer cells and hepatocellular carcinoma cells. (Figure 3F and G), indicating that the RAD001-induced Similarly, the results of the present experiment showed that apoptosis was enhanced by AZD6244. Furthermore, the RAD001 treatment of RCC cells resulted in the upregulation flow cytometry results showed that pretreatment with of LC3-II/I and downregulation of p62, which are typical RAD001 resulted in a higher rate of apoptosis in both cell features of autophagy induction, suggesting that autophagy lines, compared with no RAD001 exposure, suggesting by 786-O and A498 cells was induced by RAD001 treat- that RAD001 did indeed induce cell apoptosis. Moreover, ment. So, high concentrations of RAD001 were used in this when these cell lines were treated with both AZD6244 and experiment due to the grade of the RCC cell lines 786-O RAD001, this RAD001-induced apoptosis effect was further and A498. In addition, it is reported that RAD001 did submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Dovepress attenuation of everolimus-induced cytotoxicity by involving erK activation $% 2 & $=' ± ±  ± ± ±  ±  ± ± 5$' S(5. FKDQJH (5. S(5.(5.IROG &HOOYLDELOLW\ β$FWLQ 2 $ 2 $ &RQ 5$' $=' 5$' $=' $ 2 $ '( 2 ± ±  ± ± $=' ±  ±  ± ± 5$' /&, /&,, OHYHOV OHYHOV 3URWHLQUHODWLYH 3URWHLQUHODWLYH β$FWLQ /&,,, S /&,,, S &RQ 5$' $=' 5$' $=' 2 $ 2 $ IROG  IROG $=' ± ±  ± ± 5$' ±  ±  ±  ± $53 $53 3 $53 &OHDYHG3 $53 β$FWLQ &OHDYH3 DFWLQ &OHDYH3 DFWLQ FKDQJHRYHU FKDQJHRYHU &RQWURO &RQWURO 5$' 5$' $=' $=' 5$' 5$' $=' $=' $=' ± ± 5$' ±  ± 34 34 34 34 34 34 34 34 34 34  34 34  34 34  34 34 3, $SRSWRVLV 34 34 34 34 34 34 34 34 SURSRUWLRQ &RQ 5$' $=' 34  34 34  34 34  34 34  34 $='5$' $QQH[LQ9 ),7& Figure 3 Selumetinib (AZD6244) significantly enhances RAD001-induced apoptosis of RCCs. Notes: 786-O and a498 cells were pretreated with aZD6244 (1 μmol/l) for 30 minutes and then treated with raD001 for 8 hours. ( A) immunoblotting showed that AZD6244 significantly inhibited the activation of ERK pathway in 786-O and A498 cells. ( B) Quantification of the immunoblots. ( C) MTT assay detected the effect of aZD6244 on cell viability of 786-O and a498 cells, which suggested that aZD6244 effectively blunts raD001-induced cell viability. ( D) immunoblotting demonstrates that aZD6244 could effectively restore upregulation of lc3-ii/i and downregulation of p62 induced by raD001 in 786-O and a498 cells. ( E) Densitometric analysis was performed to quantify the immunoblots. (F) immunoblot analysis indicated that combination treatment of raD001 with aZD6244 could increase cleave-ParP level in 786-O and a498 cells, compared to the raD001 group. ( G) Densitometric analysis was performed to quantify the cleave-ParP immunoblots. ( H) The difference of apoptosis determined by flow cytometry showed that AZD6244 significantly increased RAD001-induced cells apoptosis in 786-O and A498 cell lines. ( I) Quantification of the apoptosis proportion. *P,0.05 versus control group. P,0.05 versus raD001 group. n =3. Abbreviations: rccs, renal cell carcinoma cells; erK, extracellular signal-regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ParP, poly ADP ribose polymerase; FITC, fluorescein isothiocyanate; PI, propidium iodide; RAD001, everolimus; AZD6244, selumetinib; Con, control. not induce autophagy in RCC cells at low concentrations to the ability of CQ to effectively block autophagy at the 25 26 such as 0.1 μM. Our data showed that the combination lysosomal degradation step. However, currently available of RAD001 with CQ restored downregulation of p62, but autophagy blockers are highly unspecific and lack potency, 27–30 further promoted the upregulation of LC3-II/I, which is due and are limited by the severe toxicity of these compounds. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress β Zeng et al Dovepress $ 2 $ 5$' —PRO/ K K K K K K K K K K %HFOLQ S%FO %FO β$FWLQ % 2 $ OHYHOV OHYHOV 3URWHLQUHODWLYH 3URWHLQUHODWLYH %HFOLQ S%FO%FO %HFOLQ S%FO%FO K K K K K & 2 $ $=' ± ±   ± ± 5$' ±  ±  ±  ± %HFOLQ S%FO %FO β$FWLQ ' 2 $ OHYHOV OHYHOV 3URWHLQUHODWLYH 3URWHLQUHODWLYH %HFOLQ S%FO%FO %HFOLQ S%FO%FO &RQ 5$' $=' 5$' $=' Figure 4 erK mediated autophagy through upregulating Beclin-1 and p-Bcl-2 expression. Notes: (A, B) 786-O and a498 cells were treated by raD001 for 0, 1, 2, 4, and 8 hours. ( A) immunoblotting showed that raD001 induced upregulation of downstream signaling molecules of the erK pathway including Beclin-1 and Bcl-2 in 786-O and a498 cells in a time-dependent manner. ( B) Densitometric analysis was performed to quantify the immunoblots. *P,0.05 versus 0 hour group. n=3. (C, D) 786-O and a498 cells were pretreated with aZD6244 (1 μmol/l) for 30 minutes and then treated with raD001 for 8 hours. ( C) Immunoblotting showed that AZD6244 significantly decreased protein levels of Beclin-1 and Bcl-2 in 786-O and A498 cells. ( D) Densitometric analysis was performed to quantify the immunoblots. *P,0.05 versus control group. P,0.05 versus raD001 group. n =3. Abbreviations: erK, extracellular signal-regulated kinase; raD001, everolimus. Collectively, RAD001-induced LC3 upregulation was due necessary to further explore the effectiveness of this novel to the excessive production and accumulation of LC3-II. combination therapy with RAD001. In addition, autophagy inhibition enhanced RAD001-induced Emerging evidence shows that the JNK, p38, and ERK sig- 31–33 apoptosis of RCC cells. However, due to the clinical naling pathways are involved in cell death and autophagy. limitations of the autophagy inhibitor CQ in RCC cells, it is Previous studies have indicated that ERK activation mediates submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Dovepress attenuation of everolimus-induced cytotoxicity by involving erK activation phosphorylation of Bcl-2, leading to dissociation of the Bcl-2/ References Beclin-1 complex, which releases Beclin-1 and triggers 1. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mor- 34,35 autophagy induction. In the present study, ERK, but not tality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359–E386. the p38 and JNK pathways, was activated in RCC cells after 2. Fisher R, Gore M, Larkin J. 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A phase I/II trial of hydroxy- cell carcinoma. Int J Oncol. 2012;41:712–720. chloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme. Autophagy. 2014;10:1359–1368. Drug Design, Development and Therapy Dovepress Publish your work in this journal Drug Design, Development and Therapy is an international, peer- has also been accepted for indexing on PubMed Central. The manu- reviewed open-access journal that spans the spectrum of drug design script management system is completely online and includes a very and development through to clinical applications. Clinical outcomes, quick and fair peer-review system, which is all easy to use. Visit patient safety, and programs for the development and effective, safe, http://www.dovepress.com/testimonials.php to read real quotes from and sustained use of medicines are the features of the journal, which published authors. 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