Puquitinib mesylate (XC-302) induces autophagy via inhibiting the PI3K/AKT/mTOR signaling pathway in nasopharyngeal cancer cells

Wang,Ke-Feng; Yang,Hang; Jiang,Wen-Qi; Li,Su; Cai,Yu-Chen

doi:10.3892/ijmm.2015.2378

Puquitinib mesylate (XC-302) induces autophagy via inhibiting the PI3K/AKT/mTOR signaling pathway in nasopharyngeal cancer cells

Authors:
- Ke-Feng Wang
- Hang Yang
- Wen-Qi Jiang
- Su Li
- Yu-Chen Cai
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Affiliations: State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong 510060, P.R. China
Published online on: October 16, 2015 https://doi.org/10.3892/ijmm.2015.2378
Pages: 1556-1562
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

There are numerous studies that demonstrate the anti-neoplastic activity of phosphatidylinositol 3-kinase (PI3K) inhibitors and the mechanisms of inducing autophagy in cancer cells. The new anticancer drug puquitinib mesylate (XC-302) is a molecular-targeted drug, which suppresses the activity of PI3K directly. However, it remains unclear whether XC‑302 can develop an antitumor effect by inducing autophagy in nasopharyngeal cancer cells. The MTT assay was used to study the anti‑proliferative effects of XC‑302. Subsequently, autophagy was determined by monodansylcadaverine (MDC) staining, punctate localization of green fluorescent protein (GFP)‑light chain 3 (LC3), LC3 protein blotting and electron microscopy. The expression levels of beclin 1, p62, protein kinase B (AKT), phospho (p)‑AKT, mechanistic target of rapamycin (mTOR) and p‑mTOR in XC‑302‑induced autophagy were detected. Autophagy inhibition was assayed by 3‑methyladenine (3‑MA) or small interfering RNA (siRNA) silencing of beclin 1. XC‑302 inhibited the viability of CNE‑2 in a dose‑dependent manner and the IC50 of 72 h was 5.2 µmol/l. After cells were exposed to XC‑302 for 24 h, MDC‑labeled autophagolysosomes were evident in CNE‑2 cells by fluorescence microscope. Autophagosomes and autolysosomes were identified by transmission electron microscopy. Following transfection with GFP‑LC3, XC‑302 induced a significant accumulation of GFP‑LC3, as monitored by a confocal microscope, which was reduced by 3‑MA. XC‑302 induced the formation of LC3‑II, increased beclin 1 levels and decreased the expression of p62. Additionally, the expression levels of p‑AKT and p‑mTOR were reduced with the elevation of XC‑302. Knockdown of beclin 1 with siRNA or co‑treatment with 3‑MA enhanced significantly the survival of CNE‑2 and promoted the ability of clone formation. XC‑302 also induced apoptosis in CNE‑2, and when autophagy was inhibited by 3‑MA, the apoptosis rate was decreased. The present data provides the evidence that XC‑302 can induce autophagy in CNE‑2, which promotes the program of cell death and inhibits the PI3K/AKT/mTOR signaling pathway. Furthermore, XC‑302 also promoted apoptosis in CNE‑2 cells, which could be reduced when autophagy was suppressed, meaning that autophagy may interact with apoptosis to induce cell death.

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1	Cantley LC: The phosphoinositide 3-kinase pathway. Science. 296:1655–1657. 2002. View Article : Google Scholar : PubMed/NCBI
2	Luo J, Manning BD and Cantley LC: Targeting the PI3K-Akt pathway in human cancer: Rationale and promise. Cancer Cell. 4:257–262. 2003. View Article : Google Scholar : PubMed/NCBI
3	Courtney KD, Corcoran RB and Engelman JA: The PI3K pathway as drug target in human cancer. J Clin Oncol. 28:1075–1083. 2010. View Article : Google Scholar : PubMed/NCBI
4	Chang ET and Adami HO: The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev. 15:1765–1777. 2006. View Article : Google Scholar : PubMed/NCBI
5	Spratt DE and Lee N: Current and emerging treatment options for nasopharyngeal carcinoma. Onco Targets Ther. 5:297–308. 2012.PubMed/NCBI
6	Lin S, Pan J, Han L, Guo Q, Hu C, Zong J, Zhang X and Lu JJ: Update report of nasopharyngeal carcinoma treated with reduced-volume intensity-modulated radiation therapy and hypothesis of the optimal margin. Radiother Oncol. 110:385–389. 2014. View Article : Google Scholar : PubMed/NCBI
7	Xiao WW, Huang SM, Han F, Wu SX, Lu LX, Lin CG, Deng XW, Lu TX, Cui NJ and Zhao C: Local control, survival, and late toxicities of locally advanced nasopharyngeal carcinoma treated by simultaneous modulated accelerated radiotherapy combined with cisplatin concurrent chemotherapy: Long-term results of a phase 2 study. Cancer. 117:1874–1883. 2011. View Article : Google Scholar : PubMed/NCBI
8	Gupta AK1, McKenna WG, Weber CN, Feldman MD, Goldsmith JD, Mick R, Machtay M, Rosenthal DI, Bakanauskas VJ, Cerniglia GJ, et al: Local recurrence in head and neck cancer: relationship to radiation resistance and signal transduction. Clin Cancer Res. 8:885–892. 2002.PubMed/NCBI
9	Liu T, Sun Q, Li Q, Yang H, Zhang Y, Wang R, Lin X, Xiao D, Yuan Y, Chen L, et al: Dual PI3K/mTOR inhibitors, GSK2126458 and PKI-587, suppress tumor progression and increase radio-sensitivity in nasopharyngeal carcinoma. Mol Cancer Ther. 14:429–439. 2014. View Article : Google Scholar
10	Yang F, Qian XJ, Qin W, Deng R, Wu XQ, Qin J, Feng GK and Zhu XF: Dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 has a therapeutic potential and sensitizes cisplatin in nasopharyngeal carcinoma. PLoS One. 8:e598792013. View Article : Google Scholar : PubMed/NCBI
11	Liu Y, Chen LH, Yuan YW, Li QS, Sun AM and Guan J: Activation of AKT is associated with metastasis of nasopharyngeal carcinoma. Tumour Biol. 33:241–245. 2012. View Article : Google Scholar
12	Hanahan D and Weinberg RA: Hallmarks of cancer: The next generation. Cell. 144:646–674. 2011. View Article : Google Scholar : PubMed/NCBI
13	Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, Dawson TM, Dawson VL, El-Deiry WS, Fulda S, et al: Molecular definitions of cell death subroutines: Recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ. 19:107–120. 2012. View Article : Google Scholar :
14	Deng Q, Yu X, Xiao L, Hu Z, Luo X, Tao Y, Yang L, Liu X, Chen H, Ding Z, et al: Neoalbaconol induces energy depletion and multiple cell death in cancer cells by targeting PDK1-PI3-K/Akt signaling pathway. Cell Death Dis. 4:e8042013. View Article : Google Scholar : PubMed/NCBI
15	Zhao YY, Tian Y, Zhang J, Xu F, Yang YP, Huang Y, Zhao HY, Zhang JW, Xue C, Lam MH, et al: Effects of an oral allosteric AKT inhibitor (MK-2206) on human nasopharyngeal cancer in vitro and in vivo. Drug Des Devel Ther. 8:1827–1837. 2014. View Article : Google Scholar : PubMed/NCBI
16	Munafó DB and Colombo MI: A novel assay to study autophagy: Regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci. 114:3619–3629. 2001.PubMed/NCBI
17	Ma BB, Lui VW, Hui EP, Lau CP, Ho K, Ng MH, Cheng SH, Tsao SW and Chan AT: The activity of mTOR inhibitor RAD001 (everolimus) in nasopharyngeal carcinoma and cisplatin-resistant cell lines. Invest New Drugs. 28:413–420. 2010. View Article : Google Scholar
18	Ma BB, Lui VW, Hui CW, Lau CP, Wong CH, Hui EP, Ng MH, Tsao SW, Li Y and Chan AT: Preclinical evaluation of the AKT inhibitor MK-2206 in nasopharyngeal carcinoma cell lines. Invest New Drugs. 31:567–575. 2013. View Article : Google Scholar
19	Morrison JA, Gulley ML, Pathmanathan R and Raab-Traub N: Differential signaling pathways are activated in the Epstein-Barr virus-associated malignancies nasopharyngeal carcinoma and Hodgkin lymphoma. Cancer Res. 64:5251–5260. 2004. View Article : Google Scholar : PubMed/NCBI
20	Huang XM, Dai CB, Mou ZL, Wang LJ, Wen WP, Lin SG, Xu G and Li HB: Overproduction of cyclin D1 is dependent on activated mTORC1 signal in nasopharyngeal carcinoma: Implication for therapy. Cancer Lett. 279:47–56. 2009. View Article : Google Scholar : PubMed/NCBI
21	Chen J, Hu CF, Hou JH, Shao Q, Yan LX, Zhu XF, Zeng YX and Shao JY: Epstein-Barr virus encoded latent membrane protein 1 regulates mTOR signaling pathway genes which predict poor prognosis of nasopharyngeal carcinoma. J Transl Med. 8(30)2010. View Article : Google Scholar
22	Liu P, Cheng H, Roberts TM and Zhao JJ: Targeting the phosphoinositide 3-kinase pathway in cancer. Nat Rev Drug Discov. 8:627–644. 2009. View Article : Google Scholar : PubMed/NCBI
23	Tekirdag KA, Korkmaz G, Ozturk DG, Agami R and Gozuacik D: MIR181A regulates starvation- and rapamycin-induced autophagy through targeting of ATG5. Autophagy. 9:374–385. 2013. View Article : Google Scholar : PubMed/NCBI
24	Mizushima N: Methods for monitoring autophagy. Int J Biochem Cell Biol. 36:2491–2502. 2004. View Article : Google Scholar : PubMed/NCBI
25	Fujiwara K, Iwado E, Mills GB, Sawaya R, Kondo S and Kondo Y: Akt inhibitor shows anticancer and radiosensitizing effects in malignant glioma cells by inducing autophagy. Int J Oncol. 31:753–760. 2007.PubMed/NCBI
26	Hung JY, Hsu YL, Li CT, Ko YC, Ni WC, Huang MS and Kuo PL: 6-Shogaol, an active constituent of dietary ginger, induces autophagy by inhibiting the AKT/mTOR pathway in human non-small cell lung cancer A549 cells. J Agric Food Chem. 57:9809–9816. 2009. View Article : Google Scholar : PubMed/NCBI
27	Shin SY, Lee KS, Choi YK, Lim HJ, Lee HG, Lim Y and Lee YH: The antipsychotic agent chlorpromazine induces autophagic cell death by inhibiting the Akt/mTOR pathway in human U-87MG glioma cells. Carcinogenesis. 34:2080–2089. 2013. View Article : Google Scholar : PubMed/NCBI
28	Seglen PO and Gordon PB: 3-Methyladenine: Specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci USA. 79:1889–1892. 1982. View Article : Google Scholar : PubMed/NCBI
29	Lum JJ, Bauer DE, Kong M, Harris MH, Li C, Lindsten T and Thompson CB: Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell. 120:237–248. 2005. View Article : Google Scholar : PubMed/NCBI
30	Eisenberg-Lerner A and Kimchi A: The paradox of autophagy and its implication in cancer etiology and therapy. Apoptosis. 14:376–391. 2009. View Article : Google Scholar : PubMed/NCBI
31	Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD and Levine B: Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell. 122:927–939. 2005. View Article : Google Scholar : PubMed/NCBI
32	Levine B, Sinha S and Kroemer G: Bcl-2 family members: Dual regulators of apoptosis and autophagy. Autophagy. 4:600–606. 2008. View Article : Google Scholar : PubMed/NCBI
33	Chang NC, Nguyen M, Germain M and Shore GC: Antagonism of Beclin 1-dependent autophagy by BCL-2 at the endoplasmic reticulum requires NAF-1. EMBO J. 29:606–618. 2010. View Article : Google Scholar :
34	Marquez RT and Xu L: Bcl-2:Beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch. Am J Cancer Res. 2:214–221. 2012.PubMed/NCBI
35	Mukhopadhyay S, Panda PK, Sinha N, Das DN and Bhutia SK: Autophagy and apoptosis: where do they meet? Apoptosis. 19:555–566. 2014. View Article : Google Scholar : PubMed/NCBI
36	Tolkovsky AM, Xue L, Fletcher GC and Borutaite V: Mitochondrial disappearance from cells: A clue to the role of autophagy in programmed cell death and disease? Biochimie. 84:233–240. 2002. View Article : Google Scholar : PubMed/NCBI