| Background and objectiveAutophagy is a catabolic process for the degradation and recycling of macromolecules and organelles, which is activated during stress conditions. Autophagy is initiated by the formation of double-membraned vesicles called autophagosomes, which fuse with lysosomes to form autophagolysosomes in which lysosomal hydrolases digest the vesicle contents for recycling. The formation of autophagosomes is a three-step process characterized by nucleation, elongation, and completion of an isolation membrane or phagophore.Autophagy is normally kept in base level in order to maintain cellular metabolism balance. When the cells suffer nutritional deficiencies, lack of oxygen, and other stresses, autophagy will be activated as a pro-survival mechanism. Autophagy can be regulated by a variety of factors and signaling pathways in cells, and is closely linked with apoptosis and other cellular activities. The micro-environment of the rapid growth tumor often occurs hypoxia, high acid and lack of nutrition, so tumor cells may maintain cellular homeostasis by autophagy when facing all kinds of unfavorable factors, such as metabolism stress, endoplasmic reticulum stress, especially in the use of chemotherapy drugs.Anticancer drugs can induce and regulate autophagy in cells, furthermore may lead to apoptosis. Anticancer drugs induced autophagy is related to the type of drug, concentration and time of the drug, the type of tumor cells, and other factors. Due to the characteristics of autophagy, anticancer drugs induced autophagy will lead to two different results: one is to prevent the cell from the damage in the surrounding environment; the other is to initiate type II cell death program. Though there is no a clear clue to explain why it can lead to two different results, in combination with various conventional chemotherapeutic drugs, inhibition of autophagy has emerged as an attractive and promising approach to sensitize malignancies to chemotherapy in cancer therapy, which is based on the fundamental premise that autophagy play a protective role in the anti-cancer action of these chemotherapeutic agents. However, recent report has shown that there are at least 4 functional forms of autophagy that may occur in response to chemotherapeutic drugs in a context(cancer cell line and agent) specific manner: cytoprotective, cytostatic, cytotoxic, and nonprotective. Therefore, it is essential to recognize the functional type of autophagy induced by ISO and to further determine the underlying mechanism prior to its application in clinical trial.Gnetum cleistostachyum, a Chinese herb that grows in the YunNan province of Southwestern of China, has been used for treatment of arthritis, bronchitis, cardiovascular system disease, and several cancers including bladder cancer for hundreds of years. ISO is a new derivative of stilbene, recently isolated from the Gnetum cleistostachyum. Increasing attention has been given to elucidating anti-cancer activity of natural oligostilbenes because more and more of their multifaceted biological properties are being identified. The antiproliferative and anticarcinogenic effects of ISO involved various signaling mechanisms including induction of apoptotic responses and inhibition of anchorage-independent by down-regulation of XIAP expression,or induction of cell cycle G0–G1 arrest and inhibition of anchorage-independent growth of cancer cell lines through downregulation of cyclin D1 expression at the transcriptional level. However, the signaling pathways mediated by ISO to induce cancer cell autophagy effects in cancer cells remain incompletely elucidated.Sestrin2(SESN2) encodes a member of the sestrin family of PA26-related proteins, which plays major roles in suppression of oxidative stress and regulation of adenosine monophosphate-dependent protein kinase(AMPK)-mammalian target of rapamycin(mTOR) signaling. SESN2 protein may be involved in complex regulation of cell viability in response to different stress conditions. In the present study, we found that ISO treatment effectively induced autophagy in a SESN2-dependent manner, and such ISO-induced autophagy was required for the ISO inhibition of anchorage-independent cancer cell growth, demonstrating that autophagy induced by ISO is cytotoxic to human bladder cancer cells. We also found that SESN2 was dramatically down-regulated in human bladder cancer tissues, whereas SESN2 protein expression was elevated in bladder tissues in the mice receiving ISO in drinking water as compared with the mice given BBN drinking water only. The purpose of this research is to investigate the machenism that ISO induced autophagy and cell growth inhibition in human bladder cancer cells on the basis of the existing research results, for providing a new molecular basis for the anti-cancer properties of ISO. Reseach content and method 1. Western blotting analysis was used to detect the expression of LC3, a marker protein of autophagy, for a preliminary understanding of the effective dose of ISO and the extent of induced autophagy; to detect the expression of autophagy passway protein after ISO treatment; to detect the expression of SESN2 promoter transcription factors; to detect the expression of phospho-C-JUN, SESN2 and LC3in TAM67 and shC-JUN UMUC3; to detect the expression of phospho-JNKT183/Y185, JNK, phospho-C-JUN S63, C-JUN, SESN2 and LC3 in shJNK1UMUC3 or Hela and their nonsense control cells after ISO treatment; to detectSESN2 expression in human bladder cancer tissues and corresponding adjacentnormal bladder tissues. 2. PolyJet transfection technology was used to establish a stable GFP-LC3transfected cell lines, and indirect immunofluorescence staining was applied toobserve the punctuate dots of GFP-LC3 for further confirming autophagy effect ofISO; to establish stable shBECN1 and shSESN2 transfected cell lines, for detectingISO induced autophagy after the inhibition of autophagy passway protein; toestablish a stable overexpressing TAM67 UMUC3 by transfecting awell-characterized C-JUN dominant negative mutant TAM67 and SESN2-luc toUMUC3 to detect AP-1 and SESN2 promoter activity by luciferase reporter assaysafter ISO treatment; to establish a stable shJNK1 UMUC3 and transfect SESN2-lucto detect SESN2 promoter activity. 3. The effects of BAF on cell colony formation in UMUC3 after ISO treatment andthe effects of ISO on cell colony formation in UMUC3 shRNA SESN2 andnonsense control cell line were detected by anchorage-independent growth assay; tocompare ISO effects on cell colony formation after ISO treatment in UMUC3TAM67, shC-JUN and shJNK1 UMUC3 and their vector control cells. 4. RT-PCR was used to detect the mRNA level of SESN2 after ISO treatment inUMUC3 cells; and to detect the mRNA level of SESN2 in UMUC3 TAM67,shJNK1 UMUC3 and their vector control cells. 5. The chromatin immunoprecipitation(ChIP) was used to detect the binding oftranscription factor C-JUN and specificity SESN2 promoter sequence. 6. Immunohistochemistry(IHC) was used to detect the protein expressing level ofSESN2 in bladder cancer mice induced by BBN. Result 1. ISO induced autophagy in human cancer cells. ISO can initiate autophagy in human cancer cells UMUC3, T24 T and human cervical cancer Hela cells. ISO treatment resulted in a significant increase in the conversion of LC3 from LC3-I to LC3-II in a dose-dependent manner. Following the treatment of UMUC3-GFP-LC3 cells with various concentrations of ISO, GFP-LC3 puncta increased along with the doses of ISO treatment. ISO treatment led to further increase of LC3-II and GFP-LC3 puncta level in the presence of BAF, suggesting that ISO increases autophagy flux level. BAF also obviously abolished the inhibitory effects of ISO on anchorage-independent growth of UMUC3 cells. 2. ISO induced autophagy in a SESN2-dependent manner. At the autophagic induction doses, ISO treatment increased Beclin 1 and SESN2 expression levels in UMUC3 cells. However, knockdown Beclin 1 by stably transfecting shRNA into UMUC3 cells failed to block autophagy induction as indicated by LC3-II conversion, while we employed shRNA SESN2 to knockdown its expression in UMUC3 cells, and found that upon SESN2 depletion, induction of LC-3 II was attenuated obviously. Anchorage-independent cell growth was carried out in shRNA SESN2 and nonsense control cells. In nonsense control cells ISO treatment at 5 and 10 μM caused obvious reduction in colony formations; while in shRNA SESN2 transfectants, ISO treatment at the same doses failed to inhibit colony formations in soft agar. 3. C-JUN plays an essential role in ISO-mediated induction of SESN2 expression and cell growth inhibition. ISO treatment increased SESN2 mRNA expression in a dose-dependent manner. In accordance, SESN2 transcription also increased following ISO treatment. The expressional changes of several putative transcription factors in the proximal promoter region of SESN2 were analyzed by Western Blotting following ISO treatment. The expression levels of phospho-NFκB, total NFκB, SP-1 and E2F1 were merely changed by ISO treatment. In contrast, phospho-C-JUN, the active form of C-JUN, was obviously upregulated. Ectopic expression of C-JUN dominant negative mutant TAM67 obviously blocked AP-1 and SESN2 promoter activity. RT-PCR and luciferase reporter assay further revealed that TAM67 efficiently blocked SESN2 induction at transcriptional level. SESN2 promoter was found to interact with C-JUN and this interaction was enhanced by ISO treatment by Chromatin-IP. The blockade of endogenous C-JUN activation by TAM67 or shRNA specific targeting human C-JUN can significantly attenuated the increased effects of ISO on phospholation of C-JUN, protein expression of SESN2, LC3-II and the inhibitory effects of ISO on anchorage-independent growth. 4. JNK was the upstream kinase being responsible for C-JUN-dependent SESN2 induction Activation of JNK was observed in accordance with C-JUN activation as well as LC3-II conversion. ShRNA JNK1 UMUC3 not only blocked C-JUN activation, but also attenuated SESN2 induction and LC3-II formation by ISO treatment. And shRNA JNK1 also totally blocked SESN2 mRNA induction as well as promoter activation by ISO treatment. Anchorage-independent cell growth was carried out in UMUC3 shJNK1 and its nonsense control cell. In vector and nonsense control cells ISO treatment at 5 μM and 10 μM caused obvious reduction in colony formations; while in shJNK1 cells, ISO treatment at the same doses failed to inhibit colony formations in soft agar. 5. SESN2 was downregulated in human bladder cancer tissues and ISO treatment can regulate SESN2 expression in BBN-induced mouse bladder tumor. Using human bladder cancer samples in comparison with the adjacent normal bladder tissue, SESN2 expression was dramatically down-regulated or even lost in human bladder cancer tissues as compared to their paired adjacent normal tissues. SESN2 protein level was remarkably elevated in the mouse bladder tissues followed BBN plus ISO treatment in comparison with the bladder tissues of the mice received BBN exposure alone. Conclusion 1. ISO of sub-lethal dose can induce a dose-dependent autophagy in human tumor cell line, which might be associated with its inhibition of anchorage-independent growth of human bladder cancer cells. 2. ISO induced autophagy in a SESN2-dependent manner. ISO increased SESN2 mRNA expression and enhanced its transcription activity. 3. ISO treatment induced autophagy via a C-JUN N-terminal kinase(JNK)/C-JUN-dependent mechanism, in which ISO triggered JNK1-dependent C-JUN activation and facilitated the binding of C-JUN to consensus AP-1 binding site in the SESN2 promoter region, thereby led to a significant transcriptional induction of SESN2 and induction of autophagy. 4. SESN2 expression was dramatically down-regulated or even lost in human bladder cancer tissues as compared to their paired adjacent normal tissues, while ISO treatment was capable of elevating SESN2 expression effectively in BBN-induced mouse bladder tumors in vivo. |
PDF Full Text Request