| Malignant tumor has become one of the main diseases that cause human death around the world. Curcumin is a major polyphenol compound in turmeric, which is used extensively in traditional Chinese medicine. Curcumin has many biological activities, such as anti-cancer, anti-inflammation, anti-oxidation, anti-atherosclerosis and anti-depression, with good safety. Curcumin is a hot spot for the research and development of natural anticancer drugs in the world for its cancer cell-selective killing property. Macroautophagy (briefed as autophagy hereafter) is a cellular biological machinery for cell defense and regulation of stress response developed in eukaryotes.Recent studies also show that curcumin induce autophagy. But its underlying molecular mechanisms, as well as the related significance for this induction in the anticancer therapeutic effects of curcumin, has not been elucidated yet. p53 is one of the most important tumor suppressor genes.Its deletion or lost-of-function mutation has been demonstrated to be involved in nearly all cancers. Recent findings reveal that p53 also acts as an important transcriptional factor for diverse autophagy-related genes (ATGs), thereby actively engaging in the autophagy modulation under diverse cellular stress conditions. These novel findings thus make the role of p53 in cancers as well as therapeutic responses much more complicated. CSN5 is a major component of COP9 signalosome (CSN) protein complex. One of the known functions for CSN5 has been demonstrated as a cellular intrinsic p53 inhibitor, by binding to promote p53 protein into the ubiquitin-proteasom-mediatied degradation pathway. CSN5 is also an established specific down-regulating protein target of curcumin. Deregulated over-expression of CSN5 has been detected in multiple different human cancers. It has been found that curcumin treatment results in CSN5 down-regulation concomitantly associated with p53 accumulation and apoptosis induction in cancer cells. But the function for curcumin-mediated CSN5/p53 in anticancer effects, its relationship with curcumin-induced autopahgy, and the significance in the treatment of cancers are currently poorly understood.This thesis provided research overview on the anti-cancer therapy of curcumin and autophagy. Importantly, this thesis investigated the molecular mechanisms of curcumin in the regulation of autophagy and the significances for its anticancer therapy in cell and animal models.This thesis confirmed that 30 μM curcumin triggered an acute autophagy induction in HepG2 liver cancer cells and HeLa cervical cancer cells in time-dependent manner, as judged by the down-regulation of p62 protein, the conversion of microtubule-associated protein 1 light chain 3 (LC3)-Ⅰ to LC3-Ⅱ form and autophagosome formation. This thesis further observed that, being different to other clinical anticancer drugs like etoposide (50 and 100 μM,12 h), cisplatin (15 and 30 μM,12 h) and fluorouracil (5-FU,200 and 400μM,12 h), only curcumin could down-regulate CSN5 protein levels in HepG2 cells, indicating the specific CSN5 downregulation effect of curcumin. Meanwhile, curcumin increased p53 protein accumulation in HepG2 cells. CSN5 siRNA transfection also induced p53 protein accumulation, accompanied with the autophagy induction in HepG2 cells. However, infection with V5-tagged CSN5 (CSN5-V5 tag) lentivirus, thereby stably expressed in HepG2 cells, significantly repressed curcumin (15 and 30 μM,6 h)-induced p53 protein accumulation and autophagosome formation in HepG2 cells. These observations indicate that curcumin may induce CSN5-controlled p53-mediating autophagy activation pathway in cancer cells.Thus, this thesis first explored the relationship of curcumin or CSN5 siRNA-induced autophagy and p53 protein. p53 siRNA significantly inhibited curcumin (15 and 30μM) or CSN5 siRNA-induced p62 protein down-regulation and autophagosome formation in HepG2 cells, indicating that curcumin or CSN5 siRNA depended on p53 protein expression. To address these findings, the pair of the p53-positive wild-type HCT116wt and the p53-null HCT116p53-/- human colonic cancer cell lines was respectively treated with 15 and 30μM curcumin. Curcumin induced p62 down-regulation and autophagosome formation only in HCT116wt cells but not HCT116p53-/- cells. These results indicate that curcumin induces CSN5 degradation, and then increases p53 protein accumulation, thus causes autophagy in cancer cells.Next, this thesis explored whether curcumin-induced autophagy depended on p53 transcriptional activity. p53 transcriptional activity induced by curcumin or etoposide was detected. Dual luciferase reporter gene analysis found that both 30 μM curcumin and 80 μM etoposide triggered an inducible p53 accumulation in HepG2 cells in time-dependent manner (3,6,9,12 and 18 h). Although curcumin-induced p53 accumulation was more rapidly and robustly than etoposide-induced ones, etoposide significantly increased p53 transcriptional activity in HepG2 cells; however, curcumin failed to cause these significant effects. RT-PCR analysis showed that mRNA levels of p53 target gene p21 were significantly increased in etoposide-treated HepG2 cells for almost 2-fold, but remained basically unchanged in curcumin-treated cells at the same 12 h post-treatment. CSN5 siRNA also did not change p53 transcriptional activity in HepG2 cells. Incubation with chemical p53 transcriptional activity inhibitor pifithrin-α (PFTα,10 μM,6 h) did not affect curcumin-induced p53 accumulation, p62 protein down-regulation and autophagosome formation in HepG2 cells. These results indicated that curcumin-induced autophagy had no relationship with p53 transcriptional activity. HCT116p53-/- cells infected with flag-p53r or flag-p53rR273H mutant were able to restore p62 protein down-regulation and autophagosome formation under curcumin treatment. These data thus clearly demonstrate that curcumin or CSN5 siRNA-induced autopahgy depends on p53 protein expression independent of its transcription activity.Furthermore, this thesis next elaborated the significances for curcumin mediated p53 induced autophagy in anti-cancer therapeutic response to curcumin. Treatment with 30μM curcumin for 6 h induced more cleaved-PARP protein production, less living cells and fewer living clones in HCT116p53-/- cells compared with HCT116wt cells. HCT116p53-/- cells infected with flag-p53r or flag-p53rR273H lentivirus were able to induce less cleaved-PARP protein production, more living cells and more survival clones during and after 30μM curcumin treatment for 6 h. Supporting this notion, HCT116wt or HCT116p53-/- cells (5×106) treated with 30μM curcumin for 6 h were injected subcutaneously into left or right flank of each individual female BALB/c Null mice, after 2 weeks, the tumors of HCT116p53/" cells were smaller than that of HCT116wt cells. These results indicate curcumin is more effective in killing HCT116p53-/- cells, and curcumin mediates p53 induced survival autophagy in cancer cells.Additionally, transfection of ATG5 siRNA also induced more cleaved-PARP protein levels and apoptosis cells in HepG2 and HeLa cells under curcumin treatment (30 pM, 6 h). These data also indicated that autophagy inhibition sensitizes cancer cells to the killing effects of curcumin. For further identification, HCT116wt and HCT116p53-/-were pre-treated with 100μM established autophagy inhibitor chloroquine (CQ) for 2 h, then treated with 30 μM curcumin for 6 h. Curcumin and CQ were more effective in killing HCT116wt cells while made no difference in HCT116p53-/- cells compared with HCT116p53-/- cells treated with curcumin only. We next proved these results in vivo, HCT16wt or HCT116p53-/- cells (5×106) were injected subcutaneously into left or right flank of each individual BALB/c Null mice. Xenograft tumors with roughly the same sizes were presented one week post-injection, and the tumor bearing mice were randomly divided into three groups for treating the tumors by direct injection every 2 days, with control (100 μL 0.01%), curcumin (100 μL,100 μM), and 100 μL the combination of curcumin (100μM) with CQ (100 μM) for 3 weeks. Compared with HCT116wt cell tumors in Null mice, HCT116p53-/- cell tumors showed sensitivity to curcumin treatment in Null mice. Regarding to the HCT116wt tumors, curcumin treatment alone only modestly inhibited the tumor growth, but in combination with CQ produced a significant inhibition on HCT116wt cell tumors in Null mice. Combination with CQ didn’t further improve the effects of curcumin in HCT116p53-/-cell tumors in Null mice. Therefore, p53-positive tumors display less sensitivity to curcumin treatment in vivo due to the autophagy, and in combination with autophagy inhibitor consequently improves the therapeutic effect of curcumin on these tumors. For further clarify, this thesis tested autophagy change in vivo in HCT116wt and HCT116p53-/- cell tumors. Curcumin treatment induced p62 down-regulation and the conversion of LC3-I to LC3-II form only in HCT116wt tumors but not in HCT116p53-/-tumors. Compared to HCT116wt tumors, curcumin treatment alone only modestly induced the cleaved-PARP protein levels, but in combination with CQ produced a significant up-regulation on cleaved-PARP protein levels in HCT116wt cell tumors. These results further clarify that curcumin mediated p53 induced autophagy plays an important role in cell apoptosis induced by curcumin treatment.Finally, this thesis determined whether CSN5-controlled p53-mediating autophagy activation also presented in human normal cells. As predicted,30μM curcumin caused CSN5 downregulation, p53 protein accumulation, p62 protein down-regulation, LC3-I to LC3-II conversion and autophagosome formation at 6 h in normal BJ fibroblasts cells. Autophagosome induction was also observed in CSN5 siRNA-transfected BJ cells. Furthermore, pre-transfection with p53 siRNA was able to block p62 protein down-regulation and autophagosome formation induced by curcumin and CSN5 siRNA in BJ cells. But contrast to the observations in cancer cells, transfected with either ATG5 siRNA or p53 siRNA did not sensitize normal BJ cells to the killing effect of curcumin since no inducible cleaved-PARP protein was observed in BJ cells under 30 μM curcumin treatment for 6 h. Thus, these results indicate that curcumin can induce CSN5-controlled p53-mediating autophagy activation both in human cancer and normal cells, and this pathway may be inherent in human normal and cancer cells responsible for curcumin treatment.In conclusion, this thesis found that curcumin specifically induced CSN5 down-regulation and stabilized p53 protein, and then promoted autophagy in human cells. This novel CSN5-controlled p53 model participated in cellular autophagy via an action independent of its classic transcriptional factor function. This thesis demonstrated this autophagy induction as an important survival mechanism in therapeutic effect of curcumin treatment on cancer cells. Therefore, p53 express or not may be considered as a useful predictive biomarker for cancer sensitivity to curcumin treatment. Curcumin may have better therapeutic effects on p53-negative tumors, and also suggested the combination with pharmacological autophagy inhibitor would benefit the therapeutic effects of curcumin on p53-positive cancers. And inhibition of autophagy didn’t affect the survival of normal cells in response to curcumin treatment. This thesis revealed the new molecular mechanism for anticancer effects of curcumin. The results from this thesis provide the experimental basis for the clinical treatment of tumors by curcumin and increase the sensitivity of drugs to cancers, having important scientific value and guiding significance. |
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