| Prostate cancer is the most common nonskin cancer in old men in the Western world. While improved early detection significantly decreased mortality, PCa still remains the second leading cause of cancer-related death in Western men. Although the incidence of prostate cancer was lower than the Western countries’, in recent years confirmed PCa, especially for advanced or metastatic PCa, represents an obviously rising trend in China with the aging and the diet habit changing.PCa is initially androgen-dependent with complicated pathogenesis. In addition to genetic factors, the incidence of prostate cancer is related with high levels of androgens, race, diet and lifestyle. PCa depends largely on androgen receptor (AR) signaling for growth and maintenance. Androgen deprivation therapy has become the standard first-line treatment for advanced hormone naive PCa. Unfortunately, the development of hormone-refractory prostate cancer (HRPC) has been observed to occur within a few years after hormonal deprivation therapy and is associated with poor prognosis, high apoptosis-resistance and high mortality. There are few efficacious treatment options available for curing, or even improving the survival and quality of life in patients with HRPC. Complicated factors and signals involves in the development of HRPC. Nevertheless, the underlie mechanism of PCa is still remains elusive. Therefore, novel strategies targeting the molecular basis of PCa progression and producing synergies of different signals are highly desirable. Part I Marchantin M inhibits the activities of the proteasome and triggering ER stress and apoptosisUbiquitin-proteasome system (UPS) is one of protein quality control system in eukaryotic cells. UPS works mainly through ubiquitination of the target protein and degradation of abnormal proteins and short-lived protein by proteasome. UPS plays an essential role in cell proliferation, cell cycle regulation, immune response, signal transduction and cell death. Recent studies have validated that proteasome activities in malignant tumors are much higher than that in non-malignant cells, conferring tumor cells more dependent on the UPS to maintain cellular homeostasis. Therefore, tumor cells are more sensitive to UPS inhibitors. UPS has gradually become a new target for cancer therapy, and Reagents targeting at proteasome are expected to become promising anticancer drugs. Natural products have gained much attention in their unique structures and diverse bioactivities.Macrocyclic bisbibenzyls are a series of phenolic natural products isolated from liverwort, which exert a variety of biological activities, including antifung, antioxidation and cytotoxicity. Our previous research demonstrates its inhibitory effect on PCa cells via activation of caspase pathway.Based on the nature of its structure, Marchantin M (Mar) was somewhat similar to tea polyphenols, bearing with phenol groups which may act as tyrosine mimic binding to β5subunit of proteasome. We speculate that Marchantin M may be a novel proteasome inhibitor. Therefore, we discuss the inhibitory effect of Marchantin M on proteasome in prostate cancer cells.一、Mar is a novel reversible inhibitors targeting proteasome activities1. Mar dramatically inhibits ChT-L and PGPH activities of proteasome.Mar inhibits ChT-L and PGPH activities of proteasome of20S proteasome in a dose-dependent manner in vitro. The IC50values for ChT-L and PGPH were6.99and5.33μM, respectively. However, Marchantin M hardly inhibited Try-L activity of the 20S proteasome. The Similar results were observed in Mar-treated PCa cells. Analysis of gene chip shows that Mar inhibits proteasome activities rather than suppresses expression of UPS components. The results indicate Mar is a novel inhibitor.2. Mar is a reversible inhibitor of proteasome.Since the ChT-L and PGPH activities were mediated by the β5and β1subunits of proteasome, we docked the Mar molecule into β5and β1subunits to investigate their interactions by autodock vina. The docking results revealed that Mar bound to P5and β1subunits at the active sites with a conformation suitable for proteasome inhibition. The docked free energy was-7.4kcal/mol and-6.5kcal/mol, respectively. The docking images also indicated that Marchantin M interacted with β1and β5subunits through hydrogen and hydrophobic interactions. Lineweaver-Burk double-reciprocal plots for the PGPH activity displayed characteristics of non-competitive inhibition, the Km value was determined to be48.52μM. The data clearly demonstrates that Mar is a novel reversible inhibitor of proteasome.3. In accordance with proteasome inhibition, Marchantin M caused dose-and time-dependent accumulation of ubiquitinated proteins in PCa cells. The ubiquitinated proteins were noticeable as early as1h after treatment and sustained at high levels up to9h, then dropped down following18h of exposure in PC3and DU145cells.二、Mar disrupts ER-associated degradation (ERAD) trigerring ER stress and apoptosis1. Mar-mediated proteasome Inhibition inhibits ERAD resulting in ER stress.Mar dramatically increased SPCΔ4levels in PCa cells transfected with SPCA4, a specific substrate of ERAD. Similar to the observation in MG132treatment, whereas neither Mar nor MG132affected SPCwt level. Effect of Mar on ERAD was also monitored by the localization of a green fluorescent protein in PC3cells transfected with pGFP-CFTRΔF508, another substrate of ERAD. In the absence of Mar, GFP in transfected cells was detected in both cytoplasm and ER, while Mar-treated cells displays primarily green fluorescence in the ER. Similar observations were shown in cells treated with MG132. These results indicated that Marchantin M exerted anti-proteasome activity and prevented ERAD. The transmission electron microscopy (TEM) revealed that the ER was moderately dilated in cells exposed to5μM Mar. However, the vacuolation of ER during high dose Mar treatment was observed in PC3and DU145cells. The expressin of the GRP78increased markedly following a short exposure to Marchantin M, and was sustained at high levels throughout the duration of treatment in three PCa cell lines. PERK and eIF2a phosphorylation was up-regulated in response to Mar exposure in three PCa cell lines, however their total protein level was not affected by Mar. To further investigate the effects of Marchantin M on the ER stress, four important ER stress response transducers XBP1, ATF6, ATF4and its downstream ATF3were also examined in Marchantin M-treated cells. The spliced form of XBP1mRNA increased in PC3cells exposed to Mar as early as1h, then decreased with longer treatment. However, the activated XBP1sustained for24h in DU145and LNCaP. Real time PCR analysis revealed that the activating transcription factor4(ATF4) and ATF3mRNA levels were largely increased by Mar and sustained up to48h during treatment, and the levels of activating transcription factor6(ATF6) was slightly increased in Mar-treated cells. The above data indicated that inhibition of proteasome by Mar disrupted EARD and resulted in prolonged ER stress.2. Mar induced apoptosis in PCa cells.Mar inhibitis cell viability and promotes apoptosis in PCa cells in a dose-dependent fashion. However, the apoptotic rate was much higher in hormone-dependent prostate cancer LNCaP than that of in hormone-independent prostate cancer PC3and DU145. Western blotting analysis displayed that Mar activated ER specific caspase-4resulting in the induction of caspase3cleavage to proteolytic fragment in PC3cells.3. caspase-independent cell death involes in Mar-induced cell death in HRPCTo investigate the role of apoptosis in Mar-induced cell death in PCa cells, z-VAD-fmk, a pan inhibitor of caspase was pretreated in presence and absence of Mar. The data showed z-VAD-fmk dramatically rescued cell death in LNCaP cells, however, partial rescution was observed in hormone refractory prostate cancer PC3and DU145cells, which indicated caspase-dependent and independent cell death involes in Mar-induced cell death. Part II Marchantin M inhibits AR function through up-regulating stress responsive gene of ATF3Androgen receptor (AR), a nuclear receptor, dissociated from heat shock protein when it bind its ligand, which in turn promotes AR nuclear location and dimerization. Activated AR binds to AR response elements (AREs) and regulates gene transcription,such as PSA. Androgen and its receptor plays key role in maintaining development, proliferation and differentiation in prostate gland. Previous report displayed that AR promoted development from hormone-dependent prostate cancer to horomone-independent prostate cancer. Abnormal androgen signaling due to aberrant expression mutations, or dysregulation of the AR gene has been linked to prostate tumorigenesis, and progression of prostate cancer into advanced, castration-resistant disease.Activating transcription factor3(ATF3) belongs to one of ATF/CREB family. ATF3bound to ATF/CREB element and regulates gene expression by its bZIP domain. ATF3was dramatically induced to maintain cellular homeostasis when cells were exposed to genetic toxins, oxidative stress and ER stress. However, ATF3mediated apoptosis during lasted and overloaded stress.Our data of Part I showed that marchantin M mainly induced apoptosis in LNCaP cell accompanying ATF3upregulation. Previous study disclosed forced expression of ATF3disrupting AR function. So we investigated whether upregulation of ATF3involved in inhibiting AR function and triggering apoptosis in LNCaP cell.一、Mar M raised ATF3expression by ER stress signal of PERK/eIF2α/ATF41. Mar up-regulated ATF3expression by transcriptional activation.Mar significantly increased ATF3promoter activity and promote its transcription in LNCaP cells. The MatInspector software was used to search for the regions of conserved transcription factor-binding site within the-1850to+34regions. The ATF3gene promoter (pATF3-1850/+34) contained multiple potential transcription factor-binding sites including ATF (-22/-18). To investigate which regions Mar affects transcriptional regulation of the ATF3gene, promoter activity was measured using five serial deletion constructs, the point mutation and deletion mutation constructs. Our data demonstrated that ATF binding sites was crucial to the ATF3transcription activation.2. ER stress-mediated PERK/eIF2a/ATF4involved in Mar-induced ATF3expression.Inhibition of PERK activation by dominant negative or ATF4expression by targeting siRNA dramatically decreased ATF3expression.二、Mar induced apoptosis by up-regulation of ATF3in LNCaP cell1. The inhibition of ATF3expression promoted cell survival. Knocking-down ATF3expression by siRNA significantly decreased apoptosis.2. Mar-induced ATF3triggered apoptosis in LNCaP cell in a caspase-dependent manner. Depletion of ATF3expression suppressed activation of caspase3resulting in decreasing cleavage of PARP, a substrate of active caspase3.The above results showed that the Mar promoted ATF3expression and induced canonical apoptosis in LNCaP cell.三、ATF3inhibited AR function through protein interaction and promoted apoptosis in LNCaP1. Mar significantly raised ATF3protein level in hormone-dependent prostate cancer.Mar improved ATF3transcription in three prostate cacer cells. However the protein level of ATF3in hormone-dependent LNCaP cells was much higher than in hormone-independent prostate cancer cells PC3and DU145.2. ATF3interacted with AR and inhibited its functionCo-precipitation assay showed that ATF3interacted with AR in LNCaP cells. In LNCaP cells, overexpression of ATF3significantly decreased PSA promoter activity and its mRNA levels. Other AR targeting genes, such as TMPRSS23and NKX3.1, were also significantly decreased. However, the genes expression restored when depleted ATF3by siRNA. pression. When AR, PSA promoter and different doses of ATF3expression vector were transfected into PC3cells, PSA promoter activity decreased with increasing amount of ATF3expression. The above results indicated that Mar-induced ATF3promotes apoptosis by interacting with AR and inhibiting its function in LNCaP cells.Part III Marchantin M induces autophagic cell death in prostate cancersInactivation of caspase by z-VAD-fmk partially rescued marchantin-induced cell death in hormone refractory prostate cancer, which suggests that caspase-independent mechanisms may also contribute to its cytotoxic effect on PCa cells. Besides triggering endoplasmic reticulum stress (ER stress) and promoting apoptosis, proteasome inhibition also induces autophagy.Autophagy is a catabolic pathway to maintain cellular homeostasis by eliminating injured or aging organelles and unwanted proteins, and mediating turnover of long-lived proteins. In addition to UPS, autophagy is another protein quality control system in eukaryotic cells. When cells suffers extra or intra-cellular stress, the level of autophagy could be dramatically stimulated as a cytoprotective response resulting in adaptation and survival. The hydrolytic products by enzymes of lysosome can be reused by cells. UPS and autophagy are considered the two major routes of protein degradation in eukaryotic cells and their mutual-exclusiveness and inter-dependence.Uusually proteasome inhibition activates autophagy. However, dysregulated or excessive autophagy could cause autophagic cell death, the type II programmed cell death. Several chemotherapeutic agents in mammalian cells have been shown to induce autophagic cell death.一、 Proteasome inhibition paralleled with autophagy induction1. Mar inhibited proteasome activities accompanying with ER stress and autophagyMar suppressed proteasome activities at1h in Mar-treated cells,consequrently, GRP78and LC3BII increased at2h after Mar treatment, which suggested that Mar triggered ER stress and autophagy by proteasome inhibition. 2. Mar dramatically induced autophagy in HRPCMar rapidly up-regulated autophagy levels in treated cells, as judged by the accumulation of autophagy marker protein LC3B and the accumulation of lipidated LC3BⅡ in a concentration-dependent manner. Electron microscopy of Marchantin M-treated PC3cells showed the formation of double-or multi-membranes engulfing high electron-density substances, evidenced as autohagosomes or autolysosomes. Marchantin M induced autophagy was also judged by punctate dots of a green fluorescent protein tagged form of LC3B (GFP-LC3B). The cells transfected with GFP-LC3B expression plasmid showed diffuse green fluorescence, while Marchantin M treatment caused significantly punctated green fluorescence in cytosol. The similar results were observed in GFP-LC3ransfected U87cells.3. Mar promoted autophagic flux in HRPCData of gene chip and qRT-PCR demonstated that Mar only dramatically induced LC3B expression, and other ATG genes shwoed insignificant difference. Western blotting displayed Mar not only induced LC3B expression but promotes turnover of LC3BⅠ. As increased LC3BⅠ to LC3BⅡ conversion could occur when autophagy was either stimulated or blocked. To further explore if autophagic flux can be induced by Marchantin M, we chose chloroquine (CQ), an agent that prevents the formation of autolysosomes by inhibiting autophagosome-lysosome fusion and LC3BⅡ degradation. The result showed that Marchantin M-stimulated processing of LC3B was enhanced in the presence of CQ, whereas no detectable change was observed in cells exposed to CQ alone, suggesting that Marchantin M has the potential to promote autophagosome formation. Thus, Marchantin M was effective in inducing a prolonged autophagy.二、Mar induced autophagic cell death in hormone-independent prostate cancer cells1. Pharmacological blockade of autophagy rescued Mar-mediated cell deathMar treatment led to the accumulation of LC3BⅡ, whereas this effect was significantly blocked in the presence of3-MA, and accompanied with an increase in cell viability and reduction in cell death. Similarly, Mar-mediated induction of LC3BⅡ was enhanced in combination with E-64D/Pepstatin A, another autophagy inhibitor that prevents lysosomal enzyme activation, and cell proliferation was significantly restored and cell death was markedly decreased in co-treatment.2. knock-down of LC3B, Atg5and Atg7attenuated Marchantin M-mediated cell death Knockdown of endogenous levels of Atg5by Atg5-targeting siRNA dramatically increased cell viability and blocked cell death after24and48h transfection followed by Mar treatment. Mar-induced LC3BII levels were markedly suppressed when Atg5was down-regulated by siRNA. Similar to the observations of Atg5, reduction of LC3B or Atg7by siRNA significantly alleviated cytotoxic activity of Mar. Taken together, our data clearly emonstrated that Mar activated an autophagic flux and promoted autophagy-dependent cell death.3. Combined inhibition of caspases and autophagy almost completely rescued Mar-induced cell deathCaspase inhibitors only partially reversed to Mar-induced cell death in hormone-independent prostate cancer cells. Depletion of Atg5expression by siRNA in conjunction with the caspase inhibitor almost completely blocked the cell death induced by mar, which indicated both autophagic cell death and caspase-dependent apoptosis involved in cytotoxicity to hormone-independent prostate cancer cells.三、PERK/PERK/eIF2α'PI3K/Akt/mTOR involved in Mar-mediated autophagy1. Mar inhibited PI3K/Akt/mTOR signal by preventing Aktl transcription resulting in autophagy activation.Western blotting results showed that Mar decreased the phosphorylation level of PDK1, Akt and total Akt protein resulting in reducing phospho-mTOR and phospho-p70S6K. The attenuation of AKT by Mar was further confirmed at mRNA level in PC3cells and the expression of AKT3decreased much lower than that of AKT1/2. Forced expression of AKT-wt was unable to increase the phosphorylation level of GSK3β, a downstream target of AKT, as well as the cell viability exposed to Mar. Unlike to the AKT-wt, PC3cells transfected with AKT-myr showed significant increase in phospho-GSK3β level and cell viability, accompanied with a decrease in the LC3BII. The results showed that Mar inhibited expression and activation of Akt leading to mTOR inactivation and inducing autophagy.2. Mar-induced ER stress signaling promotes autophagyBlockade of JNK/c-Jun signal with SP600125hardly affected Mar-induced LC3BII expression and cell death. To explore a link between PERK/eIF2a signaling and autophagic activation in response to proteasome inhibition by Mar, we performed transient transfection with dominant negative PERK (PERK-DN) expression plasmid into cells to impair the function of PERK. The results displayed that Mar-induced eIF2a phosphorylation was blunted by inactivation of PERK. More importantly, co-treatment of cells with Mar and PERK-DN significantly caused the blocking of LC3BII accumulation and partial restoration of viable cells as well as decreased cell death. These results indicate that endoplasmic reticulum stress signal PERK/eIF2a was involved in Mar-mediated autophagy and cell death.Part IV Conclusions and Innovation一、Conclusions1. Mar inhibited proteasome activities in vitro and established PCa cell lines, which indicated Mar was a novel revesible inhibitor of proteasome.2. Mar disrupted endoplasmic reticulum associated degradation pathway by inhibiting proteasome activity resulting in ER stress, by which promoted cell death in PCa cells.3. Mar up-regluated ATF3, a stress mediator, in hormone-dependent prostate cancer, which in turn inhibits AR function and promoted apoptosis in LNCaP cells.4. Mar M indces caspase-dependent and independent cell death in androgen-independent prostate cancer cells by induction of ER stress and autophagy.二、Innovation and defects1. We Firstly reported Mar a macrocyclic bisbibenzyls targeted the proteasome activities and showed its inhibitory effect in a reversible manner. Since irreversible inhibitors of proteasome usually cause normal cell apoptosis and death, reversible inhibitors are more valuable in drug discovery. Our data suggests that Mar may represent a promising compound in the future development to treat PCa.2. We firstly reported Mar prevented AR function by up-regulation of stress mediator ATF3and promoted apoptosis in LNCaP. Since AR in LNCaP cells was mutant, the characteristics of Mar will be beneficial for the treatment of prostate cancer expression constitutive activation of the AR.. However, the effect of Mar on wild-type AR needed to be investigated.3. Mar triggered autophagic cell death in hormone-independent prostate cancer. Since most chemotherapy agents such as paclitaxel induced autophagic protection in tumor cells, which in turn weakened its anti-tumor effect. Moreover hormone-independent PCa cells showed more potential to anti-apoptosis. However the IC50values of Mar are similar between hormone-dependent and hormone-independent PCa cells and Mar induced autophagic cell death in hormone-independent PCa cells, which conferred Mar a promising anticancer drugs. But animal pharmacodynamics of the compounds needed to be testified. |
PDF Full Text Request