Marchantin M Down-regulates Inflammation And Inhibits Cell Invasion And Migration In Prostate Cancer

Prostate cancer (PCa) is the second most frequently diagnosed cancer in males. There exist apparent regional variations in incidence rates. It mostly occurred in western developed countries. Although the incidence of prostate cancer in China was largely lower than western countries in past decades years, an obviously rising trend was observed in some developed regions of China with the gap about aging and diet habit between them and western countries is becoming narrowing.PCa is initially androgen-dependent cancer because it depends largely on androgen receptor (AR) signaling for growth and maintenance. Therefore, androgen deprivation therapy by orchiectomy or utilizing anti-androgen drugs has become the standard first-line treatment for advanced patients. Unfortunately, the development of hormone-refractory prostate cancer (HRPC) has been observed within a few years after androgen deprivation therapy. Once PCa progressed to HRPC, it obtains more apparent dangerous characteristics, such as high apoptosis-resistance, easy to invasion and migration and poor prognosis. Although people have paid great attention to the research of HRPC, the specific mechanism still remains unclear. Therefore, it is required urgently to find novel strategies targeting the molecular basis of PCa progression.Part I The down-regulation of inflammatory factors mediated by Marchantin M contributes to sensitization of prostate cancer cells to docetaxelBy2000, there were already data indicate that tumor-associated inflammatory response had the unanticipated effect of promoting tumorigenesis and progression. In the past decade years, research on the intersections between inflammation and cancer pathogenesis has blossomed. Inflammation can contribute to tumor progression by releasing bioactive molecules to the tumor microenvironment, including growth factors and pro-angiogenic factors that sustain cell proliferation and facilitate tumor angiogenesis, invasion, and metastasis, respectively. Therefore, some progress has been made in clinical treatment by utilizing traditional anti-inflammatory drugs. For example, non-steroidal anti-inflammatory drugs (NSAIDs) can help to reduce the risk of prostate cancer occurrence, and the COX-2inhibitor was found to reduce the risk of tumor progression and metastasis.Bisbibenzyls are naturally occurring macrocyclic compounds isolated from liverwort plants, and exhibit versatile biological activities, including antifungal, antioxidant and cytotoxic activities due to its diverse structures. We have previously identified several potential compounds, such as Riccardin D (Ric D), Marchantin M (Mar M), and Marchantin C (Mar C), which exert antitumor effects in vitro and in vivo. It should also be noted that our previous study has identified Mar M as a novel reversible proteasome inhibitor in prostate cancer cells based on much attention has been paid to the function of proteasome inhibitor. Analysis of-60naturally occurring bisbibenzyls and synthesized bisbibenzyl derivatives yielded Mar M, a potent chemical showing predominant anti-inflammatory activity.1. Mar M significantly inhibits the expression of inflammatory factors.Anti-inflammatory screening result indicated that compounds grouped in Marchantin (Mar group) markedly suppressed LPS-induced expression of IL6in HUVECs. Among them, the inhibition rate of IL6mediated by Mar M was34.3%. ELISA and Real-Time PCR to validate the anti-inflammation activity of Mar M carried out further study. The results indicated that Mar M not only significantly down-regulates the expression of LPS-stimulated IL1β, IL6and CCL2in HUVECs and PBMCs but also inhibits the expression of IL1β, IL6and TNFa in many malignant cells including PC3, DU-145, H460, HT-29, and K562, in a dose-and time-dependent manner. MTT assay detected in HUVECs indicated cell proliferation almost remained unchanged in response to Mar M at concentrations used for inflammation assays, supporting the observations that Mar M indeed repressed inflammatory mediators as opposed to merely slowing cell proliferation.2. Mar M inhibits NF-κB/STAT3pathway.(1) The DNA-binding activity of LPS-activated p50and p65were significantly Impaired by Mar M in HUVECs.(2) Mar M inactivated NF-κB signaling pathway in PC3, where NF-κB is Constitutively expressed at high level. Western blotting results indicated that both phosphor-p65and phosphorylation of IκBα was notably down-regulated by Mar M. In HT-29cells and H460cells, while phosphor-p65and phosphor-Iκ B a was induced by LPS, treatment with Mar M significantly reduced this effect.(3) Down-regulation of phosphor-p65may be partially due to suppression of IKKs activity by Mar M, because genetic depletion of IKB a slightly impaired Mar M-mediated inhibition of p65when compared to the cells transfected with control siRNA in the presence of Mar M.(4) Mar M suppressed the expression of phosphor-STAT3. STAT3is an important downstream mediator of NF-κB/IL6signaling. Western blotting results indicated that Mar M not only in PC3but also in LPS-stimulated H460and HT-29exerted the inhibition effect on phosphor-STAT3. Knockdown IKBa slightly recovered phosphor-STAT3in the presence of Mar M, indicating that STAT3may be partially affected by Mar M as a consequence of the inhibition of NF-κB signaling pathway.3. The potential anti-inflammatory effect of Mar M contributes to the sensitization of prostate cancer cells to docetaxel.Apoptosis assays indicated that co-incubation of PC3and DU-145cells with docetaxel and Mar M could profoundly up-regulate an apoptotic response, while blocking IL6moderately sensitized cells to docetaxel. Cell proliferation assays indicated that PC3and DU-145cells were inhibited with increasing doses of docetaxel, and that a combined application of Mar M and docetaxel achieved an additive or synergistic inhibitory effect on cell viability.Part II Mar M exerts synergistic inhibitory effect with RGS4on the invasion and migration of prostate cancer cells by down-regulating MMP7and MMP16After accepted androgen deprivation therapy, the late-stage prostate cancer often developed to castrate-resistant prostate cancer (CRPC), along with migrated to other organs. We defined this case as metastatic castrate-resistant prostate cancer (mCRPC). Docetaxel-based chemotherapy is the standard treatment of CRPC but it is associated with dose limiting toxicities. Moreover, clinical data indicated that the overall survival usually less than two years and about50%of mCRPC patients have no response to docetaxel and develop resistance to it. Although people have carried out depth systematic study on the mechanism of docetaxel resistance and much progress has been made, there still exists big debate on the feasibility of new promising therapy strategies. Therefore, novel drugs targeting the sensitization of docetaxel and significantly inhibit the invasion and migration of metastatic prostate cancer are highly desirable. Mar M has been experienced much depth study in consideration of its’striking anti-tumor effect. We have demonstrated in the first part that Mar M mediated anti-inflammatory activity could contribute to the sensitization of prostate cancer to docetaxel. In this part, we pay attention to and make initially explanation about the effect of Mar M on the invasion and migration of prostate cancer.In recent years, a great deal of studies indicated that regulators of G-protein signaling (RGS) are closely related to many activities of cancer, including angiogenesis, migration and cell apoptosis and so on. Among them, RGS4was reported that its’expression level might correlates with the malignancy degree of cancer cells in breast cancer and ovarian cancer. In addition, RGS4could notably suppress the migration of breast cancer cells MDA-MB-231. Up to now, it has not been articles report the function of RGS4in prostate cancer cells. In this part, we studied the effect of Mar M on RGS4and account for how RGS4inhibits the invasion and migration of prostate cancer cells.1. Mar M remarkable inhibits the invasion and migration of PC3.Wound healing assay and transwell invasion assay indicated that compared to docetaxel and cisplatin, Mar M could remarkable inhibited the distance of cell migration and the numbers of cell invaded to the lower chamber.2. Mar M significantly up-regulates the expression of RGS4and the expression level of RGS4varies in different prostate cancer cells.Microarray analysis results indicate that Mar M exerts obvious effect on18kinds of RGS. It should be noted that Mar M mediated RGS4expression up to2.26times. We then validated it by real-time PCR and the result indicated that Mar M indeed notably stimulated the expression of RGS4. Western blotting and real-time PCR results indicated that the highest levels of RGS4protein and mRNA were expressed in normal prostate epithelia RWPE1, and the higher and lower levels were expressed in hormone-dependent prostate cancer cells LNCaP and hormone-independent prostate cancer cells PC3and DU-145, respectively. The results suggest that the expression of RGS4may correlates with the malignancy degree of prostate cancer cells.3. Mar M remarkable un-regulated RGS4meanwhile down-regulated MMP7and MMP16.The down-regulation of MMP7and MMP16in both protein and mRNA levels were observed in PC3and DU-145cells treated with10uM or15uM Mar M. Meanwhile, we detected that RGS4was significantly up-regulated.4. Mar M exerts synergistic inhibitory effect with RGS4on MMP7and MMP16.We transfected RGS4-expressing plasmid into PC3cells in which the expression level of RGS4is low. We find that the over-expression of RGS4could inhibit MMP7and MMP16and the inhibition would be more obvious when combined with Mar M, along with the promoting of RGS4.5. Mar M exerts synergistic inhibitory effect with RGS4on the invasion and migration of PC3cells.Wound healing assay and transwell invasion assay indicated that RGS4 dramatically inhibits cell migration and invasion. Moreover, the distance of cell migration and numbers of cells invaded into lower chamber were being inhibited more obviously when combined with Mar M treatment, suggesting that Mar M exerts synergistic inhibitory effect with RGS4on the invasion and migration of PC3cells.Part Ⅲ Conclusions and Innovation一、Conclusions1. The compounds of bisbibenzyls grouped in Marchantin (Mar group) possess significantly anti-inflammatory activity.2. Mar M exerts remarkable anti-inflammatory effect in both normal endothelial cells and many malignant cells.3. Mar M notably inhibits NF-κB/IL6/STAT3pathway.4. Anti-inflammatory effect of Marchantin M contributes to sensitization of prostate cancer cells to docetaxel.5. Mar M inhibits the invasion and migration of PC3cells by down-regulate MMP7and MMMP16.6. RGS4inhibits the invasion and migration of PC3cells by down-regulate MMP7and MMMP16.二、Innovation and defects1. We firstly reported Mar M possess notably anti-inflammatory activity, and the Mar M-mediated anti-inflammatory effect may be achieved through disruption of NF-κB/STAT3signaling, leading to enhancement of efficiency of docetaxel. But animal pharmacodynamics of the compound needs to be testified.2. We firstly reported Mar M inhibits the invasion and migration of PC3cells by down-regulate MMP7and MMMP16. However, the mechanism needs to be further studied.3. We firstly reported RGS4inhibits the invasion and migration of PC3cells by down-regulate MMP7and MMMP16. However, the mechanism needs to be further studied.