| Prostate cancer (prostate cancer, PCa) is the most common malignant cancer and the second cause of death in men in Europe and America countries. Recently, prostate cancer has become more common in our country.PCa is an androgen-dependent tumor, and androgen ablation serves as an standard treatment for patients who present advanced, androgen-dependent metastatic PCa for many years. However, the high failure rate for castration causes progresses to hormone refractory prostate cancer (HRPC), that is resistant to radiation, surgery, and chemotherapy. It is the major cause of the mortality associated with HRPC. Despite numerous advances including aberrant regulation of androgen/androgen receptor leading to constitutive active androgen receptor, activation of MAPK, PI3K/Akt, JAK-STAT signaling pathways which contribute cell proliferation in addition to androgen stimuli in PCa, abnormal control of apoptosis cascade such as alterations of the expression of Bcl2, Bax resulting in apoptosis resistance, the high mortality rate occurs because the treatment of this stage of PCa has been largely ineffective, eventually the patients would die of an HRPC.To date, chemotherapy is still a common clinical strategy for HRPC. For example, the pacitaxel, Present clinical drugs has their own chemical and biological characteristics, yet treatment outcomes are not satisfactory. The mechanism includes two aspects. (1)Patients with advanced prostate cancers undergo distant metastasis, and have many complications. (2) Despite rapid symptoms improved and lifetime prolonged, serious side effects of chemical drugs cause damage of body, repress immune system and result in poor prognosis. For example, Chepatotoxicity though with the advantages of wide spectrum anti-tumor effects and strong effects; Cyclophosphamide exhibits bone marrow depression; docetaxel triggers cancer cells to produce drug resistance. Therefore, it is required urgently to find novel drugs with high effects and low toxicity to treat the androgen-independent PCas.Ubiquitin-proteasome system (UPS), the major proteolytic enzyme system in mammalian cells, plays critical roles in basic cellular functional process, including proliferation, differentiation and proliferation, and so on. As a proteolytic enzyme complex, UPS is an ATP dependent proteolytic complex, which is comprised of 20 S catalytic particle, 11S regulatory factor and two 19S regulatory particles. Maintaining the activity of UPS is greatly involved in keeping the maintenance of cell basic functions. UPS precisely degrades target proteins in mammalian cells, and then participates in the regulation of gene transcription and cell cycle, as well as in the cellular physical process which includes receptor-mediated endocytosis and antigen processing. Therefore, UPS will be an important target for drug treatment. Recent studies have validated that malignant tumors are more sensitive to UPS inhibitors compared to non-malignant tumors. Our study found that Marchantin M could inhibit the activity of UPS and the proliferation of PC3 cells.At present, natural compound with anticancer constituents is highly concerned. liverworts contains a number of natural compound, including terpenoid, phenols and bisbibenzyls, among which bisbibenzyls with unique chemical structure and wide biological property receive great attention. We screened many bisbibenzyls candidates and found that Marchantin M exhibited inhibiting effect of proteasome activity and had significantly anticancer activity of PCa PC3 cells. The mechanism by which Marchantin M inhibited proliferation of PC3 cells and activity of proteasome was discussed in our study.Cell proliferation assay, a method to test antitumor activity of Marchantin M, showed that Marchantin M remarkably repressed proliferation of adenocarcinoma cells including breast cancer MCF-7 cells, prostate cancer LNCaP, DU145 and PC3 cells. This effect was especially evident in androgen-independent PCa PC3 cells. Additionally, Marchantin M was of low toxicity in normal human retinal epithelium cells. Marchantin M significantly depressed PC3 cells in the concentration of 20μmol/L. Yet, in this concentration, it hardly had repression in hTERT-RPE1. Cell morphological alterations were observed by Gimsa staining and double staining (PI and Hoechest33342) methods. The results indicated that cells remarkably dropped off and cell morphology exhibited the characteristic changes of apoptosis. Next, we tested cell cycle by flow cytometry and western blotting, and found that apoptosis rate increased dramatically compared to the control group and cells were apparently arrested in G0/G1 phase. Changes of apoptosis related proteins by western bolting exhibited that anti-apoptosis protein Bcl2 was down-regulated whereas pro-apoptosis was significantly increased in treatment group. In the concentration of 10μmol/L, we found activation of Caspase 3, cleavage of PARP, and activation of Caspase 4 and Caspase 9. However, obvious activation of Caspase 8 was not observed. These data indicated that Marchantin M inhibited proliferation of PC3 cells by inducing apoptosis of them. Further study was made on the mechanism of apoptosis in PC3 cells by Marchantin M. Structure of Marchantin M has been validated to be similar to a proteasome inhibitor (tea polyphenol) in recent studies. So we supposed that Marchantin M might induce apoptosis of PC3 cells by inhibiting activity of proteasome. we detected activity of proteasome and found Marchantin M could inhibit enzymolysis activity of proteasome in vitro and also repress the enzymolysis activity of proteasome for total proteins in PC3 cells. This was consistent with our hypothesis.In summary, our study showed that Marchantin M could significantly inhibit proliferation of androgen-independent PCa PC3 cells and increase apoptosis of PC3 cells. The potential mechanism is that repression of UPS lead to cell apoptosis. In our study, the novel proteasome inhibitor to cope with PCa will give new hope for treatment of human PCa.
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