Novel Cyclic Bisbibenzyls Display Growth Inhibitory Effects In Human Prostate Cancer Cells Leading To Induction Of Cell Cycle Arrest And Apoptosis

Compelling evidence indicates that chemotherapy is clinically efficacy to reduce cancer morbidity and mortality. Numerous efforts have been made to decrease side-effects of these anti-tumor drugs by understanding of molecular mechanisms of tumorigenesis, metastitis and development of cancers, identifying of novel targets and signaling pathways and analyzing of structure-activity based on clinical trials. Naturally occurring products are receiving more attention in chemotherapy because they are a rich source of antitumor compounds. A variety of secondary plant metabolites have been developed as antitumor drugs, including camptothecin, vinblastine, polyphenols (tea polyphenols, flavones), taxol. Recently, a nature compound docetaxel derived from taxol is emerging as an active chemotherapeutic agent to improve quality of life and survival conditions in patients with metastatic cancers.This promising result, together with epidemiologic studies and numerous basic laboratory findings, provide evidence that natural phytochemicals with pharmacologic properties play the potential role in the prevention and treatment of various cancersProstate carcinoma (PCa) remains the most common malignancy and the second leading cause of cancer death in Western populations. One of the most troubling aspects of PCa is that, after androgen ablation therapy, androgen-dependent PCa inevitably progresses to an androgen independent state or hormone refractory prostate cancer(HRPC), for which no effective treatment has been developed. Cytotoxic chemotherapy and radiotherapy provide limited benefit to patients with HRPC. Presently, some taxane drugs, especially docetaxel serves as a stabilizing effector on microtubulin and apoptotic inducer, have shown efficacy and provided survival benefits for patients with advanced prostate cancer. This encourages us to develop nontoxic alternatives to decrease the risk of PCa by investigation of numerous chemicals in our traditional medicine herbs.The cyclic bisbibenzyls are particularly interesting because they constitute a class of natural products with unique structural frameworks in liverworts, and exhibit a diversity of biological activities as indicated by their antimicrobial, antifungal and cytotoxic activities [7-13]. However, the reports regarding of action mechanisms are not available now. Whether the bisbibenzyls exhibit inhibitory effects on cancer cells remained to be investigated.In collaborative work aimed at developing safe and effective natural products for cancer chemotherapy, we examined more than 50 bisbibenzyl compounds from liverworts by dose-screen assays. The results show that these bisbibenzyls exerts antiproliferative effecs on cancer cell lines. Analysis of the structure of bisbibenzyls and their inhibitory effects, we observed a possible structure-activity relationship among them. These observations led to the selection of Riccardin C(Ric), Pakyonol(Pak), Plagiochin E(Pla) as potent cytotoxic agents. We first investigated the effects of Ric, Pak, and Pla on cancer cell proliferation, the kinetic changes of cell growth were measured by MTT assays. The results revealed that treatment of Ric, Pak, and Pla led to inhibition on 293 cells, breast cancer MCF-7 cells, androgen-depedent prostate cancer LNCaP cells, androgen-independent prostate cancer PC3 and DU145 cells in a dose-dependent manner. A significant time-and dose-dependently inhibitory effect of these chemicals was observed in PC3 cells. The IC50 value of Ric, Pak, and Pla was 3.22μMol/L,7.98μMol/L and 5.99μMol/L in PC3 cells exposed to chemicals for 48h. Based on observed results, we were interested in the effects of these three chemicals on androgen-independent prostate cancer PC3 cells for further mechanistic studies with doses ranged from 5 to 20μM. However, these three compounds were somewhat less cytotoxic on hepatoma HepG2 cells and leukemia K562 cells, and limited to higher doses compared to that on PC3 cells. In contrast, normal human retina pigment epithelial RPE1 cells presented a little of cell growth inhibition in the presence of Ric, Pak, and Pla, with the IC50 value 65.97μMol/L,58.62μMol/L, and 57.09μMol/L respectively, In our preliminary study, we provide evidence that the bisbibenzyls inhibited chemo-and apopto-resistant PC3 cell growth, suggesting that bisbibenzyls may be potential candidates for treatment of HRPC. Additionally, cytotoxicity was quantified by the release of lactate dehydrogenase (LDH) from cells. As expected, the cytotoxicity of Ric, Pak, and Pla on PC3 cells was observed after 24h incubation. The cytotoxicity caused by Ric was more evident than that of other two compounds. We next examined the change in cell cycle progression exposed to the bisbibenzyls. Ric treatment for 48 h resulted in significant increases in the G2/M cell population in PC3, while Pak caused an increased cell number in G0/G1. In the case of Pla, it induced an accumulation of cells in G0/G1 phase with 5μMol/L, and S phase arrest at concentration of 10μMol/L. More importantly, cell cycle deregulation leading to apoptosis was occurring when PC-3 cells were treated with bisbibenzyls, as determined by induction of the apoptotic subGl population by flow cytometry assay. We observed bisbibenzyls-induced apoptosis in a dose dependent manner. Morphological changes of apoptotic cells after exposed to bisbibenzyls were determined by staining cells with Giemsa, PI/Hochest33342, and Annexin V/PI. In contrast to control cells, Ric and Pla treatment caused a great dose-related increase in fraction of apoptotic cells with condensed and fragmented DNA, apoptotic bodies and tranlocation of PS compared with nonapoptotic cells, whereas Pak caused a modest induction of apoptosis compared to Ric and Pla under same conditions.Apoptosis is characterized by changes in expression and activity of several apoptotic markers. Western blotting analysis showed that the proapoptotic marker Bax markedly increased with exposure to the three bisbibenzyls, whereas the expression level of Bcl-2 in the Ric-or Pla-treated cells was significantly decreased, but not in Pak treatment. Furthmore, we determined the effect of bisbibenzyls on the activation of caspase-3 and PARP by Western blot analysis. the results indicated that no detectable changes in the level of procaspase 3 were observed in bisbibenzyl-treated cells at lower doses, while the decreased expression of procaspase 3 was noticeable in Ric-, and Pla-treated cells at 20μM, and 10μM, respectively, but not for Pak. Consistent with caspase-3 activity, a significant decrease in the native form of PARP was obvious and corresponded to 20μM,20μM, and 5μM exposure to Ric, Pak and Pla treatment, respectively. To confirm that the apoptotic effect of bisbibenzyls was associated with caspase-3 activation, we analyzed caspase-3 activity based on enzymatic assay. The results showed that the level of caspase-3 activation was substantially higher in cells exposed to the chemicals for 24 h than the cells exposed to the vehicle control, consistent with the result in western blot assay. These results indicated that bisbibenzyl-related cell death was associated with changes in apoptosis-related gene expressions and activities.Collectively, an inhibition in androgen-independent PC-3 cell viability by Ric, Pak and Pla may be associated with an induction of cell cycle arrest and apoptosis. Alterations in the expression and activity of Bcl2/Bax and caspase-3 signaling contribute to the bisbibenzyl-mediated inhibition on prostate cancer cells.