The Development Of Anti-SMO Antibody And Its Effects On DU145 Androgen Independent Prostate Cancer Cells

Background:Prostate cancer is currently the most frequent malignancy in men and represents the second leading cause of cancer-related deaths. While early-stage prostate cancer can be cured with surgery or radiation therapy, this is not the case for metastatic disease. The mainstay of treatment for metastatic prostate cancer is androgen deprivation.Unfortunately, most of men ultimately develop androgen-independent prostate cancers (AIPC), unresponsive to hormonal manipulations. Recently, docetaxel has replaced mitoxantrone as the standard of care for patients with nonlocalized AIPC.The docetaxel-based chemtherapies has been reported to improve the quality of life for patients, offering pain relief. However,the side-effects are significant.In addition, it is clear that, while docetaxel is effective, this efficacy is modest and better therapies are definitely needed.Therefore, novel therapeutic strategies that target the molecular basis of androgen resistance are required.Hedgehog(Hh) is a developmental signaling pathway that regulates embryonic cell growth, body pattern formation and organogenesis in certain vertebrate tissues. Hh signaling is involved in normal prostate development and there is mounting evidence that dysregulated Hh signaling plays some role in prostate cancer. This evidence includes reports that human prostate tumors express Hh ligand and Hh-target genes and other reports citing experimental evidence that a chemical inhibitor of Hh signaling, cyclopamine, suppresses the growth of cultured and xenografted human prostate cancer cell lines. Collectively, this evidence implies that autocrine Hh signaling has some role in prostate cancer tumor growth or progression. The validity of this hypothesis is contradicted, however, by more recent experimental studies that failed to find a canonical signaling effect of Hh-activators or inhibitors in the most commonly utilized prostate cancer cell lines. Moreover, there are increasing concerns that cyclopamine might have off-target effects in cancer cells that belie its specificity as an Hh signaling inhibitor.Antibody is a favorable molecule for drug development due to its high binding specificity and affinity to target. Smoothened (SMO) is an important member of the Hedgehog signaling pathway. Antibodies which recognize an oligopeptide of SMO on the cancer cell surface and have the ability to silence Shh stimuli were raised in the present study.The aim was to find a new strategy for controling prostate cancer and other Hh dependent carcinomas by precisely controling the Hh activity to avoid the side effects.Methods and Results:To prepare and characterize the polyclonal antibody against SMO,a polypeptide was synthesized based on the bioinformatics analysis of SMO protein and coupled with keyhole limpet hemocyanin(KLH) for immunization. The antiserum containing specific polyclonal antibodies were prepared by immunizing healthy male rabbits,about 4-month-old and 3 kilogram weight. Antibodies were collected after the fourth immunization injection. The titre of the antiserum was detected with enzyme-linked immunosorbent assay (ELISA). The antiserum was purified with immuno-affinity chromatography. Western blot was performed with the purified antiserum on the cell lysates of DU145 cells.The antiserum had titres of 1:240,000 in ELISA. The results obtained by Western blot analysis showed that the antiserum could react with SMO with highly specific and sensitive affinities. So,the specific antibody against human SMO (anti-SMO-antibody) was obtained,which will be useful for further investigation.The effect of anti-SMO-antibody on Hh signaling activity in AIPC cell line DU145 was analyzed by RT-PCR.Because GLI1 and PTCH1 are not only the components of Hh signaling but also target genes of GLI1 trans-activation, the mRNA levels of GLI1 and PTCH1 as markers of the activity of Hh signaling pathway were examined. Anti-SMO-antibody suppressed the expression levels of both GLI1 and PTCH1 when compared with a treatment with normal rabbit IgG. To evaluate the antiproliferative effect of drugs on AIPC cells, the growth-inhibitory effects of anti-SMO-antibody was evaluated on DU145 prostate cancer cells. The flowcytometric analyses were made to determinate the percentage of apoptotic cell death induced by anti-SMO-antibody. On the other hand,to estimate the inhibitory effect of the anti-SMO-antibody on the invasiveness of prostate cancer cells, the in vitro invasive potential of untreated or drug-treated DU145 cells was evaluated by their ability to penetrate a Matrigel invasion chamber.The results showed that anti-SMO-antibody inhibited the proliferation of DU145 cells, down-regulated cell invasiveness and induced apoptosis.To establish whether the anti-proliferative effect of docetaxel on AIPC cell proliferation may be improved by combined use with anti-SMO-antibody, the growth-inhibitory effects either of single agent or in combination, were evaluated on the DU145 prostate cancer cells.To estimate the beneficial effect of combining anti-SMO-antibody for improving the docetaxel-based therapeutic regimens, the percentage of apoptotic cell death induced by docetaxel, either alone or in drug combination, were estimated by the flow cytometric analyses. Importantly, to estimate the inhibitory effect of the drugs, alone or in combination, on the invasiveness of the AIPC cells, the invasive potential of untreated or drug-treated DU145 cells was evaluated by their ability to penetrate a Matrigel-invasion chamber. Synergistic effects were observed when anti-SMO-antibody and docetaxel were used together.The results revealed that the drugs, alone or in combination,at lower concentrations inhibited the growth of androgen-independent DU145 cells. Moreover, the combined docetaxel and anti-SMO-antibody also caused a higher rate of apoptotic death of prostate cancer cells compared with individual agents. Additionally,the combined agents were more effective at suppressing the invasiveness of DU145 cells through Matrigel in vitro than the single drugs. It appeared that combined treatment with docetaxel caused additive and/or synergistic cytostatic effects on prostate cancer cells.Conclusion:Androgen-independent prostate cancer cell proliferation was associated with activity of the Hedgehog signalling pathway. Autonomous Hedgehog signalling was detectable in DU145 prostate cancer cells.The other findings also indicate that the anti-SMO-antibody alone or in combination with docetaxel could represent two promising strategies for improving the treatment in patients with metastatic and androgen-independent prostate cancers.