| Background and Objective:Prostate cancer is one of the most common malignant tumor in men, on a global scale, the morbidity and mortality of prostate cancer in the men all malignant tumors of the 2nd and 6th of respectively, the American cancer society's latest estimated that in 2015 the United States new cancer the first new prostate cancer in men, prostate cancer deaths among new second cancer deaths,due to changes in diet and lifestyle, recently the incidence of prostate cancer also showed a trend of obvious rise in China, according to the statistics respectively accounted for the morbidity and mortality of prostate cancer in the men all malignant tumors of the 6nd and 9th of respectively, It serious harm people's health. Despite high cure rates with surgery and/or radiation in early stage prostate cancer,but still have 30-40% of patients will eventually develop to advanced cancers. Docetaxel is one of the most effective chemotherapeutic agents for prostate cancer with a long history in prostate cancer therapeutics. However, development of docetaxel resistance is a general rule within only months.and Maken the treatment of advanced prostate cancer in trouble again. At present, a series of new drug of research and development, For example, cabazitaxel, Sipuleucel-T, radionuclide radium 223, abiraterone acetate and MDV3100 and so on, although they have certain curative effect to CRPC, but increasing survival of patients is not satisfactory, because Docetaxel still is one of the most basic chemotherapy drugs treatment CRPC, Combination therapies have been proposed to improve the therapeutic potential of docetaxel in prostate cancer. So seeking efficient agents for treatment of prostate cancer especially docetaxel resistant prostate cancer becomes urgent. IGF1R and IR are important RTKs, IGF1R and IR are overexpressed in prostate cancers. Inhibition of either single or dual or multiple RTKs might provide a potential therapeutic strategy for prostate cancers especially resistant prostate cancers. IGF1R/IR-mediated signaling pathways promote proliferation and survival of a series of cancer cells including prostate cancer cells, and inhibition of IGF1R/IR kinases provides therapeutic opportunities in cancer patients. These signaling pathways have been linked to a risk of prostate cancer as well as development of resistance in prostate cancers. Numerous studies showed that inhibitors of IGF1R/IR or antibodies against IGF1R/IR exhibited potential efficiency in therapeutics of a series of cancers including prostate cancers especially docetaxel-resistant prostate cancers, Considering the critical roles of IGF1R and IR in prostate tumor growth, metastasis as well as development of resistance, we have investigated the anti-cancer activities of GSK1838705A, an IGF1R/IR inhibitor, in prostate cancer especially docetaxel-resistant prostate cancer both in vitro and in vivo. to demonstrated the anti-cancer activities of GSK1838705A in therapy of prostate cancers especially those developed resistance towards docetaxel.Methods:1. The cultivation of the PC-3 human prostate cancer cells and the Docetaxel resistant cell line PC-3R was obtained by culturing PC-3 cells with gradually increased doses of docetaxel. All the cells were cultured in DMEM medium with 10% fetal bovine serum (FBS),100 units/ml penicillin and 100?g/ml streptomycin. Cells were maintained at 37? in an atmosphere comprising 95% air and 5% CO2.2. PC-3 and PC-3R cells were plated in 96-well white plates at 5×103 cells/well. Docetaxel (12.5-400nM) and GSK1838705A (0.0625-2?M) were then added and incubated for 72hr, then a CellTiter-Glo kit (Promega, Madison, WI) was used to assess cell viability.3. Flow cytomery analysis, Cell nuclei staining, western blot and else assays were used to assess cell apoptosis ability, we observed the subG1 DNA content in PC-3R cells after treatment with GSK1 838705A compared with control group. We also observed the nucleus morphology changes in PC-3R cells after treatment with GSK1838705A. including chromatin condensation and nuclear fragmentation, we also by TUNEL staining assay to further verified GSK1838705A induced PC-3R cells apoptosis. Furthermore, western blot analysis was used to research the levels of cleaved caspase-3 in the resistant cells after GSK1838705A treatmented PC-3R cells, which further confirmed GSK1838705A-induced cell apoptosis in the resistant cells.4. We next observed the effect of GSK1838705A on phosphorylation of IGF1R and IR in PC-3R cells:PC-3R cells were seeded in 6-well plates. After 24 hr, the medium was replaced with serum-free medium, and GSK1838705A (0.5 and 2 ?M) was added and incubated for 4 hr, then cells were stimulated with 30ng/mL IGF-1 or 3?g/mL insulin for 20min, followed by western blot analysis with the indicated antibodies.5. By Transwell assay to observed GSK1838705A reduced the migration of PC-3R cells in a transwell model compared with control group:PC-3R cells were treated with GSK1838705A (4,20 or 100nM) for 8hr. The non-migrated cells on the upper surface of the filter were removed, and the migrated cells on the lower side were stained and photographed. The migrated cells were lysed and the colorimetric determination was made at 595nm to obtain the quantitation result of the inhibition.6. PC-3R cells were injected subcutaneously into the axillary regions of selected nud/nud mice (4×106 cells/100?L/mouse). Cells were allowed to grow to reach a volume of 50mm3 before mice were randomized to the following experimental groups: GSK1838705A (20mg/kg), GSK1838705A (60mg/kg) and control. All groups had six animals per condition. Tumors were measured on alternating days with a microcaliper along with body weight. Total tumor volumes were calculated as follows:(mm3)= width × width × length × 0.5. GSK1838705A induced apoptosis of PC-3R tumor cells in vivo examined with TUNEL assay and the nuclei were stained with Hoechst.to evaluate the efficacy of GSK1838705A (intraperitoneal injection) on docetaxel resistant PC-3R tumor growth in mice.Results:1. GSK1838705A significantly reduced viability of both docetaxel sensitive(PC-3) and resistant (PC-3R) lines similarly in a concentration-dependent manner. (Fig.1A and 1B);2. The subGl DNA content in PC-3R cells significantly increased after treatment with GSK1838705A compared with control group (P<0.01)(Fig.2A and 2B). We also observed the nucleus morphology changes in PC-3R cells after treatment with GSK1838705A. Consistently, after treatment with GSK1838705A, the PC-3R cell nuclei had morphological changes including chromatin condensation and nuclear fragmentation, both of which are indicators of cell apoptosis (Fig.2C and D). GSK1838705A induced cell apoptosis was also confirmed by TUNEL staining assay (Fig.2C). These structural alterations indicate that treatment with GSK1838705A generated apoptosis in docetaxel resistant PC-3R cells. Furthermore, western blot analysis showed that GSK1838705A treatment significantly increased the levels of cleaved caspase-3 in the resistant cells (Fig.2E), which further confirmed GSK1838705A induced cell apoptosis in the resistant cells.3. GSK1838705A could significantly inhibit phosphorylation of IGF1R and IR. While there were no significant changes in total protein levels (Fig.3).4. GSK1838705A dramatically reduced the migration of PC-3R cells in a transwell model compared with control group (P<0.01)(Fig.4A and 4B).5. The results showed that low dose of GSK1838705A (20mg/kg) culminated in an intermediate degree of tumor growth suppression (P<0.05). Contrastingly, a high dose of GSK1838705A (60mg/kg) led to a significant inhibition of PC-3R tumor growth compared with control group(P<0.01)(Fig.5A). It should be noted that administration of GSK1838705A was well-tolerated by mice, who presented with no measurable signs of overt toxicity or decreases in body weight (P>0.05) (Fig.5C).Conclusion:GSK1838705A could reduces viability of both docetaxel sensitive and resistant prostate cancer cells, induces apoptosis of PC-3R cells, reduces PC-3R cells migration in vitro and suppresses PC-3R tumor growth in vivo. All these might open another avenue for overcoming docetaxel resistance in prostate cancers. |
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