| Prostate cancer(PC) is the most common malignancy and the second leading cause of cancer-related deaths among male patients. Although the incidence of PC in China is much lower than that in Western countries, it keeps rising rapidly in recent years. And PC seriously endangering the health of old man. So how to prevent and cure this malignant disease has been the focus of urological cancer researchers.Radiotherapy is one of the most important clinical therapies for PC patients, especially those castration refractory prostate cancer over T3 grade. Although the efficacy of radiotherapy is satisfactory initially, it is gradually weaken during cancer progression because of the radio-tolerance.The main mechanism of radiotherapy is the induction of multiple types of DNA damage, including single-strand breaks(SSB), double-strand breaks(DSB), base alterations, DNA-DNA, and DNA-protein crosslinks. Without proper DNA repair procedure, cells undergo apoptosis, mitotic catastrophe, autophagy, cellular senescence, or even carcinogensis. However, in tumor cells after radiation therapy, enhanced DNA repair capacity,such as homologous recombination repair(HRR) or non-homologous end joining(NHEJ), leads to resistance of radiotherapy. If DNA repair procedure of the cancers is targeted, the efficacy of radiotherapy will be significantly increased. By specific inhibition of DNA repair, sensitivity to radiotherapy could be greatly inhanced. This concept will become a new direction to international cancer research. It will provide a promising adjuvant combined clinical therapeutic approach to improve the outcome of PC patients to radiation treatment.Insulin-like growth factor receptor(IGF1R) is one of the members in insulin-like growth factor system(IGFS). IGFS signaling pathway is closely related to the oncogenesis of a great variety of tumors. For most cells, IGF-1 is a potent mitogen, which involved in at least two anti-apoptotic signal pathways. One is phosphatidylinositol cyclohexanone six ol-3- kinase(PI3K/AKT) pathway, another is regulated by Ras, Raf kinase and extracellular signal(Extracellular-signal-regulated kinase, ERK) / mitogen-activated protein kinase(mitogen-activated protein kinase, MAPK) pathways. IGF-1 can promote cell proliferation, improve cell survival, and it plays an important role in inhibition of cell apoptosis. Current studies had proved that serum IGF-1 levels elevation is positively correlated with greater incidence of pancreatic cancer, breast cancer, endometrial cancer and other tumors. On the other hand, IGF-1 can also promote metastasis of tumor cells.Epidermal growth factor receptor(EGFR) is the expressed protein of c-erbB1 gene. It is a family member of epidermal growth factor receptor, which is widely distributed in epithelial cells, fibroblasts, glial cells, skin cells of mammalian. EGFR pathway plays an important role in cell growth, proliferation and differentiation. Studies have shown that EGFR overexpression or aberrant expression will be related with cell proliferation, tumor angiogenesis, invasion, metastasis and inhibition of apoptosis in many solid tumors.Previous studies have confirmed that IGF1 R and EGFR overexpression can activate DNA damage repair procedure through the PI3K/AKT signaling pathway in PC cells, which is closely related to tumor radiation resistance. However, single inhibition of IGF1 R or EGFR will have poor clinical therapeutic effects. Although we found that the targeting of IGF1 R or EGFR alone can increase radiosensitization by inhibiting DNA repair, prolonged suppression of IGF1 R could activate EGFR by singal crosstalk, and vise versa. However, combined inhibition of IGF1 R and EGFR could synergisticly inhibit mutiple intra-cellular signal pathways, such as PI3K/AKT and MAPK, which had been linked with DNA damage repair procedure in PC cells. PurposeTo study the sensitization of radiation therapy after combined targeting of the IGF1 R and EGFR in PC cell, and its correlated molecular mechanisms. It may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment. MethodsThe human castration refractory PC cells DU145, PC3 were cultured and treated with AG1024 and / or Erlotinib, the radiosensitivity of these cell lines was observed 1 h after γ-irradiation. After Erlotinib AG1024 treatments, clonogenic cytotoxicity assays was performed on these cell lines to explore the possible synergistic inhibitive effect. Cell cycle and apoptosis assays were performed with flow cytometry to measure the cytotoxic effect of combined AG1024 and erlotinib treatment before radiation. Wstern-blots for phosphorylated AKT, pAKT were conducted after treatment with Erlotinib and AG1024 with or without radiation. The γH2AX and Rad51 foci formation was investigated with the immunofluorescence method by the laser confocal microscope to clarify the possible mechanism of radiosensitivity enhancement by using these two inhibitors. Animal xenograft model was established to observe the sensitivity of the tumor volume after radiation after drugs treatment and proliferation index in vivo was measured. Results(1) The tumor cell viability was significantly reduced and tumor cell apoptosis was greatly enhanced by irradiation following either Erlotinib or AG1024 treatment alone, as compared with the vehicle-treated control cells. AG1024 or Erlotinib significantly reduced tumor cell viability in epithelial PC cell lines(P < 0.05), yet the most robust growth-inhibitive effect after irradiation was achieved with simultaneous application of both inhibitors(P<0.05).(2) Combined Erlotinib and AG1024 treatment after irradiation was shown to synergisticly induce cell apoptosis in DU145 and PC3 cells.(3) The flow cytometry assay showed that the radiosensitivity of DU145, PC3 and ARCaPE cells was enhanced following increasing doses of AG1024 and/or Erlotinib, while ARCaPM was only sensitive to AG1024, but not to Erlotinib. When comparing DU145 cells with PC3 cells, we found that the former are more sensitive to Erlotinib than the latter. Both AG1024 and Erlotinib was shown to have pro-apoptotic effect of γ-irradiation, which was highest in the combined AG1024 and Erlotinib treatment group.(4) In the Western-blot assay, it was shown that AKT expression was not altered in any group. Irradiation can induce AKT phosphorylation.Both these two inhibitors could inhibit the AKT phosphorylation procedure, while the combined drug treatment could inhibit AKT phosphorylation in a synergistic way.(5) For DU145 cells, combined treatment of AG1024 and Erlotinib potently reduced HRR level when compared with the vehicle control or each agent alone(P<0.05). In contrast, the nuclear extract treated with any of the inhibitor or both were unable to alter the NHEJ related DNA repair proteins. All of these suggesting that effects of EGFR and IGF1 R inhibitors on impairment of DSB repair through inhibition of HRR, but not of NHEJ.(6) γ-irradiation significantly reduced in vivo tumor growth of parental DU145(P<0.05, as compared to the control mice), which was further reduced by treatment with AG1024 or Erlotinib(P<0.05, as compared to c-irradiation mice), and by co-treatment with AG1024 plus Erlotinib(P<0.05, as compared to γ-irradiation plus AG1024 orγ-irradiation plus Erlotinib group). Although tumor growth was not dramatically affected by irradiation over time with DU145-IIRS cell injections, treatment with AG1024, Erlotinib, or both sensitized the tumor cells to γ-irradiation. Combined treatment with both inhibitors significantly inhibited tumor growth, comparing with any one of these inhibitor treatment, indicating the synergistic growth inhibitive effect. ConclusionsTaken together, in castration refractory PC cells, the experiments provide us the evidence that the effects of EGFR and IGF1 R inhibitors on the impairment of DSB repair through inhibition of HRR DNA repair process, but not of NHEJ DNA repair process. Co-treatment with EGFR and IGF1 R inhibitors was able to suppress their crosstalk and in turn block their downstream signaling, including PI3K/AKT pathway and MAPK pathway. Crosstalk between EGFR and IGF1 R contributes to radiation-induced DSB repair through the suppression of HRR via IRS1/Rad51 signal pathway. |
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