| Prostate cancer has been recognized as one of the most common genitourinary cancer hazard to human health all over the world. The vast majority of early prostate cancer patients receiving androgen deprivation therapy for some time, the patient began to tolerance, further progress of the disease. In order to investigate the likely causes, we conducted this research.We firstly explored the relationship among ADT, EMT and the generation of CD44 + cancer cells in prostate cancer. Hormone-sensitive, three months after ADT therapy and tumor tissue samples from castration-resistant prostate cancer patients were collect and analyzed by immunohistochemical staining changes in epithelial and mesenchymal markers. We hypothesized that ADT treatment promotes EMT phenomenon. To test this hypothesis, we used a mouse orthotopic LNCaP prostate tumor model and castration-resistant prostate TRAMP mouse orthotopic tumor models. Establishing LNCaP prostate cancer orthotopic mouse model including hormone-sensitive stage three days of ADT treatment and castration resistance orthotopic mouse models, and then collecting LNCaP tumor tissue samples were used to detect changes in the epithelial and mesenchymal markers. Establish TRAMP mouse model and take castration treatment in the first 12 weeks, collecting the first 16 weeks, 20 weeks and 24 weeks of tumor tissue, immunohistochemistry staining and immunohistochemical staining method were used to analyze the changes of epithelial and mesenchymal markers, as well as the expression of CD44 and CK5/CK8. We found that E-Cadherin expression decreased while N-Cadherin and Vimentin expression was increased in prostate cancer patients after three months’ ADT therapy. With the progression of prostate cancer, the expression of epithelial markers decreased while mesenchymal markers increased. Successful establishment of hormone-dependent, ADT treatment after three days of non-dependent LNCaP tumors in nude mice and immunohistochemical methods confirmed the expression of E-Cadherin decreased, N-Cadherin and Vimentin increased. TRAMP mouse model confirmed prostate cancer progression is accompanied by the occurrence of EMT and an increase in CD44 + tumor cells.Furthermore, we investigated the mechanism of CD44 + stem/progenitor cells promoted invasion and metastasis in prostate cancer by TGFβ1-EMT signaling pathway. We also collected hormone-sensitive, three months after ADT therapy and castration-resistant tumor tissues from prostate cancer patients, which were used to analyze the changes of TGF-β1 by immunohistochemical staining. Established castration-resistant TRAMP mouse model, and collected the first 16 weeks and 24 weeks tumor tissues, using immunohistochemical staining to analyze the changes of TGF-β1. The LNCaP and CWR22RV1 prostate cancer cell lines were treated with 5ng/mL TGF-β1 at 0 hours, 3 hours, 6 hours, 9 hours and then using flow cytometry to detect the proportional change of CD44 + cells. CD44, C-Met, Nanog, Oct-4 and Sox-2 and other stem cell markers, and the expression levels of E-Cadherin, Vimentin by RT-PCR in 5ng/m L TGF-β1 treated LNCaP cell lines. Colony-forming experiment was performed to evaluate the number of cloned ball formation before and after 5ng/mL TGF-β1 treatment in LNCaP cell lines. Western blot was used to analyze the expression levels of CD44 in TGF-β1 treated two prostate cancer cell lines. Transwell invasive experiment was used to analyze the role of CD44 in the invasion of prostate cancer. Immunohistochemical staining was also used to evaluate the CD44 expression level and the role of CD44 in prostate cancer metastasis. We found that the expression of TGF-β1 increased after ADT therapy in human prostate cancer tissue specimens. With the progression of prostate cancer, its expression gradually increased. These results further validated in 16 and 24 weeks after castrated TRAMP mouse model. The proportion of CD44 + cells increased gradually with time treated by TGF-β1 in prostate cancer cell lines with increased expression of cancer stem cell markers, and the number of clone balls were also increased significantly. The high expression level of CD44 was detected in castrated TRAMP mouse model of prostate cancer metastasis tumor tissues.In the last department, we explored on the efficacy of salinomycin targeting CD44+ stem/progenitor cells in inhibiting invasion and metastasis of prostate cancer. Western blot method was used to analyze the expression levels of CD44+ cells in two salinomycin treated LNCaP and CWR22RV1 prostate cancer cell lines. Salinomycin treated CWR22RV1 nude model tumors are smaller and less number of distant metastases than the control group; Stem cell markers of CD44, C-Met, Nanog, Oct-4 and Sox-2 expression decreased in salinomycin-treated CWR22RV1 nude models, at the same time, the expression of E-Cadherin increased and Vimentin decreased. And finally we found that, the expression of two CD44 prostate cancer cell lines salinomycin treated LNCaP and CWR22RV1 were less. The smaller salinomycin treated CWR22RV1 nude mice model of in situ tumor distant metastasis, tumor number and size was smaller than the control group; the stem cell marker CD44, salinomycin treated CWR22RV1 nude mice model of in situ tumor tissue biopsy samples C-Met, Nanog, Oct-4 and Sox-2 expression decreased, E-Cadherin expression increased and the reduced expression of Vimentin.From what has been mentiond above, we can get the following three conclusions. After ADT treatment for prostate cancer, EMT can be promoted and the number of CD44 + stem/progenitor cells can be increased. CD44 + stem/progenitor cells promote the invasion and metastasis of prostate cancer by TGFβ1-EMT signaling pathway. Salinomycin inhibits the invasion and metastasis of prostate cancer by targeting the CD44 + stem/progenitor cells, which is expected to become a new drug for prostate cancer treatment. |
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