| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 94 === Prostate carcinoma is the most common malignancy in males and is the second leading cause of cancer mortality in the United States after lung cancer. Now prostate cancer occurrence increased recently in Taiwan and became the seventh leading cause of cancer mortality. Invasion and metastasis is still the greatest obstacles to the successful treatment of patients with prostate cancer. Chemotherapy is the choice of therapy for genital urinary tract cancer, when they were unresectable, recurrent or metastasis. But prostate cancer always possesses drug-resistance in this situation after hormonal therapy is of no use. Therefore, the molecular mechanisms of drug-resistance derived from hormone refractory should be investigated to find out a new target of effective alternative therapy for the treatment of prostate cancer.
Our laboratory has developed cDNA microarrays to screen the potential genes that involve the development of drug resistance of prostate cancer and try to find a new efficient therapy target for clinic treatment. There are several genes up-regulated in multidrug resistance cell line of rat prostate cancer when compared with native cells. We narrowed to three highly potential genes including macrophage migration inhibitory factor (MIF), glutathione S-transferases (GSTs), and Inhibition of Differentiation-1 (Id-1) because they are reported to associated with tumorgenesis or metastasis.
At first, we identified the relationship between these genes and acquired drug resistance of prostate cancer, we established the prostate cancer transfectants that overexpressed Id-1, MIF and GSTpi, respectively, and tested the inhibitory effect of the cytotoxicity to different chemotherpeutic agents such as doxorubicin, paclitaxel and cyclophosphamide. The cytotoxity assay results demonstrated that cells overexpressing the Id-1 gene can increase in their resistance to doxorubicin, paclitaxel and cyclophosphamide. MIF expression can drive cells increase in their resistance to paclitaxel, and GSTpi expression confers drug resistance to doxorubicin and cyclophosphamide. In addition, the induction of mdr1 gene expression was noted in upregulation of MIF and GSTpi.
To further investigate the relationship between MIF as well as GSTpi and the expression of mdr-1 gene in MDR subline of prostate cancer cells, we used these stable transfectants to determine whether the expression of mdr-1 should be modulated by MIF or GSTpi expression and whether the overproduction of p-glycoprotein driving by MIF or GSTpi confers the resistance to prostate cancer cells. The results showed that the production of gp-170 increased in MDR sublines of prostate cancer accompanying the upregulation of MIF and GSTpi in protein level. The expression of mdr-1 gene and the production of pg-170 increased in either MIF or GSTpi stable transfectants when compared with vector control. The human embryonic kidney (HEK) cells transiently transfected with the eukarytonic expression vector driving MIF or GSTpi protein presented the increased production of gp-170 protein using flow cytometric analysis. The MTT results demonstrated that the reduced chemocytotoxicity was correlated with the increased production of gp-170 protein in MIF and GSTpi transfectants.
To determine the molecular mechanism of Id-1 conferring the drug resistance in prostate cancer, we used the Id-1 stable transfectants to investigate and determine the effect of Id-1 expression and its underlying pathways on the development of multidrug resistance of prostate cancer. We found that Id-1 overproduction drove AT3 cells to become resistant to chemotherapeutic agents such as doxorubicin, taxol and cyclophosphamide but not due to the expression of mdr-1. The p38MAPK and c-jun N-terminal kinase (JNK) pathways were suppressed, and this correlated with increased expression of Id-1 after doxorubicin challenge. Treatment of the Id-1 expressing cells with p38MAPK and JNK inhibitors resulted in decreased doxorubicin-induced apoptosis. In contrast, Id-1 expressing cells treated with extracellular signal-regulated kinase (ERK) inhibitor made cells more sensitive to drug-induced apoptosis. In conclusion, overexpression of Id-1 can affect drug resistance of prostate cancer through the inhibition of p38MAPK and JNK pathways. In addition, sustained activation of ERK could provide cellular resistance to drug-induced cytotoxicity.
In conclusion, our data have provided following important informations: 1) using this MDR cells model combined with cDNA microarray method, we have successfully screened three important genes that overexpressed in MDR cell lines and its overexpression confer drug resistance in prostate cancer cells; 2) although overexpression of MIF and GSTpi genes induced mdr1 genes expression, they were unable to carry out all of the drug resistance phenotypic characteristics of MDR prostate cancer cells. It indicated there were alternative mechanisms that also confer drug resistance in prostate cancer; 3) Id-1, MIF and GSTpi may confer drug resistance not only due to the induction of mdr1 gene expression but also through their individual biological function. 4) overexpression of Id-1 in cells may lead to sustained activation of the ERK accompanying inhibition of the JNK and p38MAPK that results in promoting cells to be more resistant to drug-induced apoptosis. However, the cooperation of these three genes in the acquisition of drug resistant in prostate cancer cell line will be further addressed in animal models.
|
|---|
