| Colorectal cancer is the second leading cause of cancer deaths after lung cancer in Western countries. In China, colon cancer is also one of the most common cancers. Conventional treatment including surgery and/or chemotherapy and radiotherapy can partly relieve patients'pain. However, the results of chemotherapy and surgery for advanced stage patients are usually not satisfactory. Therefore, new therapeutic strategy is needed.Apoptosis is a ubiquitous physiological function, and plays an important role in cell proliferation, differentiation and tumor development. Many evidences have demonstrated that the inhibition of cells apoptosis was one important mechanism of tumor development. So apoptosis induction becomes a substaintial strategy to treat tumors. GC induce massive apoptosis in immature thymocytes, ALL cells, lymphoma and myeloma cells via the interaction of GC with its receptor-GR. Glucocorticoid-induced apoptosis has been exploited in the therapy of lymphoid malignancies.Glucocorticoids (GCs) are effective in inducing apoptosis via the uncharacterized mechanisms in many hematological malignancies. GC-induced apoptosis is initiated by, and strictly dependent upon, the interaction of GC with its receptor, the GR. Moreover, the level of GR expression in tumor cells is a critical determinant for GC sensitivity. Depending upon regulation of the expression or inhibition of apoptotic genes including NF-κB, AP-1, bcl-2 gene family, caspases and c-myc etc, GC both induced cell death and supported survival in lymphoid malignancies.Alford demonstrated 23% of conlon cancer samples were positive for glucocorticoid receptors in 1979. Theocharis immunohistochemically examined the GR expression in human colon cancer, presenting positive GR immunostaining in 48% of the examined cases. GR expression was statistically significantly correlated with the expression of cell cycle-related molecules, pRb and p16. Dexamethasone could induce a G1 arrest and increase the expression of p21WAF1/CIP1 in human colorectal Colo320 HSR carcinoma cells in vitro. Do GC have actually effects of antitumor and increase chemosensitivity in colon cancer? It is still diverse and even contradictory.Objectives:In this study, the GR expression in human colon cancer samples and colon cancer cell lines was detected and compared with that in clinic samples of lymphoma, which is a different type of tumor; Of colon cancer cells and lymphoma cells, the sensitivity to Dex as well as to the chemotheraputic agnets 5-Fu and L-OHP in the present of Dex were detected; Furthermore the activity of NF-κB and the expression of GR isoforms, IκB and Bcl-2 genes in Dex-treated colon cancer cells and lymphoma cells were measured. The different mechanisms of GC in colon cancer and lymphoma in the response to chemotheraputic agents were proposed and the feasibility of clinic application to the treatment of colon cancer were discussed.Methods:1. Immunohistochemistry was used to examine the expression of GR, NF-κB and Bcl-2 in colon cancer and lymphoma samples.2. The expression of GR in four colon cancer lines (Lovo, HCT-116, HT-29 and SW-480) were detected by RT-PCR and Western blotting. Cell lines negative for GR expression are treated with 5-Aza and the methylation in the promoter region of GR gene was detected by MSP-PCR.3. MTT assay was employed to detect the cell survival of colon cell lines treated with GC alone or in combination with 5-Fu and L-OHP in a way of GC pretreatment or co-administration.4. Flow cytometry, nucleus staining with Hoechst33342 and DNA Ladder were exploited to detect GC-induced apoptosis5. The activity of NF-κB and expression of GRα, GRβ, IκB and BCL-2 of colon cancer cell and lymphoma cell during the course of GC treatment are measured by RT-PCR and Western blotting. 6. GRαand GRβwere silenced by RNAi technique in order to study their effect on Dexamethasone-induced apoptosis of colon cancer cells.Results:1. Positive GR expression was found in 57.3% of cases of colon cancer, lower than that of lymphoma samples. In human colon cancer cases the expression of GR positively correlated with the degree of differentiation of cancer cells (x2 test,p=0.029), and with the expression of NF-κB (r = 0. 352, p =0. 005), not bcl-2. The expression of GR of the colon cancer was not related to either patient's gender, age, tumor location, Dukes'stage or lymph node metastasis.2. The glucocorticoid receptor was determined in Lovo and HCT-116 cells, not in HT-29 and SW-480 cells. The expression of GR in HT-29 and SW-480 cells could be partially reversed after treated with 5-Aza. Methylation in the promoter region of the GR gene was not detected by MSP-PCR.3. The proliferation of GR-expressed Lovo and HCT-116 cells could be inhibited by 1～3×10-4 mol/L Dex . The rates of inhibition were 40.2%, 46.9% and 52.6% in Lovo cells and 41.8%,49.3% and 58.8% in HCT-116 cells respectively, higher than those of cells treated with 1×10-5 mol/L Dex (p<0.01). The Dex-induced apoptosis was confirmed by hoechst33342 staining and DNA Ladder. Apoptosis were detected by flow cytometer in Lovo and HCT-116 cells 72 hours after 1×10-4mol/L Dex challenging, the rates of apoptosis was 34.8±1.9% in Lovo and 33.6±1.4% in HCT-116, higher than those in the control groups without Dex (p<0.01). In contrast, the rates of apoptosis were low in GR-negative cells, HT-29 and SW-480 even treated with 1×10-4mol/L Dex for 72 hours. The rate of apoptosis in the Jurkat treated with 1×10-5mol/L Dex for 36 hours was 50.3±2.4%, which was higher than those of colon cancer lines.4. Pretreatment and co-administration for Lovo cells with 1×10-4mol/L Dex could decreased the IC50 of 5-Fu from 72.2±8.1μg/ml to 21.1±4.1μg/ml and 18.6±4.0μg/ml respectively, and the IC50 of L-OHP from 13.7±1.3μg/ml to 5.9±0.6μg/ml and 4.8±0.7μg/ml respectively.5. The supression of NF-κB activity, up-regulation of the mRNA and proteins expression of GRαand IκB occurred in Lovo and Jurkat cells 6, 12 and 24 hours after Dex treatment. The expression of BCL-2 was not changed in two cell lines. But the mRNA expression of GRβ6 hours after Dex treatment was greatly increased in Lovo cells, not in Jurkat cells.6. The mRNA and protein expression of GRαof Lovo cells that transfected with GRαRNAi2 silencing vector were inhibited 73.7% and 59% of the level of original cells respectively. And the rate of Dex-induced apoptosis descended from 22.8±2.5% to 6.5±0.5% ( t=29.1, p <0.01). The mRNA expression of GRβdecreased 67.3% in Lovo cells with liposome-mediated transfection of synthesized GRβsiRNA and its rate of Dex-induced apoptosis increased from 34.7±1.8% to 45.4±4.3% (p <0.05).Conclusions:1. Positive GR expression was found in 57.3% of cases of colon cancer. In human colon cancer cases the expression of GR positively correlated with the degree of differentiation of cancer cells and the expression of NF-κB, not related with either bcl-2 expression, patient's gender, age, tumor location, Dukes'stage or lymph node metastasis.2. Methylation in the promoter region of the GR gene was less likely to play a role in the reduction of GR expression in colon cancer cell lines. The GR expression of HT-29 and SW-480 may be regulated by the methylation of genes regulating GR expression.3. Dex treatment could cause the inhibition of proliferation and induction of apoptosis in the GR positive colon cancer cell lines, but to a much less extent than in Jurkat cells.4. Pretreatment for 24h and co-administration with Dex increased the chemosen sitivity of Lovo cells to 5-Fu and L-OHP.5. The supression of NF-κB activity, up-regulation of GRαand IκB may be the common mechanisms for Dex-induced apoptosis in both colon cancer and lymphoma cells. Increased GRβexpression was probably one important reason involving the higher Dex-sensitivity to GC in Jurkat than in Lovo. |
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