Cellular Adhesion Molecules Variation Of Colon Cancer Cells Treated By Hyperthermia And Chemotherapy

To understand the mechanism of tumor metastasis, we studied the variation of cellular adhesion molecules, cell cycle and cell apoptosis in colon cancer cells treated with hyperthermia and chemotherapy. We used HT29, HCT8, LoVo, SW620 and T84 colon cancer cells. Using Immunohistochemistry, we selected the cells that expressed cellular adhesion molecules E-Cadherin, CD44V6 and ICAM-1, and the nm23 gene. HT29, HCT8 and LoVo were selected and exposed to 43C for 60 minutes. We also used immunohistochemistry to detect E-Cadherin, CD44V6 and ICAM-1 protein expression, and quantitative RT-PCR to investigate the mRNA transcription level. We also flow cytometry to investigate to examine the changes in the cell cycle and cell apoptosis.Mytomicin C was used for chemotherapy and hyperthermic chemotherapy to treat HT29, HCT8 and LoVo cells and also to investigate the changes of proteins, mRNA transcription, cell cycle and apoptosis.We have four groups for this study: control group, hyperthermia group (divided in 8 post hyperthermia subgroups Oh, 2h, 4h, 8h, 12h, 24h, 48h and 72h), chemotherapy group and hyperthermic-chemotherapy group.This thesis was divided in to four parts:1. Selection of colon cancer cells2. Effect of hyperthermia and chemotherapy on cellular adhesion molecules expression in colon cancer cells3. Cytotoxicity in colon cancer cells treated with Mytomicin C and hyperthermic-chemotherapy4. Cell cycle and cell apoptosis changes in colon cancer cells treated by hyperthermia and chemotherapyPart 1: Selection of colon cancer cellsBackground: To select the colon cancer cells, which can express the cellular adhesion molecules E-Cadherin, CD44V6 and ICAM-1, and the nm23 gene. The selected cells will be used for hypothermia study.Methods: An immunohistochemistry SABC technique was used to investigate the expression of cellular adhesion molecules and the nm23 gene on the 5 colon cancer cell lines HT29, HCT8, LoVo, SW620 and T84.Results: 1. All 5 cell lines expressed E-Cadherin: HT29 and HCT8 (40-70%); LoVo, SW620 and T84 (10-40%). 2. CD44V6 expression was 70-100% on HT29 and LoVo; 40-70% on HCT8, SW620 and T84. 3. ICAM-1 expression was 40-70% on HT29, LoVo, SW620 and T84; 10-40% on HCT8. 4. HCT8 cells showed positive nm23 staining but none was found in cells HT29, LoVo, SW620 and T84. Conclusion: We selected HT29, HCT8 and LoVo for hypothermia study.Part 2: Effect of hyperthermia and chemotherapy on cellular adhesionmolecules expression in colon cancer cellsBackground: To investigate the effect of hyperthermia and hyperthermic chemotherapy on cellular adhesion cells E-Cadherin, CD44V6 and ICAM-1 in 3 colon cancer cell lines HT29, HCT8 and LoVo.Methods: We used immunohistochemistry for cellular adhesion molecules protein expression, and quantitative RT-PCR to investigate the mRNA transcription level for each subgroup at Oh, 2h, 4h, 8h, 12h, 24h, 48h and 72h.Results: 1. After hyperthermia, E-Cadherin mRNA transcription level was different among 3 cell lines (P<0.05), HT29 was higher than HCT8 and LoVo; LoVo was higher than HCT8. The transcription level of E-Cadherin was not significantly different between hyperthermia and hyperthermic chemotherapy (P>0.05). 2. CD44V6 mRNA transcription level was different among the 3 cells (P<0.05): LoVo was higher than HT29 and HCT8; HT29 was higher than HCT8. The transcription level was also different between hyperthermia and hyperthermic chemotherapy (P<0.05) 3. ICAM-1 mRNA transcription level was different among the 3 cells (P<0.05), HT29 and LoVo was higher than HCTS.The transcription level was significantly different between hyperthermia and hyperthermic chemotherapy (P<0.05).Conclusion: We concluded that hyperthemia up regulated E-Cadherin expression and down regulated CD44V6 and ICAM-1. The expression level was different among 3 colon cancer cell lines.Part 3: Cytotoxicity on colon cancer cells treated by Mitomycin C andhyperthermic-chemotherapyBackground: To investigate on colon...