Effects Of Pemetrexed And Gefitinib On Human Colon Cancer Cells

Objectives1. To study the antitumor effects of pemetrexed on human colon cancr cells and the correlation of antitumor effects of pemetrexed and expressions of genes related with pemetrexed in human colon cancer cells.2. To study the antitumor effects of gefitinib on human colon cancer cells and the correlation of antitumor effects of gefitinib and EGFR signal pathway gene mutations in human colon cancer cells.3. To study the pharmacodynamic effects of pemetrexed combined with gefitinib on human colon cancer cells.4. To study the effects of pemetrexed, gefitinib, and their combination on cell cycles and apoptosis cell percentages in human colon cancer cells.5. To study the effects of pemetrexed, gefitinib, and their combination on expressions of genes related with pemetrexed in human colon cancer cells.6. To study the effects of pemetrexed, gefitinib, and their combination on the expressions of proteins of EGFR signal pathway in human colon cancer cells.Materials and Methods1. Antitumor effects of pemetrexed on human colon cancer cells HT-29, LoVo, SW620, HCT116, SW1116 and WiDr were detected by drug sensitivity test in vitro (MTT method); the mRNA expressions of RFC, FPGS, TS, and DHFR genes related with pemetrexed in six human colon cancer cells were detected by Real-Time Quantitive RT-PCR method; the correlation of antitumor effects of pemetrexed and expressions of genes related with pemetrexed in human colon cancer cells were analyzed.2. Antitumor effects of gefitinib on human colon cancer cells HT-29, LoVo, SW620, HCT116, SW1116 and WiDr were detected by drug sensitivity test in vitro (MTT method); EGFR and K-RAS gene mutations of six human cancer cells were detected by gene sequences detection; the correlation of antitumor effects of gefitinib and EGFR and K-RAS gene mutations in human colon cancer cells were analyzed.3. The pharmacodynamic effects of concurrent pemetrexed and gefitinib and sequential pemetrexed followed by gefitinib in human colon cancer cells HT-29, LoVo, SW620 and SW1116 cells were detected by drug sensitivity test in vitro (MTT method).4. The effects of pemetrexed, gefitinib, concurrent pemetrexed and gefitinib and sequential pemetrexed followed by gefitinib on cell cycles and apoptosis cell percentages of HT-29 and LoVo cells were studied by Flow cytometry detection,5. The effects of pemetrexed, gefitinib, concurrent pemetrexed and gefitinib on the mRNA expressions of RFC, FPGS, TS, and DHFR genes of HT-29 and LoVo cells were detected by Real-time quantitive RT-PCR method.6. The effects of pemetrexed, gefitinib, concurrent pemetrexed and gefitinib on the protein expressions of pEGFR, pAKT and pERK genes of HT-29 and LoVo cells were detected by western blot method.Results1. Pemetrexed inhibited the growth of HT-29, LoVo, SW620, HCT116, SW1116 and WiDr cells (P<0.01). The antitumor effects of pemetrexed were negatively correlated with TS gene expressions in six human colon cells (r=-0.886, P<0.05)2. Gefitinib inhibited the growth of HT-29, LoVo, SW620, HCT116, SW1116 and WiDr cells (P<0.01). Sequence analysis showed that the exon 19 of EGFR gene were deleted in LoVo cells, and exon 20 of EGFR gene in HT-29, SW1116 and WiDr cells were mutated. Because of cells having mutation are less, we did not find correlation between gefitinib's effect on and EGFR and K-RAS gene mutations of six colon cancer cells (P>0.05).3. Pemetrexed combined with gefitinib showed synergistic effects on human colon cancer cells HT-29, LoVo, SW620 and SW1116 (P<0.01).4. Pemetrexed, gefitinib, concurrent pemetrexed combined with gefitinib and sequential pemetrexed followed by gefitinib affected the cell cycles and apoptosis of HT-29 and LoVo cells(P<0.01).5. Gefitinib decreased TS and DHFR gene expressions of HT-29 and LoVo cells (P<0.01).6. Pemetrexed increased pEGFR, pAKT and pERK protein expressions of HT-29 and LoVo cells (P<0.01).Conclusions1. Pemetrexed can inhibit the growth of human colon cancer cells. The sensitivities to pemetrexed are negatively correlated with TS gene expressions in colon cancer cells.2. Gefitinib can inhibit the growth of human colon cancer cells.3. Pemetrexed combined with gefitinib has synergistic effects on human colon cancer cells.4. Pemetrexed, gefitinib and their combination affect the cell cycles and apoptosis of human colon cancer cells.5. Gefitinib reduces the gene expressions of TS and DHFR, and increases the sensitivities of human colon cancer cells to pemetrexed.6. Pemetrexed increases the protein expressions of pEGFR, pAKT and pERK, and gefitinib can reduce the increase caused by pemetrexed.