Cell And Breast Cancers And Colorectal Cancer - 1161 Words

An antimetabolite, 5FU is a pyrimidine analog that irreversibly inhibits TS. Thymidine is a nucleoside and a major component of the DNA and is hence required by cells for proliferation. Deoxyuridine monophostate (dUMP) upon methylation by TS generates thymidine monophostate (dTMP). 5FU interrupts the activity of TS and creates a shortage in the levels of dTMP. Thus the rapidly proliferating cells undergo death due to lack of thymidine nucleoside. The drug has successful applications in colorectal and breast cancers and is used in various combination therapies with methotrexate (Maddur et al., 2009). The concentration used for the experiments is 10  µM Oxaliplatin is an antineoplastic platinum drug. Platinum drugs target DNA synthesis by†¦show more content†¦215 cells have high proliferating rates as compared to 253 cells. To generate metformin resistant cells, 215 and 253 were cultured 4-8 weeks in the presence of 3mM of metformin. Cells were considered resistant after 3 serial passages in vitro. For the generation and enrichment of cancer stem cells, PDAC cells at 106 cells/ml concentration were grown in a serum free Dulbecco’s Modified Eagle’s Medium (DMEM-F12) complemented with B-27 and FGF. The cells were seeded in ultra-low attachment flasks. This aids in the development and the expansion of the PDAC spheres (cancer stem cells). Metformin was added (at 1:1000 dilution) in case of culturing metformin resistant spheres. All the cells were contained in a T-75 flask and incubated at 37 °C. The regular cancer cells were seeded at 106 cells/ml concentration in RPMI containing 10% (v/v) FBS. The cells were cultured in regular attachment T-75 flasks. Metformin at 1:1000 dilutions was added in case of culturing metformin resistant PDAC cells. After incubating for 7 days, spheres increase in size and range from 40-120  µm. For serial passaging, spheres were harvested using 40  µm cell strainers (filters), trypsinised to dissociate into single cells and then grown again for 4 days in the same conditions. Growth media was aspirated and the flask was washed with PBS. The cells were trypsinised and kept in an incubator at 37 °C until the cells