Effects Of PI3K/AKT Pathway On The Proliferation And Apoptosis Of Human Colorectal Cancer SW620^Lgr5+CSCs In The Inflammatory Environment

Objective To detect the correlation between Lgr5 and tumor malignancy and the dryness of Cancer Stem Cells?CSCs?,the Lgr5⁺CSCs model of human colorectal Cancer was established to explore the effect of inflammatory environment on the proliferation and apoptosis of CSCs and its molecular mechanism.Methods 1)The expression levels of Lgr5 in SW620?SW480?HT29 and HCT116human colon cancer cells were analyzed by Western blot analysis,and SW620 cells were used to analyzed the proportion of Lgr5⁺cells in FACS.SW620 spheroid cells were obtained by serum-free suspension culture,SW620^Lgr5+CSCs were isolated by FACS.The biological characteristics of Lgr5⁺CSCs were assessed by the colony formation assay and 5-FU chemotherapy sensitivity assay.2)CCK-8 assay was used to determine the appropriate dose and time of TNF-?on SW620^Lgr5+CSCs.The experiment was divided into blank Control group?Control?,TNF-?group,MK2206 group,and TNF-?group+MK2206 group.The colony formation assay and FACS to detect the proliferation and apoptosis of SW620^Lgr5+colon cancer CSCs.Western blot was used to detect the expressions of PI3K/AKT pathway related proteins AKT,p-AKT,GSK-3?,p-GSK-3?and apoptotic proteins Bax and Bcl-2 to study the effect of TNF-?on the proliferation and apoptosis of SW620^Lgr5+CSCs.Results 1)The present results showed that the protein expression level of Lgr5 in SW620 was higher than that of other cell lines by western blot.FACS results showed that the proportion of Lgr5⁺cells in SW620 was 6.9%,and the proportion of Lgr5⁺cells in SW620 spherical cells obtained from serum-free suspension culture increased significantly to 34.5%.SW620^Lgr5+CSCs were obtained by FACS,in which the ability of colony formation and 5-FU resistance were significantly higher than that of human colorectal cancer cells SW620.2)Treatment with 1 ng/mL TNF-?for 24 h resulted in the maximum increase of the viability of SW620^Lgr5+CSCs,then all further experiments were conducted for this duration.TNF-?significantly enhanced the colony formation capacity of SW620^Lgr5+CSCs,but had no significant impact on the cell apoptosis.MK2206 significantly inhibited the cell proliferation and promoted the cell apoptosis.TNF-?activated the PI3K/AKT pathway,and improved the phosphorylation level of AKT and GSK-3?.MK2206 counteracted TNF-?-induced activation of PI3K/AKT pathway in SW620^Lgr5+CSCs,reduced the expression of Bcl-2,and increased the expression of Bax.Conclusion 1.SW620^Lgr5+CSCs with Lgr5 as a CSCs marker has higher proliferation and drug resistance,suggesting that Lgr5 can be used as a therapeutic target for CRC tumor stem cell markers and has potential clinical application value.2.Inflammatory factors promote the proliferation of CSCs by activating the PI3K/AKT pathway;Inhibition of this pathway can inhibit the proliferation of CSCs and promote their apoptosis.It is suggested that the inflammatory environment plays an important role in the existence of CSCs,which provides a certain theoretical basis for the clinical treatment of CRC.