The Role Of P62 On β-escin-induced Apoptosis Incolorectal Cancer Cell And Its Potential Molecular Mechanisms

Aim: To investigate the effect of β-escin on p62 and autophagy expression and their roles in chemo-sensitivity of β-escin in colorectal cancer cells.To explore the potential molecular mechanisms of the effect of p62 expression in colorectal cancer cell chemo-sensitivity to β-escin.Methods: The proliferation effect of β-escin on colorectal cancer cells was measured by CCK8 assay and colony formation assay.The expression of p62 after β-escin treatment was observed with Western blot.The m RNA level of p62 was detected by PCR.The expression of β-escin-induced p62 after NAC addition was detected with Western blot.Western blot and FCM were used to observe cell apoptosis after β-escin treatment.The degree of DNA damage was detected with Western blot and Immunofluorescence.Transient transfection of plasmid FLAG-p62 and p62 si RNA were used to increase and decrease p62 expression,respectively,and then CCK8 assay was used to detect cell proliferation after β-escin treatment;FCM was used to analyze the rate of cell apoptosis after β-escin treatment;Western blot was used to observe apoptosis associated protein and DNA damage protein after β-escin treatment.Subcutaneous tumorigenesis assay in nude mice was used to assess the function of p62 in the β-escin antitumor effect in vivo.Western blot and FCM were used to analyze p62 knockdown with β-escin and ATM inhibitor treatment.The effect of β-escin on cell autophagy evaluated through Western blot.CCK8 assay and Western blot were used to measure cell proliferation,DNA damage and apoptosis after knockdown of Atg5 with β-escin treatment.Results:(1)β-escin inhibited cell viability and colony formation in a concentrationand time-dependent manner by CCK8 assay and colony formation assay in HCT116 and HCT8 cells.(2)β-escin induced intracellular ROS generation.β-escin induced DNA damage in a concentration-and time-dependent manner as shown by Western blot and Immunofluorescence.Meanwhile,β-escin increased the expression of p62 in tha same way,which had a protective effect against β-escin-induced DNA damage.β-escin-induced p62 upregulation occurred at the transcriptional level and was eliminated by NAC.(3)Knockdown of p62 using two si RNAs apparently enhanced β-escin-induced DNA damage,whereas overexpression of p62 reduced β-escin-induced DNA damage.(4)In addition,β-escin induced concentration-and time-dependent apoptosis with FCM and Western blot.Similarly,knockdown of p62 significantly increased β-escin-induced cell proliferation inhibition and apoptosis in vitro.Overexpression of p62 decreased the rate of cell proliferation inhibition and apoptosis.(5)The in vivo studies showed that knockdown of p62 slightly reduced tumor growth and increased chemo-sensitivity of β-escin.(6)Mechanism studies showed that the functions of p62 in β-escin-induced DNA damage were associated with β-escin-induced apoptosis,and p62 knockdown combined with the ATM inhibitor KU55933 augmented β-escin-induced DNA damage and further deteriorated β-escin-induced apoptosis.(7)Furthermore,β-escin activated autophagy and inhibition of autophagy by knockdown of Atg5 exaggerated β-escin-induced cell proliferation inhibition,DNA damage and apoptosis.Conclusion:(1)β-escin induced up-regulation of p62,autophagy and DNA damage in a concentration-and time-dependent manner in colorectal cancer cells.(2)p62 and autophagy decreased the chemo-sensitivity of β-escin treatment in colorectal cancer cells.The molecular mechanisms of cell apoptosis and DDR were involved.(3)p62 regulating ATM/γH2AX pathway and β-escin-mediated autophagy may be the therapeutic targets for the antitumor effect of β-escin which provides theoretical basis for anti-tumor therapy of β-escin in colorectal cancer.