Molecular Mechanism Of Lidamycin Hypersensitivity In Human Colon Carcinoma HCT116 Cells By Targeting Silence Of Chk1

Cell cycle checkpoints are essencial to maintain the integrity of the genome and ensure the precise progression of cell cycle.Disruptions in cell cycle checkpoints(e.g., p53 mutation) are closely associated with tumorigenesis and proposed to make tumor cells vulnerable to antitumor agents that cause DNA damage.Recent advances in cell cycle regulation have led to a suggestion of therapeutically targeting checkpoint pathways in tumor cells to increase the toxicity of DNA-damaging agents.Lidamycin (LDM,originally named C-1027),a member of the enediyne antibiotic family,is a potent DNA damaging agent to induce higher ratio of DNA double strand breaks than that of single DNA strand breaks,currently being evaluated in phaseⅡclinical trials. LDM has been reported to induce cell cycle arrests and apoptosis in p53-dependent manner in various tumor cells.In this study,we investigated whether knockdown of checkpoint kinases Chkl and/or Chk2 by RNA interfering affected LDM-induced cell cycle arrest and apoptosis in human colon carcinoma HCT116 cell line and its isogenic subline in which p53 has been inactivated by targeted homologous recombination.1.Effects of Chk1 and/or Chk2 knockdown by shRNAs on growth of HCT116 cells with different p53 status.(1) Selection of siRNA secquences targeting Chk1 and Chk2.RNA interfering is a method that inhibits the expression of target gene in mRNA by exogenously importing double strand RNAs.Based on the principles of siRNA designment,we designed and synthesized three siRNA sequences targeting Chk1 and three sequences targeting Chk2. HCT116 cells were transfected with siRNAs,mRNAs and proteins of Chk1 and Chk2 were detected by RT-PCR and western blot,respectively.The siRNA sequences with more effective knockdown were choosen for the constructions of shRNA plasmids.(2) Construtions of shRNA plasmid that knock down Chk1 and/ or Chk2.The pCD-shRNA vector was used to form pCD-shChk1(Chk1 shRNA),pCD-shChk2 (Chk2 shRNA),pCD-mock(mock shRNA) and pCD-shChk1/2(Chk1/2 shRNA) vector. RT-PCR and western blot assays showed that Chkl shRNA and Chk2 shRNA selectively down-regulated the expressions of Chk1 and Chk2 respectively,and Chk1/2 shRNA knocked down both of Chk1 and Chk2.(3) Cell growth curve assay showed the effects of the shRNAs on the proliferation of HCT116 cells with different p53 status.As shRNA control,mock shRNA affected neither the expression of these kinases nor the proliferation of cells.Chk1 shRNA and Chk1/2 shRNA inhibited cell proliferation both in HCT116 cells and HCT116 p53-/-cells compared with control mock.However,Chk2 shRNA hardly inhibited the cell growth in p53^wt or p53^-/- HCT116 cells.These results supported that Chk1 plays an important roles in cell growth.2.Knockdown of Chk1 remarkably enhanced LDM-induced growth inhibition of p53^-/- cells.(1) SRB assay showed that LDM markedly inhibited cell growth in dose dependent manner and the IC50 values of LDM were 0.7 nM for HCT116 cells and 2.8 nM for HCT 116 p53^-/- cells,respectively.LDM inhibited cell proliferation to a greater extent in p53^wt cells than in p53^-/- cells,consistent with previous study.(2) LDM-induced growth inhibition was markedly enhanced in Chkl shRNA- and Chk1/2 shRNA-transfected HCT116 p53^wt or p53^-/- cells.The potentiation of LDM cytotoxicity by Chkl shRNA was more remarkable in p53^-/- cells than in p53^wt cells. The percent of inhibition by 0.5 nM LDM was 69%in p53^wt cells and 77%in p53^-/- cells,increased 37%and 74%compared with mock cells,suggesting that Chk1 played a essential role in HCT116 p53^-/- cell proliferation.On the contrary,Chk2 knockdown had less effect on LDM cytotoxicity.Dual knockdown of Chkl and Chk2 achieved similar potentiation of LDM cytotoxicity as knockdown of Chkl alone.The percent of inhibition by 0.5 nM LDM was 75%in p53^wt cells and 79%in p53^-/- cells,increased 42%and 74%compared with mock cells.These results indicated that knockdown of Chkl is necessary for sensitization of HCT 116 cells to LDM.3.The effects of knockdowns of Chk1/Chk2 on LDM- induced cell cycle arrests in HCT116 cells with different p53 status.(1) LDM-induced cell cycle arrests in HCT116 cells were influenced by p53.FACS analysis showed that LDM induced G1 and G2/M arrests in HCT116 p53^wt cells,and G2/M arrest in p53^-/- cells.(2) Knockdowns of Chk1 and/or Chk2 did not alter cell cycle distributions of HCT116 cells and HCT116 p53^-/- cells in the absence of DNA damage,consistent with results from other cell lines.In p53^wt cells,knockdown of Chk1 moderately abrogated the LDM-induced G2/M arrest by decrease of G2/M population from 43%to 30%, resulting in a limited increase of sub-G1 DNA content from 17%to 25%.In p53^-/- cells, Chkl knockdown markedly decreased G2/M population from 63%to 39%and dramatically increased sub-G1 content from 6%to 27%.Chk2 knockdown did not affect the G2/M arrest or sub-G1 population in both cell lines treated with LDM. Similar to Chk1 knockdown,dual knockdown of Chk1 and Chk2 decreased the G2/M-phase arrest and increased sub-G1 population.These data suggested that Chk1 but not Chk2 is critical for G2/M checkpoint induced by LDM.(3) LDM induced a moderate increase of phosphorylation of histone H2AX (γ-H2AX),a marker for DNA damage,in control mock cells,which was enhanced by knockdown of Chk1,suggesting the increase of DNA damage by knockdown of Chk1. Phosphorylation of histone H3(Ser 10),a marker for mitotic phase,was down-regulated by LDM in mock cells,indicating a decrease of mitotic cells resulted from LDM-induced G2 arrest.However,knockdown of Chkl rescued the phosphorylation of H3,indicating the successful abrogation of G2 checkpoint and renewal of mitotic progression.In contrast,knockdown of Chk2 failed to rescue phosphorylation of H3 and enhance phosphorylation of H2AX.Dual knockdown of Chk1 and Chk2 showed similar efficacy as knockdown of Chk1 alone.The data suggested that knockdown of Chk1 abrogated LDM-induced G2 checkpoint and forced the cells into premature cell cycle progression to exacerbate the DNA damage.(4) Western blot analysis showed that LDM induced phosphorylations of Chk1 (Ser317),Chk2(Thr68),Cdc25C(Set216) and Cdc2(Tyr15) in HCT116 p53 wt and p53-/- cells.The LDM-induced phosphorylations of Cdc25C and Cdc2 were reduced by knockdown of Chk1 in the two cell lines,suggesting that LDM-induced G2 arrest was associated with Chk1-Cdc25c-Cdc2 pathway.Moreover,LDM induced Chk1- and Chk2- independent increases of p53 and p21,and decrease of Cdk2 in p53 wt cells but not in p53-/- cells,verifying that LDM-induced G1 arrest is dependent on p53- p21-Cdk2 pathway in HCT116 cells.4.Enhancement of LDM-indueed apoptosis by Chk1 knockdown(1) To confirm the augmentation of LDM-induced apoptosis by knockdown of Chkl,we performed annexin V/PI staining assay.Consistent with above results,LDM induced more apoptosis of p53^wt cells than that of p53^-/- cells.Knockdown of Chk1 enhanced LDM-induced apoptosis more remarkable in p53^-/- cells(increasing from 4% to 18%) than in p53^wt cells(increasing from 11%to 18%),while Chk2 shRNA failed to potentiate LDM-induced apoptosis in p53 wt and p53^-/- cells.Dual knockdown of Chk1 and Chk2 increased more LDM-induced apoptosis in p53^-/- cells(from 4%to 20%) than that in p53^wt cells(from 11%to 19%).The enhancement of LDM-induced apoptosis by dual knockdown of Chk1 and Chk2 was similar to knockdown of Chk1 alone.(2) Western blot showed that LDM activated caspase-3 and sequentially cleaved downstream target PARP in p53- dependent manner.However,LDM limitedly induced cleavages of caspase-3 and PARP in p53^-/- cells compared with p53^wt cells,suggesting LDM-induced apoptosis in p53- dependent manner.The cleaved caspase-3 and PARP in LDM-treated p53^wt cells were moderately enhanced by knockdown of Chk1, consistent with the potentiation of LDM-induced apoptosis by knockdown of Chk1. Intriguingly,knockdown of Chk1 markedly increased cleavages of caspase-3 and PARP as well as caspase-2 in LDM-treated p53^-/- cells,while caspase-2 was not activated by LDM in either p53^wt cells or p53^-/- cells,suggesting the involvement of caspase-2 in the apoptotic response of LDM-treated p53^-/- cells to knockdown of Chk1.On the contrary, knockdown of Chk1 failed to cleave caspase-2 in LDM-treated p53 wt cells.In the same conditions,knockdown of Chk2 did not affect the cleavages of the caspases in both HCT116 cell lines treated with LDM.Taken together,knockdown of Chkl triggered a p53- independent apoptotic pathway that involved caspase- 2 and caspase- 3 in LDM-treated p53-/- cells.5.ConclusionsTo our knowledge,it is the first time to report the contribution of Chk1 to the sensitivity of tumor cells to enediyne antibiotic LDM.Knockdown of Chkl,but not Chk2,is able to improve the sensitivity of HCT116 cells to LDM by abrogating G2/M arrest,inducing premature cell cycle progression entry and increasing apoptosis.The effects of Chk1 knockdown on LDM are more remarkable in p53^-/- cells than those in p53^wt cells.Moreover,Chk1 knockdown can enhance the activity of LDM in inducing apoptosis in the absence of p53 through an apoptotic mechanism that is associated with caspase-2 and cspase-3.Finally,our data demonstrate that checkpoint kinase Chk1 may be one of useful targets in combination with LDM for colon carcinoma treatment.