The Molecular Basis And Clinical Significance Of CK2 Inhibitor CX-4945 Reversing Radiation Resistance In Cisplatin Resistant Gastric Cancer Cells

Part1 The molecular mechanism of CK2 inhibitor CX-4945 reversing radiation resistance in cisplatin resistance of gastric cancer cellsGastric cancer is the third leading cause of cancer-related deaths worldwide.With an overall five-year survival rate of only 20%,it becomes a major cause of both morbidity and mortality.Current strategies for treatment of gastric cancer include surgery,chemotherapy and radiotherapy.However,the cross-resistance to chemotherapy and radiotherapy lead to the failure of treatment.Cisplatin is a widely used chemotherapeutic drug for treating gastric cancer.Cisplatin kills cancer cells by inducing DNA double strand breaks(DSB)through crosslinking of the DNA.However,cancer cells often develop enhanced DNA repair capacity to overcome cisplatin induced DNA damage,leading to drug resistance.Radiotherapy is employed to kill cancer cells also by inducing DNA double strand breaks;therefore,the research for the molecular basis of the cross-resistance to chemotherapy and radiotherapy may provide new strategy to reverse the cross-resistance and improve the effect of treatment of gastric cancer.CK2(casein kinase 2)plays a central role in the regulation of gene expression and protein synthesis/degradation by acting at different levels on the complex apoptotic machinery.CX-4945,a first-in-class clinical stage inhibitor of CK2,was found to block the cisplatin induced DNA repair response by decreasing the phosphorylation of XRCC1 at CK2-specific phosphorylation sites.However,whether CX-4945 sensitizes cisplatin resistant cells to radiotherapy was still unknown.Recently,we reported that two cisplatin resistant cell lines BGC823/DDP and SGC7901/DDP was established by chronic low dose cisplatin exposure of primary sensitive gastric cancer cell lines BGC823 and SGC7901.We found that up-regulated levels of CK2 and P-XRCC1(518S/519T/523T)in cisplatin resistant cells,and CX-4945 selectivity killed cisplatin resistant cells by inhibiting CK2 activated p-XRCC1(518S/519T/523T)phosphorylation.XRCC1 is an important molecule to involve in repairing the DNA damage induced by radiation.Thus,cisplatin resistance of gastric cancer cells may have cross resistance to radiation therapy.CX-4945 may reverse radiation resistant in cisplatin resistant cells by inhibiting CK2 activated p-XRCC1 phosphorylation.Objective:The objectives of the present study were to investigate the radiation resistance and reversing radiation resistance in cisplatin resistance of gastric cancer cells and to elucidate the underlying mechanisms of action.Methods:DNA damage repair ability and XRCC1 expression between the established resistant cells and their parent sensitive cells were detected by relative assays.CK2,XRCCI and other molecules expression relationship were used to explore the molecule mechanism of radiation resistance in cisplatin resistance of gastric cancer cells.Results:1.XRCC1 is required for cross-resistance to cisplatin and radiotherapy in human gastric cancer cells.DNA damage of cisplatin resistant cells were significantly lower than the sensitive cells in same dose treatment of high-energy X-ray treatment.Meanwhile,P-XRCC1 protein levels were up-regulated in cisplatin resistant gastric cancer cell lines.Knockdown of XRCC1 expression by transfection of XRCC1 siRNA resulted in decreased P-XRCC1 protein levels,but increased yH2AX protein levels in cisplatin resistant gastric cancer cell lines treated with X-ray.In contrast,overexpression of XRCC1 by transfection of GFP-XRCC1 plasmid resulted in increased P-XRCC1 protein levels,but decreased yH2AX protein levels in SGC7901 cells treated with X-ray.These results suggested that XRCC1 is required for repair of X-ray radiation induced DSBs and plays a critical role in human gastric cancer cells cross-resistance to cisplatin and radiotherapy.2.CX-4945 enhanced DNA damage induced by X-ray radiation via inhibits phosphorylation of XRCC1.Cisplatin resistant cells SGC7901/DDP and BGC823/DDP were more sensitive to CX-4549 than their parental cisplatin sensitive cells SGC7901 and BGC823.CX-4945 via inhibits phosphorylation of XRCC1 enhanced DNA damage induced by X-ray radiation.3.CX-4945 sensitized cisplatin resistant cells to radiation induced cytotoxicity.The colony formation assays showed that the surviving fractions of cisplatin resistant cells treated with different doses of X-Ray radiation were much higher than their parental cisplatin sensitive cells.In line with CX-4945 enhanced cisplatin induced DNA damage,the colony numbers were significantly decreased in scisplatin resistant cells by treatment with a combination of CX-4945 and different doses of X-Ray radiation.These results indicated that CX-4945 sensitized cisplatin resistant cells to X-ray radiation induced cytotoxicity.UV-B radiation was used to treat cells to induce DSBs.CCK-8 results showed that surival rates of cisplatin resistant cells after treated with UV-B were higher than that of sensitive cells.However,the UV-B induced cytotoxicity was increased in cisplatin resistant cells treated with a combination of CX-4945 and UV-B.In line with these,the UV-B induced yH2AX protein levels and yH2AX positive cells were increased in cisplatin resistant cells treated with a combination of CX-4945 and UV-B.These results indicated that CX-4945 sensitized X-Ray and UV treatment.Conclusion:Taken together,CX-4945 enhanced DNA damage induced by X-ray and UV-B radiation via inhibits phosphorylation of XRCC1.The colony formation and cytotoxic assay indicated that CX-4945 sensitized cisplatin resistant cells to X-ray and UV-B radiation induced cytotoxicity.We report for the first time that CX-4945 is a valuable targeted reagent for sensitizing radiotherapy in gastric cancer.These findings suggest that CX-4945 in gastric cancer may have clinical value.Further evaluation of these biomarkers in prospective clinical studies is warranted.Part 2 Relationship between CK2? expression and radiotherapy curative effect in gastric cardia cancer tissueObjective:The objectives of the present study were to investigate the expression of CK2 and XRCC1 in gastric cardia cancer tissue by using immunohistochemical method,and to discuss the relevance of CK2 and XRCC1 and radiotherapy curative effect and clinical pathological factors and the prognosis of gastric cardia cancer.Methods:1.A total of forty-six cases of gastric cardia cancer without surgery treatment and radiation relative contraindications,and with age 50 or more,KPS score 70 points or more,were gathered from January 2010 to December 2013,who were treated only with radiotherapy and had complete information.Thirty-six male and 10 female patients were diagnosed through fiberoptic gastroscope with biopsy.The average age was 71.8 ± 10.23 years,and with a median age of 71.2 years.On the basis of the clinical pathological stage,stage I:0 cases,stage II:22 cases,stage III:18 cases,stage IV:6 cases(4 cases with hepatic metastasis;2 cases left supraclavicular lymph node metastasis)2.Immunohistochemistry was used to detect the expression of CK2 and XRCC1 of gastric cardia cancer tissues.The correlation between CK2 and XRCC1 expression was analyzed with clinical factors and prognosis.3.To analyze the data by PASW Statistics 18.According different statistical data though X2 test,Kaplan-Meier survival analysis,P<0.05 was considered statistical difference.Results:1.Relationship between CK2 expression and clinical-pathological parameters in gastric cardia cancer tissue.The CK2 expression was shown high expression in cell cytoplasm in tissue of gastric cardia cancer.The positive expression rate was 45.7%(21/46).According to the results of X2 test,the positive expression of CK2 was related with the family history,lymph node metastasis and clinical staging(P<0.05).Positive expression rate of CK2 in the patients with family history of gastric cancer was 75.0%(9/12);56.3%(18/32)in lymph node metastasis group and 21.4%(3/14)in group without lymph node metastasis.And 27.3%(6/22)and 66.7%(12/18)and 50.0%(3/6)in II,III and IV stage group,respectively.However,there was no significance with gender,age,KPS score,chronic stomach disease,main symptoms(epigastric pain,dysphagia,emaciation),HGB,tumor differentiation and distant metastasis.2.Relationship between XRCC1 expression and clinical-pathological parameters in gastric cardia cancer tissue.The positive expression rate was 65.2%(30/46).According to the results of X2 test,the positive expression of XRCC1 was related with the lymph node metastasis and clinical staging,with statistical significance(P<0.05).Positive expression rate of XRCC1 was 78.1%(25/32)in lymph node metastasis group and 35.7%(5/14)in group without lymph node metastasis.And 45.5%(10/22)and 88.9%(16/18)and 66.7%(4/6)in II,III and IV stage group,respectively.However,there was no significance with gender,age,KPS score,chronic stomach disease,family history,main symptoms(epigastric pain,dysphagia,emaciation),HGB,tumor differentiation and distant metastasis.The co-expression of CK2 and XRCC1 was 18 cases in gastric cardia cancer.There was a close relationship between the expression of CK2 and XRCC1 in the gastric cardia cancer tissues(x2=4.267,P<0.05).3.The relationship between the expression of CK2 and XRCC1 and radiotherapy effect and prognosis of gastric cancer.The high expression of CK2 was 21/46 cases of gastric cardia cancer patients,and 5/46 cases of CR after radiotherapy,2/46 cases with PR.The total effective rate(CR+PR)was 33.3%(7/21).The low expression of CK2 was 25/46 cases,and 12/46 cases of CR after radiotherapy,8/46 cases with PR,respectively.The total effective rate(CR+PR)was 80.0%(20/25).The CK2 expression was related with radiotherapy effect,and lower expression,higher radiation efficiency.(x2= 0.252,P<0.05).The high expression of XRCC1 was 30/46 cases of gastric cardia cancer patients,and 9/46 cases of CR after radiotherapy,and 4 cases with PR.The total effective rate(CR+PR)was 43.3%(13/30).The low expression of XRCC1 was 16 cases,8 cases of CR after radiotherapy,and 6 cases with PR.The total effective rate(CR+PR)was 87.5%(14/16).The 1,2 year survival rates of CK2 high expression were 33.3%(7/21)and 14.3%(3/21),but low expression were 68.0%(17/25)and 48.0%(12/25).Similarly,the 1,2 year survival rates of XRCC1 high expression were 40.0%(12/30)and 23.3%(7/30),but low expression were 75.0%(12/16)and 50.0%(8/16).Kaplan-Meier survival curve analysis indicated that the expression of CK2 and XRCC1 was negatively related with survival rate(P<0.05).Conclusion:1.The expression of CK2 and XRCC1 in gastric cardia cancer tissue were high,and significantly associated with the lymph node metastasis and clinical staging.The later clinical stage of cases with the higher positive expression of these markers in cancer tissues.The expression of CK2 and XRCC1 associated with family history of gastric cancer.However,the expression of CK2 and XRCC1 was unrelated with gender,age,KPS score,chronic stomach disease,main symptoms(epigastric pain,dysphagia,emaciation),HGB,tumor differentiation and distant metastasis.2.The radiotherapy curative effect in the cases with high expression of CK2 and XRCC1 were poorer than low expression ones.The 1,2 year survival rates of cases with high expression of CK2 and XRCC1 were significantly lower than low expression ones.