Synergistic Anti-cancer Interactions Between LY2603618,a CHK1 Selective Inhibitor,and Gemcitabine In Human Pancreatic Cancer Cells

Pancreatic cancer is a malignant tumor of digestive tract with the characteristics of lacking of early detection methods,so pancreatic cancer is often detected at an advanced stage and the prognosis is poor.The incidence is steadily increasing year by year in China.Surgery is the first chance,but less than 20% of patients are suitable for surgery,thus chemotherapy is the main treatment for patient now.Gemcitabine(2',2'-difluorodeoxycytidine,d Fd C),a nucleoside analogue,is the standard first-line drug for treating patients with advanced pancreatic cancer though its efficacy is very limited.Therefore,new therapeutic approaches are urgently needed.Checkpoint kinase 1(CHK1)is one of the most important protein of DNA damage response(DDR)network.It involves to regulate and control all the cell cycle checkpoints,DNA repair,the progression of the cell cycle and the cell apoptosis.When there is an endogenous or exogenous stimulate in the cells,DNA damage occurs,which activates CHK1.CHK1 phosphorylates CDC25 A and CDC25 C phosphatases,inhibiting activation of CDK1/CDK2 and arresting the cell cycle progression allowing for DNA repair.More and more studies show that drugs targeting the DNA damage response route can enhance the sensitivity of the DNA damage drugs.LY2603618 is a novel small molecule compound designed to inhibit the activity of CHK1.We investigated the anti-tumor activity of LY2603618 alone and in combination with gemcitabine in pancreatic cancer cells.First,we studied the growth inhibitory effects of LY2603618 in pancreatic cancer cells.The results showed that LY2603618 can markedly inhibit the proliferation of five pancreatic cancer cell lines with cell cycle arrest at S and G2/M phase,but cause minor pancreatic cancer cell death.Then we found that LY2603618 inhibited the activity of the CHK1-CDC25-CDK network,and induced DNA damage.The results showed that LY2603618 had two functions at cell cycle checkpoints: inhibiting the function of DNA damage repairing through inhibiting CHK1 and inducing DNA damage to activate cell cycle checkpoints.The arrest of the cell cycle at S and G2/M phases showed that the function of LY2603618 is to activate cell cycle checkpoints by inducing DNA damage.At the same time,we found that the anti-tumor activity of LY2603618 was related to the activity of CDK.All the results provided the evidences that LY2603618 in combination with DNA damage-inducing drugs may cooperatively impede the growth of pancreatic cancer cells.Then we explored the mechanisms of synergistic anti-cancer interactions between LY2603618 and gemcitabine in pancreatic cancer lines.As expected,combination of LY2603618 with gemcitabine synergistically induced pancreatic cancer cells death and weakened the arrest of cell cycle at S and G2/M phases induced by gemcitabine.At the same time,we found that LY2603618 decreased gemciatbine-induced activation of cell cycle checkpoints and RNR expression and increased gemciatbine-induced DNA damage.These results indicate that LY2603618 enhances the anti-tumor activity of gemciatbine by inhibiting DNA damage repair,promoting cell cycle progression,and reducing cellular d NTP pools.Then we found the anti-tumor activity of combination of LY2603618 with gemcitabine was also related to the activity of CDK.In summary,our study provides evidence that gemcitabine synergizes with LY2603618 to inhibit the growth of pancreatic cancer cells,and shows that targeting CHK1 can enhance the anti-tumor activity of DNA damage-inducing drugs in pancreatic cancer.Our results support the clinical development of LY2603618 for pancreatic cancer alone or combination.