The Anti-tumor Efficiency Of Histidine Triad Nucleotide-binding Protein 2 In Pancreatic Cancer And Its Molecular Mechanism

Pancreatic cancer is one of the most common malignant tumors in the world.According to our "Cancer statistics in China",pancreatic cancer ranks the top 10 in the mortality rate of malignant tumors in China,while in the megalopolises even higher.The 5-year survival rate of pancreatic cancer patients is less than 6%,Radical resection is the only possible way to cure this disease.But only a few patients were diagnosed with surgical resection chance.Besides,pancreatic cancer is always aggressive and of metastastic capability.Micrometastases are often present even though nothing in imaging tests suggested.All these lead to difficult to achieve R0 resection and postoperative recurrence rate is over 85%.Pancreatic cancer is susceptible to drug resistance in chemotherapy such as gemcitabine.As in biological medicine,although Ras and other impressive genes have been found which can promote the development of pancreatic cancer occurrence,but the molecular targeted drugs are difficult to obtain or in poor effect.There was no significant improvement in postoperative median survival of pancreatic cancer patients among these 30 years.In conclusion,the key to treatment of pancreatic cancer is to look for novel molecular to improve the chemotherapeutic drug resistance and find out more targets for biological treatment of pancreatic cancer.Histidine triad nucleotide-binding protein 2（HINT2）is of nucleotide transferase and hydrolase activity and belongs to the histidine triad（HIT）superfamily,which widely involved in cell proliferation,apoptosis pathways and DNA,RNA and carbohydrate metabolism.HINT2 was reported located in mitochondria and mainly found in liver,pancreas and adrenal gland of mammalian animals.Further research suggested it sensitized HepG2 cells to mitochondrial apoptosis.So we make the hypotese that it might play an important role in pancreatic cancer too.PartⅠ Histidine triad nucleotide-binding protein 2（HINT2）effects as tumor suppressor in pancreatic cancerAims:Through the experiments to determine the anti-tumor effect of HINT2 in pancreatic cancer and its possible mechanism.Methods:The expression of HINT2 in 180 pancreatic cancer and adjacent tissue samples was determined by immunohistochemical staining.The results were statistically analyzed with clinical data to ensure the relationship between the expression of HINT2 and the development of pancreatic cancer as well as survival prognosis.In BxPC-3 L3.6pl cell lines used adenovirus to increase HINT2 expression,and then the change of cell function,as cell cycle,apoptosis,cell growth,migration and invasion,was determined via flow cytometry,CCK8 experiment,wound healing experiment and transwell experiment.Western blot was used to ensure the change of protein expression level.HINT2 overexpressed cell lines,which was built via lentivirus transfection,was applied for clony formation and subcutaneous xenograft model to determine whether HINT2 influences the ability of cell clony formation and tumor formation in vivo or not.Results:The results of immunohistochemistry staining showed that the expression level of HINT2 in pancreatic cancer tissue and adjacent tissue samples was significantly different（P<0.001）.HINT2 was relative low-expressed in pancreatic cancer tissue compared to adjacent tissue.After survival curve analysis,it is suggested that survival time of patients in HINT2 high-expressed group was statistically more than in HINT2 low-expressed group（P<0.05）.Through the analysis of clinical data,it was found that the expression of HINT2 related to pathological grade and lymphnode metastasis（P<0.05）.Conclusion:HINT2 would cause the calcium excessive uptaken in mitochondria through affecting the gate-keeper molecules of the mitochondrial calcium uniporter complex,followed by reduction of mitochondrial membrane potential,increase of reactive oxygen species（ROS）and lead to cell death via mitochondrial apoptosis pathway finally.Part Ⅱ HINT2 induces apoptosis through interaction with mitochondrial calcium uniporter complex（MCU）Aims:Our research outcomes suggested that histidine triad nucleotide-binding protein 2（HINT2）cause calcium ion(Ca²⁺)overloaded in mitochondria through interaction with mitochondrial calcium uniporter complex（MCU）and lead to cell apoptosis.Methods:BxPC-3 cell line was transfected with adenovirus to overexpress HINT2.HINT2 overexpressed cells were treated with Ruthenium Red（RR）for 24-48h to inhibit mitochondrial calcium uniporter complex（MCU）.And then Annexin V/PI kit,ROS kit and mitochondrial membrane potential（△Ψm）reagent JC-1 was used to determine the difference between control group,HINT2 overexpression group and RR group.Flow cytometry（FCM）was used to determine the fluorescence.Mitochondrial Ca²⁺ was specificly dyed by Rhod-2 AM and then detected by FCM.The expression level of components of mitochondrial calcium uniporter complex（MCU）was determined by western blot.Results:The apoptotic cells of HINT2 overexpression group was obvious more than control group.So was the increase of ROS and decrease of △Ψm with statistical difference（P<0.05）.However,the apoptotic cells reduced after RR treatment,which was the inhibitor of MCU,and the increase of ROS and decrease of △Ψm was blunted.The results of western blot showed that overexpression of HINT2 would cause relative low expression of "gatekeeper" of MCU,i.e.MICU1 and MICU2,while EMRE was relative high expressed.Stained by Rhod-2 AM,it is found that mitochondrial Ca²⁺ was obviously increase,especially after the simultaneous treatment of gemcitabine,but all these can be inhibited by RR.Conclusion:The results proved that HINT2 can cause excessive uptake of Ca²⁺ in mitochondria and thus lead to MMP reduce,ROS increase and finally pancreatic cancer cells apoptosis through influence of the MCU "gatekeeper".Part Ⅲ HINT2 has synergistic effect with gemcitabine on apoptosis in pancreatic cancer cellsAims:To determine the synergistic effect of HINT2 and gemcitabine on apoptosis in pancreatic cancer cells.Methods:BxPC-3 cells were seeded and separated in 4 groups,2 groups were transfected with control adenovirus while the other 2 with HINT2-adenovirus,then treated with PBS or 1μM gemcitabine for 24-28h.Annexin V/PI kit was used to determine the cell apoptosis rate through the detection of fluorescence by FCM.BxPC-3 cells was seeded in 96-well plates and treated with control adenovirus and HINT2-adenovirus,respectively.Then they were treated with gemcitabine in different concentration for 24h in order to make CCK8 test.Through statistical analysis we got dose-effect curve and IC50.Western blot was used to ensure the change of protein’s expression after treatment of gemcitabine,especially HINT2 and other apoptosis related proteins.Results:The co-treatment of HINT2 and gemcitabine for pancreatic cancer cells increases apoptosis rate dramatically and better than single factor effect.It is suggested that these two factors have synergistic effect on apoptosis of pancreatic cancer cells.IC50 of gemcitabine in HINT2 group is obvious lower than control group.It suggested that HINT2 can sensitize the pancrestic cells to gemcitabine.Western blot showed that HINT2 expression was increased after transfection with HINT2 adenovirus and even more when co-treated with gemcitabine,so it was apoptosis related protein.Conclusion:Our results demonstrated that HINT2 has synergistic effect with gemcitabine on apoptosis in pancreatic cancer cells.Furthermore,gemcitabine can promote the expression of HINT2,and HINT2 can also sensitize pancreatic cancer cells to gemcitabine.