The Influences On Claudin-18.2 Protein Expression Of Human Pancreatic Cancer PANC-1 Cells Mediated By Hyperthermia Therapy

Backgroud: Pancreatic cancer is one of the worst malignant alimentary canal cancers,which incidence rate is rising higher nowadays.Pancreatic cancer has the characteristics of late detection,poor prognosis,short survival time,and low 5-years survival rate.Nowadays,the research about targeted therapy on pancreatic cancer is proceeding,and surgery,radiotherapy and chemotherapy are still the main therapy strategies for pancreatic cancer.Tumor hyperthermia therapy,which is a kind of technology makes the temperature rise of tumor lesion by heat to a certain extent,so as to kill tumor cells of a treatment.Recently,with the development of hyperthermia therapy equipment and technique,hyperthermia therapy is extensively used in the cancer treatment and identified effective.Claudin protein is a main component of tight junction structure.Claudin-18.2,a protein of claudin family,only expressed in a low lever in differentiated gastric parietal cells under normal physical condition,however,the obvious up-regulation is seen in many kinds of cancer diseases,including 80% gastrointestinal adenomas and 60% pancreatic tumor.Its function may be associated with cell adhesion.Phase II clinical trial result showed that IMAB362,an antibody targeted on claudin-18.2protein,combined with chemotherapy could obviously improve gastric cancer patients’ progression-free survival and overall survival,compared to chemotherapy.This clinical trial result provided a new hope for developing the treatment of cancer patients.Therefore,claudin-18.2 protein became a hot topic in current cancer treatment.Objective: This research aimed at observing claudin-18.2 protein expression on PANC-1 cells and the effects of hyperthermia therapy and chemotherapy for claudin-18.2 protein expression of PANC-1 cells by immunocytochemistry and western blotting,to discover the therapy influence of hyperthermia therapy on pancreatic cancer and the function of hyperthermia therapy combined with chemotherapy therapy on human pancreatic cancer PANC-1 cells,providing more theoretical support for the clinical use of hyperthermia therapy and chemotherapy,and the development of antibody IMAB362.Methods: This research used the human pancreatic cancer PANC-1 cells for following experiment:(1)Detection for inhibition rate of cells: the cells was divided into control group,hyperthermia therapy group,gemcitabine group,5-fluorouracil group,combination chemotherapy group and hyperthermia therapy combined with combination chemotherapy group.Comparing the influences on inhibition rate of human pancreatic cancer PANC-1 cells,and these 6 groups would be also used in the other experiments.(2)Hematoxylin-eosin(HE)staining: observed the morphology of PANC-1 cells and the influence of hyperthermia therapy and chemotherapy on the cellular morphology(3)Based on the claudin-18.2 protein expression of human pancreatic cancer PANC-1 cells by immunocytochemistry,analysing the influences on claudin-18.2 of hyperthermia therapy and chemotherapy.(4)Western Blot: claudin-18.2protein expression of human pancreatic cancer PANC-1 cells was semi-quantitatively detected.Results:(1)MTT result showed that hyperthermia therapy had a certain inhibitory effect on cell proliferation PANC – 1(P<0.05).Hyperthermia therapy combined with combination chemotherapy group had the best effect on cell inhibition.Combination chemotherapy was the second and chemotherapy had better effects than hyperthermia therapy.(2)HE staining result showed that the growth of cell clusters was good,and the nuclear bias were observed in the control group.After hyperthermia therapy treatment,the size of human pancreatic cancer PANC-1 cells became larger;gemcitabine group,5-fluorouracil group and combination chemotherapy group showed vacuoles in cytoplasm;hyperthermia therapy combined with combination chemotherapy group showed that vacuoles in cytoplasm increased,indicating that the damage of cancer cells got more serious.(3)Immunocytochemistry result: claudin-18.2 protein showed the clear immune response on the PANC-1 cells’ membrane,accounting for about 70%.Hyperthermia therapy could obviously increase the claudin-18.2 protein expression,but gemcitabine,5-fluorouracil and combination chemotherapy group decreased the claudin-18.2 protein expression.However,the claudin-18.2 protein expression of hyperthermia therapy combined with combination chemotherapy group increased a little higher than combination chemotherapy group.(4)Western Blot showed similar result with immunocytochemistry.Conclusion: Human pancreatic cancer PANC-1 cells expressed claudin-18.2 protein.Hyperthermia therapy could promote the expression of claudin-18.2 protein,and gemcitabine or fluorouracil decreased the expression.Hyperthermia therapy combined with chemotherapy therapy had a further improved therapy effect on pancreatic cancer.Therefore,we hypothesize that hyperthermia therapy can improve the tight junctions between cells,the clinical effect of IMAB362 antibody targeted therapy on pancreatic cancer will be strengthen when combined with hyperthermia therapy.