| Background: Pancreatic cancer is estimated to be the fourth leading cause of cancer-related in the United States.The unique biology of the disease makes it difficult to be detected and diagnosed in the early stage of disease development and rapidly fatal: overall five-year survival rate approximately 6%(ranges from 2% to 9%).Most pancreatic cancer patients have no obvious symptoms until the disease progresses to an advanced stage.Although tests of biomarkers can help clinical work prompting the possibility of pancreatic cancer,large samples and rigorous validation are still needed before clinical implementation.Surgical resection is considered the first option to cure the disease,but less than 20% of patients qualify for surgery.About 70%-90% of patients eventually have systemic recurrence and disease although undergoing potentially curative resection.For most patients with pancreatic cancer,especially those with locally advanced and metastatic diseases,systemic chemotherapy is the preferred treatment option.Gemcitabine,as an anti-tumor drug,approved as a first-line treatment for pancreatic cancer in 1996.As a new cytosine nucleoside derivative,it is activated by deoxycytosine kinase and metabolized by cytosine nucleoside deaminase.It is mainly used in G1 / S phase,and it can also inhibit nucleotide reductase,lead to the decrease of deoxynucleoside triphosphate in cells,and can inhibit deoxycytosine deaminase to reduce the degradation of metabolites in cells,which has a self synergistic effect.It is illuminated that gemcitabine was superior to fluorouracil(5-FU)in median survival and overall survival.In a randomized controlled trial(RCT)in 1996,gemcitabine has become a standard regimen for pancreatic cancer patients with locally advanced and metastasis.Subsequently,some studies have established gemcitabine centered combination therapy,such as gemcitabine combined with albumin paclitaxel,erlotinib,capecitabine,fluorouracil,etc.Despitethe rapid progress,improvements in overall and median survival rates are not encouraging.In recent years,research direction has begun to aim at a complicated FOLFIRINOX regimen.In the FOLFIRINOX regimen,the synergistic effect of three drugs offers a good performance status for patients younger than 76 years but increases toxicity simultaneously.In order to explore more therapeutic options,many clinical trials have been tried in the United States and other countries,and various combination therapies may become possible in the future.Hyperthermia is considered to be an effective adjuvant therapy and has been studied for decades.Its non-invasive method is to raise the temperature of tumor tissue approximately 41-42℃ and present the direct cell-killing effect consequently.Due to the microenvironment among various tumors is distinct,the thermal dose-response relation can be changed among different cell lines.The mechanism of hyperthermia may be targeting cell structures such as membranes,the cytoskeleton,synthesis of macromolecules and interfere with the process and function of them but still,require further investigation.Besides,the expression of several genes can be altered by heat such as heat-shock proteins(HSP).According to corresponding reporting,mild hyperthermia will reinforce the anticancer effect and sensitivity of chemotherapy in gastric cancer cells through producing reactive oxygen species-induced autophagic death.It is reported that whole-body hyperthermia may contribute to the natural killer cells and elevated plasma levels of various types of interleukins and other cytokines which strongly associated with organism immunity.Based on these characteristics,hyperthermia is combined with multiple different therapies,such as chemotherapy,surgery,monoclonal antibodies and immunotherapy,in order to obtain a better therapeutic effect.In this study,we treated pancreatic cancer cell PANC-1 with gemcitabine(Gemcitabine hydrochloride),hyperthermia or gemcitabine combined with hyperthermia,and then analyzed the anti-tumor effects of three treatments in terms of cell proliferation,cell apoptosis,the formation of ROS and proteins expression associated with apoptosis,proliferation and formation of hyaluronic acid of tumor cells,hoping to explore the molecular mechanism of the effect of hyperthermia on pancreatic cancer cells.Objective: ⑴ Detecting the effect of gemcitabine and hyperthermia on human pancreatic cancer cell line PANC-1 and its effect on cell apoptosis;⑵ To analyze and compare the expression of anti apoptotic protein and caspase family protein in human pancreatic cancer cell line PANC-1 48 hours and 72 hours after three treatments,so as to explore the possible signal pathway and mechanism of apoptosis induced by hyperthermia.⑶ To explore the expression of hyaluronic acid synthetase and degrading enzyme on the surface of PANC-1,and to provide a general direction for the study of targeting extracellular substance.Methods: The human pancreatic cancer cell line PANC-1 was cultured in a NEST cellculture dish.Different concentrations of gemcitabine(5 μmol / L,10 μmol / L,20 μmol / L),hyperthermia for one hour and their combination treatment for 24,48,72 hours and then collect cell climbing tablets for using.MTT,H&E staining,flow cytometry and other related technologies were used to detect the difference of response of human pancreatic cancer cell line PANC-1 to three treatment methods and the production of ROS;immunocytochemistry(ICC),Western blot,TUNEL and immunofluorescence were used to detect the expression of anti apoptotic protein,caspase family executive protein and enzyme which associated with hyaluronic acid in extracellular matrix in PANC-1 after three treatment regimens.Results:(1)MTT results showed that hyperthermia enhanced the inhibitory effect of gemcitabine on the viability of PANC-1 cell line.The effect of chemotherapy alone and thermochemotherapy was gradient dependent.(2)The results of H&E staining showed that the morphology of human pancreatic cancer cell line PANC-1 treated by three methods changed in different degrees,and the number and density of cells decreased in different degrees.(3)Flow cytometry showed that the reactive oxygen species(ROS)were activated effectively in mitochondria within 48 hours after exposure to chemotherapy,hyperthermia or thermochemotherapy.After 48 hours,the number of ROS in any treatment group decreased dramatically,suggesting that the effect of chemotherapy,hyperthermia and thermochemotherapy on the induction of ROS decreased significantly after 48 hours.In the group of hyperthermia,chemotherapy or combination of hyperthermia and chemotherapy alone,pancreatic cancer cells showed different degrees of apoptosis,and the proportion and degree of apoptosis in the group of hyperthermia and chemotherapy were higher than that in any single treatment group.(4)The expression of anti apoptotic protein b-lymphoma-2(Bcl-2)and tumor specific inhibitor of apoptosis survivin in pancreatic cancer cells was down regulated after different treatment,especially in the thermochemotherapy group.Signal transducer and activator of transcription-3(Stat-3)and galectin-1 were down regulated in varying degrees,suggesting that hyperthermia may be related to nuclear transcription factors.In addition,the results of immunofluorescence and Western blot showed that caspase was also involved in the process of apoptosis induced by hyperthermia.As different roles of caspase family protein signaling pathway,caspase-3,8,9 are effectively activated in different therapies.Finally,the expression of HAS-2 and HYAL-1changed in varying degrees,suggesting that the content and concentration of hyaluronic acid is also related to the activity of enzyme on the surface of tumor cells.Conclusion:(1)Hyperthermia can effectively inhibit the cell viability of PANC-1 cell line in a short period of time,but the inhibition gradually weakened with time.The effect ofcomprehensive treatment(hyperthermia combined with chemotherapy)is better than that of any single treatment.(2)Hyperthermia can activate the production of intracellular ROS and induce apoptosis in a short period of time.The effect of this mode of apoptosis decreased significantly 48 hours later.(3)Hyperthermia can promote apoptosis of pancreatic cancer cells by inhibiting Bcl-2and survivin.(3)Stat-3 and galectin-1 are also involved in the process of apoptosis induced by hyperthermia.(4)In addition,caspase family proteins and related pathways are also related to the apoptosis induced by hyperthermia,including caspase-9 in mitochondrial pathway and caspase-8in death receptor pathway.Finally,caspase-3,the executive protein of caspase family proteins,carries out the apoptosis process.(5)The expression of HAS-1 and HYAL-1,which are highly related to the production of hyaluronic acid,decreased after thermochemotherapy,suggesting that the change of the protein level on the surface of PANC-1 may affect the structural composition of the stroma.The abnormal expression of some apoptosis related proteins above can provide a new exploration possibility for hyperthermia treating pancreatic cancer,and guide the further clinical treatment direction. |
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