The Expression Patterns And Clinical Significance Of Heat Shock Proteins (HSPs) In Gastric Cancer Cells SGC-7901after Thermostatic Heat Treatment

BackgroundGastric cancer (GC) is one of the leading causes of cancer death, and is more common in developing countries, especially in Asia. Male and female gastric cancer patients ranked second and third in overall cancer mortalities in China, respectively. Due to its insidious onset and unconspicuous early symptoms, majority of patients were diagnosed as advanced gastric cancer. Furthermore, some patients have peritoneal metastasis, and unsatisfactory efficacy was obtained even taking aggressive treatment approaches. Gastric tumor cells first spread in the mucosa, then violated and diffused in the mucous layer submucosa, muscularis, subserosa, serosa, breaking serosa further violations large and small omentum, peritoneum, liver, pancreas, transverse colon and other nearby tissues and organs in turn. When the penetrated the gastric serosa tissue, tumor cells may diffuse into and planted in the peritoneum, large and small omentum or other organ surface, forming a planting of metastatic nodules. Efficacy and prognosis of traditional regimes for peritoneal metastasis from gastric is very poor.With the understanding of mechanisms underlying the intraperitoneal tumor spread, multimodal approaches combining aggressive cytoreductive surgery (CRS), intraperitoneal hyperthermic chemotherapy (HIPEC) and systemic chemotherapy have been proposed and are actually considered as promising methods for GC treatment. HIPEC is advantaged by its ability to eradicate micro tumor nodules and micrometastases that cannot handled by conventional surgery. CRS plus HIPEC regime has been confirmed to improve loco-regional control of the disease and ultimately to increase survival.Heat shock proteins (HSPs) are response for the resistance to thermo-and chemotherapies. Heat shock proteins are a group of proteins induced by heat shock or cell stress, which prevent intracellular protein misfolding and aggregation. HSPs are expressed in an many tumor types and have survival benefits to cancer cells. They are reported to be involved in the inhibition of apoptosis in human pancreatic, prostate and gastric cancer cellsThe role of Hsp90is to bind and stabilize immature client proteins, many of which are conformationally-unstable proteins involved in signal transduction pathways important in cell development, growth, and survival, including transmembrane tyrosine kinases. The major function of HSP70is to promote cell survival by interfering with apoptosis The transient HIPEC may up-regulate the expression of Hsps, which diminishes the efficacy of further anti-cancer therapies. The monitoring of HSPs’kinetic may give a clue to minimize the thermo-and chemo-resistant of tumor cells, thus improves the outcomes of patients.ObjectiveTo investigate the expression patterns of two members of HSPs family, HSP70and HSP90in response to hyperthermic treatment.To investigate the expression patterns of HSP70and HSP90in patients who reveived HIPEC regime.To find out the best time for a second round of HIPEC treatment to minimize the tolerance of cancer cells.MethodsSGC7901cells cultured in Dulbecco’s modified Eagle medium (DMEM) with10%fetal bovine serum (FBS) at37℃in an atmosphere of5%CO2. When reached80-90%confluence, cells were digested with0.25%trypsin digest solution (Sigma, USA) and collected by centrifugation. Cells were then seeded into6-well plates (5X104cells per well) in500μL of full growth medium. Plates were cultured at37℃for4h to allow cells to adhere, followed by HIPEC-mimic hyperthermic treatment at41℃for1h using another incubator. After then, the plates were transferred to the previous incubator and resumed the normal culture at37℃. Cells were collected before and after treatment (0h,4h,8h,12h,16h,20h,24h,28h,32h,36h,40h,44h and48h). Protein and RNA were extracted for further analysis.For the immunofluorescent observation of HSPs, coverslips were first put in the wells, and cells were then seeded into6-well plates. After4h of incubation, cells were turned to HIPEC mimic hyperthermic treatment as previous described. Coverslips were then collected and fixed for immunofluorescent staining.ResultsThe transcription of HSP90was elevated at0h,12h,32h and48h after hyperthermic treatment (P<0.05), but decreased at16h after hyperthermic treatment (P<0.05). No obvious change was observed at the other time period (P>0.05). The transcription of HSP70kept at a high level within the beginning8h (P<0.05), but decreased after12h (P<0.05). The highest Hsp70level was observed at4h after treatment.The protein expressions of HSP90and HSP70in SGC7901cells with hyperthermic treatment were further analyzed by western blot and immunocytochemical staining. The protein expressions of HSP70were similar to the results of mRNA expression. However, expect16h and44h, protein expressions of HSP90with hyperthermic treatment were higher than that without treatment at different time period. Similar patterns of protein expression were also observed in immunocytochemical staining results.Serum levels of HSP90and HSP70before and after HIPEC therapy were showed in Figure4. Serum HSP70concentration was elevated after HIPEC therapy, but it recovered to normal level after24h. The highest concentration of HSP70was observed at12h after HIPEC therapy (P=0.01). Serum HSP90concentration was increased slightly after HIPEC therapy, and reached the peak at18h. It also decreased to normal level after24h. ConclusionDue to the multifactorial prosurvival roles of HSPs, it makes sense that the HSPs induced by the first round HIPEC treatment can compromised the efficacy of the following HIPEC treatment in clinical HIPEC therapies. They represent promising targets for the sensitization of cancer thermotherapy and chemotherapy. The present study reveals that HSPs have relative high expressions within the24h after hyperthermic treatment. The efficacy of further therapies within this time can be compromised. Therefore, a delayed (at least24h after the first round HIPEC treatment) HIPEC or chemotherapy is highly recommended. In future studies, HSPs inhibitors, which have already received extensive attention, can be used in the clinic as adjuncts to HIPEC therapy. Due to the transient high expression of HSPs in cancer cells, HSPs vaccines can be also promising adjuncts to HIPEC therapy.