The Study Of Hyperthermia Effect On MHCC97Cell Line

AimPrimary liver cancer is a common malignant tumor in China, mainlycomposed of hepatocellular carcainoma (HCC). It is difficult to make adiagnosis in early stage due to the deficiency of the typical clinicalmanifestations, and the combination with cirrhosis. Surgery is the most commontreatment in early stage. Non-operative treatment including generalizedchemotherapy, molecule-targeting agents, and local physical or chemicaltreatment. However, the treatment effect and prognosis is not optimistic.Hyperthermia is a new method of cancer therapy with positive clinicalefficacy. It plays important roles in carcinoma curing such as inducingcarcinoma apoptosis, suppressing the cell mitosis, enhancing the tumorimmunogenicity, and strengthening the body immunity. However, hyperthermiais difficult to be used for clinical tumor therapy due to lack of mechanism study.N-Myc down-stream regulated gene2(NDRG2) belongs to the NDRGfamily. It has been confirmed that NDRG2exists in many normal human tissues,and it is involved in many physiological processes, such as the cell growth,differentiation, apoptosis, and et al. NDRG2expressed in low level in some tumor tissues, such as colon cancer and spongiocytoma; but in high level insome tumor cells during stress response. It is speculated that NDRG2may beinvolved in tumor development as a tumor suppressor gene.Our study planned to explore the possible mechanism of hyperthermiatreatment by observing the morphology, proliferation, apoptosis and theexpression of protein NDRG2and HSP70in MHCC97cell after heated.MethodsMHCC97cells were divided into5groups (1control group and4experimental groups), separately heated at43℃for0h(control group),0.5h,1h,1.5h, and2h, then cultured at37℃. At3h,6h,9h,12h, and24h, the morphologywas observed by microscope, proliferation status was examined by MTT assay,apoptosis rate was tested by flow cytometry. Repeated hyperthermia treatmentafter we got the best hyperthermia duration. The NDRG2and HSP70expressionwas detected by Western blot. Experimental data was analyzed by SPSS12.0with factorial designed variance analysis, t-test was used in comparison betweentwo groups. The test standard were set as P<0.05.Results1. The effect of hyperthermia on cell morphologyCells grew well with polygonandfine cell boundary in control group. Ineach test group, the shape of MHCC97has been changed after hyperthermiatreatment of43℃after different times, as becoming smaller, shrunken,organelles broken down, or falling off from the bottles. The cell-shape wassignificantly changed in the group of2h-heated and24h-cultured.2. The effect of hyperthermia on cell proliferationCell proliferation was suppressed by thermotherapy. The group of1h-heatedand24h-cultured was changed most significantly (67.1%). The suppression was deeper after repeated hyperthermia (89%).3. The effect of hyperthermia on cell apoptosisApoptosis rate rose as the hyperthermia duration culture time increased,and the peak value was in the group of2h-heated and12h-cultured according tothe flow cytometry results. Repeated hyperthermia treatment increased theapoptosis rate, and got the maximum value at48h-heated and24h-cultured.4. The effect of hyperthermia on expression of NDRG2The expression of NDRG2was decreased after heated at43℃, and reachthe minimum value in1h-heated group; and it was markedly reduced as theculture time increased, and the valley value was reached after24h-cultured. Theexpression of NDRG2was also decreased after repeated hyperthermia, thoughno significant differences were found among the experimental groups.5. The effect of hyperthermia on expression of HSP70.The expression of HSP70rose after the hyperthermia treatment, and got thepeak value at2h-heated and24h-cultured; and it rose after repeatedhyperthermia, got the highest point at48h-reheated.ConclusionIt was confirmed by our study that the hyperthermia at43℃could impairMHCC97cells, suppress cell proliferation, cause apoptosis, reduce NDRG2expression, and increase the HSP70expression. Repeated hyperthermiatreatment did more damage to MHCC97. It is not supported by our experimentthat NDRG2may play a role on tumor suppressor gene in liver cancer cells afterheated. It can be implied that NDRG2may be involved in stress reaction afterheated.