Effect Of Hyperthermia On The Proliferation And Invasion Of Mouse Malignant Melanoma Cells

Background Melanoma is a cancer that arises from melanocytes, specialized pigmented cells that are found predominantly in the skin. The incidence of melanoma is rising steadily in China. Invation and metastasis is the most important biologic characteristics for malignant carcinoma, which also chiefly accounts for clinic death of cancer patients. Thus, inhibiting invation and metastasis is the key to succeed in curing cancer patient. In its early stages malignant melanoma can be cured by surgical resection, but once it has progressed to the metastatic stage it is extremely difficult to treat and does not respond to current therapies(such as radiation therapy and themotherapy).Hyperthermia is appealing because it is a physical treatment with fewer side effects than chemotherapy and radiotherapy, and repeated treatments should be feasible without concern for cumulative toxic side effects. Clinical experiments have shown that hyperthermia, as part of multimodal regimens, is a tolerable and clinically practical supplementary therapy for patients with advanced malignant tumor, recurrent tumor and metastatic disease. In recent years, magnetic targeted hyperthermia could heating at higher temperature(50℃) and avoiding nomal tissue to be damaged.Recently, it is reported that hyperthermia could delay tumor growth and inhibit lymph node metastasis in animal model. Furthermore, the expression of some genes associated with metastasis was shown to be down-regulated by heating, suggesting that hyperthermia may alter the character of tumor with high metastatic potential via regulating some metastasis related genes. However the concrete mechanism need futher research. B16F10 cells is a promising experimental model of invation for their metastasis biologic characteristics. Therefore, in order to find the appropriate temperature for melanoma treatment, we heat B16F10 cells at different temperature in vitro and in vivo then explore the effect on proliferation,invation and migration of cells.Objective To investigate the effect of hyperthermia on the proliferation and cell cycle of B16F10 cells.Methods B16F10 cells were heated at 41℃,43℃,45℃, 47℃,50℃and 37℃in temperature-controlled water bath, MTT assay, flow cytometry were applied to analyze the growth inhibition and cell cycle.Results The inhibitive rates of cell growth after 24h,48h,72h,96h at 45℃,47℃and 50℃was remarkably higher than at 41℃and 43℃(P <0.05). The inhibitive rates of cell growth after 24h,48h,72h,96h at 47℃and 50℃was remarkably higher than that of at 45℃(P< 0.05).There is no significant difference in inhibitive effect of cell growth between 47℃and 50℃(P<0.05). Results from flow cytometry indicate G0/G1 phase cells after 24h at 41℃,43℃were remarkably higher than that of the control group (P<0.05). However, S phase cells was remarkably lower as compared with the control group (P<0.05).G0/G1 phase cells after 24h at 47℃,50℃were remarkably lower than that of the control group (P<0.05) and S phase cells was remarkably higher as compared with the control group (P<0.05).G2/M phase cells after 24h at 43℃were remarkably higher than that of control group.Conclusions The hyperthermia at 45℃or higher termperature could remarkably inhibit the proliferation of B16F10 cells. The hyperthermia at 47℃or higher termperature could arrest the cells cycle in S phase.Objective To investigate the effect of hyperthermia on migration and invasion ability in vitro of B16F10 cells.Methods To observe the B16F10 cells after heating under invert microscope and transmission electron microscope. B16F10 cells were harvested after heating treatment at 43℃,45℃,47℃and 37℃in temperature-controlled water bath. The migration ability was measured by the scratch wound assay. In vitro cells invasion ability was evaluated by matrigel invasion assay. The activity of matrix metalloproteinase (MMP)-2/9 was investigated by gelatin zymographic assays.Results (1) Cells morphous and ultra-structure changed to different extent after heating at different temperature and showed temperature dependence.(2) The migration ability of B16F10 cells was reduced after heated at 43℃, and remarkably reduced at 45℃and 47℃.(3) The number of invading cells were 29.6±6.0,18.2±2.4 and 1.6±1.1 at 43℃,45℃and 47℃respectively, which were remarkably lower than that of control group (P<0.05).(4) (MMP)-2/9 secretion and enzymatic activity were suppressed at 43℃,45℃and 47℃which showed temperature dependence.Conclusions Cells morphous and ultra-structure changed after heating; In vitro migration and invasion of B16F10 cells was inhibited by heating; (MMP)-2/9 secretion and enzymatic activity were suppressed after heating.All above showed temperature dependence. Objective To study the effect of temperatures for in vitro heating treatment of B16F10 cells on the protein and mRNA expression of TGF-β1/Smad4, mRNA expression of TGF-β1/Smad4,(MMP)-2 /9 and VEGF.Methods B16F10 cells were harvested at 2h,6h,12h and 24h after heating treatment at 43℃,45℃,47℃and 37℃for 30min in temperature-controlled water bath, rt-PCR, western blotting, as well as immunohistochemical technique were employed to determine the mRNA and protein expressions of TGF-β1/Smad4,MMP-2/9 and VEGF.Results (1) Immunohistochemical analysis demonstrates that the expression of TGF-β1 and VEGF decreased with the increase of the temperature for heating treatment. (2) Compared with the control group, the mRNA expression of MMP-2,VEGF at 43℃was lower within 24h (P <0.05);expression of MMP-9,TGF-β1 at 43℃was lower from 6h to 24h with statistical significance (P<0.05); expression of TGF-β1,VEGF,MMP-2 and MMP-9 at 45℃,47℃was lower within 24h(P<0.05). (3) When compared with the control group, the protein expression of TGF-β1 was remarkably decreased and Smad4 was increased(P<0.05) from 12 h to 24 h at 43℃; the protein expression of TGF-β1 was remarkably decreased from 6 h to 24 h and Smad4 was increased from 6 h to 12 h at 45℃(P<0.05); at 47℃the protein expression of TGF-P, was remarkably decreased within 24 h and Smad4 was increased from 2 h to 6 h(P<0.05).Conclusions Protein and mRNA expression of TGF-β1/Smad4 was dependent on the temperature and culture time. The upregulation of Smad4 protein lasts only a period of very short time.The hyperthermia at 43℃or higher termperature could inhibit the mRNA expression of MMP-2/9 and VEGF, which may be associated with down regulation of TGF-β1.Objective To study the temperature effect of magnetic fluid hyperthermia (MFH) at 45℃or 50℃in a murine xenograft model of mouse melanomaMethods Murine xenograft model of mouse melanoma was established by transplanting cultured B16F10 cells into the subcutaneous tissue of the back of nude mouse. The tumor-bearing mice then underwent radiation by an alternative magnetic filed (AMF) after the Fe3O4 magnetic fluid (MF) was locally injected in the tumor area. The parameters of the AMF were carefully adjusted until a local tumor temperature (45℃or 50℃) was maintained for 30 min and 10 min respectively. The MFH was performed three times with 24h interval. The tumour volume, mice survival and the index of TGF-β1 were examined.Results MFH at 45℃or 50℃could inhibit the tumor growth. Compared with 45℃MFH,50℃MFH had a greater inhibitive effect on tumor growth (P<0.05). The survival of 50℃MFH group and 47℃MFH group were more prolonged than that of control group (P<0.05). When compared with the other groups, the expression of TGF-β1 was remarkably decreased at 50℃MFH group (P<0.05).Conclusion MFH at 45℃or 50℃could inhibit the tumor growth, prolong survival as well as decreased the expression of TGF-β1 with more remarkable effect at 50℃MFH group, in which the rate of total tumour regression was 57.1%.