Effect Of Hyperthermia On The Apoptosis And Proliferation Of Cervical Cancer Cell

Background: Cervical cancer has become one of the most prevalent gynecological neoplasma and the main cause is persistent infection of high-risk human papillovirus. The incidence of cervical cancer experiences a significant rise and sufferers become younger in average age. The primary possible treatment of advanced cervical cancer may be a combination of radiation and chemotherapeutic agent such as cisplatin. However, owing to lack of acknowledgment of tumor resistance and side effects of chemo-radiotherapy, the 5-year overall survival rate is only 50% for advanced cervical cancer. When tumors cells expose to higher thermal conditions, more apoptosis and necrosis are often induced and cells usually exhibit hypersensitivity to radiation and/or chemotherapy after heat treatment. The combined treatment of advanced cervical cancer has resulted in long-term major improvement in local control and survival without increasing late toxicity.The mechanism of hyperthermia on cervical cancer is not clear. Traditional hyperthermia believes that there is an apoaptosis transition at 43℃for majority of cells. However, recent studies found that hyperthermia at 43℃could not kill all kinds of cancer cells. The cell sensitivity to hyperthermia is correlated with category of cell and feature of gene. Investigation for cell sensitivity to hyperthermia contributes to the clinic treatment.A new nanotechnology based thermotherapy using magnetic nanoparticles, also referred to as magnetic fluid hyperthermia (MFH) was developed. Compared with traditional hyperthermia, magnetic fluid hyperthermia is a better temperature homogeneity and targeted orient in the tumor with less damnification to peripheral normal tissues. It shows significant effects in phaseâ…¡clinical trial, but there are no reports about the investigation of cervical cancer.Therefore, in order to find the appropriate temperature for treatment cervical cancer, we heat cervical cancer cells at different temperature in vitro and in vivo then explore the effect on apoptosis and proliferation of cells. Objective To investigate the effect of hyperthermia on the apoptosis, proliferation and cell cycle of Caski cells.Methods Caski cells were heated at 43℃, 45℃, 47℃and 37℃in temperature-controlled water bath, MTT assay, flow cytometry and immunohistochemical technique were applied to analyze the growth inhibition, apoptosis rate,necrosis rate, cell cycle as well as expression of PCNA.Results The inhibitive rates of cell growth after 24h, 48h, 72h, 96h at 45℃and 47℃was 76.11±5.32%, 81.08±4.03%, 74.71±3.78%, 72.16±4.75% and 88.57±3.77%, 91.33±2.43%, 91.09±1.17%, 92.05±1.67% respectively, which was remarkably higher than that of 26.57±3.46%, 25.49±2.76%, 22.80±3.16%, 26.87±4.01% at 43℃(P<0.05). There is no significant difference in inhibitive effect of cell growth between 45℃and 47℃(P<0.05). While the highest apoptosis rate happened at 45℃, the highest necrosis rate was observed at 47℃, both with statistical significance as compared with other groups (P<0.05). Results from flow cytometry indicate G₁ phase cells after 24h at 43℃, 45℃and 47℃were remarkably lower than that of the control group (P<0.05). However, S phase cells was remarkably higher as compared with the control group (P<0.05). Immunohistochemical analysis demonstrates that the expression of PCNA decreased with the increase of the temperature for heating treatment. When compared with the controlled group and 43℃group, the expression was descented at 45℃and 47℃, which was respectively 0.228±0.022 and 0.214±0.018(P<0.05).Conclusions The hyperthermia at 45℃or higher termperaturecould remarkably inhibit the proliferation of Caski cells , increase the apoptosis rates and necrosis rates, arrest the cells cycle in S phase and decrease the expression of PCNA. Objective To study the effect of temperatures for in vitro heating treatment of Caski cells on the protein and mRNA expression of p53, Smac/DIABLO, survivin and caspase-3.Methods Caski cells were harvested at 2h, 6h, 12h and 24h after heating treatment at 43℃, 45℃, 47℃and 37℃for 40min in temperature-controlled water bath, Real time PCR, western blotting, as well as immunohistochemical technique were employed to determine the mRNA and protein expressions of HPV16E6, p53, Smac/DIABLO, survivin and caspase-3.Results (1) The mRNA expression of HPV16E6, Smac/DIABLO, survivin and caspase-3 was increased within 24h at 43℃when compared with the control group (P<0.05). However, protein expression demonstrated time-dependent under the 43℃heating treatment. Expression of survivin was increased from 6h to 24h with statistical significance (P<0.05), though a decreased expression was observed at 2h. Expression of Smac/DIABLO at 6h and 24h was higher than that of the control group (P<0.05). When compared with the control group, caspase-3 was increased from 12h to 24h (P<0.05). (2) At 45℃, the heating treatment could inhibit the mRNA expression of HPV16E6 and survivin, as well as protein expression of survivin within 24h with statistical significance (P<0.05). When compared with the control group, the mRNA expression Smac/DIABLO and caspase-3 was increased 12-24h after heating treatment at 45℃(P<0.05). Meanwhile, the protein expression of Smac/DIABLO and caspase-3 was increased from 6h until 24h after heating (P<0.05). (3) When compared with the control group, the mRNA expression of HPV16E6, Smac/DIABLO and survivin was remarkably decreased within 24h at 47℃(P<0.05), but the mRNA expression of caspase-3 was increased at 24h (P<0.05). Protein expression of Smac/DIABLO at 2h and caspase-3 from 2h to 6h was increased with statistical significance after heating treatment (P<0.05). Protein expression of survivin was remarkably inhibited (P<0.05). (4) When compared with the control group, a reductive mRNA expression of wild type p53 was observed regardless of the heating temperature (P<0.05). (5) Compared with the control group and the 47℃group, AOD value of caspase-3 expression for the 43℃and 45℃groups was increased (P<0.05). Nucleus positive index of caspase-3 expression for the 45℃and 47℃groups was higher than that of the control group and the 43℃group (P<0.05).Conclusions Protein and mRNA expression of Smac/DIABLO, survivin and caspase-3 was dependent on the temperature and culture time. The hyperthermia at 45℃or higher temperature could inhibit the mRNA expression of HPV16E6. However, no noticeable change was observed on the mRNA expression of wild-type p53 subjected to heating treatment regardless of the temperature, which indicating the apoptosis induced by Smac/DIABLO after heating treatment could not depend on wild-type p53 gene. Caspase-3 translocated from cytoplasm to the nucleus was an important event for the apoptosis of the cells.Objective To study the temperature effect of magnetic fluid hyperthermia (MFH) at 43℃or 47℃in a murine xenograft model of human cervical cancer.Methods Murine xenograft model of human cervical cancer was established by transplanting minced fragment of tumours into the subcutaneous tissue of the thigh of nude mouse using a trocar. 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 (43℃or 47℃) was maintained for 30 min. The MFH was performed twice with 72h interval. The tumour volume, rate of tumor volume inhibition, mice survival and the index of PCNA were examined.Results MFH at 43℃or 47℃could inhibit the tumor growth. Compared with 43℃MFH, 47℃MFH had a greater inhibitive effect on tumor growth (P<0.05). The inhibitive rates of tumor volume from the first week to the fourth week at 43℃MFH group and 47℃MFH grou was 32.26%, 52.64%, 48.84%, 38.42% and 47.05%, 89.21%, 88.55%, 87.19% respectively. The survival of 47℃MFH group and 43℃MFH group were more prolonged than that of control group (P<0.05). When compared with the other groups, the expression of PCNA was remarkably decreased at 47℃MFH group (P<0.05).Conclusion MFH at 43℃or 47℃could inhibit the tumor growth, prolong survival as well as decreased the expression of PCNA with more remarkable effect at 47℃MFH group, in which the total tumour regression was observed.