Berberine Chloride and Hyperthermia Promote Osterix Expression and Suppress Cell Cycle Genes in Osteosarcoma Cells

Objectives: The aim of the current study was to investigate the effects of berberine chloride and various heat conditions on the gene expression of Osterix, RUNX-2, RANKL, CDK2, CDK4, IL-6 and IL-11. Methods:  Six groups of cells were treated with hyperthermia for 1 h: Two groups at mild, two at moderate and two at severe hyperthermia (39, 43 and 45°C respectively). Berberine chloride (80 µg/mL) was selected for treating one group of mild, moderate and severe hyperthermia (combination). All treated groups were recovered at 37°C for 24 h. Another exposure for hyperthermia (1 h) and recovery for 3 h at 37°C were applied. Results: The expression of Osterix was highly upregulated in all groups except in the severe hyperthermia and mild hyperthermia with berberine groups. Only the mild hyperthermia without berberine induced a slight increase in the expression of RUNX2, whereas severe hyperthermia alone suppressed the levels on a significant manner. Berberine alone was more effective in significantly up-regulating RANKL expression. On the other hand, CDK2 mRNA was downregulated in all groups. CDK4 showed a similar regulation in the mild hyperthermia group with control, but the expression was downregulated in the other groups, especially in severe hyperthermia the expression was significantly downregulated (p<0.5). IL-6 was upregulated highly and significantly in the group of berberine and all groups of combinations, whereas mild and moderate hyperthermia stimulated significant expression of IL-11 mRNA. Conclusion:  These results suggest that hyperthermia and berberine chloride can promote osteosarcoma cells differentiation and arrest cell-cycle

Human MG-63 osteosarcoma cells responses to long and short term hyper- and hypothermia stress

Hyper- and hypothermia are utilized as treatment modalities in cancer treatment or as a protection against ischemia-reperfusion induced cell damage. The under-lying mechanism of hyper- and hypothermia, on cell death in osteosarcoma cells are not well understood. The aim of this study is to investigate the short- and long-term effects of various severities of hyper- and hypothermia on osteoblast-like osteosarcoma cells (MG-63). MG-63 cells were treated with mild and severe hyper- and hypothermia for short, medium and long-term periods. Severe hypothermia and hyperthermia showed a time-dependent toxicity; hence viability was reduced in a significant manner at all time points and the cells were undergoing apoptosis. Mild hypothermia, on the other hand, showed a protective effect and long term exposure increased the cell viability. Severe hyperthermia induced significant DNA damage at all time points. Caspase 3/7 activity showed a significant increase at 1 h of severe hyperthermia and 72 h of severe hypothermia (p<0.05). Hsp90 expression was significantly increased at 72 h of mild hyperthermia (p<0.01), whereas Hsp70 showed a significant increase after 24 and 72 h (p<0.01 and p<0.001). Hsp27 mRNA was increased significantly at 24 h only under mild hyperthermia (p<0.01). In conclusion, hyperthermia especially severe hyperthermia induced cellular stress in MG-63 cells leading to apoptosis. Hypothermia, on the other hand caused severe cell stress only when the cells were challenged for a prolonged period with severe low temperatures

Mild hyperthermia (39°C) attenuates berberine chloride cytotoxicity against osteosarcoma cells

Many studies reported that antioxidants and dietary supplements increase tumour progress and reduce survival. Hyperthermia is an adjuvant treatment to sensitise cancer cells for radio- or chemotherapy. The current study was carried out to determine the effects of berberine chloride and hyperthermia on bone cancer cells. MG-63 osteosarcoma cells were treated with hyperthermia (39, 43 and 45°C, respectively) for 1 h. Then, the cells were treated with a low toxic dose of berberine chloride (80 μg/mL). After that, all treated groups were recovered at 37°C for 24 h. Finally, all groups were treated with hyperthermia (39, 43 and 45°C) for the second time (1 h) and were recovered for 3 h at 37°C. Cells exposed to hyperthermia without treatment of berberine chloride were used as hyperthermia control. Cells treated with 80 μg/mL of berberine at 37°C served as berberine control and cells incubated at 37°C were used as an untreated control. All treated groups showed significant apoptosis compared to the control group (p<0.05) except 39°C. On the other hand, mild hyperthermia treatment (39°C) resulted in a reduction of berberine-induced apoptosis (p<0.001). Severe and moderate hyperthermia did not show a significant increase in the rate of apoptosis compared to berberine treated cells. Mild hyperthermia treatment can effectively reduce berberine cytotoxicity and implying negative effects on cancer therapy

Severe Hyperthermia Induces Apoptosis Mediated by Caspases Activation and Suppression of Hsp90-alpha Expression in Osteosarcoma Cells

BACKGROUND: Hyperthermia is used as an adjuvant treatment to sensitize cancer cells to subsequent radiotheraphy or chemotherapy. The aim of this study was to study the effect of severe hyperthermia on osteosarcoma cells and its underlying causes.METHODS: Short-term (1 h) severe hyperthermia (45°C) was applied to osteoblast-like osteosarcoma cells (MG-63) and the heat shock response and cell death mechanisms were investigated after recovery at 37°C for 72 h.RESULTS: Cell viability was significantly reduced (p<0.05) and apoptosis was significantly induced by severe hyperthermia in MG-63 cells (p<0.001). Caspase 3/7, 4 and 12 activities were significantly increased after 72 h of recovery at 37°C, indicating that severe hyperthermia induced endoplasmic reticulum (ER) stress and apoptosis (p<0.05). Heat shock protein 90 alpha (Hsp90α) was significantly down regulated at the protein level after recovery, in association with apoptosis induction (p<0.01). Additionally, caspase 8 and 9 were activated, possibly as a result of ER stress or other stimuli while, B-cell leukemia 2 family protein (BCL-2) mRNA was down regulated (p<0.01).CONCLUSION: Severe hyperthermia could cause prolonged cell cytotoxicity by inducing apoptosis in association with inhibition of Hsp90α. This indicates the therapeutic potential of severe hyperthermia for the treatment of osteosarcoma.KEYWORDS: hyperthermia, apoptosis, endoplasmic reticulum, stress, heat shock proteins, osteosarcom