Introduction: Ovarian cancer is a deadly gynecological disease. Hyperthermia has been investigated as a potential treatment for cancerous tumors. However, its localized application still remains a challenge. Magnetic fluid hyperthermia (MFH) may be an alternative to surpass such challenge. Several cellular and molecular aspects of MFH have been previously elucidated, but there is still a need to further understand the implications of MFH at the cellular level and how this information could be employed to further enhance the effects of MFH. Therefore, this work focused on the examination of gene expression after MFH treatment and using such information to find target genes that when silenced or inhibited could produce an enhanced therapeutic outcome after MFH.
Materials and Methods: Ovarian cancer cell lines HeyA8, A2780 cp20 and SKOV3 were employed. Microarray analysis was performed in HeyA8 cells exposed to MFH for 30 min at 43°C. From the microarray results, PCR was performed to confirm HSPA6 and HSPA7 expression. HSPA6 gene was knocked down using Lipofectamine RNAiMax to observed MFH outcome. Cells were transfected with HSPA6 and control siRNA. MFH was applied after transfection and cells were counted after 48 hours. 2-phenylethynesulfonamide (PES), a HSP70 inhibitor, was studied with MFH. PES cytotoxicity was evaluated in 96 wells using the EUZ4 assay. For MFH, cells were incubated with the drug 2.5 hours prior to MFH for 30 min at 41 or 43 °C. Cells were tallied after 48 hours. Combination index was calculated with the model of Chou-Talay with the CompuSyn software(Chou 2006) . A subcutaneous HeyA8 tumor model was generated in athymic nude mice. HSPA6 siRNA and control siRNA were injected and mice were exposed to MFH for 30 min at a magnetic field of 62 kA/m.
Results and Discussion: Analysis revealed that heat shock protein genes, including HSPA6, were upregulated when MFH was applied at 43°C for 30 min. HSPA6 encodes the heat shock protein HSP70. HSP70 gene expression was confirmed by PCR in HeyA8 and A2780cp20 cell lines at 39, 41 and 43°C. The highest expression was observed at 1 hour after treatment. Results indicated that the effect of HSP70 inhibition in ovarian cancer cell lines during MFH could be a potential target for combined therapy. First, si-RNA inhibition of HSPA6 gene was achieved in vitro and a decrease in cell viability was observed when cells were exposed to MFH. An alternative avenue of inhibition was examined by using a small HSP70 inhibitor known as PES. An improvement in MFH efficacy was observed in various cell lines (A2780cp20, SKOV3 and HeyA8). Combination index was calculated reporting a synergistic effect. In vivo efficacy experiments were also performed in Nu/Nu mice with a HeyA8 subcutaneous tumor model. A reduction in tumor growth rate was observed in treatments with MFH and HSPA6 siRNA delivery. HSP70 inhibition appeared to be a promising target to enhance MFH therapeutic outcome.
Conclusions: HSP70 genes are upregulated when MFH is applied to ovarian cancer cells. HSP70 plays an important role in the cell and it is induced when cells are exposed to stress. HSP70 inhibition at the gene and protein level improved MFH treatment. PES showed a moderate synergistic effect with ovarian cancer cell lines. HSP70 inhibition is a promising target to improve MFH.
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