No abstract

Hyperthermia (HT) is an important modality in cancer treatment; however, the acquisition of thermal resistance in cancer cells due to the elevation of heat shock proteins (HSPs) makes HT less effective. Accumulating evidence suggests that microRNAs (miRNAs) play an important role in regulating cellular stress sensitivities, such as drug sensitivity and radio-sensitivity, in cancer cells. However, few studies have investigated the involvement of miRNAs in thermal sensitivity. The aim of this study was thus to investigate the contribution of miRNAs to the thermal sensitivity of human oral squamous cell carcinoma (OSCC) cells. When the HSC-2, HSC-3 and HSC-4 OSCC cell lines were treated with HT at 44˚C for 60 min, a significant increase in cell death was observed in HSC-2 and HSC-3 cells but not HSC-4 cells, suggesting that HSC-4 cells were thermally resistant under the present experimental conditions. Moreover, the expression levels of HSPs were most elevated in HSC-4 cells. When the basal expression levels of miRNAs were monitored using two different microarray systems in thermal-sensitive HSC-2 and HSC-3 cells and thermal-resistant HSC-4 cells, five miRNAs that were differentially expressed were identified. Among these miRNAs, the expression level of miR-27a in HSC-4 cells was markedly reducec compared to the expression levels in HSC-2 and HSC-3 cells. Interestingly, treatment of HSC-4 cells with a miR-27a mimic oligonucleotide significantly enhanced HT-induced cell death. Furthermore, the miR-27a mimic oligonucleotide suppressed the elevation of the expression of Hsp90 and Hsp110 in HSC-4 cells, suggesting that these HSPs may be involved in a mechanism of thermal resistance. From these findings, we concluded that in OSCC cells, miR-27a may contribute to thermal sensitivity by modulating the HSP expression.

Common genes that were differentially expressed and/or acted within a gene network in response to MHT in NHF cells were identified. These findings provide the molecular basis for a further understanding of the mechanisms of the MHT response in NHF cells.

The inhibition of DNA damage response pathway seems to be an attractive strategy for cancer therapy. It was previously reported that in rodent cells exposed to heat stress, cell growth was promoted by the activity of DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA non-homologous end joining (NHEJ) required for double-strand break repair. The absence of a functioning DNA-PK was associated with down regulation of heat shock protein 70 (HSP70). The objective of this study is thus to investigate the role of DNA-PK inhibition in heat-induced apoptosis in human cell lines. The inhibitors of phosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) at Ser2056, such as NU7026 and NU7441, were utilized. Furthermore, knock down of DNA-PKcs was carried out using small interfering RNA (siDNA-PKcs). For heat exposure, cells were placed in water bath at 44°C for 60 min. Apoptosis was evaluated after 24 h incubation flow cytometrically. Proteins were extracted after 24 h and analyzed for HSP70 and HSP40 expression by Western blotting. Total RNA was extracted 6 h after treatment and analyzed using a GeneChip® microarray system to identify and select the up-regulated genes (≥1.5 fold). The results showed an enhancement in heat-induced apoptosis in absence of functioning DNA-PKcs. Interestingly, the expression levels of HSP70 and HSP40 were elevated in the absence of DNA-PKcs under heat stress. The results of genetic network analysis showed that HSPs and JUN genes were up-regulated independently of DNA-PKcs in exposed parent and knock out cells. In the presence of functioning DNA-PKcs, there was an observed up-regulation of anti-apoptotic genes, such as NR1D1, whereas in the absence of DNA-PKcs the pro-apoptotic genes, such as EGR2, were preferentially up-regulated. From these findings, we concluded that in human cells, the inactivation of DNA-PKcs can promote heat-induced apoptosis independently of heat-shock proteins.

Hyperthermia (HT) has been used as a possible treatment modality for various types of malignant tumors. Due to its pleiotropic effects, its combined use with radiotherapy and/or chemotherapy has proven to be beneficial. However, the molecular mechanisms underling the cellular responses to heat stress remain unclear. Therefore, the aim of this study was to identify common gene expression patterns responsive to mild HT (MHT) in human cancer cells. HeLa human cervical squamous cell carcinoma (SCC) and HSC-3 human oral SCC cells were exposed to MHT at 41˚C for 30 min, followed by culture at 37˚C for 0-24 h. MHT did not affect cell viability or the cell cycle. GeneChip microarray analysis clearly revealed that many probe sets were differentially expressed by a factor of ≥1.5 in both cell lines following exposure to MHT. Of the many differentially expressed probe sets, 114 genes were found to be commonly upregulated in both HeLa and HSC‑3 cells, and two significant gene networks were obtained from the commonly upregulated genes. Gene network A included several heat shock proteins, as well as BCL2-associated athanogene 3 (BAG3), and was found to be mainly associated with the biological functions of cellular function and maintenance. Gene network B included several anti-cell death genes, such as early growth response 1 (EGR1) and endothelin 1 (EDN1) and was found to be associated with the biological functions of cell death and survival. Real‑time quantitative polymerase chain reaction demonstrated that the gene expression patterns of the 12 genes selected were consistent with the microarray data in four cancer cell lines. These findings may provide further insight into the detailed molecular mechanisms of the MHT response in cancer cells.