Abstract
Pre-treatment of tumors with hyperthermia is often used to increase the efficacy of radiotherapy. One of the main proteins induced in response to hyperthermia is heat shock protein 70 (HSP70). The aim of our study was to investigate up- and down-regulated genes in response to (thermo)radiotherapy in HSP70 proficient and deficient canine osteosarcoma cell line (Abrams), and functional role of HSP70 in the mechanism of thermoradiosensitization. Cells were transfected with negative control siRNA or siRNA targeting HSP70 and treated with hyperthermia (HT), radiotherapy (RT), and thermoradiotherapy (HTRT). RNA sequencing was used to analyze gene expression. Hyperthermia and thermoradiotherapy, but not radiotherapy alone, induced differential gene expression. We identified genes differentially expressed only in HSP70 knockdown (thus HSP70-dependent) cells in response to hyperthermia and thermoradiotherapy. Interestingly, cell proliferation but not clonogenicity and apoptosis/necrosis was affected by the HSP70 knockdown in response to thermoradiotherapy. The results suggest that HSP70 regulates expression of specific genes in response to hyperthermia and thermoradiotherapy. Further investigations into the role of specific genes regulated in a HSP70-dependent manner in response to thermoradiotherapy could pave a way into new, combinatorial treatment options for (canine) osteosarcoma and other cancer types.
Highlights
Pre-treatment of tumors with hyperthermia is often used to increase the efficacy of radiotherapy
The protein levels of heat shock protein 70 (HSP70) were strongly influenced by the hyperthermia and combined treatment in negative control siRNA transfected cells but the protein was absent in control, hyperthermia and combination-treated HSP70 knockdown cells (Fig. 2A)
We tried to address it by performing RNA sequencing analysis of canine osteosarcoma cells with high and low levels of HSP70 and treated withradiotherapy
Summary
Pre-treatment of tumors with hyperthermia is often used to increase the efficacy of radiotherapy. The aim of our study was to investigate up- and down-regulated genes in response to (thermo) radiotherapy in HSP70 proficient and deficient canine osteosarcoma cell line (Abrams), and functional role of HSP70 in the mechanism of thermoradiosensitization. We identified genes differentially expressed only in HSP70 knockdown ( HSP70-dependent) cells in response to hyperthermia and thermoradiotherapy. Further investigations into the role of specific genes regulated in a HSP70-dependent manner in response to thermoradiotherapy could pave a way into new, combinatorial treatment options for (canine) osteosarcoma and other cancer types. HSP70 plays and important role in maintaining cellular homeostasis and can indirectly influence gene expression (for example, by regulating protein folding of transcription factors). We were interested in the gene expression analysis of Abrams cells in response to radiotherapy, hyperthermia and combination of both in HSP70-proficient and HSP70 knockdown Abrams cells. We used RNA sequencing technology and quantitative RT-PCR to identify down- and up-regulated factors, clonogenic cell survival and proliferation assay to measure response to treatment, and apoptosis/necrosis assay to investigate cell death after treatment
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