OBJECTIVE: Modulated electro-hyperthermia (mEHT), a noninvasive complementary treatment of human chemo- and radiotherapy, can generate selective ~42°C heat in cancer due to elevated glycolysis (Warburg-effect) and electric conductivity in malignant tissues. Here we tested the molecular background of mEHT and its combination with doxorubicin chemotherapy using an in vitro model.
METHODS: C26 mouse colorectal adenocarcinoma cultures were mEHT treated at 42°C for 2 × 60 minutes (with 120 minutes interruption) either alone or in combination with 1 µmol/L doxorubicin (mEHT + Dox). Cell stress response, apoptosis, and cell cycle regulation related markers were detected using qPCR and immunocytochemistry supported with resazurin cell viability assay, cell death analysis using flow-cytometry and clonogenic assay.
RESULT: Cell-stress by mEHT alone was indicated by the significant upregulation and release of hsp70 and calreticulin proteins 3 hours posttreatment. Between 3 and 9 hours after treatment significantly reduced anti-apoptotic XIAP, BCL-2, and BCL-XL and elevated pro-apoptotic BAX and PUMA, as well as the cyclin dependent kinase inhibitor p21 mRNA levels were detected. After 24 hours, major elevation and nuclear translocation of phospho-p53(Ser15) protein levels and reduced phospho-Akt(Ser473) levels were accompanied by a significant caspase-3-mediated programmed cell death response. While mEHT dominantly induced apoptosis, Dox administration primarily led to tumor cell necrosis, and both significantly reduced the number of tumor progenitor colonies 10 days post-treatment. Furthermore, mEHT promoted the uptake of Dox by tumor cells and the combined treatment additively reduced tumor cell viability and augmented cell death near to synergy.
CONCLUSION: In C26 colorectal adenocarcinoma mEHT-induced irreversible cell stress can activate both caspase-dependent apoptosis and p21 mediated growth arrest pathways, likely to be driven by the upregulated nuclear p53 protein. Elevated phospho-p53(Ser15) might contribute to p53 escape from mdm2 control, which was further supported by reduced phospho-Akt(Ser473) protein levels. In combinations, mEHT could promote the uptake and significantly potentiate the cytotoxic effect of doxorubicin.