Modulated electro‐hyperthermia induced p53 driven apoptosis and cell cycle arrest additively support doxorubicin chemotherapy of colorectal cancer in vitro

Abstract

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 waf1 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 waf1 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.

Modulated Electro-Hyperthermia-Induced Tumor Damage Mechanisms Revealed in Cancer Models

“…SzIE, ÁOTK MÁB 26/2013 and the most recent one, no. PE/EA/633-5/2018), using mouse C26 colorectal cancer cell line allografted into BALB/c mice, are found in Vancsik et al, 2018 and 2019 [ 12 , 29 ]. In the studies, 10% neutral-buffered, formalin-fixed tumor tissues embedded in paraffin were cut for 2–3 µm thick serial sections and mounted on silanized adhesion glass slides.…”

“…Either using human or animal malignant tumor cell lines under in vitro or in vivo settings, the dominant pathway of tumor destruction after mEHT was apoptosis mediated programmed cell death as opposed to necrosis, as demonstrated mostly using annexin V–propidium iodide flow cytometry [ 29 , 42 ]. Apoptosis was also confirmed by the widespread detection of its characteristic morphological signs in hematoxylin–eosin-stained sections or cell cultures, including nuclear shrinkage and chromatin condensation (picnosis), cell membrane blebbing, and formation of apoptotic bodies ( Figure 1 d) [ 11 , 12 , 18 , 23 ].…”

“…Dividing TDR of the treated by TDR of the distant (untreated) tumor nodules in the same animals defined direct tumor destruction efficiency (TDE) of mEHT. Regulated cell death response was also supported by detecting elevated DNA fragmentation using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay; subG1-phase cell fraction with flow cytometry; and the upregulation and activation of enzymes mediating different pathways of apoptosis using quantitative RT-PCR, RNA sequencing, apoptosis protein arrays, Western blots, and immunohisto-/cytochemistry [ 11 , 29 , 37 , 43 ].…”

“…Krt8-del/met shifts the C26 phenotype towards vimentin expression and supports epithelial–mesenchymal transition (EMT). In C26 culture, a single 30 min shot of mEHT induced the early (3–9 h post-treatment) upregulation of the pro-apoptotic Bax and Puma, and reduced anti-apoptotic Xiap, Bcl-2, and Bcl-xl transcripts [ 29 , 51 , 52 ]. After 24 h, elevation and nuclear translocation of phospho-p53 (Ser15) and reduced phospho-Akt(Ser473) protein levels were detected, along with a significant caspase-3-mediated apoptosis.…”

Sub-Lethal Hyperthermia Enhances Anticancer Activity of Doxorubicin in Chronically Hypoxic HepG2 Cells Through ROS-Dependent Mechanism

“…(Fig. 1a, b) Numerous studies have shown that hyperthermia enhanced intracellular uptake of doxorubicin in different types of tumor cells [19,20]. In this study, we measured doxorubicin positive cells using ow cytometer.…”