Molecular Mechanisms Underlying Antiproliferative and Differentiating Responses of Hepatocarcinoma Cells to Subthermal Electric Stimulation

Abstract

Capacitive Resistive Electric Transfer (CRET) therapy applies currents of 0.4–0.6 MHz to treatment of inflammatory and musculoskeletal injuries. Previous studies have shown that intermittent exposure to CRET currents at subthermal doses exert cytotoxic or antiproliferative effects in human neuroblastoma or hepatocarcinoma cells, respectively. It has been proposed that such effects would be mediated by cell cycle arrest and by changes in the expression of cyclins and cyclin-dependent kinase inhibitors. The present work focuses on the study of the molecular mechanisms involved in CRET-induced cytostasis and investigates the possibility that the cellular response to the treatment extends to other phenomena, including induction of apoptosis and/or of changes in the differentiation stage of hepatocarcinoma cells. The obtained results show that the reported antiproliferative action of intermittent stimulation (5 m On/4 h Off) with 0.57 MHz, sine wave signal at a current density of 50 µA/mm2, could be mediated by significant increase of the apoptotic rate as well as significant changes in the expression of proteins p53 and Bcl-2. The results also revealed a significantly decreased expression of alpha-fetoprotein in the treated samples, which, together with an increased concentration of albumin released into the medium by the stimulated cells, can be interpreted as evidence of a transient cytodifferentiating response elicited by the current. The fact that this type of electrical stimulation is capable of promoting both, differentiation and cell cycle arrest in human cancer cells, is of potential interest for a possible extension of the applications of CRET therapy towards the field of oncology.

Electric Stimulation at 448 kHz Promotes Proliferation of Human Mesenchymal Stem Cells

“…Indeed, our late passage cells showed a number of genetic abnormalities, including aneuploidy (data not shown) that are characteristic of senescent cultures. The possibility that CRET elicits antiproliferative effects in senescent ADSC could take partial support from previous studies by our group, which report that CRET treatment causes antiproliferative effects in hepatocarcinoma HepG2 and neuroblastoma NB69 [32,33,34,35,36], two human cancer cell lines known to carry a variety of genetic alterations [52,53]. …”

“…For sham-exposure, the electrode pairs inserted in control dishes were also connected to the generator, but not energized. The stimulation pattern consisted of 5-minute pulses of 448 kHz sine wave current, at a subthermal density of 50 μA/mm 2 , separated by 4-h interpulse lapses, along a total period of 48 h. Such exposure parameters have been proven to affect cell proliferation in previous studies by our group [32,33,34,35,36]. The cultures were grown in two separate, identical CO 2 incubators (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…However, evidence exists indicating that, at least at the cellular level, the CRET effects are not limited to the thermal ones. In fact, previous studies by our group have shown that CRET stimulation at subthermal doses can induce anti-proliferative and cytotoxic responses in cultured human cancer cell lines, but not in primary cultures of human peripheral blood mononuclear cells [32,33,34,35,36]. These results can be interpreted as an experimental support to the evidence that the effects of CRET medical therapies are not due exclusively to temperature increase in the exposed tissues, but also to direct cellular responses to the electric stimulus itself.…”

First Assessment of the Proionic Effects Resulting from Non-Thermal Application of 448 kHz Monopolar Radiofrequency for Reduction of Edema Caused by Fractional CO2 Laser Facial Rejuvenation Treatments

“…Proionic effects or proionic system is the name given to the set of phenomena that take place at cellular level and lead to the restoration of the cell membrane's normal potential, as well as the ionic balance established through the cell membrane. Although radiofrequency (RF) has been and is traditionally used for its thermal effects, Hernández-Bule et al [2,3,4,5,6] have described effects resulting from subjecting various tissues to a 448 kHz electromagnetic field at intensities which are not capable of heating such tissues, showing the occurrence of molecular phenomena other than those caused by the well-known classic thermal effects. Among these, proionic phenomena stand out.…”

Synergic Carcinostatic Effects of Ascorbic Acid and Hyperthermia on Ehrlich Ascites Tumor Cell

“…Capacitive-resistive electric transfer (CRet) system applies electric currents at frequencies within the 0.4-0.6 MHz range to treatment of inflammation and musculoskeletal injuries [16][17][18][19]. It has been demonstrated that CRet system, which can produce electric stimuli and thermal effects simultaneously in tissues, can induce synergistically cytostatic and/ or cytotoxic effects in tumor tissues [20,21].…”