RESEARCH ARTICLE Apoptosis and cell cycle arrest of hepatocellular carcinoma spheroids treated by an alternating electric field Chun-Hao Huang 1 | Kin Fong Lei 2,3 | Ngan-Ming Tsang 3,4 1 Program in Biomedical Engineering, College of Engineering, Chang Gung University, Taoyuan, Taiwan 2 Graduate Institute of Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan 3 Department of Radiation Oncology, Chang Gung Memorial Hospital, Linkou, Taiwan 4 Department of Traditional Chinese Medicine, Chang Gung University, Taoyuan, Taiwan Correspondence Kin Fong Lei, Graduate Institute of Biomedical Engineering, Chang Gung University, 259 Wen-Hua 1st Road, Kweishan, Taoyuan, 333 Taiwan. Email: kflei@mail.cgu.edu.tw Funding information Chang Gung Memorial Hospital, Linkou, Grant/ Award Numbers: BMRPC05, CMRPD2H0022 Most of the current cancer therapies may induce serious side effects and affect patient quality of life. Recently, a novel treatment using an alternating low-intensity and intermediate-frequency electric field was proposed and found to be a noninvasive and minimally toxic approach. However, additional fundamental studies and scientific evidence are required to further support the development of this treatment into a standard cancer therapy. In the current work, an in-house fabricated culture plate was developed to study the responses of hepatocellular carcinoma spheroids to treatment with an alternating electric field. From the results of the viability study, the electric field was confirmed to influence the dividing cells in the spheroids. Fluorescent staining of live and dead cells revealed that a fraction of the cells were damaged in the field- treated spheroids. Moreover, flow cytometry analyses were conducted and showed that a fraction of the cells in the spheroids underwent apoptosis and cell cycle arrest. Additionally, the apoptosis pathway (Bax/caspase) and cell cycle arrest pathway (p53/p21) were found to be activated after exposure to the electric field. In summary, the results further elucidated the cellular and molecular mechanism inducing apoptosis and cell cycle arrest in the field-treated hepatocellular carcinoma spheroids. This study provides more evidence to support the efficacy of electric-field-based cancer therapy. KEYWORDS apoptosis, cancer therapy, cell cycle arrest, electrical field, hepatocellular carcinoma spheroids 1 | INTRODUCTION In the past decade, alternating electric fields with low intensity (1–3 V/cm) and intermediate frequency (100–300 kHz) have been reported to influence cell division, thereby inhibiting cell growth. 1–3 Cells under the electric field were perturbed during the transition from metaphase to anaphase. Cell apoptosis and proliferation arrest were induced by aberrant mitotic exit. 4,5 in vitro studies and animal experiments confirmed that the electric field selectively affected the rapidly proliferating cells. 1–5 A new cancer treatment technique was then developed, called tumor-treating fields (TTF). In TTF, an alternat- ing electric field is delivered by an insulated electrode array attached to the skin surrounding the tumor region. Clinical studies reported that glioblastoma multiforme (GBM) patients treated with TTF therapy had a longer overall survival than patients treated with standard therapy. 6–8 Long-term survival of recurrent GBM patients has also been reported. 9,10 An apparently increase in overall and progression- free survival was concluded in randomized clinical trials of TTF therapy in combination with anticancer drugs. 11–14 However, another randomized clinical trial reported there was no improvement in overall survival for recurrent GBM patients treated with TTF therapy alone compared with the patients treated with chemotherapy. 15 Currently, the clinical efficacy of TTF therapy alone remains controversial. Since TTF therapy has not been adopted as one of the standard options for cancer therapy, more fundamental studies and scientific evidence are required to support the use of this novel cancer therapy. The in vitro cell culture technique is a widely used method to study cellular responses to extracellular stimulation in scientific Received: 25 July 2018 Revised: 17 December 2018 Accepted: 5 February 2019 DOI: 10.1002/btpr.2787 Biotechnology Progress. 2019;35:e2787. wileyonlinelibrary.com/journal/btpr © 2019 American Institute of Chemical Engineers 1 of 9 https://doi.org/10.1002/btpr.2787