CCL2 and IL18 expressions may associate with the anti-proliferative effect of noncontact electro capacitive cancer therapy in vivo

Abstract

Background: Noncontact Electro Capacitive Cancer Therapy (ECCT) is a novel treatment modality in cancer. Chemokine (C-C motif) ligand 2 (CCL2) has a major role in the outgrowth of metastatic breast cancer. Interleukin 18 (IL18) plays a role in macrophage alteration, which leads to excessive angiogenesis. This study aims to elaborate on the association of CCL2, IL18, IL23α, and TNF-α (tumor necrosis factor-alpha) expression with the anti-proliferative effect of ECCT in rat breast tumor tissue. Methods: Low intensity (18 Vpp) and intermediate frequency (150 kHz) alternating current-electric field (AC-EF) between two capacitive electrodes were exposed as external EF to a rat cage. Twenty-four rats were divided into four groups of six replicates. Breast tumor tissues were collected from 7, 12-dimethylbenz[a]anthracene (DMBA)-induced rats. Two groups were non DMBA-induced rats without ECCT exposure (NINT) and with (NIT). The other two groups were DMBA-induced rats without ECCT exposure (INT) and with (IT). Mammary glands and breast tumor tissues were collected from each group and preserved. Hematoxylin-eosin and immunohistochemistry staining were performed on paraffin sections of tissues using anti-PCNA, anti-ErbB2, anti-Caspase3, and anti-CD68. CCL2, IL18, IL23α, and TNF-α mRNA relative expressions were analyzed using qRT-PCR. Results: ECCT exposure may cause the reduction of PCNA protein expression as well as ErbB2 on breast tumor tissues, but it causes the increase of Caspase3 and macrophage CD68 protein. In rat breast tumor tissues of IT groups, the mRNA expression of CCL2 and IL18 are significantly down-regulated, in contrast with the up-regulated expression of these cytokines in tumor tissues of the INT group. IL23α and TNF- α expression remained similar in both groups. Conclusion: CCL2 and IL18 expressions have an association with the inhibition of breast tumor cell proliferation affected by ECCT exposure

Cytotoxic T cells response with decreased CD4/CD8 ratio during mammary tumors inhibition in rats induced by non-contact electric fields

“…Semi-quantitative IHC scoring was performed using the percentage of positive area of stained cells (CD4 and CD8-positive T cells; caspase-3, ErbB2, PCNA-positive tumor cells) and the number of stained cells (CD68-positive macrophages). IHC scoring was performed in ImageJ ver.1.51, a freely available software 22 . The comparison between positive area of stained cells and total area showed the distributions of targeted antigens and cells 30 .…”

“…The data of PCNA, ErbB2, caspase-3, CD68, CD4, and CD8-positive cells and CD4/CD8 ratio were analysed quantitatively using non-parametric Mann-Whitney test, since the data were not normally distributed. All data were analysed statistically using GraphPad Prism ver.8.4.3 software 22 for Windows by the same researcher (NF). Qualitative data (morphology of mammary tumors) were analysed descriptively.…”

“…This study was conducted at the Integrated Research and Testing Laboratory (LPPT) of Universitas Gadjah Mada (UGM) and at the Animal Structure and Development Laboratory of Faculty of Biology, UGM. LPPT UGM has been awarded ISO/IEC 17025:2000 accreditation for the competence of testing and calibration 22 . Experimental protocol in this study was performed following approval by the Ethical Clearance Committee of LPPT UGM with ethical clearance number: 00015/4/ LPPT/IV/2017.…”

“…The SD rats were divided into four groups, namely control (non-induction and non-therapy/NINT), placebo (non-induction and therapy/NIT), DMBA-induced mammary tumors without therapy (induction and non-therapy/INT), and DMBA-induced mammary tumors with therapy (induction and therapy/IT) groups. The sample size in each group was calculated according to the Federer formula, where 6 biological replicates were used for each group 22 . The animals were selected randomly and assigned to control and treatment groups.…”

“…In addition, Pratiwi et al 22 reported the increased expression of CD68 and caspase-3 in rat breast tumor tissues under the exposure to 150 kHz non-contact electric fields while reducing the expression of PCNA and ErbB2 proteins. However, the study by Pratiwi et al 22 showed an increase of the tumor size during therapy. Using different intermediate frequency (100 kHz), we examined the effect of non-contact electric fields exposure on tumor growth in mammary tumorsinduced rats and observed the activation of immune cells in fighting the tumor cells.…”