Introduction

World Health Organization (WHO) defined cancer as broad spectrum of diseases which can affect almost each and every system of the body when abnormal cells grow and divide in an uncontrollable manner, move away from their usual limitations to infiltrate adjacent parts of the body, and/or travels to other organs. The later progression is called metastasizing and it is the prime causes of death from cancer.^[1]

Another definition said that “most cancer is a complex and heterogeneous set of diseases without a simple definition”. In addition, it is believed that between 5 and 10% of cancer cases are brought on by mutation and up to 15% by inflammation; a few 80% of cancer cases are "sporadic" tumors, the cause of which is yet unclear. Even mutations are late events, or epiphenomena, in a complex chain of circumstances that can explain the genesis of the majority of cancer form.^[2]

Cancer primarily develops as a result of chromosomal instability. The CBMN cytome assay, which Fenech^[3] demonstrated to be a multi-endpoint cytogenetic approach, allowed assessments of a variety of nuclear anomalies suggestive of a variety of chromosomal instabilities. Chromosomal aberrations include structural/numerical chromosome abnormalities, chromosome mal-segregation occurring throughout mitosis and manifested as micronuclei (MNi), anaphase bridge formation manifested as nucleoplasmic bridges (NPBs), and gene amplification or elimination of unresolved DNA complexes manifested as nucleoplasmic buds (NBUDs).

Plasma medicine is an interdisciplinary research field that combines plasma physics and chemistry with biology and clinical medicine to bring new modalities of cancer treatment to market. Application of plasma to cellular structures has been found to generate abundant reactive species. Their interaction with cells manipulates cellular redox signaling, ultimately resulting in changes in surface receptor function, initiation of cell cycle arrest, activation of
 

p53 by DNA damage, and subsequent p53-dependent apoptosis and other effects on various cells.^[4]

According to another viewpoint, The goal of cancer therapy is to encourage the death of malignant cells while sparing healthy cells from too much harm (selectivity).^[5] As well, the absence of functionality of tumor-suppressor gene or the overexpression of an anti-apoptotic protein are both important pathways in cancer development^[6] P53 referred to as "the guardian of the genome."^[7] In addition, The Bcl2 family of proteins is widely studied for its role in apoptosis.^[8] Even the absence of apoptosis leads to cancer.^[9]

All regulation and communication between our body and mind go in a bidirectional flow of neuropeptides, cytokines, and hormones.^[10] Cytokines are the major mediators of communication, particularly regarding inflammation and infection.^[11] Cytokines play a highly critical position in system homeostasis at some point of immune challenge.^[12]

Additionally, they claimed that DNA damage responses could be in charge of the start of senescence after inflammatory mediators released a variety of cytokines, including IL-1, IL-6, IL-8, and TGF-β.^[13] Pro-inflammatory cytokines like TNF-α, IL-1, and IL-6, as well as the anti-inflammatory IL-10 family, cause up- and down-regulation of cellular activity.^[14]

CAPPJ efficacy has been estimated in a variety of medical conditions such as osteosarcoma, glioblastoma, melanoma, pancreatic, ovarian, breast, and cervical cancer ...etc. it’s entirely believed that the major mechanism of the plasma’s efficacy is facilitated through the stimulation of debauchery of energy via radiation and/or chemical energy.^[15]

The goal of the current study is to try to explain how plasma affects cells accurately through its roles in oxidative stress, changes in redox state pathways, or direct impact on DNA (epigenetically), Then, it tries to interpret the interaction between plasma and DNA and its reflection on the realization of the concept for cancer definition factors and its treatment.

Materials and Methods

Experimental design

The presented work is designed to clarify the following: Direct and indirect (irradiated cultures) effects of CAPPJ exposure on the normal whole blood samples and breast cancerous cells. harmless and effective exposure doses of CAPPJ for normal and cancerous cells respectively and the relationship between genetic damage and triggering of the immunological cytokines.

Characters of the plasma jet supplied by the tesla coil

A cold plasma jet's characteristics may be crucial for having a better impact in the biological application, according to Elaragi^[16] who created a CAPPJ powered by a Tesla coil. As a result of the breakdown of a brief spark gap, the capacitor in Tesla's coil transformer connected to a coil with a few turns, creating a resonant circuit with an oscillation frequency that was typically between 20 and 100 kHz and was controlled by the capacitance of the capacitor and the inductance of the coil. A plasma jet generator, gas flow circuit, and high voltage, low current, and high frequency AC make up the experimental system as shown in (Figure 1).

Chemicals

Chemicals for blood culture were purchased from Gibco (Carlsbad, CA, USA) and heat-inactivated fetal calf serum (FCS) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Determination of IL-1β: Human (IL-1β) ELISA Kit was purchased from CUSABIO with Catalog Number: CSB-E08053h. IL-6: Human (IL-6) ELISA Kit was purchased from RayBio with Catalog Number: ELH-IL6-001. IL-10: Human (IL-10) ELISA Kit was purchased from CUSABIO. TNF-α: Human TNF-α ELISA Kit was purchased from ALPCO with Catalog Number: 45-TNFHU-E01. Finally, anti- P53 primary antibody was purchased from Abcam, ab131442, and anti- Bcl2 primary antibody from Abcam, ab131442.

Subjects and blood sampling

Subjects

Normal human blood samples: Eight healthy volunteers (4 males and 4 females) with blood samples were collected in order to reduce any potential inter-individual heterogeneity in treatment response. The donors matched for age, smoking, and environment.

Breast cancer cell line: it was purchased from the Genetic Engineering Unit, National Research Centre, Egypt.

All human blood samples had been drawn under sterile conditions and placed in a heparinized vacutainer tube (v = 5 ml; Becton Dickinson, USA).

Exposure doses

Normal whole blood samples were taken from everyone and split into two categories: the first was for direct CAPPJ exposure to four dose groups (20 (Bl-D1), 40 (Bl-D2), 60 (Bl-D3), and 120 (Bl-D4) sec, respectively) at a distance of 3 cm from the blood surface, and the second was for an unexposed group (control group). The second exposure was indirect and involved normal blood that was grown on CAPPJ-exposed media with the same 4 dosages (20 (Cl-D1), 40 (Cl-D2), 60 (Cl-D3), and 120 (Cl-D4) sec, respectively. The identical doses were administered directly after conception to the Breast Cancer Cell Line (BCC).

Blood culture

Before beginning culture and immunological investigations, blood samples incubated for 20 hours at 37 °C. For each sample, triple blood cultures established for 72 hours in accordance with the Evans and O'Riordan methodology.^[17]

Cytokinesis-block micronucleus cytome assay, immunological parameters, gene expressions, and cell viability assay

Cytokinesis-block micronucleus cytome assay was carried out as described by Fenech. ^{[3, 18]} The assay evidenced mitotic alert (mono-, bi-, tri-, and quadrinucleated cells), cytotoxicity (MNi frequencies, NPBs, and nuclear buds), necrotic and apoptotic cells. An enzyme-linked immunosorbent assay for the quantitative measurement of human IL-1β, IL-6, IL-10, and TNF-α concentrations in cell culture supernatants and plasma. P53 and Bcl2 gene expressions were observed using electrophoresed proteins on SDS-PAGE in accordance with Horiuchi et al.^[19]

Statistical analysis

The statistical analysis was performed using the Statistical Package for Social Science (SPSS) software version 20 for Windows, a one-way analysis of variance (ANOVA), and Tukey multiple comparison tests (P values of 0.05 were considered significant.^[20]

Results and Discussion

Table 1 represents the cytogenetic data of the direct effect of CAPPJ on the normal whole blood; Table 2 indicates indirect CAPPJ effects on the normal whole blood.

a: significant when compared with control gp

b: significant when compared with Bl-D1 gp

c: significant when compared with Bl-D2 gp

d: significant when compared with Bl-D3 gp

a: significant when compared with control gp

b: significant when compared with Cl-D1 gp

c: significant when compared with Cl-D2 gp

d: significant when compared with C-D3 gp

Samples exposed to CAPPJ with different doses (Control, Bl-D1, Bl-D2, Bl-D3, and Bl-D4 groups) illustrated in Table 1. The same previous parameters were recorded for normal human blood cultivated in irradiated culture with the same CAPPJ doses (Cl-D1, Cl-D2, Cl-D3, and Cl-D4) in Table 2.

Table 1 showed that, a significant decrease in the genetic damage for both doses (Bl-D2 and Bl-D3) groups (mono + 1 Mn, bi+ 1Mn, and nuclear buds) when compared with control and Bl-D1, where the Bl-D4 group recorded significantly high incidences of Mni in mono- and binucleated cells.

Table 1 illustrated a gradually significant increase in the incidence of apoptotic cells from group Bl-D1 to Bl-D4 in comparison with the control group. There are no significant differences for mono+2MN, bi+2MN, bi+3MN, trinucleated, quadrinucleated, and nucleoplasmic bridge when its values are compared between all groups. Necrotic cells in Bl-D4 recorded a high frequency of incidence in comparison with the other groups.

There is a significant difference between Bl-D2, Bl-D3, and Bl-D4 in mononucleated and binucleated cell counts. The data in Table 2 is opposed to that obtained in Table 1. There is a high incidence of frequencies of Mni in both mono- and binucleated cells and nuclear buds. Also, necrotic and apoptotic cells recorded high significant frequencies, especially Cl-D4 when compared to control and Cl-D1groups. But, Cl-D3 and Cl-D4 groups displayed significant differences in trinucleated and quadrinucleated counts when compared with control and other groups.            

Table 3 illustrated BCC viability percentages and protein expression of Bcl2 and P53 genes. The BCC - Control group is considered a reference for cell viability with a value of 100%. There are gradual decreases in the cell line from the BCC-D1 group to the BCC-D4 group (82%, 64%, 51%, and 10%) respectively. While results recorded a decrease in Bcl2 protein expression there are increasesinP53 gene expressions.

In Table 4 the most effective dose was observed in the Bl-D2 group for the Bcl2 gene expression and their significant difference between all exposed groups when compared with the control group. On the other hand, there is a significant increase for (Bl-D2, Bl-D3, and Bl-D4) when compared with control and Bl-D1 groups for P53 gene expression.

It is also displayed a regular decrease in protein expression of Bcl2 genes from Cl-D1 to Cl-D4 groups when compared with the control group, which opposes the regular elevation of protein expression of P53 from Cl-D1 to Cl-D4 groups when compared with the control group.

(Figure 2a) illustrated that the first dose group BL-D1 had a non-significant difference in the concentrations of IL-1β and TNF-α compared to the control group. While there are differences in levels of IL-6 and IL-10 that were significantly increased. At BL-D2, BL-D3, and BL-D4, there is a progressive and significant increase in concentrations of IL-1β and IL-6 compared to controls and BL-D1. Whereas, IL-10 levels were raised in the BL-D2 group and gradually decreased in the BL-D3 and BL-D4 groups. TNF-α recorded increasing levels through BL-D2 and BL-D3 groups but significantly decreased in the BL-D4 group. The corresponding attitude occurs for the same rating data from (Figure 2b) except for the IL-1β values.

(Figure 3) indicated data of irradiated culture media before cultivation and demonstrated that IL-1β, IL-6, IL-10, and TNF-α concentrations values were opposed to those recorded for all groups in (Figures 2a and 2b) that there are gradually decreasing through the four doses groups.

(Photomicrographs illustrated the different forms of the cell types of cytome assay) (Figure 4).