Proto-Oncogenes and Cell Cycle Gene Expression in Normal and Neoplastic Oral Epithelial Cells Stimulated With Soluble Factors From Single and Dual Biofilms of Candida albicans and Staphylococcus aureus

Abstract

This study was aimed at analyzing proto-oncogenic signaling pathway activation in normal oral keratinocytes (NOK-si) and neoplastic cell lines (SCC 25 and Detroit 562) stimulated with metabolites (soluble factors) from single and dual biofilms of Candida albicans and Staphylococcus aureus. Soluble factors (SF) from early (16-h) and mature (36-h) biofilms of C. albicans and S. aureus were collected and incubated with cell cultures, which were subsequently evaluated using gene expression via RT-qPCR, cell viability via AlamarBlue^TM, and flow cytometry cell cycle analysis. In general, exposure to the SF of early and mature biofilms from C. albicans and dual species caused a major reduction in NOK-si cell viability and enhanced the sub G0 phase. This led to a decrease in gene expression. However, in this cell line, SF of S. aureus biofilms upregulated the CDKN1A gene followed by the maintenance of cell viability and a significant increase in the G2/M population. For tumor cells, SCC 25 and Detroit 562, the stimuli of SF biofilms upregulated oncogenes such as hRAS and mTOR, as well as Bcl-2 and CDKN1A. SCC 25 and Detroit 562 cells could survive even after 24 h of stimuli from both SF (early and mature). This occurred without significant changes taking place in the cell cycle progression for SCC 25, and with a significant tendency to increase the G2/M phase for Detroit 562. These results point to the fact that metabolites from prevalent clinical fungal and bacterial biofilms, C. albicans and S. aureus, can disrupt the homeostasis of normal and neoplastic oral epithelial cells. This changes proto-oncogenes' expression, specifically PI3KCA, hRAS, mTOR, BRAF, and cell cycle genes CDKN1A and Bcl-2, thus causing a disturbance in cell viability, survival, and the cell cycle profile.

Keywords: Candida albicans; Staphylococcus aureus; biofilms; gene expression; metabolites; oral cancer.

Figure 1

Cell viability profile. AlamarBlue^TM assay performed to evaluate the viability profile of cell lines NOK-si (A), SCC 25 (B), and Detroit 562 (C) upon 4 h and 24 h of stimulation with soluble factors (SF) from C. albicans (Ca), S. aureus (Sa), and dual species (D) of early (16-h) and mature (36-h) biofilms. One-way analysis of variance, Kruskal-Wallis test, and Dunn's Multiple Comparison Test [*compared with the control (Ct) without a stimulus]. **p < 0.01, ***p < 0.001.

Figure 2

Cell cycle distribution index. NOK-si (A), SCC 25 (B), and Detroit 562 (C) cell lines stimulated with soluble factors (SF) from C. albicans (Ca), S. aureus (Sa), and dual species (D) from early (16-h) (A, C, E) and mature (36-h) (B, D, F) biofilms. Cells were fixed and labeled with propidium iodide, and their DNA content was measured using flow cytometry. The cell cycle distribution index (SubG0, G0/G1, S, G2/M) was analyzed after 4 and 24 h of stimulation.

Figure 3

Cell cycle distribution histogram. The distribution rate of the cell cycle populations G0/G1 (blue), S (yellow), and G2/M (green) were analyzed for cell lines NOK-si (A, B), SCC 25 (C, D), and Detroit 562 (E, F) upon 4 h and 24 h of stimulation with soluble factors (SF) of C. albicans (Ca), S. aureus (Sa), and Dual species (D) from early (16-h) (A, C, D) and mature (36-h) (B, D, F) biofilms. The box inside of each histogram brings the distribution rate (%) of each cell cycle population. Sub G0/G1: population seen to the left of the G0/G1 peak. The Watson Pragmatic model was performed using the FlowJo ™ Software v10 program.

Figure 4

Proto-oncogenes and cell cycle gene expression upon SF from early biofilms. Gene expression was evaluated using the RT-qPCR analysis of NOK-si, SCC 25, and Detroit 562 upon 4 h of stimulation with soluble factors (SF) of C. albicans (Ca) (□), S. aureus (Sa) (•), and dual species (D) (Δ) from early 16-h biofilms. Data are shown as fold change values compared with cells stimulated with RPMI-1640 medium (control – black line) supplemented using the 2^-ΔΔCt method (HPRT was used as the internal control). *IC 95% means a significant difference vs. the control.

Figure 5

Proto-oncogenes and cell cycle gene expression upon SF from mature biofilms. Gene expression was evaluated by the RT-qPCR analysis of NOK-si, SCC 25, and Detroit 562 upon 4 h of stimulation with soluble factors (SF) of C. albicans (Ca) (□), S. aureus (Sa) (•), and dual species (D) (Δ) from mature 36-h biofilms. Data are shown as fold change values compared with cells stimulated with RPMI-1640 medium (control – black line) supplemented using the 2^−ΔΔCt method (HPRT was used as internal control). *IC 95% means a significant difference vs. the control.