Rhopalurus junceus scorpion venom induces G2/M cell cycle arrest and apoptotic cell death in human non-small lung cancer cell lines

Abstract

Background: Non-small cell lung cancers (NSCLC) represent the primary cause of cancer-related deaths worldwide. Rhopalurus junceus venom has been shown to exert cytotoxic effects against a panel of epithelial cancer cells in vitro and suggested that NSCLC was the subtype most susceptible to the treatment.

Methods: This study evaluated the effect of Rhopalurus junceus scorpion venom on cell viability, in non-cancerous (MRC-5, lung; CHO-K1, ovary) and NSCLC (A549; NCI-H460) cell lines. The effects on cell cycle, apoptosis, and cell signaling-related proteins were determined by flow cytometry and WB. Protein fractions responsible for the observed effect were identified using HPLC.

Results: Scorpion venom was more effective against NSCLC than non-cancerous cells. E_max values were 20.0 ± 5.8% and 22.47 ± 6.02% in A549 and NCI-H460 cancer cells, respectively, as compared to 50 ± 8.1% in MRC-5 and 54.99 ± 7.39% in CHO-K1 cells. It arrested NSCLC cells in the G2/M phase, while non-cancerous cells were arrested in the S (MRC-5) or G0/G1 (CHO-K1) phases. No changes were observed in the Bax/Bcl-2 or the cleaved-caspase 3/Total caspase 3 ratios in cells treated with venom. Likewise, the scorpion venom treatment did not affect p-ERK, p-AKT, or p-38MAPK protein levels. In contrast, scorpion venom treatment increased the cytosolic apoptosis-inducing factor (AIF) in A549 cells, indicating caspase-independent apoptosis. Additionally, combined etoposide/venom exposure provoked G2/M arrest and apoptosis in NSCLC more strongly than either substance alone. Furthermore, upon crude venom fractioning through RP-HPLC, we found two soluble fractions with high cytotoxic effects.

Conclusion: The present study concludes that a specific fraction of Rhopalurus junceus venom reduces cell viability of NSCLC cells. The AIF protein plays a key role in mediating caspase-independent apoptotic cell death. These findings suggest that Rhopalurus junceus venom enhances the anticancer effect of etoposide in vitro by causing cell cycle arrest and caspase-independent apoptosis.

Keywords: Apoptosis; Cell cycle arrest; Rhopalurus junceus; Scorpion venom; Synergism.

Figure 1.. (A) Morphological effects in cancerous (A549, NCI-H460) and non-cancerous (MRC-5, CHO-K1) cells treated with R. junceus scorpion venom. (B) Cells were seeded in 96-well plates and treated for 72 h with increasing concentrations from 0.062 to 2 mg/mL. Dose-response curves were determined by MTT assay. The culture media was used as negative control. Results are expressed as the percentage of control. (C) IC₅₀ values obtained from dose-response curves fitted to the Hill equation. (D) Maximum cell viability inhibition was obtained from the fit to the Hill equation (E_max) in all cell lines at the highest scorpion venom concentration. *p < 0.05 compared to non-cancerous cells from Kruskal-Wallis non-parametric test. The experiments were performed three times with five technical replicates. Data values are expressed as mean ± SE. Scale bar: 100 µm.

Figure 2.. Effect of R. junceus scorpion venom on the cell cycle in non-cancerous and cancerous cells. (A) Representative histograms of the cell cycle in the MRC-5 cell line, after scorpion venom treatment for 48 h. Bar graph summary representing the percentage of cells in G0/G1, S, and G2/M in MRC-5 cells. (B) Representative histograms of the cell cycle in the CHO-K1 cell line, after scorpion venom treatment for 48 h. Bar graph summary representing the percentage of cells in G0/G1, S, and G2/M in CHO-K1cells. (C) Representative histograms of the cell cycle in the A549 cell line, after scorpion venom treatment for 48 h. Bar graph summary representing the percentage of cells in G0/G1, S, and G2/M in A549 cells. (D) Representative histograms of the cell cycle in the NCI-H460 cell line, after scorpion venom treatment for 48 h. Bar graph summary representing the percentage of cells in G0/G1, S, and G2/M in NCI-H460 cells. Cells (5 × 10⁵) were plated in 60 mm dishes and treated with ½IC₅₀, IC_50, or 2xIC₅₀ venom for 48 h. The cells were stained with propidium iodide, and the cell cycle was analyzed by flow cytometry. Each bar represents mean ± SE (n = 6). *p < 0.05 compared to control (the culture media in absence of scorpion venom) from Kruskal-Wallis non-parametric test.

Figure 3.. Analysis of apoptotic cell death in NSCLC A549 and NCI-H460 cells treated with R. junceus scorpion venom. Cells (5 × 10⁵) were plated in 60 mm dishes and treated with ½IC₅₀, IC_50, or 2xIC₅₀ venom for 48 h. Cells were stained with FITC-conjugated Annexin V and PI for flow cytometric analysis. (A) Representative scatter plot of PI (y-axis) and Annexin-V (x-axis) as measurements of apoptotic cell death. (B) Bar graph of the percentage of total apoptosis determined by flow cytometry (n = 6). *p < 0.05 compared to the control (the culture media in absence of scorpion venom) from the ANOVA test. Cells in the lower right (Annexin V⁺/PI^-) represent early apoptosis and the upper right (Annexin V⁺/PI⁺) represent late apoptosis.

Figure 4.. Analysis of cell cycle and apoptosis in serum-starved A549 cells treated with R. junceus scorpion venom (2xIC₅₀) for 24 h, and 48 h after 10% FBS release. (A) Representative histograms of the cell cycle in A549 cells, after scorpion venom treatment. (B) Bar graph summary representing the percentage of cells in G0/G1, S, and G2/M. Bars represent an average of six measurements. (C) Representative scatter plot of PI (y-axis) and Annexin-V (x-axis) as measurements of apoptotic cell death. (D) Bar graph of the percentage of total apoptosis determined by flow cytometry (n = 6). (E) Bar graph of the percentage of necrosis determined by flow cytometry (n = 6). *p < 0.05 compared to control (the culture media in absence of scorpion venom) from the ANOVA test. Cells in the lower right (Annexin V⁺/PI^-) represent early apoptosis and the upper right (Annexin V⁺/PI⁺) represent late apoptosis.

Figure 5.. Relative protein expression of target proteins in A549 cells following R. junceus scorpion venom treatment at ½IC₅₀, IC₅₀, 2xIC₅₀ for 48 h. Total cell lysates were obtained and then subjected to western blot analysis to measure the expression levels of proteins. (A) Western blot of apoptosis-related proteins Bax and Bcl-2. (B) Western blot of apoptosis-related protein caspase 3. (C) Western blot of cleaved-AIF protein. (D) Bar graph from Bax. (E) Bar graph from Bcl-2. (F) Bar graph from Bax/Bcl-2 ratio. (G) Bar graph from cleaved caspase 3/total caspase 3 ratio. Each bar represents mean ± SE (n = 3). (H) Bar graph of mean fold change from cleaved AIF protein. The mean fold change was plotted for each sample on a bar graph. *p < 0.05 compared to the control (the culture media in absence of scorpion venom) from Kruskal-Wallis non-parametric test. Bax and Bcl-2 signals were individually normalized to (-tubulin. AIF signal was normalized to β-Actin.

Figure 6.. Relative protein expression of cell signaling-related proteins in A549 cells following R. junceus scorpion venom treatment at ½IC₅₀, IC₅₀, 2xIC₅₀ for 48 h. (A) Western blot and bar graph from phosphorylated p38/total p38 ratio. (B) Western blot and bar graph from phosphorylated p42/44/total p42/44 ratio. (C) Western blot and Bar graph from phosphorylated pAKT/total AKT ratio. Each bar represents mean ± SE (n = 3). *p < 0.05 compared to control (the culture media in absence of scorpion venom) from Kruskal-Wallis non-parametric test.

Figure 7.. (A) Concentration-response curve from etoposide treatment in A549 cells. (B) Bar graph showing the cell viability determined by MTT assay in A549 cells at different concentrations of R. junceus venom (½IC₅₀, IC₅₀, 2xIC₅₀) combined with etoposide (IC₅₀). Untreated cells represent 100% cell viability. The culture medium was used as the negative control. *p < 0.05 respect to etoposide as a single treatment. The experiments were performed three times with five technical replicates. (C) Normalized isobologram of in vitro drug-to-drug interaction between R. junceus venom (RjSV) and etoposide (Eto) in A549 cancer cell line based on CompuSyn analysis from MTT data. (D) Representative cell cycle histogram and bar graph summary representing the percentage of cells in G0/G1, S, and G2/M after 48-h treatment. Bars represent an average of six measurements. (E) Representative scatter plot of PI (y-axis) and Annexin-V (x-axis) as measurements of apoptotic cell death and bar graph of the percentage of total apoptosis determined by flow cytometry (n = 6). *p < 0.05 compared to the control (the culture media in absence of scorpion venom) from the ANOVA test.

Figure 8.. Relative protein expression of target proteins in A549 cells following R. junceus scorpion venom (IC₅₀), etoposide (Eto, IC₅₀), and combined treatment. (A) Western blot of apoptosis-related proteins Bax and Bcl-2. (B) Western blot of apoptosis-related protein caspase 3. (C) Bar graph from Bax. (D) Bar graph from Bcl-2. (E) Bar graph from Bax/Bcl-2 ratio. (F) Bar graph from cleaved caspase 3/total caspase 3 ratio. Each bar represents mean ± SE (n = 3). *p < 0.05 compared to control (the culture media in absence of scorpion venom) from Kruskal-Wallis non-parametric test. Bax and Bcl-2 signals were individually normalized to (-tubulin.

Figure 9.. Reverse phase-HPLC chromatogram and MTT assay of Rhopalurus junceus scorpion venom fractions. (A) Reverse phase-HPLC chromatogram of R. junceus venom. Venom was injected into the C18-reverse phase column and run in HPLC. The run conditions were as follows: phase A - 0.1% TFA in HPLC grade water; phase B - 0.1% TFA in acetonitrile. Scorpion venom was eluted at 1 mL/min flow rate using a linear gradient of 0-45% of solvent B for 45 min. Fractions indicated with an arrow were collected individually from each run. (B) Evaluation of selected fractions at a unique concentration of 500 µg/mL, against MRC-5 and A549 cells. Cells were seeded in 96-well plates, treated for 72 h, and analyzed by MTT assay. (C) Dose-response curves of cells seeded in 96-well plates, treated for 72 h with increasing concentrations (15.1, 31.25, 62.5, 125, 250, 500, 1000 µg/mL) of fractions F4 and F5 and determined by MTT assay. IC₅₀ values were obtained from dose-response curves fitted to the Hill equation. The culture medium was used as the negative control. Results were expressed as the percentage of control. *p < 0.05 compared to non-cancerous cells from Kruskal-Wallis non-parametric test. The experiments were performed three times with three technical replicates. Data values were expressed as mean ± SE.