Anticancer effects of combinational treatment with BRAFV600E siRNA and PI3K pathway inhibitors in melanoma cell lines harboring BRAFV600E

Abstract

In the present study, the anti-tumor effects of combination treatment with an siRNA targeting B-Raf proto-oncogene serine/threonine kinase (BRAF)^V600E and phosphoinositide 3-kinase (PI3K) signaling pathway inhibitors was investigated in melanoma cell lines harboring BRAF^V600E. Human melanoma A375 and WM115 cells were treated with siRNA targeting to BRAF or BRAF^V600E, combined with treatment with PI3K signaling pathway inhibitors. CCK-8 and EdU proliferation assays were performed to assess cell viability and proliferation, respectively, following treatment. In addition, flow cytometry analysis was performed to determine cell cycle distribution, and western blot analysis was performed to analyze the activity of the extracellular signal-regulated kinase (ERK) and PI3Ksignaling pathways following treatment. Targeting BRAF^V600E using small interfering (si)RNA significantly decreased cell viability and DNA replication in tumor cell lines that harbor oncogenic BRAF^V600E. Inhibition of BRAF^V600E by siRNA combined with treatment with PI3K or mammalian target of rapamycin signaling pathway inhibitors significantly decreased cell viability and proliferation compared with siRNA or inhibitor treatment alone. Concomitant BRAF^V600E and PI3K inhibition led to G₁/S phase arrest in melanoma cells. However, melanoma cells in which oncogenic BRAF^V600E is not highly expressed (WM115 cells) were not sensitive to BRAF^V600E targeted therapy. The PI3K signaling pathway inhibitors were more effective in this cell line. The results from the present study provide an insight into the potential effectiveness of combination therapy and personalized cancer treatments.

Keywords: BRAFV600E siRNA; ERK pathway; PI3K pathway; combination therapy; melanoma.

Figure 1.

BRAF and BRAF^V600E-targeted siRNAs significantly decrease A375 cell viability. Viability of (A) A375 and (B) HEK293A cells following treatment with 30 nmol/l of siWTM, siMB3, siMEK1, siMEK2 or siNC siRNA for 48 h. (C) DNA replication was assessed by the EdU method in melanoma A375 cells. Cells were treated with or without 30 nM of siWTM, siMB3, siMEK1 +siMEK2, and siControl for 48 h. EdU was stained cells (red) and Hochest 33342 (blue) was stained the nuclei of total cells. (D) The DNA replication measurement on A375 and HEK293A cells with 30 nM of siWTM or siMB3. EdU was stained (red) following 48 h treatment. Data are represented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001, compared with the corresponding control. siRNA, small interfering RNA; EdU, 5-ethynyl-2′-deoxyuridine; BRAF, B-Raf proto-oncogene serine threonine kinase; siWTM, siRNA targeting wild type BRAF and mutant BRAF^V600E; siMB3, siRNA targeting mutant BRAF^V600E; MEK, mitogen-activated protein kinase kinase; NC, negative control.

Figure 2.

Expression of total and mutant BRAF is increased in A375 compared with HEL293A and WM115 cells. (A) Measurement of mRNA level of mutant BRAF^V600E in melanoma A375, WM115 and HEK293A cell lines. (B) Measurement of the protein expression of BRAF, pERK1/2, pAKT(S473), pS6 in A375 and WM115 melanoma cell lines by western blot analysis. (C) Cell viability of A375 and WM115 cells, measured using a CCK-8 assay following treatment with 5 nM of siMB3 for 72 h. p, phosphorylated; ERK, extracellular signal-regulated kinase; AKT, RAC-α serine-threonine-protein kinase; S6, S6 ribosomal protein.

Figure 3.

Cell-growth response curves for melanoma cell lines A375 and WM115. Cells were treated with the indicated concentrations of PI-103, AZD8055, ZSTK474, or GSK6906930 for 72 h and cell viability was measured using a CCK-8 assay. Results represent the mean ± standard deviation of three independent experiments.

Figure 4.

Combination treatment of siMB3 with phosphoinositide 3-kinase/RAC-α serine-threonine-protein kinase/mammalian target of rapamycin inhibitors significantly decreases cell viability compared with siMB3 or inhibitor alone. Cells were treated with the indicated concentrations of AZD8055, PI-103, ZSTK474, or GSK690693 alone or with 5 nM of siMB3 for 72 h. Cell viability was measured using a CCK-8 assay. Results represent the mean of three replicates ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 compared with the corresponding control.

Figure 5.

DNA replication assay in A375 cells following treatment with siMB3 in combination with AZD8055 or with siMB3 or inhibitor alone. (A) Representative images and quantification of DNA replication of A375 cells treated with siMB3, AZD8055, and siMB3/AZD8055 combination using the EdU method. A375 cells were incubated with or without 5 nM siMB3, 100 nM AZD8055 or siMB3/AZD8055 combination, and EdU was used to stain cells (red) following 48 or 72 h treatment. Cell nuclei were stained with Hoechst 33342 (blue). Graphs represent the percentage of total cells with EdU-positive nuclei. (B) Cell cycle assay of siMB3, AZD8055, and siMB3/AZD8055 combination at 48 or 72 h. A375 cells were incubated with or without 5 nM of siMB3, 100 nM of AZD8055, and siMB3/AZD8055 combination, stained with propidium iodide and subjected to flow cytometry analysis 48 or 72 h following treatment.

Figure 6.

Representative western blot analysis of expression levels of BRAF, pERK1/2 (240/244), pS6 (202/204) and pAKT(S473) in A375 cells following 2, 8, 12, 24, 48 and 72 h treatment with 5 nM of siMB3, 100 nM of AZD8055 or siMB3/AZD8055 combination. The expression levels of GAPDH and β-actin were included as loading controls. The results are representative of three independent experiments.

Figure 7.

PI3K/RAC-α serine-threonine-protein kinase/mammalian target of rapamycin signaling pathway modulation in A375 cells following BRAF^V600E knockdown and PI3K inhibition. (A and B) Western blot analyses of A375 cells treated with siMB3 and PI-103 alone or in combination after (A) 48 or (B) 72 h treatment. GAPDH was used as a loading control. (C) Viability of A375 cells following treatment with siMB3/PI-103 combination for 72 h using aCCK-8 assay. (D and E) Western blotting analyses of A375 cells treated with siMB3 and ZSTK474 alone or in combination after (D) 48 or (E) 72 h treatment. GAPDH was used as a loading control. (F) Viability of A375 cells following treatment with siMB3/ZSTK474 combination for 72 h using a CCK-8 assay. *P<0.05 **P<0.01, **P<0.001, compared with the corresponding control.

Figure 8.

DNA replication assay in A375 cells treated with siMB3, PI-103, ZSTK474, siMB3/PI-103 or siMB3/ZSTK474 combination using the EdU method at 48 or 72 h following treatment. Cell nuclei were stained with Hoechst 33342 (blue).