Propranolol enhanced the anti-tumor effect of sunitinib by inhibiting proliferation and inducing G0/G1/S phase arrest in malignant melanoma

Abstract

Both sunitinib, a multi-target tyrosine kinase inhibitor (TKI) and propranolol, a non-selective β-blocker, have proven therapeutic effects on malignant melanoma (MM). This study reports a synergistic effect of propranolol and sunitinib upon A375, P8 MM cell lines and mice xenografts. Cell viability assays detected a significant decrease of sunitinib IC50 in combination with propranolol, which was confirmed by a colony formation assay. Western blot showed that propranolol and sunitinib combination significantly down-regulated phospho-Rb, phospho-ERK, Cyclin D1, and Cyclin E, but had no effect on Bax, Bcl-2, or cleaved PARP expression. The average tumor size of propranolol and low-dose sunitinib (Sun L) combination treated mice was reduced and similar to high-dose sunitinib treated A375 xenografts. The Ki67 index was significantly reduced in propranolol and Sun L combination treated group compared with single Sun L treated group. This synergistic effect between propranolol and sunitinib to inhibit MM proliferation was through suppressing ERK/Cyclin D1/Rb/Cyclin E pathways and inducing G0/G1/S phase arrest, rather than by inducing tumor cell apoptosis.

Keywords: cell cycle; combination treatment; malignant melanoma; propranolol; sunitinib.

Figure 1. Sunitinib and propranolol effects on cell proliferation in A375 and P8 melanoma cell lines

(A-D) MTS assay measured cell viability after increasing suninitib (1, 5, 10, 15, 20 µM) and propranolol (25, 50, 100, 150, 200 µM) concentrations at 24, 48, and 72H MTS. Relative growth rate was calculated as the ratio of treated to untreated cells at each dose for each replicate. Sunitinib IC50 and propranolol, after incubation for 24, 48, and 72H, was 9.24, 5.3, 3.24µM, and 135, 98.52, 77.3 µM, respectively, in the A375 cell lines. It was 12.03, 7.22, 6.00 µM and 115.6, 76.29 60.3 µM in P8 cell lines respectively.

Figure 2. C-PST effects on cell proliferation in melanoma cell lines

(A-F) An MTS assay calculated survival rate from the combined treatment for sunitinib (1, 2.5, 5 µM) and propranolol (50, 100 µM) concentrations at 24, 48, 72H. Results are presented as mean ± SD. Significant differences were evaluated using one-way ANOVA, and the asterisk (^***) indicates a significant difference compared the same concentration of sunitinib or propranolol alone with sunitinib 2.5 µM and propranolol 50 µM combination group using Dunnett’s multiple comparison test(P<0.001). Pro, propranolol.

Figure 3. C-PST significantly reduced colony growth in A375 and P8 cell lines

A colony formation assay measured A375 cell (A) and P8 lines (B). Cell seed concentration was 2^*10³ and 1^*10³ cells. Cultivation lasted 7 and 9 days respectively. PBS and DMSO, in equal volumes, were mixed as negative controls and treated with propranolol, sunitinib, and the C-PST. (C) Colony formation efficiency is described in A. Error bars represent the standard deviation. Results are presented as mean ± SD, ^***P<0.001.

Figure 4. Cell-cycle analysis (G0/G1, S, and G2/M) of A375 and P8 cell lines

(A-D) Flow cytometric analysis of A375 and P8 human MM cells exposed to propranolol 50 µM, sunitinib 2.5 µM, and C-PST after 24 and 48H cultivation to establish cell percentages in G0/G1, S, and G2/M phases of the cell cycle. Data are presented as mean ± SD.

Figure 5. Cell cycle related western blot analysis of C-PST in MM cells

(A)(F) C-PST suppressed ERK1/2 and Rb phosphorlate levels. Cyclin D1 and Cyclin E expression levels were suppressed in both A375 and P8 cell lines. ERK, p-ERK, Cyclin D1, Rb, p-Rb, and Cyclin E protein fold levels changed in propranolol 50 µM, sunitinib 2.5 µM, the C-PST and the control groups in (B-E) A375 cells and (G, H) P8 cells. Results were determined by three independent experiments and normalized by total protein level. Data are presented as mean ± SD. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. Ctrl.

Figure 6. C-PST inhibited in vivo tumor development

(A) A375 untreated mice (PBS and citrate buffer mixed group) xenograft excised tumors and mice treated with propranolol 2mg/k^*day: Pro, low-dose sunitinib 40mg/kg^*day: Sun L, Sun L+Pro, high-dose sunitinib 80mg/kg^*day: Sun H. (B) Schematic plan for the administration of control (PBS and citrate buffer mixed group), Pro, Sun L, Sun L+Pro or Sun H to tumor-bearing mice. (C) Tumor growth curves measured by average volume of 5 to 7 tumors for each group. (D) All the tumors were removed and measured by average weight for each group at day 15. (E) A375 model mice body weights were measured at the beginning of the test and every three days there after. Results are presented as mean ± SD, ^***P<0.001.

Figure 7. C-PLST inhibited xenograft tumor development

(A) An immunohistochemistry assay assessed Ki67. (B) The Ki67 index. Results are presented as mean ± SD, ^***P<0.001.