Rapamycin inhibits B16 melanoma cell viability invitro and invivo by inducing autophagy and inhibiting the mTOR/p70‑S6k pathway

Abstract

Rapamycin is an immunosuppressant that has been shown to prevent tumor growth following organ transplantation. However, its exact mode of antitumor action remains unknown. The present study used the B16-F10 (B16) murine melanoma model to explore the antitumor mechanism of rapamycin, and it was revealed that rapamycin reduced B16 cell viability in vitro and in vivo. In addition, in vitro and in vivo, the results of western blotting showed that rapamycin reduced Bcl2 expression, and enhanced the protein expression levels of cleaved caspase 3 and Bax, indicating that it can induce the apoptosis of B16 melanoma cells. Furthermore, the results of cell cycle analysis and western blotting showed that rapamycin induced B16 cell cycle arrest in the G₁ phase, based on the reduction in the protein expression levels of CDK1, cyclin D1 and CDK4, as well as the increase in the percentage of cells in G₁ phase. Rapamycin also significantly increased the number of autophagosomes in B16 melanoma cells, as determined by transmission electron microscopy. Furthermore, the results of RT-qPCR and western blotting showed that rapamycin upregulated the protein expression levels of microtubule-associated protein light chain 3 (LC3) and Beclin-1, while downregulating the expression of p62 in vitro and in vivo, thus indicating that rapamycin could trigger cellular autophagy. The present study revealed that rapamycin in combination with chloroquine (CQ) further increased LC3 expression compared with that in the CQ group, suggesting that rapamycin induced an increase in autophagy in B16 cells. Furthermore, the results of western blotting showed that rapamycin blocked the phosphorylation of p70 ribosomal S6 kinase (p70-S6k) and mammalian target of rapamycin (mTOR) proteins in vitro and in vivo, thus suggesting that rapamycin may exert its antitumor effect by inhibiting the phosphorylation of the mTOR/p70-S6k pathway. In conclusion, rapamycin may inhibit tumor growth by inducing cellular G₁ phase arrest and apoptosis. In addition, rapamycin may exert its antitumor effects by inducing the autophagy of B16 melanoma cells in vitro and in vivo, and the mTOR/p70-S6k signaling pathway may be involved in this process.

Keywords: B16 melanoma cells; apoptosis; autophagy; cell cycle; mTOR/p70-S6k signaling pathway; rapamycin.

Figure 1.

RAPA inhibits B16 cell viability in vitro. Cells were treated with RAPA for 48 h. (A) Cell viability was detected by MTT assay. (B) IC₅₀ value of RAPA in B16 cells. Data are presented as the mean ± SD logC: for 10⁻³, 10⁻², 10⁻¹, 10°, 10¹, 10², 10³, 10⁴ and 10⁵ nM take the logarithm of the base 10. (n=3). **P<0.01 and ****P<0.0001 vs. RAPA 0 nM group. B16, B16-F10; IC₅₀, half-maximal inhibitor concentration; ns, not significant; RAPA, rapamycin.

Figure 2.

RAPA induces the apoptosis of B16 cells. B16 cells were treated with RAPA for 48 h. (A) Effects of RAPA on cell apoptosis were assessed through flow cytometry. (B) Statistical analysis of apoptosis rates for each group in (A). (C) Cleaved caspase 3, Bax and Bcl2 protein expression levels were detected by western blotting. (D) Relative expression of cleaved caspase 3 protein. (E) The ratio of Bcl2 to Bax protein expression. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. RAPA 0 nM group. B16, B16-F10; ns, not significant; RAPA, rapamycin.

Figure 3.

Effects of RAPA on the cell cycle progress of B16-F10 cells. (A) Cell cycle distribution was assessed by flow cytometry. (B) Results of statistical analysis of cell rates at different time periods for each group in (A). (C) CDK1, cyclin D1, CDK4, CDK6, cyclin E1 and CDK2 protein expression levels were detected by western blotting. Relative expression of (D) CDK1, (E) Cyclin D1, (F) CDK4, (G) CDK6, (H) Cyclin E1 and (I) CDK2 proteins. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. RAPA 0 nM group. ns, not significant; RAPA, rapamycin.

Figure 4.

RAPA induces autophagy in B16 cells. (A) B16 cells were treated with 0, 1 and 10 nM RAPA for 48 h. The formation of autophagic vesicles was observed by transmission electron microscopy. M indicates mitochondrial structure, N the nucleus, C the cytoplasm, and red triangles indicate autophagic lysosomes. Magnification, ×8,000; scale bar, 2.0 nm. (B) Expression levels of autophagy-related LC3, p62 and Beclin-1 in B16 cells were detected by western blotting. Relative expression of (C) LC3II, (D) Beclin-1 and (E) p62 proteins. Relative mRNA expression levels of (F) LC3 II, (G) Beclin-1 and (H) p62. (I) B16 cells were treated with rapamycin (100 nM) and/or CQ (10 µM) for 48 h. The protein expression levels of LC3 and p62 were detected by western blotting. (J) Relative expression of LC3II protein. (K) Relative expression of LC3II protein. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. RAPA 0 nM group or as indicated. B16, B16-F10; CQ, chloroquine; LC3, microtubule-associated protein light chain 3; ns, not significant; RAPA, rapamycin.

Figure 5.

RAPA inhibits the mTOR/p70-S6k signaling pathway in B16 cells. B16 cells were treated with 0, 1, 10 and 100 nM RAPA for 48 h. (A) Western blotting results showed that RAPA inhibited the expression of p-mTOR and p-p70-S6k compared with in the control group. Statistical analysis of the (B) p-mTOR/mTOR ratio, (C) the p-p70-S6k/p70-S6k ratio and (D) the p-4E-BP1/4E-BP1 ratio at the protein level. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. RAPA 0 nM group. 4E-BP1, eukaryotic translation initiation factor 4e-binding protein 1; B16, B16-F10; mTOR, mammalian target of rapamycin; ns, not significant; p70-S6k, p70 ribosomal S6 kinase; p-, phosphorylated; RAPA, rapamycin.

Figure 6.

RAPA induces autophagy and inhibits growth of B16-F10 melanoma cells in vivo. (A) On day 14 of tumor growth, the mice were executed and the tumors were collected and photographed (n=8 mice per group). (B) Tumor volumes were measured using Vernier calipers and were calculated every day. (C) Tumor weight was measured. Data are presented as the mean ± SD (n=8). (D) Relative protein expression levels of LC3 and p62 were detected in tumors by western blotting. (E) Relative expression of LC3 II protein in tumor tissues. (F) Relative expression of p62 protein in tumor tissues. (G) Expression levels of LC3 and p62 in each group were detected by immunohistochemistry (magnification, ×40; scale bar, 20 µm. (H) Statistical analysis of the area of LC3 protein-positive regions. (I) Statistical analysis of the area of p62 protein-positive regions. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. RAPA 0 mg/kg/day group. LC3, microtubule-associated protein light chain 3; RAPA, rapamycin.

Figure 7.

RAPA induces apoptosis in tumors. (A) TUNEL fluorescence staining of tumor cell death detected by fluorescence microscopy (magnification, ×100; scale bar, 10 µm). (B) Western blotting results showed that RAPA significantly increased the expression levels of cleaved caspase 3 and Bax, and decreased the expression levels of Bcl2. (C) Results of relative expression analysis of Cleaved caspase 3 protein in tumor tissues. (D) Statistical analysis of the ratio of the protein Bcl2/Bax in tumor tissues. Data are presented as the mean ± SD (n=3). **P<0.01 and ***P<0.001 vs. RAPA 0 mg/kg/day group. RAPA, rapamycin.

Figure 8.

RAPA inhibits the mTOR/p70-S6k signaling pathway in tumors. Proteins were extracted from tumors. (A) Western blotting results showed that RAPA significantly inhibited the expression levels of p-mTOR, p-4E-BP1 and p-p70-S6k compared with in the control group in tumor tissues. Statistical analysis of the (B) p-mTOR/mTOR ratio, (C) the p-p70-S6k/p70-S6k and (D) the p-4E-BP1/4E-BP1 ratio in tumor tissues at the protein level. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. RAPA 0 mg/kg/day group. 4E-BP1, eukaryotic translation initiation factor 4e-binding protein 1; mTOR, mammalian target of rapamycin; p70-S6k, p70 ribosomal S6 kinase; p-, phosphorylated; RAPA, rapamycin.