Antitumor effect of sunitinib in human prostate cancer cells functions via autophagy

Abstract

The aim of the present study was to explore sunitinib-induced autophagic effects and the specific molecular mechanisms involved, in vitro, using PC-3 and LNCaP human prostate cancer cell lines. Cells were exposed to escalating doses of sunitinib treatment and subsequent cell viability and cell cycle analyses were performed to evaluate the inhibitory effect of sunitinib in vitro. Immunofluorescence staining of microtubule associated protein 1A/1B-light chain 3 (LC3) puncta was employed to assess autophagy levels after sunitinib treatment. Western blot analysis was performed to evaluate variations in the levels of LC3, sequestosome-1, extracellular signal regulated kinase 1/2 (ERK1/2), mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 kinase (p70S6K) and cleaved caspase-3 proteins. The present study revealed that sunitinib treatment inhibited cell growth and triggered autophagy in a dose-dependent manner in both cell lines. In addition, sunitinib activated ERK1/2 and inhibited mTOR/p70S6K signaling. Sunitinib-induced autophagy was notably reversed by ERK1/2 kinase inhibitor, U0126. Furthermore, inhibition of sunitinib-induced autophagy by 3-methyladenine enhanced apoptosis and exhibited improved cell viability, which indicated that sunitinib induces not only apoptosis but also autophagic cell death in prostate cancer cell lines. These results may lead to an improved understanding of the mechanism of sunitinib's cytotoxic action and may provide evidence that combined sunitinib autophagy-regulating treatment may be of benefit to anti-prostate cancer therapy.

Keywords: apoptosis; autophagy; extracellular signal-regulated kinases 1/2; mechanistic target of rapamycin; prostate cancer cells; sunitinib.

Figure 1.

Effect of sunitinib on the proliferation in PC-3 and LNCaP cells. Following 5, 10 or 20 µmol/l sunitinib treatment in PC-3 and LNCaP cells for 24 h, cell viability was measured using a Cell Counting Kit-8 kit assay. Sunitinib inhibited the proliferation of (A) PC-3 and (B) LNCaP cells in a dose-dependent manner. Data are presented as mean ± standard deviation (n=4). *P<0.05 and **P<0.01 vs. Con. Con, control.

Figure 2.

Cell-cycle (G0/G1, S and G2/M) analysis of PC-3 and LNCaP cells exposed to sunitinib (10 µM) or DSMO (control) for 48 h. Flow cytometric analysis was performed using PC-3 and LNCaP human PCa cells to establish the percentage of cells in G0/G1, S and G2/M phases of the cell cycle. (A) Control PC-3 cells treated with DSMO. (B) PC-3 cells treated with 10 µM sunitinib. (C) Control LNCaP cells treated with DSMO. (D) LNCaP cells treated with 10 µM sunitinib. Data are presented as mean ± standard deviation (n=4). *P<0.05 vs. control. DSMO, dimethyl sulfoxide.

Figure 3.

Effect of sunitinib on the formation of LC3 puncta in PC-3 and LNCaP cells. (A) Fluorescence images obtained from sunitinib-treated and control groups of prostate cancer cells, PC-3 and LNCaP. Cells treated with DMSO were set as control groups, the numbers of LC3 puncta per cell were counted manually. The number of LC3 puncta (green fluorescent) per cell in sunitinib-treated groups increased in a dose dependent manner (magnification, ×630). (B) Limited numbers of punctas were located in control group PC-3 and LNCaP cells. The numbers of LC3 puncta per cell in PC-3 cells of 5, 10 and 20 µM sunitinib-treated groups were 23.29±3.36, 59.74±6.48 and 113.71±7.52, respectively. The numbers of LC3 puncta per cell in LNCaP cells of 5, 10 and 20 µM sunitinib-treated groups were 28.90±6.34, 87.56±6.45 and 198.56±5.33, respectively. Data are presented as means ± standard deviation (n=4). *P<0.05 and **P<0.01, vs. Con. DSMO, dimethyl sulfoxide. LC3, microtubule associated protein 1A/1B-light chain 3; Con, control; DAPI, 4′,6-diamidino-2-phenylindole; DIC, differential interference contrast.

Figure 4.

Western blotting analysis of sunitinib-induced autophagy in prostate cancer cells. (A) Following sunitinib treatment at different concentrations for 24 h, increased levels of LC3-II protein expression and decreased levels of SQSTM1/p62 protein expression were observed in both PC-3 and LNCaP cells. (B) Fold changes of LC3-II/ LC3-I and (C) the expression level of SQSTM1/p62 between the treatment and the control groups were determined by three independent experiments and normalized by the total protein level. Data are presented as mean ± standard error. *P<0.05 and **P<0.01, vs. Con. LC3, microtubule associated protein 1A/1B-light chain 3; Con, control; SQSTM1/p62, sequestosome-1.

Figure 5.

Effect of sunitinib on mTOR and ERK signaling in PC-3 and LNCaP cells. (A) Sunitinib treatment suppressed the phosphorylated levels of mTOR and p70S6K and activated the phosphorylated level of ERK1/2, as demonstrated by western blotting. Fold changes in p-mTOR, mTOR, p-p70S6K and p-ERK proteins in 5, 10, or 20 µM sunitinib-treated groups and control groups in (B) PC-3 cells and (C) LNCaP cells. Results were determined by three independent experiments and normalized by the total protein level. Data are presented as mean ± standard deviation. *P<0.05 and **P<0.01, vs. Con. mTOR, mechanistic target of rapamycin; p70S6K, ribosomal protein S6 kinase beta-1; ERK 1/2, extracellular signal-regulated kinases 1/2; Con, control.

Figure 6.

Inhibition of ERK1/2 by U1026 effects on the autophagy level induced by sunitinib. (A) Inhibition of the phosphorylation of ERK1/2 by U0126 (10 µM) in PC-3 and LNCaP cells suppressed the increased fold changes of LC3II/I levels triggered by sunitinib, as demonstrated by using western blotting. (B) Fold changes of LC3II/I expression levels in sunitinib-treated groups and the control groups were determined by three independent experiments and normalized by the total protein level. Data are presented as mean ± standard deviation, *P<0.05 and **P<0.01, vs. control. ERK 1/2, extracellular signal-regulated kinases 1/2; LC3, microtubule associated protein 1A/1B-light chain 3.

Figure 7.

Inhibition of autophagy by 3-MA effects on sunitinib-induced cell apoptosis. (A) Flow cytometric analysis indicated inhibition of autophagy by 3-MA (5 µM) increased the rate of apoptosis induced by sunitinib. (B) Percentage of apoptotic cells in control, sunitinib-treated, 3-MA-treated and sunitinib + 3-MA-treated groups in PC-3 and LNCaP cells (C) Following sunitinib treatment, caspase-3 was activated and inhibition of autophagy enhanced the activation of caspase-3. Data in panel (B) represent the mean ± standard deviation of three independent experiments, *P<0.05 and **P<0.01. 3-MA, 3-methyladenine; Con, control.

Figure 8.

Inhibition of autophagy by 3-MA effects on sunitinib-induced apoptosis. Sunitinib-induced cell death was reversed by inhibition of autophagy with 3-MA (5 µM) in PC-3 cells, which was similarly observed in LNCaP cells; however, no statistical significance was detected in LNCaP cells. Data are presented as mean ± standard deviation. *P<0.05 and **P<0.01. 3-MA, 3-methyladenine.