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. 2019 Jun;18(12):1379-1392.
doi: 10.1080/15384101.2019.1618119. Epub 2019 May 29.

Capsaicin induces cytotoxicity in human osteosarcoma MG63 cells through TRPV1-dependent and -independent pathways

Zhengqi Bao  1 , Xiusong Dai  1 , Peter Wang  2 , Yisheng Tao  3 , Damin Chai  3
Affiliations

Capsaicin induces cytotoxicity in human osteosarcoma MG63 cells through TRPV1-dependent and -independent pathways

Zhengqi Bao et al. Cell Cycle. 2019 Jun.

Abstract

An accumulating body of evidence has shown that capsaicin induces apoptosis in various tumor cells as a mechanism of its anti-tumor activity. However, the effects of capsaicin on osteosarcoma have not been studied extensively. In the current study, we explore the molecular mechanism of capsaicin-mediated tumor suppressive function in osteosarcoma. We found that capsaicin-induced apoptosis and the activation of transient receptor potential receptor vanilloid 1 (TRPV1) in a dose- and time-dependent manner in human osteosarcoma MG63 cells in vitro. Blocking TRPV1 using capsazepine attenuated the capsaicin-induced cytotoxicity, mitochondrial dysfunction, overproduction of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity. In addition, the results demonstrated that capsaicin induced the activation of adenosine 5'-monophosphate-activated protein kinase (AMPK), p53 and C-jun N-terminal kinase (JNK). In addition, Compound C (antagonist of AMPK) attenuated the activation of p53, which appeared to be TRPV1 independent. Taken together, the present study suggests that capsaicin effectively causes cell death in human osteosarcoma MG63 cells via the activation of TRPV1-dependent (mitochondrial dysfunction, and overproduction of ROS and JNK) and TRPV1-independent (AMPK-p53) pathways. Thus, capsaicin may be a potential anti-osteosarcoma agent.

Keywords: Osteosarcoma; TRPV1; apoptosis; capsaicin; mitochondria.

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Figures

Figure 1.
Figure 1.
Capsaicin reduces cell viability and induces apoptosis in human osteosarcoma MG63 cells. (a) Capsaicin-induced dose-dependent decreases in cell viability following 48 h of drug treatment. Significant effects were observed with 20 or 40 μM of capsaicin. (b) Time course of capsaicin-induced cell death following treatment with 20 μM of capsaicin. Significant effects were observed following after 48 or 72 h of capsaicin treatment. (c) Capsaicin-induced dose-dependent apoptosis following 48 h of treatment. Significant effects were observed with 20 or 40 μM of capsaicin. (d) Time course of capsaicin-induced apoptosis. Significant effects were observed following 48 or 72 h of capsaicin treatment. (e) Changes in apoptosis-associated proteins including cleaved PARP, PARP, cleaved caspase-3, caspase-3, Cytochrome C and Bcl-2 were detected by western blotting following MG63 cells treatment with 20 μM capsaicin for various lengths of time. (f) Quantification of the phosphorylation levels of cleaved-PARP, cleaved caspase-3, Cytochrome C and Bcl-2. Data are expressed as the mean ± SD. *, p < 0.05, compared with control.
Figure 2.
Figure 2.
TRPV1 activation is required for capsaicin-induced mitochondrial dysfunction in human osteosarcoma MG63 cells. (a) Time course of the capsaicin-induced expression of TRPV1 mRNA expression. Capsaicin inducedan increase in TRPV1 mRNA expression at 4–12 h following the capsaicin (20 μM) treatment. (b) Capsaicin inducedsimilar increases in TRPV1 protein expression, although a slightly delayed time course was observed; TRPV1 protein increased 8–12 h following the treatment. (c-e) The Capsaicin-induced effects were TRPV1-dependent: (c) TRPV1 protein expression induced by Ctr (vehicle), 20 μM of capsaicin, 20 μM of capsazepine (Cpz, TRPV1 antagonist), or capsaicin + Cpz; (d) cell viability induced by the same treatments; and (e), apoptosis levels induced by the same treatments. (f-g) Capsaicin induced changes in apoptosis-associated proteins were also TRPV1-dependent: (f) Representative Western blotting presenting the levels of cleaved PARP, PARP, cleaved caspase-3, caspase-3, Cytochrome C and Bcl-2 as well as DM1A (α-tubulin) from the same experiment; (g) Quantification of the protein levels detected in (f). The data in (f) and (g) are representatives of the results from three separate experiments. Data are expressed as the mean ± SD. *, p < 0.05, compared with control; #, p < 0.05, compared with capsaicin group.
Figure 3.
Figure 3.
TRPV1 activation is required for capsaicin-induced mitochondrial dysfunction in human osteosarcoma MG63 cells. (a-c) Time course of capsaicin-induced reductions in MMP (a), complex I (b), and ATP levels (c). Significant effects were observed between 8 and 12 h following the capsaicin treatment. (d-f) Effects of capsazepine (Cpz) on capsaicin-induced decrease of MMP (d), complex I (e) and ATP (f), respectively. Blocking TRPV1 using Cpz attenuated the capsaicin-induced decrease in MMP, complex I activity and ATP content in MG63 cells. The values are expressed as the mean ± SD. *, p < 0.05, compared with control, #, p < 0.05, compared with capsaicin group.
Figure 4.
Figure 4.
TRPV1 activation is required for capsaicin-induced Cytochrome C release into the cytoplasm from the mitochondria and the decrease in Bcl-2 in the mitochondria of human osteosarcoma MG63 cells. (a) Time course of capsaicin-induced Cytochrome C release into the cytoplasm; significant effects were observed at 48 and 72 h following treatment. (b) Increased levels of Cytochrome C in the cytoplasm induced by capsaicin were blocked by the TRPV1 antagonist capsazepine. (c) Time course of the capsaicin-induced reduction of mitochondrial Bcl-2; significant effects were observed at 48 and 72 h following the capsaicin treatment. (d) The capsaicin-induced decrease in the mitochondrial Bcl-2 was blocked by capsazepine. Cytochrome C levels in the cytoplasm were normalized to DM1A, and Bcl-2 in the mitochondria was normalized to COX IV. The values are expressed as the mean ± SD. *, p < 0.05, compared with control; #, p < 0.05, compared with capsaicin group.
Figure 5.
Figure 5.
TRPV1 activation is required for the capsaicin-induced overproduction of ROS and the decrease in SOD activity in human osteosarcoma MG63 cells. (a) Time course of capsaicin-induced ROS generation. Capsaicin (20 μM) caused significant ROS production 48 and 72 h following the capsaicin treatment. (b) Time course of capsaicin-induced decrease in SOD activity. Significant effects were observed at 48 and 72 h following the capsaicin treatment. (c) Effects of capsazepine (20 μM) on the capsaicin-induced increase of ROS generation. (d) Effects of capsazepine on the capsaicin-induced decrease in SOD activity. The values are expressed as the mean ± SD. *, p < 0.05, compared with control, #, p < 0.05, compared with capsaicin group.
Figure 6.
Figure 6.
Roles of AMPK in the capsaicin-induced effects. (a-c) Time courses of capsaicin-induced phosphorylation of AMPKα (a), p53 (b), and JNK (c) detected by western blotting analysis. The phosphorylated AMPKα, p53, and JNK were normalized, respectively, to the total level of each corresponding protein. (d) MG63 cells were treated with capsaicin (20 μM), Compound C (20 μM), capsaicin + Compound C (1 h prior to administration of capsaicin) for 48 h, respectively. Treatment with the AMPK antagonist compound C (CC) blocked the capsaicin-induced activation of p53. (e) MG63 cells were treated with capsaicin (20 μM), capsazepine (20 μM), and capsaicin plus capsazepine (1 h prior to addition of capsaicin) for 48 h, respectively. (f) Treatment with capsazepine blocked capsaicin-induced activation of JNK. The values are expressed as the mean ± SD. *, p < 0.05, compared with control; #, p < 0.05, compared with the capsaicin group. **, p<0.01, compared wuth control; ##, p<0.01, compared with the capsaicin group.
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This work was supported by the Nature Science Major and Key Program of College and University of Anhui Province (No. KJ2018ZD024 and KJ2016A460), Key Projects of Support Program for Outstanding Young Talents in Colleges and Universities of Anhui Province (No. gxyq2017032), and the Nature Science Foundation of Bengbu Medical University (No.BYKF1771 and No. BYKF1708ZD).

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