Marsdenia tenacissima Extract Induces Autophagy and Apoptosis of Hepatocellular Cells via MIF/mToR Signaling

Abstract

Hepatocellular carcinoma (HCC) seriously endangers humans. In traditional Chinese medicine, Marsdenia tenacissima (MTE) has anti-inflammatory, antiasthmatic, antihypertensive, and anticancer effects. This study reveals the antiproliferative effect of MTE on the HCC cells in vitro and provides a theoretical basis for the development and clinical application of anti-HCC agents. Methods. MHCC-97H and HepG2 cells were cultured in vitro and exposed to various concentrations and durations of MTE, and an MTT assay was used to detect the effects of MTE on cell proliferation. Transmission electron microscopy revealed the morphological changes in the two cell lines after MTE stimulation. The MTE effects on the apoptosis and cell cycle distribution of the cell lines were detected by flow cytometry. Western blotting and qRT-PCR were used to detect target gene expression at the protein and mRNA levels, respectively. Results. MTE reduced the viability of the MHCC-97H and HepG2 cells in a dose- and time-dependent manners (P < 0.05). Autophagic vesicles and apoptotic bodies were found in the MHCC-97H and HepG2 cells after MTE incubation, and the Annexin V-PI assay showed that the apoptotic rates of the cell lines increased with increasing MTE concentration (P < 0.05). Autophagy inducer rapamycin promoted the MTE-induced apoptotic rates of the cell lines, whereas autophagy inhibitor chloroquine inhibited the apoptotic rates. More cells in the S phase were found in the two cell lines after MTE treatment (P < 0.05). After MTE incubation, MIF, CD47, and beclin-1 protein levels significantly increased. Furthermore, in the MTE group, Akt, mTOR, and caspase3 expressions decreased; however, LC 3 expression increased, which was significantly different from the control group (P < 0.05). Conclusions. MTE inhibited proliferation and induced autophagy, apoptosis, and S phase cell cycle arrest in the MHCC-97H and HepG2 cells. These effects might be related to the activation of MIF and mTOR signaling inhibition.

Figure 1

MTE suppressed the proliferation of the MHCC-97H and HepG2 cells in a dose- and time-dependent manner. (a, b) The MHCC-97H and HepG2 cells were stimulated with various concentrations of MTE (0, 12.5, 25, 50, 100, 200, and 400 mg/mL) for 24 h, 48 h, and 72 h. The cell viabilities of (a) the MHCC-97H and (b) HepG2 were assessed by MTT assay. (c) The cellular morphologies of the MHCC-97 cells in the control and MTE treatment group (35 mg/mL) were observed using electron microscopy (×40). (d) The cellular morphologies of the HepG2 cells in the control and MTE treatment group (50 mg/mL) were observed using electron microscopy (×40). n = 5.

Figure 2

MTE induced autophagy and apoptosis of the MHCC-97H and HepG2 cells. (a) Normal morphologies of the MHCC-97H and HepG2 cells were observed using transmission electron microscopy. (b) 35 mg/mL and 50 mg/mL MTE treatment induced the formation of autophagosomes and autophagosome-lysosome structures in the MHCC-97H and HepG2 cells, respectively. (c) 35 mg/mL and 50 mg/mL MTE treatment induced the formation of apoptotic bodies in the MHCC-97H and HepG2 cells, respectively.

Figure 3

MTE induced apoptosis of the MHCC-97H and HepG2 cells in a dose-dependent manner. (a) Apoptotic rates for the MHCC-97H cells after MTE treatment (17.5 mg/mL, 35 mg/mL, and 70 mg/mL) were detected by FCM. (b) Quantitative data for (a). (c) Apoptotic rates for the HepG2 cells after MTE treatment (25 mg/mL, 50 mg/mL, and 100 mg/mL) were detected by FCM. (d) Quantitative data for (c). n = 3.

Figure 4

Relationship between autophagy and apoptosis induced by MTE treatment. (a) The MHCC-97H cells were treated with Rapa (10 μg/mL), CQ (8 μmol/L), MTE (35 mg/mL), Rapa (10 μg/mL) plus MTE (35 mg/mL), or CQ (8 μmol/L) plus MTE (35 mg/mL). The apoptotic cells were detected by FCM. (b) Quantitative data for (a). (c) The HepG2 cells were treated with Rapa (10 μg/mL), CQ (8 μmol/L), MTE (50 mg/mL), Rapa (10 μg/mL) plus MTE (50 mg/mL), or CQ (8 μmol/L) plus MTE (50 mg/mL). The apoptotic cells were detected by FCM. (d) Quantitative data for (c). n = 3.

Figure 5

MTE induced the S cell cycle arrest in the MHCC-97H and HepG2 cells. (a) Cell cycle distributions of the MHCC-97H cells after MTE treatment (17.5 mg/mL, 35 mg/mL, and 70 mg/mL) were detected by FCM. (b) Quantitative data for (a). (c) The cell cycle distributions of the HepG2 cells after MTE treatment (25 mg/mL, 50 mg/mL, and 100 mg/mL) were detected by FCM. (d) Quantitative data for (c). n = 3.

Figure 6

MTE induced the autophagy of HCC cells by inhibiting the Akt/mTOR pathway via MIF. (a) LC3 I, LC3 II, caspase3, cleaved caspase3, MIF, CD74, Beclin-1, and β-actin protein levels in MTE (17.5 mg/mL, 35 mg/mL, and 70 mg/mL)-treated MHCC-97H cells were examined by WB assay. (b) Akt, mTOR, caspase 3, and LC 3 mRNA levels in MTE (17.5 mg/mL, 35 mg/mL, and 70 mg/mL)-treated MHCC-97H cells were detected by qRT-PCR assay. (c) The mRNA levels for Akt, mTOR, caspase 3, and LC 3 genes in MTE (25 mg/mL, 50 mg/mL, and 100 mg/mL)-treated HepG2 cells were detected by qRT-PCR assay. (d) The mTOR, Akt, and GAPDH protein levels in MTE (17.5 mg/mL, 35 mg/mL, and 70 mg/mL)-treated MHCC-97H cells were detected by WB assay. (e) The mTOR, Akt, and GAPDH protein levels in MTE (17.5 mg/mL, 35 mg/mL, and 70 mg/mL)-treated HepG2 cells were detected by WB assay. n = 3.