Uncovering the Mechanisms of Cryptotanshinone as a Therapeutic Agent Against Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a fatal and dominant form of liver cancer that currently has no effective treatment or positive prognosis. In this study, we explored the antitumor effects of cryptotanshinone (CPT) against HCC and the molecular mechanisms underlying these effects using a systems pharmacology and experimental validation approach. First, we identified a total of 296 CPT targets, 239 of which were also HCC-related targets. We elucidated the mechanisms by which CPT affects HCC through multiple network analysis, including CPT-target network analysis, protein-protein interaction network analysis, target-function network analysis, and pathway enrichment analysis. In addition, we found that CPT induced apoptosis in Huh7 and MHCC97-H ells due to increased levels of cleaved PARP, Bax, and cleaved caspase-3 and decreased Bcl-2 expression. CPT also induced autophagy in HCC cells by increasing LC3-II conversion and the expression of Beclin1 and ATG5, while decreasing the expression of p62/SQSTM1. Autophagy inhibitors (3-methyladenine and chloroquine) enhanced CPT-induced proliferation and apoptosis, suggesting that CPT-induced autophagy may protect HCC cells against cell death. Furthermore, CPT was found to inhibit the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Interestingly, activation of PI3K by insulin-like growth factor-I inhibited CPT-induced apoptosis and autophagy, suggesting that the PI3K/AKT/mTOR signaling pathway is involved in both CPT-induced apoptosis and autophagy. Finally, CPT was found to inhibit the growth of Huh7 xenograft tumors. In conclusion, we first demonstrated the antitumor effects of CPT in Huh7 and MHCC97-H cells, both in vitro and in vivo. We elucidated the potential antitumor mechanism of CPT, which involved inducing apoptosis and autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway. Our findings may provide valuable insights into the clinical application of CPT, serving as a potential candidate therapeutic agent for HCC treatment.

Keywords: apoptosis; autophagy; cryptotanshinone; hepatocellular carcinoma; system pharmacology.

Figure 1

A schematic diagram showing the systems pharmacology approach for investigating the therapeutic mechanisms of cryptotanshinone (CPT) on human hepatocellular carcinoma (HCC) by integrating target identification, network analysis, and experimental validation.

Figure 2

Cryptotanshinone (CPT)-CPT target network. The network includes CPT and 296 CPT targets.

Figure 3

Protein-protein interaction (PPI) network. The nodes get larger with increasing degree. Edges: PPIs between targets of cryptotanshinone (CPT) and their interaction partners; purple circle nodes: common targets of CPT and human hepatocellular carcinoma (HCC); dark purple circle nodes: hub targets of CPT and HCC (Degree ≥ 80).

Figure 4

Target-function network. The functional module is connected to the target when the target participates in that biological process.

Figure 5

Integrated human hepatocellular carcinoma (HCC)-pathway and functional modules.

Figure 6

Cryptotanshinone (CPT) inhibits the proliferation of Huh7 and MHCC97-H cells. (A) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis of human hepatocellular carcinoma (HCC) cell lines, including Huh7 and MHCC97-H cells, treated with the indicated doses of CPT for 24 and 48 h. These experiments were independently repeated three times. (B) A colony formation assay was performed for Huh7 and MHCC97-H cells incubated with various doses (0, 3, 6, and 12 μM) of CPT. Colony survival relative to control groups was calculated and presented as mean ± standard deviation (SD) (n = 3). (C) Proliferative capacity was measured by EdU assay in Huh7 and MHCC97-H cells treated with the indicated concentrations of CPT (0, 3, 6, and 12 μM) for 24 h. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control group.

Figure 7

Cryptotanshinone (CPT) induces apoptosis in Huh7 and MHCC97-H cells. (A) Huh7 and MHCC97-H cells were treated with CPT (0, 3, 6, and 12 μM) for 24 h and analyzed using the Annexin V-FITC apoptosis detection kit. (B) TUNEL assay in Huh7 and MHCC97-H cells after treatment with CPT (0, 3, 6, and 12 μM). (C) Expression levels of PARP, cleaved PARP, caspase-3, cleaved caspase-3, Bcl-2, and Bax proteins in Huh7 and MHCC97-H cells following CPT (0, 3, 6, and 12 μM) treatment were detected by western blotting. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control group.

Figure 8

Cryptotanshinone (CPT) stimulates autophagy in Huh7 and MHCC97-H cells. (A) Western blot analysis of LC3-I, LC3-II, p62/SQSTM1, Beclin1, and ATG5 in human hepatocellular carcinoma (HCC) cells treated with the indicated concentrations of CPT (0, 3, 6, and 12 μM) for 24 h. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control group. (B) Western blot analysis of LC3-I conversion to LC3-II in Huh7 and MHCC97-H cells treated with 6 μM CPT in the absence or presence of 10 μM chloroquine (CQ) or 5 mM 3-methylamphetamine (3-MA) for 24 h was conducted. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the CPT group. (C) Huh7 and MHCC97-H cells were treated with or without 6 μM CPT for 24 h. LC3B puncta were then stained with anti-LC3B antibody by immunofluorescence and imaged with a confocal microscope. (D) Cells were treated with or without 6 μM CPT for 24 h and the changes in ultrastructure were detected by transmission electron microscopy. Arrows: autophagosomes/autolysosomes. Scale bar: 1 µm.

Figure 9

Inhibition of cryptotanshinone (CPT)-induced autophagy promotes cell apoptosis in Huh7 and MHCC97-H cells. (A) Huh7 and MHCC97-H cells were treated with different concentrations of CPT alone or in combination with 10 μM chloroquine (CQ) or 5 mM 3-methylamphetamine (3-MA) for 24 h. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control group. (B) Huh7 and MHCC97-H cells were treated with 6 μM CPT alone or in combination with 10 μM CQ or 5 mM 3-MA for 24 h. Proliferative capacity was measured using a colony formation assay. **P < 0.01 versus the CPT group. (C) Flow cytometric analysis of apoptosis in Huh7 and MHCC97-H cells exposed to 6 μM CPT alone or in combination with autophagy inhibitors (3-MA or CQ) for 24 h. **P < 0.01, ***P < 0.001 versus the CPT group. (D) Western blot analysis of PARP, cleaved-PARP, Bcl-2, and Bax in human hepatocellular carcinoma (HCC) cells treated with 6 μM CPT alone or in combination with autophagy inhibitors (3-MA or CQ) for 24 h.

Figure 10

Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway is involved in cryptotanshinone (CPT)-induced autophagy and apoptosis. (A) Western blot analysis of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR in human hepatocellular carcinoma (HCC) cells treated with different doses of CPT (0, 3, 6, and 12 μM) for 24 h. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control group. (B) HCC cells were exposed to 6 μM CPT alone or in combination with 50 ng/ml insulin-like growth factor-I (IGF-I). The expression levels of LC3-I, LC3-II, Beclin1, Bcl-2, and Bax proteins were assessed by western blotting.

Figure 11

Cryptotanshinone (CPT) inhibits the growth of Huh7 cell xenografts in nude mice. (A) Photograph of extracted tumors from each group of mice (n = 6). Statistical analysis of tumor volumes (C), body weight (D), and tumor weight (E) in each group of mice (n = 6). (B) TUNEL assays were conducted to measure apoptosis in tumor tissues using immunochemistry and immunofluorescence analyses. (F) The binding mode of CPT with phosphatidylinositol 3-kinase (PI3K) determined by molecular docking simulation. CPT formed stable hydrogen bonds with PI3K at SER-854 and VAL-851. (G) Electrostatic surfaces of CPT and PI3K obtained using Pymol. Lower electrostatic potential denotes better binding ability. (H) Tumor tissue lysates were extracted from the control and CPT-treated groups and the expression levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, PARP, cleaved PARP, caspase-3, cleaved caspase-3, Bcl-2, Bax, LC3-I, LC3-II, p62/SQSTM1, and Beclin1 proteins were assessed by western blotting.

Figure 12

Working model of the experimental validation. Diagram indicating cryptotanshinone (CPT)-induced autophagy and apoptosis in human hepatocellular carcinoma (HCC) via inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway.