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. 2024 Mar 30;29(7):1557.
doi: 10.3390/molecules29071557.

Study on the Antitumor Mechanism of Tanshinone IIA In Vivo and In Vitro through the Regulation of PERK-ATF4-HSPA5 Pathway-Mediated Ferroptosis

Chunxiang Guo  1 , Wei Zhao  1   2 , Wei Wang  1   2 , Zheng Yao  1   2 , Wenhui Chen  1   2 , Xiaoyi Feng  1   2
Affiliations

Study on the Antitumor Mechanism of Tanshinone IIA In Vivo and In Vitro through the Regulation of PERK-ATF4-HSPA5 Pathway-Mediated Ferroptosis

Chunxiang Guo et al. Molecules. .

Abstract

As a traditional Chinese medicine, Salvia miltiorrhiza Bunge was first recorded in the Shennong Materia Medica Classic and is widely used to treat "the accumulation of symptoms and masses". The main active ingredient of Salvia miltiorrhiza Bunge, Tanshinone IIA (TIIA), has shown anti-inflammatory, antitumor, antifibrosis, antibacterial, and antioxidative activities, etc. In this study, the results showed that TIIA could inhibit the proliferation and migration of HepG2 cells and downregulate glutathione (GSH) and Glutathione Peroxidase 4 (GPX4) levels; besides, TIIA induced the production of Reactive Oxygen Species (ROS), and upregulated the total iron content. Based on network pharmacology analysis, the antitumor effect of TIIA was found to be focused on the endoplasmic reticulum (ER)-mediated ferroptosis signaling pathway, with protein kinase R (PKR)-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-heat shock 70 kDa protein 5 (HSPA5) as the main pathway. Herein, TIIA showed typical ferroptosis characteristics, and a ferroptosis inhibitor (ferrostatin-1) was used to verify the effect. The antitumor effects of TIIA, occurring through the inhibition of the PERK-ATF4-HSPA5 pathway, were further observed in vivo as significantly inhibited tumor growth and the improved pathological morphology of tumor tissue in H22-bearing mice. In summary, the antitumor mechanism of TIIA might be related to the downregulation of the activation of PERK-ATF4-HSPA5 pathway-mediated ferroptosis.

Keywords: PERK-ATF4-HSPA5 pathway; Tanshinone IIA; anti-hepatoma; endoplasmic reticulum stress; ferroptosis.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Tanshinone IIA inhibited the proliferation and migration of HepG2 cells. TIIA-L, low concentration of TIIA (2.5 μM); TIIA-H, high concentration of TIIA (10 μM); ADR (2 μM). (a) HepG2 cells were treated with different concentrations of TIIA (IC50 = 4.17 ± 0.27 μM), ADR (IC50 = 1.45 ± 0.10 μM); L02 cells were treated with different concentrations of TIIA (IC50 = 13.55 ± 1.32 μM). (b) The morphology of HepG2 cells treated with TIIA for 24 h. (c) The colony tests of HepG2 cells with TIIA treatments was conducted for 7 days. (d) The transwell of HepG2 cells treated with TIIA for 48 h. (e) The scratch experiments of HepG2 cells treated with TIIA for different time periods (0, 36, and 72 h). The red arrow indicates the distance of the scratch. *** p < 0.001 vs. control group.
Figure 2
Figure 2
Compound target network of TIIA and signal pathway enrichment. Using the Venny online tool, (a) 296 targets of TIIA and (b) 69 targets of TIIA and hepatocellular carcinoma were screened out. (c) The size and color of the circle are arranged according to the degree of correlation, with the 14 most likely targets forming a ring on the left side. Circles that are darker in color and larger circles indicate a higher correlation. (d) GO functional enrichment analysis and (e) KEGG enrichment analysis based on the DAVID database.
Figure 3
Figure 3
TIIA promoted ferroptosis of HepG2 cells. TIIA-L, low concentration of TIIA (2.5 μM); TIIA-H, high concentration of TIIA (10 μM); ADR (2 μM). HepG2 cells treated with TIIA for 24 h. (a) Flow cytometry assay double-stained with Annexin V and PI. Histogram statistics of cell death. The number of cell clusters increases, and the image color changes from blue to red. (b) Determination of the total iron content. (c) The expression of GPX4 was detected using Western blot. (d) Fluorescence staining of ROS in HepG2 cells. Green fluorescence represents the production of ROS. Data were presented as mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control group.
Figure 4
Figure 4
TIIA inhibited the PERK-ATF4-HSPA5 signaling pathway. TIIA-L, low concentration of TIIA (2.5 μM); TIIA-H, high concentration of TIIA (10 μM); ADR (2 μM). HepG2 cells treated with TIIA for 24 h. (a) Expression levels of PERK mRNA. (b) The expressions of PERK, ATF4, HSPA5, GPX4, and p-PERK were measured using Western blot. (c) The fluorescence image of the laser confocal detection of the expression of PERK (green) on the HepG2 cell membrane and ATF4 (green) in HepG2 cells (10 × 60). Blue pseudocolor is fluorescent DNA dye (DAPI). Scale bar = 50 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control group.
Figure 5
Figure 5
Ferrostatin-1 blocked the antitumor effect of TIIA. TIIA-L, low concentration of TIIA (2.5 μM); TIIA-H, high concentration of TIIA (10 μM); ADR (2 μM). PERK, ATF4, HSPA5, GPX4, and p-PERK expression in HepG2 cells measured by Western blotting after ferrostatin-1 was added for 1 h, the culture medium containing ferrostatin-1 was discarded, and treatment took place with TIIA for 24 h. Data are presented as mean ± SD.* p < 0.05, ** p < 0.01, and *** p < 0.001 vs. C group. # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. C + F group.
Figure 6
Figure 6
TIIA binds to PERK and increases the denaturation temperature. HepG2 cells were treated with 10 μM TIIA for 1 h. (a) The structure complex of PERK-TIIA. TIIA is depicted in red, and the chains of PERK subunits are represented by green, cyan blue, and magenta. Data deposition: the crystallography, atomic coordinates, and structural factors were deposited in the Protein Data Bank, www.pdb.org (accessed on 14 December 2023) (PDB ID code 4G34). (b) The CETSA binding assay of PERK and β-actin in the presence or absence of TIIA (20 µM) at different temperatures was detected using Western blot. The temperature-dependent melting curves and the apparent aggregation temperature were calculated by nonlinear regression. Values represent the mean ± SD (N = 3 replicates).
Figure 7
Figure 7
TIIA-inhibited tumor growth in H22 mice. M: saline model group, TIIA-L: low dosage of TIIA (20 mg/kg), TIIA-H: high dosage of TIIA (50 mg/kg), Sorafenib (60 mg/kg). (a) The tumor volume and body weight were calculated every two days. (b) The tumor removal weight. (c) The morphology of tumors was assayed with H&E staining. The red arrow indicates that multi-level and asymmetric division phenomena can be observed in the tumor cells of the model group. * p < 0.05 vs. model group.
Figure 8
Figure 8
TIIA inhibited the PERK-ATF4-HSPA5 signaling pathway and decreased the GPX4 level in tumor tissue. M: saline model group, TIIA-L: low dosage of TIIA (20 mg/kg), TIIA-H: high dosage of TIIA (50 mg/kg), Sorafenib (60 mg/kg). (a) The expressions of PERK, ATF4, HSPA5, GPX4, and p-PERK were detected using Western blot. (b) GSH level of tumor tissue. (c) The immunohistochemistry was analyzed. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. model group.
Figure 9
Figure 9
The possible anticancer mechanism of TIIA.
See this image and copyright information in PMC

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Grants and funding

This research was funded by the National Natural Science Foundation of China (No. 82060737 and No. 82160898), the Science and Technology Planning Project of Yunnan Province (No. 202301AT070253), and the Yunnan Provincial Science and Technology Talents and Platform Plan Project (No. 202305AC060041).