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Cryptotanshinone increases the sensitivity of liver cancer to sorafenib by inhibiting the STAT3/Snail/epithelial mesenchymal transition pathway
doi: 10.2478/fzm-2022-0016
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Abstract:
Objective Sorafenib resistance has been a major factor limiting its clinical use as a targeted drug in liver cancer. The present study aimed to investigate whether cryptotanshinone can enhance the sensitivity of liver cancer and reduce the resistance to sorafenib. Methods Sorafenib-resistant cells were established based on HepG2 and Huh7 cell lines. And the anti-tumor effect of sorafenib combined with cryptotanshinone on the sorafenib-resistant cells was verified by MTT, colony formation, transwell assays and tumor growth xenograft model. Moreover, the effects of the combined treatment on the expression of phosphorylated (p)-STAT3, as well as epithelial mesenchymal transition (EMT) and apoptosis related proteins of cells were evaluated by western blot analysis. Results It was identified that cryptotanshinone inhibited the viability of both HepG2 and Huh7 cells in a dose- and time-dependent manner, and decreased p-STAT3 expression rather than total STAT3 expression at a concentration of 40 njmol/L. In the sorafenib-resistant cells, sorafenib in combination with cryptotanshinone markedly inhibited cell viability, invasion and migration compared with sorafenib alone. In contrast, increased p-STAT3 level by colivelin led to the inhibition of the synergistic effect of cryptotanshinone and sorafenib not only on cell viability, but also on EMT and apoptosis, suggesting that cryptotanshinone and sorafenib may act by downregulating STAT3 signaling. Further, the inhibition of carcinogenicity effect was also verified in xenografted tumor models. Conclusion The present results indicated that cryptotanshinone could synergize with sorafenib to inhibit the proliferative, invasive, and migratory abilities of sorafenib-resistant cells by downregulating STAT3 signaling. -
Key words:
- cryptotanshinone /
- sorafenib /
- sorafenib-resistance /
- STAT3 signaling /
- liver cancer
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Figure 1. Cryptotanshinone inhibits the proliferation and STAT3 activation of liver cancer cells.
(A) HepG2 and Huh7 cells were exposed to different concentrations (0, 5, 10, 20, 40, 80, 160 and 320 μmol/L) of cryptotanshinone for 24, 48 and 72 h. (B, C) The expression levels of p-STAT3 and STAT3 were measured via western blotting. The band density of p-STAT3 was normalized to STAT3. The data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2. Sorafenib inhibits STAT3 activation in sorafenib-sensitive cells but not in sorafenib-resistant liver cancer cells.
The expression levels of p-STAT3 and STAT3 were measured by western blot analysis. (A) Sorafenib-resistant cells and parental cells were incubated with or without 10 μmol/L sorafenib for 24 h. (B, C) Sorafenib-resistant cells and parental cells were incubated with sorafenib at serial concentrations of 0, 2, 5 or 10 μmol/L for 24 h. The protein levels were detected via western blot analysis. GAPDH served as the loading control. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3. Cryptotanshinone synergizes with sorafenib to inhibit the malignant biological behavior of sorafenib-resistant cells.
(A) HepG2-SR and Huh7-SR cells were exposed to different concentrations (0, 20, 40 and 80 μmol/L) of cryptotanshinone and/or different concentrations (0, 2, 5 and 10 μmol/L) of sorafenib for 24 h. Cell viability was assessed and normalized to the values from control cells. (B) Huh7-SR and HepG2-SR cells were incubated for 48 h with sorafenib (10 μmol/L), cryptotanshinone (40 μmol/L) or a combination. Cell viability was assessed and normalized to control cells. ***P < 0.001 vs. Control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Cryptotanshinone; & & & P < 0.001 vs. Sorafenib alone. (C, D) Cells described in panel (A) were subjected to Transwell assay for 48 h, and cell migration and invasion were evaluated.***P < 0.001 vs. Control; #P < 0.05, ###P < 0.001 vs. Cryptotanshinone; & & & P < 0.001 vs. Sorafenib alone.
Figure 4. Cryptotanshinone synergizes with sorafenib to inhibit p-STAT3, EMT and promote apoptosis in sorafenib-resistant cells.
Western blot analysis of protein levels of p-STAT3 (A), and EMT-(B) and apoptosis-associated markers (C) in Huh7-SR and HepG2-SR. Band densities were normalized to GAPDH. Data represent three independent experiments. **P < 0.01, ***P < 0.001 vs. Control; ##P < 0.01, ###P < 0.001 vs. Cryptotanshinone; & & & P < 0.001 vs. Sorafenib alone.
Figure 5. Activation of STAT3 enhances the malignant biological behavior of sorafenib-resistant cells.
(A) HepG2-SR and Huh7-SR cells were incubated with 0 or 10 μmol/L colivelin for 0.5 h, and then incubated with 40 μmol/L cryptotanshinone and/or 10 μmol/L sorafenib for 24 h. Cell viability was assessed and compared with the corresponding untreated cells. *P < 0.05, ***P < 0.001. (B) Cells were subjected to colony formaiton assay. *P < 0.05 vs. Control; #P < 0.05 vs. Cryptotanshinone; & P < 0.05 vs. Sorafenib; %P < 0.05 vs. combination. (C, D) The cells were subjected to Transwell assay for 48 h, and then cell migration and invasion were measured. *P < 0.05, ***P < 0.001 vs. Control; #P < 0.05, ##P < 0.01 vs. Cryptotanshinone; & P < 0.05, & & & P < 0.001 vs. Sorafenib; %P < 0.05, %%P < 0.01, %%%P < 0.001 vs. combination.
Figure 6. Activation of STAT3 enhances EMT activation and inhibits apoptosis of the sorafenib-resistant liver cancer cells.
Western blot analysis of the protein levels of p-STAT3 (A) and EMT-(B) and apoptosis-associated markers (C) in Huh7-SR and HepG2-SR. Band densities were normalized to GAPDH. Data represent three independent experiments. ***P < 0.001.
Figure 7. Cryptotanshinone increases the sensitivity to sorafenib in sorafenib-resistant cells in vivo.
(A) Xenograft tumor images from different groups (N = 3). (B) Measurement of tumor volume (N = 3). (C) Measurement of the tumor weight of different groups (N = 3). *P < 0.05, **P < 0.01 vs. Control; #P < 0.05, ##P < 0.01 vs. Cryptotanshinone; & P < 0.05, & & P < 0.01 vs. Sorafenib alone.
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