Carnosic acid increases sorafenib-induced inhibition of ERK1/2 and STAT3 signaling which contributes to reduced cell proliferation and survival of hepatocellular carcinoma cells

Abstract

Hepatocellular carcinoma (HCC) has increasing worldwide incidence but when unresectable lacks curative options. Treatment with a kinase inhibitor Sorafenib (Sf), while initially effective, results in only short increases in patient survival, thus there is a need for improved treatment regimens. Numerous treatment regimens have been explored wherein Sf is combined with other agents, such as non-toxic botanicals like Curcumin or Silibinin. Recently, we have shown that carnosic acid (CA), a component of the food preservative Rosemary Extract, can markedly enhance the cytotoxic actions of Sf in several cell lines derived from HCC, but not in the cell line Hu1545 derived from normal hepatocytes. CA has been shown to enhance Sf-induced cell death in the neoplastic cell lines, principally due to the composite of increased apoptosis and cytotoxic autophagy. In the present study we focused on the mechanisms that underlie the reduced proliferation and survival of HCC cells when CA is added to Sf and how this relates to the increase in Sf-induced DNA damage as well as to the elevation of cytoplasmic levels of reactive oxygen species (ROS). Importantly, the elevation of ROS levels induced by Sf was increased by adding CA. We found that CA enhanced Sf-induced prolongation of cell cycle, and the overall decrease in cell growth was associated with reduced activation of both STAT3 transcription factor (TF) and extracellular signal-regulated protein kinase (Erk)1/2. Our data suggest that a regimen incorporating CA, an inexpensive and non-toxic food additive, in the treatment of advanced HCC merits clinical evaluation.

Keywords: ERK1/2; STAT3; carnosic acid; hepatoma; sorafenib.

Figure 1. Carnosic acid potentiates sorafenib-induced elevation of cytosolic ROS levels in HCC cells.

(A and B) Mean fluorescence intensity of the DCFH-DA oxidized product dichlorofluorescein (DCF) was measured in Huh7 (A) and HerpG2 (B) cells following treatment with the indicated agents for 24 h. Cells exposed to H₂O₂ for 30 minutes were used as the positive control. The data are the means ± SD (n = 7 for Huh7; n = 6 for HepG2). ^* p < 0.05 vs. control; ^# p < 0.05; and ^## p < 0.01 vs. Sf alone.

Figure 2. Comet assays of DNA damage in sorafenib and/or carnosic acid-treated HCC cells.

Representative images of tailing by damaged DNA in Huh7 (A) and HepG2 (B) cells show that treatment with carnosic acid (CA) alone for 24 hours has no apparent effect on both cell lines (left-side panels). As expected, there is DNA damage when the cells are exposed to 1 μM sorafenib (Sf) alone, for 24 hours. DNA damage is increased when Sf is combined with CA (right-side panels). Quantitation of comet tails were shown in the bar charts, as described in Materials and Methods. ^* p < 0.05 vs. control; ^# p < 0.05 vs. Sf alone; n = 3.

Figure 3. Combination of carnosic acid with sorafenib increases the expression of proteins related to DNA damage.

Huh7 and HepG2 cells were treated with the indicated agents for 24 or 48 hours. The levels of proteins related to DNA damage, P-H2AX, GADD45, ATM, and Chk2, were determined by western blots. β-actin was used as the loading control. Average Integrated Density Values (IDV) from three separate experiments are shown in bar charts above each blot. ^* p < 0.05 vs. control; ^# p < 0.05 vs. Sf alone.

Figure 4. Carnosic acid enhances the sorafenib-induced retardation of cell proliferation and cell death.

The total cell number of Huh7 and HepG2 cells and the number of dead cells were enumerated by the hemocytometer following treatment with the indicated agents for 24 hours, as detailed in Materials and Methods. (A) Cell proliferation (CP) was calculated as the percent of an increase in the total cell number over the baseline cell number relative to CP of the control sample. (B) The percent of dead cells was calculated as the number of trypan blue-positive cells relative to the total number of cells. (C, and D) Combination of CA (10 μM) and Sf (1 μM) significantly decreases cell proliferation and viability in both HCC cell lines. Cell cycle distribution was determined by Propidium Iodide staining, and the ratios of G1/S and G2/S are demonstrated in the bar charts. ^* p < 0.05; ^** p < 0.01 vs. control; ^# p < 0.05 vs. Sf alone; n = 3.

Figure 5. Carnosic acid enhances the sorafenib-induced increase in the expression of proteins related to DNA damage and cell cycle arrest.

Following treatment of Huh7 and HepG2 cells with the indicated agents for 24 or 48 hours. The levels of proteins related to DNA damage (ATR and Chk1) and cell cycle progression inhibitors (p21 and p27) were determined by western blots. β-actin was used as the loading control. Average Integrated Density Values from three separate experiments are shown in the bar charts above each blot. ^* p < 0.05 vs. control; ^# p < 0.05 vs. Sf alone.

Figure 6. Carnosic acid enhances the sorafenib-induced reduction of P-ERK1/2 and P-STAT3 levels in HCC cells.

Huh7 and HepG2 cells were treated with the indicated agents for 24 hours. The protein levels of P-ERK1/2 (Thr202/Tyr204), P-STAT3 (Tyr605), and total (T) ERK1/2 and STAT3 were determined by western blots. The ratios of P-ERK1/2 vs. T-ERK1/2 and P-STAT3 vs. T-STAT3 are shown in the table below. CA further enhances Sf-induced reduction of activation of both ERK1/2 and STAT3.

Figure 7. The effects of inhibition of ERK1/2 or STAT3 by pharmacological inhibitors on cell death and cell cycle related proteins.

HCC cells were pretreated with either MEK1 inhibitor U0126 (1 μM) or the STAT3 inhibitor Stattic (20 μM) for 1 hour, and then treated with indicated agents for another 24 hours. The protein levels of P-ERK1/2, P-STAT3, and cell death- and cell cycle-related targets were determined by western blotting. β-actin was used as the loading control. The blots shown illustrate one of three individual experiments, and Integrated Density Values are shown under each blot.