EM23, a natural sesquiterpene lactone, targets thioredoxin reductase to activate JNK and cell death pathways in human cervical cancer cells

Abstract

Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants and found to have potential anticancer activities. However, the intracellular molecular targets of SLs and the underlying molecular mechanisms have not been well elucidated. In this study, we observed that EM23, a natural SL, exhibited anti-cancer activity in human cervical cancer cell lines by inducing apoptosis as indicated by caspase 3 activation, XIAP downregulation and mitochondrial dysfunction. Mechanistic studies indicated that EM23-induced apoptosis was mediated by reactive oxygen species (ROS) and the knockdown of thioredoxin (Trx) or thioredoxin reductase (TrxR) resulted in a reduction in apoptosis. EM23 attenuated TrxR activity by alkylation of C-terminal redox-active site Sec498 of TrxR and inhibited the expression levels of Trx/TrxR to facilitate ROS accumulation. Furthermore, inhibition of Trx/TrxR system resulted in the dissociation of ASK1 from Trx and the downstream activation of JNK. Pretreatment with ASK1/JNK inhibitors partially rescued cells from EM23-induced apoptosis. Additionally, EM23 inhibited Akt/mTOR pathway and induced autophagy, which was observed to be proapoptotic and mediated by ROS. Together, these results reveal a potential molecular mechanism for the apoptotic induction observed with SL compound EM23, and emphasize its putative role as a therapeutic agent for human cervical cancer.

Keywords: ROS; apoptosis; sesquiterpene lactone; thioredoxin; thioredoxin reductase.

Figure 1. EM23 induced cell apoptosis and cell cycle arrest

(A) Chemical structure of EM23. (B) Effects of EM23 on the growth of the following human cancer cell lines: lung cancer cell line A549; breast cancer cell line MCF-7; esophageal cancer cell lines TE-1, EC109, and EC9706; cervical cancer cell lines CaSki and SiHa; and leukemia cell lines HL-60 and K562. Cells were treated with various concentrations of EM23 for 24 h, and cell viability was measured by MTT assay. (C) Effects of EM23 on cell cycle distribution. CaSki and SiHa cells were treated with 0, 5, 15, and 20 μM EM23 for 24 h, and cell cycle distribution was measured by flow cytometry after PI staining. (D) Induction of apoptosis by EM23 in CaSki and SiHa cells. Cells were treated as above and index of apoptotic cells were analyzed by flow cytometry after Annexin-V-FITC/PI staining. All data are presented as the mean ± SD of three independent experiments. *P < 0.5 and **P < 0.01.

Figure 2. Effects of EM23 on caspase 3 and mitochondrial membrane potential (MMP)

(A) Western blotting analysis of the expression levels of the following: caspases 3, cleaved caspases 3, PARP and XIAP. (B) Effect of EM23 on MMP in CaSki and SiHa cells with the treatment of 0, 5, 15, and 20 μM EM23 for 24 h. The MMP were analyzed by flow cytometry after JC-1 staining. Cells with MMP loss were gated. All data are representatives of three independent experiments or presented as the mean ± SD of three independent experiments. *P < 0.5 and **P < 0.01.

Figure 3. The role of ROS production in EM23-induced cell apoptosis

(A) EM23-induced ROS production in CaSki and SiHa cells treated with 15 μM EM23 for 1-4 h. Cells were stained with DCFH-DA, and analyzed for fluorescence by flow cytometry at 525 nm. (B) EM23-induced growth inhibition in CaSki and SiHa cells was inhibited by NAC pre-treatment. Cells were pretreated with 10 mM NAC, and then treated with 15 μM EM23 for 24 h. Cell viability was then analyzed by MTT assay. (C) EM23-induced apoptosis was inhibited by NAC pre-treatment. CaSki and SiHa cells were treated as above and index of apoptotic cells were analyzed by flow cytometry after Annexin-V-FITC/PI staining. (D) EM23-induced MMP disruption was inhibited by NAC pre-treatment. Cells were treated as above and index of MMP were analyzed by flow cytometry after JC-1 staining. All values are presented as the mean ± SD of three independent experiments. *P < 0.5 and **P < 0.01.

Figure 4. EM23 inhibited TrxR activity by binding to the selenocysteine site

(A) NADPH-reduced TrxR was incubated with various concentrations of EM23 in cell-free system at room temperature for 5 min. TrxR activity was measured by DTNB reduction assay. (B) NADPH-reduced TrxR was incubated with10 μM EM23 for 5, 15, 30 and 90 min. TrxR activity was measured by DTNB reduction assay. (C) Different concentrations of EM23 were added to reduced TrxR and incubated at room temperature for 1 h, then subjected to BIAM alkylation assays. The same amounts of DMSO were added to the control groups. (D) TOF LC/MS analysis of the adduct formed by TrxR C-terminal peptide Ac-Gly-[Cys-Sec]-Gly-NH₂ and EM23. The m/z signal peaks corresponding to EM23, tetrapeptide of C-terminal motif of TrxR and adduct were indicated by arrows, respectively. Up right, the zoomed in signal peak of m/z 810.2. All data are representatives of three independent experiments.

Figure 5. The binding mode research between EM23 and TrxR by docking simulations

(A) The MOLCAD surface of the binding pocket of docked TrxR-EM23 complexes was displayed in Fast Connolly pattern. (B) Binding interactions with selected residues of the active site of TrxR for EM23.

Figure 6. Effects of EM23 on the expression levels of Trx/TrxR system

(A) CaSki and SiHa cells were treated with 0, 5, 15 and 20 μM EM23 for 24 h, and Trx and TrxR expression levels were analyzed by western blotting. (B) The mRNA levels of Trx and TrxR were analyzed by RT-PCR; GAPDH was used as an internal control. Data are presented as the mean ± SD of three independent experiments. *P < 0.5 and **P < 0.01.

Figure 7. Effect of Trx or TrxR knock down on EM23-induced apoptosis

CaSki and SiHa cells were transfected with Trx or TrxR siRNA. (A) Protein lysates were prepared 24 h after transfection, and the expression levels of Trx and TrxR were analyzed by western blotting. (B) After transfection for 24 h, CaSki and SiHa cells were treated with 15 μM EM23 for another 24 h. Apoptotic cells were then analyzed by flow cytometry after Annexin-V-FITC/PI staining. Data are presented as the mean ± SD of three independent experiments. *P < 0.5 and **P < 0.01.

Figure 8. Effects of EM23 on ASK1, JNK and ERK signaling pathways

(A) CaSki and SiHa cells were treated with 15 μM EM23 for 8 h. ASK1-Trx complex was then precipitated by Protein A beads conjugated with Trx antibodies and analyzed by western blotting with ASK1 and Trx antibodies. (B) CaSki and SiHa cells were treated with 0, 5, 15 and 20 μM EM23 for 24 h, the expression levels of ASK1, p-ASK1, JNK, p-JNK, ERK and p-ERK were analyzed by western blotting analysis. (C) Effect of ROS scavenger NAC on JNK signaling pathway and PARP. CaSki and SiHa cells were pre-treated with 10 mM NAC followed by 15 μM EM23 for 24 h. Protein lysates were prepared and expression levels of JNK, p-JNK and PARP were analyzed by western blotting analysis. All data are representatives of three independent experiments.

Figure 9. Effects of ASK1 and JNK inhibitor on EM23-induced apoptosis

Cells were pre-treated with 0.6 and 0.2 μM ASK1 and JNK inhibitor, respectively, and then incubated with 15 μM EM23 for 24 h. Index of apoptosis in CaSki (A) and SiHa (B) cells were analyzed by flow cytometry after Annexin-V-FITC/PI staining. Data are presented as the mean ± SD of three independent experiments. *P < 0.5 and **P < 0.01.

Figure 10. EM23 induced autophagy and inhibited Akt/mTOR signaling pathway in CaSki and SiHa cells

(A) Microstructure of cells treated with 15 μM EM23 for 24 h; transmission electron microscopy shows multiple vacuoles. (B) Western blotting analysis of expression levels of LC3 I/II in CaSki and SiHa cells treated with 0, 5, 15, and 20 μM EM23 for 24 h. (C) The expression levels of p-Akt, Akt, p-mTOR, mTOR, p-4E-BP1, p-P70S6K, p-S6, and S6 in CaSki and SiHa cells. Cells were treated with 0, 5, 15, and 20 μM EM23 for 24 h and then subjected to western blotting analysis. (D) CaSki and SiHa cells were pretreated with 10 mM NAC and treated with 15 μM EM23 for 24 h, protein lysates were then prepared and the expression levels of LC3 I/II were analyzed by western blotting analysis. All data are representatives of three independent experiments.

Figure 11. Effects of 3-MA on EM23-induced cell growth inhibition and apoptosis

CaSki and SiHa cells were pre-treated with 5 mM 3-MA for 1 h, followed by 15 μM EM23 for 24 h. Cell viability was analyzed by MTT assay (A) and the apoptotic index cells were analyzed by flow cytometry after Annexin-V-FITC/PI staining (B). All data are presented as the mean ± SD of three independent experiments. *P < 0.5 and **P < 0.01.

Figure 12. EM23-mediated intracellular signaling elicits apoptosis in human cervical cancer cells