Licochalcone A induces autophagy through PI3K/Akt/mTOR inactivation and autophagy suppression enhances Licochalcone A-induced apoptosis of human cervical cancer cells

Abstract

The use of dietary bioactive compounds in chemoprevention can potentially reverse, suppress, or even prevent cancer progression. However, the effects of licochalcone A (LicA) on apoptosis and autophagy in cervical cancer cells have not yet been clearly elucidated. In this study, LicA treatment was found to significantly induce the apoptotic and autophagic capacities of cervical cancer cells in vitro and in vivo. MTT assay results showed dose- and time-dependent cytotoxicity in four cervical cancer cell lines treated with LicA. We found that LicA induced mitochondria-dependent apoptosis in SiHa cells, with decreasing Bcl-2 expression. LicA also induced autophagy effects were examined by identifying accumulation of Atg5, Atg7, Atg12 and microtubule-associated protein 1 light chain 3 (LC3)-II. Treatment with autophagy-specific inhibitors (3-methyladenine and bafilomycin A1) enhanced LicA-induced apoptosis. In addition, we suggested the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of mTOR pathway by LicA. Furthermore, the inhibition of PI3K/Akt by LY294002/si-Akt or of mTOR by rapamycin augmented LicA-induced apoptosis and autophagy. Finally, the in vivo mice bearing a SiHa xenograft, LicA dosed at 10 or 20 mg/kg significantly inhibited tumor growth. Our findings demonstrate the chemotherapeutic potential of LicA for treatment of human cervical cancer.

Keywords: Licochalcone A; apoptosis; autophagy; cervical cancer.

Figure 1. The ability of LicA to induce apoptosis in SiHa cervical cancer cells

A. The molecular structure of LicA. B. SiHa and C. HeLa cells were incubated with various concentrations (0, 10, 30, 50, 70, and 100 μM) of LicA for 24 and 48 hours. Cell viability was determined by using an MTT assay. SiHa cells were treated with various concentrations (0∼50 μM) of LicA and 50 μM LicA for 0, 6, 12, and 24 hours. D. Then examined by annexin V/PI double stained assay. E. Cell lysates were subjected to Western blotting. SiHa and HeLa cells were pretreated with Z-VAD-FMK (25 μM), for 2 hours and then incubated with LicA (50 μM) for 24 hours. F. Cell viability was determined by using MTT assay. G. The apoptotic cells were measured by flow cytometry. **p < 0.01, untreated cells or LicA plus Z-VAD-FMK versus LicA-treated cells. Data are presented as the mean ± SE of at least three independent experiments.

Figure 2. The ability of LicA to induce autophagy in SiHa and HeLa cervical cancer cells

A. SiHa and HeLa cells were treated with various concentrations (0∼50 μM) of LicA and 50 μM LicA for 0, 6, 12, and 24 hours. B. Cell lysates were subjected to Western blotting with anti-LC3-II and anti-β-actin antibodies. C. Cells were fixed and immunostained with anti-LC3-II antibody (red), and cell nuclei were counterstained with DAPI reagent. D. Effects of LicA on expression of autophagy regulatory proteins. E. HeLa cells expressed GFP-LC3 and acidic vesicular organelles were measured by fluorescence microscope and western blotting after the cells were treated with LicA. Data are presented as the mean±SE of at least three independent experiments. **p < 0.01, compared with that of the untreated control (0 μM or 0 hours).

Figure 3. Autophagy decreased LicA-induced apoptosis in SiHa cervical cancer cells

SiHa cells were pretreated with an autophagy inhibitor, 3-MA (10 mM) and bafilomycin A (BA; 10 nM) for 2 hours and then incubated with LicA (50 μM) for 24 hours. A. Cell lysates were subjected to Western blotting. B. Cell viability was determined by using MTT assay. C. The apoptotic cells were measured by flow cytometry. D. Cells expressed production of acidic vesicular organelles and quantification were examined by fluorescence microscope and flow cytometry. E. SiHa cells treated with the combination of LicA and siAtg12/siBeclin-1 duplexes. Cell viability was assayed or F. annexin V-positive cells were quantitatively analyzed. **p < 0.01, untreated cells or LicA plus 3-MA, BA, or siAtg12/siBeclin1 versus LicA-treated cells. Data are presented as the mean ± SE of at least three independent experiments.

Figure 4. LicA induced apoptosis via the PI3K/Akt signaling pathway in SiHa and HeLa cells

SiHa and HeLa cells were treated with various concentrations (0∼50 μM) of LicA and 50 μM LicA for 0, 6, 12, and 24 hours. A. Cell lysates were subjected to Western blotting. B. SiHa and HeLa cells were treated with LicA in the absence or presence of LY294002 (15 μM) and siAkt (100 nM) by Western blotting. C. Cell viability was measured by MTT assay and D. the proportion of annexin V-positive cells was analyzed by flow cytometry. **p < 0.01, untreated cells or LicA plus LY294002 or siAkt versus LicA-treated cells. Data are presented as the mean ± SE of at least three independent experiments.

Figure 5. LicA-induced apoptosis and autophagy via the mTOR pathway

SiHa cells were treated with various concentrations (0∼50 μM) of LicA and 50 μM LicA for 0, 6, 12, and 24 hours. A. Cell lysates were subjected to Western blotting. B. SiHa cells were treated with LicA in the absence or presence of rapamycin (100 nM) by Western blotting. C. Cell viability was measured by MTT assay and D. the proportion of annexin V-positive cells was analyzed by flow cytometry. **p < 0.01, untreated cells or LicA plus rapamycin versus LicA-treated cells. Data are presented as the mean ± SE of at least three independent experiments.

Figure 6. LicA inhibited the tumor progression of cervical cancer in vivo

A. Nude mice inoculated with SiHa cervical cancer cells were fed with 0, 10, or 20 mg/kg LicA continuously until study termination. B. The growth of tumors was monitored in terms of tumor volume every week. The weights of the mice C. and the tumors D. were determined. E. IHC stainings of cleaved-caspase-3, cleaved-PARP, LC3-II and Ki-67 in untreated and LicA-treated SiHa xenografts. **p < 0.01, compared with that of the untreated control (DMSO).

Figure 7. Overview of pathways for LicA-inhibited the phosphorylation of PI3K-Akt-mTOR axis and autophagy inhibition enhances LicA-induced apoptosis in human cervical cancer cells