Cycloartobiloxanthone Induces Human Lung Cancer Cell Apoptosis via Mitochondria-dependent Apoptotic Pathway

Abstract

Background: Lung cancer is one of most malignant types of cancer and new anticancer agents are still required. Cycloartobiloxanthone, a flavonoid isolated from stem bark of Artocarpus gomezianus, has potential for being developed for anticancer therapy.

Materials and methods: Cytotoxicity of cycloartobiloxanthone was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay against four human lung cancer cell lines (H23, H460, H292 and A549) and their half-maximal inhibitory concentrations (IC₅₀) were assessed. Apoptotic induction in H460 cells was investigated by Hoechst 33342/propidium iodide (PI) staining assay and protein hallmarks of mitochondria-dependent apoptotic pathway were examined by western blot analysis.

Results: Cycloartobiloxanthone exhibited potent cytotoxic effect on both small and non-small cell lung cancer cells. Nuclear Hoechst/PI staining revealed that apoptotic cell death was the main mechanism of toxicity of cycloartobiloxanthone. The apoptosis-inducing potency of cycloartobiloxanthone was comparable to those of standard anticancer drugs cisplatin and etoposide at the same concentration. Protein analysis further showed that apoptosis was mediated via mitochondria-dependent pathway. p53 was activated in cells treated with cycloartobiloxanthone. Subsequently, pro-apoptotic protein B-cell lymphoma 2 (BCL2)-associated X protein (BAX) was found to be significantly increased, concomitantly with the decrease of anti-apoptotic proteins BCL2 and myeloid cell leukemia 1 (MCL1). Moreover, markers of the intrinsic apoptosis pathway, namely activated caspase-9, activated caspase-3, and cleaved poly(ADP-ribose)polymerase (PARP), dramatically increased in cycloartobiloxanthone-treated cells compared to the non-treated controls.

Conclusion: Cycloartobiloxanthone has anticancer activity against human lung cancer cells by triggering mitochondrial apoptotic caspase-dependent mechanism. This compound might have promising effects for cancer therapy.

Keywords: BAX; Cycloartobiloxanthone; MCL1; apoptosis; lung cancer; mitochondria-dependent apoptotic pathway.

Figure 1. Structure of cycloartobiloxanthone.

Figure 2. Cytotoxic effect of cycloartobiloxanthone on H460, H292, A549 and H23 cancer cells. Each cell lines were treated with 0-100 μM of cycloartobiloxanthone for 24 h. Cell viability was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay. Data was presented as mean±SD (n=3). The half maximal inhibitory concentration (IC50) of each cell line was calculated.

Figure 3. A: Apoptotic and necrotic cell death after 24 h of cycloartobiloxanthone treatment were examined by Hoechst 33342/propidium iodide (PI) co-staining. B: Percentage of apoptotic and necrotic nuclei in cycloartobiloxanthone-treated cells. Data are presented as the mean±SD (n=3). *Significantly different at p<0.05 compared to the untreated control group.

Figure 4. A and B: Apoptotic and necrotic cell death after 24 h of 50 μM cisplatin, etoposide and cycloartobiloxanthone treatments were examined by Hoechst 33342/propidium iodide (PI) co-staining. C: Percentage of apoptotic and necrotic nuclei in each treatment group. Data are presented as the mean±SD (n=3). *Significantly different at p<0.05 compared to the untreated control group.

Figure 5. Western blot analysis of proteins involved in mitochondria-mediated apoptotic cell death. H460 cells were treated with different doses of cycloartobiloxanthone for 24 h. B: Relative protein levels were quantified by densitometry. Data represent the mean±SD (n=3). *Significantly different at p<0.05 compared to the untreated control group.

Figure 6. Possible mechanism of action of cycloartobiloxanthone-induced apoptosis. Once cycloartobiloxanthone enters the cancer cell, it activates p53 and inhibits anti-apoptotic B-cell lymphoma 2 (BCL2) family proteins, BCL2 and myeloid cell leukemia 1 (MCL1), resulting in up-regulation of pro-apoptotic BCL2 family protein, BCL2-associated X protein (BAX). Alteration of the balance between these pro- and anti-apoptotic proteins triggers mitochondria-mediated apoptotic pathway through the caspase cascade. Cytochrome c released from mitochondria and apoptotic protease activating factor 1 protein forms the apoptosome with activated caspase- 9 (cleavage form). Cleaved caspase-9 then cleaves caspase-3, which is a key caspase that triggers apoptotic processes, one of which is inhibition of DNA repair by poly (ADP-ribose) polymerase (PARP) activity.