A Marine Terpenoid, Heteronemin, Induces Both the Apoptosis and Ferroptosis of Hepatocellular Carcinoma Cells and Involves the ROS and MAPK Pathways

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of death, resulting in over 700 thousand deaths annually worldwide. Chemotherapy is the primary therapeutic strategy for patients with late-stage HCC. Heteronemin is a marine natural product isolated from Hippospongia sp. that has been found to protect against carcinogenesis in cholangiocarcinoma, prostate cancer, and acute myeloid leukemia. In this study, heteronemin was found to inhibit the proliferation of the HCC cell lines HA22T and HA59T and induce apoptosis via the caspase pathway. Heteronemin treatment also induced the formation of reactive oxygen species (ROS), which are associated with heteronemin-induced cell death, and to trigger ROS removal by mitochondrial SOD2 rather than cytosolic SOD1. The mitogen-activated protein kinase (MAPK) signaling pathway was associated with ROS-induced cell death, and heteronemin downregulated the expression of ERK, a MAPK that is associated with cell proliferation. Inhibitors of JNK and p38, which are MAPKs associated with apoptosis, restored heteronemin-induced cell death. In addition, heteronemin treatment reduced the expression of GPX4, a protein that inhibits ferroptosis, which is a novel form of nonapoptotic programmed cell death. Ferroptosis inhibitor treatment also restored heteronemin-induced cell death. Thus, with appropriate structural modification, heteronemin can act as a potent therapeutic against HCC.

Figure 1

The cytotoxicity of heteronemin against HCC cell lines. The viability of (a) HA22T and (b) HA59T cells was determined 24 and 48 hours after the heteronemin treatment. ^∗∗p < 0.01, ^∗p < 0.05 compared with the control group; all data are presented as the mean ± S.D. of three independent experiments. (c) The morphological changes of HA22T and HA59T cells after 24 hours of heteronemin treatment. Magnification: 100x and 200x.

Figure 2

Heteronemin induces cell apoptosis via the caspase cascade. (a) HA22T and HA59T cells were treated with control or 5, 10, 20, or 30 μM heteronemin for 24 hours and stained with annexin V/7AAD to analyze apoptotic cells. (b) and (c) Quantification of apoptotic (annexin V⁺) cells in (a). ^∗p < 0.05, ^∗∗∗∗p < 0.0001 compared with the control. (d) and (e) Quantification of nonapoptotic (annexin V^-/7AAD⁺) cells in (a). ^∗∗p < 0.01 compared with the control. #p < 0.05 compared with 20 μM and 30 μM heteronemin-treated cells. (f) and (g) Cell viability of HA22T and HA59T cells pretreated with 20 μM Z-VAD-FMK, a pan-caspase inhibitor, for 4 hours and treated with 20 μM heteronemin for 24 hours. ^∗∗∗p < 0.001. (h) and (i) Western blot analysis of the Bax expression in heteronemin-treated HA22T and HA59T cells. All data are presented as the mean ± S.D. of three independent experiments.

Figure 3

ROS formation is associated with heteronemin-induced cell death. The number of H₂O₂-positive cells in (a) HA22T and (b) HA59T was detected with DCFDA and analyzed by flow cytometry. In addition, O₂∙^–-positive cells in (c) HA22T and (d) HA59T cells were detected with DHE and analyzed by flow cytometry. Western blot analysis of the SOD1 and SOD2 expression in (e) HA22T and (f) HA59T cells after the heteronemin treatment. (g) HA22T and (h) HA59T cells were treated with NAC (10 mM) for 2 hours before being treated with 20 μM heteronemin, and cell viability was measured after 24 hours. All data are presented as the mean ± S.D. of three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001; all data are presented as the mean ± S.D. of three independent experiments.

Figure 4

The MAPK signaling pathway regulates the heteronemin-mediated cell death. Western blot analysis of ERK1/2 expression in (a) HA22T and (b) HA59T cells after heteronemin treatment. (c) HA22T and (d) HA59T cells were pretreated with 30 μM SP600125, a JNK inhibitor, for 1 hour before being treated with 20 μM heteronemin, and cell viability was observed. (e) HA22T and (f) HA59T cells were pretreated with 30 μM SB203580, a p38 inhibitor, for 1 hour before being treated with 20 μM heteronemin, and cell viability was analyzed. ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; all data are presented as the mean ± S.D. of three independent experiments.

Figure 5

Heteronemin initiates ferroptosis, which is associated with heteronemin-induced cell death. Western blot analysis of ferroptosis markers and the reduction in GPX4 in (a) HA22T and (b) HA59T cells after heteronemin treatment. Liproxstatin and ferrostatin were used to determine the effect of ferroptosis on heteronemin-associated cell death. (c) HA22T and (d) HA59T cells were cotreated with 5 μM liproxstatin and 20 μM heteronemin, and cell viability was measured. (e) HA22T and (f) HA59T cells were cotreated with 15 μM ferrostatin and 20 μM heteronemin treatment, and cell viability was measured. (g) HA22T was cotreated with 15 μM ferrostatin and 20 μM heteronemin, and apoptosis was measured with annexin V/7AAD double staining. ^∗p < 0.05, ^∗∗p < 0.01; all data are presented as the mean ± S.D. of three independent experiments.

Figure 6

The potential anticancer mechanism of heteronemin. Heteronemin was found to induce ROS formation, resulting in p38/JNK activation and caspase-associated apoptosis and ferroptosis and leading to cancer cell death.