{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T05:09:47Z","timestamp":1789016987211,"version":"build-2803163510"},"reference-count":38,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T00:00:00Z","timestamp":1788998400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Med."],"abstract":"<jats:sec>\n                    <jats:title>Background and aims<\/jats:title>\n                    <jats:p>\n                      Hepatocellular carcinoma (HCC) is a cause of cancer-related mortality, and therapeutic efficacy is constrained by treatment resistance and toxicity. This study evaluated the anticancer activity and underlying molecular mechanisms of the\n                      <jats:italic>Chiliadenus iphionoides<\/jats:italic>\n                      ethanolic extract in hepatocellular carcinoma cells.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Hep3B cells were treated with the\n                      <jats:italic>C. iphionoides<\/jats:italic>\n                      ethanolic extract (10\u2013100\u2005\u00b5g\/mL) for 24\u2005h. Cell viability, tumor-associated markers, intracellular reactive oxygen species (ROS), oxidative DNA damage, apoptosis, mitochondrial membrane potential, intrinsic apoptotic signaling, cell-cycle regulatory genes, and cell-cycle distribution were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), ELISA, RT-qPCR, fluorometric assays, and flow cytometry. ROS dependency was examined by N-acetyl-L-cysteine (NAC) pretreatment. Key findings were evaluated in HepG2 and Huh-7 HCC cells, and non-tumorigenic THLE-3 hepatocytes.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      <jats:italic>C. iphionoides<\/jats:italic>\n                      reduced Hep3B viability in a concentration-dependent manner (IC50\u2009=\u200935.2\u2005\u00b5g\/mL; 95% CI, 31.85\u201338.47) and suppressed the expression of alpha-fetoprotein, GPC-3, and VEGF-A. Treatment-induced ROS accumulation was accompanied by increased 8-hydroxy-2'-deoxyguanosine (8-OHdG) and\n                      <jats:italic>\u03b3<\/jats:italic>\n                      H2AX levels, consistent with oxidative DNA damage. Mitochondrial depolarization was associated with Bax activation, increased caspase-9 activity, enhanced cleavage of caspase-3 and PARP-1, reduced BCL-2 expression, and increased Annexin V-positive apoptotic cells, indicating the activation of the intrinsic mitochondrial apoptotic pathway. Cyclin D1 mRNA was downregulated, whereas p21 mRNA increased despite the TP53-null background of Hep3B cells. Concordantly, G0\/G1-phase accumulation increased from 50.1\u2009\u00b1\u20090.9% to 76.5\u2009\u00b1\u20091.4%, while S-phase cells decreased from 31.5\u2009\u00b1\u20090.7% to 11.2\u2009\u00b1\u20090.9%. NAC pretreatment substantially attenuated ROS accumulation, oxidative DNA damage, mitochondrial dysfunction, and apoptotic signaling, supporting an upstream role for ROS. Comparable responses were observed in HepG2 and Huh-7 cells, whereas THLE-3 hepatocytes were less affected, indicating the preferential rather than absolute sensitivity of malignant hepatocytes.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      <jats:italic>C. iphionoides<\/jats:italic>\n                      ethanolic extract exhibits anticancer activity in HCC cells\n                      <jats:italic>in vitro<\/jats:italic>\n                      through ROS-associated oxidative DNA damage, mitochondrial apoptosis, and p53-independent G0\/G1 arrest.\n                    <\/jats:p>\n                  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