The purified extract of Cremastrae pseudobulbus pleiones pseudobulbus (EPC) induces mitochondrial apoptosis in hepatocellular carcinoma cells through inhibiting a key oncogenic signaling pathway

Background: Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers and is associated with high incidence rate and high mortality rate. Cremastrae pseudobulbus pleiones pseudobulbus (CPPP) is commonly used in the clinical treatment of HCC, and our preliminary studies have identified four phenanthrenes and one bibenzyl as its primary active components.

Purpose: This study aimed to establish an extraction and purification process for the active components of CPPP, obtain its purified extract (EPC), and further elucidate the anti-tumor effects of EPC against HCC and its underlying mechanisms.

Methods: Orthogonal design experiment and macroporous resin chromatography were employed to establish the optimal extraction and purification conditions for EPC. The anti-HCC effects of EPC were evaluated through a comprehensive series of assays including CCK-8 assay, EdU incorporation, and wound healing in vitro, alongside a mouse xenograft model in vivo. The mode of cell death was determined using specific inhibitors, flow cytometry, and Western blot analysis. Mitochondrial morphology and function were assessed using transmission electron microscopy, ROS, ATP and CA²⁺ assay kits. The molecular mechanisms were elucidated through thermal proteome profiling, and western blot analysis, with subsequent verification using specific target and pathway activators.

Results: The content of 5 active components in the purified EPC was 4.74 times higher than that before purification, and its inhibitory effect on HCC cells viability was significantly enhanced. Pharmacological experiments confirmed that EPC could inhibit the proliferation and migration of HCC cells as well as the growth of HCC tumors. Furthermore, EPC induced Apoptosis by impairing mitochondrial morphology and function through increasing the protein expression levels of Bax/Bcl-2, cleaved Caspase-3, and cleaved Caspase-9 proteins. Mechanistic analysis revealed that this anti-HCC effect was largely dependent on suppression of the KRAS/PI3K/Akt/mTOR pathway by EPC. Activation of either KRAS protein or the PI3K/Akt/mTOR pathway attenuated the therapeutic efficacy of EPC.

Conclusions: EPC effectively alleviates HCC progression by predominantly triggering intrinsic, mitochondria-related Apoptosis, which is mediated through the suppression of the KRAS/PI3K/Akt/mTOR signaling pathway. Our findings provide evidence for the pharmacological basis of CPPP and identify EPC as a promising purified lead extract warranting further pharmacokinetic and safety evaluation for HCC.