The antitumor effects of lupenone on colon cancer and its mechanistic insights

Abstract

Background: Lupenone, a natural constituent derived from medicinal plants and fruits, is classified as a lupane-type triterpenoid and is known for its diverse biological activities, including anti-diabetic, anti-cancer, and anti-inflammatory effects. However, the specific impact of lupenone on colon cancer has not been fully elucidated.

Purpose: The purpose of this research was to explore the anti-cancer effects of lupenone monotherapy and in combination with chemotherapy agents in colon cancer.

Method: In vitro study, the cytotoxic effects of lupenone on CT26 and MC38 colon cancer cells were examined using CCK8 assay and colony formation assay. The anti-proliferation properties of lupenone were assessed via Ki67 staining, while cell apoptosis was analyzed using flow cytometry. The anti-migratory effects of lupenone were also detected using scratch wound healing assay and transwell assay. Reactive oxygen species (ROS) levels and endoplasmic reticulum (ER) stress pathways were measured using live-cell probes and real-time PCR, respectively. Autophagy and autophagy flux were evaluated through western blotting and immunofluorescence staining. In vivo experiment, the anti-tumor effects of lupenone, both as a monotherapy and in combination with cisplatin, were further examined in a CT26 tumor-bearing mouse model. Tumor volume and protein expression were used to evaluate anti-tumor efficacy, while histological staining assessed the safety of lupenone. RNA sequencing was performed to clarify the mechanisms of the combination of lupenone and cisplatin.

Results: Lupenone significantly inhibited the growth of CT26 and MC38 colon cancer cells in a concentration-dependent manner, reducing cell proliferation and migration while promoting apoptosis. lupenone increased ROS levels and induced ROS-dependent ER stress in colon cancer cells. Additionally, lupenone triggered autophagy and inhibited autophagy flux, exerting anti-colon cancer effects that were attenuated by the autophagy inhibitor 3MA. Molecular docking revealed that lupenone has the potential to bind to mTOR, and the mTOR activator MHY1485 partially reversed lupenone-induced autophagy. Additionally, the scavenging of ROS and the inhibition of ER stress partially reversed the autophagic effects of lupenone. In vivo studies revealed that lupenone suppressed subcutaneous tumor growth without causing significant weight loss or damage to major organs. The combination therapy with lupenone and cisplatin enhanced anti-tumor efficacy, with RNA sequencing analyses indicating regulation of cancer cell metabolism, T cell differentiation and activation, extracellular matrix remodeling, and the immune-inflammatory microenvironment.

Conclusions: Our study indicated lupenone monotherapy inhibited the growth of colon canceris and was non-toxic to major organs, while the combination of lupenone and chemotherapy drug could enhance the anti-cancer efficacy of chemotherapy drug. These findings highlight the potential of lupenone as a novel therapeutic option for the treatment of colon cancer.

Keywords: Autophagy; Colon cancer; ER stress; Lupenone; ROS.