The Role of Autophagy and Mitophagy in Synthetic Lethality of KRAS Mutant Human Colorectal Cancer Cells by Phytochemical Formosanin C

Synthetic lethality refers to the interaction between two co‐essential genes such that inhibiting the function of either gene separately results in cell survival, but inhibiting the function of both genes results in cell death. Targeting synthetic lethal interactions may spare normal cells while selectively killing cancer cells. Formosanin C (FC) is an anti‐cancer diosgenin saponin isolated from Paris formosana Hayata (Liliaceae) which is a perennial herb grown in the humid and dark forests of Taiwan at about the level of 1000 m. The plat has been used as a folk remedy for snake‐bite inflammation and tumors. FC is also a component of “Yunnan Bai Yao”, a traditional Chinese hemostatic medicine. Mutation of KRAS, a RAS superfamily of protein, affects the growth and spread of tumors. Nearly 35–45% of colorectal cancer (CRC) has the KRAS mutations which failed to the targeted therapies and led to poor prognosis. Our previous results indicated that FC‐induced apoptosis of human CRC HT‐29 cells (KRAS wild‐type) via the changes of mitochondrial membrane potential. To determine whether FC can benefit CRC with KRAS mutations to overcome cell resistance, HT‐29 and HT‐29 KRAS (with inducible KRAS mutant gene) cells were used in the present study. MTT analysis revealed that FC induced a stronger dosage‐related growth inhibition on HT‐29 KRAS. Annexin V/propidium iodide double staining analysis further showed that both early and late apoptosis were induced to a greater extent by FC on HT‐29 KRAS than HT‐29. No induction of necrosis was observed. FC also induced a higher degree of autophagy on HT‐29 KRAS, characterized by an increase of LC3‐II expression and LC3‐II/I ratio, induction of LC3‐II puncta, and elevation of acidic vesicular organelles (AVOs) formation, representing the production of autolysosomes at the later stage of autophagic flux. It is noteworthy that administration of autophagy inhibitor Bafilomycin A1 increased FC‐induced growth inhibition and elevated apoptosis on both cells, suggesting the FC‐induced autophagy is cytoprotective. Moreover, fluorescence microscopy analysis revealed that FC induced formation of Parkin puncta in HT‐29 KRAS cells to a greater extent. Addition of mitochondrial division inhibitor‐1 (Mdivi‐1), which can selectively inhibit Drp1 to prevent mitochondrial fission and mitophagy, reversed the FC‐induced phenomena. These data suggest that FC‐induced mitophagy was via Drp1 and Parkin pathway. It is noteworthy that addition of the mitophagy inhibitor Mdivi‐1 increased FC‐induced growth inhibition on HT‐29 KRAS, implying that the FC‐induced mitophagy is cytoprotective. Overall, our data indicate that FC may target one more gene other than mutant KRAS to trigger a stronger apoptosis (synthetic lethality), and addition of either autophagy or mitophagy inhibitor further enhanced the FC‐induced cytotoxicity on HT‐29 KRAS cells, suggesting a possible role of autophagy and mitophagy on KRAS mutant CRC. Support or Funding Information (Supported in part by the Ministry of Science and Technology, Taiwan, No. MOST 105‐2320‐B‐003‐003 and National Taiwan Normal University, Taiwan, No. 10502). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .