Chun-Li Su

Ferroptosis, a form of cell death, is characterized by lipid peroxidation and is dependent on iron and reactive oxygen species (ROS). Here, through bioinformatics analysis, formosanin C was predicted to be a ferroptosis inducer in colorectal cancer (CRC) by suppressing antioxidation capacity. Indeed, formosanin C induced iron accumulation, lipid ROS formation, and ferroptosis in CRC. We found that TP53 and KRAS were the second and third most frequently mutated genes in CRC and were associated with a poor prognosis. Analyses of differentially expressed genes indicated that fatty acid and labile iron levels tended to be higher in CRC than in normal tissues, suggesting the predisposition of CRC cells to ferroptosis. Transcriptomic analyses in CRC patients further identified that wild-type TP53 and mutant KRAS separately favored ferroptosis. Likewise, p53 knockdown rendered HCT 116 cells less sensitive to ferroptosis, and KRAS HT-29 cells were more sensitive to ferroptosis compared with their parental counterparts. Moreover, formosanin C synergistically enhanced chemosensitivity to cisplatin, and this process was mediated by lipid ROS. Overall, our novel gene-expression screening platform allows for the efficient identification of the biological function of novel phytochemicals, and the data suggest that formosanin C is an effective ferroptosis inducer in CRC cells with p53 or oncogenic KRAS.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death. Sorafenib, a multikinase inhibitor, has been approved as a first‐line systemic therapeutic for HCC patients based on the results of two large clinical trials, in which sorafenib significantly increased life expectancy of patients with Child Pugh A advanced stage of liver cancer, no matter which races they were or whether being infected with hepatitis B or C virus; however, its efficacy is compromised by the resistance of the tumor cells. By using integrative bioinformatics analysis, we identified ferroptosis as a candidate to modulate sorafenib‐resistant HCC. Ferroptosis is a novel, iron‐dependent, non‐apoptotic regulated cell death with characteristics of impaired lipid peroxide repair, redox active iron, and the oxidation of polyunsaturated fatty acids. Here, glutathione peroxidase 4 (GPX4) was further identified as a favorable prognostic factor in cancer survival by analyzing data repositories. Compared to the parental human HCC Huh7 cells, lower expression of GPX4, dysregulated iron homeostasis, and higher expression of acyl‐CoA synthetase long‐chain family member 4 were observed in sorafenib‐resistant Huh7R cells, and the Huh7R cells exhibited higher sensitivity to ferroptosis induction exerted by RSL3, a GPX4 inhibitor. The RSL3‐induced ferroptosis was attenuated by lysosomal blocker bafilomycin A1, indicating that lysosomal degradation of ferritin may confer sensitivity to GPX4‐inactivation‐induced ferroptosis by providing accessible iron. Taken together, our findings demonstrate that GPX4‐inactivation‐induced ferroptosis is a promising and effective treatment option capable of overcoming sorafenib resistance in liver cancer, and our novel gene expression‐screening platform via integrated analysis of differentially expressed genes and pathways allows efficient identification of therapeutic strategies.

Drp1 is considered the master regulator of mitochondrial fission and affects the proper segregation during mitosis. Here, we identified two clusters on mitochondria, including Drp1-Ser-616/Mff/Mid51 and-Drp1-Ser637/Fis1/Mid49 for midzone fission (division) and peripheral fission signatures, respectively. This study revealed that phospho-Drp1-Ser637 is involved in mitigating mitochondrial oxidative stress, resolving a long-standing localization issue. Additionally, we demonstrated that Mid49 and Mid51 exhibit distinct functions in recruiting Drp1 for mitochondrial fission. It also confirmed the essential role of the Fis1 and Mid49 adaptors contributed into mitochondrial fission within the phospho-Drp1-Ser637/Fis1/Mid49 cluster during M-phase. Moreover, it suggested that there is mutual exclusivity between two clusters of phospho-Drp1 with four adaptors on mitochondria, as well as varied responses to specific anti-cancer drugs during mitotic arrest. Remarkably, the upregulation of the spindle assembly checkpoint (SAC) was also observed, ensuring the prolongation of M-phase with the formation of multipolar spindles. Collectively, the phospho-Drp1-637/Fis1/Mid49 cluster may not only function in peripheral fission (mitophagy) but also serve as a key SAC coordinator for the regulation of spindle assembly during mitotic arrest. These findings appear to be mutually exclusive of phospho-Drp1 with four adaptors on mitochondria and vary in response to specific anti-cancer drugs.

Formosanin C (FC) is a natural compound extracted from Paris formosana Hayata with anticancer activity. FC induces both autophagy and apoptosis in human lung cancer cells. FC‐induced depolarization of mitochondrial membrane potential (MMP) may trigger mitophagy. In this study, we clarified the effect of FC on autophagy, mitophagy, and the role of autophagy in FC‐related cell death and motility. We found FC caused the continuous increase of LC3 II (representing autophagosomes) from 24 to 72 h without degradation after treatment of lung and colon cancer cells, indicating that FC blocks autophagic progression. In addition, we confirmed that FC also induces early stage autophagic activity. Altogether, FC is not only an inducer but also a blocker of autophagy progression. Moreover, FC increased MMP accompanied by overexpression of COX IV (mitochondria marker) and phosphorylated Parkin (p‐Parkin, mitophagy marker) in lung cancer cells, but no colocalization of LC3 with COX IV or p‐Parkin was detected under confocal microscopy. Moreover, FC could not block CCCP (mitophagy inducer)‐induced mitophagy. These results imply that FC disrupts mitochondria dynamics in the treated cells, and the underlying mechanism deserves further exploration. Functional analysis reveals that FC suppresses cell proliferation and motility through apoptosis and EMT‐related pathway, respectively. In conclusion, FC acts as an inducer as well as a blocker of autophagy that results in cancer cell apoptosis and decreased motility. Our findings shed the light on the development of combined therapy with FC and clinical anticancer drugs for cancer treatment.

Targeting ferritin via autophagy (ferritinophagy) to induce ferroptosis, an iron- and reactive oxygen species (ROS)-dependent cell death, provides novel strategies for cancer therapy. Using a ferroptosis-specific inhibitor and iron chelator, the vulnerability of triple-negative breast cancer (TNBC) MDA-MB-231 cells to ferroptosis was identified and compared to that of luminal A MCF-7 cells. Saponin formosanin C (FC) was revealed as a potent ferroptosis inducer characterized by superior induction in cytosolic and lipid ROS formation as well as GPX4 depletion in MDA-MB-231 cells. The FC-induced ferroptosis was paralleled by downregulation of ferroportin and xCT expressions. Immunoprecipitation and electron microscopy demonstrated the involvement of ferritinophagy in FC-treated MDA-MB-231 cells. The association of FC with ferroptosis was strengthened by the results that observed an enriched pathway with differentially expressed genes from FC-treated cells. FC sensitized cisplatin-induced ferroptosis in MDA-MB-231 cells. Through integrated analysis of differentially expressed genes and pathways using the METABRIC patients’ database, we confirmed that autophagy and ferroptosis were discrepant between TNBC and luminal A and that TNBC was hypersensitive to ferroptosis. Our data suggest a therapeutic strategy by ferroptosis against TNBC, an aggressive subtype with a poor prognosis.

Analysis of various public databases revealed that HRAS gene mutation frequency and mRNA expression are higher in bladder urothelial carcinoma. Further analysis revealed the roles of oncogenic HRAS, autophagy, and cell senescence signaling in bladder cancer cells sensitized to the anticancer drug cisplatin using the phytochemical pterostilbene. A T24 cell line with the oncogenic HRAS was chosen for further experiments. Indeed, coadministration of pterostilbene increased stronger cytotoxicity on T24 cells compared to HRAS wild-type E7 cells, which was paralleled by neither elevated apoptosis nor induced cell cycle arrest, but rather a marked elevation of autophagy and cell senescence in T24 cells. Pterostilbene-induced autophagy in T24 cells was paralleled by inhibition of class I PI3K/mTOR/p70S6K as well as activation of MEK/ERK (a RAS target) and class III PI3K pathways. Pterostilbene-induced cell senescence on T24 cells was paralleled by increased pan-RAS and decreased phospho-RB expression. Coadministration of PI3K class III inhibitor 3-methyladenine or MEK inhibitor U0126 suppressed pterostilbene-induced autophagy and reversed pterostilbene-enhanced cytotoxicity, but did not affect pterostilbene-elevated cell senescence in T24 cells. Animal study data confirmed that pterostilbene enhanced cytotoxicity of cisplatin plus gemcitabine. These results suggest a therapeutic application of pterostilbene in cisplatin-resistant bladder cancer with oncogenic HRAS.

Ferroptosis, a recently discovered form of iron-dependent cell death, requires an increased level of lipid-reactive oxygen species (ROS). Ferritinophagy, a ferritin degradation pathway, depends on a selective autophagic cargo receptor (NCOA4). By screening various types of natural compounds, formosanin C (FC) was identified as a novel ferroptosis inducer, characterized by attenuations of FC-induced viability inhibition and lipid ROS formation in the presence of ferroptosis inhibitor. FC also induced autophagic flux, evidenced by preventing autophagic marker LC3-II degradation and increasing yellow LC3 puncta in tandem fluorescent-tagged LC3 (mRFP-GFP) reporter plasmid (ptfLC3) transfected cells when combined with autophagic flux inhibitor. It is noteworthy that FC-induced ferroptosis and autophagic flux were stronger in HepG2 cells expressing higher NCOA4 and lower ferritin heavy chain 1 (FTH1) levels, agreeing with the results of gene expression analysis using CTRP and PRISM, indicating that FTH1 expression level exhibited a significant negative correlation with the sensitivity of the cells to a ferroptosis inducer. Confocal and electron microscopy confirmed the pronounced involvement of ferritinophagy in FC-induced ferroptosis in the cells with elevated NCOA4. Since ferroptosis is a non-apoptotic form of cell death, our data suggest FC has chemotherapeutic potential against apoptosis-resistant HCC with a higher NCOA4 expression via ferritinophagy.

Formosanin C (FC) is an anti‐tumor 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. Ferroptosis is a new form of iron‐dependent cell death, which occurs via an increased labile iron pool and lipid reactive oxygen species (ROS). Ferritinophagy is a ferritin (an iron storage protein) degradation pathway, depending on a selective autophagic cargo receptor nuclear coactivator 4 (NCOA4). Increased labile iron pool by ferritinophagy has been recognized as an upstream regulator of ferroptosis. Thus, ferroptosis has been reported to be a form of autophagic cell death. Although iron is an essential nutrient that facilitates cell proliferation and growth, overexpression of ferritin and down‐regulation of ferroportin (an iron export protein) has recently been observed in human hepatocellular carcinoma (HCC), indicating the increased dependence of iron in the cancer cells. In the present study, FC caused a significant dosage‐related population growth inhibition in HCC HepG2 and Hep3B cells using SRB assay, which can be partially blocked by co‐administration of ferroptosis inhibitor ferrostatin‐1 (a scavenger of lipid ROS). FC induced elevation in lipid ROS formation can also be partially reversed by addition of either ferrostatin‐1, or the presence of antioxidant vitamin C and/or vitamin E using flow cytometry, confirming FC‐induced ferroptosis. Flow cytometric analysis also revealed that FC elevated the percentage of acidic vesicular organelles‐positive cells, representing the formation of autolysosomes and induction of autophagic flux in both cell lines. The FC‐induced autophagy was confirmed using Western blotting, characterized by the production of LC3‐II and a further elevation of LC3‐II expression by addition of autophagy inhibitor bafilomycin A1 to prevent the fusion of autophagosomes and lysosomes for cargo degradation. In the present of bafilomycin A1, accumulation of NCOA4 and ferritin was observed in both FC‐treated cell lines using Western blotting. Concurrently, increase in co‐localization of LC3 puncta with NCOA4 or ferritin was exhibited using confocal microscopy, suggesting FC‐induced ferritinophagy. It is noteworthy that HepG2 cells with a higher NCOA4 expression were more sensitive to FC‐induced ferroptosis and autophagy compared to Hep3B cells with a higher ferritin expression. Overall, our data suggest chemotherapeutic potential of phytochemical FC for treatment of HCC with higher expression of NCOA4 via ferritinophagy and ferroptosis. Support or Funding Information: Supported in part by the Ministry of Science and Technology, Taiwan, No. MOST 106‐2320‐B‐003‐006‐MY3 and 107‐2320‐B‐003‐002‐MY2