Amentoflavone induces caspase-dependent/-independent apoptosis and dysregulates cyclin-dependent kinase-mediated cell cycle in colorectal cancer in vitro and in vivo.

Colorectal cancer (CRC) is recognized as the third most common malignancy and the second most deadly in highly developed countries. Although the treatment of CRC has improved in the past decade, the mortality rate of CRC is still increasing. Amentoflavone, one of the flavonoids detected in medical plants, is reported to possess potential anticancer properties in various cancers. However, its role in CRC has not been studied. This study aimed to investigate the role and underlying mechanism of amentoflavone on CRC in vitro and in vivo. We identified the cytotoxicity, apoptosis effect, cell cycle alteration, DNA damage induction and tumor progression inhibition of amentoflavone in HT-29 model by using MTT assay, flow cytometry, immunofluorescence (IF) staining, Western blotting and animal experiments. Amentoflavone induced cytotoxicity is caused by triggering G1 arrest, DNA damage and apoptosis in HT-29 cells. The expression of cyclin D1, CDK4 and CDK6 was decreased by amentoflavone; in contrast, the phosphorylation of ATM and CHK2 and the expression of p21 and p27 were increased. The apoptosis induction of amentoflavone in CRC is not only caspase-dependent but also increases EndoG and AIF nuclear translocation in a caspase-independent manner. Importantly, the apoptosis induction of amentoflavone is not affected by the activity of p53 in CRC. Amentoflavone suppressed the progression of CRC by initiating G1 arrest and ATM/CHK2-mediated DNA damage-responsive, caspase-dependent/independent apoptotic effects. We uncovered a novel tumor-inhibitory role of amentoflavone in CRC that is not associated with p53 activity, which may serve as a potential treatment for CRC.

Novel Paired Cell Lines for the Study of Lipid Metabolism and Cancer Stemness of Hepatocellular Carcinoma.

There are few well-characterized syngeneic murine models for hepatocellular carcinoma (HCC), which limits immunological studies and the development of immunotherapies for HCC. We previously established an oncogene-induced spontaneous HCC mouse model based on transposon-mediated oncogene (AKT and NRASV12) insertion into the genome of hepatocytes to induce tumorigenesis. Two tumor clones with different levels of lipid droplets (LDs) showed similar in vitro growth but distinctive in vivo phenotypes, including divergent proliferative capability and varying induction of myeloid-derived suppressor cells (MDSCs). The two clones showed distinct gene expression related to lipid metabolism, glycolysis, and cancer stemness. Endogenous fatty acid (FA) synthesis and exogenous monounsaturated fatty acid (MUFA) consumption promoted both tumor proliferation and cancer stemness, and upregulated c-Myc in the HCC cell lines. Moreover, the LDhi HCC cell line expressed a higher level of type II IL-4 receptor, which promoted tumor proliferation through binding IL-4 or IL-13. The chromosomal DNA of two tumor clones, NHRI-8-B4 (LDhi) and NHRI-1-E4 (LDlo) showed five identical AKT insertion sites in chromosomes 9, 10, 13, 16 and 18 and two NRAS integration sites in chromosomes 2 and 3. Herein, we describe two novel HCC cell lines with distinct features of lipid metabolism related to cancer stemness and differential interplay with the immune system, and present this syngeneic HCC mouse model as a practical tool for the study of cancer stemness and discovery of new therapies targeting liver cancers.