Background: Recent studies showed that macrophages co-cultured with ovarian cancer stem-like cells (OCSLCs) induced SKOV3 cell stemness via IL-8/STAT3 signaling. Genistein (GEN) demonstrates chemopreventive activity in inflammation-associated cancers. The present study aimed to examine whether and if GEN inhibits the stemness of SKOV3 and OVCA-3R cells induced by co-culture of THP-1 macrophages and SKOV3-derived OCSLCs. Methods: The co-culture was treated with or without different concentrations (10, 20, and 40 μmol/L) of GEN for 24 h. Depletion or addition of IL-8 in Co-CM and knockdown or overexpression of STAT3 in THP-1 macrophages was performed to demonstrate the possible associated mechanisms. The combined effects of GEN and STAT3 knockdown were examined with the nude mouse modle by co-injection of SKOV3-derived OCSLCs with THP-1 macrophages. Results: Our results showed that GEN down-regulated CD163 and p-STAT3 expression of THP-1 macrophage, decreased the levels of IL-10, increased the levels of IL-12 and nitric oxide (NO) in the conditioned medium, and reduced the clonogenic and sphere-forming capacities and the expression of CD133 and CD44 in SKOV3 cells induced by co-culture of THP-1 macrophages and OCSLCs in a dose-dependent manner. Moreover, depletion or addition of IL-8 enhanced or attenuated the effect of GEN. Additionally, knockdown or overepression of STAT3 in THP-1 macrophages potentiated or attenuated the inhibitory effects of GEN. Importantly, STAT3 overexpression retrieved the effects of IL-8 combined with GEN depletion on M2 polarization of THP-1 macrophages and stemness of SKOV3 cells induced by co-culture. The combination of GEN and STAT3 knockdown cooperatively inhibited the growth of tumors co-inoculated with OCSLCs/THP-1 macrophages in nude mice in vivo through blocking IL-8/STAT3 signaling. Conclusions: In summary, our findings suggested that GEN can inhibit the increased M2 polarization of macrophages and stemness of ovarian cancer cells by co-culture of macrophages with OCSLCs through disrupting IL-8/STAT3 signaling axis. This assisted GEN to be as a potential chemotherapeutic agent in human ovarian cancer.

Intestinal L cells regulate a wide range of metabolic processes, and L-cell dysfunction has been implicated in the pathogenesis of obesity and diabetes. However, it is incompletely understood how luminal signals are integrated to control the development of L cells. Here we show that food availability and gut microbiota-produced short-chain fatty acids control the posttranslational modification on intracellular proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) in intestinal epithelial cells. Via FOXO1 O-GlcNAcylation, O-GlcNAc transferase (OGT) suppresses expression of the lineage-specifying transcription factor Neurogenin 3 and, thus, L cell differentiation from enteroendocrine progenitors. Intestinal epithelial ablation of OGT in mice not only causes L cell hyperplasia and increased secretion of glucagon-like peptide 1 (GLP-1) but also disrupts gut microbial compositions, which notably contributes to decreased weight gain and improved glycemic control. Our results identify intestinal epithelial O-GlcNAc signaling as a brake on L cell development and function in response to nutritional and microbial cues.

Manganese superoxide dismutase (MnSOD) promotes invasive and migratory activities by upregulating Forkhead box protein M1 (FoxM1) expression. The present study investigated whether modulation of MnSOD and FoxM1 expression was responsible for the antitumor effects of genistein on cancer stem‑like cells (CSLCs) derived from non‑small cell lung cancer cells (NSCLCs). Spheroids prepared from H460 or A549 cells were defined as lung cancer stem‑like cells (LCSLCs) and were treated with genistein. The Cell Counting Kit‑8 assay was performed to assess human lung fibroblast IMR‑90 cell proliferation, as well as NSCLC H460 and A549 cell proliferation following treatment with genistein. MnSOD, FoxM1, cluster of differentiation (CD)133, CD44, BMI1 proto‑oncogene, polycomb ring finger (Bmi1) and Nanog homeobox (Nanog) protein expression levels were examined via western blotting. The sphere formation assay was conducted to evaluate LCSLC self‑renewal potential, and LSCLC migratory and invasive activities were analyzed using the wound healing and Transwell invasion assays, respectively. Knockdown and overexpression of MnSOD and FOXM1 via short hairpin‑RNA or cDNA transfection were performed. The results indicated that genistein (80 and 100 µM) suppressed H460 and A549 cell viability compared with IMR‑90 cells. Sub‑cytotoxic concentrations of genistein (20 and 40 µM) inhibited sphere formation activity and decreased the protein expression levels of CD133, CD44, Bmi1 and Nanog in LCSLCs compared with the control group. Genistein also suppressed the migratory and invasive activities of LCSLCs compared with the control group. MnSOD and FoxM1 overexpression antagonized the effects of genistein (40 µM), whereas MnSOD and FoxM1 knockdown enhanced the inhibitory effects of genistein (20 µM) on CSLC characteristics of LCSLCs. Overall, the results suggested that genistein suppressed lung cancer cell CSLC characteristics by modulating MnSOD and FoxM1 expression levels.

Casticin, a polymethoxyflavone, is reported to have anticancer activities. The aim of the present study was to examine the molecular mechanisms by which casticin induces apoptosis in ovarian cancer cells. The human ovarian cancer cell lines SKOV3 and A2780 were cultured in vitro. Various molecular techniques, including histone/DNA enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), western blot analysis and gene transfection, were used to assess the expression of FOXO3a and forkhead box protein M1 (FoxM1) in casticin-treated ovarian cancer cell lines. Casticin-induced apoptotic cell death was accompanied by the activation of transcription factor FOXO3a, with a concomitant decrease in the expression levels of FoxM1 and its downstream target factors, namely survivin and polo‑like kinase 1 (PLK1), and an increase in p27KIP1. A small inhibitory RNA (siRNA) knockout of FoxM1 potentiated casticin-induced apoptosis in ovarian cancer cells. Silencing FOXO3a expression using siRNA increased FoxM1 expression levels and clearly attenuated the induction of apoptosis by casticin treatment. These results show that casticin-induced apoptosis in ovarian cancer may be caused by the activation of FOXO3a, leading to FoxM1 inhibition.

Background: Manganese superoxide dismutase (MnSOD) upregulating FoxM1 have previously been demonstrated promoting lung cancer stemness. Isovitexin exhibits antitumor activities in various cancers. This study aimed to assess whether isovitexin inhibits hepatic carcinoma stem-like cells (HCSLCs) features via regulating MnSOD and FoxM1 expression. Methods: Second-generation spheres from the hepatic carcinoma cell lines, respectively, were used as HCSLCs. Protein amounts of MnSOD, FoxM1 and stemness-associated markers (CD133, CD44, ALDH1, Bmi1, Nanog and Oct4) were determined by immunoblotting. In vitro carcinogenicity was evaluated by sphere- and colony-formation assays. The effects of isovitexin on HCSLC carcinogenicity and stemness were examined in vitro and in xenograft models. An adenoviral delivery system was employed to manipulate MnSOD and/or FoxM1. Luciferase reporter assay was performed to verify isovitexin downregulated FoxM1 by inhibiting MnSOD-mediated effects of E2F1 and/or Sp1 on activation of FoxM1 promoter. Results: FoxM1 upregulation by MnSOD contributed to carcinogenicity and stemness, with increased sphere- and colony-formation capabilities, upregulated stemness-associated markers and CD133 ⁺ subpopulation as well as elevated oncogenicity in vivo in HCSLCs compared with hepatic carcinoma cells. Isovitexin substantially decreased sphere and colony formation rates, and stemness-associated markers in cultured HCSLCs by suppressing MnSOD and FoxM1 expression. Importantly, isovitexin significantly inhibited tumor growth of in nude mice bearing HCSLCs and reduced CD133 protein expression of xenograft in nude mice. MnSOD or FoxM1 knockdown enhanced the effects of isovitexin suppression on carcinogenicity and stemness in HCSLC. MnSOD or FoxM1 overexpression attenuated the effects of isovitexin. Additionally, isovitexin and MnSOD knockdown could inhibit FoxM1 reporter activity via a decreased binding of E2F1 and/or Sp1 onto FoxM1 promoter. FoxM1 overexpression reversed the effects of isovitexin combined with MnSOD knockdown, without affecting MnSOD expression. Moreover, MnSOD knockdown plus thiostrepton, a FoxM1 specific inhibitor, cooperated with isovitexin to repress xenograft tumor growth and downregulate MnSOD and FoxM1 in nude mice bearing HCSLCs from MHCC97H cells. Conclusions: Isovitexin inhibits carcinogenicity and stemness in HCSLCs by downregulating FoxM1via inhibition of MnSOD.

Casticin is one of the main components of the fruits of Vitex rotundifolia L. Studies have shown that casticin inhibits the growth of various cancer cells, including colon cancer. In the present study, the anti‑carcinogenic effects of casticin on human colon cancer and the underlying mechanisms were investigated. The results revealed that casticin significantly induced apoptosis of HT‑29, HCT‑116, SW480 and Caco‑2 cells, induced the accumulation of reactive oxygen species (ROS) and increased the protein levels of apoptosis signal‑regulating kinase 1 (ASK1), c‑Jun N‑terminal kinase (JNK) and B‑cell lymphoma 2-interacting mediator of cell death (Bim) in HT‑29 cells. Pretreatment with N‑acetylcysteine, an antioxidant chemical compound, inhibited the activation of ASK1, JNK and Bim, as well as the apoptosis induced by casticin. Small interfering RNA targeting ASK1 significantly attenuated the induction of JNK and Bim activation and apoptotic cell death by casticin treatment. SP600125, a specific JNK inhibitor, attenuated Bim activation and apoptosis, but did not alter ASK1 phosphorylation levels. In addition, casticin treatment resulted in apoptosis by the same mechanism in HCT‑116, SW480 and Caco‑2 cells. These results suggest that casticin significantly induced apoptosis by the activation of the ASK1‑JNK‑Bim signaling cascade and the accumulation of ROS in colon cancer cells.