Nobiletin and xanthohumol sensitize colorectal cancer stem cells to standard chemotherapy

Colorectal cancer (CRC) mortality is mainly caused by patient refractoriness to common anti-cancer therapies and consequent metastasis formation. Besides, the notorious toxic side effects of chemotherapy are a concurrent obstacle to be tackled. Thus, new treatment approaches are needed to effectively improve patient outcomes. Compelling evidence demonstrated that cancer stem cells (CSCs) are responsible for treatment failure and relapse. New natural treatment approaches showed capabilities to selectively target the CSC subpopulation by rendering them targetable by standard cytotoxic compounds. Herein we show the anti-cancer properties of the polymethoxyflavones and prenylflavonoids extracted from Citrus sinensis and Humulus lupulus, respectively. The natural biofunctional fractions, singularly and in combination, reduced the cell viability of CRC stem cells (CR-CSCs) and synergized with 5-fluorouracil and oxaliplatin (FOX) chemotherapy. These phenomena were accompanied by a reduced S and G2/M phase of the cell cycle and upregulation of cell death-related genes. Notably, both phytoextracts in combination with FOX thwarted stemness features in CR-CSCs as demonstrated by the impaired clonogenic potential and decreased Wnt pathway activation. Extracts lowered the expression of CD44v6 and affected the expansion of metastatic CR-CSCs in patients refractory to chemotherapy. Together, this study highlights the importance of polymethoxyflavones and prenylflavonoids as natural remedies to aid oncological therapies

PI3K-driven HER2 expression is a potential therapeutic target in colorectal cancer stem cells

Objective: Cancer stem cells are responsible for tumour spreading and relapse. Human epidermal growth factor receptor 2 (HER2) expression is a negative prognostic factor in colorectal cancer (CRC) and a potential target in tumours carrying the gene amplification. Our aim was to define the expression of HER2 in colorectal cancer stem cells (CR-CSCs) and its possible role as therapeutic target in CRC resistant to anti-epidermal growth factor receptor (EGFR) therapy. Design: A collection of primary sphere cell cultures obtained from 60 CRC specimens was used to generate CR-CSC mouse avatars to preclinically validate therapeutic options. We also made use of the ChIP-seq analysis for transcriptional evaluation of HER2 activation and global RNA-seq to identify the mechanisms underlying therapy resistance. Results: Here we show that in CD44v6-positive CR-CSCs, high HER2 expression levels are associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which promotes the acetylation at the regulatory elements of the Erbb2 gene. HER2 targeting in combination with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) inhibitors induces CR-CSC death and regression of tumour xenografts, including those carrying Kras and Pik3ca mutation. Requirement for the triple targeting is due to the presence of cancer-associated fibroblasts, which release cytokines able to confer CR-CSC resistance to PI3K/AKT inhibitors. In contrast, targeting of PI3K/AKT as monotherapy is sufficient to kill liver-disseminating CR-CSCs in a model of adjuvant therapy. Conclusions: While PI3K targeting kills liver-colonising CR-CSCs, the concomitant inhibition of PI3K, HER2 and MEK is required to induce regression of tumours resistant to anti-EGFR therapies. These data may provide a rationale for designing clinical trials in the adjuvant and metastatic setting

In vitro antitumor effects of the cold-water extracts of Pleurotus eryngii var ferulae and Pleurotus nebrodensis on human colon cancer cells

For centuries, mushrooms have been used as folk medicines especially in Asian countries where their medicinal properties are well known. On the basis of numerous experimental evidences collected in the last decades, the immunomodulatory and anti-neoplastic properties of substances extracted from various species belonging to genera of edible mushrooms [Agaricus L., Auricularia Bull. ex Juss., Ganoderma P. Karst., Grifola Gray, Lentinus Fr., Schizophyllum Fr., Tremella Dill ex L., etc.] are extensively recognized also at scientific levels. Several works have demonstrate that anti-cancer property of these molecules is due to their ability to enhance immune system activity and/or to act directly on cancer cells. Polysaccharides are responsible of a remarkable antitumor activity in vivo and many authors demonstrated their action through screening against sarcoma 180 in mice with an intraperitoneal or oral methods of administration (Ikekawa et al., 1969; Mori et al., 1986; Mizuno et al., 1995, Wasser & Weis, 1997). Among the well-studied medicinal mushrooms there are those belonging to the genus Pleurotus (Fr.) P. Kumm., one of the widely cultivated edible mushroom. Most of them are cultivated and provide a relatively cheap food of high dietetic value through rather simple solid-state fermentation processes. In addition, Pleurotus biomass demonstrates significant medicinal effects and its bioactive compounds possess antibiotic, antitumor, hypocholesterolemic and immunomodulation properties (Gunde-Cimerman, 1999; Babitskatya et al., 2000; Gunde-Cimerman & Plemenitas, 2001; Wasser, 2002; Zhang et al., 2004; Chang, 2006; Tsai et al., 2009). Most of researches concerning the anti-tumor properties of Pleurotus spp. have been carried out by testing the methanol/ethanol/ hot water extracts of Pleurotus ostreatus (Jacq.) P. Kumm. in several in vivo and in vitro cancer models. These studies provided interesting data supporting the possibility to isolate new therapeutic agents from this mushroom, but much less is known about the biological activities of extracts from other species of genus Pleurotus. Species like P. eryngii (DC.) Quél. var. ferulae (Lanzi) Sacc. and P. nebrodensis (Inzenga) Quél. are typical Mediterranean mushrooms and are particularly popular as choice edible mushrooms (Gargano et al., 2011 Zervakis, 2001). The aim of the present research was to evaluate whether the cold-water extract of P. eryngii var. ferulae (CWE-Pef) and P. nebrodensis (CWE-Pn) can affect the tumor phenotype of human colon cancer cells. Our result demonstrate that CWE-Pef and CWE-Pn inhibited the growth of HCT116 colon cancer cells by inducing apoptosis, promoted cancer cells aggregation and inhibited the adhesion of tumor cells to endothelial cells. Furthermore, both extracts inhibited tyrosine phosphorylation signaling as well as ERK phosphorylation. These effects were specific for tumor cells since no effects were observed on non-tumor cells. Finally, our data indicated that the active compounds may be protein/peptide compounds since high temperature treatment of CWE-Pef and CWE-Pn completely eliminated their anti-proliferative activity

CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin

Limited therapeutic options are available for advanced colorectal cancer (CRC). Herein, we report that exposure to a neo-synthetic bis(indolyl)thiazole alkaloid analog, nortopsentin 234 (NORA234), leads to an initial reduction of proliferative and clonogenic potential of CRC sphere cells (CR-CSphCs), followed by an adaptive response selecting the CR-CSphC-resistant compartment. Cells spared by the treatment with NORA234 express high levels of CD44v6, associated with a constitutive activation of Wnt pathway. In CR-CSphC-based organoids, NORA234 causes a genotoxic stress paralleled by G2-M cell cycle arrest and activation of CHK1, driving the DNA damage repair of CR-CSphCs, regardless of the mutational background, microsatellite stability, and consensus molecular subtype. Synergistic combination of NORA234 and CHK1 (rabusertib) targeting is synthetic lethal inducing death of both CD44v6-negative and CD44v6-positive CRC stem cell fractions, aside from Wnt pathway activity. These data could provide a rational basis to develop an effective strategy for the treatment of patients with CRC