Overexpression of Fatty Acid Synthase Upregulates Glutamine-Fructose-6-Phosphate Transaminase 1 and O-Linked N-Acetylglucosamine Transferase to Increase O-GlcNAc Protein Glycosylation and Promote Colorectal Cancer Growth.

Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.

Perfluorooctanesulfonic acid exposure leads to downregulation of 3-hydroxy-3-methylglutaryl-CoA synthase 2 expression and upregulation of markers associated with intestinal carcinogenesis in mouse intestinal tissues.

Perfluorooctanesulfonic acid (PFOS) is a widely recognized environment pollutant known for its high bioaccumulation potential and a long elimination half-life. Several studies have shown that PFOS can alter multiple biological pathways and negatively affect human health. Considering the direct exposure to the gastrointestinal (GI) tract to environmental pollutants, PFOS can potentially disrupt intestinal homeostasis. However, there is limited knowledge about the effect of PFOS exposure on normal intestinal tissues, and its contribution to GI-associated diseases remains to be determined. In this study, we examined the effect of PFOS exposure on the gene expression profile of intestinal tissues of C57BL/6 mice using RNAseq analysis. We found that PFOS exposure in drinking water significantly downregulates mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme, in intestinal tissues of mice. We found that diets containing the soluble fibers inulin and pectin, which are known to be protective against PFOS exposure, were ineffective in reversing the downregulation of HMGCS2 expression in vivo. Analysis of intestinal tissues also demonstrated that PFOS exposure leads to upregulation of proteins implicated in colorectal carcinogenesis, including ß-catenin, c-MYC, mTOR and FASN. Consistent with the in vivo results, PFOS exposure leads to downregulation of HMGCS2 in mouse and human normal intestinal organoids in vitro. Furthermore, we show that shRNA-mediated knockdown of HMGCS2 in a human normal intestinal cell line resulted in increased cell proliferation and upregulation of key proliferation-associated proteins such as cyclin D, survivin, ERK1/2 and AKT, along with an increase in lipid accumulation. In summary, our results suggest that PFOS exposure may contribute to pathological changes in normal intestinal cells via downregulation of HMGCS2 expression and upregulation of pro-carcinogenic signaling pathways that may increase the risk of colorectal cancer development.

The role of perfluorooctane sulfonic acid (PFOS) exposure in inflammation of intestinal tissues and intestinal carcinogenesis.

PFAS (per- and polyfluoroalkyl substances) are organofluorine substances that are used commercially in products like non-stick cookware, food packaging, personal care products, fire-fighting foam, etc. These chemicals have several different subtypes made of varying numbers of carbon and fluorine atoms. PFAS substances that have longer carbon chains, such as PFOS (perfluorooctane sulfonic acid), can potentially pose a significant public health risk due to their ability to bioaccumulate and persist for long periods of time in the body and the environment. The National Academies Report suggests there is some evidence of PFOS exposure and gastrointestinal (GI) inflammation contributing to ulcerative colitis. Inflammatory bowel diseases such as ulcerative colitis are precursors to colorectal cancer. However, evidence about the association between PFOS and colorectal cancer is limited and has shown contradictory findings. This review provides an overview of population and preclinical studies on PFOS exposure and GI inflammation, metabolism, immune responses, and carcinogenesis. It also highlights some mitigation approaches to reduce the harmful effects of PFOS on GI tract and discusses the dietary strategies, such as an increase in soluble fiber intake, to reduce PFOS-induced alterations in cellular lipid metabolism. More importantly, this review demonstrates the urgent need to better understand the relationship between PFOS and GI pathology and carcinogenesis, which will enable development of better approaches for interventions in populations exposed to high levels of PFAS, and in particular to PFOS.

Açaí (Euterpe oleracea Mart.) Seed Oil Exerts a Cytotoxic Role over Colorectal Cancer Cells: Insights of Annexin A2 Regulation and Molecular Modeling.

Açaí, Euterpe oleracea Mart., is a native plant from the Amazonian and is rich in several phytochemicals with anti-tumor activities. The aim was to analyze the effects of açaí seed oil on colorectal adenocarcinoma (ADC) cells. In vitro analyses were performed on CACO-2, HCT-116, and HT-29 cell lines. The strains were treated with açaí seed oil for 24, 48, and 72 h, and cell viability, death, and morphology were analyzed. Molecular docking was performed to evaluate the interaction between the major compounds in açaí seed oil and Annexin A2. The viability assay showed the cytotoxic effect of the oil in colorectal adenocarcinoma cells. Acai seed oil induced increased apoptosis in CACO-2 and HCT-116 cells and interfered with the cell cycle. Western blotting showed an increased expression of LC3-B, suggestive of autophagy, and Annexin A2, an apoptosis regulatory protein. Molecular docking confirmed the interaction of major fatty acids with Annexin A2, suggesting a role of açaí seed oil in modulating Annexin A2 expression in these cancer cell lines. Our results suggest the anti-tumor potential of açaí seed oil in colorectal adenocarcinoma cells and contribute to the development of an active drug from a known natural product.

Mechanisms of radioresistance and the underlying signaling pathways in colorectal cancer cells.

Radiotherapy is one of the most common modalities for the treatment of a wide range of tumors, including colorectal cancer (CRC); however, radioresistance of cancer cells remains a major limitation for this treatment. Following radiotherapy, the activities of various cellular mechanisms and cell signaling pathways are altered, resulting in the development of radioresistance, which leads to therapeutic failure and poor prognosis in patients with cancer. Furthermore, even though several inhibitors have been developed to target tumor resistance, these molecules can induce side effects in nontumor cells due to low specificity and efficiency. However, the role of these mechanisms in CRC has not been extensively studied. This review discusses recent studies regarding the relationship between radioresistance and the alterations in a series of cellular mechanisms and cell signaling pathways that lead to therapeutic failure and tumor recurrence. Our review also presents recent advances in the in vitro/in vivo study models aimed at investigating the radioresistance mechanism in CRC. Furthermore, it provides a relevant biochemical basis in theory, which can be useful to improve radiotherapy sensitivity and prolong patient survival.

Long-term resistance to 5-fluorouracil promotes epithelial-mesenchymal transition, apoptosis evasion, autophagy, and reduced proliferation rate in colon cancer cells.

The drug, 5-fluorouracil (5FU) is a standard first-line treatment for colorectal cancer (CRC) patients. However, drug resistance acquisition remains an important challenge for effective clinical outcomes. Here, we established a long-term drug-resistant CRC model and explored the cellular events underlying 5FU resistance. We showed that 5FU-treated cells (HCT-116 5FUR) using a prolonged treatment protocol were significantly more resistant than parental cells. Likewise, cell viability and IC50 values were also observed to increase in HCT-116 5FUR cells when treated with increasing doses of oxaliplatin, indicating a cross-resistance mechanism to other cytotoxic agents. Moreover, HCT-116 5FUR cells exhibited metabolic and molecular changes, as evidenced by increased thymidylate synthase levels and upregulated mRNA levels of ABCB1. HCT-116 5FUR cells were able to overcome S phase arrest and evade apoptosis, as well as activate autophagy, as indicated by increased LC3B levels. Cells treated with low and high doses displayed epithelial-mesenchymal transition (EMT) features, as observed by decreased E-cadherin and claudin-3 levels, increased vimentin protein levels, and increased SLUG, ZEB2 and TWIST1 mRNA levels. Furthermore, HCT-116 5FUR cells displayed enhanced migration and invasion capabilities. Interestingly, we found that the 5FU drug-resistance gene signature is positively associated with the mesenchymal signature in CRC samples, and that ABCB1 and ZEB2 co-expressed at high levels could predict poor outcomes in CRC patients. Overall, the 5FU long-term drug-resistance model established here induced various cellular events, and highlighted the importance of further efforts to identify promising targets involved in more than one cellular event to successfully overcome drug-resistance.

Crosstalk between PI3K/Akt and Wnt/ß-catenin pathways promote colorectal cancer progression regardless of mutational status.

The PI3K/Akt and Wnt/ß-catenin pathways play an important role in the acquisition of the malignant phenotype in cancer. However, there are few data regarding the role of the interplay between both pathways in colorectal cancer (CRC) progression. The mutational status and the clinicopathological characteristics of PI3K/Akt and Wnt/ß-catenin pathways were accessed by bioinformatic analysis whereas that the impact of the interplay between the activity of both pathways to explain tumorigenic potential was performed in vitro using IGF-1 and Wnt3a treatments in CRC cell models. The mutational status of these pathways did not influence the survival of CRC patients, but an association between clinicopathological characteristics in patients with mutations in one, but not in both pathways was observed. A potentiating effect on the activation of both pathways and enhanced cellular migration and proliferation was observed when both pathways were activated simultaneously with IGF-1 and Wnt3a. In addition, these effects were hindered after pretreatment with LY294002, a specific PI3K inhibitor, suggesting some dependence between these two signaling cascades. Our findings show that, regardless of mutational status, there is an interplay between the activity of PI3K/Akt and Wnt/ß-catenin pathways that contributes to events related to CRC progression and that the reversal of such events using a PI3K inhibitor highlights the value of targeting these pathways for potential directed therapies in CRC patients.

Synergistic effect of pyrazoles derivatives and doxorubicin in claudin-low breast cancer subtype.

BACKGROUND: Breast cancer is a global public health problem. For some subtypes, such as Claudin-low, the prognosis is poorer and the treatment is still a challenge. Pyrazoles are an important class of heterocyclic compounds and are promising anticancer agents based on their chemical properties. The present study was aimed not only at testing pyrazoles previously prepared by our research group in two breast cancer cell lines characterized by intermediated response to conventional chemotherapy but also at analyzing the possible synergistic effect of these pyrazoles associated with doxorubicin. METHODS: Four 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H pyrazoles were tested for the first time in MCF-7 and MDA-MB-231 culture cells. The pyrazoles with best results in cytotoxicity were used in combination with doxorubicin and compared with this drug alone as standard. The synergic effect was analyzed using Combination Index method. In addition, cell death and apoptosis assays were carried out. RESULTS: Two pyrazoles with cytotoxic effect in MCF-7 and especially in MDA-MB-231 were identified. This activity was markedly higher in pyrazoles containing bromine and chlorine substituents. The combination of these pyrazoles with doxorubicin had a significant synergic effect in both cells tested and mainly in MDA-MB-231. These data were confirmed with apoptosis and cell death analysis. CONCLUSIONS: The synergic effect observed with combination of these pyrazoles and doxorubicin deserves special attention in Claudin-low breast cancer subtype. This should be explored in order to improve treatment results and minimize side effects.

Antitumor potential of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazoles in human bladder cancer cells.

Bladder cancer is a genitourinary malignant disease common worldwide. Current chemotherapy is often limited mainly due to toxicity and drug resistance. Thus, there is a continued need to discover new therapies. Recently evidences shows that pyrazoline derivatives are promising antitumor agents in many types of cancers, but there are no studies with bladder cancer. In order to find potent and novel chemotherapy drugs for bladder cancer, a series of pyrazoline derivatives 2a-2d were tested for their antitumor activity in two human bladder cancer cell lines 5647 and T24. The MTT assay showed that the compounds 1-thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazole (2a) and 1-thiocarbamoyl-5-(4-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (2c) decrease the cell viability of 5637 cells. Molecular modeling indicated that these compounds had a good oral bioavailability and low toxicities. Clonogenic assay and flow cytometric analysis were used to assess colony formation, apoptosis induction and cell cycle distribution. Overall, our results suggest that pyrazoline 2a and 2c, with the substituents hydrogen and chlorine respectively, may decrease cell viability and colony formation of bladder cancer 5637 cell line by inhibition of cell cycle progression, and for pyrazoline 2a, by induction of apoptosis. As indicated by the physicochemical properties of these compounds, the steric factor influences the activity. Therefore, these pyrazoline derivatives can be considered promising anticancer agents for the treatment of bladder cancer.