A combination of inhibitors of glycolysis, glutaminolysis and de�novo fatty acid synthesis decrease the expression of chemokines in human colon cancer cells

Abstract

Lonidamine, 6-Diazo-5-oxo-L-norleucine (DON) and orlistat are well known inhibitors of glycolysis, glutaminolysis and of de novo fatty acid synthesis, respectively. Although their antitumor effects have been explored in detail, the potential inhibition of the malignant metabolic phenotype and its influence on the expression of chemokines and growth factors involved in colon cancer, have not been previously reported to the best of our knowledge. In the present study, dose-response curves with orlistat, lonidamine or DON were generated from cell viability assays conducted in SW480 colon cancer cells. In addition, the synergistic effect of these compounds was evaluated in SW480 human colon cancer cells. The determination of the doses used for maximum synergistic efficacy led to the exploration of the mRNA levels of the target genes hexokinase-2 (HK2), glutaminase-1 (GLS-1) and fatty acid synthase (FASN) in human SW480 and murine CT26.WT colon cancer cells. The cell viability was evaluated following transfection with small interfering (si)RNA targeting these genes and was assessed with trypan blue. The expression levels of chemokines and growth factors were quantified in the supernatant of SW480 cells with LEGENDplex™. The combination of lonidamine, DON and orlistat resulted in a synergistic cytotoxic effect and induced the transcription of the corresponding gene targets but their corresponding proteins were actually downregulated. The downregulation of the expression levels of HK2, GLS-1 and FASN following transfection of the cells with the corresponding siRNA sequences decreased their viability. The treatment significantly reduced the expression levels of 9 chemokines [interleukin-9, C-X-C motif chemokine ligand (CXCL) 10, eotaxin, chemokine ligand (CCL) 9, CXCL5, CCL20, CXCL1, CXCL11 and CCCL4] and one growth factor (stem cell factor). These changes were associated with decreased phosphorylated-nuclear factor κB-p65. The data demonstrate that lonidamine, DON and orlistat in combination reduce the expression levels of chemokines and growth factors in colon cancer cells. Additional research is required to investigate the exact way by which both tumor and stromal cells regulate the expression levels of chemokines and growth factors.

Pharmacological inhibition of tumor anabolism and host catabolism as a cancer therapy

“…Confirming our previous findings 16 , we observed strong growth inhibitory effects in vitro which were associated with G 0 /G 1 arrest of cell cycle and increased apoptosis. As far as we know, there are no publications testing cell cycle or cell death with a combination of inhibitors of these pathways.…”

“…5 × 10 4 SW480 cells/well were seeded in 6-well plates (Costar), with 2 mL of complete medium. After 24 h of pre-incubation, cells were treated during 72 h with orlistat, lonidamine, or DON at synergistic concentrations, as stated before 16 , and with the maximum circulating concentrations of GH, insulin, or indomethacin, reported in healthy subjects [45][46][47] . The concentrations are found on Supplementary Table S1.…”

Redox Homeostasis and Metabolism in Cancer: A Complex Mechanism and Potential Targeted Therapeutics

“…Orlistat is a tetrahydrolipstatin ( Figure 6) which is an FDA approved gastric and pancreatic lipase inhibitor that works in the intestine by hindering fat absorption by up to 30%, It is used mainly as a weight loss medication in obese patients [158]. Several studies investigated the effect of Orlistat as an antitumorigenic drug in various cancers such as T-cell Leukemia [158], melanoma [159], colorectal cancer [160,161], prostate cancer [162], hepatoma [163], breast cancer, and pancreatic cancer [164]. Orlistat suppresses tumor growth by inhibiting Fatty Acid Synthase (FASN), an anabolic multifunctional enzyme responsible for endogenous fatty acid synthesis from precursors acetyl-CoA and malonyl-CoA to make the 16 carbon polyunsaturated fatty acid palmitate [165,166].…”

“…Furthermore, the study showed that the combination of both Orlistat and Paclitaxel had a strong synergistic effect on growth inhibition and cell apoptosis in Hep3B cells [163]. Additionally, when Orlistat was combined with Lonidamine and 6-Diazo-5-oxo-L-norleucine (DON) for treatment of SW480 colon cancer cells, they exhibited a synergistic cytotoxic effect with downregulation of their protein targets Hexokinase-2 (HK2), Glutaminase-1 (GLS-1), and Fatty Acid Synthase (FASN), respectively [160]. Finally, a study by Saleh et al indicated that Orlistat exerted a cytotoxic effect and induced apoptosis in human breast cancer (MCF-7) and human pancreatic cancer (PANC-1) cell lines [164].…”

Identification of Monotonically Differentially Expressed Genes across Pathologic Stages for Cancers

“…For the top colon cancer MEGs, the consistent tumor suppressor genes included MAP2K4, MAPK10, RUNX3, WNK2 (the four genes were identified by the TSGene 2.0 database), BECN1 [ 27 ], FASN [ 28 ], NAT1 [ 29 ], and NR3C2 [ 30 ]. For monotonically increasing genes, the consistent ones include CCKBR, BMP4 [ 31 , 32 ], and SLC29A1 [ 33 ] which were determined to be oncogenes by previous studies.…”