RESUMO
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.
RESUMO
The aim of the present study was to investigate in vivo the feasibility and efficacy of the combination of lonidamine (LND), 6-diazo-5-oxo-L-norleucine (DON) and orlistat to simultaneously target glycolysis, glutaminolysis and de novo synthesis of fatty acids, respectively. The doses of LND and DON used in humans were translated to mouse doses (77.7 mg/kg and 145.5 mg/kg, respectively) and orlistat was used at 240 mg/kg. Three schedules of LND, DON and orlistat at different doses were administered by intraperitoneal injection to BALB/c mice in a 21-day cycle (schedule 1: LND, 0.5 mg/day; DON, 0.25 mg/day 1, 5 and 9; orlistat, 240 mg/kg/day; schedule 2: LND, 0.1 mg/day; DON, 0.5 mg/day 1, 5 and 9; orlistat, 240 mg/kg/day; schedule 3: LND, 0.5 mg/day; DON, 0.08 mg/day 1, 5 and 9; orlistat, 360 mg/kg/day) to assess tolerability. To determine the antitumor efficacy, a syngeneic tumor model in BALB/c mice was created using colon cancer CT26.WT cells, and a xenogeneic tumor model was created in nude mice using the human colon cancer SW480 cell line. Mice were treated with schedule 1. Animals were weighed, clinically inspected during the experiment and the tumor volume was measured at day 21. The 3 schedules assessed in the tolerability experiments were well tolerated, as mice maintained their weight and no evident clinical signs of toxicity were observed. Combination treatment with schedule 1 significantly decreased tumor growth in each mouse model. No evident signs of toxicity were observed and mice maintained their weight during treatment. The triple metabolic blockade of the malignant phenotype appears feasible and promising for cancer therapy.
RESUMO
Lonidamina (1-[ 2,4-diclorofenil metil]-1H indazol-3-ácido carboxílico), (lnd), es una droga antineoplásica cuyo mecanismo de acción se ejerce sobre el metabolismo intermedio de la glucosa. Los efectos de la lnd sobre el crecimiento celular y el metabolismo celular se investigaron en las células HT- 29, línea celular de carcinoma colónico humano, que requiere altas concentraciones de glucosa para su crecimiento indiferenciado en cultivo. La lnd en dosis de 0.2 mM disminuyó significativamente el crecimiento celular y la formación de colonias en agar; con la interrupción del tratamiento se observó el restablecimiento del crecimiento celular en 24 horas. El tratamiento con lnd produce la redistribución de los glicoconjugados y el receptor de la manosa, sin afectar en forma drástica la síntesis de glucógeno ni la de proteínas. Estas posiblemente sean las causas de la rápida reversibilidad del tratamiento.
Lonidamine (1-[ 2,4-dichlorophenyl methyl]-1H indazole-3-carboxylic acid), lnd, is an antitumoral drug acting on mitochondria and glucose metabolism. Cell growth and metabolic effects of lnd and drug post-treatment effect were investigated in undifferentiated HT-29 human colonic carcinoma cell line which requires high glucose medium concentration for growth. 0.2 mM lnd significantly decreased cell spreading and growth in monolayer or agar cell culture. After drug treatment cell growth was reestablished to control value within 24 h. Ind modified glycoconjugates and mannose-receptor distribution (analyzed by confocal microscopy), while glucose-glycogen and protein synthesis were not affected, these being the possible reasons for the fast reversible effect.