Wild-type IDH1 Knockout Leads to G0/G1 Arrest, Impairs Cancer Cell Proliferation, Altering Glycolysis, and the TCA Cycle in Colon Cancer.

The isocitrate dehydrogenase (IDH), which participates in the TCA cycle, is an important key enzyme in regulating cell metabolism. The effect of the metabolic IDH enzyme on cancer pathogenesis has recently been shown in different types of cancer. However, the role of wild-type (wt) IDH1 in the development of colon cancer is still unknown. Our study investigated the role of the IDH1 enzyme in key hallmarks of colon cancer using various methods such as wound healing, cell cycle, colony formation ability, invasion, and apoptosis analysis. Furthermore, cell metabolism was investigated by pyruvate analysis, dinitrosalicylic acid, and HPLC methods. In addition, CRISPR/Cas9 tool was utilized to knockout the IDH1 gene in colon adenocarcinoma cells (SW620). Further studies were performed in two isogenic IDH1 KO clones. Our findings in both clones suggest that IDH1 KO results in G0/G1 arrest, and reduces proliferation by approximately twofold compared to IDH1 WT cells. In addition, the invasion, migration, and colony formation abilities of IDH1 KO clones were significantly decreased accompanied by significant morphological changes. In the context of metabolism, intracellular glucose, pyruvate, αKG, and malate levels were decreased, while the intracellular citrate level was increased in IDH1 KO clones as compared to IDH1 WT cells. Our results reveal that wt IDH1 knockout leads to a decrease in the aggressive features of colon cancer cells. In conclusion, we reported that wt IDH1 has an effective role in colon cancer progression and could be a potential therapeutic target.

The elevated D-2-hydroxyglutarate level found as a characteristic metabolic change of colon cancer in both in vitro and in vivo models.

The D-2-hydroxyglutarate (D-2-HG), whose normal cellular concentration is low, can be accumulated 10-100 times natural levels in some cancer types and participates in the carcinogenesis process. D-2-HG is produced by different pathways specific to cancer type. In this study, the level of significant metabolites produced in some metabolic pathways related to D-2-HG in the energy metabolism was determined in colon adenocarcinoma cell lines at different stages. Then, the differences in TCA and Cori cycle, glutaminolysis, and Glycolysis were investigated in the brain, colon, liver, and tumor tissues extracted from xenograft models. The levels of glucose, pyruvate, lactate, all TCA cycle intermediates, and D-2-HG were determined by the HPLC analysis, DNS method, and pyruvate assay. The intracellular D-2-HG level was found at 22.6 µmol/mg in primary (Caco-2) and 152.6 µmol/mg in metastatic (SW620) colon adenocarcinoma cells, whereas it could not be detected in colon epithelial cell line (CCD-18Co). In the xenograft models, D-2-HG could not be detected in CCD-18Co colon and brain tissues, whereas it was produced in Caco-2 and SW620 tissues. Most importantly, the level of D-2-HG was 7.4 and 19.9-fold increased in Caco-2 and SW620 tumor tissues compared to healthy tissue, respectively. In addition, the D-2-HG production pathways were investigated. The results revealed that the carbon source of D-2-HG is glucose, and the imbalance of wt-IDH1/2 enzymes plays a role in its production. Overall, the in vitro and in vivo results show that the enhanced production of endogenous D-2-HG is a characteristic change in the metabolism of colon cancer.

Synthesis and characterization of thiosemicarbazone-functionalized organoruthenium (II)-arene complexes: Investigation of antitumor characteristics in colorectal cancer cell lines.

In the current study, organoruthenium(II)-arene complexes (I-IV) have been prepared by the reaction of [{(η6-p-cym)RuCl}2(µ-Cl)2] with new thiosemicarbazones (TSC1-4).The isolate was analyzed using elemental analysis, FT-IR, 1H and 13C NMR spectroscopy and single-crystal XRD. Subsequently, the complexes and TSC ligands were assessed for anticancer properties in vitro against three different colorectal cancer stage's cell lines (Caco-2, DLD-1, and SW620) and a noncancerous cell line (CCD18Co). The complexes (I-IV) showed higher cytotoxicity with low IC50 values as 0.1-0.33 µM in colorectal cell lines except for SW620 (47.4-84.20 µM) than in a noncancerous cell. Complex I was 2.8 and 24.5-fold more active against Caco-2 and DLD-1 than CCD18Co, respectively. The complexes (I-IV) accumulated at a high concentration in the cell nuclei and caused cell cycle arrest by affecting the G0/G1 and/or G2/M phase and showed high binding affinity with CT-DNA (Kb = 104 M-1). The expression of Caspase-3 and Caspase-9 apoptosis-related protein levels was slightly upregulated and Atg5 autophagy-related protein level was clearly downregulated according to control and 5-FU-treated cells after complex I and II treatment. Furthermore, it was observed that cytotoxicity of the complexes is decreased while cancer progresses. Altogether, this study indicates that all organoruthenium (II)-arene complexes (particularly complex I) can be a promising alternative to platinum counterparts in cancer treatment.