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Objective To study the effect of carboxyamidotriazole-orotate(CTO) on the proliferation and fatty acid anabolism regulation of human pancreatic cancer cells. Methods Human pancreatic cancer cell lines AsPC-1, AsPC-1/GEM(AR), PANC-1 and MiaPaCa-2 were used as the study subjects; cell survival rate was detected by sulfonylrhodamine B(SRB); the mRNA level of key genes for fatty acid synthesis was detected by qPCR; the protein level of the AMPK/ACC pathway was detected by Western blot; intracellular lipid metabolites were examined by liquid chromatography-mass spectrometry(LC-MS). Results Comparing to control group, CTO significantly decreased the cell viability of AsPC-1, AR, PANC-1, and MiaPaCa-2(P＜0.05). CTO down-regulated the mRNA level of key fatty acid synthesis genes(P＜0.05). CTO significantly reduced the protein expression of AMPK, ACC and c-Myc(P＜0.05), while increasing the protein expression of p-AMPK and p-ACC(P＜0.05). CTO decreased lipid metabolite content in AR cells(P＜0.05). Conclusions CTO attenuates cellular fatty acid anabolism by inhibition of oncogene c-Myc expression and AMPK/ACC pathway, down-regulates the expression of fatty acid synthesis-related genes, and then inhibits proliferation of the human pancreatic cancer cell lines AsPC-1, AR, PANC-1 and MiaPaCa-2.

Objective To explore regulation mechanism of carboxyamidotriazole-orotate (CTO) on oncogenic phenotype of mouse glioma-associated macrophages. Methods Bone marrow-derived macrophages(BMDMs) or RAW264.7 macrophages (tumor associated macrophages,TAMs) were prepared by super-culture of mouse glioma GL261 cells. The mRNA of TAMs oncogenic mediators was detected by qPCR. The oxygen consumption rate (OCR) of TAMs was measured by Seahorse bioenergy method. The protein level of hypoxia-inducible factor-1 α(HIF-1α), hypoxia-inducible factor-2 α (HIF-2α) and peroxisome proliferator-activated receptor (PPAR) co-activator factor-1 β (PGC-1β) in TAMs was detected by Western blot. Results CTO decreased M1-related mediators such as IL-1β, IL-6 and TNF-α(P＜0.001) and M2-related mediators such as IL-10, ARG-1 and TGF-β1(P＜0.001) in TAMs. CTO significantly down-regulated OCR in TAMs (P＜0.01); The protein level of HIF-1α and of HIF-2α in TAMs was down-regulated by CTO(P＜0.01),which was contrary to the effect of carboxycyanide-4-trifluormethoxyphenylhydrazone (FCCP),which in turn promoted mitochondrial oxygen consumption. CTO significantly reduced PGC-1β protein in TAMs (P＜0.001),an effect consistent with rotenone(RTN), a mitochondrial respiratory chain inhibitor. Conclusions A decreased oxidative phosphorylation down-regulates the expression of PGC-1β and destabilizes HIF-1α and HIF-2α, thus affects the production of several M1 and M2 phenotypes of carcinogenic mediators in TAMs, which may be a potential mechanism through which CTO improves the chemotherapy efficacy of glioblastoma.

Objective To investigate the effects of carboxyamidotriazole-orotate (CTO) on the proliferation and apoptosis of mouse glioma cell line GL261. Methods The proliferation of GL261 cells was detected by live cell counting. Cell cycle of GL261 cells was detected by PI staining and flow cytometry. Apoptosis of GL261 cells was detected by annexinⅤ/PI double staining and flow cytometry; DCFH-DA staining and flow cytometry were used to detect the content of intracellular reactive oxygen species (ROS) of GL261 cells. The oxygen consumption rate (OCR) of GL261 cells was measured in Seahorse bioenergy assay. The ATP content in GL261 cells was detected by ATP detection kit. Results Compared to the control group, the proliferation of GL261 cells in CTO treatment group was significantly inhibited in a time- and dose-dependent way, the proportion of GL261 cells in S phase was significantly increased (P＜0.05) in 20 μmol/L CTO group. Compared with the control group, more apoptosis occurred in GL261 cells of CTO group, and ROS content in GL261 cells of 20 μmol/L CTO group was also significantly increased (P＜0.01). Compared with the control group, the contents of OCR and ATP in GL261 cells was signifi- cantly reduced in the CTO group(P＜0.01). Conclusions CTO inhibits proliferation of GL261 cells and promotes their apoptosis by increased production of ROS. In addition, CTO inhibits the mitochondrial respiration of GL261 cells and the production of ATP in mitochondria, thus inhibits growth of glioma cells.