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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 proliferation, apoptosis and metabolism of human pancreatic cancer gemcitabine-resistant cell line (ASPC-1-GEM, AG cells). Methods Cell viability was detected by sulforhodamine B(SRB); apoptosis was detected by annexin V/PI staining and flow cytometry; cell division rate was examined by CFSE microscopy and flow cytometry; The oxygen consumption rate (OCR) was detected by Seahorse bio-energy assay; intracellular NAD+ and NADH contents were detected with MTT and NAD+/NADH ratio was calculated. Autophagy-related protein expression was detected by Western blot. Results Comparing with the control group, the living AG cells were significantly reduced and the proportion of apoptotic cells was increased in CTO-treated group. The drug effect was time-dose dependent. The fluorescence intensity of CFSE staining of AG cells was significantly increased in the 20 μmol/L CTO group (P＜0.05); Compared with the control group, intracellular nicotinamide adenine dinucleotidehydrogen(NADH) content was increased after incubation with different concentrations of CTO treatment(P＜0.05 or P＜0.01) and no significant change in the content of nicotinamide adenine dinucleotide (NAD+) NDA+/NADH ratio was decreased(P＜0.01). The OCR in AG cells was significantly reduced and the expression of autophagy-related proteins was significantly inhibited in the CTO-treated group (P＜0.05). Conclusions CTO inhibits the proliferation of gemcitabine-resistant pancreatic cancer cells by inducing apoptosis, impairing mitochondrial respiration, and down-regulating the expression of autophagy-related proteins in AG cells.

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.

Objective To investigate the direct effects of carboxyamidotriazole(CAI)on the survival and apoptosis of colon cancer cells(MC38 and C26)and its indirect effects through enhancing the anticancer effects of spleen lymphocytes in mice. Methods MC38 and C26 cells were cultured in vitro,spleen lymphocytes of mice were isolated,MC38 and C26 cells were co-cultured with spleen lymphocytes of mice,and the cells were treated with different concentrations of CAI. CCK-8 method was used to detect cell survival,flow cytometry was used to detect cell apoptosis and the proportion of CD4+CD3+ T cells and CD8+CD3+ T cells in spleen lymphocytes of mice. Results The number of living cells of MC38 and C26 cells decreased(P＜0.05,P＜0.01 and P＜0.001)and the apoptosis rate was increased(P＜0.01 and P＜0.001)after being treated with different concentrations of CAI. The number of living cells of MC38 and C26 cells was decreased(P＜0.001)upon being co-cultured with spleen lymphocytes of mice for 48 h, and the apoptosis rate was increased(P＜0.001). The number of living cells of MC38 and C26 cells in co- culture groups treated with different concentrations of CAI for 48 h further decreased(P＜0.001)and the apoptosis rate increased(P＜0.05 and P＜0.001)as compared with those co-cultured with spleen lymphocytes of mice without CAI. The proportion of CD4+CD3+ T cells and CD8+CD3+ T cells in spleen lymphocytes of mice was significantly increased(P＜0.05 and P＜0.01)after 48 h treatment with different concentrations of CAI. Conclusions CAI can inhibit the survival and promote apoptosis of MC38 and C26 cells,and enhance the effects of spleen lymphocytes in mice on inhibiting the survival and promoting apoptosis of MC38 and C26 cells through a potential mechanism of increasing the proportion of CD4+CD3+ T cells and CD8+CD3+ T cells in spleen lymphocytes of mice.

Objective To study the effect of carboxyamidotriazole (CAI) on CD8+T cells, and to explore the critical mechanism of CAI in reinforcing cytotoxicity of activated T cells. Methods CD8+T cells of mouse were isolated by immunomagnetic beads and divided into control group, CAI group, ZK756326 (Ca2+ activator) group and CAI+ZK756326 group. Flow cytometry was used to detect the level of intracellular free calcium ions in CD8+T cells; Enzyme-linked immunosorbent assay was used to detect the expression of intracellular calcineurin (CaN); Immunoflu- orescence staining was used to detect NFAT2 nuclear transportation; Chromatin immunoprecipitation-qPCR was used to detect the expression of PD-1 regulated by NFAT2. Mice spleen cytotoxic T lymphocytes (CTLs) were isolated and examined for the expression of PD-1 in CD8+T cells by flow cytometry. Results Intracellular Ca2+ concentration in CD8+T cells significantly decreased in the CAI group (P＜0.001), while intracellular Ca2+ concentration increased in the CAI+ZK756326 group(P＜0.01); CAI significantly decreased the content of calcineurin phosphatase in CD8+T cells (P＜0.001);CAI significantly inhibited NFAT2 nuclear transportation (P＜0.001) and significantly inhibited PD-1 transcription process depend on NFAT2 (P＜0.001); CAI significantly reduced the proportion of PD-1+CD8+T cells in mice spleen CTLs cells (P＜0.001). Conclusions CAI inhibits NFAT2 nuclear translocation by decreasing the level of calcium ions in the CD8+ T cells and inhibiting the expression of calcineurin, thereby reducing the expression of PD-1 in CD8+ T cells, which facilitated immunotherapy intervention.

Objective To explore the effect of berberine (BBR) on the vulnerability of atherosclerotic plaques caused by hyperglycemia and hyperlipidemia, and to discover the potential mechanism. Methods Male ApoE-/-mice were randomly divided into the control group, hyperglycemia group (intraperitoneal injection of streptozotocin(STZ), a dose of 45 mg/(kg·d) for 5 consecutive days), and hyperlipidemia group (high-fat diet, HFD). After two weeks, 10 mice were randomly selected from the hyperglycemia group to form a drug prevention group[gavage, BBR 50 mg/(kg·d)].Four weeks after modeling,the animals in the hyperglycemia model group were randomly divided into the model (STZ) group (gavage, solvent), BBR25, 50, and 100 (STZ) group (gavage, different doses of berberine [25, 50, and 100 mg/(kg·d)], the atorvastatin group [gavage, 2.5 mg/(kg·d)], the englitazone group [gavage, 1.25 mg/(kg·d)], and the hyperlipidemia model group were randomly divided into the model (HFD) group (gavage, solvent), BBR50 (HFD) group [gavage, berberine 50 mg/(kg·d)]. After 6 weeks of continuous treatment, the aorta of the mouse was taken for pathological and immunohistological examination to evaluate the atherosclerotic plaque lesions and their vulnerability index; Thp1 cells were stimulated to differentiate into macrophages with phorbol ester (PMA) and were used to investigate the effect of berberine on autophagy and inflammatory signaling pathways; Western blot was conducted to detect the expression of MIF, as well as NLRP3, AMPK/mTOR signal pathway and autophagy-related protein level in mouse aorta and cells.ELISA was used to detect the level of IL-1β in serum and cell supernatant. Results Compared with the control group, the atherosclerotic lesions in the hyperglycemia and hyperlipidemia groups were worsened, and the plaque vulnerability index was significantly increased. After the treatment with berberine, the plaque lesions were significantly improved, and the plaque vulnerability index decreased significantly(P＜0.05).Berberine down-regulated MIF protein expression in mouse aortic tissue homogenated and Thp1 cells, promoted AMPK activation, inhibited mTOR phosphorylation, regulated autophagy-related protein expression (LC3Ⅱ/LC3Ⅰ ratio increased, SQSTM1/P62 decreased), and down-regulated NLRP3 and IL-1β expression. Conclusions Berberine can effectively inhibit formation of atherosclerotic plaque and its vulnerability in ApoE-/-mice caused by hyperglycemia and hyperlipidemia. The mechanism may be attributed to the down-regulation of MIF protein expression and the promotion of autophagy of macrophages through the AMPK/mTOR signaling pathway to inhibit the inflammatory response.

Cancer cells have a high demand for sugars and express diverse carbohydrate receptors, offering opportunities to improve delivery with multivalent glycopolymer materials. However, effectively delivering glycopolymers to tumors while inhibiting cancer cell activity, altering cellular metabolism, and reversing tumor-associated macrophage (TAM) polarization to overcome immunosuppression remains a challenging area of research due to the lack of reagents capable of simultaneously achieving these objectives. Here, the glycopolymer-like condensed nanoparticle (∼60 nm) was developed by a one-pot carbonization reaction with a single precursor, promoting multivalent interactions for the galactose-related receptors of the M2 macrophage (TAM) and thereby regulating the STAT3/NF-κB pathways. The subsequently induced M2-to-M1 transition was increased with the condensed level of glycopolymer-like nanoparticles. We found that the activation of the glycopolymer-like condensed galactose (CG) nanoparticles influenced monocarboxylate transporter 4 (MCT-4) function, which caused inhibited lactate efflux (similar to inhibitor effects) from cancer cells. Upon internalization via galactose-related endocytosis, CG NPs induced cellular reactive oxygen species (ROS), leading to dual functionalities of cancer cell death and M2-to-M1 macrophage polarization, thereby reducing the tumor's acidic microenvironment and immunosuppression. Blocking the nanoparticle-MCT-4 interaction with antibodies reduced their toxicity in glioblastoma (GBM) and affected macrophage polarization. In orthotopic GBM and pancreatic cancer models, the nanoparticles remodeled the tumor microenvironment from "cold" to "hot", enhancing the efficacy of anti-PD-L1/anti-PD-1 therapy by promoting macrophage polarization and activating cytotoxic T lymphocytes (CTLs) and dendritic cells (DCs). These findings suggest that glycopolymer-like nanoparticles hold promise as a galactose-elicited adjuvant for precise immunotherapy, particularly in targeting hard-to-treat cancers.

Finding a cancer-selective drug that avoids damaging healthy cells and organs is a holy grail in medical research. In our previous studies, gold-coated iron (Fe@Au) nanoparticles showed cancer selective anti-cancer properties in vitro and in vivo but were found to gradually lose that activity with storage or "ageing." To determine the reasons for this diminished anti-cancer activity, we examined Fe@Au nanoparticles at different preparation and storage stages by means of transmission electron microscopy combined with and energy-dispersive X-ray spectroscopy, along with X-ray diffraction analysis and cell viability tests. We found that dried and reconstituted Fe@Au nanoparticles, or Fe@Au nanoparticles within cells, decompose into irregular fragments of γ-F₂O₃ and agglomerated gold clumps. These changes cause the loss of the particles' anti-cancer effects. However, we identified that the anti-cancer properties of Fe@Au nanoparticles can be well preserved under argon or, better still, liquid nitrogen storage for six months and at least one year, respectively.

Previously, iron core-gold shell nanoparticles (Fe@Au) have been shown to possess cancer-preferential cytotoxicity in oral and colorectal cancer (CRC) cells. However, CRC cell lines are less sensitive to Fe@Au treatment when compared with oral cancer cell lines. In this research, Fe@Au are found to decrease the cell viability of CRC cell lines, including Caco-2, HT-29, and SW480, through growth inhibition rather than the induction of cell death. The cytotoxicity induced by Fe@Au in CRC cells uses different subcellular pathways to the mitochondria-mediated autophagy found in Fe@Au-treated oral cancer cells, OECM1. Interestingly, the Caco-2 cell line shows a similar response to OECM1 cells and is thus more sensitive to Fe@Au treatment than the other CRC cell lines studied. We have investigated the underlying cell resistance mechanisms of Fe@Au-treated CRC cells. The resistance of CRC cells to Fe@Au does not result from the total amount of Fe@Au internalized. Instead, the different amounts of Fe and Au internalized appear to determine the different response to treatment with Fe-only nanoparticles in Fe@Au-resistant CRC cells compared with the Fe@Au-sensitive OECM1 cells. The only moderately cytotoxic effect of Fe@Au nanoparticles on CRC cells, when compared to the highly sensitive OECM1 cells, appears to arise from the CRC cells' relative insensitivity to Fe, as is demonstrated by our Fe-only treatments. This is a surprising outcome, given that Fe has thus far been considered to be the "active" component of Fe@Au nanoparticles. Instead, we have found that the Au coatings, previously considered only as a passivating coating to protect the Fe cores from oxidation, significantly enhance the cytotoxicity of Fe@Au in certain CRC cells. Therefore, we conclude that both the Fe and Au in these core-shell nanoparticles are essential for the anticancer properties observed in CRC cells.