PREX2 contributes to radiation resistance by inhibiting radiotherapy-induced tumor immunogenicity via cGAS/STING/IFNs pathway in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) lacks established biomarkers or molecular targets for predicting or enhancing radiation response. Phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 2 (PREX2) exhibits intricate implications in tumorigenesis and progression. Nevertheless, the precise role and underlying mechanisms of PREX2 in CRC radioresistance remain unclear. METHODS: RNA-seq was employed to identify differentially expressed genes between radioresistant CRC cell lines and their parental counterparts. PREX2 expression was scrutinized using Western blotting, real-time PCR, and immunohistochemistry. The radioresistant role of PREX2 was assessed through in vitro colony formation assay, apoptosis assay, comet assay, and in vivo xenograft tumor models. The mechanism of PREX2 was elucidated using RNA-seq and Western blotting. Finally, a PREX2 small-molecule inhibitor, designated PREX-in1, was utilized to enhance the efficacy of ionizing radiation (IR) therapy in CRC mouse models. RESULTS: PREX2 emerged as the most significantly upregulated gene in radioresistant CRC cells. It augmented the radioresistant capacity of CRC cells and demonstrated potential as a marker for predicting radioresistance efficacy. Mechanistically, PREX2 facilitated DNA repair by upregulating DNA-PKcs, suppressing radiation-induced immunogenic cell death, and impeding CD8+ T cell infiltration through the cGAS/STING/IFNs pathway. In vivo, the blockade of PREX2 heightened the efficacy of IR therapy. CONCLUSIONS: PREX2 assumes a pivotal role in CRC radiation resistance by inhibiting the cGAS/STING/IFNs pathway, presenting itself as a potential radioresistant biomarker and therapeutic target for effectively overcoming radioresistance in CRC.

Effects of extracorporeal circulation with different time on platelet count after cardiac surgery: a retrospective study based on medical records.

Our objective was to observe the effects of extracorporeal circulation (ECC) with different time on platelet count in patients undergoing cardiac surgery. A total of 427 patients who underwent elective cardiac surgery under ECC in affiliated hospital of north Sichuan medical college from January 1, 2018 to July 31, 2021 were divided into three groups according to ECC time. We concluded that thrombocytopenia was common after ECC, maximum drop of the platelet counts after ECC was usually seen on the second day after ECC, and platelet counts started to recover on the fifth day after ECC. With the extension of ECC time, the drop in platelet counts is more pronounced, the volume of perioperative blood loss and blood products transfusion are more, and the recovery level and speed of platelet counts is lower.

Endothelial cell-derived S1P promotes migration and stemness by binding with GPR63 in colorectal cancer.

Hematological metastasis was the main metastatic method of colorectal cancer and the main reason for failure of radical surgery. Vascular endothelial cells played an important role in tumor hematologic metastasis. We previously performed RNA-Seq on primary and metastatic colorectal carcinoma (CRC) tissues and then identified GPR63 as a potential metastasis-promoting gene, but its role and mechanisms in the interaction between cancer cells and vascular endothelial cells were still unknown. In this study, GPR63 was significantly elevated in CRC tissues compared with paracarcinoma tissues. GPR63 expression was closely related to lymph node metastasis and distant metastasis in 147 CRC tissues. GPR63 promoted cell migration and stemness. Moreover, endothelial cell-derived S1P enhanced the migration and sphere-forming ability of CRC through activation of GPR63. Mechanistically, S1P promoted GPR63 binding to Src to activate JAK2/STAT3 pathway, and therefore promoted CRC cell migration. Our study revealed a novel mechanism by which endothelial cells promoted metastasis of CRC cells, which might have potential as a promising target for CRC therapy.

Effects of etomidate combined with dexmedetomidine on adrenocortical function in elderly patients: a double-blind randomized controlled trial.

Etomidate has been advocated to be used in anesthesia for the elderly and the critically ill patients due to its faint effect on cardiovascular system. But the dose-dependent suppression of etomidate on adrenal cortex function leads to the limitation of its clinical application. Clinical research showed that dexmedetomidine could reduce the dose requirements for intravenous or inhalation anesthetics and opioids, and the hemodynamics was more stable during the operation. The objective was to observe the effect of etomidate combined with dexmedetomidine on adrenocortical function in elderly patients. 180 elderly patients scheduled for elective ureteroscopic holmium laser lithotripsy were randomly allocated to PR group anesthetized with propofol-remifentanil, ER group anesthetized with etomidate-remifentanil, and ERD group anesthetized with dexmedetomidine combined with etomidate-remifentanil. Patients in each group whose operation time was less than or equal to 1 h were incorporated into short time surgery group (PR1 group, ER1 group and ERD1 group), and whose surgical procedure time was more than 1 h were incorporated into long time surgery group (PR2 group, ER2 group and ERD2 group). The primary outcome was the serum cortisol and ACTH concentration. The secondary outcomes were the values of SBP, DBP, HR and SpO2, the time of surgical procedure, the dosage of etomidate and remifentanil administered during surgery, the time to spontaneous respiration, recovery and extubation, and the duration of stay in the PACU. The Serum cortisol concentration was higher at t1~2 in ERD1 group compared to ER1 group (P < 0.05). The Serum cortisol concentration at t1~3 was higher in ERD2 group than in ER2 group (P < 0.05). The Serum ACTH concentration was lower at t1~2 in ERD1 group compared to ER1 group (P < 0.05). The Serum ACTH concentration at t1~3 was lower in ERD2 group compared to ER2 group (P < 0.05). The SBP at T1 and T3 were higher in ER2 and ERD2 group than in PR2 group (P < 0.05). The DBP in ER1 and ERD1 group were higher at T1 compared to PR1 group (P < 0.05). The dosage of etomidate was significantly lower in ERD1 group and ERD2 group than in ER1 group and ER2 group (P < 0.05), respectively. The administration of dexmedetomidine combined with etomidate can attenuate the inhibition of etomidate on adrenocortical function in elderly patients and maintain intraoperative hemodynamic stability.

Inhibition of NOS1 promotes the interferon response of melanoma cells.

BACKGROUND: NOS1 expression predicts poor prognosis in patients with melanoma. However, the molecular function of NOS1 in the type I IFN response and immune escape of melanoma is still unknown. METHODS: The CRISPR/Cas9 system was used to generate NOS1-knockout melanoma cells and the biological characteristics of NOS1-knockout cells were evaluated by MTT assay, clonogenic assay, EdU assay, and flow cytometric assay. The effect on tumor growth was tested in BALB/c-nu and C57BL/6 mouse models. The gene expression profiles were detected with Affymetrix microarray and RNA-seq and KEGG (Kyoto Encyclopedia of Genes and Genomes) and CLUE GO analysis was done. The clinical data and transcriptional profiles of melanoma patients from the public database TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus, GSE32611) were analyzed by Qlucore Omics Explorer. RESULTS: NOS1 deletion suppressed the proliferation of melanoma A375 cells in culture, blocked cell cycling at the G0/G1 phase, and decreased the tumor growth in lung metastasis nodes in a B16 melanoma xenograft mouse model. Moreover, NOS1 knockout increased the infiltration of CD3+ immune cells in tumors. The transcriptomics analysis identified 2203 differential expression genes (DEGs) after NOS1 deletion. These DEGs indicated that NOS1 deletion downregulated mostly metabolic functions but upregulated immune response pathways. After inhibiting with NOS1 inhibitor N-PLA, melanoma cells significantly increased the response to IFN[Formula: see text] by upregulation expression of IFN[Formula: see text] simulation genes (ISGs), especially the components in innate immune signaling, JAK-STAT, and TOLL-LIKE pathway. Furthermore, these NOS1-regulating immune genes (NOS1-ISGs) worked as a signature to predict poor overall survival and lower response to chemotherapy in melanoma patients. CONCLUSION: These findings provided a transcriptional evidence of NOS1 promotion on tumor growth, which is correlated with metabolic regulation and immune escape in melanoma cells.

Circular RNA circ_001422 promotes the progression and metastasis of osteosarcoma via the miR-195-5p/FGF2/PI3K/Akt axis.

BACKGROUND: Circular RNAs (circRNAs) are involved in diverse processes that drive cancer development. However, the expression landscape and mechanistic function of circRNAs in osteosarcoma (OS) remain to be studied. METHODS: Bioinformatic analysis and high-throughput RNA sequencing tools were employed to identify differentially expressed circRNAs between OS and adjacent noncancerous tissues. The expression level of circ_001422 in clinical specimens and cell lines was measured using qRT-PCR. The association of circ_001422 expression with the clinicopathologic features of 55 recruited patients with OS was analyzed. Loss- and gain-of-function experiments were conducted to explore the role of circ_001422 in OS cells. RNA immunoprecipitation, fluorescence in situ hybridization, bioinformatics database analysis, RNA pulldown assays, dual-luciferase reporter assays, mRNA sequencing, and rescue experiments were conducted to decipher the competitive endogenous RNA regulatory network controlled by circ_001422. RESULTS: We characterized a novel and abundant circRNA, circ_001422, that promoted OS progression. Circ_001422 expression was dramatically increased in OS cell lines and tissues compared with noncancerous samples. Higher circ_001422 expression correlated with more advanced clinical stage, larger tumor size, higher incidence of distant metastases and poorer overall survival in OS patients. Circ_001422 knockdown markedly repressed the proliferation and metastasis and promoted the apoptosis of OS cells in vivo and in vitro, whereas circ_001422 overexpression exerted the opposite effects. Mechanistically, competitive interactions between circ_001422 and miR-195-5p elevated FGF2 expression while also initiating PI3K/Akt signaling. These events enhanced the malignant characteristics of OS cells. CONCLUSIONS: Circ_001422 accelerates OS tumorigenesis and metastasis by modulating the miR-195-5p/FGF2/PI3K/Akt axis, implying that circ_001422 can be therapeutically targeted to treat OS.

The role of S-nitrosylation of PFKM in regulation of glycolysis in ovarian cancer cells.

One of the malignant transformation hallmarks is metabolism reprogramming, which plays a critical role in the biosynthetic needs of unchecked proliferation, abrogating cell death programs, and immunologic escape. However, the mechanism of the metabolic switch is not fully understood. Here, we found that the S-nitrosoproteomic profile of endogenous nitrogen oxide in ovarian cancer cells targeted multiple components in metabolism processes. Phosphofructokinase (PFKM), one of the most important regulatory enzymes of glycolysis, was S-nitrosylated by nitric oxide synthase NOS1 at Cys351. S-nitrosylation at Cys351 stabilized the tetramer of PFKM, leading to resist negative feedback of downstream metabolic intermediates. The PFKM-C351S mutation decreased the proliferation rate of cultured cancer cells, and reduced tumor growth and metastasis in the mouse xenograft model. These findings indicated that S-nitrosylation at Cys351 of PFKM by NOS1 contributes to the metabolic reprogramming of ovarian cancer cells, highlighting a critical role of endogenous nitrogen oxide on metabolism regulations in tumor progression.

The heterocyclic compound Tempol inhibits the growth of cancer cells by interfering with glutamine metabolism.

Tempol (4-hydroxy-2,2,6,6-Tetramethylpiperidine-1-oxyl, TPL), a nitroxide compound, inhibits proliferation and increases the vulnerability of cancer cells to apoptosis induced by cytotoxic agents. However, the molecular mechanism of TPL inhibiting cancer cell proliferation has not been fully understood. In this study, we evaluated the metabolic effect of TPL on cancer cells and explored its cancer therapeutic potential. Extracellular flow assays showed that TPL inhibited cellular basal and maximal oxygen consumption rates of mitochondrial. 13C metabolic flux analysis showed that TPL treatment had minimal effect on glycolysis. However, we found that TPL inhibits glutamine metabolism by interfering with the oxidative tricarboxylic acid cycle (TCA) process and reductive glutamine process. We found that the inhibitory effect of TPL on metabolism occurs mainly on the step from citrate to α-ketoglutarate or vice versa. We also found that activity of isocitrate dehydrogenase IDH1 and IDH2, the key enzymes in TCA, were inhibited by TPL treatment. In xenograft mouse model, TPL treatment reduced tumor growth by inhibiting cellular proliferation of xenograft tumors. Thus, we provided a mechanism of TPL inhibiting cancer cell proliferation by interfering with glutamine utilization that is important for survival and proliferation of cancer cells. The study may help the development of a therapeutic strategy of TPL combined with other anticancer medicines.

Microcystins-LR induced apoptosis via S-nitrosylation of GAPDH in colorectal cancer cells.

Microcystins-LR (MC-LR), a cyanobacterial toxins, initiate apoptosis in normal and tumor cells. Nitric oxide produced by iNOS is necessary for MC-LR-induced apoptosis. However, the underlying mechanism of NO mediated MC-LR cytotoxicity remains unclear. Here, we performed in vitro experiments on MC-LR cytotoxicity associated with NO induced S-nitrosyation of GAPDH in human colon cancer cells SW480. MTT assay indicated that MC-LR decreased the cellular viability by high concentration (>1 µM). Flow cytometer assay revealed that apoptosis was core mode for MC-LR cytotoxicity. Griess assay showed that MC-LR exposure increased the release of NO through the function of NOS1 and NOS2 in SW480 cells. In turn, NO stress induced the S-nitrosylated modification of GAPDH leading to its nuclear translocation following Siah1 binding. CHIP assay showed that the nuclear GADPH increased P53 transcript of a panner of apoptosis related genes. Moreover, apoptosis induced by MC-LR could be reduced by GAPDH or si-Siah1 or NOSs inhibitor, L-NAME. Thus, our study verified a molecular mechanism of NO/GAPDH/Siah1 cascade in MC-LR mediated apoptosis in colorectal cancer cells, providing a further understanding the in vitro molecular mechanism of MC-LR colorectal toxicity.

NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2.

BACKGROUND: The dysfunction of type I interferon (IFN) signaling is an important mechanism of immune escape and metastasis in tumors. Increased NOS1 expression has been detected in melanoma, which correlated with dysfunctional IFN signaling and poor response to immunotherapy, but the specific mechanism has not been determined. In this study, we investigated the regulation of NOS1 on the interferon response and clarified the relevant molecular mechanisms. METHODS: After stable transfection of A375 cells with NOS1 expression plasmids, the transcription and expression of IFNα-stimulated genes (ISGs) were assessed using pISRE luciferase reporter gene analysis, RT-PCR, and western blotting, respectively. The effect of NOS1 on lung metastasis was assessed in melanoma mouse models. A biotin-switch assay was performed to detect the S-nitrosylation of HDAC2 by NOS1. ChIP-qPCR was conducted to measure the binding of HDAC2, H4K16ac, H4K5ac, H3ac, and RNA polymerase II in the promoters of ISGs after IFNα stimulation. This effect was further evaluated by altering the expression level of HDAC2 or by transfecting the HDAC2-C262A/C274A site mutant plasmids into cells. The coimmunoprecipitation assay was performed to detect the interaction of HDAC2 with STAT1 and STAT2. Loss-of-function and gain-of-function approaches were used to examine the effect of HDAC2-C262A/C274A on lung metastasis. Tumor infiltrating lymphocytes were analyzed by flow cytometry. RESULTS: HDAC2 is recruited to the promoter of ISGs and deacetylates H4K16 for the optimal expression of ISGs in response to IFNα treatment. Overexpression of NOS1 in melanoma cells decreases IFNα-responsiveness and induces the S-nitrosylation of HDAC2-C262/C274. This modification decreases the binding of HDAC2 with STAT1, thereby reducing the recruitment of HDAC2 to the ISG promoter and the deacetylation of H4K16. Moreover, expression of a mutant form of HDAC2, which cannot be nitrosylated, reverses the inhibition of ISG expression by NOS1 in vitro and decreases NOS1-induced lung metastasis and inhibition of tumor infiltrating lymphocytes in a melanoma mouse model. CONCLUSIONS: This study provides evidence that NOS1 induces dysfunctional IFN signaling to promote lung metastasis in melanoma, highlighting NOS1-induced S-nitrosylation of HDAC2 in the regulation of IFN signaling via histone modification.

[Effect of the chemoprotectant tempol on anti-tumor activity of cisplatin].

OBJECTIVE: To investigate the effect of the chemoprotectant tempol on the anti-tumor activity of cisplatin (DDP). METHODS: The cellular toxicity of tempol in human colon cancer SW480 cells and mouse colon cancer CT26 cells were evaluated using MTT and cell counting kit-8 assays. CalcuSyn software analysis was used to determine the interaction between tempol and DDP in inhibition of the cell viability. A subcutaneous homograft mouse model of colon cancer was established. The mice were randomly divided into control group, tempol group, cisplatin group and tempol + DDP treatment group with intraperitoneal injections of the indicated agents. The tumor size, body weight and lifespan of the mice were measured, and HE staining was used to analyze the cytotoxic effect of the agents on the kidney and liver. Immunohistochemistry and Western blotting were performed to detect the expression of Bax and Bcl2 in the tumor tissue, and TUNEL staining was used to analyze the tumor cell apoptosis. The level of reactive oxygen species (ROS) in the tumor tissue was determined using flow cytometry. RESULTS: Tempol showed inhibitory effects on the viability of SW480 and CT26 cells. CalcuSyn software analysis showed that tempol had a synergistic anti-tumor effect with DDP (CI < 1). In the homograft mouse model, tempol treatment alone did not produce obvious anti-tumor effect. HE staining showed that the combined use of tempol and DDP alleviated DDP-induced fibrogenesis in the kidneys, but tempol also reduced the anti-tumor activity of DDP. Compared with the mice treated with DDP alone, the mice treated with both tempol and DDP had a significantly larger tumor size (P < 0.01) and a shorter lifespan (P < 0.05). Tempol significantly reversed DDP-induced expression of Bax and Bcl2 in the tumor tissue and tumor cell apoptosis (P < 0.001), and obviously reduced the elevation of ROS level in the tumor tissue induced by DDP treatment (P < 0.05). CONCLUSIONS: Tempol can attenuate the anti-tumor effect of DDP while reducing the side effects of DDP. Caution must be taken and the risks and benefits should be carefully weighed when considering the use of tempol as an anti-oxidant to reduce the toxicities of DDP.

Mitochondrial NOS1 suppresses apoptosis in colon cancer cells through increasing SIRT3 activity.

Previous studies have suggested that nitric oxide (NO) which is synthetized by nitric oxide synthase (NOS) is closely related to the carcinogenesis and progression of colon cancer. However, the precise physiopathological role of NO on colon cancer remains unclear, and a lot of related studies focused on NOS2 and NOS3, but little on NOS1. Here, stable overexpression NOS1 of colon cancer cells were constructed to investigate whether NOS1 plays a special role in colon cancer. We observed that NOS1 protein was presented in mitochondria. Both the basal and cisplatin-induced mitochondrial superoxide were inhibited by NOS1, and the cisplatin-induced apoptosis was also inhibited by NOS1. Geldanamycin, a Hsp90 N-terminal inhibitor, was able to impede NOS1 translocation into mitochondria and reverse NOS1-induced apoptosis resistance. Importantly, SIRT3 activity was enhanced by NOS1, which contributes to the low level of mitochondrial superoxide and apoptosis resistance. Our data suggest a link between NOS1 and apoptosis resistance in colon cancer cells through mtNOS1-SIRT3-SOD2 axis. Furthermore, NOS1-induced apoptosis resistance could be reversed by inhibiting mitochondrial translocation of NOS1.