L-arginine/nitric oxide pathway metabolites are altered in colorectal cancer (CRC). We evaluated underlying changes in pathway enzymes in 55 paired tumor/tumor-adjacent samples and 20 normal mucosa using quantitative-PCR and assessed the impact of classic and novel oxicam analogues on enzyme expression and intracellular metabolite concentration (LC-MS/MS) in Caco-2, HCT116, and HT-29 cells. Compared to normal mucosa, ARG1, PRMT1, and PRMT5 were overexpressed in both tumor and tumor-adjacent tissue and DDAH2 solely in tumor-adjacent tissue. Tumor-adjacent tissue had higher expression of ARG1, DDAH1, and DDAH2 and lower NOS2 than patients-matched tumors. The ARG1 expression in tumors increased along with tumor grade and reflected lymph node involvement. Novel oxicam analogues with arylpiperazine moiety at the thiazine ring were more effective in downregulating DDAHs and PRMTs and upregulating ARG2 than piroxicam and meloxicam. An analogue distinguished by propylene linker between thiazine’s and piperazine’s nitrogen atoms and containing two fluorine substituents was the strongest inhibitor of DDAHs and PRMTs expression, while an analogue containing propylene linker but no fluorine substituents was the strongest inhibitor of ARG2 expression. Metabolic reprogramming in CRC includes overexpression of DDAHs and PRMTs in addition to ARG1 and NOS2 and is not restricted to tumor tissue but can be modulated by novel oxicam analogues.

Juvenile neuronal ceroid lipofuscinosis ( JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3 Dex7/8 knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3Dex7/8 mice were administered 3 · 10 10 genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3Dex7/8 mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3Dex7/8 mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3 Dex7/8 mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and other diseases in this category.

Immunosuppressive interleukins (IL)-4 and 13 may directly promote cancer but neither their status nor role in gastrointestinal tract is clarified. We aim at quantifying ILs and their receptors in paired normal-tumor samples (n = 49/51) and sera (n = 263), using immunoassays and RTqPCR, and screening for their effect on colonic cancer cells. Both ILs were elevated locally at protein level in all cancers but only IL13 transcripts in colon were upregulated. Interleukin and their receptor expression reflected cancer pathology to varying degrees, with the association frequently inverse and manifested in non-cancerous tissue. Positive correlation with cancer-promoting genes BCL2, BCLxL, HIF1A, VEGFA, ACTA2, CCL2, PTGS2, and CDKN1A, but not Ki67, was demonstrated, particularly for ILs’ receptors. Circulating IL-4 was elevated in all, while IL-13 only in colorectal or esophageal cancers, reflecting their advancement. IL4Ra and IL13Ra1 transcripts were downregulated by hypoxia and, in Caco-2, also by IL-4. Interleukin stimulation slightly improved colonic cancer cell viability, weakly upregulating BCL2 and Ki67 in HCT116 and HT-29. It affected cell motility more markedly and was consistently accompanied by upregulation of claudin-2. Gastrointestinal tract cancers are associated with IL-4 and IL-13 upregulation, which may facilitate cancer growth. Targeting both interleukins as an antineoplastic strategy warrants further investigation.

The L-Arginine/NO pathway is involved in carcinogenesis and immunity. Its diagnostic and prognostic value in colorectal cancer (CRC) was determined using tandem mass spectrometry in 199 individuals (137 with CRC) and, during a three-day follow up, in 60 patients undergoing colorectal surgery. Citrulline was decreased and asymmetric (ADMA) and symmetric (SDMA) dimethylarginines and dimethylamine (DMA) were increased in CRC. The DMA increase corresponded with CRC advancement while arginine, ADMA, and SDMA levels were higher in left-sided cancers. Arginine, citrulline, ADMA, and DMA dropped and SDMA increased post incision. Females experienced a more substantial drop in arginine. The arginine and ADMA dynamics depended on blood loss. The initial SDMA increase was higher in patients requiring transfusions. Postoperative dynamics in arginine and dimethylarginines differed in robot-assisted and open surgery. Concomitant SDMA, citrulline, and DMA quantification displayed a 92% accuracy in detecting CRC. Monitoring changes in arginine, ADMA, and SDMA in the early postoperative period predicted postoperative ileus with 84% and surgical site infections with 90% accuracy. Changes in ADMA predicted operative morbidity with 90% and anastomotic leakage with 77% accuracy. If positively validated, L-arginine/NO pathway metabolites may facilitate CRC screening and surveillance, support differential diagnosis, and assist in clinical decision-making regarding patients recovering from colorectal surgery.