Nitric oxide synthase inhibitors as potential therapeutic agents for gliomas: A systematic review.

INTRODUCTION: Gliomas represent the most prevalent form of brain tumors, among which glioblastomas are the most malignant subtype. Despite advances in comprehending their biology and treatment strategies, median survival remains disappointingly low. Inflammatory processes involving nitric oxide (NO), critically contribute to glioma formation. The inducible isoform of NO synthase (iNOS) is highly overexpressed in gliomas and has been linked to resistance against temozolomide (TMZ) treatment, neoplastic transformation, and modulation of immune response. While both in vitro and in vivo studies showed the potential of iNOS inhibitors as effective treatments for gliomas, no clinical trials on gliomas have been published. This review aims to summarize the available evidence regarding iNOS as a target for glioma treatment, focusing on clinically relevant data. METHODS: Following PRISMA guidelines, we conducted a systematic review by searching PubMed/Medline, and Embase databases in May 2023. We included studies that investigated the impact of NOS inhibitors on glioma cells using L-NMMA, CM544, PBN, 1400W or l-NAME either alone or combined with TMZ. We extracted data on the NOS inhibitor used, subtype, study setting, animal model or cell lines employed, obtained results, and safety profile. Our inclusion criteria encompassed original articles in English or Spanish, studies with an untreated control group, and a primary outcome focused on the biological effects on glioma cells. RESULTS: Out of 871 articles screened from the aforementioned databases, 37 reports were assessed for eligibility. After excluding studies that did not utilize glioma cells or address the designated outcome, 11 original articles satisfied the inclusion and exclusion criteria. Although no NOS inhibitor has been tested in a published clinical trial, three inhibitors have been evaluated using in vivo models of intracranial gliomas. l-NAME, 1400W, and CM544 were tested in vitro. Co-administration of l-NAME, or CM544 with TMZ showed superior results in vitro compared to individual agent testing. CONCLUSION: Glioblastomas remain a challenging therapeutic target. iNOS inhibitors exhibit substantial potential as treatment options for oncologic lesions, and they have demonstrated a safe toxicity profile in humans for other pathological conditions. Research endeavors should be focused on investigating their potential effects on brain tumors.

Evaluation of local, regional and abscopal effects of Boron Neutron Capture Therapy (BNCT) combined with immunotherapy in an ectopic colon cancer model.

OBJECTIVE: The aim of the present study was to evaluate the local and regional therapeutic efficacy and abscopal effect of BNCT mediated by boronophenyl-alanine, combined with Bacillus Calmette-Guerin (BCG) as an immunotherapy agent in this model. METHODS: The local effect of treatment was evaluated in terms of tumor response in the irradiated tumor-bearing right hind flank. Metastatic spread to tumor-draining lymph nodes was analyzed as an indicator of regional effect. The abscopal effect of treatment was assessed as tumor growth inhibition in the contralateral (non-irradiated) left hind flank inoculated with tumor cells 2 weeks post-irradiation. The experimental groups BNCT, BNCT + BCG, BCG, Beam only (BO), BO +BCG, SHAM (tumor-bearing, no treatment, same manipulation) were studied. RESULTS: BNCT and BNCT + BCG induced a highly significant local anti-tumor response, whereas BCG alone induced a weak local effect. BCG and BNCT + BCG induced a significant abscopal effect in the contralateral non-irradiated leg. The BNCT + BCG group showed significantly less metastatic spread to tumor-draining lymph nodes vs SHAM and vs BO. CONCLUSION: This study suggests that BNCT + BCG-immunotherapy would induce local, regional and abscopal effects in tumor-bearing animals. BNCT would be the main effector of the local anti-tumor effect whereas BCG would be the main effector of the abscopal effect. ADVANCES IN KNOWLEDGE: Although the local effect of BNCT has been widely evidenced, this is the first study to show the local, regional and abscopal effects of BNCT combined with immunotherapy, contributing to comprehensive cancer treatment with combined therapies.

Flavonoid silybin improves the response to radiotherapy in invasive bladder cancer.

Conservative treatment for invasive bladder cancer (BC) involves a complete transurethral tumor resection combined with chemotherapy (CT) and radiotherapy (RT). The major obstacles of chemo-radiotherapy are the addition of the toxicities of RT and CT, and the recurrence due to RT and CT resistances. The flavonoid Silybin (Sb) inhibits pathways involved in cell survival and resistance mechanisms, therefore the purpose of this paper was to study in vitro and in vivo, the ability of Sb to improve the response to RT, in two murine BC cell lines, with different levels of invasiveness, placing emphasis on radio-sensitivity, and pathways involved in radio-resistance and survival. In vitro, Sb radio-sensitized murine invasive cells through the inhibition of RT-induced NF-κB and PI3K pathways, and the increase of oxidative stress, while non-invasive cells did not show to be sensitized. In vivo, Sb improved RT-response and overall survival in invasive murine tumors. As Sb is already being tested in clinical trials for other urological cancers and it improves RT-response in invasive BC, these results could have translational relevance, supporting further research.

FGFR3 Down-Regulation is Involved in bacillus Calmette-Guérin Induced Bladder Tumor Growth Inhibition.

PURPOSE: Bacillus Calmette-Guérin is the standard treatment for patients with nonmuscle invasive high histological grade bladder cancer. Previously we found that bacillus Calmette-Guérin induces murine bladder cancer MB49 cell death in vitro and in vivo, generating tissue remodeling, which involves the release of fibroblast growth factor (FGF)-2. MATERIALS AND METHODS: We studied the effect of bacillus Calmette-Guérin treatment on FGF-2 and FGF receptor (FGFR) expression in bladder cancer. RESULTS: In vitro FGF-2 increased MB49 cell proliferation but did not reverse bacillus Calmette-Guérin induced cell death. Increased FGF-2 expression was detected after bacillus Calmette-Guérin treatment. Moreover MB49 cells expressed high FGFR3 levels, which decreased after treatment. Similar results were observed in human T24 bladder cancer cells. In vivo MB49 tumors expressed higher FGFR3 levels than normal urothelium. Tumor FGFR3 decreased after treatment and correlated with tumor growth inhibition in response to bacillus Calmette-Guérin. In a pilot bioassay using 11 human bladder tumors treated ex vivo with bacillus Calmette-Guérin we found a subgroup of 41% of patients in whom FGFR3 was decreased after treatment. CONCLUSIONS: Based on bladder cancer murine model results we infer that down-regulation of FGFR3 is a predictive marker of a good response to bacillus Calmette-Guérin therapy. The decrease in FGFR3 in response to bacillus Calmette-Guérin occurred not only in a murine model but also in a human bladder cancer cell line and in some patient samples. More patients and increased followup are needed to establish the predictive role of FGFR3 as a marker in human bladder cancer.

Expression of inducible nitric oxide synthase in tumoral and non-tumoral epithelia from bladder cancer patients.

We have previously demonstrated that nitric oxide (NO) is elevated in the urine from bladder cancer patients. As the inducible nitric oxide synthase (iNOS) produces high NO output, the aim of this study was to examine iNOS expression and activity in tumoral (BT) and non-tumoral bladder tissue (NT). iNOS expression was determined by Western blot in 42 BT, 22 NT, and 4 normal bladders (normal B). iNOS activity was evaluated by conversion of [(14)C]l-arginine to [(14)C]l-citrulline plus NO, in additional 15 BT, 8 NT, and 1 normal B. iNOS tissue localization was studied by immunohistochemistry. iNOS expression and activity were found in almost 50% of bladder cancer patients, in both BT and in NT. A similar positive or negative iNOS expression in each pair of NT and BT tissue compared was observed, suggesting that high urine NO levels could be generated by an active iNOS present not only in the tumor but also in the non-tumoral bladder tissue. By immunohistochemistry, heterogeneous iNOS staining was detected in tumor cells from superficial and invasive tumors, while it was not evident in the normal bladder epithelium. A follow-up of 21 patients during 2 years showed recurrences in 80% with positive iNOS. On the contrary, no recurrences were observed in 73% of iNOS negative patients. Our results suggest that iNOS expression in bladder tissue may predispose to cancer recurrences.

High expression of cathepsin B in transitional bladder carcinoma correlates with tumor invasion.

BACKGROUND: Cathepsin B (CB) is a lysosomal cysteine proteinase synthesized as a zymogen of 39-47 kilodaltons (kD), which is subsequently converted into an active single- chain form of 33 kD (CB33) and, by additional processing, into the active 2-chain form containing a heavy chain of 27-29 kD (CB(27-29)) and a light chain of 4-6 kD. Increased or altered CB expression has been documented in a variety of tumor cells, but to the authors' knowledge only one study published to date has reported clinicopathologic significance for CB in transitional cell carcinoma (TCC) of the bladder. METHODS: In this work, CB expression was determined by Western blot analysis in TCC bladder tissue from 30 patients. Nontumor bladder tissue was also analyzed for CB expression. RESULTS: The study results demonstrate higher expression of CB in TCC invasive tumors than in superficial bladder carcinoma. Furthermore, whereas normal bladder only expressed the 29-kD CB protein, tumor and peritumoral tissue demonstrated the 27- to 29-kD CB form. Immunohistochemical staining did not evidence changes in CB localization between tumor and nontumor tissue. CONCLUSIONS: According to the results of the current study, bladder tumor progression appears to be associated with quantitative changes in CB protein expression, as well as with qualitative changes related to the type of CB expressed.

Nitric oxide synthase, arginase and cyclooxygenase are involved in muscarinic receptor activation in different murine mammary adenocarcinoma cell lines.

Investigations on the influence of the parasympathetic nervous system via muscarinic signaling in tumor progression have produced contradictory evidence. We investigated the expression of muscarinic acetylcholine receptors (mAchR) and their intracellular transduction pathways, in two murine mammary adenocarcinoma cell lines, LM3 and LM2 in comparison with the normal murine mammary epithelial cell line: NMuMG. Saturation binding assays with the tritiated muscarinic antagonist quinuclidinyl benzilate ([3H]-QNB) indicate that LM3 cells express higher amounts of mAchR than LM2 cells. Muscarinic receptor activation with carbachol (CARB) enhanced basal production of citrulline to a greater extent in LM3 cells than in LM2 cells. The nitric oxide synthase (NOS) inhibitor, NGmono-methyl-L-arginine (L-NMMA), blunted this effect only in LM3 cells while in LM2 cells the action of CARB was blocked by Nomega hydroxy-L-arginine (L-OH-Arg), which is known to inhibit the arginase pathway. Atropine blocks the action of CARB in both cell lines. Additionally, mAchR activation stimulates prostaglandin E2 (PGE2) synthesis only in LM2 cells. NMuMG cells show detectable basal amounts of nitric oxide and PGE2, but they did not respond to CARB. Binding experiments confirm the absence of mAchR in these cells. The findings indicate that mAchR expression in tumor cells, and its control on arginine metabolism, via NOS/arginase, and on PGE2 synthesis by COX activation, could be a switch on mechanism that might lead mammary cells from normal to malignant phenotype. Moreover, mAchR coupling to distinct effectors might be associated with differences in aggressiveness of tumor cells.

Reactivity of tumor-draining lymph nodes and the nitric oxide pathway.

Regional lymph nodes are important in the generation of tumor-directed immune responses. The relationship between nitric oxide synthase (NOS) expression and the biological behavior of tumor-draining lymph node (TDLNs) cells in vivo was determined using a spontaneously arising BALB/c mammary adenocarcinoma S13. We first demonstrated a reduction of tumor size and tumor-induced angiogenesis by blocking NOS activity in vivo. TDLNs harvested from tumor-bearing mice (TBM) on day 16 after tumor implant, showed enhanced NOS activity and NOS expression compared to control nodes. Identification of the NOS isoforms present in TDLNs resulted in expression of neuronal NOS (nNOS), endothelial NOS (eNOS) and absence of inducible NOS (iNOS). TDLN cells admixed with tumor cells and inoculated into normal mice (Winn assay) induced a reduction of tumor growth although, when inoculated alone, were able to induce the formation of new blood vessels (angiogenesis). Our data indicate that the in vivo antitumor activity of TDLN cells is modulated by a balance between angiogenesis and antitumor effectors. In our model, when trafficking of leukocytes is obviated, the control of tumor growth by TDLN cells can be explained in part by an antitumor activity great enough to exceed the angiogenic component elicited by the same cells, leading to a reduction of tumor size.

Inhibición del crecimiento tumoral por acción de mastocitos, heparina y heparinas modificadas / Inhibition of the tumoral growth by action of mastocytes, heparin and modified heparins

Es característico de los procesos neoplásicos la proliferación de mastocitos en el tejido peritumoral, si bien su significado funcional es controvertido; estas células se caracterizan por liberar el medio, luego de ser activadas, un alto número de mediadores químicos alguno de los cuales se produce por síntesis "de novo", mientras que otros se encuentran almacenados en sus gránulos. Entre estos últimos se encuentra la heparina, proteoglicano cuya acción sobre el crecimiento celular, ha sido estudiada en varios tipos celulares normales, así como en el de algunos tumorales, obsevándose distintos efectos en los distintos casos. Por tal motivo, disponiendo en nuestro laboratorio, de dos adenocarcinomas mamarios murinos de distinta capacidad metastásica [M3 y MM3] y de un tercero de mayor velocidad de crecimiento [S13], que los nombrados anteriormente, centramos nuestro análisis, en la primera instancia, en la observación de si la proliferación de mastocitos era constante o variable en función del tiempo de portación tumoral para todos los tumores. Observamos que el índice de proliferación dependió del tipo tumoral. Seguidamente estudiamos in vivo e in vitro la acción que ejercieron las células tumorales y sus medios condicionados sobre los mastocitos de cavidad peritoneal de animales normales, en lo referente a la manifestación del fenotipo mastocitario y su contenido de heparina, mediante la implementación de técnicas histoquímicas específicas para su detección, Observamos que el tumor provoca la liberación de heparina por parte de los mastocitos y los inhibe para resintetizarla. Precisamente aquellos carentes de heparina, coinyectados con células tumorales o en cocultivo, in vitro, no ejercieron acción sobre el desarrollo tumoral, mientras que los ricos en heparina inhibieron en un 70 por ciento la toma tumoral en el M3, y disminuyeron las metástasis en el MM3. Heparinas de distinta capacidad anticoagulante, inhibieron el crecimiento de ambos tumores in vitro. Su acción fue independiente de factores del suero con afinidad por la heparina, fue dosis dependiente y reversible... (TRUNCADO)

Inhibición del crecimiento tumoral por acción de mastocitos, heparina y heparinas modificadas / Inhibition of the tumoral growth by action of mastocytes, heparin and modified heparins

Es característico de los procesos neoplásicos la proliferación de mastocitos en el tejido peritumoral, si bien su significado funcional es controvertido; estas células se caracterizan por liberar el medio, luego de ser activadas, un alto número de mediadores químicos alguno de los cuales se produce por síntesis "de novo", mientras que otros se encuentran almacenados en sus gránulos. Entre estos últimos se encuentra la heparina, proteoglicano cuya acción sobre el crecimiento celular, ha sido estudiada en varios tipos celulares normales, así como en el de algunos tumorales, obsevándose distintos efectos en los distintos casos. Por tal motivo, disponiendo en nuestro laboratorio, de dos adenocarcinomas mamarios murinos de distinta capacidad metastásica [M3 y MM3] y de un tercero de mayor velocidad de crecimiento [S13], que los nombrados anteriormente, centramos nuestro análisis, en la primera instancia, en la observación de si la proliferación de mastocitos era constante o variable en función del tiempo de portación tumoral para todos los tumores. Observamos que el índice de proliferación dependió del tipo tumoral. Seguidamente estudiamos in vivo e in vitro la acción que ejercieron las células tumorales y sus medios condicionados sobre los mastocitos de cavidad peritoneal de animales normales, en lo referente a la manifestación del fenotipo mastocitario y su contenido de heparina, mediante la implementación de técnicas histoquímicas específicas para su detección, Observamos que el tumor provoca la liberación de heparina por parte de los mastocitos y los inhibe para resintetizarla. Precisamente aquellos carentes de heparina, coinyectados con células tumorales o en cocultivo, in vitro, no ejercieron acción sobre el desarrollo tumoral, mientras que los ricos en heparina inhibieron en un 70 por ciento la toma tumoral en el M3, y disminuyeron las metástasis en el MM3. Heparinas de distinta capacidad anticoagulante, inhibieron el crecimiento de ambos tumores in vitro. Su acción fue independiente de factores del suero con afinidad por la heparina, fue dosis dependiente y reversible... (TRUNCADO)(AU)