Vasopressin Analog [V4Q5]dDAVP Exerts Cooperative Anticancer Effects in Combination With Low-Dose 5-Fluorouracil on Aggressive Colorectal Cancer Models.

Background: Colorectal cancer (CRC) is a leading cause of cancer-associated mortality worldwide. Despite being an essential component of systemic chemotherapy for advanced CRC, 5-fluorouracil (5-FU) clinical use has severe limitations, such as high toxicity, low selectivity and drug resistance. [V4Q5]dDAVP (1-deamino-4-valine-5-glutamine-8-D-arginine vasopressin) is a peptide vasopressin analog and a selective agonist of the arginine vasopressin type 2 membrane receptor (AVPR2), expressed in microvascular and tumor tissue. This synthetic compound has well-proven antitumor and antimetastatic activity in different tumor types, including metastatic CRC. The objective of this work was to assess the potential combinational benefits in preclinical CRC models after [V4Q5]dDAVP addition to 5-FU. Methods: Effects on cellular viability, cell cycle progression, apoptosis and molecular mechanisms associated to [V4Q5]dDAVP treatment in combination with 5-FU were evaluated in murine CT-26 and human COLO-205 cell lines. In vivo, impact of dual therapy was explored on CRC tumor growth and metastatic spread. Results: In CRC cells, [V4Q5]dDAVP (1 µM) addition to sub-IC50 5-FU concentrations resulted in the enhancement of cytostatic effects induced by chemotherapy. Reduction of cell viability after combined treatment was associated with cell cycle arrest in the G0/G1 phase, induction of apoptosis and increased gene expression of the cyclin-dependent kinase inhibitor p21 (CDKN1A) and the tumor suppressor p53 (TP53) in malignant cells, as assessed by flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. In vivo, intravenous administration of [V4Q5]dDAVP (0.3 µg/kg) in combination with safe low doses of 5-FU (50 or 80 mg/kg for CT-26 or COLO-205 tumor models, respectively) effectively abrogated CRC growth, reducing aggressiveness of primary lesions and increasing survival of tumor-bearing mice. In addition, concomitant administration of [V4Q5]dDAVP and 5-FU inhibited pulmonary metastasis formation by CT-26 cells in immunocompetent mice, especially reducing macrometastatic disease. Conclusions: [V4Q5]dDAVP seems to enhance the efficacy of 5-FU-based chemotherapy in CRC by modulating tumor progression, as well as metastatic dissemination, suggesting its potential role as a safe and cost-effective co-adjuvant agent for the management of advanced CRC.

Cu(II)-acylhydrazone complex, a potent and selective antitumor agent against human osteosarcoma: Mechanism of action studies over in vitro and in vivo models.

Osteosarcoma (OS) is a frequent bone cancer, affecting largely children and young adults. Cisplatin (CDDP) has been efficacious in the treatment of different cancer such us OS but the development of chemoresistance and important side effects leading to therapeutic failure. Novel therapies including copper compounds have shown to be potentially effective as anticancer drugs and one alternative to usually employed platinum compounds. The goal of this work is the evaluation of the in vitro and in vivo antitumoral activity and dilucidate the molecular target of a Cu(II) cationic complex containing a tridentate hydrazone ligand, CuHL for short, H2L=N'-'-(2-hydroxy-3-methoxybenzylidene)thiophene-2-carbohydrazide, against human OS MG-63 cells. Anticancer activity on MG-63 cell line was evaluated in OS monolayer and spheroids. CuHL significantly impaired cell viability in both models (IC50 2D: 2.1 ± 0.3 µM; 3D: 9.1 ± 1.0 µM) (p < 0.001). Additional cell studies demonstrated the copper compound inhibits cell proliferation and conveys cells to apoptosis, determined by flow cytometry. CuHL showed a great genotoxicity, evaluated by comet assay. Proteomic analysis by Orbitrap Mass Spectometry identified 27 differentially expressed proteins: 17 proteins were found overexpressed and 10 underexpressed in MG-63 cells after the CuHL treatment. The response to unfolded protein was the most affected biological process. In addition, in vivo antitumor effects of the compound were evaluated on human OS tumors xenografted in nude mice. CuHL treatment, at a dose of 2 mg/kg i.p., given three times/week for one month, significantly inhibited the progression of OS xenografts and was associated to a reduction in mitotic index and to an increment of tumor necrosis (p < 0.01). Administration of standard-of-care cytotoxic agent CDDP, following the same treatment schedule as CuHL, failed to impair OS growth and progression.

In Silico and In Vitro Evaluation of Bevacizumab Biosimilar MB02 as an Antitumor Agent in Canine Mammary Carcinoma.

Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC.

Estudio exploratorio de eficacia de ivermectina combinada a α- PD-1 en cáncer colorrectal resistente a quimioinmunoterapia / EXPLORATORY EFFECTIVENESS STUDY OF IVERMECTIN COMBINED WITH α-PD-1 IN CHEMOTHERAPY RESISTANT COLORRECTAL CANCER

[RESUMEN]. Las terapias inmunológicas con inhibidores de checkpoints (ICIs) han revolucionado el abordaje del cáncer colorrectal (CCR), pero su efectividad se restringe a tumores inmunorreactivos con deficiencia en la reparación de errores tipo mismatch (dMMR). La ivermectina (IVM), un agente antiparasitario, se propone como posible estrategia terapéutica debido a su impacto en la muerte celular inmunogénica (MCI) y la reversión de resistencia a medicamentos. La investigación evaluó el efecto antineoplásico de IVM combinado con α-PD-1 empleando el modelo murino CT-26, una línea de CCR KRAS-mutada y competentes para MMR. El análisis bioinformático mediante la plataforma GEPIA2 empleandola base TCGA confirmó la expresión diferencial de blancos moleculares de IVM en tejido tumoral versus normal, siendo más alta en tumores con inestabilidad microsatelital baja (MSI-L)/microsatelital estable (MSS) que en inestabilidad microsatelital alta (MSI-H). En experimentos in vitro, CT-26 mostró alta sensibilidad a IVM (IC50: 11 µM, luego de exposición por 72 horas), alterando el metabolismo y aumentando la secreción de IL-6. El análisis proteómico identificó 17 proteínas sobreexpresadas y 8 inhibidas, relacionadas con evasión inmunológica y proliferación celular. En ratones BALB/c portadores de tumores CT-26, la terapia combinada de IVM y α-PD-1 redujo significativamente el crecimiento tumoral y la progresión metastásica. La preinmunización con células CT-26 tratadas con IVM disminuyó la incidencia y la progresión tumoral. IVM podría potenciar la respuesta a ICIs en tumores "fríos". Estos hallazgos sugieren que la combinación de IVM y α-PD-1 puede mejorar la inmunorreactividad y respuesta terapéutica en CCR.

[ABSTRACT]. Matrix Assisted Laser DesorptionIonization ­ Time of Flight ­ MassSpectrometry (MALDI-TOF-MS) has emerged as anoutstandingtechnique in thefieldofclinicalmicrobiology, with a simple methodology and deliveryof precise results in anexceptionally short timeframe. Thistechnology has garnered notable success in recentyears, establishing as a fundamental toolboth in thecharacterizationofmicroorganisms and translationalresearch.Thecombinationoftheinherentfeaturesofthistechniquewiththepotentialof machine learninganalysis has provento be ofgreatvalue in clinicalmicrobiology, particularly in theantibioticresistancefield. Itsapplication has acceleratedbacterial diagnosis and opened new perspectives in criticalareasof medicine, such sepsis and oncology. In Argentina, several research groups actively contributing to its expansión, applying MALDI-TOF-MS in the fight against infectious diseases, including the COVID-19 pandemic. Theseeffortspromiseto continue drivingresearch and clinical diagnosis in the country and worldwide.

Propranolol blocks osteosarcoma cell cycle progression, inhibits angiogenesis and slows xenograft growth in combination with cisplatin-based chemotherapy.

Osteosarcoma is still associated with limited response to standard-of-care therapy and alarmingly elevated mortality rates, especially in low- and middle-income countries. Despite multiple efforts to repurpose ß-blocker propranolol in oncology, its potential application in osteosarcoma management remains largely unexplored. Considering the unsatisfied clinical needs of this aggressive disease, we evaluated the antitumoral activity of propranolol using different in vitro and in vivo osteosarcoma preclinical models, alone or in addition to chemotherapy. Propranolol significantly impaired cellular growth in ß2-adrenergic receptor-expressing MG-63 and U-2OS cells, and was capable of blocking growth-stimulating effects triggered by catecholamines. siRNA-mediated ADRB2 knockdown in MG-63 cells was associated with decreased cell survival and a significant attenuation of PPN anti-osteosarcoma activity. Direct cytostatic effects of propranolol were independent of apoptosis induction and were associated with reduced mitosis, G0/G1 cell cycle arrest and a significant down-regulation of cell cycle regulator Cyclin D1. Moreover, colony formation, 3D spheroid growth, cell chemotaxis and capillary-like tube formation were drastically impaired after propranolol treatment. Interestingly, anti-migratory activity of ß-blocker was associated with altered actin cytoskeleton dynamics. In vivo, propranolol treatment (10 mg/kg/day i.p.) reduced the early angiogenic response triggered by MG-63 cells in nude mice. Synergistic effects were observed in vitro after combining propranolol with chemotherapeutic agent cisplatin. Sustained administration of propranolol (10 mg/kg/day i.p., five days a week), alone and especially in addition to low-dose metronomic cisplatin (2 mg/kg/day i.p., three times a week), markedly reduced xenograft progression. After histological analysis, propranolol and cisplatin combination resulted in low tumor mitotic index and increased tumor necrosis. ß-blockade using propranolol seems to be an achievable and cost-effective therapeutic approach to modulate osteosarcoma aggressiveness. Further translational studies of propranolol repurposing in osteosarcoma are warranted.

Effect of Ivermectin and Atorvastatin on Nuclear Localization of Importin Alpha and Drug Target Expression Profiling in Host Cells from Nasopharyngeal Swabs of SARS-CoV-2- Positive Patients.

Nuclear transport and vesicle trafficking are key cellular functions involved in the pathogenesis of RNA viruses. Among other pleiotropic effects on virus-infected host cells, ivermectin (IVM) inhibits nuclear transport mechanisms mediated by importins and atorvastatin (ATV) affects actin cytoskeleton-dependent trafficking controlled by Rho GTPases signaling. In this work, we first analyzed the response to infection in nasopharyngeal swabs from SARS-CoV-2-positive and -negative patients by assessing the gene expression of the respective host cell drug targets importins and Rho GTPases. COVID-19 patients showed alterations in KPNA3, KPNA5, KPNA7, KPNB1, RHOA, and CDC42 expression compared with non-COVID-19 patients. An in vitro model of infection with Poly(I:C), a synthetic analog of viral double-stranded RNA, triggered NF-κB activation, an effect that was halted by IVM and ATV treatment. Importin and Rho GTPases gene expression was also impaired by these drugs. Furthermore, through confocal microscopy, we analyzed the effects of IVM and ATV on nuclear to cytoplasmic importin α distribution, alone or in combination. Results showed a significant inhibition of importin α nuclear accumulation under IVM and ATV treatments. These findings confirm transcriptional alterations in importins and Rho GTPases upon SARS-CoV-2 infection and point to IVM and ATV as valid drugs to impair nuclear localization of importin α when used at clinically-relevant concentrations.

Anticancer activity of repurposed hemostatic agent desmopressin on AVPR2-expressing human osteosarcoma.

Osteosarcoma is the most prevalent primary bone malignancy. Due to its high aggressiveness, novel treatment strategies are urgently required to improve survival of patients with osteosarcoma, especially those with advanced disease. Desmopressin (dDAVP) is a widely used blood-saving agent that has been repurposed as an adjuvant agent for cancer management due to its antiangiogenic and antimetastatic properties. dDAVP acts as a selective agonist of the vasopressin membrane receptor type 2 (AVPR2) present in the microvascular endothelium and in some cancer cells, including breast, lung, colorectal and neuroendocrine tumor cells. Despite the fact that dDAVP has demonstrated its antitumor efficacy in a wide variety of tumor types, exploration of its potential anti-osteosarcoma activity has, to the best of our knowledge, not yet been conducted. Therefore, the aim of the present study was to evaluate the preclinical antitumor activity of dDAVP in osteosarcoma. Human MG-63 and U-2 OS osteosarcoma cell lines were used to assess in vitro and in vivo therapeutic effects of dDAVP. At low micromolar concentrations, dDAVP reduced AVPR2-expressing MG-63 cell growth in a concentration-dependent manner. In contrast, dDAVP exhibited no direct cytostatic effect on AVPR2-negative U-2 OS cells. As it would be expected for canonical AVPR2-activation, dDAVP raised intracellular cAMP levels in osteosarcoma cells, and coincubation with phosphodiesterase-inhibitor rolipram indicated synergistic antiproliferative activity. Cytostatic effects were associated with increased apoptosis, reduced mitotic index and impairment of osteosarcoma cell chemotaxis, as evaluated by TUNEL-labeling, mitotic body count in DAPI-stained cultures and Transwell migration assays. Intravenous administration of dDAVP (12 µg/kg; three times per week) to athymic mice bearing rapidly growing MG-63 xenografts, was indicated to be capable of reducing tumor progression after a 4-week treatment. No major alterations in animal weight, biochemical or hematological parameters were associated with dDAVP treatment, confirming its good tolerability and safety. Finally, AVPR2 expression was detected by immunohistochemistry in 66% of all evaluated chemotherapy-naive human conventional osteosarcoma biopsies. Taking these findings into account, repurposed agent dDAVP may represent an interesting therapeutic tool for the management of osteosarcoma. Further preclinical exploration of dDAVP activity on orthotopic or metastatic osteosarcoma models are required.

Preclinical efficacy of CIGB-300, an anti-CK2 peptide, on breast cancer metastasic colonization.

CK2 is a serine/threonine kinase that is overexpressed in breast cancer and its inhibition is associated to reduced tumor growth and disease progression. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to protein kinase CK2 catalytic subunit alpha and to CK2 substrates thus preventing the enzyme activity. Our aim was to evaluate the potential therapeutic benefits of CIGB-300 on breast cancer disease using experimental models with translational relevance. We demonstrated that CIGB-300 reduces breast cancer cell growth in MDA-MB-231, MCF-7 and F3II cells, exerting a pro-apoptotic action and cell cycle arrest. We also found that CIGB-300 decreased cell adhesion, migration and clonogenic capacity of malignant cells. Effect on experimental breast cancer lung metastasis was evaluated after surgical removal of primary F3II tumors or after tail vein injection of tumor cells, also we evaluated CIGB-300 effect on spontaneous lung metastasis in an orthotopic model. Systemic CIGB-300 treatment inhibited breast cancer colonization of the lung, reducing the size and number of metastatic lesions. The present preclinical study establishes for the first time the efficacy of CIGB-300 on breast cancer. These encouraging results suggest that CIGB-300 could be used for the management of breast cancer as an adjuvant therapy after surgery, limiting tumor metastatic spread and thus protecting the patient from distant recurrence.

Administration of the vasopressin analog desmopressin for the management of bleeding in rectal cancer patients: results of a phase I/II trial.

Purpose The vasopressin analog desmopressin (dDAVP) is known to increase plasma levels of hemostatic factors, and preclinical studies in colorectal cancer models have demonstrated that it hampers tumor vascularization and metastatic progression. We evaluated safety and preliminary efficacy of dDAVP in rectal cancer patients with bleeding, before receiving specific oncologic treatment with surgery, chemotherapy and/or radiotherapy. Methods Patients with rectal cancer having moderate or severe rectal bleeding were enrolled in an open-label, dose-finding trial. Intravenous infusions of dDAVP were administered during two consecutive days in doses from 0.25 to 2.0 µg/kg, using single or twice daily regimen. Bleeding was graded using a score based on the Chutkan scale and tumor perfusion was evaluated by dynamic contrast-enhanced magnetic resonance imaging. Results The trial accrued a total of 32 patients. Dose-limiting toxicity occurred in patients receiving 1 µg/kg or higher. The most prominent treatment-related severe adverse event was hyponatremia. Most patients receiving the maximum tolerated dose of 0.5 µg/kg showed at least a partial hemostatic response and 58% developed a complete response with absence of bleeding at day 4 and/or at the last follow-up at day 14. Tumor perfusion was decreased in two-thirds of patients after dDAVP treatment. Conclusions dDAVP appeared as a promising hemostatic agent in rectal cancer patients with bleeding. Randomized clinical trials to confirm its effectiveness are warranted.Clinical trial registration www.clinicaltrials.gov NCT01623206.

Development and therapeutic potential of vasopressin synthetic analog [V4Q5]dDAVP as a novel anticancer agent.

Since its discovery, arginine vasopressin (AVP) was subjected to several modifications with the aim of obtaining novel derivatives with increased potency and selectivity for biomedical use. Desmopressin (dDAVP) is a first generation synthetic analog of AVP with hemostatic and antimetastatic activity. dDAVP acts as a selective agonist of the arginine vasopressin type 2 receptor (AVPR2) present in microvascular endothelium and cancer cells. Considering its selective effects on AVPR2-expressing malignant and vascular tissue, and interesting antitumor profile, dDAVP was used as a lead compound for the development of novel peptide analogs with enhanced anticancer efficacy. After conducting different structure-activity relationship studies to determine key aminoacidic positions for its antitumor activity against AVPR2-expressing malignant cells, dDAVP was rationally modified and a wide panel of synthetic analogs with different sequence and structural modifications was assessed. As a result of this structure-based drug derivatization novel AVP analog [V4Q5]dDAVP (1-deamino-4-valine-5-glutamine-8-d-arginine vasopressin) was selected as the most active candidate and further developed. [V4Q5]dDAVP was evaluated in highly aggressive and metastatic cancer preclinical models deploying enhanced cytostatic, antimetastatic and angiostatic effects in comparison to parental peptide dDAVP. In addition, novel compound demonstrated good tolerability as evaluated in several toxicological studies, and cooperative therapeutic effects after combination with standard-of-care chemotherapy. In summary, due to its ability to inhibit growth and tumor-associated angiogenesis, as well as impairing progression of metastatic disease, AVP analogs such as novel [V4Q5]dDAVP are promising compounds for further development as coadjuvant agents for the management of advance or recurrent cancers.

Preclinical Efficacy of [V4 Q5 ]dDAVP, a Second Generation Vasopressin Analog, on Metastatic Spread and Tumor-Associated Angiogenesis in Colorectal Cancer.

PURPOSE: Control of metastatic spread of colorectal cancer (CRC) remains as a major therapeutic challenge. [V4 Q5 ]dDAVP is a vasopressin peptide analog with previously reported anticancer activity against carcinoma tumors. By acting as a selective agonist of arginine vasopressin type 2 membrane receptor (AVPR2) present in endothelial and tumor cells, [V4Q5]dDAVP is able to impair tumor aggressiveness and distant spread. Our aim was to evaluate the potential therapeutic benefits of [V4Q5]dDAVP on highly aggressive CRC disease using experimental models with translational relevance. MATERIALS AND METHODS: Murine CT-26 and human Colo-205 AVPR2-expressing CRC cell lines were used to test the preclinical efficacy of [V4Q5]dDAVP, both in vitro and in vivo. RESULTS: In syngeneic mice surgically implanted with CT-26 cells in the spleen, sustained intravenous treatment with [V4Q5]dDAVP (0.3 µg/kg) dramatically impaired metastatic progression to liver without overt signs of toxicity, and also reduced experimental lung colonization. The compound inhibited in vivo angiogenesis driven by Colo-205 cells in athymic mice, as well as in vitro endothelial cell migration and capillary tube formation. [V4Q5]dDAVP exerted AVPR2-dependent cytostatic activity in vitro (IC50 1.08 µM) and addition to 5-fluorouracil resulted in synergistic antiproliferative effects both in CT-26 and Colo-205 cells. CONCLUSION: The present preclinical study establishes for the first time the efficacy of [V4Q5]dDAVP on CRC. These encouraging. RESULTS: suggest that the novel second generation vasopressin analog could be used for the management of aggressive CRC as an adjuvant agent during surgery or to complement standard chemotherapy, limiting tumor angiogenesis and metastasis and thus protecting the patient from CRC recurrence.

In vitro and in vivo evaluation of desmopressin-loaded poly(D,L-lactic-co-glycolic acid) nanoparticles for its potential use in cancer treatment.

AIM: To develop and characterize the antitumor activity of poly(D,L-lactic-co-glycolic acid) nanoparticles loaded with hemostatic and anticancer drug desmopressin (dDAVP). MATERIALS & METHODS: After full physicochemical characterization, anticancer activity of dDAVP-loaded poly(D,L-lactic-co-glycolic acid) nanoparticles (NPdDAVP) was evaluated in vitro and in vivo on a highly aggressive breast cancer model. RESULTS: After efficiently loading desmopressin in poly(D,L-lactic-co-glycolic acid) matrix, NPdDAVP exhibited suitable physicochemical characteristics for biomedical applications. NPdDAVP displayed a potent cytostatic effect in vitro, inhibiting tumor cell proliferation and colony forming ability. Moreover, intravenous treatment using nanoparticulated-dDAVP inhibited tumor progression and prolonged survival in animals bearing rapidly-growing mammary tumors. CONCLUSION: Within the framework of promising dDAVP repurposing studies, these findings support further preclinical development of the NPdDAVP for the management of highly aggressive cancer.

Search of vasopressin analogs with antiproliferative activity on small-cell lung cancer: drug design based on two different approaches.

AIM: Development of compounds with therapeutic application requires the interaction of different disciplines. Several tumors express vasopressin (AVP; arginine vasopressin) receptors with contrasting effects depending on receptor subtype. Desmopressin (dDAVP) is an AVP-selective analog with antiproliferative properties. In this work, an evolutionary approach and a rational strategy were applied in order to design novel AVP analogs. RESULTS: We designed two novel analogs; dDInotocin (dDINT, insect analog), and [V4Q5]dDAVP, and demonstrated the importance of the dDAVP conformational loop for its antiproliferative activity. [V4Q5] dDAVP showed major cytostatic effect on lung cancer cells than dDAVP and its cytostatic effect was abolished by V2R blockade. CONCLUSION: Combination of these strategies could provide the basis for future studies for the development of improved compounds with potential therapeutic applications.