Cytotoxic effect of hydroalcoholic extract of Annona Muricata against a human cell line of gastric adenocarcinoma / Efecto citotóxico del extracto hidroalcoholico de la Annona Muricata frente a la línea celular humana de adenocarcinoma gástrico

Background: Therapeutic advances against cancer have not been as successful as expected and have adverse effects that patients rarely tolerate. A study in Peru identified favorable anticancer effects of Annona muricata (AM), a medicinal plant known as soursop, in C-678 mouse gastric adenocarcinoma. However, to date, no results have been reported in human cells. Objective: The objective of this study was to determine the cytotoxic effect of AM extract against a human gastric adenocarcinoma cell line (AGS). Methodology: Experimental in vitro analytical study using a hydroalcoholic extract of AM (AMOH) leaves collected in the Amazonas. Chemical functional groups were identified by phytochemical screening. To obtain the cytotoxic effect, different dilutions of extract were added to the plates containing the cell lines and the data were extrapolated to GraphPad employing an observation card. Finally, the cytotoxic effect was expressed as the half-maximal inhibitory concentration (IC50) and nonlinear regression analysis was performed to determine the growth inhibition of cancer cells. Results: Phytochemical screening showed the presence of reducing carbohydrates, alkaloids, phenols, tannins, triterpenes, steroids, saponins, flavonoids, proteins, cardiac glycosides, and anthocyanins. A calibration curve with gallic acid was used to determine the total phenol content and, quercetin was used to identify the flavonoid content. The AGS cell line showed cytotoxic activity with AMOH with an IC50 at 24 hours of 45.81 µg/mL and 19.05 µg/mL at 48 hours. Conclusion: Several chemical functional groups of AM were identified. In addition, the AMOH showed a cytotoxic effect against the AGS cell line

Antecedente: Los avances terapéuticos frente al cáncer no han tenido el éxito esperado y presentan efectos adversos pocas veces tolerados por el paciente. Un estudio en Perú identificó el efecto anticancerígeno de la Annona muricata (AM), planta medicinal conocida como guanábana, en adenocarcinoma gástrico de ratón C-678 con resultados favorables, sin embargo, no se ha encontrado evidencia previa en células humanas. Objetivo: El objetivo de este estudio fue determinar el efecto citotóxico del extracto de AM frente a la línea celular de adenocarcinoma gástrico humano (AGS). Materiales y métodos: Estudio experimental in vitro tipo analítico con extracto hidroalcohólico de hojas de AM (AMOH) recolectadas en Amazonas. Mediante screening fitoquímico se identificaron los grupos funcionales químicos. Para obtener el efecto citotóxico, se añadieron diferentes diluciones de extracto a las placas que contienen las líneas celulares y mediante una ficha de observación los datos fueron extrapolados a GraphPad. Finalmente se expresó como la concentración inhibitoria media máxima (IC50) y se hizo un análisis de regresión no lineal con la finalidad de encontrar la cantidad de inhibición de crecimiento de células oncológicas. Resultados: En el screening fitoquímico se pudo identificar la presencia de carbohidratos reductores, alcaloides, fenoles, taninos, tritérpenos y esteroides, saponinas, flavonoides, proteínas, glicósidos cardiotónicos y antocianinas. Para identificar el contenido total de fenoles se utilizó la curva de calibración con ácido gálico el cual nos comprobó la presencia de una buena cantidad de estos metabolitos. Adicionalmente se utilizó quercetina para identificar el contenido de flavonoides, obteniendo resultados favorables ya que se hizo evidente su presencia. La línea celular AGS mostró una actividad citotóxica frente al AMOH con un IC50 a las 24 horas de 45.81ug/mL y 19.05ug/mL a las 48 horas. Conclusión: Se identifica a los grupos funcionales de la AM. Además, AMOH demostró un efecto citotóxico contra la línea celular AGS

Screening for Active Compounds Targeting Human Natural Killer Cell Activation Identifying Daphnetin as an Enhancer for IFN-γ Production and Direct Cytotoxicity.

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ+ NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments.

Mitochondrial complex II in intestinal epithelial cells regulates T cell-mediated immunopathology.

Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases.

Treatment of Relapsing HPV Diseases by Restored Function of Natural Killer Cells.

Human papillomavirus (HPV) infections underlie a wide spectrum of both benign and malignant epithelial diseases. In this report, we describe the case of a young man who had encephalitis caused by herpes simplex virus during adolescence and currently presented with multiple recurrent skin and mucosal lesions caused by HPV. The patient was found to have a pathogenic germline mutation in the X-linked interleukin-2 receptor subunit gamma gene (IL2RG), which was somatically reverted in T cells but not in natural killer (NK) cells. Allogeneic hematopoietic-cell transplantation led to restoration of NK cytotoxicity, with normalization of the skin microbiome and persistent remission of all HPV-related diseases. NK cytotoxicity appears to play a role in containing HPV colonization and the ensuing HPV-related hyperplastic or dysplastic lesions. (Funded by the National Institutes of Health and the Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources.).

Dietary source of polyunsaturated fatty acids influences cell cytotoxicity in broiler chickens.

The current study aims to investigate the effects of dietary source of n-3 polyunsaturated fatty acids (PUFA) on immune response in broiler chickens, represented by cytotoxic cell activity. A total of 255 one-day-old male Cobb 500 broiler chickens were fed on fish oil (FO)-, flaxseed oil-enriched diets at 50 and 19 g/kg, respectively, in addition to the soybean-based control diet. At slaughter, samples of blood and spleen were harvested from 20 birds/treatment (n = 20). The immune tissues' fatty acid profile was analyzed by gas chromatography, and the cytotoxic cell activity was investigated. The results showed that supplementing broiler chickens with diets rich in n-3 PUFA had a substantial effect on the broiler immune tissues' fatty acid profile. Cytotoxic cell activity was significantly higher in splenocytes and peripheral blood mononuclear cells (PBMCs) from broilers fed flaxseed oil than those provided FO and the soybean control diet. These results suggest that flaxseed oil may be used to enrich chickens with n-3 PUFA and improve the immune status of chicken flocks to resist diseases.

Aerosol 1,25-dihydroxyvitamin D3 supplementation: A strategy to boost anti-tumor innate immune activity.

BACKGROUND: 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] plays a role in calcium homeostasis but can also exert immunomodulatory effects. In lungs, characterized by a particular immunosuppressive environment primarily due to the presence of alveolar macrophages (AM), 1,25(OH)2D3 has been shown to favor the immune response against pathogens. Here, we explored the ability of aerosolized 1,25(OH)2D3 to locally promote an anti-tumor phenotype in alveolar macrophages (AM) in the treatment of lung metastases. METHODS: Cytotoxicity assay has been used to assess the capability of AM, in vitro treated of not with 1,25(OH)2D3, to stimulate NK cells. Sulforhodamine B (SRB) assay has been used to assess the effect of 1,25(OH)2D3 on MC-38 and B16 tumor cells in vitro growth. 1,25(OH)2D3 was aerosolized in immunocompetent mouse models to evaluate the effect of local administration of 1,25(OH)2D3 on in vivo growth of MC-38 and B16 tumor cells within lungs and on infiltrating immune cells. RESULTS: In vitro incubation of naïve AM with 1,25(OH)2D3 improved their ability to stimulate NK cell cytotoxicity. In vivo aerosolized 1,25(OH)2D3 significantly reduced the metastatic growth of MC-38 colon carcinoma, a tumor histotype that frequently metastasizes to lung in human. Immune infiltrate obtained from digested lungs of 1,25(OH)2D3-treated mice bearing MC-38 metastases revealed an increased expression of MHCII and CD80 on AM and an up-modulation of CD69 expression on effector cells that paralleled a strong increased ability of these cells to kill MC-38 tumor in vitro. CONCLUSIONS: Together, these data show that aerosol delivery can represent a feasible and novel approach to supplement 1,25(OH)2D3 directly to the lungs promoting the activation of local immunity against cancer.

Importance of antibody isotypes in antitumor immunity by monocytes and complement using human-immune tumor models.

Monoclonal antibodies (mAbs) have revolutionized clinical medicine, especially in the field of cancer immunotherapy. The challenge now is to improve the response rates, as immunotherapy still fails for many patients. Strategies to enhance tumor cell death is a fundamental aim, but relevant model systems for human tumor immunology are lacking. Herein, we have developed a preclinical human immune - three-dimensional (3D) tumor model (spheroids) to map the efficiency of tumor-specific isotypes for improved tumor cell killing. Different anti-CD20 Rituximab (RTX) isotypes alone or in combination, were evaluated for mediating complement-dependent cytotoxicity and antibody-dependent phagocytosis by human monocytic cells in 3D spheroids, in parallel with monolayer cultures, of human CD20+ B-cell lymphomas. We demonstrate that the IgG3 variant of RTX has the greatest tumoricidal effect over other isotypes, and when combined with apoptosis-inducing RTX-IgG2 isotype the therapeutic effect can be substantially enhanced. The results show further that the treatment outcome by RTX isotypes is influenced by tumor morphology and expression of the complement inhibitor CD59. Hence, the human immune-3D tumor model is a clinical relevant and attractive ex vivo system to predict mAbs for best efficacy in cancer immunotherapy.

Grain-sized moxibustion promotes NK cell antitumour immunity by inhibiting adrenergic signalling in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related death worldwide, and non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer diagnoses. As an ancient therapy, moxibustion has been used to treat cancer-related symptoms in clinical practice. However, its antitumour effect on NSCLC remains largely unexplored. In the present study, a Lewis lung cancer (LLC) xenograft tumour model was established, and grain-sized moxibustion (gMoxi) was performed at the acupoint of Zusanli (ST36). Flow cytometry and RNA sequencing (RNA-Seq) were used to access the immune cell phenotype, cytotoxicity and gene expression. PK136, propranolol and epinephrine were used for natural killer (NK) cell depletion, ß-adrenoceptor blockade and activation, respectively. Results showed that gMoxi significantly inhibited LLC tumour growth. Moreover, gMoxi significantly increased the proportion, infiltration and activation of NK cells, whereas it did not affect CD4+ and CD8+ T cells. NK cell depletion reversed gMoxi-mediated tumour regression. LLC tumour RNA-Seq indicated that these effects might be related to the inhibition of adrenergic signalling. Surely, ß-blocker propranolol clearly inhibited LLC tumour growth and promoted NK cells, and gMoxi no longer increased tumour regression and promoted NK cells after propranolol treatment. Epinephrine could inhibit NK cell activity, and gMoxi significantly inhibited tumour growth and promoted NK cells after epinephrine treatment. These results demonstrated that gMoxi could promote NK cell antitumour immunity by inhibiting adrenergic signalling, suggesting that gMoxi could be used as a promising therapeutic regimen for the treatment of NSCLC, and it had a great potential in NK cell-based cancer immunotherapy.

Functionally Relevant Differences in Plasma Fatty Acid Composition and Expression of Cytotoxic and Inhibitory NK Cell Receptors between Healthy Young and Healthy Elder Adults.

(1) Background: In the healthy ageing, NK cell number is not modified; however, their spontaneous cytotoxicity decreases. We postulated that the age-dependent decline in metabolic activities might be responsible for this effect. (2) Methods: The fatty acid profile of 30 healthy young males (23 ± 4 years old, BMI 22.1 ± 1.3) and 30 older males (63 ± 5 years old, BMI 22.9 ± 2.5) donors were evaluated along with the expression of killing (KR) and inhibitory NK receptors (KIR) at basal level and after cultivation with fatty acids for 24 h. (3) Results: Significantly higher levels of oleic (p < 0.01), arachidonic (p < 0.001), lignoceric (p < 0.001), and nervonic acids (p < 0.0001) and significantly lower levels of docosapentaenoic and docosahexaenoic acids (p < 0.01) were found in elders as compared to young adults. At basal levels, significant (p < 0.005) differences in KR and KIR expression were encountered; 12/16 antigens. Treatment of cells with saturated fatty acids or arachidonic acid (AA) significantly enhanced KR expressions (p < 0.001). AA treatment decreased inhibitory KIR expression while docosahexaenoic, and eicosapentaenoic acid increased them. (4) Conclusions: Changes in fatty acids blood levels, and KR and KIR expression in NK cell, are age-dependent. Supplementation of NK cells with eicosapentaenoic or docosahexaenoic acid enhanced inhibitory KIR receptors' expression which may improve their cell function.

Immunogenicity and Toxicity of Different Adjuvants Can Be Characterized by Profiling Lung Biomarker Genes After Nasal Immunization.

The efficacy of vaccine adjuvants depends on their ability to appropriately enhance the immunogenicity of vaccine antigens, which is often insufficient in non-adjuvanted vaccines. Genomic analyses of immune responses elicited by vaccine adjuvants provide information that is critical for the rational design of adjuvant vaccination strategies. In this study, biomarker genes from the genomic analyses of lungs after priming were used to predict the efficacy and toxicity of vaccine adjuvants. Based on the results, it was verified whether the efficacy and toxicity of the tested adjuvants could be predicted based on the biomarker gene profiles after priming. Various commercially available adjuvants were assessed by combining them with the split influenza vaccine and were subsequently administered in mice through nasal inoculation. The expression levels of lung biomarker genes within 24 h after priming were analyzed. Furthermore, we analyzed the antibody titer, cytotoxic T lymphocyte (CTL) induction, IgG1/IgG2a ratio, leukopenic toxicity, and cytotoxicity in mice vaccinated at similar doses. The association between the phenotypes and the changes in the expression levels of biomarker genes were analyzed. The ability of the adjuvants to induce the production of antigen-specific IgA could be assessed based on the levels of Timp1 expression. Furthermore, the expression of this gene partially correlated with the levels of other damage-associated molecular patterns in bronchoalveolar lavage fluid. Additionally, the changes in the expression of proteasome- and transporter-related genes involved in major histocompatibility complex class 1 antigen presentation could be monitored to effectively assess the expansion of CTL by adjuvants. The monitoring of certain genes is necessary for the assessment of leukopenic toxicity and cytotoxicity of the tested adjuvant. These results indicate that the efficacy and toxicity of various adjuvants can be characterized by profiling lung biomarker genes after the first instance of immunization. This approach could make a significant contribution to the development of optimal selection and exploratory screening strategies for novel adjuvants.

TILs and Anti-PD1 Therapy: An Alternative Combination Therapy for PDL1 Negative Metastatic Cervical Cancer.

BACKGROUND: We investigated the efficacy of TILs and anti-PD1 combination therapy in patients with metastatic cervical cancer with low MSI expression and PDL1-negative. METHODS: A total of 80 patients were put on TILs and anti-PD1 combination therapy, and the progression-free survival time (PFS) and overall survival time (OS) were assessed by Kaplan-Meier analysis. Univariate and multivariate analyses were performed to identify factors that could predict the prognosis of metastatic cervical cancer in the previously described patients. RESULTS: The objective response rate was 25%, whereas the mPFS and mOS were 6.1 and 11.3 months, respectively. The therapeutic efficacy was influenced by the characteristics of TILs, infection with HPV, and development of fever just after the therapy. CONCLUSION: Overall, our results show that the combination therapy of TILs and anti-PD1 significantly improves the prognosis of metastatic cervical cancer.

Integrative Transcriptomic and Small RNA Sequencing Reveals Immune-Related miRNA-mRNA Regulation Network for Soybean Meal-Induced Enteritis in Hybrid Grouper, Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂.

A 10-week feeding experiment was conducted to reveal the immune mechanism for soybean meal-induced enteritis (SBMIE) in hybrid grouper, Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂. Four isonitrogenous and isolipidic diets were formulated by replacing 0, 10, 30, and 50% fish meal protein with soybean meal (namely FM, SBM10, SBM30, and SBM50, respectively). The weight gain rate of the SBM50 group was significantly lower than those of the other groups. Plica height, muscular layer thickness, and goblet cells of the distal intestine in the SBM50 group were much lower than those in the FM group. The intestinal transcriptomic data, including the transcriptome and miRNAome, showed that a total of 6,390 differentially expressed genes (DEGs) and 92 DEmiRNAs were identified in the SBM50 and FM groups. DEmiRNAs (10 known and 1 novel miRNAs) and their DE target genes were involved in immune-related phagosome, natural killer cell-mediated cytotoxicity, Fc gamma R-mediated phagocytosis, and the intestinal immune network for IgA production pathways. Our study is the first to offer transcriptomic and small RNA profiling for SBMIE in hybrid grouper. Our findings offer important insights for the understanding of the RNA profile and further elucidation of the underlying molecular immune mechanism for SBMIE in carnivorous fish.

Assessment of In Vitro Anti-melanoma Potential of Ephedranthus pisocarpus R.E.Fr.

BACKGROUND/AIM: Despite being a rare disease, melanoma is considered the most dangerous skin cancer due to its highly invasive and aggressive nature, and still requires for more effective treatments. The aim of this study was to evaluate the in vitro anti-melanoma potential of Ephedranthus pisocarpus R.E.Fr. (Annonaceae), a popular Brazilian plant with medicinal properties. MATERIALS AND METHODS: Initially, the ethanolic extract (EtOH) was obtained from E. pisocarpus leaves and later partitioned using increasing polarity solvents. The anti-melanoma potential of E. pisocarpus was assessed by spectrophotometry and its cytotoxicity determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and confocal microscopy. RESULTS: We demonstrated that the EtOH extract and fractions from E. pisocarpus had a moderate photoprotective action (FPS 3.0-5.0) against UVA radiation. Interestingly, the dichloromethane fraction presented higher anti-melanoma activity against B16-F10 (IC50=46.8 µg/ml) and SK-MEL-28 cells (IC50=40.1 µg/ml) and lesser toxicity on normal cells. Additionally, our study reported that spathulenol, one of the major constituents from E. pisocarpus, acts through an apoptosis-dependent mechanism in SK-MEL-28 cells. CONCLUSION: The present study demonstrated, for the first time, the in vitro anti-melanoma potential of E. pisocarpus against melanoma cells.

An Update on XMEN Disease.

"X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia" (XMEN) disease is an inborn error of glycosylation and immunity caused by loss of function mutations in the magnesium transporter 1 (MAGT1) gene. It is a multisystem disease that strongly affects certain immune cells. MAGT1 is now confirmed as a non-catalytic subunit of the oligosaccharyltransferase complex and facilitates Asparagine (N)-linked glycosylation of specific substrates, making XMEN a congenital disorder of glycosylation manifesting as a combined immune deficiency. The clinical disease has variable expressivity, and impaired glycosylation of key MAGT1-dependent glycoproteins in addition to Mg2+ abnormalities can explain some of the immune manifestations. NKG2D, an activating receptor critical for cytotoxic function against EBV, is poorly glycosylated and invariably decreased on CD8+ T cells and natural killer (NK) cells from XMEN patients. It is the best biomarker of the disease. The characterization of EBV-naïve XMEN patients has clarified features of the genetic disease that were previously attributed to EBV infection. Extra-immune manifestations, including hepatic and neurological abnormalities, have recently been reported. EBV-associated lymphomas remain the main cause of severe morbidity. Unfortunately, treatment options to address the underlying mechanism of disease remain limited and Mg2+ supplementation has not proven successful. Here, we review the expanding clinical phenotype and recent advances in glycobiology that have increased our understanding of XMEN disease. We also propose updating XMEN to "X-linked MAGT1 deficiency with increased susceptibility to EBV-infection and N-linked glycosylation defect" in light of these novel findings.

Andiroba oil (Carapa guianensis Aubl) shows cytotoxicity but no mutagenicity in the ACPP02 gastric cancer cell line.

Andiroba (Carapa guianensis Aubl) is an Amazonian plant whose oil has been widely used in traditional medicine for various purposes, including anti-inflammation. Research reports indicate that the oil can confer antitumor activity due to the presence of fatty acids, which can directly influence cell death mechanisms. Thus, andiroba oil (AO) has gained interest for its potential to be used in antineoplastic therapies. Here, we report an in vitro analysis of the cytotoxic and mutagenic potential of AO in the gastric cancer cell line, ACP02. Cell survival was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, differential staining with ethidium bromide and acridine orange assessed apoptosis-necrosis, and mutagenesis was assessed by the micronucleus test. The apolar oil was first diluted in 0.1% dimethyl sulfoxide (DMSO) and then further diluted to six concentrations (0.01, 0.1, 1, 10 and 100 µg/mL and 1 mg/mL) in RPMI medium. Controls included RPMI alone (negative control) and 0.1% DMSO diluted in medium (vehicle control). The MTT test showed that AO significantly reduced cell viability (P < .05) only when the highest tested concentration was applied for 48 hours. The apoptosis/necrosis test showed that the highest concentration of AO induced cell death by apoptosis at 24 and 48 hours. There was no statistically significant increase in the frequency of micronuclei. The ability of the AO to decrease the viability of ACP02 cells via apoptosis, without exerting mutagenic effects, suggests that the oil could be useful as an alternative therapeutic agent for primary tumors of stomach cancer.

Developing a 3D B Cell Lymphoma Culture System to Model Antibody Therapy.

Diffuse large cell B cell lymphoma (DLBCL) accounts for approximately 30%-40% of all non-Hodgkin lymphoma (NHL) cases. Current first line DLBCL treatment results in long-term remission in more than 60% of cases. However, those patients with primary refractory disease or early relapse exhibit poor prognosis, highlighting a requirement for alternative therapies. Our aim was to develop a novel model of DLBCL that facilitates in vitro testing of current and novel therapies by replicating key components of the tumor microenvironment (TME) in a three-dimensional (3D) culture system that would enable primary DLBCL cell survival and study ex vivo. The TME is a complex ecosystem, comprising malignant and non-malignant cells, including cancer-associated fibroblasts (CAF) and tumor-associated macrophages (TAM) whose reciprocal crosstalk drives tumor initiation and growth while fostering an immunosuppressive milieu enabling its persistence. The requirement to recapitulate, at least to some degree, this complex, interactive network is exemplified by the rapid cell death of primary DLBCL cells removed from their TME and cultured alone in vitro. Building on previously described methodologies to generate lymphoid-like fibroblasts from adipocyte derived stem cells (ADSC), we confirmed lymphocytes, specifically B cells, interacted with this ADSC-derived stroma, in the presence or absence of monocyte-derived macrophages (MDM), in both two-dimensional (2D) cultures and a 3D collagen-based spheroid system. Furthermore, we demonstrated that DLBCL cells cultured in this system interact with its constituent components, resulting in their improved viability as compared to ex-vivo 2D monocultures. We then assessed the utility of this system as a platform to study therapeutics in the context of antibody-directed phagocytosis, using rituximab as a model immunotherapeutic antibody. Overall, we describe a novel 3D spheroid co-culture system comprising key components of the DLBCL TME with the potential to serve as a testbed for novel therapeutics, targeting key cellular constituents of the TME, such as CAF and/or TAM.

Os efeitos da Punica granatum L. em diferentes concentrações sobre duas linhagens celulares: estudo in vitro / The effects of Punica granatum L. at different concentrations on two cell lines: in vitro study

Introdução: A Punica granatum L. (PG), utilizada como medicamento fitoterápico, apresenta propriedades terapêuticas, anti-inflamatórias e antioxidante. Embora diversos estudos já tenham sido realizados com este fitoterápico, seus possíveis efeitos citotóxicos nos tecidos humanos ainda não são claros. Objetivo: Avaliar a citotoxicidade da PG por meio de cultura celular com duas linhagens: fibroblastos humanos de mucosa oral (FLM) e células de carcinoma epidermoide oral humano (KB). Material e método: As células foram submetidas ao teste de viabilidade celular por 24 horas nas concentrações da PG 1%, 0,50%, 0,25%, 0,125%, 0,062% e 0,031%, e aos testes de citotoxicidade celular em 4 horas, 1, 3, 5 e 7 dias, em diferentes concentrações, realizados em triplicata. Foi utilizado um controle negativo (Triton 1%) e um controle positivo sem o extrato de PG. Os dados obtidos foram submetidos à ANOVA (p < 0,05). Resultado: Foi possível observar que o extrato da PG possui efeitos inibitórios, apresentando-se maior nas células KB em relação às FLM. Os testes de citotoxicidade e viabilidade mostraram inibição e morte das células KB e FLM nas concentrações 1%, 0,50% e 0,25%. Conclusão: O efeito inibitório da PG foi dose-dependentes, mostrando-se maior nas células KB em relação às FLM.

Introduction: Punica granatum L. (PG), used as a herbal medicine, has therapeutic, anti-inflammatory and antioxidant properties, although several studies have already been carried out with this herbal medicine, its possible cytotoxic effects on human tissues are still unclear. Objective: To evaluate the cytotoxicity of PG through cell culture with two strains: human oral mucosa fibroblasts (LFM) and human oral squamous cell carcinoma (KB) cells. Material and method: The cells were submitted to the cell viability test for 24 hours in the concentrations of PG 1%, 0.50%, 0.25%, 0.125%, 0.062% and 0.031% and the cell cytotoxicity tests in 4 hours, 1, 3, 5 and 7 days in different concentrations, performed in triplicate. A negative control (Triton 1%) and a positive control without the PG extract were used. The data obtained were submitted to ANOVA (p <0.05). Result: it was possible to observe that the PG extract has inhibitory effects, being higher in KB cells in relation to LFM. The cytotoxicity and viability tests showed inhibition and death of KB and FLM cells at concentrations of 1%, 0.50% and 0.25%. Conclusion: The inhibitory effect of PG was dose dependent, showing itself to be greater in KB cells compared to LMB.

Suppression of cardiomyocyte functions by ß-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method

Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. Methods: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. Results: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. Conclusion: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.(AU)

Phlogiellus bundokalbo spider venom: cytotoxic fractions against human lung adenocarcinoma (A549) cells

Spider venom is a potential source of pharmacologically important compounds. Previous studies on spider venoms reported the presence of bioactive molecules that possess cell-modulating activities. Despite these claims, sparse scientific evidence is available on the cytotoxic mechanisms in relation to the components of the spider venom. In this study, we aimed to determine the cytotoxic fractions of the spider venom extracted from Phlogiellus bundokalbo and to ascertain the possible mechanism of toxicity towards human lung adenocarcinoma (A549) cells. Methods: Spider venom was extracted by electrostimulation. Components of the extracted venom were separated by reversed-phase high performance liquid chromatography (RP-HPLC) using a linear gradient of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 95% acetonitrile (ACN). Cytotoxic activity was evaluated by the MTT assay. Apoptotic or necrotic cell death was assessed by microscopic evaluation in the presence of Hoechst 33342 and Annexin V, Alexa FluorTM 488 conjugate fluorescent stains, and caspase activation assay. Phospholipase A2 (PLA2) activity of the cytotoxic fractions were also measured. Results: We observed and isolated six fractions from the venom of P. bundokalbo collected from Aurora, Zamboanga del Sur. Four of these fractions displayed cytotoxic activities. Fractions AT5-1, AT5-3, and AT5-4 were found to be apoptotic while AT5-6, the least polar among the cytotoxic components, was observed to induce necrosis. PLA2 activity also showed cytotoxicity in all fractions but presented no relationship between specific activity of PLA2 and cytotoxicity. Conclusion: The venom of P. bundokalbo spider, an endemic tarantula species in the Philippines, contains components that were able to induce either apoptosis or necrosis in A549 cells.(AU)