Melanoma-Derived Extracellular Vesicles Induce CD36-Mediated Pre-Metastatic Niche.

CD36 expression in both immune and non-immune cells is known to be directly involved in cancer metastasis. Extracellular vesicles (EVs) secreted by malignant melanocytes play a vital role in developing tumor-promoting microenvironments, but it is unclear whether this is mediated through CD36. To understand the role of CD36 in melanoma, we first analyzed the SKCM dataset for clinical prognosis, evaluated the percentage of CD36 in lymphatic fluid-derived EVs (LEVs), and tested whether melanoma-derived EVs increase CD36 expression and induce M2-macrophage-like characteristics. Furthermore, we performed a multiplex immunofluorescence (MxIF) imaging analysis to evaluate the CD36 expression and its colocalization with various other cells in the lymph node (LN) of patients and control subjects. Our findings show that cutaneous melanoma patients have a worse clinical prognosis with high CD36 levels, and a higher percentage of CD36 in total LEVs were found at baseline in melanoma patients compared to control. We also found that monocytic and endothelial cells treated with melanoma EVs expressed more CD36 than untreated cells. Furthermore, melanoma-derived EVs can regulate immunosuppressive macrophage-like characteristics by upregulating CD36. The spatial imaging data show that cells in tumor-involved sentinel LNs exhibit a higher probability of CD36 expression than cells from control LNs, but this was not statistically significant. Conclusively, our findings demonstrated that CD36 plays a vital role in controlling the immunosuppressive microenvironment in the LN, which can promote the formation of a protumorigenic niche.

Prevalence of Single-Sided Deafness in a Tertiary Healthcare Hospital in Mumbai.

PURPOSE: The current study was taken up to estimate the prevalence of Single Sided Deafness (SSD) in a tertiary healthcare hospital in Mumbai. Also to determine the acceptance of Contralateral routing of offside signal (CROS) devices by individuals with SSD. METHOD: A retrospective study design was followed to collect data from September 2020 to September 2022. The data were collected by reviewing the files of the cases diagnosed with Single-Sided Deafness in a tertiary health care hospital in Mumbai. RESULT: The prevalence of SSD was found to be 24% for the given period. It was observed that 50 out of 4456 took a free trial and 2 bought the device. CONCLUSION: The poor acceptance and purchase of CROS are attributed to the cost, and missed benefits of true binaural hearing in localization and hearing in noise. These benefits can be achieved by using CI, however, the cost and fear of surgery refrained the participants. Also, participants were observed to learn great use of communication repair strategies. Therefore, it is required to counsel individuals with SSD for CI and provide knowledge about care for the better with regular audiological follow-up.

Melanoma-derived mediators can foster the premetastatic niche: crossroad to lymphatic metastasis.

The natural history of advanced malignant melanoma demonstrates that, in most cases, widespread tumor dissemination is preceded by regional metastases involving tumor-draining lymph nodes [sentinel lymph nodes (SLNs)]. Under physiological conditions, LNs play a central role in immunosurveillance to non-self-antigens to which they are exposed via afferent lymph. The dysfunctional immunity in SLNs is mediated by tumor secretory factors that allow the survival of metastatic melanoma cells within the LN by creating a premetastatic niche (PMN). Recent studies outline the altered microenvironment of LNs shaped by melanoma mediators. Here, we discuss tumor secretory factors involved in subverting tumor immunity and remodeling LNs and highlight emerging therapeutic strategies to reinvigorate antitumoral immunity in SLNs.

Improving combination therapies: targeting A2B-adenosine receptor to modulate metabolic tumor microenvironment and immunosuppression.

BACKGROUND: We investigated the role of A2B-adenosine receptor in regulating immunosuppressive metabolic stress in the tumor microenvironment. Novel A2B-adenosine receptor antagonist PBF-1129 was tested for antitumor activity in mice and evaluated for safety and immunologic efficacy in a phase I clinical trial of patients with non-small cell lung cancer. METHODS: The antitumor efficacy of A2B-adenosine receptor antagonists and their impact on the metabolic and immune tumor microenvironment were evaluated in lung, melanoma, colon, breast, and epidermal growth factor receptor-inducible transgenic cancer models. Employing electron paramagnetic resonance, we assessed changes in tumor microenvironment metabolic parameters, including pO2, pH, and inorganic phosphate, during tumor growth and evaluated the immunologic effects of PBF-1129, including its pharmacokinetics, safety, and toxicity, in patients with non-small cell lung cancer. RESULTS: Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial inorganic phosphate emerged as a correlative and cumulative measure of tumor microenvironment stress and immunosuppression. A2B-adenosine receptor inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ectonucleotidases, increased expression of adenosine deaminase, decreased tumor growth and metastasis, increased interferon γ production, and enhanced the efficacy of antitumor therapies following combination regimens in animal models (anti-programmed cell death 1 protein vs anti-programmed cell death 1 protein plus PBF-1129 treatment hazard ratio = 11.74 [95% confidence interval = 3.35 to 41.13], n = 10, P < .001, 2-sided F test). In patients with non-small cell lung cancer, PBF-1129 was well tolerated, with no dose-limiting toxicities; demonstrated pharmacologic efficacy; modulated the adenosine generation system; and improved antitumor immunity. CONCLUSIONS: Data identify A2B-adenosine receptor as a valuable therapeutic target to modify metabolic and immune tumor microenvironment to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies.

Metabolic reprogramming by adenosine antagonism and implications in non-small cell lung cancer therapy.

Non-small cell lung cancer (NSCLC) is a heterogeneous disease with genetic and environmental parameters that influence cell metabolism. Because of the complex interplay of environmental factors within the tumor microenvironment (TME) and the profound impact of these factors on the metabolic activities of tumor and immune cells, there is an emerging interest to advance the understanding of these diverse metabolic phenotypes in the TME. High levels of adenosine are characteristic of the TME, and adenosine can have a significant impact on both tumor cell growth and the immune response. Consistent with this, we showed in NSCLC data from TCGA that high expression of the A2BR leads to worse outcome and that expression of A2BR may be different for different mutation backgrounds. We then investigated the metabolic reprogramming of tumor cells and immune cells (T and dendritic cells) by adenosine. We used A2AR and A2BR antagonism or agonism as well as receptor knockout animals to explore whether these treatments altered specific immune compartments or conferred specific therapeutic vulnerabilities. Using the seahorse assay, we found that an A2BR antagonist modulates oxidative stress homeostasis in NSCLC cell lines. In addition, we found distinct metabolic roles of A2AR and A2BR receptors in T cell activation and dendritic cell maturation. These data suggest potential mechanisms and therapeutic benefits of A2 receptor antagonist therapy in NSCLC.

Phospho-proteomic analysis of primary human colon epithelial cells during the early Trypanosoma cruzi infection phase.

The protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, causes severe morbidity and mortality in afflicted individuals. About 30% of T. cruzi-infected individuals present with cardiac, gastrointestinal tract, and/or neurological disorders. Megacolon, one of the major pathologies of Chagas disease, is accompanied by gastrointestinal motility disorders. The molecular mechanism of T. cruzi-mediated megacolon in Chagas disease is currently unknown. To decipher the molecular mechanism of T. cruzi-induced alteration in the colon during the early infection phase, we exposed primary human colonic epithelial cells (HCoEpiC) to invasive T. cruzi trypomastigotes at multiple time points to determine changes in the phosphoprotein networks in the cells following infection using proteome profiler Human phospho-kinase arrays. We found significant changes in the phosphorylation pattern that can mediate cellular deregulations in colonic epithelial cells after infection. We detected a significant increase in the levels of phosphorylated heat shock protein (p-HSP) 27 and transcription factors that regulate various cellular functions, including c-Jun and CREB. Our study confirmed significant upregulation of phospho (p-) Akt S473, p-JNK, which may directly or indirectly modulate CREB and c-Jun phosphorylation, respectively. We also observed increased levels of phosphorylated CREB and c-Jun in the nucleus. Furthermore, we found that p-c-Jun and p-CREB co-localized in the nucleus at 180 minutes post infection, with a maximum Pearson correlation coefficient of 0.76±0.02. Increased p-c-Jun and p-CREB have been linked to inflammatory and profibrotic responses. T. cruzi infection of HCoEpiC induces an increased expression of thrombospondin-1 (TSP-1), which is fibrogenic at elevated levels. We also found that T. cruzi infection modulates the expression of NF-kB and JAK2-STAT1 signaling molecules which can increase pro-inflammatory flux. Bioinformatics analysis of the phosphoprotein networks derived using the phospho-protein data serves as a blueprint for T. cruzi-mediated cellular transformation of primary human colonic cells during the early phase of T. cruzi infection.

Metabolic fingerprinting in breast cancer stages through 1H NMR spectroscopy-based metabolomic analysis of plasma.

Breast cancer (BC) is one of the most common malignancies among women worldwide, which is indeed associated with metabolic reprogramming. However, BC is a very complex and heterogeneous disease, which can relate with the changes in metabolic profiles during BC progression. Hence, investigating the metabolic alterations during BC stage progression may reveal the deregulated pathways and useful metabolic signatures of BC. To demonstrate the metabolic insights, we opted 1H NMR spectroscopy based metabolomics of blood plasma of early and late stage BC (N = 72) with age and gender matched healthy subjects (N = 50). Further, the metabolic profiles were analyzed to delineate the potential signatures of BC by performing multivariate and nonparametric statistical analysis in early and late stages of BC in comparison with healthy subjects. Sixteen metabolites levels were differentially changed (p < 0.05) in the early and late stages of BC from healthy subjects. Among them, the levels of hydroxybutyrate, lysine, glutamate, glucose, N-acetyl glycoprotein, Lactate were highly distinguished in BC stages and showed a good biomarker potential using receiver-operating curves based diagnostic models. Furthermore, the significant modulation and good diagnostic performances of glutamate, N-acetyl glycoprotein and Lactate in LBC as compared to EBC give their significance in the BC progression. In general, our observations demonstrate that these panels of metabolites may act as vital component of the metabolism of early to late stage BC progression. Our results also open new avenue towards early and late stage BC diagnosis and intervention implying metabolomics approaches.

Evaluation of growth inhibitory response of Resveratrol and Salinomycin combinations against triple negative breast cancer cells.

Resveratrol (RSVL) a dietary phytochemical showed to enhance the efficacy of chemotherapeutic drugs. Recently, Salinomycin (SAL) has gained importance as cancer therapeutic value for breast cancer (BC), however, its superfluxious toxicity delimits the utility. Taking the advantage of RSVL, the therapeutic efficacy of RSVL and SAL combination was studied in vitro and in vivo system. Firstly, the synergistic combination dose of RSVL and SAL was calculated and further, the efficacy was examined by wound healing, and Western blots analysis. Further, in vivo study was performed to confirm the effect of colony formation and apoptosis detection by flow cytometry based assays. Further, the molecular mode of action was determined at both transcript and translational level by quantitative Real Time PCR combination in Ehrlich ascitic carcinoma model.The combination of IC20 (R20) of RSVL and IC10 (S10) dose of SAL showed best synergism (CI<1) with ∼5 fold dose advantage of SAL. Gene expression results at mRNA and protein level revealed that the unique combination of RSVL and SAL significantly inhibited epithelial mesenchymal transition (Fibronectin, Vimentin, N-Cadherin, and Slug); chronic inflammation (Cox2, NF-kB, p53), autophagy (Beclin and LC3) and apoptotic (Bax, Bcl-2) markers. Further, i n vivo study showed that low dose of SAL in combination with RSVL increased life span of Ehrlich ascitic mice. Overall, our study revealed that RSVL synergistically potentiated the anticancer potential of SAL against triple negative BC.

Toxicoproteomics in human health and disease: an update.

INTRODUCTION: Toxicoproteomics is an emerging area of omics, intended to explore the changes in protein expression and modifications in biological samples exposed to toxicants. The development of techniques that utilize sophisticated instruments in proteomics has facilitated the exploration of a wide-range of protein coverage and assisted the quantitative and qualitative evaluation of protein changes as a result of the toxic effects of toxic substances. Areas covered: Studies on toxicoproteomics have an immense potential to explore the molecular mechanism of action of a variety of toxic substances through deciphering the proteomic map altered as a result of toxicant exposure. Here, we provide an overview of toxicoproteomic approaches and the current paradigm of toxicoproteomics. Expert commentary: Research in this area continues to increase our understanding of the role of toxicants in worsening human health and toxicity driven diseases. The progress in toxicoproteomics may realize the development of novel biomarkers, drug targets and personalized medicines by incorporating the advanced proteomic applications in this field.

Diallyl Sulfide and Its Role in Chronic Diseases Prevention.

Diallyl sulfide (C6H10S, DAS) is one of the novel natural organosulfur compounds, which is mostly obtained from the genus Allium plants. Numerous studies have revealed several unique properties of DAS in terms of its health-promoting effects. DAS has proved to be anticancer, antimicrobial, anti-angiogenic, and immunomodulatory like unique functions as demonstrated by the multiple investigations. Diallyl sulfide can also impede oxidative stress and chronic inflammation as suggested by the literature. Studies also explored that DAS could thwart the development of chronic diseases like cancer, neuronal, cardiovascular disease through modulating mechanistic pathways involved in pathogenesis. In this book chapter, we have attempted to give the comprehensive view on DAS about the physiochemical and biological properties, and its preventive role in chronic diseases with a mechanistic overview.

Quantitative proteomics revealed novel proteins associated with molecular subtypes of breast cancer.

UNLABELLED: The early diagnosis and successful treatment of breast cancer (BC) is still a challenging task due to the diverse origin and functional heterogeneity of cancer cells. The heterogeneity of BC may likely to explained by molecular BC subtypes, comprises Luminal-A (LA), Luminal-B (LB), Triple-negative (TN) and HER2-positive (HP), which are governed by a variety of cancer associated pathways. To identify protein signatures in different BC subtypes, we performed isobaric tag for absolute and relative quantitation (iTRAQ) of enriched blood plasma samples of BC subtypes (N=32) and healthy subjects (N=8). After analyses of data, 58 proteins were found to be modulated in BC subtypes from healthy subjects (p<0.05) and among these; Fibronectin (FN1), Alpha-2-macroglobulin (A2M), and Complement component-4-binding protein-alpha (C4BPA) and Complement factor-B (CFB) were selected for validation in BC subtypes and healthy subjects in the independent set of blood plasma (N=100) and tissue samples (N=25). Statistical analysis showed the significant modulation of FN1 and C4BPA in LB, and A2M in TN patients in both plasma as well as tissues comparatively control (p<0.05). Further, FN1 and C4BPA in LB subtype revealed a good diagnostic accuracy in plasma level validation. The receiver operating characteristic (ROC) curve and regression analysis demonstrated that these proteins with associated criterion of expression could act as discriminating signatures among BC subtypes with diagnostic and prognostic relevance. SIGNIFICANCE: The heterogeneity of breast cancer (BC) has gained many challenges for successful management of BC, thus, the delineating proteomic alterations BC subtypes may provide great clinical values in diagnostic, prognostic and therapeutics of BC. The findings from the present quantitative proteomic study have deciphered the altered proteomic patterns and their possible molecular interactions in each BC subtype. The study showed a strong association of FN1, A2M, C4BPA and CFB in molecular subtypes of BC, in which, C4BPA and A2M demonstrated a potent signature in blood plasma and tissue samples of LB and TN subtypes in BC patients, respectively. The findings also revealed the altered level expressions of these selected proteins could classify BC subtypes through plasma and tissue based expression analysis in patients and control samples. Hence, these proteins could have clinical importance for the diagnosis and prognosis purposes among molecular BC subtypes.

Autoantibodies against TYMS and PDLIM1 proteins detected as circulatory signatures in Indian breast cancer patients.

PURPOSE: Breast cancer (BC) is the most common invasive cancer in women worldwide. Autoantibodies (AAbs) to tumor-associated antigens (TAAs) have a great potential for the development of diagnostic biomarkers in cancer. This study was performed to identify AAbs and cognate TAAs that may improve detection of this deadly disease. EXPERIMENTAL DESIGN: Serological proteome analysis of plasma samples of BC patients (N = 30) and healthy controls (N = 30) was performed to identify TAAs. Expressions of selected TAAs were also determined in breast tumor tissues (N = 10) by immunohistochemistry. An independent validation cohort (N = 124) was tested to determine diagnostic accuracy of selected AAbs titer by ELISA. RESULTS: Thymidylate synthase (TYMS) and C-terminal LIM domain protein 1 (PDLIM1) were found to react more specifically with plasma samples of BC patients. Both TAAs were also found to be significantly over expressed (p < 0.001) in breast tumor tissues compared to adjacent normal tissues. TYMS AAbs response was positively correlated (r = 0.778, p < 0.008) with TYMS overexpression in BC tissues. TYMS and PDLIM1 AAbs titers discriminated BC from controls with a sensitivity/specificity of 57.81%/95% and 73.44%/58.33%, respectively. CONCLUSION AND CLINICAL RELEVANCE: High titers of both TYMS and PDLIM1 AAbs were significantly more prevalent in BC cases than controls. Our data recommends further investigations for evaluating their potential for BC detection.

Resveratrol improves the anticancer effects of doxorubicin in vitro and in vivo models: A mechanistic insight.

BACKGROUND: Resveratrol (RSVL), a well known dietary compound and in combination with doxorubicin (DOX) has gained a global importance for cancer prevention. However, mechanism of action by this combination is not well understood till date. HYPOTHESIS: The synergistic combination of RSVL and DOX might be more effective in anti-cancer activity by modulating the diverse cancer signaling pathways as compared to their alone treatments. METHODS: The cytotoxicity of alone and combination doses of RSVL and DOX were analyzed by colorimetric MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) cell proliferation assay. The migration and colony forming abilities were evaluated by wound healing and clonogenic assays. Apoptosis was detected by Annexin V/PI and DAPI stainings. The cell cycle and intracellular reactive oxygen species (ROS) generation were measured by flow cytometry. The differential expression of genes and proteins were measured by qRT-PCR and western blotting analyses. Finally, in-vivo studies were performed in Ehrlich ascitic carcinoma (EAC) mouse model. RESULTS: The synergistic combination of DOX (IC20) and RSVL (IC30) was selected based on the combination index values in MCF-7 and MDA-MB-231 cell lines. This combination showed potent growth inhibition with ∼2.5 fold of dose advantage and also significantly decreased the wound healing and clonogenic potential of breast cancer cells. The combination treatment was also found to inhibit the inflammatory response (NF-kB, COX-2), autophagic flux (LC3, Beclin-1), redox regulation (Nrf2) and induces apoptosis (BAX: BCL-2 ratio and Caspase-9) in breast cancer cells. Further, combined dosages of DOX (5 mg/kg b.wt) and RSVL (10 mg/kg b.wt) inhibited tumor volume with increased life span (139%, p value<0.05) in Ehrlich ascitic carcinoma (EAC) cells bearing mice. CONCLUSION: In brief, our results suggested that resveratrol chemosensitizes doxorubicin in combination, through inhibiting breast cancer cells proliferation and invasion, and inducing apoptosis via suppression of chronic inflammation and autophagy.

Current perspectives of molecular pathways involved in chronic inflammation-mediated breast cancer.

Inflammation has multifaceted role in cancer progression including initiation, promotion and invasion by affecting the immune surveillance and associated signaling pathways. Inflammation facilitates the over-expression of cytokines, chemokines and growth factors involved in progression of different cancers including breast cancer progression. Deregulation of biological processes such as oxidative stress, angiogenesis, and autophagy elicit favorable immune response towards chronic inflammation. Apart from the role in carcinogenesis, chronic inflammation also favors the emergence of drug resistance clones by inducing the growth of breast cancer stem-like cells. Immunomodulation mediated by cytokines, chemokines and several other growth factors present in the tumor microenvironment regulate chronic inflammatory response and alter crosstalk among various signaling pathways such as NF-κB, Nrf-2, JAK-STAT, Akt and MAPKs involved in the progression of breast cancer. In this review, we focused on cellular and molecular processes involved in chronic inflammation, crosstalk among different signaling pathways and their association in breast cancer pathogenesis.

Sulfur alleviates arsenic toxicity by reducing its accumulation and modulating proteome, amino acids and thiol metabolism in rice leaves.

Arsenic (As) contamination of water is a global concern and rice consumption is the biggest dietary exposure to human posing carcinogenic risks, predominantly in Asia. Sulfur (S) is involved in di-sulfide linkage in many proteins and plays crucial role in As detoxification. Present study explores role of variable S supply on rice leaf proteome, its inclination towards amino acids (AA) profile and non protein thiols under arsenite exposure. Analysis of 282 detected proteins on 2-DE gel revealed 113 differentially expressed proteins, out of which 80 were identified by MALDI-TOF-TOF. The identified proteins were mostly involved in glycolysis, TCA cycle, AA biosynthesis, photosynthesis, protein metabolism, stress and energy metabolism. Among these, glycolytic enzymes play a major role in AA biosynthesis that leads to change in AAs profiling. Proteins of glycolytic pathway, photosynthesis and energy metabolism were also validated by western blot analysis. Conclusively S supplementation reduced the As accumulation in shoot positively skewed thiol metabolism and glycolysis towards AA accumulation under AsIII stress.

Circulating miRNAs revealed as surrogate molecular signatures for the early detection of breast cancer.

The miRNAs have well studied roles in cancer. Here, we identified altered miRNA expression by global miRNA profiling in peripheral blood mononuclear cells (PBMCs) of breast cancer (n = 15) and healthy subjects (n = 15), and further validated the selected miRNAs in PBMCs (n = 45), blood plasma (n = 45) and breast tissue samples (n = 09). The expression of altered miRNAs was also evaluated in PBMCs among early stage (n = 32), advanced stage (n = 13), triple positive (n = 5) and triple negative (n = 5) breast cancer patients. Results showed differential pattern of expressions of these miRNAs in multiple cohorts, however in early stage breast cancer, miR-106a-5p and miR-454-3p were upregulated (p < 0.05), miR-195-5p and miR-495 were downregulated (p < 0.05) in PBMCs. In addition, these miRNAs were also significantly associated with cancer and ErbB signaling pathways. Multiple regression analysis and receiver-operative curve (ROC) analysis of miR-195-5p and miR-495 with area under curve (AUC) of 0.901 showed best discriminating combination for early stage breast cancer detection. In summary, the present study delineated the importance of miR-195-5p and miR-495 miRNAs as prospective circulating surrogate molecular signatures for early detection of breast cancer.

New Enlightenment of Skin Cancer Chemoprevention through Phytochemicals: In Vitro and In Vivo Studies and the Underlying Mechanisms.

Skin cancer is still a major cause of morbidity and mortality worldwide. Skin overexposure to ultraviolet irradiations, chemicals, and several viruses has a capability to cause severe skin-related disorders including immunosuppression and skin cancer. These factors act in sequence at various steps of skin carcinogenesis via initiation, promotion, and/or progression. These days cancer chemoprevention is recognized as the most hopeful and novel approach to prevent, inhibit, or reverse the processes of carcinogenesis by intervention with natural products. Phytochemicals have antioxidant, antimutagenic, anticarcinogenic, and carcinogen detoxification capabilities thereby considered as efficient chemopreventive agents. Considerable efforts have been done to identify the phytochemicals which may possibly act on one or several molecular targets that modulate cellular processes such as inflammation, immunity, cell cycle progression, and apoptosis. Till date several phytochemicals in the light of chemoprevention have been studied by using suitable skin carcinogenic in vitro and in vivo models and proven as beneficial for prevention of skin cancer. This revision presents a comprehensive knowledge and the main molecular mechanisms of actions of various phytochemicals in the chemoprevention of skin cancer.