Promising anticancer activity of cromolyn in colon cancer: in vitro and in vivo analysis.

PURPOSE: Colon cancer is a prevalent cancer globally, representing approximately 10% of all cancer cases and accounting for 10% of all cancer-related deaths. Therefore, finding new therapeutic methods with high efficiency will be very valuable. Cromolyn (C), a common anti-allergic and mast cell membrane stabilizing drug, has recently shown valuable anti-cancer effects in several studies. This study was designed to investigate the anti-cancer activity of cromolyn on colon cancer in vitro and in vivo and to determine values such as selectivity index and survival effect. METHODS: HT-29 (colon cancer) and MCF-10 (normal epithelial) cell lines were treated with C and Doxorubicin (DOX; Positive control). IC50 values and the effects of C and DOX on apoptosis were explored using methyl thiazole diphenyl-tetrazolium bromide (MTT) assay and Annexin V/PI Apoptosis Assay Kit. To investigate in an animal study, colon cancer was subcutaneously induced by CT26 cells (mouse colon cancer) in bulb/c mice. Mice were treated with 0.05 LD50 intraperitoneal every other day for 35 days. After the death of mice, tumor volume, tumor weight, and survival rate were evaluated. RESULTS: C selectively and significantly suppressed the proliferation of cancer cells in a dose-dependent manner. The IC50 values for the MCF-10 and HT29 cell lines were 7.33 ± 0.78 µM and 2.33 ± 0.6 µM, respectively. Notably, the selective index (SI) highlighted that C displayed greater selectivity in inhibiting cancer cell growth compared to DOX, with SI values of 3.15 and 2.60, respectively. C exhibited higher effectiveness and selectivity in inducing apoptosis in cancer cells compared to DOX, with a significant p-value (61% vs. 52%, P-value ≤ 0.0001). Also, in mice bearing colon cancer, C reduced the tumor volume (6317 ± 1685mm3) and tumor weight (9.8 ± 1.6 g) compared to the negative control group (weight 12.45 ± 0.9 g; volume 7346 ± 1077) but these values were not statistically significant (P ≤ 0.05). CONCLUSION: Our study showed that cromolyn is a selective and strong drug in inhibiting the proliferation of colon cancer cells. Based on our results, the efficacy of C in vitro analysis (MTT assays and apoptosis), as well as animal studies is competitive with the FDA-approved drug doxorubicin. C is very promising as a low-complication and good-efficacy drug for cancer drug repositioning. This requires clinical research study designs to comprehensively evaluate its anti-cancer effects.

Evaluation of the genetic diversity of KIR genes in patients with endometrial cancer.

Endometrial cancer (EC) constitutes more than half of all genital cancers in women, with an increasing incidence in different countries. Natural killer cells (NK cells) are kinds of innate immune cells that are controlled by sets of receptors, such as killer cell Ig-like receptors (KIRs), which can inhibit or activate NK cells. In this study, we evaluated the diversity and genetic association of KIRs in confirmed cases of endometrial cancer compared to healthy controls. A total of 151 women with EC and 167 age/race-matched healthy controls were analyzed for KIR genes. Demographic and histopathologic data were gathered in questionnaires, and 16 KIR genes along with two variants of KIR2DS4 (KIR2DS4fl and KIR2DS4del), were genotyped by usingsequence specific primers-polymerase chain reaction (SSP-PCR) method. A comparison between cases and controls revealed that although there were not any significant differences in A haplotype associated genes and also the variants of KIR2DS4 (p >0.05), B haplotype associated genes such as KIR2DS2 and KIR2DL2 decreased significantly in EC patients in comparison with healthy controls (p=0.03 and p=0.01, respectively). Furthermore, we found that EC mostly developed in cases with the AA genotype; however, the carriers of Bx and C4T4 genotypes were less frequent in patients with EC. Our results revealed that KIR2DS2 and KIR2DL2, along with Bx and C4T4 genotypes, have a protective impact against developing endometrial cancer in Iranians.

Serum Interleukin-38 and Tumor-Infiltrating Lymphocytes in Primary Brain Tumors.

Background: Tumor-infiltrating lymphocytes (TILs) and brain stromal cells produce immunosuppressive cytokines, contributing to an immunosuppressive tumor microenvironment (TME). Interleukin-38 (IL-38) is a novel anti-inflammatory cytokine and a natural modulator of the innate and adaptive immune system. However, its biological roles in brain tumors are not well defined. Objective: To assess the serum levels of IL-38 and the percentages of TILs in the tumor tissues of patients with primary brain tumors and to determine their associations with the pathological features of the disease. Methods: IL-38 was evaluated in sera using the enzyme-linked immunosorbent assay (ELISA). Hematoxylin and eosin (H&E)-stained sections were scored to determine the percentages of TILs in four different areas: the invasive margin, central tumor, perivascular and perinecrotic areas. Results: IL-38 serum levels were significantly higher in low- and high-grade tumors than in healthy individuals, meanwhile, its levels remained consistent between these two grades. Although no significant difference was found in IL-38 serum levels between different histological subtypes of brain tumors, its levels were significantly higher in intra-axial brain tumors than in extra-axial ones. Additionally, a significant positive correlation was observed between serum levels of IL-38 and tumor size in patients with low-grade tumors. TILs were detected in at least one of the four examined areas; however, no statistically significant correlation was found between IL-38 levels and TILs. Conclusion: Our data may suggest a connection between IL-38 and immune suppression and tumor progression in primary brain tumors. Further investigation is needed to uncover the role of IL-38 in the brain tumor microenvironment.

Frequent expression of CD45RO memory T cell marker as well as low to high expression of PD-1 and PD-L1 inhibitory molecules in seminoma and dysgerminoma.

BACKGROUND: Seminoma and dysgerminoma are rare testicular and ovarian germ cell tumors characterized by a significant infiltration of immune cells in the tumor microenvironment. According to the failure of conventional treatments in some patients, it is crucial to identify novel prognostic and therapeutic biomarkers for these patients. The objectives of this study were to evaluate the expression of CD45RO and PD-1/PD-L1 and investigate their association with the clinicopathological characteristics of the patients. METHODS: Immunohistochemistry was performed to assess the expression of CD45RO, PD-1, and PD-L1 in tumor-infiltrated lymphocytes (TILs), and tumor cells in 33 seminoma and 31 dysgerminoma patients. The expression levels were evaluated using a semiquantitative approach, weighted histoscore, which considers both the intensity and extent of staining. RESULTS: All seminoma and dysgerminoma patients exhibited CD45RO expression in TILs, with 66.7 % and 90.3 % displaying high levels of expression, respectively. PD-1 expression in TILs was observed at low levels in 81.8 % and 77.4 % and at high levels in 18.2 % and 19.4 % of seminoma and dysgerminoma patients, respectively. Likewise, low expression of PD-L1 in tumor cells was detected in 63.6 % of seminoma and 61.3 % of dysgerminoma patients, while none of the patients exhibited high expression of PD-L1. In seminoma patients, a positive correlation was observed between PD-1 expression in TILs and CD45RO expression and between PD-L1 expression in tumor cells and TILs score. CONCLUSION: The frequent infiltration of CD45RO, along with variable expression of PD-1 and PD-L1 on TILs and tumor cells, could impact the effectiveness of anti-tumor responses and immunotherapy.

Molecular identification of neuraminidase gene mutations in influenza A/H1N1 and A/H3N2 isolates of Mazandaran province, north of Iran.

OBJECTIVES: The neuraminidase (NA) mutations causing resistance to NA inhibitors (NAIs) mostly compromise the fitness of influenza viruses. Considering the importance of these mutations, constant monitoring of the effectiveness of available drugs is critical. This study aimed to identify NA mutations in the influenza A/H1N1 and A/H3N2 subtypes in the samples of Mazandaran, Iran from 2016 to 2020. METHODS: In this cross-sectional study, 20 influenza A/H1N1 and 20 influenza A/H3N2 samples were included in the study. After design of appropriate primers for NA gene, all samples subjected to RT-PCR and electrophoresis. Then the PCR product was sequenced to determine the mutations. RESULTS: In the present study, no oseltamivir resistance-related mutations were detected. Still, NA gene showed variations compared to the vaccine strains. In A/H1N1, a total of 43 mutations were detected. Similarly, in A/H3N2, a total of 66 mutations were observed. In all isolates of H1N1, N200S, N248D and I321V mutations were detected in the antigenic site of NA protein, which can affect vaccine incompatibility and virus escape from the host's immune system. Also, H150R mutation was observed in the NA active site of H3N2, which is the cause of agglutination by NA protein. Also, S245N mutation was identified as a new N-Glycosylation site of H3N2 subtype. CONCLUSIONS: The study of NA gene sequences revealed no oseltamivir resistance mutations. In H1N1 isolates, ca. 97% identities and in the H3N2 subtype, 96% identities were observed compared to reference isolate of 2009, which indicates the importance of constant monitoring of the emergence of the drug resistance mutations.

In vitro and in vivo anticancer activity of mebendazole in colon cancer: a promising drug repositioning.

Colon cancer is one of the most common cancers and one of the main causes of death worldwide. Therefore, new treatment methods with better efficiency and fewer risks are very necessary. Mebendazole (MBZ), a drug commonly used for helminthic infections, has recently received attention as a suitable candidate for the treatment of various cancers. This study aimed to investigate, in vitro and in vivo, anticancer activity and selectivity Index of MBZ on colon cancer. HT-29 (human colorectal adenocarcinoma) and MCF-10 (non-tumorigenic epithelial) cell lines were treated with MBZ and Doxorubicin (DOX; positive control drug). IC50 values were estimated using methyl thiazole diphenyl-tetrazolium bromide (MTT) assay. We employed flow cytometry using annexin V-FITC and propidium iodide dyes. For the animal study, colon cancer was subcutaneously induced by CT26 cells (mouse colon cancer) in Bulb/C mice. The mice were treated with 0.05 of LD50, intraperitoneal, every other day for 35 days. Finally, the survival rate, tumor volume, and tumor weight were calculated. Our results demonstrated that IC50 values after 72 h for HT29 and MCF-10 cell lines were 0.29 ± 0.04 µM and 0.80 ± 0.02 µM, respectively. MBZ was more selective than DOX in inhibiting the proliferation of cancer cells compared to normal cells (2. 75 vs. 2.45). Annexin V/PI staining demonstrated that MBZ treatment at IC50 concentrations induced (78 ± 12%) apoptosis in the HT29 cancer cell line after 48 h (P ≤ 0.0001). Also, in mice bearing colon cancer, MBZ significantly reduced the tumor volume (1177 ± 1109 mm3; P ≤ 0.001) and tumor weight (2.30 ± 1.97 g; P ≤ 0.0001) compared to the negative control group (weight 12.45 ± 2.0 g; volume 7346 ± 1077). Also, MBZ increases mean survival time (MST) and increase life span (ILS) percentage in the animal study (51.2 ± 37% vs 93%, respectively). This study suggests that mebendazole strongly and selectively inhibits proliferation and induces apoptosis in colon cancer cells. It may be, accordingly, a promising drug for clinical research and application.

The antitumor effect of oncolytic respiratory syncytial virus via the tumor necrosis factor-alpha induction and ROS-bax-mediated mechanisms.

BACKGROUND: Cervical cancer represents one of the most prevalent cancers among women worldwide, particularly in low- and middle-income nations. Oncolytic viruses (OVs) can infect cancer cells selectively and lethally without harming normal cells. Respiratory syncytial virus (RSV) is an oncolytic virus for anticancer therapy because of its propensity to multiply within tumor cells. This research aimed to assess the in vitro antitumor activities and molecular basis processes of the oncolytic RSV-A2 on the TC-1 cancer cells as a model for HPV­related cervical cancers. METHODS: Cellular proliferation (MTT) and lactate dehydrogenase (LDH) release assays were used to investigate the catalytic impacts of RSV-A2 by the ELISA method. Real-time PCR and flow cytometry assays were utilized to assess apoptosis, autophagy, intracellular concentrations of reactive oxygen species (ROS), and cell cycle inhibition. RESULTS: Our MTT and LDH results demonstrated that TC-1 cell viability after oncolytic RSV-A2 treatment was MOI-dependently and altered significantly with increasing RSV-A2 virus multiplicity of infection (MOI). Other findings showed that the RSV-A2 potentially resulted in apoptosis and autophagy induction, caspase-3 activation, ROS generation, and cell cycle inhibition in the TC-1 cell line. Real-time PCR assay revealed that RSV-A2 infection significantly elevated the Bax and decreased the Bcl2 expression. CONCLUSIONS: The results indicated that oncolytic RSV-A2 has cytotoxic and inhibiting effects on HPV-associated cervical cancer cells. Our findings revealed that RSV-A2 is a promising treatment candidate for cervical cancer.

Implementation of an In-House Platform for Rapid Screening of SARS-CoV-2 Genome Variations.

BACKGROUND: Global real-time monitoring of SARS-CoV-2 variants is crucial to controlling the COVID-19 outbreak. The purpose of this study was to set up a Sanger-based platform for massive SARS-CoV-2 variant tracking in laboratories in low-resource settings. METHODS: We used nested RT-PCR assay, Sanger sequencing and lineage assignment for 930-bp of the SARS-CoV-2 spike gene, which harbors specific variants of concern (VOCs) mutations. We set up our platform by comparing its results with whole genome sequencing (WGS) data on 137 SARS-CoV-2 positive samples. Then, we applied it on 1028 samples from March-September 2021. RESULTS: In total, 125 out of 137 samples showed 91.24% concordance in mutation detection. In lineage assignment, 123 out of 137 samples demonstrated 89.78% concordance, 65 of which were assigned as VOCs and showed 100% concordance. Of 1028 samples screened by our in-house method, 78 distinct mutations were detected. The most common mutations were: S:D614G (21.91%), S:P681R (12.19%), S:L452R (12.15%), S:T478K (12.15%), S:N501Y (8.91%), S:A570D (8.89%), S:P681H (8.89%), S:T716I (8.74%), S:L699I (3.50%) and S:S477N (0.28%). Of 1028 samples, 980 were attributed as VOCs, which include the Delta (B.1.617.2) and Alpha (B.1.1.7) variants. CONCLUSION: Our proposed in-house Sanger-based assay for SARS-CoV-2 lineage assignment is an accessible strategy in countries with poor infrastructure facilities. It can be applied in the rapid tracking of SARS-CoV-2 VOCs in the SARS-CoV-2 pandemic.

Comparative proteomics analysis in different stages of urothelial bladder cancer for identification of potential biomarkers: highlighted role for antioxidant activity.

BACKGROUND: Non-muscle-invasive bladder cancer (NMIBC) has a high recurrence rate and muscle-invasive bladder cancer (MIBC) has unfavorable outcomes in urothelial bladder cancer (UBC) patients. Complex UBC-related protein biomarkers for outcome prediction may provide a more efficient management approach with an improved clinical outcome. The aim of this study is to recognize tumor-associated proteins, which are differentially expressed in different stages of UBC patients compared non-cancerous tissues. METHODS: The proteome of tissue samples of 42 UBC patients (NMIBC n = 25 and MIBC n = 17) was subjected to two-dimensional electrophoresis (2-DE) combined with Liquid chromatography-mass spectrometry (LC-MS) system to identify differentially expressed proteins. The intensity of protein spots was quantified and compared with Prodigy SameSpots software. Functional, pathway, and interaction analyses of identified proteins were performed using geneontology (GO), PANTHER, Reactome, Gene MANIA, and STRING databases. RESULTS: Twelve proteins identified by LC-MS showed differential expression (over 1.5-fold, p < 0.05) by LC-MS, including 9 up-regulated in NMIBC and 3 up-regulated in MIBC patients. Proteins involved in the detoxification of reactive oxygen species and cellular responses to oxidative stress showed the most significant changes in UBC patients. Additionally, the most potential functions related to these detected proteins were associated with peroxidase, oxidoreductase, and antioxidant activity. CONCLUSION: We identified several alterations in protein expression involved in canonical pathways which were correlated with the clinical outcomes suggested might be useful as promising biomarkers for early detection, monitoring, and prognosis of UBC.

Polymorphism Analysis of Interleukin-18 and Interleukin-37 Genes in Hepatitis B Infections with Different Outcomes: A Preliminary Report from an Iranian Population.

OBJECTIVE: Given the vital role of cytokines in influencing the outcomes of hepatitis B virus (HBV) infections, this study aimed to investigate the association between polymorphisms of interleukin (IL)-18 and IL-37 and the outcomes of HBV infection. METHODS: In this study, we enrolled 300 subjects with chronic HBV infection, including those with cirrhosis/hepatocellular carcinoma (C/HCC), chronic active hepatitis B (CAH) infection, or asymptomatic carriers (AC), and 58 individuals whose infection was spontaneously cleared (SC). Genomic DNA was extracted, and IL-18/IL-37 genotyping was performed using PCR-RFLP and ARMS-PCR. RESULTS: The frequency of genotypes and alleles of IL-18 single nucleotide polymorphisms (SNPs) at positions rs1946519, rs1946518, and rs187238 and IL-37 at position rs4241122 were not statistically different among the four studied groups (P>0.05). Furthermore, the frequency of different haplotypes was similar among the studied groups (P>0.05). CONCLUSIONS: Polymorphisms of IL-18 SNPs at positions rs1946519, rs1946518, and rs187238 and variation of IL-37 at position rs4241122 do not appear to influence the outcome of HBV infection.

Proteomics strategies for urothelial bladder cancer diagnosis, prognosis and treatment: Trends for tumor biomarker sources.

Urothelial bladder cancer (UBC) is a heterogeneous multifactorial malignancy with a high recurrence rate. Current procedures for UBC diagnosis suffering from the lack of clinical sensitivity and specificity screening tests. Therefore, biomarkers have promising values to predict pathological conditions and can be considered as effective targets for early diagnosis, prognosis and antitumor immunotherapy. Recently, researchers have been interested for tumor proteins as biomarkers for different diseases. At present, proteomics methods have rapidly progressive that has potential identified biomarkers of UBC. Specifically, there has been several studies on the potential application of proteomics for the identification, quantification, and profiling of proteins for UBC in different sources. Based on these studies, using the panel of biomarkers as proteomic patterns may achieve higher sensitivity and specificity than single proteins in the diagnosis of UBC. In the present review, we evaluate recent literature related to the UBC proteome focusing especially on new proteomics techniques. Moreover, we classify UBC tumor biomarkers as diagnostic, prognostic, and therapeutic targets based on their sources (urine, serum/plasm, cell line, and tumor tissue) and we also discuss the advantages and limitations of each source. In this manner, this review article provides a critical assessment presentation of the advances in proteomics for all aspects of UBC diagnosis, prognosis, and treatment based on sources.

Role of MexAB-OprM efflux pump in the emergence of multidrug-resistant clinical isolates of Pseudomonas aeruginosa in Mazandaran province of Iran.

BACKGROUND: Multidrug-resistant clinical isolates can cause many therapeutic problems. The MexAB-OprM efflux pump plays a significant role in expelling toxins and drugs from the bacterial cells resulting in multidrug-resistant Pseudomonas aeruginosa isolates. PURPOSE: This study aimed to investigate the effect of the MexAB-OprM efflux pump in the emergence of multidrug-resistant clinical isolates of P. aeruginosa. METHODS AND RESULTS: For the present study, 100 clinical isolates of P. aeruginosa were collected from different wards of teaching hospitals (2018-2019). After confirmation and detection of bacteria by standard methods, the antibiotic resistance pattern of the isolates was determined by the disk agar diffusion method. Also, the minimum inhibitory concentration (MIC) of ciprofloxacin was measured in the presence and absence of phenylalanine arginine beta-naphthylamide by the broth microdilution method. Then, the real-time PCR was used to investigate the expression level of the mexB gene compared to the standard PAO1 strain. Forty-one/100 isolates exhibited multidrug-resistant phenotype (MDR), while piperacillin-tazobactam and levofloxacin were the most and least effective antibiotics tested, respectively. Also, 54/100 isolates showed no increased expression of mexB gene compared to the standard PAO1 strain. However, among the 41 MDR isolates, 12 (29.26%) showed a more than three-fold increase in the expression level of the mexB gene. In this study, a significant relationship was observed between the resistance to tested antibiotics in MDR strains and the increased expression of the mexB gene. CONCLUSION: We found that increasing the expression of the mexB gene can cause the emergence of multidrug-resistant strains by increasing the minimum inhibitory concentration of the antibiotics. Then, we need to evaluate the resistance mechanisms separately in different area of a country to improve the antibiotic stewardship.

Immunological and biological dissection of normal and tumoral salivary glands.

Salivary glands naturally play central roles in oral immunity. The salivary glands microenvironment inevitable may be exposed to exogenous factors consequently triggering the initiation and formation of various malignant and benign tumors. Mesenchymal stem cells are recruited into salivary gland microenvironment, interact with tumor cells, and induce inhibitory cytokines as well as cells with immunosuppressive phenotypes such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). The immune components and tumor immune responses in malignant and benign SGTs are still under investigation. Immune responses may directly play a limiting role in tumor growth and expansion, or may participate in formation of a rich milieu for tumor growth in cooperation with other cellular and regulatory molecules. Immune checkpoint molecules (e.g. PDLs, HLA-G and LAG3) are frequently expressed on tumor cells and/or tumor-infiltrating lymphocytes (TILs) in salivary gland microenvironment, and an increase in their expression is associated with T cell exhaustion, immune tolerance and tumor immune escape. Chemokines and chemokine receptors have influential roles on aggressive behaviors of SGTs, and thereby they could be candidate targets for cancer immunotherapy. To present a broad knowledge on salivary glands, this review first provides a brief description on immunological functions of normal salivary glands, and then describe the SGT's tumor microenvironment, by focusing on mesenchymal stem cells, immune cell subsets, immune checkpoint molecules, chemokines and chemokine receptors, and finally introduces immune checkpoint inhibitors as well as potential targets for cancer therapy.

Simultaneous Expression of PD-1 and PD-L1 in Peripheral and Central Immune Cells and Tumor Cells in the Benign and Malignant Salivary Gland Tumors Microenvironment.

BACKGROUND: To investigate the differential expression of PD-1 and PD-L1 in salivary gland tumors (SGTs, malignant and benign subtypes) and determine their association with the clinicopathological characterization of the patients. METHODS: The immunohistochemistry was used to examine PD-1 and PD-L1 expression in specimens from 83 patients with primary SGTs including salivary ductal carcinoma (SDC), adenoid cystic carcinoma (AdCC), acinic cell carcinoma (ACC), mucoepidermoid carcinoma (MEC), warthin's tumors (WT), poleomorphic adenoma (PA) and other subtypes. RESULTS: The expression of PD-1 in peripheral and central immune cells (ICs) of MEC, and peripheral ICs of ACC was significantly higher than those with AdCC (P = 0.02, P = 0.02, P = 0.03, respectively). Interestingly, the expression of PD-1 was also observed in peripheral and central malignant tumor cells (TCs), particularly in SDC and ACC. Despite no significant difference in PD-L1 expression of TCs among malignant subtypes, the peripheral and central ICs of ACC and MEC were revealed to express PDL-1 significantly more than those with AdCC (P < 0.05). WTs were rich in PD-1/PD-L1 expressing ICs. However, the tumor microenvironment of PA generally had low levels of PD-1/PD-L1 expression. In general, the expression of PD-1 in peripheral and central TCs was found to be significantly higher in malignant tumors than in benign ones (P = 0.002 and P = 0.003, respectively). CONCLUSION: The simultaneous presentation of PD-1 and PD-L1 in TCs and ICs of SGTs, their significant association with disease severity as well as the positive correlation between these immune checkpoints may suggest the therapeutic potential of anti-PD-1 and anti-PDL-1 combinational immunotherapy for SGTs.

Clinical significance of IL-37 serum level and polymorphism in patients with endometrial cancer.

Endometrial cancer (EC) is recognized as the second most common type of cancer among women. Interleukin-37 (IL-37) is a recently discovered member of the IL-1 cytokine family characterized by its anti-inflammatory properties, which are believed to have both anti-tumour and tumorigenic effects. However, the precise role of IL-37 in the development of EC remains largely unknown. In the current study, we aimed to explore genotype and allele frequencies of the IL-37 gene (rs4241122) and measure IL-37 protein levels in patients with EC, with a view to determining the clinical significance in these patients. A total of 105 patients with confirmed EC and 105 healthy controls, aged 31-73, participated in the study. IL-37 serum levels were investigated using an ELISA method, while the frequency of genotypes and alleles of the IL-37 gene was determined using the ARMS-PCR method. The findings demonstrate a significant increase in IL-37 serum levels in EC patients compared to controls (p<0.0001). Moreover, higher levels of IL-37 were strongly associated with unfavourable indices, such as EC grade III, poorly differentiated tumours, and regional spread of tumour cells (p<0.05). However, genotyping of the IL-37 gene revealed no significant difference between the two groups, and there was no association between IL-37 genotype and IL-37 protein level or clinicopathological characteristics (p>0.05). The results of this study suggest that elevated serum levels of may contribute to tumour progression, probably through its immune suppressive activity. Clinically, IL-37 may serve as a promising factor and/or therapeutic target for EC management, although, further studies are warranted.

Evaluation of morphology and angiogenesis of breast cancer in BALB/c mice using trypsin inhibitor from Cucumis melo seeds. In vitro and in vivo study.

Introduction: Melon seeds, as an excellent source of protease inhibitors, may have a protective role against tumor progression and angiogenesis. However, their effects on angiogenesis and the mechanism of their action against cancer progression remain elusive. This study aimed to investigate the effect of bioactive compounds of melon seed on the expression of angiogenesis genes in BALB/c mice with breast cancer. Material and methods: Trypsin inhibitor (TI) was purified from the seed powder of Cucumis melo. Half- maximal inhibitory concentration was determined for TI, extract of melon seed powder (EXT), and tamoxifen (TAM) by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Also, breast tumor was induced by subcutaneous injection of MC4-L2 cells in BALB/c inbred mice breast tissue. After tumor growth, mice were treated with TI, EXT, and TAM to examine their effects on the tumor characteristics and expression of angiogenesis-related genes including MMP-2, MMP-9, and vascular endothelial growth factor (VEGF) using the reverse transcription polymerase chain reaction method. Results: Trypsin inhibitor, EXT, TAM, and adjuvant treatment of TI + TAM resulted a reduction in expression of MMP-2, MMP-9, and VEGF. All treatments improved the breast tumor characteristics and the necrosis. The real-time polymerase chain reaction method verified the positive effects of the treatments on the breast cancer cell line and tumors. Conclusions: The results indicated that treatments with TI purified from Cucumis melo seeds and also combination therapy of TI and TAM can be considered as an alternative therapy in breast cancer patients. Further studies are warranted.

Simultaneous Increase in Serum Levels of IL-37 and IL-18 Binding Protein In Low-Grade and High-Grade Brain Tumors.

BACKGROUND: IL-18binding protein (IL-18BP) might play a role in tumor escape from immune surveillance through interacting with IL-37. Such interactions modulate the antitumor activity of IL-18 and affect regulatory T cell (Treg) function. However, the biological roles of IL-37 and IL-18BP have not yet been explored in brain tumors. This study aimed to investigate serum levels of IL-37 and IL-18BP in high-grade and low-grade brain tumors and determine their associations with pathological characteristics of the patients. SUBJECTS AND METHODS: This case-control study consisted of 60 patients with brain tumors (40 low-grade and 20 high-grade) and 30 healthy controls. Enzyme-linked immunosorbent assay (ELISA) kits were used to measure the levels of IL-37 and IL-18BP in serum. RESULTS: Our results indicated that serum levels of IL-37 and IL-18BP were significantly higher in patients with brain tumors (109.02, 426.37 pg/mL), high-grade (104.44, 428.87 pg/mL), and low-grade (113.88, 426.37 pg/mL) tumors in compared to healthy controls (35.03, 362.00 pg/mL), (P<0.05). Interestingly, our results revealed a significant positive correlation between IL-37 and IL-18BP serum levels in brain tumors (n=60, R=0.42, P=0.001). Our study also showed that serum levels of IL-37 and IL-18BP in glioblastoma grade IV were approximately similar to those in astrocytoma grade II, meningioma type I, and pituitary adenoma. Furthermore, no significant differences were found in serum levels of IL-37 and IL-18BP between patients with low-grade and high-grade tumors (P=0.24 and P=0.61, respectively). CONCLUSION: The simultaneous increase in IL-37 and IL-18BP serum levels and their positive correlation may facilitate disease progression in low-grade and high-grade brain tumors by inhibiting antitumor immune responses.

Immunosuppressive Effects and Potent Anti-tumor Efficacy of mTOR Inhibitor Everolimus in Breast Tumor-bearing Mice.

To investigate the effects of everolimus, a mechanistic/mammalian target of rapamycin (mTOR) inhibitor, on tumor growth and immune response in a mouse model of breast cancer. Human hormone receptor-positive (HR+)/human epidermal growth receptor 2-negative (HER2-) MC4-L2 cell line was used to establish a mouse model of breast cancer. The inhibitory effects of high (10 mg/kg) and low (5 mg/kg) doses of everolimus were investigated on tumor growth. Additionally, the frequency of CD4+Foxp3+ regulatory T cells (Tregs), CD8+Foxp3+ Tregs, and CD4+ and CD8+ T cells expressing cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) was explored by flow cytometry in bone marrow, lymph nodes, and spleen. Our results showed that both 10 mg/kg and 5 mg/kg doses of everolimus efficiently inhibited tumor growth, resulting in reduced breast tumor volume. In addition, it was revealed that everolimus-treated mice induced a higher frequency of CD4+Foxp3+ Tregs, CD8+Foxp3+ Tregs, and CD4+Foxp3+CTLA-4+ Tregs as well as CD4+ and CD8+ T cells expressing CTLA-4 in their bone marrow, lymph nodes, and spleen compared with standard control (vehicle-treated) in a dose-dependent manner. Furthermore, we found that everolimus treatment with 10 mg/kg and 5 mg/kg increased the frequency of Helios+Foxp3+ Tregs in the bone marrow of treated mice compared with the control group. Our results indicate that treatment with everolimus not only inhibits tumor growth but also exerts an immunomodulatory effect by inducing Tregs in the lymphoid organs of breast cancer-bearing mice. The combination of therapy with other anti-cancer agents may negate immune suppression and improve the efficacy of mTOR-targeted breast cancer therapy.

Proteomics Study of Mesenchymal Stem Cell-Like Cells Obtained from Tumor Microenvironment of Patients with Malignant and Benign Salivary Gland Tumors.

Objective: Salivary gland tumors (SGTs) show some aggressive and peculiar clinicopathological behaviors that might be related to the components of the tumor microenvironment, especially mesenchymal stem cells (MSCs)-associated proteins. However, the role of MSCs-related proteins in SGTs tumorigenesis is poorly understood. This study aimed to isolate and characterize MSCs from malignant and benign tumor tissues and to identify differentially expressed proteins between these two types of MSCs. Materials and Methods: In this experimental study, MSC-like cells derived from benign (pleomorphic adenoma, n=5) and malignant (mucoepidermoid carcinoma, n=5) tumor tissues were verified by fluorochrome antibodies and flow cytometric analysis. Differentially expressed proteins were identified using two-dimensional polyacrylamide gel electrophoresis (2DE) and Mass spectrometry. Results: Results showed that isolated cells strongly expressed characteristic MSCs markers such as CD44, CD73, CD90, CD105, and CD166, but they did not express or weakly expressed CD14, CD34, CD45 markers. Furthermore, the expression of CD24 and CD133 was absent or near absent in both isolated cells. Results also discovered overexpression of Annexin A4 (Anxa4), elongation factor 1-delta (EF1-D), FK506 binding protein 9 (FKBP9), cytosolic platelet-activating factor acetylhydrolase type IB subunit beta (PAFAH1B), type II transglutaminase (TG2), and s-formylglutathione hydrolase (FGH) in MSCs isolated from the malignant tissues. Additionally, heat shock protein 70 (Hsp70), as well as keratin, type II cytoskeletal 7 (CK-7), were found to be overexpressed in MSCs derived from the benign ones. Conclusion: Malignant and benign SGTs probably exhibit a distinct pattern of tissue proteins that are most likely related to the metabolic pathway. However, further studies in a large number of patients are required to determine the applicability of identified proteins as new targets for cancer therapy.