Aquaporins in colorectal cancer: exploring their role in tumorigenesis, metastasis, and drug response.

Aquaporins (AQPs) are small, integral proteins facilitating water transport across plasma cell membranes in response to osmotic gradients. This family has 13 unique members (AQP0-12), which can also transport glycerol, urea, gases, and other salute small molecules. AQPs play a crucial role in the regulation of different cellular processes, including metabolism, migration, immunity, barrier function, and angiogenesis. These proteins are found to aberrantly overexpress in various cancers, including colorectal cancer (CRC). Growing evidence has explored AQPs as a potential diagnostic biomarker and therapeutic target in different cancers. However, there is no comprehensive review compiling the available information on the crucial role of AQPs in the context of colorectal cancer. This review highlights the significance of AQPs as the biomarker and regulator of tumor cells metabolism. In addition, the proliferation, angiogenesis, and metastasis of tumor cells related to AQPs expression as well as function are discussed. Understanding the AQPs prominent role in chemotherapy resistance is of great importance clinically.

Unraveling the potential of CD8, CD68, and VISTA as diagnostic and prognostic markers in patients with pancreatic ductal adenocarcinoma.

Introduction: Pancreatic cancer is a truculent disease with limited treatment options and a grim prognosis. Immunotherapy has shown promise in treating various types of cancer, but its effectiveness in pancreatic cancer has been lacking. As a result, it is crucial to identify markers associated with immunological pathways in order to improve the treatment outcomes for this deadly cancer. The purpose of this study was to investigate the diagnostic and prognostic significance of three markers, CD8, CD68, and VISTA, in pancreatic ductal adenocarcinoma (PDAC), the most common subtype of pancreatic cancer. Methods: We analyzed gene expression data from Gene Expression Omnibus (GEO) database using bioinformatics tools. We also utilized the STRING online tool and Funrich software to study the protein-protein interactions and transcription factors associated with CD8, CD68, and VISTA. In addition, tissue microarray (TMA) and immunohistochemistry (IHC) staining were performed on 228 samples of PDAC tissue and 10 samples of normal pancreatic tissue to assess the expression levels of the markers. We then correlated these expression levels with the clinicopathological characteristics of the patients and evaluated their survival rates. Results: The analysis of the GEO data revealed slightly elevated levels of VISTA in PDAC samples compared to normal tissues. However, there was a significant increase in CD68 expression and a notable reduction in CD8A expression in pancreatic cancer. Further investigation identified potential protein-protein interactions and transcription factors associated with these markers. The IHC staining of PDAC tissue samples showed an increased expression of VISTA, CD68, and CD8A in pancreatic cancer tissues. Moreover, we found correlations between the expression levels of these markers and certain clinicopathological features of the patients. Additionally, the survival analysis revealed that high expression of CD8 was associated with better disease-specific survival and progression-free survival in PDAC patients. Conclusion: These findings highlight the potential of CD8, CD68, and VISTA as diagnostic and prognostic indicators in PDAC.

Evaluation of tumor-educated platelet long non-coding RNAs (lncRNAs) as potential diagnostic biomarkers for colorectal cancer.

INTRODUCTION: Cancer-derived circulating components are increasingly considered as candidate sources for non-invasive diagnostic biomarkers. This study aimed to investigate the expression of tumor-educated platelet (TEP) long non-coding RNAs (lncRNAs) in colorectal cancer (CRC) patients and determine whether it could be served as a potential tool for CRC diagnosis. MATERIALS AND METHODS: Relative quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of three cancer-related platelet-derived lncRNAs CCAT1, HOTTIP, and XIST in 75 CRC patients and 42 healthy controls. Quantitative data were analyzed by SPSS (IBM Corp., Armonk, NY, USA) for comparison of cancer and non-cancer individuals. The receiver operating characteristic (ROC) curve analysis was further performed to assess the diagnostic values of lncRNAs within the CRC patients. RESULTS: The expression levels of lncRNAs colon cancer associated transcript 1 (CCAT1) (P = 0.006) and HOXA transcript at the distal tip (HOTTIP) (P = 0.049), but not X-inactive specific transcript (XIST) (P = 0.12), were significantly upregulated in CRC patients compared to healthy individuals. However, there were no significant correlations between platelet lncRNAs and clinicopathological characteristics, including sex, age, tumor location, differentiation, and size (all at P > 0.05). The area under the ROC curve (AUC) of the lncRNA CCAT1 was 0.61 (sensitivity, 71%; specificity, 50%). CONCLUSION: TEP lncRNA CCAT1 is detectable in the circulation of CRC patients and could be considered as a potential diagnostic biomarker.

Catalase-gold nanoaggregates manipulate the tumor microenvironment and enhance the effect of low-dose radiation therapy by reducing hypoxia.

Radiotherapy as a standard method for cancer treatment faces tumor recurrence and antitumoral unresponsiveness. Suppressive tumor microenvironment (TME) and hypoxia are significant challenges affecting efficacy of radiotherapy. Herein, a versatile method is introduced for the preparation of pH-sensitive catalase-gold cross-linked nanoaggregate (Au@CAT) having acceptable stability and selective activity in tumor microenvironment. Combining Au@CAT with low-dose radiotherapy enhanced radiotherapy effects via polarizing protumoral immune cells to the antitumoral landscape. This therapeutic approach also attenuated hypoxia, confirmed by downregulating hypoxia hallmarks, such as hypoxia-inducible factor α-subunits (HIF-α), vascular endothelial growth factor (VEGF), and EGF. Catalase stability against protease digestion was improved significantly in Au@CAT compared to the free catalase. Moreover, minimal toxicity of Au@CAT on normal cells and increased reactive oxygen species (ROS) were confirmed in vitro compared with radiotherapy. Using the nanoaggregates combined with radiotherapy led to a significant reduction of immunosuppressive infiltrating cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (T-regs) compared to the other groups. While, this combined therapy could significantly increase the frequency of CD8+ cells as well as M1 to M2 macrophages (MQs) ratio. The combination therapy also reduced the tumor size and increased survival rate in mice models of colorectal cancer (CRC). Our results indicate that this innovative nanocomposite could be an excellent system for catalase delivery, manipulating the TME and providing a potential therapeutic strategy for treating CRC.

The immunomodulatory role of exosomal microRNA networks in the crosstalk between tumor-associated myeloid-derived suppressor cells and tumor cells.

Myeloid-derived suppressor cells (MDSCs) are considered a heterogeneous group of immature myeloid cells engaging in aggressive tumor progression and metastasis in the tumor microenvironment (TME) of patients diagnosed with cancer, through downregulation of anti-tumor immune responses. Exosomes are small vesicles carrying specific cargos, including proteins, lipids, and MicroRNA (miRNAs). Such exosomal miRNAs delivered by MDSCs and tumor cells are short noncoding RNAs mediating some of the immunosuppressive characteristics of MDSCs in the TME. However, when it comes to cancer diseases, how these miRNAs interact with MDSCs and encourage MDSCs differentiation and function need further investigations. In this review, we discuss MDSC-derived exosomal miRNAs and those derived from tumor cells (TDE) could modulate anti-tumor immunity and regulate the interaction between tumor cells and MDSCs in the TME. Afterward, we focus on dividing miRNAs, as an important substance interacting with MDSCs and tumor cells in the TME, into those have an immunosuppressive or stimulating effect not only on MDSCs expansion, differentiation, and suppressive function but also on tumor evasion.

Breast metastasis from gastric carcinoma: A case report.

BACKGROUND: There are rare cases of breast metastasis from gastric carcinoma origin. In this regard, we presented a case of signet ring cell stomach cancer with metastasis to the breast. CASE PRESENTATION: The case was a 44-year-old female with a history of gastric cancer and chief complaint of progressive bilateral and gradual breast enlargement and mass palpation 6 years after stomach surgery. An excisional biopsy of the right breast was performed at the early phase of her clinical symptoms with the pathology at the early phase of fibroadenoma and sclerosing adenosis. Given the persistent right breast thickening and enlargement, the ultrasonography and MRI together showed right breast large masses, ductal enhancement in left retro areolar space beside the bilaterally enlarged axillary lymph nodes after 6 months. In this phase, the core needle biopsy showed right breast mass adenocarcinoma consistently with metastatic gastric carcinoma and also core needle biopsy of both axillary lymph nodes indicating the involvement by tumor consistently with metastatic gastric carcinoma. In the IHC, breast tumor cells were negative for ER, PR, HER2/ neu, GATA3, GCDFP15 and CK20, but positive for CK7, CK19, and CDx2. CONCLUSION: The diagnosis of breast metastasis of gastric carcinoma was confirmed according to the past history of patient, histological finding, and immune-histological markers.

Placental Extract and Exosomes Derived from Pregnant Mice Attenuate the Development of Experimental Autoimmune Encephalomyelitis.

Placental extract (PE) and exosomes from pregnant mice appear to have immunomodulatory and neuroprotective effects. In this study, we assessed the potential therapeutic effects of PE and exosomes obtained from pregnant mice in experimental autoimmune encephalomyelitis (EAE) mouse models. C57BL/6 mice, 8 to 12 weeks of age, were prepared and administered PE, exosomes, and glatiramer acetate (GA), as an FDA-approved treatment for multiple sclerosis (MS), after EAE induction. Thereafter, the therapeutic effects of treatment were evaluated by measuring the clinical courses of the mice as well as determining the number of regulatory T (Treg) cells using flow cytometry, cytokine levels, and microRNA-326 expression via real-time PCR. GA, PE, and exosomes reduced clinical severity, the extent of spinal cord demyelination, and the infiltration of inflammatory cells into the spinal cord. The frequency of CD4+CD25+FoxP3+ Treg cells increased after treatment of EAE mice with GA, PE, and exosomes. The mRNA expression of the inflammatory cytokines (interleukin-17  and interferon-gamma), as well as miR-326 expression, decreased significantly in the EAE mice after treatment with GA and exosomes. PE and exosomes from pregnant mice are involved in the modulation of Treg/Th17 balance and provide a therapeutic approach for MS. Further clinical studies will hopefully confirm the safety and efficacy of such treatments in MS patients.

Bioassay-guided Isolation of Flavonoids from Caesalpinia bonduc (L.) Roxb. and Evaluation of Their Cytotoxicity.

Cancer is one of the most important causes of death all around the world. Screening plants and their secondary metabolites as cytotoxic agents is one of the common methods for identifying new compounds used in chemotherapy and inhibition cancer process. Caesalpinia bonduc (L.) Roxb. from the Fabaceae family was used for improving wound, fever, tumor, hydrocele, hernia, smallpox, toothache, inflammation, and as astringent, anthelmintic, antidiabetic, and antimalarial agent in traditional medicine. A bioassay-guided study of this species led to the isolation of three flavonoids. At first, the cytotoxicity of methanol extract of aerial parts (leaves and stems), seeds, and legumes of this plant was tested against MCF-7 and PC-3 by MTT assay. The methanol extract of legumes showed better inhibitory activities (IC50 < 500 µg/mL). As a result, this extract was selected for fractionation. In the next step, the ethyl acetate (EtOAc) fraction was selected for phytochemical analysis based on the inhibitory activity (IC50 = 170 ± 0.9 µg/mL). In this way, total phenol content (625 ± 7.2 GAE/g extract) and antioxidant activity (IC50 = 6.1 ± 0.3 µg/mL) was compared by BHT (IC50 = 13.5 ± 0.7 µg/mL). Finally, three compounds including, quercetin-3-methyl ether (1), kaempferol (2), and kaempferol-3-O-α-L-rhamnopyranosyl-1→2)-ß-D-xylopyranoside (3) were isolated from EtOAc fraction, and all isolated compounds were tested for their cytotoxicity and compound 1 showed better inhibitory activity than other two compounds. This study suggests that Caesalpinia bonduc could be considered for further investigations as a natural source of biological compounds.

Vitamin E and selenium improve mesenchymal stem cell conditioned media immunomodulatory effects.

BACKGROUND: Mesenchymal stem cells (MSCs) with immunoregulatory properties affect immune systems. Many studies showed that antioxidants such as vitamin E (Vit E) and selenium (Se) could improve stem cells survival. This study aims to investigate the effects of MSC conditioned media (CM) treated with Vit E and Se on immune cells. METHODS: MSCs were isolated and cultured with Vit E and Se. Immature dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) were cultured with MSC CM treated with Vit E and Se. The expression of HLA-DR, CD86, CD40, and CD83 on mature DC were evaluated. DC supernatant and PBMCs supernatant was collected for the study of TGF-ß, IL-10, and IL-12. PBMCs evaluated for the expression of T-bet, GATA3, RORγt, and FOXP3. RESULTS: MSC CM increased CD40 on myeloid DC (mDC). CD40 has been decreased in DC treated with MSC (Vit E) and MSC (Se) CM. HLA-DR expression on DCs and IL-12 level were significantly reduced in MSC (Vit E) CM. IL-10 concentration increased in DCs treated with MSC (Vit E) and MSC (Se) CM. Treatment of PBMCs with MSC CM decreased IL-10 level, FOXP3, and RORγt expression. On the other hand, the MSC (Vit E) CM and MSC (Se) CM decreased the IL-10 level and increased IL-12, T-bet, and RORγt. CONCLUSIONS: According to the results, the treatment of MSC with Vit E and Se enhanced the ability of MSCs to inhibit DCs and improved immunomodulatory effects. Concerning the effect of MSC on PBMC, it seems that it increased RORγt expression through monocytes.

Breast cancer immunotherapy: Current and novel approaches.

The crucial role of the immune system in the progression/regression of breast cancer (BC) should always be taken into account. Various immunotherapy approaches have been investigated for BC, including tumor-targeting antibodies (bispecific antibodies), adoptive T cell therapy, vaccines, and immune checkpoint blockade such as anti-PD-1. In addition, a combination of conventional chemotherapy and immunotherapy approaches contributes to improving patients' overall survival rates. Although encouraging outcomes have been reported in most clinical trials of immunotherapy, some obstacles should still be resolved in this regard. Recently, personalized immunotherapy has been proposed as a potential complementary medicine with immunotherapy and chemotherapy for overcoming BC. Accordingly, this review discusses the brief association of these methods and future directions in BC immunotherapy.

Immune and metabolic checkpoints blockade: Dual wielding against tumors.

Recent advances in cancer immunotherapy have raised hopes for treating cancers that are resistant to conventional therapies. Among the various immunotherapy methods, the immune checkpoint (IC) blockers were more promising and have paved their way to the clinic. Tumor cells induce the expression of ICs on the immune cells and derive them to a hyporesponsive exhausted phenotype. IC blockers could hinder immune exhaustion in the tumor microenvironment and reinvigorate immune cells for an efficient antitumor response. Despite the primary success of IC blockers in the clinic, the growing numbers of refractory cases require an in-depth study of the cellular and molecular mechanisms underlying IC expression and function. Immunometabolism is recently found to be a key factor in the regulation of immune responses. Activated or exhausted immune cells exploit different metabolic pathways. Tumor cells can suppress antitumor responses via immunometabolism alteration. Therefore, it is expected that concurrent targeting of ICs and immunometabolism pathways can cause immune cells to restore their antitumor activity. In this review, we dissected the reciprocal interactions of immune cell metabolism with expression and signaling of ICs in the tumor microenvironment. Recent findings on dual targeting of ICs and metabolic checkpoints have also been discussed.

Breast cancer: Biology, biomarkers, and treatments.

During the past recent years, various therapies emerged in the era of breast cancer. Breast cancer is a heterogeneous disease in which genetic and environmental factors are involved. Breast cancer stem cells (BCSCs) are the main player in the aggressiveness of different tumors and also, these cells are the main challenge in cancer treatment. Moreover, the major obstacle to achieve an effective treatment is resistance to therapies. There are various types of treatment for breast cancer (BC) patients. Therefore, in this review, we present the current treatments, novel approaches such as antibody-drug conjugation systems (ADCs), nanoparticles (albumin-, metal-, lipid-, polymer-, micelle-based nanoparticles), and BCSCs-based therapies. Furthermore, prognostic and predictive biomarkers will be discussed also biomarkers that have been applied by some tests such as Oncotype DX, Mamm αPrint, and uPA/PAI-1 are regarded as suitable prognostic and predictive factors in breast cancer.

Candida albicans Beta-Glucan Induce Anti- Cancer Activity of Mesenchymal Stem Cells against Lung Cancer Cell Line: An In-Vitro Experimental Study.

OBJECTIVE: ß-glucan, glucopyranosyl polymers of fungi cell wall, represent an immune stimulating effects with potential anti-cancer activity. Mesenchymal stem cells (MSC) have immunomodulating properties in cancer microenvironment. The aim of this study was to investigate the anti-cancer effect of Candida albicans (C. albicans) beta-glucan on MSCs supernatant for apoptosis assay of lung cancer cells in vitro. METHODS: Beta-glucan was extracted from cell wall of C.albicans. MSC isolated from adipose tissue of patients and confirmed using specific surface markers expression which examined by flow cytometry. MSCs treated with various concentrations of ß-glucans for 48 hours. Cytotoxic effect of ß-glucans was evaluated using MTT assay. MSC and lung cancer line cocultured and treated with ß-glucans and apoptosis assay was done by flow cytometry. RESULTS: Cytotoxicity findings showed a significant decrease in MSC viability during 48h, however it was dose-dependent (P<0.05). According to the obtained findings, supernatant of mesenchymal stem cells treated with ß-glucans increased cancer cells apoptosis (P<0.05). CONCLUSION: Beta glucan may highlight a potential and novel promising candidate in future strategies to cause apoptosis of cancer cells and consider as therapeutic  agent against tumor growth as well. Definitely, more in vitro and in vivo studies are required to understand its functions.

Evaluation of the frequency of CD5+ B cells as natural immunoglobulin M producers and circulating soluble CD5 in patients with bladder cancer.

Bladder cancer is the most common malignancy of the genitourinary tract. It is the fourth most common malignancy in men and the fifth most common malignancy in the general population, with a high recurrence rate. CD5+ B lymphocytes are a subset of B lymphocytes, which contribute to innate immune responses. These cells are involved in the spontaneous production of self-reactive natural antibodies. On the other hand, natural antibodies can recognize tumor-associated antigens, including proteins or carbohydrates, and eliminate these cells in a complement-dependent manner or via induction of apoptosis. Besides surface CD5, the soluble form of this molecule is involved in the regulation of immune system. Considering the role of CD5+ B cells in the production of natural immunoglobulin M (IgM) and role of these antibodies in antitumor responses, in this study, we aimed to investigate the frequency of CD5 in B cells and to evaluate the diagnostic potential of these cells and also soluble CD5 (sCD5) in patients with bladder cancer. Blood specimens were collected from 40 patients with bladder cancer, who were referred to Sina Hospital in Tehran, IRAN. The levels of CD5+ and CD5- B lymphocytes were measured in the peripheral blood via flow cytometry, and the levels of sCD5 and total IgM were investigated in the serum by ELISA and nephlometry techniques, respectively. The frequency of CD5+ and CD5- B cells was significantly lower in patients, compared with the healthy controls. Detectable levels of sCD5 were found in two patients (5%), while total IgM showed no significant difference between the patient and control groups. The present results suggest that B cell subsets may be affected by malignancy. Therefore, further research is needed to identify B cells and their soluble markers for diagnosis of patients with bladder cancer.

Clinical Value of Serum S100A8/A9 and CA15-3 in the Diagnosis of Breast Cancer.

BACKGROUND AND OBJECTIVE: S100A8/A9 is a heterodimer calcium-binding protein which is involved in tumor cell proliferation, adhesion and invasion, and is proposed as a biomarker for better diagnosis and prognosis in many cancers. The aim of this study was to evaluate the simultaneous serum-based level of S100A8/A9 and CA15-3 as well-illustrated cancer biomarkers, as well as their prognostic value in breast cancer patients and healthy matched controls. MATERIAL AND METHODS: Thirty breast cancer patients at different stages of disease and healthy matched controls with no history of inflammatory, autoimmune diseases, or cancer, were enrolled in the study. The levels of S100A8/A9 and CA15-3 were assessed serologically using the Enzyme-linked immunosorbent assay (ELISA) method, and the relevance of these markers with patients' clinicopathological features were subsequently assessed. RESULTS: Based on our data, the serum levels of both S100A8/A9 and CA15-3 were significantly higher in patients compared to the healthy controls, and thus positively correlated with tumor size. Also, statistical analysis shows that the serum level of S100A8/A9 has 100% specificity and sensitivity (AUC = 1.00, 95% CI) for the diagnosis of breast cancer patients. CONCLUSION: According to our data as well as other observations, the S100A8/A9 heterodimer can be considered as a potential biomarker for the proper diagnosis and prognosis of breast cancer.

Myeloid-derived suppressor cells and tumor: Current knowledge and future perspectives.

The immunosuppressive features of tumor lesions participate not only as one of the major players inducing cancer progression but also a big challenge for effective immunotherapy. It has been found that immunosuppression associated with chronic inflammatory factors, such as growth factors, cytokines, and chemokines is generated by stroma and tumor cells. Chronic and exhaustive secretion of these mediators triggers the generation of myeloid-derived suppressor cells (MDSCs) demonstrating one of the key players engaged in tumor immunosuppression. In point of fact, direct cell-to-cell contact is a prerequisite for immunosuppressive functions of MDSCs. From the clinical perspective, the frequency of peripheral blood MDSCs is correlated with clinical stage and therapeutic response in various cancers. Furthermore, MDSCs are involved in chemoresistant settings. Altogether, it is a rational therapeutic approach to block the fierce cycle in which MDSCs are developed and infiltrated to favor cancer progression. In this review, we will summarize recent findings of MDSCs in tumor progression and discuss potential therapeutic strategies that could be evaluated in future clinical trials.

Dkk-3 as a potential biomarker for diagnosis and prognosis of colorectal cancer.

Background: The Dickkopf 3 (Dkk-3) protein is a member of the Dkk family known as Wnt signaling inhibitor. The level of DKk-3 changes in a wide range of cancers, such as colorectal cancer, lung cancer, prostate cancer, and bladder cancer, is proposed as a biomarker for diagnosis and prognosis of many cancers. The present study was conducted to evaluate the serum level of Dkk-3 as a cancer biomarker and to determine their prognostic value in colorectal cancer (CRC) patients and healthy matched controls. Methods: A total of 30 colorectal cancer patients at different stages of the disease and healthy matched controls with no history of inflammatory and autoimmune disease or cancer were enrolled in the study. The level of Dkk-3 was assessed serologically using enzymelinked immunosorbent assay (ELISA) method, moreover, relevance of these markers with patients' clinicopathological features was subsequently assessed. Means comparison and ROC curves analysis were done using SPSS software. P-value ˂0.05 was considered significant in all the tests. Results: In this study, it was revealed that serum level of Dkk-3 was significantly (p<0.001) lower in patients compared to the healthy controls. Statistical analysis showed that serum level of Dkk-3 has 78% specificity and 77% sensitivity (AUC= 0.782, 95% CI) for diagnosis of colorectal cancer. Conclusion: Dkk-3 protein can be considered as a potential biomarker for diagnosis and possibly the prognosis of colorectal cancer.

Shark cartilage 14 kDa protein as a dendritic cells activator.

Low molecular weight components of shark cartilage are reported to have anti-tumor as well as immuno-stimulating effects. Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that have a key role in establishment of anti-cancer immune response. In this study, the effect of 14 kDa protein from shark cartilage was investigated on stimulation and maturation of dendritic cells. The isolated 14 kDa protein from shark cartilage extract was added to DCs medium during overnight culture and their maturation and T cells stimulation potential was investigated. The majority of shark-cartilage-treated DCs expressed higher levels of maturation markers and were more effective in stimulation of allogenic T cells compared with non-treated DCs (p < 0.05). Our results showed that shark cartilage 14 kDa protein can potentially be used in DC-mediated T-cells stimulation and induction of desirable immune responses in clinical trials such as cancer immunotherapy. However, further studies are required to examine this proposal.

Cytotoxic Effect of Immunotoxin Containing The Truncated Form of Pseudomonas Exotoxin A and Anti-VEGFR2 on HUVEC and MCF-7 Cell Lines.

OBJECTIVE: Immunotoxins (ITs) have been developed for the treatment of cancer, and comprise of antibodies linked to toxins. Also vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis, and the blockade of VEGF receptor-2 (VEGFR2) inhibits angiogenesis and tumor growth. The aim of this study was to produce anti-VEGFR2/rPE (Pseudomonas exotoxin) 38 IT to test its cytotoxic activity and mechanism of action. MATERIALS AND METHODS: In this basic research and experimental study, at first, DNA that encodes recombinant PE38 protein was inductively expressed in Escherichia coli (E.coli) and purified by nickel-sepharose chromatography and further analyzed by western blot. Then, for production of IT, rPE38 was chemically conjugated to anti- VEGFR2. The cytotoxicity response of IT treatment was evaluated by 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) test in Human Umbilical Vein Endothelial Cell (HUVEC) and Michigan Cancer Foundation-7 (MCF-7) (VEGFR2+) cell lines. The mechanism of IT cytotoxicity was observed by Annexin V staining and flow cytometry. Continuous variables were compared with the analysis of variance (ANOVA; for all groups). P values less than 0.05 were considered statistically significant. RESULTS: SDS-PAGE showed 98% purity of rPE38 and IT. In vitro dose-dependent cytotoxicity assay demonstrated that anti-VEGFR2/PE38 is toxic to VEGFR2-positive cells. IT treatment significantly inhibited proliferation of HUVEC and MCF-7 in a VEGFR2-specific manner as compared with the control groups (p<0.05). Flow cytometry showed that the mechanism of IT induced cell death is mediated by apoptosis. CONCLUSION: IT treatment also caused remarkable synergistic cytotoxicity characterized by decreased cell viability, and an increased apoptotic index by both anti-VEGFR2 and PE38. Thus these results raise the possibility of using anti-VEGFR2/PE38 IT for cancer therapy because nearly all tumors induce local angiogenesis with high VEGFR expression.

Evaluation of the Effect of the 47 kDa Protein Isolated from Aged Garlic Extract on Dendritic Cells.

OBJECTIVES: Garlic (Allium sativum) is known as a potent spice and a medicine with broad therapeutic properties ranging from antibacterial to anticancer, and anticoagulant. One of the major purified garlic protein components is the 47 kDa protein. In this study, the effect of 47 kDa protein extracted from aged garlic (AGE) was evalua. MATERIALS AND METHODS: Forty seven kDa protein was purified from AGE by ammonium sulfate precipitation and gel filtration. SDS-PAGE was used to determine the molecular weight and purity of the isolated protein. DCs were purified from spleen of BALB/c mice by Nycodenz centrifugation and their adhesiveness to the plastic dish. The 47 kDa protein isolated from AGE was added to DCs medium during the overnight culture and the expression of DC surface markers was assessed via flowcytometry. RESULTS: The 47 kDa protein-treated DCs lowered the expression of DC maturation markers including: CD40, CD86 and MHC-II in comparison with non-treated DCs; (median of 41% versus 47%, 84% versus 91% and 83% versus 90%, respectively) but we observed no statistical difference between the two groups. CONCLUSION: Upon treatment with DCs with 47 kDa protein, DCs down regulated the expression of costimulatory and MHC-II surface molecules, which is similar to tolerogenic DC phenotype. According to the results of the present study, we found that 47 kDa protein purified from AGE can be considered as a potential candidate to generate tolerogenic DCs in vitro.