Overexpression of Transmembrane Phosphatase with Tensin homology (TPTE) in prostate cancer is clinically significant, suggesting its potential as a valuable biomarker.

PURPOSE: Cancer testis antigens (CTAs) are a family of proteins typically expressed in male testicles but overexpressed in various cancer cell types. Transmembrane Phosphatase with Tensin homology (TPTE) is expressed only in the testis of healthy individuals and is a member of the family of CTAs. The current study, for the first time, examined the significance of TPTE expression in prostate cancer (PCa) tissues by generating a novel antibody marker targeting TPTE protein. METHODS: Polyclonal antibodies were prepared for TPTE-p1 and TPTE-p2 peptides, which are derived from the extracellular domains of TPTE. Anti-TPTE-p2 antibody was then used to study the extent and pattern of TPTE expression in 102 PCa and 48 benign prostatic hyperplasia (BPH) tissue samples by immunohistochemistry. The viability of cancer cell lines (PC-3 and MCF-7 cells) was also evaluated in the presence of anti-TPTE-p2 antibody using the MTT test. RESULTS: The immunohistochemical analysis demonstrated a significant increase in cytoplasmic and membrane TPTE expression in the PCa samples compared to the BPH group (both P < 0.0001). Cytoplasmic TPTE expression was positively correlated with Gleason score and PSA levels (P = 0.03 and P = 0.001, respectively). Significant correlations were identified between the levels of PSA and perineural invasion and the membrane expression (P = 0.01, P = 0.04, respectively). Moreover, anti-TPTE-p2 antibody inhibited PC-3 and MCF-7 cells proliferation compared to the control group for 24 h (P < 0.001 and P = 0.001, respectively) as well as for 48 h (P = 0.001 and P = 0.001, respectively). CONCLUSION: Our findings indicate that increased TPTE expression is associated with progression of disease. The ability of anti-TPTE-p2 antibody to recognize and target the TPTE protein makes it a potential biomarker to assess and/or target the PCa.

Impaired monocyte-derived dendritic cell phenotype in prostate cancer patients: A phenotypic comparison with healthy donors.

BACKGROUND: Dendritic cells (DCs) play a crucial role in immunity. Research on monocyte-derived DCs (Mo-DCs) cancer vaccines is in progress despite limited success in clinical trials. This study focuses on Mo-DCs generated from prostate cancer (PCA) patients, comparing them with DCs from healthy donors (HD-DCs). METHODS: Mo-DCs were isolated from PCA patient samples, and their phenotype was compared to HD-DCs. Key parameters included monocyte count, CD14 expression, and the levels of maturation markers (HLA-DR, CD80, CD86) were assessed. RESULTS: PCA samples exhibited a significantly lower monocyte count and reduced CD14 expression compared to healthy samples (p ⟨ 0.0001). Additionally, PCA-DCs expressed significantly lower levels of maturation markers, including HLA-DR, CD80, and CD86, when compared to HD-DCs (p = 0.123, p = 0.884, and p = 0.309, respectively). CONCLUSION: The limited success of DC vaccines could be attributed to impaired phenotypic characteristics. These observations suggest that suboptimal characteristics of Mo-DCs generated from cancer patient blood samples might contribute to the limited success of DC vaccines. Consequently, this study underscores the need for alternative strategies to enhance the features of Mo-DCs for more effective cancer immunotherapies.

Development of dendritic cell loaded MAGE-A2 long peptide; a potential target for tumor-specific T cell-mediated prostate cancer immunotherapy.

BACKGROUND: Prostate cancer (PCa) is the second leading cause of cancer-related deaths among men worldwide. Immunotherapy is an emerging treatment modality for cancers that harnesses the immune system's ability to eliminate tumor cells. In particular, dendritic cell (DC) vaccines, have demonstrated promise in eliciting a tumor-specific immune response. In this study, we investigated the potential of using DCs loaded with the MAGE-A2 long peptide to activate T cell cytotoxicity toward PCa cell lines. METHODS: Here, we generated DCs from monocytes and thoroughly characterized their phenotypic and functional properties. Then, DCs were pulsed with MAGE-A2 long peptide (LP) as an antigen source, and monitored for their transition from immature to mature DCs by assessing the expression levels of several costimulatory and maturation molecules like CD14, HLA-DR, CD40, CD11c, CD80, CD83, CD86, and CCR7. Furthermore, the ability of MAGE-A2 -LP pulsed DCs to stimulate T cell proliferation in a mixed lymphocyte reaction (MLR) setting and induction of cytotoxic T cells (CTLs) in coculture with autologous T cells were examined. Finally, CTLs were evaluated for their capacity to produce interferon-gamma (IFN-γ) and kill PCa cell lines (PC3 and LNCaP). RESULTS: The results demonstrated that the antigen-pulsed DCs exhibited a strong ability to stimulate the expansion of T cells. Moreover, the induced CTLs displayed substantial cytotoxicity against the target cells and exhibited increased IFN-γ production during activation compared to the controls. CONCLUSIONS: Overall, this innovative approach proved efficacious in targeting PCa cell lines, showcasing its potential as a foundation for the development and improved PCa cancer immunotherapy.

Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli.

Purpose: Medical usage of L-asparaginase (ASNase), the first-line of acute lymphoblastic leukemia treatment, is linked to allergic responses and toxicities, which necessitates the development of new bio-better ASNases. The aim of the current study was in silico design of a novel ASNase with predicted improved enzymatic properties using strategies encompassing sequence-function analysis of known ASNase mutants. Additionally, current study aimed to show that the new enzyme is active. Methods: Based on 21 experimentally reported mutations for ASNase, a virtual library of mutated enzymes with all 7546 possible combinations of up to 4 mutations was generated. Three-dimensional models of proposed mutant enzymes were built and their in silico stabilities were calculated. The most promising mutant was selected for preparing a genetic construct suitable for expression of the designed ASNase in bacterial cells. Results: Computational study predicted that Y176F/S241C double mutation of Escherichia coli ASNase may increase its folding stability. The designed ASNase was expressed in two different E. coli strains (Origami B(DE3) and BL21(DE3)pLysS) and then the soluble fractions prepared from the cell lysates of the host cells were used in enzyme activity assay. Results showed that enzyme activity of soluble fraction from Origami (95.4 ± 7.5 IU/0.1 mL) was four times higher than that of soluble fraction from pLysS (25.8 ± 2.5 IU/0.1 mL). Conclusion: A novel functional double mutant ASNase with predicted improved enzymatic properties was designed and produced in E. coli. The results of the current study suggest a great commercial potential for the identified enzyme in pharmaceutical and industrial applications.

Designing and developing a sensitive and specific SARS-CoV-2 RBD IgG detection kit for identifying positive human samples.

BACKGROUND: More than 3 y into the coronavirus 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to undergo mutations. In this context, the Receptor Binding Domain (RBD) is the most antigenic region among the SARS-CoV-2 Spike protein and has emerged as a promising candidate for immunological development. We designed an IgG-based indirect enzyme-linked immunoassay (ELISA) kit based on recombinant RBD, which was produced from the laboratory to 10 L industry scales in Pichia pastoris. METHODS: A recombinant-RBD comprising 283 residues (31 kDa) was constructed after epitope analyses. The target gene was initially cloned into an Escherichia coli TOP10 genotype and transformed into Pichia pastoris CBS7435 muts for protein production. Production was scaled up in a 10 L fermenter after a 1 L shake-flask cultivation. The product was ultrafiltered and purified using ion-exchange chromatography. IgG-positive human sera for SARS-CoV-2 were employed by an ELISA test to evaluate the antigenicity and specific binding of the produced protein. RESULTS: Bioreactor cultivation yielded 4 g/l of the target protein after 160 h of fermentation, and ion-exchange chromatography indicated a purity > 95%. A human serum ELISA test was performed in 4 parts, and the ROC area under the curve (AUC) was > 0.96 for each part. The mean specificity and sensitivity of each part was 100% and 91.5%, respectively. CONCLUSION: A highly specific and sensitive IgG-based serologic kit was developed for improved diagnostic purposes in patients with COVID-19 after generating an RBD antigen in Pichia pastoris at laboratory and 10 L fermentation scales.

Evaluation of G2013 (α-L-guluronic acid) efficacy on PC-3 cells through inhibiting the expression of inflammatory factors.

Given multiple treatment strategies for prostate cancer, its mortality rate is still high; therefore, novel treatment strategies seem necessary. G2013 or α-L-guluronic acid is a new patented drug with immunomodulatory and anti-inflammatory properties. This study aimed to evaluate the property of G2013 on inflammatory molecules involved in tumorigenesis of prostate cancer. MTT assay was used to assess the effect of the drug on the proliferation of PC-3 cells. Expression of interleukin 8 (IL-8), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), myeloid differentiation factor 88 (MYD-88), cyclooxygenase 2 (COX-2), matrix metalloproteinase-2 (MMP-2), and MMP-9 genes were studied in the PC-3 cells treated with 25 (low dose) or 50 (high dose) µg/mL of G2013 for 24 h using quantitative real-time polymerase chain reaction (qRT-PCR) technique. Protein expression of NF-κB and protein activities of MMP-2 and MMP-9 were assayed using flow cytometry and gelatin zymography, respectively. The expression of COX-2 (p = 0.007 at low dose), MMP-2 (p = 0.023 at low dose, p = 0.002 at high dose), NF-κB (p = 0.004 at low dose) and IL-8 (p < 0.0001 in both doses) genes, NF-κB protein (p < 0.0001 in both doses), and MMP-2 activity (p < 0.0001 in both doses) were significantly reduced in the presence of G2013 as compared to the control group. Cancer cell proliferation was also inhibited under 10-500 µg/mL G2013 treatment. Our results revealed that G2013 has the potential to inhibit PC-3 cell proliferation and reduce the expression of tumour-promoting mediators, COX-2, MMP-2, NF-κB, and IL-8 involved in the progression and metastasis of prostate cancer.

Anti-tumor effect of M2000 (ß-d-mannuronic acid) on the expression of inflammatory molecules in the prostate cancer cell.

Aim: The importance of chronic inflammation during the progression of prostate cancer (PCa) is well-known. M2000 (ß-d-mannuronic acid) is a novel anti-inflammatory drug. According to its potential capacity for the inhibition of molecules involved in creating conditions of inflammation, it is reasonable to assess the anti-inflammatory role of M2000 in PCa cells.Methods: MTT assay was performed to determine the cytotoxicity of M2000 in PC3 cells. Correspondingly, these cells were cultured and then treated with low (25 µg/ml) and high (50 µg/ml) doses of M2000 as optimal doses. Thereafter, real-time RT-PCR, flow cytometry analysis, and zymography were performed to evaluate the expressions of MYD-88, NF-kB, IL-8, COX-2, MMP-2, and MMP-9 molecules. Results: Of note, the M2000 at the concentration of ≤200 µg/ml had no cytotoxicity effect on the cells. MYD-88 gene expression was significantly down-regulated at both low and high doses in the M2000-treated cells compared to the control (p = .017 and p = .001, respectively). The expression of the NF-kB was also reduced at both the gene and protein levels (all p values were <.001). The expression of IL-8 and COX-2 genes was also down-regulated in the high dose of M2000 (p<.001, p = .001, respectively). The decreased expression of the MMP-9 gene was observed at both doses (both p values were <.001).Conclusion: Inhibitory effects of M2000 on the activity of MMPs in the LPS/M2000-treated cells were evident, but not in the M2000-treated cells. M2000 as a new anti-inflammatory drug appears to constitute a potential agent for down-regulation of inflammatory molecules in the PCa cells.

The Anti-Proliferative Effect of a Newly-Produced Anti-PSCA-Peptide Antibody by Multiple Bioinformatics Tools, on Prostate Cancer Cells.

BACKGROUND: Prostate Stem Cell Antigen (PSCA) is a small cell surface protein, overexpressed in 90% of prostate cancers. Determination of epitopes that elicit an appropriate response to the antibody generation is vital for diagnostic and immunotherapeutic purposes for prostate cancer treatment. Presently, bioinformatics B-cell prediction tools can predict the location of epitopes, which is uncomplicated, faster, and more cost-effective than experimental methods. OBJECTIVE: We aimed to predict a novel linear peptide for Prostate Stem Cell Antigen (PSCA) protein in order to generate anti-PSCA-peptide (p) antibody and to investigate its effect on prostate cancer cells. METHODS: In the current study, a novel linear peptide for PSCA was predicted using in silico methods that utilize a set of linear B-cell epitope prediction tools. Polyclonal antibody (anti-PSCA-p antibody "Patent No. 99318") against PSCA peptide was generated. The antibody reactivity was determined by the Enzyme-Linked Immunosorbent Assay (ELISA) and its specificity by immunocytochemistry (ICC), immunohistochemistry (IHC), and Western Blotting (WB) assays. The effect of the anti-PSCA-p antibody on PSCA-expressing prostate cancer cell line was assessed by Methylthiazolyldiphenyl- Tetrazolium bromide (MTT) assay. RESULTS: New peptide-fragment of PSCA sequence as "N-CVDDSQDYYVGKKN-C" (PSCA-p) was selected and synthesized. The anti-PSCA-p antibody against the PSCA-p showed immunoreactivity with PSCA-p specifically bound to PC-3 cells. Also, the anti-PSCA-p antibody strongly stained the prostate cancer tissues as compared to Benign Prostatic Hyperplasia (BPH) and normal tissues (P < 0.001). As the degree of malignancy increased, the staining intensity was also elevated in prostate cancer tissue (P < 0.001). Interestingly, the anti-PSCA-p antibody showed anti-proliferative effects on PC-3 cells (31%) with no growth inhibition effect on PSCA-negative cells. CONCLUSION: In this study, we developed a new peptide sequence (PSCA-p) of PSCA. The PSCA-p targeting by anti-PSCA-p antibody inhibited the proliferation of prostate cancer cells, suggesting the potential of PSCA-p immunotherapy for future prostate cancer studies.

Opportunities and challenges of the tag-assisted protein purification techniques: Applications in the pharmaceutical industry.

Tag-assisted protein purification is a method of choice for both academic researches and large-scale industrial demands. Application of the purification tags in the protein production process can help to save time and cost, but the design and application of tagged fusion proteins are challenging. An appropriate tagging strategy must provide sufficient expression yield and high purity for the final protein products while preserving their native structure and function. Thanks to the recent advances in the bioinformatics and emergence of high-throughput techniques (e.g. SEREX), many new tags are introduced to the market. A variety of interfering and non-interfering tags have currently broadened their application scope beyond the traditional use as a simple purification tool. They can take part in many biochemical and analytical features and act as solubility and protein expression enhancers, probe tracker for online visualization, detectors of post-translational modifications, and carrier-driven tags. Given the variability and growing number of the purification tags, here we reviewed the protein- and peptide-structured purification tags used in the affinity, ion-exchange, reverse phase, and immobilized metal ion affinity chromatographies. We highlighted the demand for purification tags in the pharmaceutical industry and discussed the impact of self-cleavable tags, aggregating tags, and nanotechnology on both the column-based and column-free purification techniques.

Epitope Prediction by Novel Immunoinformatics Approach: A State-of-the-art Review.

Immunoinformatics is a science that helps to create significant immunological information using bioinformatics softwares and applications. One of the most important applications of immunoinformatics is the prediction of a variety of specific epitopes for B cell recognition and T cell through MHC class I and II molecules. This method reduces costs and time compared to laboratory tests. In this state-of-the-art review, we review about 50 papers to find the latest and most used immunoinformatic tools as well as their applications for predicting the viral, bacterial and tumoral structural and linear epitopes of B and T cells. In the clinic, the main application of prediction of epitopes is for designing peptide-based vaccines. Peptide-based vaccines are a considerably potential alternative to low-cost vaccines that may reduce the risks related to the production of common vaccines.

In-vitro prostate cancer biomarker detection by directed conjugation of anti-PSCA antibody to super paramagnetic iron oxide nanoparticless.

Background: The main property of a successful conjugation of antibodies to nanoparticles is keeping the potency of antibody for binding the antigen, and an oriented conjugation can do that. Under such ground, this study was carried out to explore the efficiency of two conjugation methods in binding iron nanoparticles to an antibody produced against PSCA (prostate stem cell antigen) using in vitro labeling of PC3 cells. Methods: In this experimental study, we conjugated dextran-superparamagnetic iron oxide nanoparticles (dexSPIONs) to anti-PSCA antibody by two different methods, including targeting carbohydrate moieties in FC domain and the free amine group of amino acid side chains. Ultimately, Iron staining was done by anti-PSCA antibody-dexSPIONs in PC3 cells to detect antibody binding to the cells. Results: A strong blue dye was induced by iron staining in conjugated dexSPIONs on the membrane of PC3 cells by the former method than the second one. Moreover, cells treated with 20 nm diameters of dexSPIONs showed higher resolution of blue color than those treated with 100 nm nanoparticles. Conclusion: This oriented conjugation method promoted the efficiency of targeting tumor antigens, and the presence of iron particles might enhance MRI image intensity in vivo by targeting PSCA-overexpressing cells in future studies.

Co-expression of TLR-9 and MMP-13 is associated with the degree of tumour differentiation in prostate cancer.

In vitro experiments demonstrated that stimulation of Toll-like receptor 9 (TLR-9) by synthetic TLR-9 ligands induces the invasion of TLR-9-expressing prostate cancer cells through matrix metalloproteinase 13 (MMP-13). However, the clinical value of TLR-9 and MMP-13 co-expression in the pathophysiology of the prostate is unknown. In the study, we evaluated the expression levels and clinical significance of the TLR-9 and MMP-13 in a series of prostate tissues. One hundred and eighty prostate tissues including prostate cancer (PCa) (n = 137), high-grade prostatic intraepithelial neoplasia (HPIN) (n = 18) and benign prostatic hyperplasia (BPH) (n = 25) were immunostained for the TLR-9 and MMP-13 markers. Subsequently, the correlation between the TLR-9 and MMP-13 staining scores and clinicopathological parameters was obtained. Higher expressions of TLR-9 and MMP-13 were found in PCa and high-grade prostatic intraepithelial neoplasia compared to benign prostatic hyperplasia tissues. Among PCa samples, a positive relationship was revealed between the MMP-13 expression and Gleason score (P < 0.001). There was a significant correlation between TLR-9 expression and regional lymph node involvement (P = 0.04). The expression patterns of TLR-9 and MMP-13 markers demonstrated a reciprocal significant correlation between the two markers in the same series of prostate samples (P < 0.001). Furthermore, the Gleason score of TLR-9high /MMP-13high and TLR-9low /MMP-13low phenotypes showed a significant difference (P = 0.002). Higher expressions of TLR-9 and MMP-13 can confer aggressive behaviour to PCa. Therefore, these markers may be used as a valuable target for tailored therapy of PCa.

Autoantibody against new gene expressed in prostate protein is traceable in prostate cancer patients.

AIM: We assessed an autoantibody against new gene expressed in prostate (NGEP) protein for prostate cancer (PCa) that may better diagnosis and prognosis approaches in the patients with PCa. METHODS: Autoantibodies against NGEP were measured in sera of PCa patients by ELISA. RESULTS: The autoantibody against NGEP is present in a significantly higher proportion in the sera of PCa patients as compared with healthy controls (p < 0.001). An inverse significant correlation was found between seropositive patients and Gleason score (p < 0.05) and serum prostate-specific antigen (recombinant NGEP; p < 0.05). CONCLUSION: The data showed that measurement of autoantibody against NGEP as a novel prostate-specific antigen in sera can be used as a potential biomarker to discriminate well-differentiated PCa patients from normal subjects.

Anti-Oxidants as Chemopreventive Agents in Prostate Cancer: A Gap Between Preclinical and Clinical Studies.

BACKGROUND: Tumor cells may be expressed as a result of oxidative stress. The extent of oxidative stress correlates with the aggressive and metastatic potency of cancer. OBJECTIVE: One simple way to control prostate cancer is through chemoprevention which refers to the administration of natural or synthetic agents to block, reverse, or delay the process of carcinogenesis. The most chemopreventive agents are antioxidants in nature. METHODS: In this review, we summarized the effects of dietary antioxidants with a focus on their molecular mechanisms and possible roles in the treatment of prostate cancer cells. We also reported the recent outcomes of laboratory and/or clinical trials of antioxidants in prostate cancer patients. RESULTS: Numerous pre-clinical studies showed that antioxidants protect DNA against being damaged by Reactive Oxygen Species (ROS), thereby genetic mutations causing cancer are likely to be prevented. However, the clinical trial results showed that antioxidants have yielded mixed outcomes or benefitted only a subgroup of the population. CONCLUSION: A greater understanding of the molecular events associated with antioxidants will enhance the development of treatment and could result in better strategies for the chemoprevention of prostate cancer. Recent patents also suggest that anti-oxidant compounds can be effective for the prevention and the treatment of prostate cancer.

Repair of rat critical size calvarial defect using osteoblast-like and umbilical vein endothelial cells seeded in gelatin/hydroxyapatite scaffolds.

The present study used a previously developed three-dimensional Gelatin/Hydroxyapatite (Gel/HA) homogeneous nanocomposite scaffold with porosity of 82% and interconnecting pores ranging from 300 to 500 µm. Cell-seeded scaffolds were used to evaluate bone regeneration of rat critical-size calvarial defect. Totally, 36 male Wistar rats were randomly divided into four experimental groups, including blank defect (defects without any graft), blank scaffold (defects filled with Gel/HA scaffold without cells), and two groups of cell-seeded scaffolds (defects filled with either Gel/HA scaffold seeded with osteoblast-like and endothelial cells or osteoblast-like cell-seeded constructs). After 1, 4, and 12 weeks of scaffold implantation, rats were sacrificed and the calvaria were harvested for histological, immunohistochemical and histomorphometric analysis. In vitro tests showed that scaffolds were nontoxic to cells and promoted ideal cellular attachment. In vivo investigation on scaffold revealed that blank calvarial defects indicated incomplete tissue coverage and little evidence of bone healing. However, blank scaffold and cell-seeded scaffolds significantly promoted osteoconduction and ostegogenesis. Taken together, pre-seeded Gel/HA nanocomposite scaffold with osteoblasts and endothelial cells presented an effective combination to improve osteogenesis in the engineered bone implant. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1770-1778, 2016.

Establishment of a new immunological method for direct detection of Mycobacterium in solution.

BACKGROUND/PURPOSE: Tuberculosis (TB) is a crucial health problem. Prevention of the disease requires rapid diagnosis. Rapid liquid culture systems, nucleic acid amplification tests, and high-performance liquid chromatography (HPLC) are among the rapid tests used for detecting Mycobacterium species. However, these tests are expensive and require extensive equipment and expertise, which is hardly affordable in resource-poor countries. Although direct microscopy is performed routinely as an initial step for detection of the bacteria, it is not sufficiently sensitive. As a result, we thought of establishing a low-cost immunological test that can potentially replace direct microscopy with higher sensitivity and specificity. METHODS: The assay is based on pre-incubation of biotinylated rabbit antibody against Antigen 60 (A60) with a solution containing Bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis (MTB) followed by incubation with a streptavidin-alkaline phosphatase (STA-ALP) conjugate. The test is devised in enzyme-linked immunosorbent assay (ELISA) and non-ELISA formats, therefore it does not require extensive facilities and expertise. RESULTS: The ELISA format showed a 100-fold improvement in the lower detection limit of BCG compared with direct microscopy. With the non-ELISA formats, there was a 2- and 16-fold improvement for the cartridge assay and the microfuge tube assay, respectively. CONCLUSION: In conclusion, we successfully detected BCG and MTB in solution using the new immunological method. Our results are very promising and the new immunological method could potentially replace direct microscopy with higher sensitivity and specificity.

Preparation of Immunotoxin Herceptin-Botulinum and Killing Effects on Two Breast Cancer Cell Lines.

BACKGROUND: Worldwide, breast cancer is the most common cancer diagnosed among women and a leading cause of cancer deaths. The age of onset in Iran has become reduced by a decade for unknown reasons. Herceptin, a humanized monoclonal antibody, is a target therapy for breast cancer cells with over expression of HER2- neu receptors, but it is an expensive drug with only 20% beneficial rate of survival. This study introduces a novel approach to enhance the efficacy of this drug through immunoconjugation of the antibody to botulinum toxin. Decreasing the cost and adverse effects of the antibody were secondary goals of this study. MATERIALS AND METHODS: Botulinum toxin was conjugated with Herceptin using heterobifunctional cross linkers, succinimidyl acetylthiopropionate (SATP) and sulfo-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) according to the supplier's guidelines and tested on two breast cancer cell lines: SK-BR-3 and BT-474. Toxin and Herceptin were also used separately as controls. The cytotoxicity assay was also performed using the new bioconjugate on cultured cells with Alamar blue and a fluorescence plate reader. RESULTS: Herceptin-Toxin bioconjugation significantly improved Herceptin efficacy on both breast cancer cell lines when compared to the control group. CONCLUSIONS: Toxin-Herceptin bioconjugation can be a potential candidate with increased efficiency for treating breast cancer patients with over expression of the HER2 receptor.

Study of NGEP expression in androgen sensitive prostate cancer cells: A potential target for immunotherapy.

BACKGROUND: Prostate cancer is one of the leading causes of cancer deaths among men. New gene expressed in prostate (NGEP), is a prostate-specific gene expressed only in normal prostate and prostate cancer tissue. Because of its selective expression in prostate cancer cell surface, NGEP is a potential immunotherapeutic target. To target the NGEP in prostate cancer, it is essential to investigate its expression in prostate cancer cells. METHODS: In the present study, we investigated NGEP expression in LNCaP and DU145 cells by real time and RT-PCR, flow cytometric and immunocytochemical analyses. RESULTS: Real time and RT-PCR analyses of NGEP expression showed that NGEP was expressed in the LNCaP cells but not in DU145 cells. The detection of NGEP protein by flow cytometric and immunocytochemistry analyses indicated that NGEP protein was weakly expressed only in LNCaP cell membrane. CONCLUSION: Our results demonstrate that LNCaP cell line is more suitable than DU145 for NGEP expression studies; however, its low-level expression is a limiting issue. NGEP expression may be increased by androgen supplementation of LNCaP cell culture medium.

Study of NGEP expression pattern in cancerous tissues provides novel insights into prognostic marker in prostate cancer.

AIM: The aim of this study was to produce a novel polyclonal antibody against extracellular domain of NGEP protein and explore its role in prognosis of prostate cancer. MATERIALS & METHODS: Polyclonal antibodies against two peptides (NGEP-p1 and NGEP-p2) derived extracellular domains of NGEP were prepared and the intensity and distribution of NGEP expression analyzed in large series of prostate tissue specimens. RESULTS: We found a significant inverse correlation between NGEP expression and prognostic features such as Gleason score, pathologic tumor stage and prostate-specific antigen level using anti-NGEP-p2 antibody. CONCLUSION: The results indicate that the high level of expression could be associated with good prognosis in prostate cancer. However, additional studies are required to evaluate NGEP as an independent prognostic factor for prostate carcinoma.

Leucine-rich amelogenin peptide (LRAP) as a surface primer for biomimetic remineralization of superficial enamel defects: An in vitro study.

This study was carried out to obtain more information about the assembly of hydroxyapatite bundles formed in the presence of Leucine-Rich Amelogenin Peptide (LRAP) and to evaluate its effect on the remineralization of enamel defects through a biomimetic approach. One or 2 mg/mL LRAP solutions containing 2.5 mM of Ca(+2) and 1.5 mM phosphate were prepared (pH = 7.2) and stored at 37 °C for 24 h. The products of the reaction were studied using atomic force microscopy (AFM), transmission electron microscopy (TEM), and selected area electron diffraction (SAED). Vickers surface microhardness recovery (SMR%) of acid-etched bovine enamel, with or without LRAP surface treatment, were calculated to evaluate the influence of peptide on the lesion remineralization. Distilled water and 1 or 2 mg/mL LRAP solution (pH = 7.2) were applied on the lesions and the specimens were incubated in mineralization solution (2.5mM Ca(+2) , 1.5mM PO4 (-3) , pH = 7.2) for 24 h. One-way ANOVA and Tukey's multi-comparison tests were used for statistical analysis. The pattern of enamel surface repair was studied using FE-SEM. AFM showed the formation of highly organized hierarchical structures, composed of hydroxyapatite (HA) crystals, similar to the dental enamel microstructure. ANOVA procedure showed significant effect of peptide treatment on the calculated SMR% (p < 0.001). Tukey's test revealed that peptide treated groups had significantly higher values of SMR%. In conclusion, LRAP is able to regulate the formation of HA and enhances the remineralization of acid-etched enamel as a surface treatment agent.