A comparative study of long interspersed element-1 protein immunoreactivity in cutaneous malignancies.

BACKGROUND: Skin cancer is the most common cancer worldwide and commonly classified into malignant melanoma (MM) and Nonmelanoma skin cancers (NMSCs), which mainly include basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). The extent to which Long Interspersed Element-1 (LINE-1, L1) ORF1p is expressed in cutaneous malignancies remains to be evaluated. This study aimed to assess LINE-1 ORF1p immunoreactivity in various skin cancer subtypes. METHOD: The expression level of LINE-1 ORF1p was evaluated in 95 skin cancer specimens comprising 36 (37.9%) BCC, 28 (29.5%) SCC, and 31 (32.6%) melanoma using the tissue microarray (TMA) technique. Then the association between expression of LINE-1 encoded protein and clinicopathological parameters was analyzed. RESULTS: We showed that LINE-1 ORF1p expression level was substantially higher in BCC and SCC patients compared with melanoma samples (p < 0.001). BCC cases had a higher LINE-1 histochemical score (H-score) compared with SCC cases (p = 0.004). In SCC samples, a lower level of LINE-1 ORF1p expression was associated with age younger than the mean (p = 0.041). At the same time, no significant correlation was found between LINE-1 ORF1p expression and other clinicopathological parameters (all p > 0.05). CONCLUSIONS: According to our observation, LINE-1 ORF1p immunoreactivity in various skin tumor subtypes extends previous studies of LINE-1 expression in different cancers. LINE-1ORF1p overexpression in NMSCs compared with MM can be considered with caution as a tumor-specific antigen for NMSCs.

Evidence for embryonic stem-like signature and epithelial-mesenchymal transition features in the spheroid cells derived from lung adenocarcinoma.

Identification of the cellular and molecular aspects of lung cancer stem cells (LCSCs) that are suggested to be the main culprit of tumor initiation, maintenance, drug resistance, and relapse is a prerequisite for targeted therapy of lung cancer. In the current study, LCSCs subpopulation of A549 cells was enriched, and after characterization of the spheroid cells, complementary DNA (cDNA) microarray analysis was applied to identify differentially expressed genes (DEGs) between the spheroid and parental cells. Microarray results were validated using quantitative real-time reverse transcription-PCR (qRT-PCR), flow cytometry, and western blotting. Our results showed that spheroid cells had higher clonogenic potential, up-regulation of stemness gene Sox2, loss of CD44 expression, and gain of CD24 expression compared to parental cells. Among a total of 160 genes that were differentially expressed between the spheroid cells and the parental cells, 104 genes were up-regulated and 56 genes were down-regulated. Analysis of cDNA microarray revealed an embryonic stem cell-like signature and over-expression of epithelial-mesenchymal transition (EMT)-associated genes in the spheroid cells. cDNA microarray results were validated at the gene expression level using qRT-PCR, and further validation was performed at the protein level by flow cytometry and western blotting. The embryonic stem cell-like signature in the spheroid cells supports two important notions: maintenance of CSCs phenotype by dedifferentiating mechanisms activated through oncogenic pathways and the origination of CSCs from embryonic stem cells (ESCs). PI3/AKT3, as the most common up-regulated pathway, and other pathways related to aggressive tumor behavior and EMT process can confer to the spheroid cells' high potential for metastasis and distant seeding.

Differential Expression of Cancer Stem Cell Markers ALDH1 and CD133 in Various Lung Cancer Subtypes.

Cancer stem cells (CSCs) are hypothesized to be the main culprit of lung cancer progression. Clinicopathological significance of stem cell markers CD133 and ALDH1 in a large group of lung cancer patients was evaluated. ALDH1 and CD133 had higher expression levels in the NSCLC compared to the SCLC. Over-expression of both ALDH1 and CD133 markers was exclusively found in SCC and ADC. Low level of ALDH1 expression was strongly correlated with poor differentiation in ADC cases. Thus, ALDH1(high)/CD133(high) phenotype can be considered as a CSC marker in some lung cancer subtypes.

Comparative evaluation of SARS-CoV-2 IgG assays against nucleocapsid and spike antigens.

BACKGROUND: There are few studies to compare antibody response against anti-spike (S) and anti- nucleoprotein (N) SARS-CoV-2. OBJECTIVE: The aim of this study was to evaluate the IgG antibody production against S and N antigens of the virus and their correlation with the time and severity of the disease. METHODS: The IgG antibodies against S and N antigens of SARS-CoV-2 in serum specimens of 72 symptomatic patients who tested real-time reverse transcription polymerase chain reaction positive for SARS-CoV-2 were detected using the ELISA technique. Different antibody response was compared and the correlation with the time from disease onset and the severity was evaluated. RESULTS: Forty-eight of 72 (67%) patients tested positive for anti-SARS-CoV-2 antibodies, while 24 (33%) did not have detectable antibodies. Comparison of antibody levels for N and S antibodies showed that they correlate with each other well (r= 0.81; P< 0.001). However, sensitivity of anti-S SARS-CoV-2 IgG and anti-N SARS-CoV-2 IgG was 30% and 60%, during the first 7 days after symptom onset (r= 0.53; P= 0.111), but increased to 73% and 68% at more than 1-week post symptom onset (r= 0.89, P= 0.111), respectively. Cases with positive IgG response showed a decreased CD8+ T cells percentage compared to the negative IgG groups (26 ± 14 vs. 58 ± 32, p= 0.066 in anti-N IgG group and 28 ± 15 vs. 60 ± 45, p= 0.004 in anti-S IgG group, respectively). CONCLUSION: Nearly one-third of the confirmed COVID-19 patients had negative serology results. Lower percent positivity at early time points after symptom onset (less than 1 week) was seen using anti-S SARS-COV-2 IgG kit compare to the anti-N SARS-CoV-2 IgG; therefore, clinicians should interpret negative serology results of especially anti-S SARS-CoV-2 IgG with caution.

Evaluating the Correlation Between the Survival Rate of Patients with Esophageal Squamous Cell Carcinoma and Expression of p53 and Cyclin D1 Biomarkers Along with Other Prognostic Factors.

OBJECTIVE: In the present study, we aimed to evaluate the relationship between the survival rate of patients with esophageal squamous cell carcinoma (SCC) and expression of two biomarkers along with age, gender, tumor margin, depth of invasion, site of tumor, tumor diameter, tumor grade, number of involved nodes, and vascular invasion. MATERIALS AND METHODS: In this retrospective survey, medical records of patients referred to the Shohada-e Tajrish hospital during 2001 to 2005 were reviewed and subjects with definite diagnosis of SCC were included. Required data were extracted from the patients' records, and their prepared paraffin-embedded tissue blocks were collected under supervision of two pathologists. Immunohistochemistry (IHC) analysis was performed at the Firoozgar hospital in Tehran, Iran. RESULTS: The studied population included 20 men (74%) and 7 women (26%). The mean age at diagnosis was 58 ± 22. Results showed significantly higher survival rates in women compared to men (85.7 vs. 40%) (p = 0.001) and in patients with well-differentiated tumors compared to poor-differentiated cases (20 vs. 5%) (p = 0.004). No significant relationship was found between p53 expression and prognostic factors like age, gender, the site, grade, and size of the tumor, depth of invasion, involvement of lymph nodes, and vascular invasion. CONCLUSION: Positivity of p53 and cyclin D1 was not found to be predictive of survival in patients with esophageal SCC which might be due to the small sample size of the present survey. Further investigations with larger sample populations and longer follow-ups are required to evaluate this correlation.

Osteogenic potential of stem cells-seeded bioactive nanocomposite scaffolds: A comparative study between human mesenchymal stem cells derived from bone, umbilical cord Wharton's jelly, and adipose tissue.

Bone regeneration is considered as an unmet clinical need, the aim of this study is to investigate the osteogenic potential of three different mesenchymal stem cells (MSCs) derived from human bone marrow (BM-MSCs), umbilical cord Wharton's jelly (UC-MSCs), and adipose (AD-MSCs) seeded on a recently developed nanocomposite scaffold (bioactive glass/gelatin) implanted in rat animal models with critical size calvarial defects. In this study, after isolation, culture, and characterization, the MSCs were expanded and seeded on the scaffolds for in vitro and in vivo studies. The adhesion, proliferation, and viability of the cells on the scaffolds evaluated in vitro, showed that the scaffolds were biocompatible for further examinations. In order to evaluate the scaffolds in vivo, rat animal models with critical size calvarial defects were randomly categorized in four groups and treated with the scaffolds. The animals were sacrificed at the time points of 4 and 12 weeks of post-implantation, bone healing process were investigated. The histological and immunohistological observations showed (p < 0.01) higher osteogenesis capacity in the group treated with BM-MSCs/scaffolds compared to the other groups. However, the formation of new angiogenesis was evidently higher in the defects filled with UC-MSCs/scaffolds. This preliminary study provides promising data for further clinical trials. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 61-72, 2018.

MicroRNA-31 inhibits RhoA-mediated tumor invasion and chemotherapy resistance in MKN-45 gastric adenocarcinoma cells.

microRNAs are small single-stranded non-coding RNA molecules which modify gene expression by silencing potential target genes. The aberrant expression of RhoA, a small GTPase protein of Rho family, is involved in gastric cancer tumorigenesis. Since miR-31 is a pleomorphic molecule, we evaluated the miR-31/RhoA axis in inducing the malignant phenotype of gastric cancer cells MKN-45. Also, the clinicopathological significance of RhoA was investigated in a well-defined collection of gastric carcinomas which were embedded in tissue microarray blocks. Induction of miR-31 in MKN-45 followed by suppression of RhoA expression resulted in increased sensitivity to 5-fluorouracil, inhibition of cell proliferation, and invasion compared to the control groups. Immunohistochemical analysis in gastric adenocarcinoma patients' samples showed significantly higher expression of RhoA in diffuse versus intestinal subtype tumors ( P = 0.009), poorly differentiated versus well and moderately differentiated tumors ( P = 0.03) and the presence of vascular invasion versus the absence of vascular invasion ( P = 0.04). Our findings suggest a critical role for miR-31, as a tumor suppressor gene, in gastric cancer tumorigenesis by targeting the RhoA. Impact statement Gastric cancer ranks as the third leading cause of cancer-associated deaths worldwide. The RhoA gene encodes a small GTPase protein of Rho family (RhoA) that its dysregulation is associated with cell motility and invasion. A strong line of evidence supports the regulation of RhoA by a number of miRs, including miR-31 in tumors. Our findings revealed that miR-31 is involved in gastric cancer tumorigenesis as a tumor suppressor gene. Through down-regulation of RhoA, miR-31 decreased cell proliferation, migration, and invasion in gastric cancer cells. In addition, induction of miR-31 increased sensitivity to 5-FU; thus, increasing its tissue concentrations could be a potential target for treatment of gastric cancer in the future.

Lgr5High/DCLK1High phenotype is more common in early stage and intestinal subtypes of gastric carcinomas.

BACKGROUND: Gastric carcinoma is the third most common malignancy and is one of the main causes of cancer deaths worldwide. Cancer stem cells (CSCs) are a subpopulation of tumour cells capable of self-renewal and differentiation, likely responsible for the initiation, recurrence, metastasis and chemo/radio-resistance. OBJECTIVE: This study was conducted to evaluate the expression patterns and clinicopathologic significance of putative CSC markers, Lgr5 and DCLK1, in gastric carcinoma. METHOD: The expression levels of Lgr5 and DCLK1 were examined in a well-defined series of gastric carcinoma tissues, including 75 (80%) from intestinal and 19 (20%) from diffuse subtypes, using tissue microarray (TMA). In addition, the correlation of the expression of these markers with clinicopathological factors was explored. RESULTS: Higher expressions of Lgr5 and DCLK1 were mainly detected in intestinal subtypes of gastric carcinomas compared to diffuse subtypes (P= 0.005 and P= 0.050, respectively). We also found a higher expression of Lgr5 and DCLK1 more frequently in well-differentiated gastric carcinoma cases (P< 0.001 and P= 0.007). The combined analysis demonstrated that the co-expression of Lgr5 and DCLK1 (Lgr5High/DCLK1High) was more common in intestinal subtypes (P= 0.025) and well-differentiated gastric carcinoma samples (P< 0.001). Interestingly, there was a significant correlation between Lgr5High/DCLK1High phenotype and early-stage gastric carcinoma specimens (P= 0.045). CONCLUSION: Our findings indicated that the Lgr5High/DCLK1High expression pattern may be considered as a signature phenotype for intestinal subtypes of gastric carcinoma.

Differential role of Wnt signaling and base excision repair pathways in gastric adenocarcinoma aggressiveness.

Aberrant activation of Wnt and base excision repair (BER) signaling pathways are implicated in tumor progression and chemotherapy resistance in gastric adenocarcinoma. This study was conducted to clarify the role of E2F6 and RhoA, components of the Wnt signaling pathway, and SMUG1, a component of the BER pathway in gastric adenocarcinoma. Expression levels and clinicopathological significance of three biomarkers, namely E2F6, RhoA, and SMUG1, as potential signaling molecules involved in tumorigenesis and aggressive behavior, were examined using tissue microarray. Our analysis showed a relative increase in the expression of E2F6 in gastric adenocarcinoma with no lymph node metastasis (χ 2, P = 0.04 and OR, P = 0.08), while overexpression of RhoA and SMUG1 was found more often in the diffuse subtype of gastric adenocarcinoma as compared to the intestinal subtype (χ 2, P = 0.05, OR, P = 0.08 and χ 2, P = 0.001, OR, P = 0.009, respectively). Higher expression of RhoA was frequently seen in tumors with vascular invasion (χ 2, P = 0.01 and OR, P = 0.01). In addition, increased expression of SMUG1 was found more often in poorly differentiated tumors (χ 2, P = 0.01 and OR, P = 0.01). The distinct phenotype of E2F6Low/SMUG1High was more common in poorly differentiated tumors (P = 0.04) and with omental involvement (P = 0.01). The RhoAHigh/SMUG1High expression pattern was significantly more often found in diffuse subtype compared to the intestinal subtype (P = 0.001) as well as in poorly differentiated tumors (P = 0.004). The E2F6Low/SMUG1High and RhoAHigh/SMUG1High phenotypes can be considered as aggressive phenotypes of gastric adenocarcinoma. Our findings also demonstrated the synergistic effect of RhoA and SMUG1 in conferring tumor aggressiveness in diffuse subtype of gastric adenocarcinoma.

Fabrication and in vivo evaluation of an osteoblast-conditioned nano-hydroxyapatite/gelatin composite scaffold for bone tissue regeneration.

In this study, the effects of osteoblast-conditioning on mechanical behavior, biocompatibility, biodegradation and osteoinductive properties of a nano-hydroxyapatite/gelatin (HA/GEL) nanocomposite scaffold was investigated. The scaffold was fabricated using the layer solvent casting combined with the freeze-drying and lamination techniques. The scaffolds were conditioned by culture of osteoblasts on their surface and their elimination by a repeated freeze-thawing process. The potential of the osteoblast-conditioned HA/GEL (HA/GEL/OC) scaffold to support cell adhesion and growth and its cytotoxicity was assessed in vitro using rat mesenchymal stem cells. For in vivo studies, the HA/GEL/OC nanocomposite was implanted in the critical size bone defect created on rat calvarium and studied after 7, 30 and 90 days. The results showed that mechanical and in vitro biological properties of the scaffold were not affected by the process of conditioning. However, in vivo studies demonstrated that osteoblast-conditioning enhanced biocompatibility and osteoinductivity and of the nanocomposite scaffold. The osteoblast conditioning also accelerated collagen content during the bone healing. In the experimental group that received the HA/GEL/OC and MSCs, the newly formed bone occupied almost the entire defect (93.4 ± 3.3%) within 3 months. In conclusion, this study indicates that osteoblast-conditioning is a viable strategy for the development of bone tissue engineering scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2001-2010, 2016.

Clinical significance of putative cancer stem cell marker CD44 in different histological subtypes of lung cancer.

BACKGROUND: According to the cancer stem cell theory, tumors originate from a subset of cells known as cancer stem cells (CSCs) that are responsible for tumor initiation, resistance and relapse. CD44 is a cell adhesion molecule that can aid in the identification of CSCs in various malignancies. OBJECTIVE: The purpose of the current study is to evaluate the expression level and clinical significance of CD44 in lung cancer samples. METHODS: One hundred and ninety-five lung tumor samples including 74 (38%) squamous cell carcinomas (SCC), 61 (31%) adenocarcinomas (ADC), 23 (12%) large cell carcinoma (LCC) in non-small cell lung cancer (NSCLC) group and 37 (19%) small cell lung cancer (SCLC) samples were examined for the expression of CD44 using immunohistochemistry method. The correlation of CD44 expression with clinicopathological parameters as well as Ki-67 status was also assessed. RESULTS: Univariate analysis demonstrated that CD44 expression was significantly higher in NSCLC compared to SCLC (P < 0.001). Among NSCLC, higher level of CD44 expression was found in SCC compared to ADC (P< 0.001) and LCC (P=0.046). Increased expression of CD44 was significantly correlated with higher grade tumors which correspond to poor prognosis in SCC (P=0.012) and the lower level of CD44 expression was more often found in well differentiated ADC tumors (P=0.03). In addition, high expression of CD44 was significantly associated with decreased level of proliferative marker Ki-67 (P=0.04). CONCLUSIONS: CD44 could be a valuable tool for the study of lung CSCs and provide a novel therapeutic target for treatment of the patients with lung cancer in combination with conventional therapy.