Exp Mol Pathol [journal]
- Chlamydia and ocular adnexal lymphomas: An Indian experience. [JOURNAL ARTICLE]
- Exp Mol Pathol 2016 Jul 16.
AN INDIAN EXPERIENCE: Ocular adnexal lymphomas (OALs) are a heterogeneous group of malignancies, majority being extranodal mucosa-associated lymphoid tissue (MALT) type. Different geographical regions have reported association of Chlamydia with OALs (MALT type). In India, role of Chlamydia in OALs remains unexplored. The aim of this study was to detect Chlamydia and to correlate with clinicopathological features of OALs in India. The clinicopathological features of 41 OAL cases were studied prospectively. Chlamydia DNA was detected by genus specific PCR amplifying major outer membrane protein (MOMP) gene followed by DNA sequencing. Chlamydia immunoexpression was evaluated by immunofluorescence and immunohistochemistry. The results were correlated with clinicopathological features including follow-up and survival. Chlamydia genome was detected in 3/41 (7.3%) OAL cases by PCR. Direct sequencing revealed C. trachomatis in 3 positive cases. Immunofluorescence and immunohistochemistry showed Chlamydia antigen in 5/41 and 1/41 cases respectively. Immunofluorescence demonstrated higher sensitivity than immunohistochemistry. A significant association was observed between Chlamydia positivity and orbital location (P=0.05). Follow-up revealed relapse in 2 Chlamydia positive cases (P=0.056). Our results demonstrate for the first time presence of C. trachomatis genome in 7.3% OAL cases in India. As no other reports are documented, more detailed studies from different regions within India are needed to explore status of Chlamydia in OALs.
- Upregulation of autophagy components in alcoholic hepatitis and nonalcoholic steatohepatitis. [JOURNAL ARTICLE]
- Exp Mol Pathol 2016 Jul 16; 101(1):81-88.
There are many homeostatic mechanisms for coping with stress conditions in cells, including autophagy. In many studies autophagy, as an intracellular pathway which degrades misfolded and damaged protein, and Mallory-Denk Body (MDB) formation have been shown to be protective mechanisms against stress such as alcoholic hepatitis. Alcohol has a significant role in alteration of lipid homeostasis, sterol regulatory element-binding proteins (SREBPs) and peroxidase proliferator-activated receptors through AMP-activated protein kinase (AMPK)-dependent mechanism. AMPK is one of the kinases that regulate autophagy through the dephosphorylation of ATG1. Activation of ATG1 (ULK kinases family) activates ATG6. These two activated proteins relocate to the site of initial autophagosome and activate the other downstream components of autophagocytosis. Many other proteins regulate autophagocytosis at the gene level. CHOP (C/EBP homologous protein) is one of the most important parts of stress-inducible transcription that encodes a ubiquitous transcription factor. In this report we measure the upregulation of the gene that are involved in autophagocytosis in liver biopsies of alcoholic hepatitis and NASH. Electron microscopy was used to document the presence of autophagosomes in the liver cells. Expression of AMPK1, ATG1, ATG6 and CHOP in ASH were significantly (p value<0.05) upregulated in comparison to control. Electron microscopy findings of ASH confirmed the presence of autophagosomes, one of which contained a MDB, heretofore undescribed. Significant upregulations of AMPK-1, ATG-1, ATG-6, and CHOP, and uptrending of ATG-4, ATG-5, ATG-9, ATR, and ATM in ASH compared to normal control livers indicate active autophagocytosis in alcoholic hepatitis.
- Overexpression of DLX2 is associated with poor prognosis and sorafenib resistance in hepatocellular carcinoma. [JOURNAL ARTICLE]
- Exp Mol Pathol 2016 Jun 11; 101(1):58-65.
The mechanism underlying poor prognosis and sorafenib resistance in patients with hepatocellular carcinoma (HCC) is unknown and, to date, no useful predictive biomarkers of sorafenib resistance have been identified. Distal-less homeobox 2 (DLX2) is a transcription factor involved in cell cycle regulation that is closely correlated with cancer prognosis. In this study, we showed that DLX2 is overexpressed in HCC tissues and cell lines and that the level of DLX2 overexpression is positively correlated with histological grade, metastasis and Ki67 expression, which are indicators of poor prognosis. We also found that DLX2 accumulates in proliferating HCC cells, where it is associated with the expression of proliferating cell nuclear antigen (PCNA), Cyclin D1 and Cyclin A. Flow cytometry and cell counting kit-8 (CCK-8) assays indicated that DLX2 depletion causes cell cycle arrest at the G1 phase and hinders cell proliferation. Moreover, the sensitivity of HCC cells to sorafenib is restored when the DLX2 gene is knocked down using a short interfering RNA. We demonstrated that DLX2 facilitates sorafenib resistance by promoting the expression of markers of epithelial-mesenchymal transition and by activating the extracellular signal-regulated protein kinase pathway. Our findings reveal that DLX2 plays a regulatory role in HCC cell proliferation and suggests that targeting DLX2 represents a novel strategy to increase sorafenib efficacy in the management of HCC. In conclusion, DLX2 is a novel marker of poor prognosis and sorafenib resistance in patients with HCC.
- c-Yes enhances tumor migration and invasion via PI3K/AKT pathway in epithelial ovarian cancer. [JOURNAL ARTICLE]
- Exp Mol Pathol 2016 Jun 8; 101(1):50-57.
Overexpression of c-Yes has been noted to correlation with several human cancers. However, the effects of c-Yes on epithelial ovarian cancer (EOC) development remain unclear. The aim of this study is going to prove the effects of c-Yes and related mechanisms in proliferation, metastasis and invasion of EOC. Immunohistochemical analysis was performed in 119 human EOC samples, and the data was correlated with clinic pathologic features. Furthermore, western blot analysis is performed for c-Yes in EOC samples and cell lines to evaluate their protein levels and molecular interaction. Kaplan-Meier survival analysis shows that the strong expression of c-Yes exhibited a significant correlation with poor prognosis in human EOC (P<0.01(⁎)). Meanwhile, we found that knockdown of c-Yes by shRNA inhibited the ability of migration and invasion in EOC cells via the PI3K/AKT pathway. In a word, these results suggested that c-Yes plays an important role in migration and invasion of EOC.
- Hyperoxia disrupts the intestinal barrier in newborn rats. [JOURNAL ARTICLE]
- Exp Mol Pathol 2016 Jun 4; 101(1):44-49.
Animal studies have demonstrated that neonatal hyperoxia injures the distal small intestine. This study aimed to determine the effects of neonatal hyperoxia exposure on the intestinal morphology and intestinal barrier integrity in newborn rats. Sprague-Dawley rat pups were exposed to either ambient air or hyperoxia. The ambient air and normobaric hyperoxia groups were maintained in room air and 85% O2 for 2weeks, respectively. The rats were euthanized on Postnatal Day 14, and the terminal ileum was collected for histological analyses and oxidative stress measurements. The generation of reactive oxygen species was evaluated by measuring the production of 8-hydroxy-2'-deoxyguanosine (8-OHdG). The expression and localization of epithelial injury markers [intestinal fatty acid binding protein (I-FABP)] and intestinal barrier proteins [occludin and zonula occludens (ZO)-1] were analyzed through immunofluorescence staining and western blotting. The body weight at birth was comparable between the two groups. On Postnatal Day 14, the rats in the hyperoxia group exhibited significantly lower body weight, a higher serum interleukin-6 level, a higher intestinal injury score, higher 8-OHdG and I-FABP expression, and lower occludin and ZO-1 protein expression than did those in the ambient air group. Hyperoxia exposure injured the distal small intestine and disrupted the intestinal barrier in newborn rats. This may be attributable to oxidative stress during the postnatal period.
- Alterations in glucose metabolism proteins responsible for the Warburg effect in esophageal squamous cell carcinoma. [JOURNAL ARTICLE]
- Exp Mol Pathol 2016 May 31; 101(1):66-73.
Esophageal squamous cell carcinoma (ESCC) is the most frequent esophageal tumor in the world. ESCC presents late diagnosis, highly aggressive behavior and poor survival. Changes in tumor cell energy metabolism appear to have a prominent role in malignant transformation. Tumor cells consume glucose avidly and produce lactic acid, even under normoxia. Among the factors that may contribute to the stimulation of glycolysis in tumor cells, there are changes in the glycolytic pathway enzymes such as: pyruvate kinase M1 and M2 (PKM2 and PKM1), hexokinase II (HKII), glucose transporter isoform 1 (GLUT-1), and transcription factor induced by hypoxia (HIF1α), responsible for the transcription of proteins cited. The objective of this study is to evaluate the alterations of these proteins and their association with clinicopathological data in ESCC. We performed immunohistochemistry to determine HIF-1α, GLUT-1, PKM1, PKM2, HK2 and Ki67-expression in ESCC patients and controls. Also, we used RT-qPCR to evaluated mRNA expression of GLUT-1 in esophageal mucosa of individuals without cancer, but are alcohol drinkers and tobacco smokers. Our results showed the exclusively expression of GLUT-1 in tumors cells and dysplastic samples. We also observed a compartmentalization of the expression of PKM1 and PKM2 in relation to tumor cells and stroma associated to tumor areas. All of the proteins evaluated, excepted GLUT-1, were frequently detected in normal mucosa. No correlations between clinicopathological features and protein expressions were observed. GLUT-1 expression appears in initial tumor lesions and is maintained through ESCC evolution. We reported for the first time PKM1 staining in normal esophagus and ESCC, being mostly present in more differentiated cells.
- The suppressor of cytokine signaling SOCS1 promotes apoptosis of intestinal epithelial cells via p53 signaling in Crohn's disease. [JOURNAL ARTICLE]
- Exp Mol Pathol 2016 May 25; 101(1):1-11.
The suppressor of cytokine signaling SOCS1 is a member of the cytokine signaling pathway inhibitor family, which is induced by the IFN-γ induced JAK signaling pathway. The expression of SOCS1 has been found to increase in Crohn's disease (CD) patients, but the role of SOCS1 in intestinal epithelium is unclear. This study was designed to investigate whether SOCS1 has a role in the death of intestinal epithelial cells and intestinal injury. The results showed that the expression of SOCS1 increased in CD patients, and the expression of SOCS1, p-p53 and PUMA increased in the mouse TNBS induced colitis model. Using IFN-γ treated HT-29 cells as an apoptotic model of intestinal epithelial cells in vitro, we confirmed that SOCS1 promoted apoptosis of intestinal epithelial cells by activating p53. In HT-29 cells which were treated with IFN-γ, the interaction between p53 and SOCS1 and phosphorylation of p53 were significantly higher than untreated cells. When knocking SOCS1 down by using SOCS1 siRNA, phosphorylation of p53 and apoptosis of intestinal epithelial cells which was induced by IFN-γ were significantly inhibited. In summary, our findings suggest that SOCS1 may promote apoptosis of intestinal epithelial cells at least partly through mediating p53 signaling.
- Exosomal formulation enhances therapeutic response of celastrol against lung cancer. [JOURNAL ARTICLE]
- Exp Mol Pathol 2016 May 24; 101(1):12-21.
Celastrol (CEL), a plant-derived triterpenoid, is a known inhibitor of Hsp90 and NF-κB activation pathways and has recently been suggested to be of therapeutic importance in various cancers. However, the molecular mechanisms of celastrol-mediated effects in lung cancer are not systematically studied. Moreover, it suffers from poor bioavailability and off-site toxicity issues. This study aims to study the effect of celastrol loaded into exosomes against two non-small cell-lung carcinoma (NSCLC) cell lines and explore the molecular mechanisms to determine the proteins governing the cellular responses. We observed that celastrol inhibited the proliferation of A549 and H1299 NSCLC cells in a time- and concentration-dependent manner as indexed by MTT assay. Mechanistically, CEL pre-treatment of H1299 cells completely abrogated TNFα-induced NF-κB activation and upregulated the expression of ER-stress chaperones Grp 94, Grp78, and pPERK. These changes in ER-stress mediators were paralleled by an increase in apoptotic response as evidenced by higher annexin-V/PI positive cells evaluated by FACS and immunoblotting which showed upregulation of the ER stress specific pro-apoptotic transcription factor, GADD153/CHOP and alteration of Bax/Bcl2 levels. Exosomes loaded with CEL exhibited enhanced anti-tumor efficacy as compared to free CEL against lung cancer cell xenograft. CEL did not exhibit any gross or systemic toxicity in wild-type C57BL6 mice as determined by hematological and liver and kidney function test. Together, our data demonstrate the chemotherapeutic potential of CEL in lung cancer and that exosomal formulation enhances its efficacy and reduces dose related toxicity.
- Effects of IL-33/ST2 pathway in acute inflammation on tissue damage, antioxidative parameters, magnesium concentration and cytokines profile. [JOURNAL ARTICLE]
- Exp Mol Pathol 2016 May 21; 101(1):31-37.
The aim of this study was to examine the role of IL-33/ST2 pathway in a pathogenesis of acute inflammation and its effects on tissue damage, antioxidative capacity, magnesium concentration and cytokine profile in acutely inflamed tissue.Male mice were randomly divided in four groups: wild-type control group (WT-C), ST2 knockout control group (KO-C), wild-type inflammatory group (WT-I), and ST2 knockout inflammatory group (KO-I). Acute inflammation was induced in WT-I and KO-I by intramuscular injection of turpentine oil, while mice in WT-C and KO-C were treated with saline. After 12h, animals were euthanized, and blood was collected for determination of creatine kinase (CK) and aspartate transaminase (AST) activity. The treated tissue was used for histopathological analysis, determination of volume density of inflammatory infiltrate (Vdii) and necrotic fiber (Vdnf), gene expression of interleukin (IL)-33, ST2, tumor necrosis factor alpha (TNF-alpha), IL-6, IL-12p35, and transforming growth factor beta (TGF-beta), concentration of magnesium (Mg), copper (Cu), selenium (Se), manganese (Mn) and reduced glutathione (GSH), and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity.Presence of inflammatory infiltration and necrosis in the treated tissue was histopathologically confirmed in WT-I and KO-I. Vdii was significantly higher in WT-I when compared to KO-I, whereas Vdnf did not significantly differ between WT-I and KO-I. CK and AST significantly increased in both inflammatory groups when compared to corresponding control groups. However, the values of CK and AST were significantly higher in WT-I than in KO-I. Mg in the treated tissue was significantly lower in WT-I in comparison to WT-C and KO-I, while there was no significant difference between KO-C and KO-I. There was no significant difference in Cu, Se, and Mn in the treated tissue between WT-C, KO-C, WT-I and KO-I. Gene expression of IL-33 in the treated tissue increased in both inflammatory groups when compared to the corresponding control groups, but it was significantly higher in KO-I than in WT-I. Gene expression of ST2 in the treated tissue was significantly higher in WT-I than in WT-C. Gene expression of TNF-alpha, IL-6, and IL-12p35 in the treated tissue was significantly higher in WT-I and KO-I than in the corresponding control groups, and IL-6 was significantly higher in KO-C than in WT-C. TGF-beta gene expression in the treated tissue was significantly higher in KO-I when compared to WT-I, while there was no difference between WT-C and KO-C. SOD activity decreased at the site of acute inflammation in both inflammatory groups, while the GPx activity increased. GSH in the treated tissue was significantly higher in KO-I than in KO-C or WT-I.The results of our study have indicated, to our knowledge for the first time, that IL-33/ST2 pathway plays a role in enhancing inflammation and tissue damage at the site of acute inflammation by affecting the concentration of magnesium and GSH, important for antioxidative capacity, as well as gene expression of anti-inflammatory cytokine TGF-beta.