Placental Expression and Relative Role of Anti-inflammatory Annexin A1 and Animal Lectin Galectin-3 in the Pathogenesis of Preeclampsia.

Preeclampsia (PE) remains the major cause for maternal and foetal mortality and morbidity all over the world. Preeclampsia is associated with maternal, placental aggravated inflammatory response and generalized endothelial damage. AnnexinA1 (AnxA1) is glucocorticoid regulated protein regulates a wide range of cellular and molecular steps of the inflammatory response and is implicated in resolution of inflammation. Galectin-3 (Gal-3), ß-galcotoside-binding lectin participates in many functions, both intra- and extracellularly. Recently it has been shown that galectin-3 modulates the inflammation. Role of AnxA1 and Galectin-3 is poorly studied in context with human reproductive disease like Preeclampsia. Therefore, the present study examined the expression of AnxA1 and Gal-3 which are involved in modulation of inflammation and their association in the placental bed of pregnancy with and without PE. The study group consisted of placental bed biopsy tissues obtained from pregnancies with PE (n = 30) and without (n = 30) PE. The expression of AnxA1 and Gal-3 in the placental bed tissues was evaluated quantitatively using Immunohisto-chemistry (IHC), western blot and mRNA expression analysis by quantitative RT-PCR. Our IHC, western blot and RT PCR analyses showed the increase in the expression of AnxA1 and Gal-3 in PE group compared with the normotensive control group (P < 0.001). The increased expression of AnxA1 and Gal-3 in placental bed may be associated with a systemic inflammatory response in PE, suggesting role of AnxA1 and Gal-3 in PE pathogenesis.

Reciprocal regulation of pro-inflammatory Annexin A2 and anti-inflammatory Annexin A1 in the pathogenesis of rheumatoid arthritis.

Annexin A2 has been implicated in several immune modulated diseases including Rheumatoid arthritis (RA) pannus formation. The most relied treatment option for RA pathogenesis is glucocorticoids. Glucocorticoids regulate the synthesis, phosphorylation and cellular deposition of Annexin A1. This annexin mediates the anti-inflammatory actions of glucocorticoids. These two first characterized members of annexin superfamily proteins acts reciprocally, one as an anti-inflammatory and the other proinflammatory in nature. The possibility of these molecules as soluble biomarkers and as an upstream regulator of major cytokine devastation at RA microenvironment has not been previously explored. Current study elucidates the reciprocal regulation of these two annexins in RA pathogenesis. These Annexin A2/A1 and downstream cytokines in RA serum were analysed by ELISA. Western blot, Immunocytochemistry, immunoprecipitation and Immunohistochemistry were adapted to analyse these molecules in tissue and synovial fibroblasts and also in different experimental conditions. Significant increase in the level of Annexin A2 was noticed in naïve RA patients compared to controls (14.582 ± 1.766 ng/ml vs. 7.37 ± 1.450 ng/ml; p ≤ 0.001). In remission cases significant low levels was detected. On the contrary, significant decrease in the level of Annexin A1 was noticed in naïve RA patients compared to healthy controls (12.322 ± 2.91 vs. 16.998 ± 4.298 ng/ml; p ≤ 0.001), wherein remission cases serum Annexin A1 was significantly high. The knockdown of proinflammatory Annexin A2 by siRNA/antibody treatment could mimic the glucocorticoid treatment as which induced cellular Annexin A1 and membrane translocation resulting in the terminal action. Current data elucidating the regulatory interplay between Annexin A2 and Annexin A1 in RA pathogenesis.

Cell surface interaction of annexin A2 and galectin-3 modulates epidermal growth factor receptor signaling in Her-2 negative breast cancer cells.

Overexpression and activation of tyrosine kinase receptors like EGFR and Src regulate the progression and metastasis of Her-2 negative breast cancer. Recently we have reported the role of cell membrane interaction of phospholipid-binding protein annexin A2 (AnxA2) and EGFR in regulating cellular signaling in the activation of angiogenesis, matrix degradation, invasion, and cancer metastasis. Beta-galactoside-specific animal lectin galectin-3 is an apoptosis inhibitor, and cell surface-associated extracellular galectin-3 also has a role in cell migration, cancer progression, and metastasis. Similar expression pattern and membrane co-localization of these two proteins made us to hypothesize in the current study that galectin-3 and AnxA2 interaction is critical for Her-2 negative breast cancer progression. By various experimental analyses, we confirm that glycosylated AnxA2 at the membrane surface interacts with galectin-3. N-linked glycosylation inhibitor tunicamycin treatment convincingly blocked AnxA2 membrane translocation and its association with galectin-3. To analyze whether this interaction has any functional relevance, we tried to dissociate this interaction with purified plant lectin from chickpea (Cicer arietinum agglutinin). This highly specific 30 kDa plant lectin could dissociate AnxA2 from endogenous lectin galectin-3 interaction at the cell surface. This dissociation could down-regulate Bcl-2 family proteins, cell proliferation, and migration simultaneously triggering cell apoptosis. Targeting this interaction of membrane surface glycoprotein and its animal lectin in Her-2 negative breast cancer may be of therapeutic value.

Association of pro-fibrinolytic receptor AnnexinA2 with tissue plasminogen activator/Inhibitor-1 in pre-eclampsia.

Background: The clinical manifestations of pre-eclampsia are related to placental anti-angiogenic factor alteration. These variations are mainly due to the alteration of plasminolytic components. The study aims to compare the expression of plasminolytic components in the placenta of women with and without pre-eclampsia. Material and Methods: The study included pregnant women with pre-eclampsia as PE group (n = 30) and without pre-eclampsia as a control group (n = 30). Placental bed biopsy tissues were collected. AnxA2, tPA, PAI-1 expression in the placental villous tissue was quantitatively evaluated using immunohistochemistry, western blot, and real time-PCR analysis. Results: The results of the study showed a significant decrease in the expression of ANXA2 and increased expression of tPA and PAI-1 in PE group compared to control group (p<0.005). AnxA2 expression showed positive correlation with tPA (r=+0.895, p=0.002) and negative correlation with PAI-1(r=-0.905, p=0.020) in control group whereas in the PE group AnxA2 expression was negatively correlated with tPA ((r=-0.801, p=0.016) and PAI-1 (R=-0.831, P=0.010). Conclusion: Decreased AnxA2 with increased expression of PAI-1 and tPA may be responsible for the altered fibrinolytic activity and play a significant role in pre-eclampsia pathogenesis.

Ubiquitin Mediated Degradation of EGFR by 17 ß-estradiol in Triple Negative MDA-MB-231 (TNBC) Breast Cancer Cells Line.

BACKGROUND: Triple Negative Breast Cancer (TNBC) commonly displays Epidermal growth factor receptor (EGFR). Effective EGFR degradation results in the suppression of tumor in various models. Studies have addressed the relevance of this strategy in the treatment of TNBC. In the present study, we examined the effect of 17 ß- estradiol on EGFR expression in MDA-MB-231 (TNBC) cell line and assessed whether 17 ß-estradiol degrades EGFR by ubiquitination pathway. OBJECTIVES: The objective of this study is to treat MDA-MB-231 cell lines with Cycloheximide with or without 17ß-estrdiol to observe whether 17ß-estradiol leads to EGFR degradation and to treat with MG-132 to assess whether degradation occurs through ubiquitination pathway. METHODS: MDA-MB-231 cells were treated with 17ß-estradiol (E2) and EGFR expression was studied by western blotting at different intervals by using Cycloheximide chase. To assess ubiquitination pathway of degradation of EGFR in MDA-MB-231 cell line, MG-132 was used. RESULTS: EGFR expression was reduced with ß-estradiol treatment in MDA-MB-231 cell line with Cycloheximide chase. Upon Treatment with MG-132 and E2, EGFR expression did not reduce, suggesting that Estrogen degrades EGFR by ubiquitination pathway. CONCLUSION: Estrogen degrades EGFR in MDA-MB-231 cells and this degradation occurs by ubiquitination.

Induction of Endometrial HOXA 10 Gene Expression by Vitamin D and its Possible Influence on Reproductive Outcome of PCOS Patients Undergoing Ovulation Induction Procedure.

Background and Objectives: Polycystic ovarian syndrome (PCOS) is one of the most common endocrinopathies in women frequently presenting with anovulatory infertility. Low successful pregnancy and live birth rates even after successful ovulation induction (OI) and in vitro fertilization (IVF) in these patients indicate that endometrial dysfunction may be another important factor contributing to infertility. Vitamin D acting through nuclear receptors induces the expression of various genes required for cell growth and differentiation and plays a crucial role in reproduction. Homeobox 10 (HOXA10) may be one of the potential targets for vitamin D action. HOXA10 gene product promotes the differentiation of endometrial cells, making the endometrium receptive for implantation. The present study was undertaken to determine the effect of circulating vitamin D levels on HOXA10 gene expression in endometrial tissues and its possible influence on the reproductive outcome of PCOS patients undergoing OI procedure. Materials and Methods: A prospective cohort study was conducted on 110 infertile PCOS patients. The patients were divided into two groups: Group 1: Vitamin D ³20 ng/ml, Group 2: Vitamin D <20 ng/ml. Endometrial samples were obtained from 22 patients using pipelle biopsy, used to determine HOXA10 mRNA (messenger ribonucleic acid) expression by quantitative RT-PCR (reverse transcription-polymerase chain reaction) and protein expression by Western blotting. OI was performed using Clomiphene citrate or Letrozole from the 3rd day of the cycle, and patients were followed up for a maximum of five cycles. Attainment of successful pregnancy was considered a positive outcome. Results: Both the groups were similar in mean age and other endocrine parameters. Serum vitamin D levels were significantly low (P < 0.001), and BMI (body mass index) was significantly high (P = 0.032) in group 2 compared to group 1. Endometrial HOXA10 mRNA (by quantitative rtPCR) and protein expression (by western blotting) were significantly low in group 2 compared to group 1. The clinical pregnancy rate was low in group 2 (28.6%) compared to group 1 (42.3%), but this difference was not significant (P = 0.22). On regression analysis adjusted for age and BMI, vitamin D was an independent predictor of successful pregnancy after OI (P = 0.09). Conclusion: Circulating vitamin D levels influence the endometrial HOXA10 gene expression, and this may be reflected on the reproductive outcome of infertile PCOS patients undergoing OI.

Decreased expression of annexin A2 and loss of its association with vascular endothelial growth factor leads to the deficient trophoblastic invasion in preeclampsia.

OBJECTIVES: Preeclampsia (PE) remains the major cause for maternal and foetal mortality and morbidity. Invasion of endovascular trophoblast and remodelling of spiral artery are crucial actions of normal placental development. Non-fulfilment of these processes plays a leading role in the development of preeclampsia. Vascular endothelial growth factor (VEGF) is produced by extravillous trophoblastic tissue and decidual cell population is a well-known angiogenic growth which plays a fundamental role in placental pathogenesis of PE. Annexin A2 (ANXA2) is a profibrinolytic protein receptor required for plasminolysis, which is an important step in the formation of new blood vessel along with VEGF. Role of ANXA2 is poorly studied in context with human reproductive disease like preeclampsia. The purpose of the present study is to examine the expression and association of VEGF and ANXA2 in the term placentas of pregnancies with and without PE. METHODS: The study group comprised of placental tissues procured from gestations with PE (n=30) and without (n=20) PE. The expression of VEGF and ANXA2 in the placental villous tissue was evaluated quantitatively by means of IHC, western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Our IHC, western blotting and RT-PCR analysis illustrated the significant decrease in the expression of VEGF and ANXA2 in PE group compared with the normotensive control group (p<0.005). We observed statistically significant positive correlation among the expression of ANXA2 and VEGF in placentas of normotensive control group (p<0.0001). CONCLUSIONS: The diminished expression of VEGF and ANXA2 in placenta may be associated with the defective angiogenesis and which may possibly play a vital role in PE pathogenesis.

Estimation and correlation of serum and salivary glucose and immunoglobulin A levels and salivary candidal carriage in diabetic and non-diabetic patients.

Background. A correlation has been noted between diabetes mellitus (DM) and changes in the oral cavity. The present study aimed to estimate, compare, and correlate serum and salivary glucose and IgA levels and salivary candidal carriage in diabetic and non-diabetic individuals. Methods. Eighty-eight subjects were categorized into three groups: group 1 (controlled DM; n=27), group 2 (uncontrolled DM; n=32) and group 3 (non-diabetics; n=29). Serum and salivary glucose levels were estimated by glucose oxidase/peroxidase method, serum and salivary IgA by a diagnostic kit, and candidal colonization by inoculating samples into Sabouraud dextrose agar plate. Statistical analyses were carried out by one-way ANOVA, post hoc Tukey tests, and Pearson's correlation coefficient. Results. Significant elevation of serum IgA levels was observed in group 2 compared to group 3 and significant decreases in salivary IgA levels in groups 1 and 2. The candidal carriage was significantly higher in group 2 compared to group 3. Serum glucose and salivary IgA levels showed a significant correlation in group 1. There was a positive correlation between serum/ salivary glucose and serum/salivary IgA levels in group 2. In addition, there was a significant correlation between serum glucose and serum IgA levels in group 3. Conclusion. Saliva could be a potential, non-invasive diagnostic tool to estimate glucose levels. The evaluation of salivary components, like IgA, might be useful in diagnosing and managing oral manifestations in diabetic individuals. Elevated salivary glucose levels contribute to elevated candidal carriage, making individuals susceptible to oral candidiasis.

Protein Phosphatase 1 Regulatory Subunit SDS22 Inhibits Breast Cancer Cell Tumorigenesis by Functioning as a Negative Regulator of the AKT Signaling Pathway.

Protein phosphatases play a crucial role in cell cycle progression, cell survival, cellular signaling, and genomic integrity. The protein phosphatase 1 (PP1) regulatory subunit SDS22 plays a significant role in cell cycle progression. A recent study showed that SDS22 plays a vital role in epithelial integrity and tumor suppression in Drosophila. However, its tumor suppressive activity remains obscure in the mammalian system. Here, for the first time, we show that SDS22 inhibits the growth of breast cancer cells through induction of apoptosis. SDS22 negatively regulates the AKT kinase signaling pathway through PP1. SDS22 associates predominantly with AKT and dephosphorylates the phospho Thr308 and phospho Ser473 through PP1 and hence abrogates the cell migration, invasion, and tumor growth. Thus, our study deciphers the long-standing question of how PP1 negatively regulates the AKT signaling pathway. Further, we observed a significant converse correlation in the expression levels of SDS22 and phospho form of AKT with reduced levels of SDS22 in the higher grades of cancer. Overall, our results suggest that SDS22 could be a putative tumor suppressor and replenishment of SDS22 would be an important strategy to restrict the tumor progression.

Culture and characterization of human dental pulp­derived stem cells as limbal stem cells for corneal damage repair.

Limbal stem cell deficiency (LSCD) is one of the leading causes of corneal damage. Injury or inflammation in the cornea causes LSCD, which may be unilateral or bilateral depending upon the cause. Limbal epithelial cell implants successfully improve vision in patients with chemical injury­induced LSCD. Transplantation of cultured epithelial stem cells has become a treatment of choice for numerous patients with LSCD. Bilateral LSCD is frequently observed in the general population, where no residual stem cells are available for ex vivo culture. Allografts are associated with a high risk of rejection, neoplasia, and disease transmission. In this respect, allogenic cell populations from other regions in the patient may substitute for allogenic material. In the present study, dental pulp stem cells were cultured in limbal stem cell media and these cells were characterized against limbal stem cells, revealing the significance of using dental pulp stem cell treatment in bilateral LSCD. The morphology and culture pattern of both limbal and dental pulp stem cells grown in limbal stem­specific media were similar. Polymerase chain reaction analysis revealed that stem cell markers were highly expressed in limbal stem cells compared to in dental pulp stem cells, regardless of the medium and scaffold in which they were grown. Although dental pulp stem cell molecular expression is quite low at the transcript level, the functional protein level according to immunocytochemistry and western blot analyses demonstrated that stem cells and corneal differentiation molecule levels were quite high, indicating their potential as limbal stem cells in the respective microenvironment.

Differential expression pattern of annexin A2 during nephrogenesis and kidney carcinoma.

The creation of a cancer cell could be due to reactivation of repressed gene in the process of normal embryonic development. The differences in embryonic origins and functions of various components of nephron may contribute to the diversity of morphological patterns, molecular and immunohistochemical phenotypes of common renal neoplasms. Renal cell carcinomas (RCCs) are the most common amongst the genitourinary cancers. Annexin A2 (AnxA2) is a multifunctional calcium-regulated phospholipids-binding protein found in a subset of renal neoplasms. Since the tumor cells usually recapitulate embryonic cells, we studied the ontogeny of AnxA2 in developing renal tissues and compared it with those of normal adult RCCs, to better understand their role in renal development and tumorigenesis. AnxA2 immunoexpression was evaluated by immunohistochemistry from various autopsied fetuses, mature kidney and renal cancer tissue specimens. The study showed moderate membranous AnxA2 immunoexpression in the ureteric buds and collecting tubules of fetal kidneys (in all gestational ages) and in the collecting ducts of adult normal renal tissues. It is not often expressed in the proximal convoluted tubules of normal adult kidney; however, younger fetal kidneys show moderate AnxA2 immunoexpression in the proximal convoluted tubules (thought to be the origin of RCC) and the reappearance of strong membranous AnxA2 immunoexpression in the clear cell carcinoma is suggesting a deregulation of the gene during tumorigenesis. The understanding of the AnxA2 molecular immunoexpression pattern during development, its specific function and deregulated immunoexpression in different renal carcinoma types indicates the decisive role of AnxA2 in the cancer progression.

Comparative expression analysis of phospholipid binding protein annexina1 in nephrogenesis and kidney cancer.

Background The expression in the glomerular mesangial cells, papillary, and collecting duct cells demonstrated annexin A1 (AnxA1)'s role in specific renal functions. With varying concentrations of calcium (Ca2+), it is considered to regulate cellular processes such as cell proliferation, apoptosis, and clearance of apoptotic cells by forming ceramides, a key lipid mediator of apoptosis. It also participates in tumorigenesis based on its location. On account of these features, we investigated the expression of this apoptosis-associated protein in fetal kidneys at different gestational periods, mature kidneys and in kidney cancer tissues in order to localize and possibly characterize its role during nephrogenesis and renal tumors. Methods AnxA1 expression was evaluated by an immunohistochemistry technique in "paraffin-embedded" renal tissue sections from autopsied fetuses at different gestational ages, in mature kidneys and renal cancer tissues. Results The current study data demonstrated that AnxA1 is expressed in the mesangial cells and podocytes of maturing glomeruli in the developing renal cortex of fetal kidneys at 14 to 19 weeks of gestation. The expression in the mesangial cells declined in later weeks of gestation and persisted into adulthood. AnxA1 expression increased with the progression of clear cell renal cell carcinoma (CCRCC) and also in other cancer types indicating a potential role of the protein in tumorigenesis. Conclusions We presume that AnxA1 in the podocytes and mesangial cells play important roles in various signaling pathways in the functioning of the glomerulus. These results and concepts provide a framework to further dissect its biological properties and thereby develop diagnostic, prognostic, and therapeutic strategies targeting the molecule in various renal pathologies.

A MicroRNA/Ubiquitin Ligase Feedback Loop Regulates Slug-Mediated Invasion in Breast Cancer.

The transformation of a normal cell to cancer requires the derail of multiple pathways. Normal signaling in a cell is regulated at multiple stages by the presence of feedback loops, calibration of levels of proteins by their regulated turnover, and posttranscriptional regulation, to name a few. The tumor suppressor protein FBXO31 is a component of the SCF E3 ubiquitin ligase and is required to arrest cells at G1 following genotoxic stresses. Due to its growth-suppression activity, it is underexpressed in many cancers. However, the molecular mechanism underlying the translational regulation of FBXO31 remains unclear. Here we show that the oncogenic microRNAs miR-93 and miR-106a repress FBXO31, resulting in the upregulation of Slug, which is involved in epithelial-mesenchymal transition and cell invasion. FBXO31 targets and ubiquitylates Slug for proteasomal degradation. However, this mechanism is repressed in breast tumors where miR-93 and miR-106a are overexpressed. Our study further unravels an interesting mechanism whereby Slug drives the expression of miR-93 and miR-106a, thus establishing a positive feedback loop to maintain an invasive phenotype. Together, these results establish the presence of interplay between microRNAs and the ubiquitination machinery, which together regulate cancer cell invasion.

Annexin A2 and its downstream IL-6 and HB-EGF as secretory biomarkers in the differential diagnosis of Her-2 negative breast cancer.

Background AnnexinA2 (AnxA2) membrane deposition has a critical role in HB-EGF shedding as well as IL-6 secretion in breast cancer cells. This autocrine cycle has a major role in cancer cell proliferation, migration and metastasis. The objective of the study is to demonstrate annexinA2-mediated autocrine regulation via HB-EGF and IL-6 in Her-2 negative breast cancer progression. Methods Secretory annexinA2, HB-EGF and IL-6 were analysed in the peripheral blood sample of Her-2 negative ( n = 20) and positive breast cancer patients ( n = 16). Simultaneously, tissue expression was analysed by immunohistochemistry. The membrane deposition of these secretory ligands and their autocrine regulation was demonstrated using triple-negative breast cancer cell line model. Results Annexina2 and HB-EGF expression are inversely correlated with Her-2, whereas IL-6 expression is seen in both Her-2 negative and positive breast cancer cells. RNA interference studies and upregulation of annexinA2 proved that annexinA2 is the upstream of this autocrine pathway. Abundant soluble serum annexinA2 is secreted in Her-2 negative breast cancer (359.28 ± 63.73 ng/mL) compared with normal (286.10 ± 70.04 ng/mL, P < 0.01) and Her-2 positive cases (217.75 ± 60.59 ng/mL, P < 0.0001). In Her-2 negative cases, the HB-EGF concentrations (179.16 ± 118.81 pg/mL) were highly significant compared with normal (14.92 ± 17.33 pg/mL, P < 0.001). IL-6 concentrations were increased significantly in both the breast cancer phenotypes as compared with normal ( P < 0.001). Conclusion The specific expression pattern of annexinA2 and HB-EGF in triple-negative breast cancer tissues, increased secretion compared with normal cells, and their major role in the regulation of EGFR downstream signalling makes these molecules as a potential tissue and serum biomarker and an excellent therapeutic target in Her-2 negative breast cancer.