Gestational hyperglycemia on diet and medication: impact on placental pathology and pregnancy outcomes

Background: To evaluate the placental morphology and perinatal outcome in patients with gestational hyperglycemia on diet and medication.Methods: Placental examinations performed at the Department of Pathology between August 2016 to August 2018 were retrospectively reviewed. Of the received 140 placentas, 35 of gestational diabetes (GDM) and pre gestational diabetes were identified and segregated into hyperglycemia on diet and on medication. The clinical details, placental findings and perinatal outcome of patients in both the groups (gestational hyperglycemia on diet and medication) were collected and analyzed.Results: Among the 35 cases, there were 24 cases of mild gestational hyperglycemia controlled with diet and 11 cases of hyperglycemia on medication (oral hypoglycemic drugs ± insulin).Most of the placentae in both the groups weighed less than tenth centile. The cord abnormalities such as hyper coiling, velamentous /marginal insertion and furcate cord were observed more in women with GDM on diet. There was no significant gross placental lesion in those on medication. Placental histological features most consistently associated with both the groups include, disturbances of villous maturation (DVM), Derangements in uteroplacental / foetoplacental circulation and villous capillary lesions. Small for gestational age and intrauterine foetal death were found in both the groups, but more commonly in patients with hyperglycemia on medication.Conclusions: Villous maturation defects, uteroplacental / foetoplacental malperfusion are the essential placental changes which can result in adverse perinatal outcomes in women with hyperglycemia irrespective of the diabetic control

Antibacterial properties of Passiflora foetida L. – a common exotic medicinal plant

Passiflora foetida L. (stinking passion flower) is an exotic medicinal vine. The antibacterial properties of leaf and fruit (ethanol and acetone) extracts were screened against four human pathogenic bacteria i.e. Pseudomonas putida, Vibrio cholerae, Shigella flexneri and Streptococcus pyogenes by well-in agar method. The results showed the leaf extract having remarkable activity against all bacterial pathogens compared to fruits. This study supports, the traditional medicines (herbal extracts) to cure manydiseases like diarrhea, intestinal tract, throat, ear infections, fever and skin diseases

Anti-Angiogenesis Effect Associated with Inhibition of VEGFR by F16 Treatment in U87 cell line and Glioblastoma Xenograft Tumors

Objective. This study was conducted to determine the effectiveness of the Anti-angiogenic Agent F16 treatment using glioblastoma cell line and xenograft tumor implanted in nude mice. Background. Glioblastoma multiforme (GBM) tumors are highly vascularized tumors, and their growth is angiogenesis-dependent; antagonizing tumor angiogenesis by using angiogenesis inhibitors is considered as one of the promising approaches. Methods. In this context, intensive pre-clinical evaluation of a novel small molecule named F16 has exhibited potent anti-angiogenic and anti-tumor activities by selectively antagonizing Vascular Endothelial Growth Factor Receptor (VEGFR) in the in vitro and in vivo models. Results. Our in vitro studies have clearly demonstrated the ability of F16 to inhibit U87MG cell survival, migration, and invasion. Furthermore, pharmacokinetic evaluation of F16 with tissue distribution analysis has shown that this molecule is transported across the blood-brain barrier and accumulates in the brain regions with no signs of neurotoxicity. Therefore, we conducted experiments to determine the efficacy of F16 in delaying glioblastoma progression via inhibiting tumor angiogenesis using the xenograft model. Our in vivo studies with the subcutaneously implanted tumors indicated that F16 is efficacious in delaying tumor growth by blocking tumor angiogenesis. Conclusion. Our in vitro and in vivo results strongly demonstrated that F16 has significant cytotoxic effects on U87MG cells and the host mice also showed a good level of tolerability for F16 treatments. Further studies are underway to complete the pre-clinical testing of F16. Grants. This research was supported by the Royal Dames of Cancer Research Inc. Ft. Lauderdale, F

Effect of SAHA on the Expression of Chromatin Modifying Enzymes in Prostate and Breast Cancer Cells

Objective. Our study analyzed the effects of HDAC inhibitor SAHA treatment on gene expressions of Chromatin Modifying Enzymes. Background. Histone deacetylase (HDAC) inhibitors are one of the important epigenetic regulators that have enormous therapeutic potential in various diseases, including cancers. For example, SAHA (Suberoylanilidehydroxamic acid) has been known as a potent inhibitor of histone deacetylases that eventually lead to differentiation, growth arrest, and apoptosis of various cancer cells. Methods. In our study, we utilized the RT2 Profiler PCR Array that was specific for the Human Epigenetic Chromatin Modifying Enzymes. We examined the impact of SAHA (7.5 µM) treatment on gene expression patterns of LNCaP (prostate cancer cells) and MCF-7 (breast cancer) cells. Results. As a result of SAHA treatment, the expression levels of AURKB (0.11), SUV39H1 (0.23), AURKA (0.4), and SETD7 (0.49) were found to be significantly down-regulated compared to the control in the LNCaP cells. In addition, the mRNA level of KDM6B was also up-regulated (by 2.4 folds) after SAHA treatment. On the other hand, in the MCF-7 cells PAK1 (0.06), NSD1 (0.19), SETD7 (0.24), DNMT3A (0.31), NEK6 (0.34), SETD6 (0.38), PRMT1 (0.4), AURKB (0.4) and SUV39H1 (0.45) were found to be significantly down-regulated after 24 hr of SAHA treatment. Conclusion. Our results offer evidence that SAHA can impact the gene expression profile of epigenetic chromatin modification enzymes and exert its anti-cancer effect in both prostate and breast cancer cells. Grants. The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged

Designing Potent Inhibitors Of Human p38γ For Effective Breast Cancer Therapy

Oncogenic constitutive enzyme human p38γ is a serine/threonine protein kinase, activated through phosphorylation by environmental stress and pro-inflammatory cytokines responses. Breast cancer, hepatoma, colon cancer, atherosclerotic lesion (coronary artery lesion / hardening of artery), hypertension and inflammations are some of the diseases caused by human p38γ due to over expression. Over expression of the protein in turn induces anti-apoptosis and inflammatory responses, increased malignant transformation and cell differentiation. Thus, designing potent inhibitors against p38γ would be highly practicable for development of novel means of breast cancer therapeutics. Extensive preclinical data and proteomic analysis support the involvement of human p38γ protein in MAPK signaling pathway in cancer biology and it can be used as a potential therapeutic target in human cancers. Phylogenetic analysis delineated the close relationship of human p38γ with other 27 human oncogenes involved in cell signaling pathway. The pathway analysis also clearly explains the formation of anti-apoptosis and inflammatory responses. It is relevant and attractive drug target for cancer diseases. Computational method for drug designing was practiced here to explore lead molecules targeting human p38γ. Amino acid residues viz. Lys56, Pro110, Met112, Asp115, Gly157, Asn158, Asp171 and water molecules viz. W2111, W2039 and W2152 were observed to have important role in ligand binding in human p38γ crystal structure (PDB ID: 1CM8) through PyMol. BIRB796, SB202190, 3-[4-[2-(cyclopentylamino) pyrimidin-4-yl]-1H-pyrazol-5-yl] cyclohexan-1-ol, (R)-2-(sec-butyl amino)-N-(2-methyl-5-(methyl carbamoyl) phenyl) thiazole-5-carboxamide are the four published inhibitors and PDB ligand phosphoaminophosphonic acid-adenylate ester (ANP) were selected and an in house library of 1909 lead molecules was generated through Ligand.info Metadata base. Maestro 9.0 virtual screening protocol was practiced to determine the binding affinity of 1909 compounds into p38γ active site grid generated around the centroid of binding site residues. The Glide computational docking method of Maestro virtual screening protocol had generated 18 lead molecules with good binding affinity towards human p38γ. The best ranked lead molecule 3-DEAZA-ADENOSINE (Lead ‘1’) docked with least docking score -10.22 kcal/ mol representing better binding affinity than the existing published inhibitors and other 17 lead molecules proposed against human p38γ in the present study. Analysis of binding orientations of the docking complex revealed four amino acid residues Pro110, Met112, Asp115, Asn158 and W2039 of active site were directly involved in formation of hydrogen bond network which complements well with previous crystallographic reports of human p38γ –ANP inhibitor complex. 3-DEAZA-ADENOSINE was also observed to be involved in good van der Waal interaction with Lys56 that is highly important for ATP binding and subsequent activation of human p38γ. Thus, 3-DEAZA-ADENOSINE might be encouraging for new directions as a drug for human p38γ protein for the novel class treatment of breast cancer

Effect of the HDAC Inhibitor on Histone Acetylation and Methyltransferases in A2780 Ovarian Cancer Cells

Background andObjective: Epigenetic modifications are believed to play a significant role in the development of cancer progression, growth, differentiation, and cell death. One of the most popular histone deacetylases inhibitors (HDACIs), suberoylanilide hydroxamic acid (SAHA), also known as Vorinostat, can directly activate p21WAF1/CIP1 gene transcription through hyperacetylation of histones by a p53 independent mechanism. In the present investigation, we evaluated the correlation between histone modifications and DNA methyltransferase enzyme levels following SAHA treatments in A2780 ovarian cancer cells. Materials and Methods: Acetylation of histones and methyltransferases levels were analyzed using RT2 profiler PCR array, immunoblotting, and immunofluorescence methods in 2D and 3D cell culture systems. Results: The inhibition of histone deacetylases (HDAC) activities by SAHA can reduce DNA methyl transferases / histone methyl transferases (DNMTs/HMTs) levels through induction of hyperacetylation of histones. Immunofluorescence analysis of cells growing in monolayers and spheroids revealed significant up-regulation of histone acetylation preceding the above-described changes. Conclusions: Our results depict an interesting interplay between histone hyperacetylation and a decrease in methyltransferase levels in ovarian cancer cells, which may have a positive impact on the overall outcomes of cancer treatment

Effect of HDAC Inhibitor on DNA Methylation and Cell Cycle Regulation in Prostate Cancer

Objective: Our study was aimed to analyse the expression of methyltransferase levels in LNCaP (prostate Cancer) cells during SAHA treatment. Background: Prostate cancer is the second leading cause of death in men after lung cancer in the US. Nearly 1 in 8 men will be diagnosed of prostate cancer in their lifetime, and the risk increases significantly once the men cross the age of 70. Recognizing ways to reduce the death of prostate cancer is therefore a top research priority. Epigenetic regulation of gene plays an important role in the controlling cell cycle and tumor growth in various cancers. Epigenetic changes generally occur through alterations in DNA and Histone modification such as acetylation, methylation, phosphorylation, and ubiquitination. SAHA (Suberoylanilide Hydroxamic Acid) is a broad spectram inhibitor of histone deacetylase (HDAC), which is used to modify the status of Histone Acetylation during cancer treatments. However, the impact of SAHA on methyltransferase levels or methylation status of DNA has not been studied in detail. Methods: LNCaP cells were cultured in complete RPMI-1640 growth medium and treated with SAHA (7.5 uM) for 24 hours. Western blot technique was used to analyze the expression levels of DNMT3A, SUV39H1, PRMT1, and p21. Results: Our experimental results have shown that SAHA treatment reduce the levels of the methyltransferase enzymes listed above. Furthermore, SAHA treatment increased the protein levels of p21, which is a CDKI (cyclin-dependent kinase inhibitor). Conclusion: Methylation is an important modification of DNA that can regulate gene expressions. Our results indicate that SAHA treatment, which is known to regulate histone acetylation, can impact the methylation status also through an indirect mechanism and allow for the control of transcription of tumor suppressor genes. Acknowledgement: This Research was supported by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida

The Emerging Role of GSK-3 Inhibitors as Promising Drug Candidates in NSCLC

Objective. This study was conducted to assess the apoptotic role of GSK-3 inhibitors: BIO and CHIR 98014 against H460 K-Ras mutant (mut) and H1975 K-Ras wild type (wt) Non-small cell lung cancer (NSCLC) cells. Background. NSCLC accounts for 80% - 85% of lung cancers, with mutation of KRAS being the most frequent aberration. Our study was designed to determine the use of GSK-3 inhibitors as apoptotic inducers against NSCLC cells. Methods. In our study, the cell viability and cell proliferation of H460 and H1975 were measured using MTT and BrdU assay after 24, 48, and 72 h of BIO and CHIR 98014 treatments. Imaging studies to assess Reactive oxygen species (ROS), Mitochondrial Membrane Potential (MMP), and Caspase-3/7 cleavage were conducted. The trans-endothelial migratory assay was conducted to assess the potential of BIO and CHIR 98014 for inhibiting cancer metastasis. Western blot analysis was conducted for measuring pGSK-3, phospho-p53, p21, XIAP, BAX, LC3B, Caspase-3, and Caspase-9 levels. Results. GSK-3 inhibitors significantly reduced the cell viability after 24 h treatment in H1975 compared to H460 cells. In addition, BIO and CHIR 98014 demonstrated significant upregulation of ROS levels, while decreasing its mitochondrial membrane potential, leading to cleavage of Caspase-3, 7, and 9. Interestingly, a significant elevation of phospho-p53, p21, and LC3B levels was observed with BIO and CHIR 98014 treatments. Conclusion. Our results indicated that GSK-3 inhibitors were able to induce cell death by activating both extrinsic and intrinsic apoptotic pathways. Grants. This study was funded by the PFRDG grant 334818 and the financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida

Analysis of Epigenetic Modifications in HCC827 and LNCaP cells

Objective: Our study was aimed to examine the expression levels of DNA methyltransferase in HCC827 (Lung Cancer) cells during SAHA (Suberoylanilide Hydroxamic Acid) treatment. Background: Epigenetic changes in chromatin have been found to regulate oncogenesis and cancer cell growth. DNA methylation and Histone modification are some of the most important changes found during cancer growth. Epigenetic therapy using histone deacetylase inhibitors (HDACi) SAHA can induce cell cycle arrest, differentiation, suppressed cell growth, and cell death of cancer cells. DNA methyl transferases are suspected to be involved in SAHA induced cell death. Methods: HCC827 cells were treated with SAHA (7.5µM) for 24 hrs. Microarray and western blotting analysis were used to assess the exoression levels of the DNA methyltrasnferase. Results: We found that SAHA treatment reduced the expression level of SUV39H1, DNMT3A, and PRMT1 after 24 hrs of treatment using Western blot as well as Microarray analysis. Conclusion: These results provide important information regarding that the interplay between histone acetylation and de-methylation under the same treatment, leading to two opposite effects: demethylation of DNA and upregulation of tumor suppressor genes. Grants: The Royal Dames of Cancer Research Inc. of Fort Lauderdale, Florida is gratefully acknowledged for their generous support