DNA Ladder Assay Research Articles

Preparation, characterization and anti-proliferative effects of sclareol-loaded solid lipid nanoparticles on A549 human lung epithelial cancer cells

Here, sclareol-loaded solid lipid nanoparticles (sclareol-SLNs) was formulated and tested for potential geno-cytotoxicity upon A549 lung cancer cells. Sclareol-SLNs was formulated using hot homogenization. Different physicochemical features including size, zeta potential, dispersity, drug loading (DL), and encapsulation efficiency (EE) as well as in vitro drug release behavior were determined. Genotoxicity and cytotoxicity of the developed sclareol-SLNs were evaluated using DNA ladder and MTT assay, respectively. The cell death types were determined by detection of phosphatidylserine, dsDNA integrity, and proportion of different amounts in the associated cell cycle. Electron microscopy and particle size analysis showed dispersed and homogenous NPs with an average diameter of 88 nm and a high percent of EE (89%) and drug loading (42.47 mg/g). Sustained drug release was achieved at a physiologic pH, and long-term stability in terms of the size, zeta, dispersity, and EE of NPs was achieved. Cytotoxicity assay results showed that the plain sclareol inhibited A549 growth with an IC50 value of 19 μg/mL after 24 h, yet sclareol-SLNs inhibition was perceived after 48 h. DNA fragmentation assay using gel electrophoresis showed clear fragmentation in the chromatin within the cell's treated by sclareol and NPs. Flow cytometry analyses determined early and late apoptosis in sclareol and sclareol-SLNs treated cells.

Do protons and X-rays induce cell-killing in human peripheral blood lymphocytes by different mechanisms?

PurposeSignificant progress has been made in the technological and physical aspects of dose delivery and distribution in proton therapy. However, mode of cell killing induced by protons is less understood in comparison with X-rays. The purpose of this study is to see if there is any difference in the mode of cell-killing, induced by protons and X-rays in an ex vivo human peripheral blood lymphocyte (HPBL) model. Materials and methodsHPBL were irradiated with 60 MeV proton beam or 250-kVp X-rays in the dose range of 0.3–4.0 Gy. Frequency of apoptotic and necrotic cells was determined by the Fluorescein (FITC)-Annexin V labelling procedure, 1 and 4 h after irradiation. Chip-based DNA Ladder Assay was used to confirm radiation-induced apoptosis and necrosis. Chip-based DNA Ladder Assay was used to confirm radiation-induced apoptosis. ResultsEx vivo irradiation of HPBL with proton beams of 60 MeV or 250 kVp X-rays resulted in apoptotic as well as necrotic modes of cell-killing, which were evident at both 1 and 4 h after irradiation in the whole dose and time range. Generally, our results indicated that protons cause relatively higher yields of cell death that appears to be necrosis compared to X-rays. The analysis also demonstrates that radiation type and dose play a critical role in mode of cell-killing. ConclusionObtained results suggest that X-rays and protons induce cell-killing by different modes. Such differences in cell-killing modes may have implications on the potential of a given therapeutic modality to cause immune modulation via programmed cell death (X-rays) or necrotic cell death (proton therapy). These studies point towards exploring for gene expression biomarkers related necrosis or apoptosis to predict immune response after proton therapy.

Exopolysaccharide from Marine Bacillus velezensis MHM3 Induces Apoptosis of Human Breast Cancer MCF-7 Cells through a Mitochondrial Pathway

ObjectiveThe production of new natural pharmaceutical agents that increase the efficiency of chemotherapy without affecting the normal cells is the goal of all researchers. Therefore, the present study expects to evaluate the antioxidant and anticancer studies against MCF-7 cell lines of EPS produced by novel Egyptian marine bacterial strain.MethodsMarine bacterium was isolated, purified and identified by 16S rRNA gene amplification and sequence analyses. MHMEPS (the produced EPS) was analyzed by Fourier Transform Infra-red (FTIR), monosugars identification by HPLC, molecular weight estimation and sulfur content were determined. While, in-vitro antioxidants characters was determined using various methods and anticancer studies against MCF-7 cell lines.ResultsBacillus velezensis MHM3 produced 5.8 g/L of MHMEPS. The chemical analysis of MHMEPS showed 24% uronic acid and 18.19% sulfate and monosugars glucuronic acid, glucose, fructose and rhamnose with molar ratio of 4.00: 2.00: 1.00: 0.13, correspondingly, with an overall weight average molecular weight Mw of 1.145×104 g/mol and the number average of molecular weights Mn of 5.155 ×103 g/mol. The FTIR analysis and periodate oxidation indicate the existence of β-(1–4) linkage acidic polysaccharide. MHMEPS showed antioxidant scavenging activity against DPPH•, H2O2 and Metal chelating activity, respectively. So, reducing power method give high activity at 500 µg/ml. MHMEPS hinder the proliferation of MCF-7 cells at 5-80 μg/ml compared to the control group. Moreover, induced apoptosis was associated with activation of caspase-3. Also increased cytochrome C levels significantly in a dose-dependent manner compared with the control. The Caspase-3 activity was raised in MHMEPS treated MCF-7 cells compared with the control (p<0.05) in a dose-dependent manner. Therefore, the result of DNA fragmentation was confirmed by DNA ladder assay. We presume that MHMEPS has high potential at its low concentration, as a novel restorative agent for the treatment of MCF-7 cells, with no cytotoxicity against normal cells.

Radioprotective and immunomodulatory effects of Mesua ferrea (Linn.) from Western Ghats of India., in irradiated Swiss albino mice and splenic lymphocytes

The present study reports the efficacy of Mesua ferrea (Linn.) extract in mitigating radiation induced toxicity along with immunomodulatory property. Swiss albino mice, splenic lymphocytes and plasmid pBR322 DNA were used to evaluate the radioprotective effect by exposing them to 6Gy EBR or 4 Gy gamma radiation. In this study, Mesua ferrea methanolic (MfM) or aqueous extracts (MfA) significantly protected pBR322 DNA against radiation induced strand breaks. Both the extracts significantly offered protection against radiation-induced apoptosis as indicated by propidium iodide staining and DNA ladder assay. MfM or MfA significantly scavenged radiation derived free radicals indicating their antioxidant potential. MfM or MfA were orally administered to Swiss albino mice at 250 or200 mg/kg body weight respectively for 7 days. The study showed significant increase in the levels of glutathione, and activities of endogenous antioxidant enzymes superoxide dismutase & catalase. Administration of MfA or MfM to mice significantly reduced electron beam radiation (EBR; 6Gy) induced increase in MDA levels. Immunomodulatory efficacy of MfM and MfA was evaluated using concanavalin-A (Con-A) induced proliferation of CFSE labelled splenic lymphocytes. Both the extracts significantly reduced proliferation in a dose-dependent manner. Further, MfM or MfA treatment prevented EBR induced histopathological changes in jejunum, spleen, liver and kidney. This demonstrates that the plant Mesua ferrea has promising antioxidant, radioprotective and immunomodulatory activity which may be attributed to the active constituents present in it.

Two non-cytotoxic type 2 ribosome-inactivating proteins (Sambucus Sieboldiana lectin and Sambucus Nigra lectin) lead neurosphere cells to caspase-independent apoptosis

We found a new phenomenon of cell damage and/or cell death of almost all types of neurosphere cells derived from telencephalons of mouse embryos. The cell damage/death was induced by non-cytotoxic type 2 ribosome-inactivating proteins: Sambucus Sieboldiana lectin (SSA) and Sambucus Nigra lectin (SNA). Self-renewal and aggregation of the neurosphere cells were inhibited under the presence of SSA or SNA. These lectins led the neurosphere cells to cell damage by making them lose their proliferation capacity. It was found that Siaα2-6Galβ1-4GlcNAc or Siaα2-6Gal on the membrane of neurosphere cells play an important role in the induction of cell damage of neurosphere cells. Moreover, despite the fact that most of the neurosphere cells cultured under the presence of SSA or SNA were Annexin V positive, these cells were not identified by DNA ladder assays. Additionally, the cells were not rescued from cell death even under the presence of a pan-caspase inhibitor. These findings imply that cell death of neurosphere cells caused by SSA or SNA is caspase-independent apoptosis via an exogenous pathway. SSA and SNA recognition molecules were detected as 39 and 42 kDa glycoproteins on the membrane of neurosphere cells.

Olive leaf extract counteracts cell proliferation and cyst growth in an in vitro model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive enlargement of kidney cysts, leading to chronic kidney disease. Since the available treatment for ADPKD is limited, there is emerging interest for natural compounds as potential therapeutic candidates. The aim of our study was to investigate whether an olive leaf extract may be able to counteract the cyst growth in an in vitro model of ADPKD. We treated WT9-12 cells with an olive leaf extract (OLE). In monolayer culture we evaluated cell viability by the MTT assay, protein expression by western-blot analysis and apoptosis by DNA laddering and TUNEL assays. For functional studies we used transient transfection and ChIP assays. Intracellular calcium measurement was performed with a spectrofluorimeter using a fluorescent probe. 3D-cell-culture was used for cyst growth studies. OLE reduced the WT9-12 cell growth rate and affected intracellular signaling due to high c-AMP levels, as OLE reduced PKA levels, enhanced p-AKT, restored B-Raf-inactivation and down-regulated p-ERK. We elucidated the molecular mechanism by which OLE, via Sp1, transactivates the p21WAF1/Cip1 promoter, whose levels are down-regulated by mutated PKD1. We demonstrated that p-AKT up-regulation also played a crucial role in the OLE-induced anti-apoptotic effect and that OLE ameliorated intracellular calcium levels, the primary cause of ADPKD. Finally, using a 3D-cell-culture model we observed that OLE reduced the cyst size. Therefore, multifaceted OLE may be considered a new therapeutic approach for ADPKD treatment.

Degradation of Poly(ε-caprolactone) and bio-interactions with mouse bone marrow mesenchymal stem cells

Bio-inspired scaffolds in bone tissue engineering using multipotential mesenchymal stem cells grow at a rapid rate found its successful use in orthopedic injury treatment. Poly(ε-caprolactone)/PCL is widely used in medical devices, tissue engineering, and drug delivery systems. Most desirable property of biodegradable polymer to be employed in medical application is synchronization of degradation with functional tissue regeneration. Limited studies have incorporated the degradation kinetics and implication of degradation products of pure unmodified PCL. The present study analyzes short term in vitro degradation profile of PCL films in physiological condition. The study reports weight loss, changes in molecular weight distribution and morphological variation in PCL thin film over a period of 90-day degradation. When the degradable material is in contact with host tissue, there exists robust and dynamic microenvironment controlling the cell functionality. To comprehend the biocompatibility aspects of polymer material, the study considered mouse bone marrow mesenchymal stem cells (BMSCs) as model system mimicking in vivo. There was no indication of toxicity revealed with MTT, LDH leakage, direct contact assay and clonogenic assay. Absence of oxidative stress and apoptosis denotes BMSCs functional integrity sustained upon exposure to PCL degradation products. Cell cycle analysis and DNA ladder assay confirmed cell survival and genomic stability. The study revealed that the topography of pure unmodified PCL surface is suitable for cell adhesion. It was also observed that the viability of differentiated cells (osteoblasts) was maintained in presence of PCL extract. Furthermore, polymer and its degradation products were proved to be hemocompatible. These results synergistically suggest that pure unmodified PCL and its degradation products are non-toxic at molecular level.

Isolation and screening of rare Actinobacteria, a new insight for finding natural products with antivascular calcification activity.

Vascular calcification (VC) is a significant pathological process in some life-threatening diseases. Several pathological mechanisms, including transdifferentiation of vascular smooth muscle cells to osteoblast-like cells and apoptosis are involved in VC. Compounds with an inhibitory effect on these processes are potentially efficient medications. In consideration of the multiple biological activities of Actinobacteria, this research was aimed at finding anti-VC metabolite-producing Actinobacteria. After the isolation and identification of Actinobacteria, the effect of their fermentation broth extracts on the apoptosis rate was measured using various methods, for example, ethidium bromide/acridine orange staining, DNA laddering and diphenylamine assays. The effect of the most effective fermentation broth extract of Actinobacteria (FBEA) on the mRNA expression of runt-related transcription factor 2 (Runx2) and osteopontin (OPN) was examined. Finally, the most effective FBEA was fractionated and the chemical composition of anti-VC fractions was analysed using GC-MS. Various VC inhibition rates were observed in the tested FBEA (20μgml-1 ; 17·9-60·15%). The inhibition of DNA fragmentation was 7-48%. The FBE with the greatest anticalcification activity belonged to Kribbella sp. UTMC 267 and, according to 16S rRNA analysis, Kribbella sancticallisti with a similarity of 98·53% is its nearest neighbour. The FBE of Kribbella sp. UTMC 267 reduced Runx2 mRNA expression by 2·95-fold and OPN mRNA expression by 28·57-fold, both of which are considered significant (P<0·05). Finally, GC-MS analysis showed the existence of potent anti-oxidative and anti-inflammation agents in FBE of Kribbella sp. UTMC 267. Actinobacterial metabolites can provide a new strategy for treating VC diseases by reducing the expression of osteogenic genes, the apoptosis rate and oxidative stress. This study highlights the therapeutic potential of Kribbella sp. metabolites and Actinobacteria as a new natural source for drug discovery programs in the nonantibiotic bioactivity field.

Myricetin enhances on apoptosis induced by serum deprivation in PC12 cells mediated by mitochondrial signaling pathway

Polyphenols have many beneficial effects and an effective disease therapeutic auxiliary drug. Previously, myricetin, a polyphenol, had been reported to possess various biological effects on human physiology. However, mechanism of myricetin on apoptosis induced in PC12 cells is still unclear. PC12 cells were treated with myricetin in two concentration levels comprising 0.1 and 1 μM under serum-free condition. As a result, morphological changes were observed using trypan blue assay. DNA fragmentation was determined by DNA ladder assay to evaluate DNA damage levels. Western blotting results showed that cytosolic cytochrome c which was released from mitochondria. Subsequently, tumor suppressor gene p53, pro-apoptotic and anti-apoptotic Bcl-2 family proteins Bax and Bcl-2 were expressed. The caspase cascade reaction was induced through caspase 3 and 9 expression. From these results, it is suggested that myricetin significantly enhanced the apoptosis induced by serum deprivation in a dose-dependent manner in PC12 cells.

Leuconostoc mesenteroides-derived anticancer pharmaceuticals hinder inflammation and cell survival in colon cancer cells by modulating NF-κB/AKT/PTEN/MAPK pathways

Promising results from different studies on the effect of probiotics in cancer prevention and therapy have so far been reported. However, the molecular mechanism of the interaction of probiotics with cancer cells is yet to be fully understood. In the present study, Leuconostoc mesenteroides was isolated from traditional dairy products, and its probiotic characteristics were determined. HT-29 cells were treated with conditioned-medium of designated bacteria and the cell apoptosis was studied at cellular and molecular level using DAPI staining, flow cytometry, DNA ladder assays, and real-time quantitative-PCR (q-PCR). Based on our findings, L. mesenteroides promoted apoptosis in colon cancer cell line by upregulation of MAPK1, Bax, and caspase 3, and downregulation of AKT, NF-κB, Bcl-XL expressions and some key oncomicroRNAs such as miRNA-21 and miRNA-200b significantly (p≤0.03). The results indicated the likelihood of the examined probiotic as an alternative or complementary treatment modality in signaling–targeted cancer therapy.

Hyperoside protects against hypoxia/reoxygenation induced injury in cardiomyocytes by suppressing the Bnip3 expression

AimsRole of hyperoside in protecting cardiomyocytes from ischemia/reperfusion induced injury has been proved. However, possible protecting mechanisms remain unclear. To fix the problem, an essential pro-apoptotic protein Bnip3 was studied in our experiments. Methods and resultsNeonatal rat cardiomyocytes were used and submitted to hypoxia for 8h followed by reoxygenation for 2h to simulate the ischemia/reperfusion injury. Hypoxia/reoxygenation(H/R) induced damage to cardiomyocytes and the protective effect of hyperoside were examined by means of MTT assay. H/R-induced apoptosis was assessed by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL) and DNA Ladder assay. mRNA expression of Bnip3 was determined by use of quantitative real-time reverse transcription polymerase chain reaction assay. Protein levels of Bnip3, Bax, Bcl-2 and cleaved caspase-3 were examined using western-blot assay. Our results showed that H/R caused great damage to cardiomyocytes, upregulated the protein expressions of Bnip3, Bax, cleaved caspase3, and decreased the expression of the anti-apoptotic protein of Bcl-2. Whereas, compared with the H/R group, a decrease in activities of Bnip3, Bax, cleaved caspase3, and a promoting expression of Bcl-2 were detected in the H/R goup pretreated with hyperoside. ConclusionIt was concluded in our study that H/R-induced apoptotic effect in cardiomyocytes could be attenuated by hyperoside, and the protective role of hyperoside, if not completely, could be partly through the suppression of the pro-apoptotic gene Bnip3.

Biopolymer mediated nanoparticles synthesized from Adenia hondala for enhanced tamoxifen drug delivery in breast cancer cell line

Silver nanoparticles (AgNPs) are an important class of nanomaterials, which have used as antimicrobial and disinfectant agents due to their detrimental effect on target cells. In the present study it was explored to deliver a novel tamoxifen drug system that can be used in breast cancer treatment, based on chitosan coated silver nanoparticles on MCF-7 human breast cancer cells. AgNPs synthesized from Adenia hondala tuber extract were used to make the chitosan coated AgNPs (Ch-AgNPs), in which the drug tamoxifen was loaded on chitosan coated silver nanoparticles (Tam-Ch-AgNPs) to construct drug loaded nanoparticles as drug delivery system. The morphology and characteristics of the Ch-AgNPs were investigated by UV, FTIR, zeta potential and FESEM. Furthermore, the toxicity of AgNPs, Ch-AgNPs, Tam-Ch-AgNPs was evaluated through cell viability, lactate dehydrogenase leakage, reactive oxygen species generation, caspase-3, DNA laddering, and TUNEL assay in human breast cancer cells (MCF-7) and HBL-100 continuous cell line as a control. Treatment of cancer cells with various concentrations of AgNPs, Ch-AgNPs, Tam-Ch-AgNPs for 24 h revealed that Tam-Ch-AgNPs could inhibit cell viability and induce significant membrane leakage in a dose-dependent manner. Cells exposed to Tam-Ch-AgNPs showed increased reactive oxygen species and hydroxyl radical production when compared to AgNPs, Ch-AgNPs. Furthermore, the apoptotic effects of AgNPs, Ch-AgNPs, Tam-Ch-AgNPs were confirmed by activation of caspase-3 and DNA nuclear fragmentation. The present findings suggest that Tam-Ch-AgNPs could contribute to the development of a suitable anticancer drug delivery.

Wogonin, a natural flavonoid, intercalates with genomic DNA and exhibits protective effects in IL-1β stimulated osteoarthritis chondrocytes

Wogonin has recently been shown to possess anti-inflammatory and chondroprotective properties and is of considerable interest due to its broad pharmacological activities. The present study highlights that Wogonin binds DNA and exerts chondroprotective effects in vitro. Wogonin showed strong binding with chondrocytes genomic DNA in vitro. The mode of binding of Wogonin to genomic-DNA was assessed by competing Wogonin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. EtBr fluorescence reduced significantly with increase in Wogonin concentration suggesting possible DNA intercalation of Wogonin. Further, in silico molecular docking of Wogonin on mammalian DNA also indicated possible intercalation of Wogonin with DNA. The denaturation and FRET studies revealed that Wogonin prevents denaturation of DNA strands and provide stability to genomic DNA against a variety of chemical denaturants. The cellular uptake study showed that Wogonin enters osteoarthritis chondrocytes and was mainly localized in the nucleus. Wogonin treatment to OA chondrocytes protects the fragmentation of genomic DNA in response to IL-1β as evaluated by DNA ladder and TUNEL assay. Treatment of chondrocytes with Wogonin resulted in significant suppression of IL-1β-mediated induction of ROS. Further, Wogonin exhibited protective potential through potent suppression of extrinsic and intrinsic apoptotic pathways and induction of anti-apoptotic proteins in IL-1β-stimulated osteoarthritis chondrocytes. Our data thus suggest that DNA intercalation by Wogonin may result in the stabilization of genomic DNA leading to protective activity.

Abstract 4083: Basic mechanisms of Vernonia amygdalina exert antitumor activities in breast cancer cells

Abstract Breast cancer continues to represent the largest cause of mortality in the world especially among women. It is estimated that cancer kills more women in the USA than any other illness and it is also the leading cause of death among all Americans. Traditional medicine represents the first-choice of healthcare treatment for at least 80% of people living in developing countries. Preliminary studies from our laboratory have shown that a novel natural product, extracts of Vernonia amygdalina (VA) leaf exerts DNA-damaging anticancer activities against breast cancer. There is a need to develop targeted approaches for the treatment of human breast cancer. Therefore, the goal of this research was to determine the therapeutic mechanisms of V. amygdalina leaf extracts as anti-cancer agent in the management of breast cancer. To achieve our goal, cell viability, live and death cells were determined by trypan blue exclusion test. The extent of oxidative cell/tissue damage was determined by measuring malondialdehydes (MDA) levels. Expression of p53 tumor suppressor gene was assessed both by immunofluorescent and western blot analyses. Cell apoptosis was measured both by flow cytometry analysis and DNA laddering assay. Data obtained from the Trypan blue indicated that V. amygdalina significantly reduced the viability of MCF-7 cells in a dose-dependent response, showing gradual increase in the loss of viability in V. amygdalina-treated cells due to membrane damage. We observed an up-regulation of p53 tumor suppressor gene in V. amygdalina-treated cells compared to the control. Flow cytometry data showed a strong dose-response relationship between V. amygdalina exposure and caspase-3 positive cells. These results were confirmed by data of DNA laddering assay showing a clear evidence of nucleosomal DNA fragmentation in V. amygdalina-treated cells. Collectively, data generated from the present study demonstrated that V. amygdalina destroys breast cancer cells by reducing the percentage of cell viability and inducing apoptosis through up-regulation of p53 tumor suppressor gene, activation of caspase-3, and induction of nucleosomal DNA fragmentation associated with the formation of MDA, a by-product of lipid peroxidation and biomarker of oxidative stress. This result suggests that V. amygdalina treatment may be a good anti-cancer candidate for the treatment of breast cancer. Research supported by NIH Grant No. NIMHDG12MD007581) Citation Format: Clement G. Yedjou, Paul B. Tchounwou. Basic mechanisms of Vernonia amygdalina exert antitumor activities in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4083. doi:10.1158/1538-7445.AM2017-4083

Abstract 4105: Diosgenin and temozolamide: A potential combinatorial chemotherapy to overcome temozolamide resistance in glioblastoma multiforme

Abstract Introduction: Glioblastoma multiforme (GBM) accounts for the most aggressive form of tumor showing poor prognosis. Prevailing treatment modality includes chemotherapy with temozolomide (TMZ) concomitant with surgical resection and/or irradiation. However, it has been observed that a number of patients are developing resistance to TMZ owing to its high dosage regimen. The aim of this study is to examine the effects of diosgenin (DSG), a natural steroidal saponin obtained from fenugreek, in combination with TMZ in human GBM cells and TMZ resistant GBM cells. Methodology: The potential of combinatorial chemotherapy for overcoming TMZ resistance was evaluated through development of TMZ resistant GBM clones. These clones were generated by treatment of GBM cells with sub-lethal dose of TMZ over several cycles. Cellular effects were studied by viability assay, flow cytometry and wound healing assay on both single and combined drug treated GBM cells and TMZ resistant cells, respectively. The morphological study and cellular uptake in combined or individual drug treated cells was assessed by microscopy and immunofluorescence staining. The time dependant effect of drugs on invasive and migratory potential of GBM cells and TMZ resistant cells was analysed through zymography for matrix metalloproteinases (MMPs) activity and western blot for apoptotic proteins, epithelial and mesenchymal (EMT) markers, MMPs and VEGF expression. Further, the drug induced apoptosis was also assessed through chromatin condensation and DNA laddering assay. Results: Our investigation shows that co-administration of DSG and TMZ resulted in a substantial increase in GBM cell apoptosis and marked inhibition of cell growth in vitro. Anti-angiogenic and anti-invasive potential of DSG and TMZ were assessed through in vitro studies. At molecular level, DSG and TMZ synergistically lower XIAP expression and cleavage of intracellular death substrates such as PARP thereby shifting the balance from survival to apoptosis as indicated by the rise in the sub-G1 cell population. This combination also alters EMT markers, downregulates the expression of Bcl-xL, Bcl-2, Mcl1, MMPs and VEGF and induces expression of Bax, AIF, cytochrome C. Conclusion: The results suggested that DSG in combination with TMZ reduced the dose of TMZ, when administered singly, and also inhibited the migration and invasion of GBM and TMZ resistant cells. It induced apoptosis and altered the expression of EMT markers. These findings reveal a new therapeutic potential for overcoming TMZ resistance in GBM therapy. This novel modality may be a promising tool for GBM treatment. Citation Format: Y Rajesh, Angana Biswas, Subhayan Das, Mahitosh Mandal. Diosgenin and temozolamide: A potential combinatorial chemotherapy to overcome temozolamide resistance in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4105. doi:10.1158/1538-7445.AM2017-4105

Mechanism underlying methyl eugenol attenuation of intestinal ischemia/reperfusion injury.

Intestinal ischemia/reperfusion (I/R) injury is associated with a high risk of mortality in the clinical situation. Many factors are involved in I/R, including reactive oxygen species, cytokine release, and apoptosis. We aimed to determine whether a pure methyl eugenol (ME) given before intestinal ischemia, protects against intestinal I/R injury and the possible mechanism involved in this protection. Rat received ME (100 mg/kg) for 30 days then underwent intestinal I/R with 30 min ischemia and 60 min reperfusion. Serum lactate dehydrogenase (LDH) level, tissue malondialdehyde (MDA), as well as some antioxidant biomarkers were assessed, while the serum level of tumor necrosis factor alpha (TNF-α) was determined by ELISA. The change in TNF-α and interleukin 6 (IL-6) gene expressions were evaluated and confirmed by assessing protein level of TNF-α in the intestinal tissue by immunohistochemistry. Apoptosis was evaluated using DNA-laddering assay and by detecting caspase-3 immunohistochemically. Administration of ME prior to I/R injury resulted in a modulation of the production of MDA, LDH, and nitric oxide and restoration of the tested oxidative stress biomarkers. Pretreatment with ME downregulated messenger RNA of TNF-α and IL-6 inflammatory cytokines and their protein expressions in I/R rats. Marked inhibition of the apoptotic DNA and improvement of the architectures of small intestine were observed after pretreatment with ME. ME exhibits a protective effect against intestinal I/R via amelioration of the oxidative stress and inflammatory cytokines gene expression. Therefore, the supplementation of ME prior to intestinal I/R might be helpful in the attenuation of I/R complications.

Bi-directionally protective communication between neurons and astrocytes under ischemia

The extensive existing knowledge on bi-directional communication between astrocytes and neurons led us to hypothesize that not only ischemia-preconditioned (IP) astrocytes can protect neurons but also IP neurons protect astrocytes from lethal ischemic injury. Here, we demonstrated for the first time that neurons have a significant role in protecting astrocytes from ischemic injury. The cultured medium from IP neurons (IPcNCM) induced a remarkable reduction in LDH and an increase in cell viability in ischemic astrocytes in vitro. Selective neuronal loss by kainic acid injection induced a significant increase in apoptotic astrocyte numbers in the brain of ischemic rats in vivo. Furthermore, TUNEL analysis, DNA ladder assay, and the measurements of ROS, GSH, pro- and anti-apoptotic factors, anti-oxidant enzymes and signal molecules in vitro and/or in vivo demonstrated that IP neurons protect astrocytes by an EPO-mediated inhibition of pro-apoptotic signals, activation of anti-apoptotic proteins via the P13K/ERK/STAT5 pathways and activation of anti-oxidant proteins via up-regulation of anti-oxidant enzymes. We demonstrated the existence of astro-protection by IP neurons under ischemia and proposed that the bi-directionally protective communications between cells might be a common activity in the brain or peripheral organs under most if not all pathological conditions.

A novel spiroindoline targets cell cycle and migration via modulation of microtubule cytoskeleton.

Natural product-inspired libraries of molecules with diverse architectures have evolved as one of the most useful tools for discovering lead molecules for drug discovery. In comparison to conventional combinatorial libraries, these molecules have been inferred to perform better in phenotypic screening against complicated targets. Diversity-oriented synthesis (DOS) is a forward directional strategy to access such multifaceted library of molecules. From a successful DOS campaign of a natural product-inspired library, recently a small molecule with spiroindoline motif was identified as a potent anti-breast cancer compound. Herein we report the subcellular studies performed for this molecule on breast cancer cells. Our investigation revealed that it repositions microtubule cytoskeleton and displaces AKAP9 located at the microtubule organization centre. DNA ladder assay and cell cycle experiments further established the molecule as an apoptotic agent. This work further substantiated the amalgamation of DOS-phenotypic screening-sub-cellular studies as a consolidated blueprint for the discovery of potential pharmaceutical drug candidates.

Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B.

ObjectiveThe present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B.Methodology/Principal findingsWe studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2− (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further, study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophage.ConclusionsKalsomeTM10 induces apoptotic-like cell death in L. donovani parasites to exhibit its anti-leishmanial function.

Hepatoprotective effect of the solvent extracts of Viola canescens Wall. ex. Roxb. against CCl4 induced toxicity through antioxidant and membrane stabilizing activity

BackgroundViola canescens Wall. ex. Roxb. exhibits analgesic, antimalarial and antispasmodic activities. It is used folklorically for the treatment of liver diseases, hypertension, malaria and cancer. The current study investigates phytochemical constituents, antioxidant and hepatoprotective activity of solvent extracts of whole plant of Viola canescens.MethodsPhytochemicals, acute toxicity study and antioxidant activity of Viola canescens methanolic extract (VCME), ethyl acetate fraction (EAF), and partially purified EAF (90% EAF and combination of 80% EAF + 20% methanol fraction (EAF + Me) was carried out. Hepatoprotective activity of VCME, EAF (200 and 400 mg/kg body weight) and partially purified EAF (50 mg/kg body weight) was investigated in carbon tetrachloride (CCl4) intoxicated BALB/c mice for 7 days. Membrane stabilization effect was determined by hypotonic solution induced hemolysis while DNA ladder assay was carried out by polyacrylamide gel electrophoresis.ResultsPhytochemical screening of VCME showed the presence of alkaloids, phenols, flavonoids, saponins, carbohydrates, tannins and triterpenes. VCME, EAF (at 200 and 400 mg/kg body weight) and partially purified EAF (90% EAF and EAF + Me) at 50 mg/kg body weight significantly reduced the level of ALT, ALP, total bilirubin and restored the level of serum protein in comparison to CCl4 treated group. A significant reduction in malondialdehyde (MDA) and elevation in catalase (CAT) and superoxide dismutase (SOD) level was observed in extract treated animals as compared to CCl4 (p < 0.05). The IC50 values in membrane stabilization potential for VCME, EAF and sodium salicylate were 3.7 ± 0.11, 3.4 ± 0.15 and 3.2 ± 0.09 mg/ml, respectively. Similarly, CCl4 induced degradation of DNA was counteracted by VCME and EAF. The liver biopsy of mice treated with the solvent extracts showed remarkable restoration of normal histological archeitecture.ConclusionsViola canescens showed significant hepatoprotective potential due to its antioxidant and membrane stabilization effect.