Trypan Blue Dye Exclusion Method Research Articles

Synthesis, characterization and cytotoxicity of zinc oxide nanoparticles by green synthesis method

The use of engineered nanoparticles (NPs) has increased considerably because of their almost universal applications, such as electronics, optical, pharmaceutical, biomedical and energy fields, which have been expanding rapidly during the past few decades. Due to the widespread applications of zinc oxide nanoparticles (ZnONPs), the potential exposure of workers, consumers and scientists to these particles has increased. We conducted a ZnONPs synthesis through a low cost, eco-friendly and sustainable method, where Beta vulgaris extract was utilized as a reducing agent. Characterization of ZnONPs was conducted by High-Resolution Transmission Electron Microscopy (HRTEM), Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray diffraction analysis (XRD) to obtain the morphological properties. In this study, we evaluate ZnONPs toxicity in vitro in two mouse fibroblast cell lines C2C12 and L929 myoblast cell lines and human lymphocytes from peripheral blood (HLPB), using Trypan blue dye exclusion method. MTT assay method only in cell lines C2C12 and L929 after 24 h of exposure. The results show the ZnONPs size was between 16 and 24 nm, with hexagonal crystal structure and semi-spherical morphology. The toxic effects of ZnONPs were analyzed in the two cell lines after a 24 and 48 h incubation period. The value of TC50 ZnONPs was calculated after a 24 h exposure, which was found to be between 28 and 37 μg/mL and 20–30 μg/mL at a 48 h exposure. The viability value was reported non toxic at concentrations of 1 × 10−6-1x10−3 μg/mL.

Fluorescent di-(2-picolyl)amine based drug-like ligands and their Re(CO)3 complexes towards biological applications

Herein we report two new fac-Re(I) tricarbonyl complexes, fac-[Re(CO)3(N(SO2)(1-nap)dpa)]PF6 (C1) and fac-[Re(CO)3(N(SO2)(2-nap)dpa)]PF6 (C2), of two naphthalene derivatized tridentate ligands (N(SO2)(1-nap)dpa (L1) and N(SO2)(2-nap)dpa (L2)) and one reported Re(I) complex, fac-[Re(CO)3(N(SO2Me2Nnap)dpa)]PF6 (C3), of a dansyl appended di-2-picolylamine ligand (N(SO2Me2Nnap)dpa (L3)). The properties of the compounds were elucidated using spectrophotometric measurements (UV–Vis, FTIR and 1H NMR). A single crystal X-ray study was carried out for the three ligands. The 1H NMR spectra of the three complexes obtained in DMSO‑d6 displayed two doublets (exo-CH and endo-CH) for the magnetically inequivalent methylene protons, compared to their uncoordinated ligands where the methylene protons show a singlet peak. The formation of the metal complexes was further supported by FTIR spectra in which the SN stretching band for the metal complexes appears at lower wavenumbers compared to that of the corresponding free ligands. In comparison with the uncoordinated ligands, the Re(I) complexes, C1 and C3, displayed a bathochromic shift while C2 showed a hypsochromic shift in the absorption spectra in methanol. The fluorescent maxima and the fluorescence quantum yield (ΦF) of L1 and L2 were 338 nm (ΦF = 0.056) and 343 nm (ΦF = 0.039), respectively. Interestingly, all the compounds except C2 showed excellent fluorescent emissions. Biologically, the compounds were investigated for their cytotoxicity in vitro on human lymphocytes following the Trypan blue dye exclusion method. It was observed that both Re(I) complexes as well as the ligands show low toxicity towards human lymphocytes at working concentrations below 10 mg/ml (L1, L2: 0.026 M, L3: 0.023 M, C1, C2, C3: 0.012 M). In silico studies revealed that the ligands are potential candidates for anti-inflammatory agents and this was further supported by molecular docking studies, in which they showed a higher affinity for Prostaglandin synthase 2. These drug-like molecules, having an affinity to bovine serum albumin, are thus potential drug leads for anti-inflammatory agents.

Cesium suppresses fibroblast proliferation and migration.

During wound healing, fibroblasts proliferate from the margin, and migrate into the provisional matrix where they differentiate into myofibroblasts resulting in wound contraction; however, fibroblasts are hyperproliferative during chronic tissue damage. We previously reported that cesium chloride inhibited a human cancer cell proliferation; therefore, cesium is also presumed to suppress fibroblast proliferation. We here investigated the effects of cesium chloride on the proliferation and migration of murine embryotic fibroblast cells, NIH/3T3 cells. Cultured NIH/3T3 cells with 0-10 mM sodium and cesium chloride were counted using trypan blue dye-exclusion method, then cell growth and viability were evaluated. The percentage of wound closure was calculated by scratch assay. The number of the cells was decreased by application of 1-10 mM cesium in a dose-dependent manner, whereas the viability of the cells was unchanged. The treatment with 3-10 mM cesium inhibited the proliferation rate and % of wound closure compared with controls. These results suggested that cesium inhibits the proliferation and migration of fibroblast cells. This study indicates a possible therapeutic role of cesium chloride in the treatment of wound healing and fibrosis.

Synthesis, Spectroscopic Characterization, Antibacterial and Short Term in vitro Cytotoxicity Studies of Copper(II) Complexes of Novel Tridentate N,N,S Donor Ligand 2-Benzoylpyridine-N(4),N(4)-(N,N-diethyl-N-methylamine-2,2'-diyl)thiosemicarbazone

A tridentate N,N,S-donor ligand, 2-benzoylpyridine-N(4),N(4)-(N,N-diethyl-N-methylamine-2,2'-diyl)thiosemicarbazone (Hbptsc) has been synthesized and characterized by elemental CHN analysis, UV-visible, FT-IR and 1H NMR spectroscopy. Copper(II) complexes of the ligand, Hbptsc synthesized have been characterized by elemental analysis, UV-visible spectra, FTIR spectra and EPR spectroscopic simulation. The complexes hold the stoichiometry of the type [CuLX] where X= Cl (1), NO3 (2), SO4 (3), N3 (4), SCN (5) confirmed by the molar conductivity studies of 10-3 M solutions in DMF at room temperature. The EPR spectra of the complexes recorded in DMF at 77 K shows an axial type spectra with two distinct g-values, g|| and g⊥ indicating a four coordinated planar geometry. The antimicrobial studies of the copper(II) complexes shows an appreciable activity against both gram positive and gram negative bacteria using streptomycin as positive control. The short term in vitro cytotoxicity studies following trypan blue dye exclusion method exhibits pronounced activity against the Dalton’s Lymphoma Ascites tumour cells extruded from the peritoneal cavity of mice.

Influence of LBP alone or Combined with TRAIL on Apoptosis of MLL Rearranged Leukemic Cells

To investigate the effect of lycium barbarum polysaccharide (LBP) alone or combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the apoptosis of leukemia cell lines with MLL gene-rearrangement, and to explore the cell apoptotic pathway after the combined action. MLL-ALL cell line KOCL44 and KOCL45 were selected as the research object, then the control and experimental groups were set up. The cell survival rate was measured by the trypan blue dye exclusion method, the cell early apoptosis and expression of death receptors on the cell surface were detected by flow cytometry with Annexin-V/PI double staining. The protein level of caspase-8, BID, caspase-3, caspase-9, BAD, BCL-2, as well as mitochondrial and cytosol Cyto-C were detected by Western blot. LBF combined with TRAIL inhibited the growth of KOCL44 and KOCL-45 cells and showed the synergistic effect, the results of flow cytometry with Amnexiu V/PI double staining were consistent with above-mentioned results. After treatment of KOCL44 and KOCL45 cells with LBF plus TRAIL, the significant expression of DR4 on cell surface was not found, while the expression of DR4 receptor was enhanced significantly, the pro-apoptotic proteins including caspase-8, BID, caspase-3, caspase-9 and BAD were activated significantly and BCL-2 was suppressed significantly with time-dependent manner. The expression of mitochondria cyto-C in KOCL44 and KOCL45 decreased along with prolonging of treatment time (r=-0.95, r=-0.866), while the expression of cytosol cyto-C in KOCL44 and KOCL45 increased along with prolonging of treatment time (r=0.883, r=0.903). The combination of LBP and TRAIL significantly increases the apoptosis of KOCL44 and KOCL45, and the LBP and TRAIL can up-regulate the expression of TRAIL death receptor-DR5 on the cell surface, activate the pathway of caspase and mito-chrondia mitachondria, thus enhance the sensitivity of KOCL44 and KOCL45 to TRAIL induced apoptosis through both mitochondrial and apoptotic pathway.

A polyherbal formulation, HC9 regulated cell growth and expression of cell cycle and chromatin modulatory proteins in breast cancer cell lines

Ethnopharmacological relevanceHC9, a polyherbal formulation, is based upon a traditional Ayurvedic formulation, Stanya Shodhana Kashaya (SSK, having 10 plant materials), formulated on Stanyashodhana gana, explained by Charaka in Charakasaṃhita Sutrasthana IV and mentioned in other texts as well. Stanyasodhana is the Sanskrit name for a group of medicinal plants, classified for “improving the quality of milk”. SSK is used by Ayurvedic practitioners for the cleansing and detoxification of breast milk in lactating mothers as well as for the management of various clinical conditions. HC9 is composed of equal ratios of nine different medicinal plants that include Picrorhiza kurroa Royle ex Benth., Cyperus rotundus L., Zingiber officinale Roscoe, Cedrus deodara (Roxb. ex D.Don) G.Don, Tinospora cordifolia (Willd.) Miers, Holarrhena antidysenterica (Roth) Wall. ex A.DC., Swertia chirata Buch.-Ham. ex Wall., Cissampelos pareira L. and Hemidesmus indicus (L.) R. Br. ex Schult.. It differs from the SSK formulation by having one ingredient [Marsdenia tenacissima (Roxb.)Moon (Murva)] less, due to its unavailability since it is mostly found in tropical hilly tracts of peninsular India and Vindhya ranges as well as in lower Himalayan tracts. All the medicinal plants in the formulation have reported activity against different types of cancers. Aim of the studyThe present study is aimed at evaluating the anticancer activity of the polyherbal formulation (HC9) and its mechanism of action against breast cancer cell lines. Materials and MethodsThe effect of HC9 on the viability of breast cancer (MCF-7 and MDAMB231) and non-cancerous (MCF-10A) cell lines was evaluated by MTT assay. The effect on cell growth and colony formation potential of cancer cells was determined by trypan blue dye exclusion method and soft agar assay, respectively. Cell cycle arrest was determined by propidium iodide (PI) staining and analyzed by flowcytometer. Scratch wound assay was used for studying cell migration. Cell invasion was determined by using BD BioCoat Matrigel invasion chambers. The gene expression of HIF-1α was examined by RT-PCR. The expression of p53, SMAR1, p16, MMP-2, CDP/Cux, p21, Rb, phospo-Rb (ppRb), VEGF, NFқB and COX-2 proteins was determined by western blotting. ResultsHC9 significantly altered growth of breast cancer cell lines, MCF-7 and MDA MB-231. It blocked the cell cycle progression at S phase in MCF-7 by up regulating the expression of p53, p21 and p16 proteins. In MDA MB-231, HC9 induced G1 phase arrest by up regulating the expression of p53, p21 and pRb proteins with simultaneous decrease in ppRb. It significantly reduced migration and invasion in both the cell lines, accompanied by decrease in the expression of MMP-2/9, HIF-1α and VEGF. HC9 decreased the expression of inflammatory markers (NF-қB, COX-2), and modulated the expression of chromatin modulators (SMAR1 and CDP/Cux) in both MCF-7 and MDA MB-231. ConclusionsHC9 exhibited potent anticancer activity against breast cancer cells, thereby warranting further pre-clinical and clinical studies in future.

Effects of SAHA and RG7388 on p21WAF1/CIP1 and p27KIP1 mediated Pathways in Cancer Cells

Epigenetic modifications can lead to significant influence on cancer development, growth and progression. The main epigenetic modifications observed in human are methylation and acetylation. Histone acetylations are largely regulated by histone deacetylases (HDACs) and, several HDAC inhibitors (HDACi) such as SAHA (Vorinostat), which are already approved for human use or currently in different stages of clinical trials, belong to the new class of drugs with promising effects on the cancer growth and metastatic process. The small molecule RG7388 is a newly developed inhibitor that is specific for an oncogene‐derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. One of the unique characteristics of these two drugs is their ability to induce p21 expression through distinct mechanisms. A clear understanding related to the molecular mechanism whereby SAHA can induce cell cycle arrest and trigger necrosis, apoptosis or necroptosis is still evolving. Similarly, the ability of RG7388 for producing anticancer effect is undergoing thorough investigation, since it can produce p53 dependent and p53 independent effects. So far, our experiments with MCF‐7 breast cancer cells and LNCaP prostate cancer cells have confirmed the abilities of these two drugs to induce p21 expression through distinct mechanisms. In addition, these two drugs were expected trigger cell cycle arrest and cell death through mechanisms that are similar. Therefore, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 by using the MCF‐7 and LNCaP cells. The cytotoxicity, cell cycle arrest and apoptosis/necroptosis effects of the treatments were assessed by using Trypan Blue Dye Exclusion (TBDE) method, and fluorescence assay with caspase 3/7 specific DEVD‐amc fluorogenic substrate. In addition, cell cycle and apoptosis related proteins were analyzed by immunoblotting methods. To this point, our results from MCF‐7 and LNCaP cells suggest that the cell death caused by SAHA treatment in both cell lines are through induction of p21WAF1/CIP1 and p27Kip1 levels, that could be independent of p53. On the other hand, treatment of LNCaP cells with RG7388 was able to induce p21WAF1/CIP1 expression through inhibition of MDM2 that was also causing significant cell death. However, the RG7388 treatment was not able to elevate p21WAF1/CIP1 in MCF‐7 cells, even though there was clear evidence of p53 elevation in these cells. Hence, we are suspecting that, there is some level of uncoupling of p53 mediated transcriptional induction of p21WAF1/CIP1 in MCF‐7 cells. As of now, our studies confirm that two different drugs, which have the ability to induce p21 expression, exhibit distinct mechanisms of cell death through p53 dependent and independent pathways. Since it points to an interesting interplay between multiple pathways, confirmation of the actual cell death mechanism induced by RG7388 in MCF‐7 and LNCaP cancer cells requires additional exploration.Support or Funding InformationThis research was supported by the Royal Dames of Cancer Research Inc., Fort Lauderdale, FloridaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cell Cycle Arrest and Cytotoxic Effects of SAHA and RG7388 Mediated through p21WAF1/CIP1 and p27KIP1 in Cancer Cells.

Background and Objective: Alterations in gene expressions are often due to epigenetic modifications that can have a significant influence on cancer development, growth, and progression. Lately, histone deacetylase inhibitors (HDACi) such as suberoylanilide hydroxamic acid (SAHA, or vorinostat, MK0683) have been emerging as a new class of drugs with promising therapeutic benefits in controlling cancer growth and metastasis. The small molecule RG7388 (idasanutlin, R05503781) is a newly developed inhibitor that is specific for an oncogene-derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. These two drugs have shown the ability to induce p21 expression through distinct mechanisms in MCF-7 and LNCaP cells, which are reported to have wild-type TP53. Our understanding of the molecular mechanism whereby SAHA and RG7388 can induce cell cycle arrest and trigger cell death is still evolving. In this study, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 using MCF-7 and LNCaP cells. Materials and Methods: The cytotoxicity, cell cycle arrest, and apoptosis/necroptosis effects of the SAHA and RG7388 treatments were assessed using the Trypan Blue dye exclusion (TBDE) method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence assay with DEVD-amc substrate, and immunoblotting methods. Results: The RG7388 treatment was able to induce cell death by elevating p21WAF1/CIP1 through inhibition of MDM2 in LNCaP, but not in MCF-7 cells, even though there was evidence of p53 elevation. Hence, we suspect that there is some level of uncoupling of p53-mediated transcriptional induction of p21WAF1/CIP1 in MCF-7 cells. Conclusion: Our results from MCF-7 and LNCaP cells confirmed that SAHA and RG7388 treatments were able to induce cell death via a combination of cell cycle arrest and cytotoxic mechanisms. We speculate that our findings could lead to the development of newer treatments for breast and prostate cancers with drug combinations including HDACi.

Evaluation of Cytotoxic Activity of Annona muricata Fruits and Leaves

Introduction: Screening of various medicinal plants for biological active compounds has become a vital source of cancer related drugs. The present study was designed to evaluate and compare the cytotoxicity of Annona muricata fruits and leaves. Method: The leaves and fruits are dried and standardised by performing ash value, extractive value and phytochemical screening, the dried fruits and leaves of Annona muricata were defatted with petroleum ether and extracted using methanol as solvent. The extracts were evaluated for in-vitro cytotoxic study by MTT assay using Hep G2 cell lines and trypan blue dye exclusion method using DLA and EAC cells. Haemolytic activity was performed to determine the toxicity of extracts to normal cells. Results: The qualitative analysis of phytochemical extracts and parts possesses carbohydrates, cardiac glycosides, alkaloids, flavones and tannins. In MTT assay, while comparing the cytotoxic action of methanolic extracts of Annona muricata leaves and fruits of (MEAML and MEAMF) with standard curcumin (18.6µg/ml), MEAMF (IC50=12.4 µg/ml) showed more cytotoxic action to Hep G2 cell lines than MEAML (IC50=82.5µg/ml). In dye exclusion test, MEAML showed an IC50 value of about 140.3µg/ml, 137.2µg/ml for DLA and EAC respectively, whereas 179.4µg/ml, 165.2µg/ml in MEAMF. None of the extract possessed any haemolytic activity revealing their non-toxicity on normal cells. Conclusion: Methanolic extracts of Annona muricata fruits and leaves possess cytotoxic activity with no significant haemolytic activity towards normal erythrocytes.

An in vivo immunomodulatory and anti-inflammatory study of fermented Dendropanax morbifera L\xe9veille leaf extract

BackgroundMedicinal plants represent a source of new drugs for the prevention and treatment of infectious diseases. Dendropanax morbifera Léveille is an economically and medicinally important subtropical tree that has various biological activities. However, its ability to affect immune responses in vivo is unknown. Hence, this study was designed to examine the immunomodulatory activity of fermented D. morbifera extract in BALB/c mice.Methodsfive-week-old female BALB/c mice were arranged in six groups and kept under a standard laboratory condition. Splenocyte counts were determined using the trypan blue dye exclusion method, and splenic lymphocyte proliferation was determined using concanavalin A and lipopolysaccharide (LPS). Flow cytometric analysis was performed to phenotype T-lymphocytes. Next, cytokine and immunoglobulin quantitation was performed using sandwich ELISA.ResultsThe results showed an increase in spleen cells by 71 and 67% in mice treated with 125 and 250 mg/kg of D. morbifera, respectively. In addition, splenocyte proliferation was increased 58.7% in response to concanavalin A treatment, while LPS treatment induced a 73.3% increase in mice treated with 125 mg/kg. T-cell phenotypic analysis indicated that D. morbifera-treated groups showed higher CD8a+, CD11b and CD3+ T-cell expression. However, the treatment groups showed suppression of IL-1α, Il-1β and IL-4. In addition, the IgG super-family was downregulated in a dose-dependent manner by 4.5% up to 43.7%.ConclusionsTaken together, we show that D. morbifera increases the number and proliferation of T- and B-lymphocytes. Moreover, these effects may play a role in boosting non-specific immunity, while suppressing proinflammatory cytokines and immunoglobulins after a single antigen exposure.

High glycine concentration increases collagen synthesis by articular chondrocytes in vitro: acute glycine deficiency could be an important cause of osteoarthritis

Collagen synthesis is severely diminished in osteoarthritis; thus, enhancing it may help the regeneration of cartilage. This requires large amounts of glycine, proline and lysine. Previous works of our group have shown that glycine is an essential amino acid, which must be present in the diet in large amounts to satisfy the demands for collagen synthesis. Other authors have shown that proline is conditionally essential. In this work we studied the effect of these amino acids on type II collagen synthesis. Bovine articular chondrocytes were cultured under a wide range of different concentrations of glycine, proline and lysine. Chondrocytes were characterized by type II collagen immunocytochemistry of confluence monolayer cultures. Cell growth and viability were assayed by trypan blue dye exclusion method. Type II collagen was measured in the monolayer, every 48 h for 15 days by ELISA. Increase in concentrations of proline and lysine in the culture medium enhances the synthesis of type II collagen at low concentrations, but these effects decay before 1.0 mM. Increase of glycine as of 1.0 mM exceeds these effects and this increase continues more persistently by 60–75%. Since the large effects produced by proline and lysine are within the physiological range, while the effect of glycine corresponds to a much higher range, these results demonstrated a severe glycine deficiency for collagen synthesis. Thus, increasing glycine in the diet may well be a strategy for helping cartilage regeneration by enhancing collagen synthesis, which could contribute to the treatment and prevention of osteoarthritis.

Effects of Vorinostat, Letrozole and RG7388 Treatments on Cell Cycle Arrest and Survival of Breast and Prostate Cancer Cells

Alterations in gene expressions are often due to epigenetic modifications that can lead to significant influence on cancer development, growth, and progression. In this respect, HDAC inhibitors such as Vorinostat, which can exert epigenetic alterations, are emerging as a new class of drugs with promising impact on the cancer growth and metastatic process. Letrozole is one of the commonly used aromatase inhibitors that can elicit strong anti‐cancer effects on breast cancers through direct as well as indirect mechanisms. On the other hand, RG7388 is a newly developed inhibitor for an oncogene‐derived protein called MDM2, which is in clinical trials for the treatment of various cancers. Our aim for this study was to understand the molecular mechanism whereby Vorinostat can induce cell cycle arrest and trigger necrosis or apoptosis, when used in monotherapy or in combination with Letrozole or RG7388. We performed assays to measure the cell cycle arrest effects of individual drug treatments alongside of combination treatments with Vorinostat + Letrozole, and Vorinostat + RG7388 using MCF‐7 and LNCaP cancer cells. The cytotoxicity, cell cycle arrest and pro‐apoptotic effects of the treatments were assessed by using Trypan Blue dye exclusion method, MTT assay, immunoblotting, RT‐PCR, and qRT‐PCR methods. Our results from MCF‐7 and LNCaP cells confirmed that Vorinostat, Letrozole and RG7388 treatments were able to induce cell death via combination of cell cycle arrest and cytotoxic mechanisms in a dose and time dependent manner. The IC50 value for Vorinostat and RG7388 were determined to be around 7.5 μM and 2 μM respectively. Although, Letrozole showed stronger cytotoxic effects with the IC50 value of 100 nM in MCF‐7 and LNCaP cells, combination of Letrozole with SAHA did not produce synergy or additive effects. On the other hand, when SAHA was used in combination with RG7388, a significant increase in the cytotoxic effect was obtained. Induction of cell cycle arrest in the cancer cells were evidenced by the elevation of p21 protein levels following SAHA and RG7388 treatments. In addition, the SAHA treatment in LNCaP cells showed significant up regulation in the expression of other cell cycle related genes such as, p27, and p53 and down regulation of AURKB, CDC25C, and CDK1. The effects of SAHA monotherapy and combinations were assessed further by using SYTOX and Ac‐DEVD‐amc [Ac (N‐acetyl)‐DEVD‐AMC (7‐amino‐4‐methylcoumarin)] staining method, to determine the extent of necrotic cell death or apoptosis. Our results confirmed that the cell death caused by SAHA treatment was primarily through induction of necrosis. On the other hand, the RG7388 treatment was able to induce apoptosis by elevating Caspase 3 activity. It appears that, during combination treatments with SAHA and RG7388, two parallel pathways are induced simultaneously that could lead to increased cancer cell death. It has been reported in the literature that the elevated levels of acetylated histones (H2 and H3) and consequent activation of intracellular signals are responsible for the cell cycle arrest and necrosis that are typically observed when HDAC inhibitors (HDACi) are used. So far, the results of SAHA and RG7388 combination experiments have confirmed the abilities of these two agents for eliciting additive or synergistic effects in MCF7 and LNCaP experimental models. We are speculating that our findings could lead to the development of newer treatments for breast and prostate cancers using this type of combinations.Support or Funding InformationThis research was supported by the Royal Dames of Cancer Research, Fort Lauderdale, Florida, USAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phytochemical characterization and biological activity evaluation of ethanolic extract of Cinnamomum zeylanicum

Ethnopharmacological relevanceIndia being a multicultural nation, every region of the country offers a distinct culinary flavor and taste. These flavors are attributed to spices and condiments which form the mainstay of Indian cuisine. Most of these spices and condiments are derived from various biodiversity hotspots in India and form the crux of India's multidiverse and multicultural cuisine. Apart from their varying aromas, flavors and tastes, these spices and condiments are known to possess several medicinal properties also. Most of these spices find considerable mention in Ayurveda, the indigenous system of medicine, as panaceas for several aliments. Cinnamomum zeylanicum (CZ), belonging to family Lauraceae and commonly known as cinnamon is one such spice known to have diverse medicinal properties since time immemorial. Aim of the studyIn the present study, apoptotic and anti-microbial activity of ethanolic extract of CZ was evaluated against human breast cancer cell line MDA-MB-231 and compared for its effect on normal kidney epithelial cell line Vero. Materials and methodsEthanolic extract of tree bark of CZ was used to determine the cytotoxic effect on MDA-MB-231 using Trypan blue dye exclusion method and cytometry. The tested dose of the extract was 10–100 µg/mL. Antibacterial activity was determined using disc diffusion method against Staphylococcus aureus and Escherichia coli in the range 2–10 mg/mL. Apoptotic activity was determined using DNA fragmentation assay. ResultsEthanolic extract of CZ was found to have an IC50 value of 25 µg/mL against MDA cell line. On the other hand, CZ extract did not have any significant effect on Vero cells even at 100 µg/mL (IC50 > 100 µg/mL). The ethanolic extract of CZ bark showed significant antibacterial activity against S. aureus at 10 mg/mL while no appreciable activity was detected against E. coli. DNA isolated from extract treated cancer cells showed a fragmentation pattern characteristic of apoptosis. However, no DNA fragmentation was observed in DNA isolated from extract treated Vero cells. ConclusionEthanolic bark extract of CZ could be potentially beneficial in treating breast cancer and may be of interest for future studies in developing integrative cancer therapy against proliferation, metastasis, and migration of breast cancer cells.

Bardoxolone methyl induces neuritogenesis in Neuro2a cells.

Bardoxolone methyl (RTA 402, CDDOMe) has been long known for its anti-inflammatory and exceptional cytotoxic activity. The biological responses to CDDOMe are truly dose dependent. And owing to the structural modifications introduced in its parent molecule oleanolic acid, CDDOMe is able to form reversible adducts with cellular proteins containing redox sensitive cysteine residues. This nature of CDDOMe makes it a multifunctional molecule targeting multiple signaling pathways. This study was initiated to study the response of Neuro2a, a mouse neuroblastoma cell line to CDDOMe. Neuro2a cells were treated with CDDOMe and all trans retinoic acid (ATRA) for 4days and observed for neurite outgrowth. The neurite length was estimated using ImageJ software (Neuron growth plugin). Cell viability was investigated using MTT dye reduction and trypan blue dye exclusion method. Gene expression of differentiation markers was analyzed using quantitative PCR. Cellular localization of Tuj1 and synaptophysin in differentiated Neuro2a cells was observed using immunofluorescence. CDDOMe ceased proliferation and induced dramatic neurite outgrowth in Neuro2a cells. These morphological changes were accompanied by time dependent increase in the mRNA levels of tyrosine hydroxylase, neurofilament 200 and synaptophysin. Besides, cytoskeleton protein Tuj1 and the synaptic vesicle protein synaptophysin were also observed to be localized in the neurites induced by CDDOMe. These early shreds of evidence suggest that CDDOMe induces differentiation in Neuro2a cells at concentrations ranging from 0.2 to 0.4μM and indeed contributes the existing knowledge on CDDOMe induced activities in cells.

Evidence that Extreme Dilutions of Paclitaxel and Docetaxel Alter Gene Expression of In Vitro Breast Cancer Cells.

Gene expression analysis of cells treated with extreme dilutions or micro amounts of drugs has been used to provide useful suggestions about biological responses. However, most of the previous studies were performed on medicines being prepared from a variety of herbal and metal sources. This study investigated the effects of ultramolecular dilution of the taxane anti-cancer drugs, which are not commonly used in homeopathic medicines, on mRNA expression profiles of five key genes (p53, p21, COX-2, TUBB2A and TUBB3) in the breast cancer cell line MCF-7. MCF-7 cells were exposed to paclitaxel (Taxol) or docetaxel (Taxotere) preparations (6X, 5C and 15C dilutions prepared from pharmacological concentration of 25 nmol/L) for 72 hours. The cell culture groups were evaluated with the trypan blue dye exclusion method for the proliferation/cytotoxicity rates, immuno-staining β-tubulin for microtubule organization, and reverse transcription polymerase chain reaction for gene expression levels.Fold-change in gene expression was determined by the ΔΔCt method. The administration of diluted preparations had little or no cytotoxic effect on MCF-7 cells, but altered the expression of genes analyzed with a complex effect. According to the ΔΔCt method with a five-fold expression difference (p < 0.05) as a cut-off level, ultra-high dilutions of paclitaxel and docetaxel showed differential effects on the studied genes with a concentration-independent activity. Furthermore, the dilutions disrupted the microtubule structure of MCF-7 cells, suggesting that they retain their biological activity. Despite some limitations, our findings demonstrate that gene expression alterations also occur with ultra-high dilutions of taxane drugs.

Abstract A56: Effect of long-chain omega-3 polyunsaturated fatty acids on cisplatin chemoresistance in murine and human melanoma cells

Abstract The incidence of melanoma is continuously growing worldwide, and an effective therapy for the metastatic disease still does not exist. Moreover, an intrinsic/extrinsic resistance to conventional chemotherapeutics, such as cisplatin (CPt), often arises in this tumor. The increased expression of a number of DNA repair genes, including the Excision Repair Cross-Complementation group 1 (ERCC1), has been related to the melanoma resistance to CPt. ERCC1 is a component of an important chemoresistance pathway [tyrosine-phosphatase dual-specificity phosphatase-6 (DUSP6)/extracellular signal-regulated kinase phosphorylation (p-ERK)/ERCC1]. New therapeutic strategies are under study, and one possibility is the administration of natural products combined with CPt therapy, in order to revert the CPt resistance. Many experimental in vitro and in vivo studies have contributed to support the antineoplastic properties of long-chain omega-3 polyunsaturated fatty acids (LC-omega-3 PUFA), especially docosahexaenoic acid (DHA), in tumor cells of different origin, including melanoma. In the present work the effects of CPt treatment combined with LC-omega-3 PUFA have been evaluated in highly metastatic murine (B16F10) and human (WM266-4) melanoma cell lines. Melanoma cells were exposed to increasing concentrations of CPt (1, 2.5 and 5 μM), alone or in combination with DHA (10 μM). Cell viability was analysed by the Trypan blue dye exclusion method 24, 48, and 72 h after treatment. The in vitro invasive ability of melanoma cells was assessed by the Wound Healing assay. The effects of increasing DHA concentrations combined or not with CPt on the expression of several proteins (ERCC1, pERK and DUSP6) related to CPt chemoresistance in melanoma cells were also analyzed by Western Bbotting. The data obtained demonstrated that the CPt/DHA combined treatment, performed by adding suboptimal concentrations of both the compounds, was significantly more efficient in inhibiting cell growth than the treatments with each of the two compounds administered alone. We confirmed that CPt was able to increase ERCC1 and decrease DUSP- 6 in a dose-dependent manner in both WM266-4 and B16F10 cell lines. Moreover, in the present work it was observed that DHA was able to revert the effects of CPt on ERCC1, DUSP6, and p-ERK expression. Similarly to what was observed with DHA, the LC-omega-3 PUFA eicosapentaenoic acid (EPA) exerted similar effects, even though at lower extent, on ERCC1, p-ERK, and DUSP6 expression in the same experimental conditions. Overall, the findings indicate that the combination of CPt with a dietary supplementation with LC-omega-3 PUFA could potentially represent a new, promising, complementary therapeutic strategy in order to overcome CPt resistance in melanoma. Moreover, the results suggest that the CPt/LC-omega-3 PUFA combination may allow the use of lower and suboptimal doses of CPt, thus probably reducing the undesirable side effects often accompanying the treatment with this antineoplastic drug. Citation Format: Renata O. Vasconcelos, Simona Serini, Ana Paula S. Votto, Gilma S. Trindade, Gabriella Calviello. Effect of long-chain omega-3 polyunsaturated fatty acids on cisplatin chemoresistance in murine and human melanoma cells [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A56.

Phytochemical standardization of panchavalkala: An ayurvedic formulation and evaluation of its anticancer activity in cervical cancer cell lines

Background: Cervical cancer is the most common malignant disease affecting women worldwide. The currently available therapies for cancer, even though effective, affect the patient's health severely due to the associated side effects. Thus, nowadays, complementary/alternative medicines are being extensively researched upon for their use as an adjunct therapy. Panchavalkala, an Ayurvedic formulation, is traditionally being used as a douche in leukorrhea and other gynecological diseases. Objective: The objective of the study was to phytochemically standardize aqueous extract of Panchavalkala (PVaq) and evaluate its anticancer activity against human cervical cancer cell lines. Materials and Methods: The phytochemical characterization of PVaq was done by liquid chromatography–mass spectrometry (LCMS) technique. The effect of PVaq on the viability of SiHa and HeLa cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide dye assay. The effect of the extract on growth kinetics was evaluated by trypan blue dye exclusion method and soft agar assay. Results: LCMS analysis showed presence of 77 compounds, of which 15 major compounds included proanthocyanidin B1, chlorogenic acid, caffeic acid, epicatechin, leucopelargonidin 3-O-alpha-L-rhamno-beta-D-glucopyranoside, leucocyanidin, naringenin-7-o-glucoside, mesoinositol, catechin, vogelin E, mesoinositol, behenic acid, bergenin, acacetin, and gallic acid. PVaq significantly (P Abbreviations used: PVaq: Aqueous extract of Panchavalkala; LCMS: Liquid chromatography–mass spectrometry; HPV: Human papillomavirus; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

In vitro evaluation of equine fibroblast-like synoviocytes viability treated with doxycycline

Objective- The purpose of present study was to investigate the viability of equine fibroblast-like synoviocytes (FLSs) treated with doxycycline. Design- Experimental study. Sample population- FLSs from metacarpophalangeal joints of six skeletally mature horses. Methods- FLSs were established from synovial fluids of healthy joints. The cells were treated with different concentrations (1, 5, 10, 50, 100, 150, 300, 400 µg/ml) or without doxycycline for 48-hour. Viability of FLSs was determined by MTT assay and the Trypan blue dye exclusion method. Results- No significant differences were observed between viability of FLSs cultures treated with doxycycline until 150 µg/ml and control group (P>0.05). Doxycycline at 300 and 400 µg/ml significantly decreased FLSs viability (P<0.05). FLSs viability were 74.28% and 59.07% in 300 and 400 µg/ml, respectively, when measured by the MTT assay. Also FLSs viability at 300 and 400 µg/ml of doxycycline were 68.10% and 43.26%, respectively with Trypan blue exclusion method. Conclusion and Clinical Relevance- These findings demonstrate that doxycycline was not toxic for equine FLSs at concentration equal or less than 150 µg/ml in vitro. Further studies are needed to investigate the safety, efficacy and detrimental effects of doxycycline in equine joints.

Evaluation of apoptotic activity of Withania coagulans methanolic extract against human breast cancer and Vero cell lines

BackgroundThe genus Withania (Family: Solanaceae) holds an important position in Ayurveda, the Indian traditional system of medicine. Withania somnifera Dunal and Withania coagulans Dunal have been documented in folklore as panaceas for various ailments since time immemorial. W. coagulans (WC), commonly called as Indian cheese maker is used for fermenting milk for cheese production in various parts of India. ObjectivesIn the study, in vitro cytotoxicity of methanolic extract of dried fruits (berries) of WC was evaluated in a dose dependent manner using trypan blue dye exclusion method against human breast cancer cell line MDA-MB-231 and normal kidney epithelial cell line Vero in the range 20–200 μg/ml. Material and methodsThe percentage viability of the cell lines was determined by using MTT assay and cytometry. ResultsMethanolic extract of WC showed significant anticancer activity against MDA-MB-231 cell line. Cell viability was reduced to about 50% at 40 μg/ml of methanolic extract in 50% DMSO. Cytotoxicity of the extract was lower in 10% and 1% DMSO. On the other hand, methanolic extract of WC did not exhibit any significant in vitro activity against Vero cells at 170 and 200 μg/ml. AGE of isolated DNA from treated cancer cells revealed characteristic ladder like fragmentation, a hallmark of apoptosis. HPLC profiling was carried out for identification of the active components, which demonstrated the presence of Withaferin A in the methanolic extract. ConclusionMethanolic extract of WC possesses apoptotic activity against human breast cancer cells in vitro albeit lower in comparison to W. somnifera and warrants further investigation.

Chemical Modification of Ginsenoside on Cell Viability and Cytokine Secretion.

Rb1 is a ginsenoside steroid glycoside found exclusively in the plant Panax ginseng. In an earlier report, we showed that Rb1 increased cell proliferation and reduced VEGF (vascular endothelial growth factor) secretion by human retinal pigment epithelial (ARPE19) cells. In the present study, we hypothesized that chemical modification of Rb1 changes the level of VEGF secretion by ARPE19 cells. Three derivatives of Rb1 were chemically synthesized by hydrogenation (Rb1-H2), acetylation (Rb1-Acyl), and epoxidation (Rb1-Epoxy). Structural modifications were confirmed by 1H Nuclear Magnetic Resonance (NMR) spectra and Mass Spectrometry (MS). To test the biological activity, chemically modified compounds were added to cell culture media and incubated for 72 hours at a concentration of 250 nM at 37°C. Conditioned media were collected and cells were harvested/ counted after treatment. Viable cell numbers were determined by the trypan blue dye exclusion method and VEGF levels by Enzyme-Linked Immunosorbent Assays (ELISA). Consistent with the prior report, results of the present study show Rb1 increased cell proliferation and decreased VEGF secretion. Similar to Rb1's effect on cell proliferation, treatment with Rb1-H2, Rb1-Acyl and Rb1-Epoxy resulted in an increase in cell numbers. In contrast to Rb1- induced decrease in VEGF secretion, treatment with Rb1-H2, Rb-Acyl and Rb1-Epoxy resulted in increased VEGF levels. Chemical modifications of the ginsenoside Rb1 significantly affect the biological activity of VEGF secretion by ARPE19 cells. Additional SAR (Structure Activity Relationship) experiments will be conducted to study the detailed mechanisms by which how specific modifications of Rb1 functional groups alter biological activities.