Trypan Blue Dye Exclusion Assay Research Articles

Evaluation of mutagenic, cytotoxic, mitochondrial dysfunction, apoptotic activity, and acute toxicity of ethanolic extract of Cissus quadrangularis

Recent studies have focused on exploring the efficacy of Cissus quadrangularis extract (EECQ) against various metabolic disorders involving the liver as the prime target organ, suggesting a considerable threat of hepatotoxicity in the person encountering it. Consequently, the current study was aimed to unravel the mutagenic, cytotoxic, mitochondrial dysfunction, apoptotic activity in HepG2 cells, and acute toxicity of EECQ. MTT, SRB, trypan blue dye exclusion, and lactate dehydrogenase (LDH) assay were performed in HepG2 cell lines to determine the cytotoxicity of the extract. The mutagenic potential was determined by the Ames test using various strains of Salmonella typhimurium. Acute toxicity was done at a dose of 2000 mg/kg in Sprague Dawley rats. MTT and SRB cytotoxicity assays demonstrated dose-dependent cytotoxicity of extract. The three highest noncytotoxic doses from the above assay, investigated by trypan blue dye exclusion and LDH assay, did not reveal cytotoxicity. Besides, mitochondrial dysfunction was determined by measuring cellular and mitochondrial ROS, ATP, NAD, mitochondrial membrane potential, Bax/Bcl2 ratio, mitochondrial and cytoplasmic cytochrome c, and apoptosis-inducing factor, were found to be equivalent in both extract exposed and unexposed cells. Moreover, the apoptotic cell morphology and the expression of pro-apoptotic mRNAs and proteins were equivalent in both the group. In acute toxicity, EECQ in rats did not cause any significant change in body weight, liver index, and liver function test. All-encompassing, the present study unraveled that EECQ is not mutagenic, cytotoxic, nor apoptotic in human hepatic cells, as well as neither acute toxicity.

The effect of natural polyphenols Resveratrol, Gallic acid, and Kuromanin chloride on human telomerase reverse transcriptase (hTERT) expression in HepG2 hepatocellular carcinoma: role of SIRT1/Nrf2 signaling pathway and oxidative stress.

There is evidence that low doses or physiological concentrations of certain natural polyphenols enhance the activity of telomerase. However, the precise mechanism by which natural polyphenols regulate telomerase activity remains unclear. Recent research indicates that NF-E2 related factor 2 (Nrf2) and silent information regulator 1 (SIRT1) are involved in human telomerase reverse transcriptase (hTERT) regulation. Thus, in order to better comprehend the mechanism by which polyphenols regulate hTERT, the present study investigated the effects of the natural polyphenols Resveratrol, Gallic acid, and Kuromanin chloride on hTERT, Nrf2, and SIRT1 expression as well as oxidative stress in HepG2 hepatocellular carcinoma. The trypan blue dye exclusion assay was used to assess cell viability. The level of mRNA for hTERT, Nrf2, and SIRT1 was then determined using real-time PCR. A spectrophotometric analysis was conducted to quantify oxidative stress markers. The results demonstrated that Resveratrol induces the expression of hTERT and the SIRT1/Nrf2 pathway in a dose-dependent manner. Gallic acid at concentrations of 10 and 20μM also increased the expression of the hTERT and SIRT1/Nrf2 pathway. Furthermore, dose-dependent overexpression of hTERT and Nrf2 was induced by Kuromanin chloride at 10 and 20µM. Moreover, we found that Resveratrol and Kuromanin chloride ameliorated oxidative stress, whereas Gallic acid exacerbated it. This study demonstrates that low doses of polyphenols (Resveratrol, Gallic acid, and Kuromanin chloride) upregulate the expression of the hTERT gene in the HepG2 hepatocellular carcinoma cell line, possibly via induction of the SIRT1/Nrf2 signaling pathway. Therefore, by targeting this pathway or hTERT, the anti-cancer effect of polyphenols can be enhanced.

Use of gold nanoparticle-silibinin conjugates: A novel approach against lung cancer cells.

Lung cancer presents one of the most challenging carcinomas with meager 5-year survival rates (less than 20%), high metastasis and high recurrence due to chemo- and radio- resistance. An alternative or complementation to existing prognosis modalities is the use of phytochemicals such as silibinin, which targets essential cytokines, angiogenic factors and transcription factors for a profound anti-tumor effect. However, the problems of low solubility in an aqueous physiological environment, poor penetration, high metabolism and rapid systemic clearance limit the therapeutic use of silibinin. Conjugation of gold nanoparticles (GNPs) with silibinin may overcome the above challenges along with distinct advantages of biocompatibility, optical properties for monitoring and causation of cytotoxicity in cancer cells. The current study thus aims to develop silibinin conjugated gold nanoparticles (Sb-GNPs) with pH responsive release in the cancer microenvironment, optimizing several parameters for its higher activity and further evaluate the nanoplatform for their efficacy in inducing cell death in vitro against A549 lung cancer cells. GNPs was synthesized using trisodium citrate dihydrate as the reducing agent and further used for the conjugation of silibinin. The synthesized GNPs were found to be monodispersed and spherical in shape. The silibinin was successfully conjugated with gold nanoparticles and long-term stability of GNPs and Sb-GNPs nanoconjugates in suspension phase was confirmed by FTIR and DLS. Anticancer properties of Sb-GNPs were confirmed by different assay using MTT, Trypan blue dye exclusion assay and cell cycle analysis assay. After conjugation of silibinin with GNPs, the efficacy of silibinin increased 4–5 times in killing the cancer cells. This is the first report on using silibinin gold nanoconjugate system for lung cancer therapy with promising future applications.

Combinatorial Effect of Arsenic and Herbal Compounds in Telomerase-Mediated Apoptosis Induction in Liver Cancer.

Tumour illness and its resistance against existing anticancer therapies pose a serious health concern globally despite the progressive advancement of therapeutic options. The prevailing treatment of HCC using numerous antitumor agents has inflated long-lived complete remissions, but a percentage of individuals still die due to disease recurrence, indicating a need for further exploration of possible anti-tumour regimes. We aim to boost the effectiveness of the HCC treatment by conducting current investigations evaluating the effect of arsenic trioxide (ATO) with different herbal compounds like quercetin and aloe-emodin against liver tumour via inhibition of telomerase, a pro-cancer enzyme. The anticancer activity of ATO with herbal compounds was investigated in human control liver cell line (Wrl-68) and cancer liver cell line (HepG2) at different time intervals. Viability and cytotoxicity in response to combinatorial drugs were assessed in vitro by trypan blue dye exclusion assay and MTT and WST assay. Apoptosis was analysed by annexin V/PI assay, and the expression of telomerase and apoptosis-regulating proteins was evaluated by immunoblotting and qRT-PCR. Arsenic trioxide in combination with quercetin and aloe-emodin reduced cell viability in cancerous cells compared to normal cells by inducing apoptosis, downregulating telomerase and Bcl-2 (anti-apoptotic protein) and upregulating the expression of Bax (pro-apoptotic protein). ATO exhibited significant anticancer effects due to the synergistic effects of quercetin and aloe-emodin in liver tumour cells. The current study data collectively suggest that a successful inhibition of cancer growth by the combination of ATO and tested herbal medicines against liver tumour growth is via the inhibition of telomerase activity.

Atorvastatin attenuates NS1 (Non-structural protein-1) of dengue type-2 serotype-induced expressions of matrix metalloproteinases in HL-60 cells, differentiated to neutrophils: Implications for the immunopathogenesis of dengue viral disease

BackgroundThe dengue is a vector borne viral infection in humans. Bite of mosquito infected with a dengue virus transmits the disease. The neutrophils support more to the innate immune response by switching to infected tissues and triggering immunomodulatory mechanisms including the release of proteases and host defence peptides. MethodsCell viability by MTT and trypan blue dye exclusion assay, bright field microscopy for assessment of cell morphology, cytokines measurements by ELISA, estimation of protein by Bradford assay were done. Assessments of matrix metalloproteinase genes mRNA expressions were done using real-time PCR. ResultsIn the present study, we have for the first time unveiled that, NS1 antigen of dengue type-2 serotype, induce and stimulate the neutrophils cells to express high levels of matrix metalloproteases. NS1 exposure of HL-60 cells differentiated to neutrophils affected cell morphology and in 24 h of exposure. We have demonstrated that, the NS1 antigen has induced MMP-2, MMP-14 and MMP-9 expressions in neutrophils in a 24hrs exposure time. NS1 exposure has also further upregulated MMP-1, MMP-13, and MMP-8 expressions in neutrophils in a 24hrs exposure time. Notably, treatment with atorvastatin concentrations downregulated the expression profile of the all matrix metalloprotease significantly. Importantly, NS1 antigen has significantly increased the IL-6, IL-13 release by the HL,60 cells which was reversed by atorvastatin. On the other hand, NS1 exposure enhanced the mRNA expressions of VEGF-A and VEGF-D which was reversed by atorvastatin. However, we found that, NS1 exposure reduced the mRNA expressions profile of VEGF-C, which was reversed by atorvastatin. ConclusionIn conclusion, we report that, neutrophils associated matrix metalloprotease are involved in the pathogenesis of dengue viral disease. VEGF growth factors may also be released by the neutrophils which may subsequently participate in the endothelial dysfunctions leading to dengue shock syndrome.

The γ-Glutamylcyclotransferase Inhibitor Pro-GA Induces an Antiproliferative Effect Through the Generation of Mitochondrial Reactive Oxygen Species.

γ-Glutamylcyclotransferase (GGCT) is up-regulated in a broad range of cancers, including breast cancer, and GGCT inhibition has been shown to be a promising strategy for therapy. Herein, we evaluated the efficacy and mechanism of action of pro-GA, a GGCT enzymatic inhibitor, in MCF7 breast cancer cells. Proliferation was evaluated by WST-8 and trypan blue dye exclusion assays. Western blot analysis was conducted to examine the expression of cyclin-dependent kinase inhibitors (CDKI), including p21, p27, and p16. Induction of senescence was assessed by senescence-associated β-galactosidase staining. Generation of mitochondrial superoxide reactive oxygen species (ROS) was assessed using flow cytometry. The effect of N-acetylcysteine (NAC) on pro-GA dependent inhibition of proliferation, ROS generation, and senescence was also studied. The efficacy of systemic administration of pro-GA was evaluated in a MCF7 xenograft mouse model. Treatment with pro-GA inhibited proliferation of MCF7 cells, increased CDKI expression and mitochondrial ROS, and induced cellular senescence. We found that cotreatment with NAC restored proliferation in pro-GA treated cells. NAC similarly suppressed CDKI expression, mitochondrial ROS generation, and senescence induced by pro-GA. Furthermore, the systemic administration of pro-GA in an MCF7 xenograft model had significant antitumor effects without toxicity. Pro-GA may be a promising therapeutic agent for the treatment of breast cancer.

Identification and biotin receptor-mediated activity of a novel seleno-biotin compound that inhibits viability of and induces apoptosis in ovarian cancer cells

A series of seleno-biotin analogs were synthesized and their anticancer activity and mode of action were assessed using ovarian cancer cells. Compound 2, out of the other analogs, in direct comparison to biotin alone, more effectively reduced the cell viability and induced apoptosis in ovarian cancer cell lines in a dose dependent manner as demonstrated by the cell viability assay, trypan blue dye exclusion assay, Annexin V/7-AAD, and Caspase 3/7 apoptosis assays. Furthermore, compound 2 showed efficacy better than 5-fluorouracil (5-FU) and similar to cisplatin, in vitro; notably it was more cytotoxic to drug-resistant Hey A8 cells than cisplatin. The cytotoxicity of compound 2 was primarily mediated by reactive oxygen species (ROS) as demonstrated by DCFDA based ROS estimation. Biotin receptors (BR) saturation and the use of a BR negative cell line showed a significant decline in the cytotoxic ativity of the compound 2, confirming that its activity is BR-mediated. These experiments demonstrated that selenium modified biotin which contains an ester linked redox cycling selenocyanate group has the potential for human therapeutic applications against ovarian and other cancers over-expressing BR.

In vivo and in vitro Study to Evaluate the Anti-osteoporotic Activity of Punica granatum Seed, Bambusa arundinaceae Leaves and Trichosanthes diocio Fruit Ethanolic Extract

Background: In the present study, it was aimed at evaluating the efficacy of ethanolic extract of Punica granatum seed, Bambusa arundinaceae leaves and Trichosanthes diocio fruit on their anti-osteoporotic activity in ovariectomized (OVX) rats and mcf-7 cell line to identify their osteoprotective role. Methods: Thirty six female albino wistar OVX rats were treated with the ethanolic extract of Punica granatum seed, Bambusa arundinaceae leaves and Trichosanthes diocio fruit for 45 days. The ethanolic extracts were given in different concentrations for 24 h and a cytotoxicity study was carried out by trypan blue dye exclusion assay. Result: The ethanolic extract of Punica granatum seed, Bambusa arundinaceae leaves and Trichosanthes diocio fruit treated notably averted an OVX-induced increase in body weight. Moreover, exposure to ethanolic extract of Bambusa arundinacea significantly decreased lipid peroxidation and protein carbonyl formation. Treated mcf-7 cell line viability was also increased. All these finding suggest that the ethanolic extract of Punica granatum seed, Bambusa arundinaceae leaves and Trichosanthes diocio fruit have an important role in reducing osteoporosis.

Abstract 5452: Discovery of a novel seleno-leonurine analog as a potential therapeutic for ovarian cancer

Abstract Leonurine is an active alkaloid that is extracted from Traditional Chinese Medicine Herba leonuri. It has been reported to be beneficial for cardiovascular diseases, including atherosclerosis, acute and chronic myocardial infarction (MI), and ischemic stroke. Mao et al. illustrated that leonurine could up-regulate the expression of phosphorylated p38 and down-regulate phosphorylated Akt expression through inhibiting mitochondrial pathway, thus suppressing the proliferation and promoting lung cancer cell apoptosis. Accumulating evidence suggest that the extract of Leonurus sibiricus transgenic roots could induce apoptosis in different grades of human glioma cells and significantly promotes cisplatin sensitivity in cervical cancer cells via repressing cell proliferation, improving intrinsic cell apoptosis and inhibiting the expression of P-Gp and MRP1 protein, which has potential therapeutic value in cervical cancer treatment. In the present study, we designed and synthesized several selenium analogs of leonurine and their cytotoxicity were assessed using four different ovarian cancer cell lines viz. OVCAR-3, OVCAR-10, ES2, and drug resistant HeyA8 cells. Compared to the control and leonurine group, compound 834 inhibited the ovarian cancer cell viability in dose- and time-dependent manner. The IC50 values of compound 834 was 4.6 µM, 3.8 µM, 6.2 µM, and 5.7 µM for OVCAR-3, HeyA8, OVCAR-10, and ES2 cells, respectively, while for leonurine the IC50 value was >500 µM for 48 h treatment. To further acquaint the effect of 48 h treatment on cell proliferation, we performed trypan blue cell viability assay, trypan blue dye exclusion assay, Annexin V/7-AAD, Caspase 3/7 apoptosis assays and Western blotting of apoptotic proteins involved in apoptosis. Compound 834 significantly induced apoptosis in OVCAR-3 and HeyA8 cells in dose dependent manner. Upregulation of several apoptotic proteins, as evident by Western blot, suggested that 834 causes induction of apoptotic pathways efficiently. Further, the cell cycle analysis showed a significant arrest of OVCAR-3 and HeyA8 cells in the S-phase, which was further confirmed by the analysis of cyclin A2, cyclin-E1, p21 and p27 proteins expression. Conclusively, our research revealed that leonurine analog 834 significantly induces apoptosis in ovarian cancer cells via repressing cell proliferation, improving intrinsic cell apoptosis and inhibiting the expression of several signaling proteins, which has potential therapeutic value in ovarian cancer treatment. Citation Format: Asif Raza, Jacek Krzeminski, Shantu Amin, Arun Sharma. Discovery of a novel seleno-leonurine analog as a potential therapeutic for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5452.

Abstract 5449: Novel seleno-biotin compound inhibits viability of and induces reactive oxygen species (ROS)-mediated apoptosis in ovarian cancer cells

Abstract Ovarian cancer is one of the most common cancers in women and even with the currently available treatment options, including surgery, radiation, and chemotherapy, it caused 13,940 deaths in US alone in 2020. Thus, there is a continuous need for developing novel approaches for the treatment of ovarian cancer, particularly drug resistant ovarian cancer, as ovarian cancer most adversely affects the lives patients. Here in, we have designed seleno-biotin derivatives as potential therapeutics for ovarian cancer. The rational for designing these hybrid small molecules is two fold: (i) The biotin receptors are up-regulated in various carcinomas including ovarian cancer cells while being expressed at low levels in normal cells. Due to the high affinity of biotin towards BR, biotin has been used as a conjugate for several targeted imaging, sensing, and delivery in vitro and in vivo. (ii) Organoselenium compounds with appropriate selenium moieties have shown therapeutic efficacy in several pre-clinical cancer models. Therefore, we designed several analogs of biotin by conjugating selenium moieties connected through ester, selenoester, or amide functionalities and their cytotoxicities were assessed using four different ovarian cancer cell lines. Ester-linked biotin-selenocyanate compound 2, out of the other analogs, in direct comparison to biotin alone, more effectively reduced the cell viability (IC50 9.8-18.5 µM against all the tested cell lines) and induced apoptosis in ovarian cancer cells in dose dependent manner as demonstrated by the cell viability assay. It showed three times more potency (IC50 14.08 µM) than standard drugs 5-FU (IC50 48.4 µM) and cisplatin (IC50 41.0 µM) against drug-resistant Hey A8 cancer cells and three times more activity with an IC50 value of 9.8 µM as compared to 5-FU (IC50 25.92 µM) against ES-2 cells. The above findings were supported by trypan blue dye exclusion assay, Annexin V/7-AAD, Caspase 3/7 apoptosis assays and Western blotting of apoptotic proteins. Compound 2 effectively caused cell cycle arrest of the treated OVCAR-3 cells in S-phase of the cell cycle, thus causing regulatory changes in the cell cycle proteins, as evident by Western blotting. In addition, compound 2 redox cycles was demonstrated by DCFDA based reactive oxygen species estimation. These experiments demonstrate that selenium modified biotin which contains a covalently attached redox cycling selenide group has the potential for human therapeutic applications against ovarian and other cancers over-expressing biotin receptors. Citation Format: Asif Raza, Amandeep Singh, Amin Shantu, L. Mallory Boylan, Julian E. Spallholz, Arun Sharma. Novel seleno-biotin compound inhibits viability of and induces reactive oxygen species (ROS)-mediated apoptosis in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5449.

Detection of Virulence-Associated Genes and in vitro Gene Transfer From Aeromonas sp. Isolated From Aquatic Environments of Sub-himalayan West Bengal.

Aeromonas is omnipresent in aquatic environments and cause disease within a wide host range. A total of thirty-four isolates from water samples of small fish farms were identified as Aeromonas based on biochemical characteristics and 16S rRNA gene sequence. A total of six virulent factors were analyzed which indicated 100% of isolates as beta-haemolytic and proteolytic, whereas 44.1, 38.2, and 70.6% of isolates produced DNAse, siderophore, and amylase, respectively. Studies on the occurrence of four genetic determinants of virulence factors revealed that aer/haem (haemolytic toxin) and flaA (polar flagella) genes were present in 44.1% of strains whereas ascV (type 3 secretion system) and aspA (serine protease) genes were detected in 21.5 and 8.82% of strains, respectively. Fish (Anabas testudineus) challenge studies showed that the isolate GP3 (Aeromonas veronii) bearing five virulent factors with the combination of aer/haem+/ascV+/fla+ genes induced severe lesions leading to 100% of mortality. In contrast, RB7 possessing four virulence factors and three genes (aer/haem+/ascV+/aspA+) could not produce severe lesions and any mortality indicating the absence of correlation between the virulence factors, its genes, and the pathogenicity in fishes. GP3 was cytotoxic to human liver cell line (WRL-68) in trypan blue dye exclusion assay. The 431 bp aer/haem gene of GP3 was transferable to E. coli Dh5α with a conjugational efficiency of 0.394 × 10–4 transconjugants per recipient cell. The transfer was confirmed by PCR and by the presence of 23-kb plasmids in both donor and transconjugants. Therefore, the occurrence of mobile genetic elements bearing virulence-associated genes in Aeromonas indicates the need for periodic monitoring of the aquatic habitat to prevent disease outbreaks.

Diospyros malabarica Fruit Extract Derived Silver Nanoparticles: A Biocompatible Antibacterial Agent

Biogenic silver nanoparticles demonstrate excellent antibacterial activity against a broad range of bacteria. Herein, aqueous biogenic silver nanoparticles (Aq@bAgNPs) and ethanolic biogenic silver nanoparticles (Et@bAgNPs) were synthesized using aqueous as well as ethanolic extracts of Diospyros malabarica fruit, respectively. The as-prepared biogenic silver nanoparticles (bAgNPs) were characterized using UV-Vis, FTIR as well as energy dispersive X-ray (EDS) spectroscopy, electron microscopy, dynamic light scattering spectroscopy (DLS), and zetasizer. The zeta potentials of Aq@bAgNPs and Et@bAgNPs were −9.8 ± 2.6, and −12.2 ± 1.9 mV, respectively. The antibacterial activity of bAgNPs was investigated against seven bacterial strains (i.e., pathogenic and nonpathogenic) and Et@bAgNPs exhibited the highest antibacterial propensity (i.e., 20 nm in diameter) against Bacillus subtillis through disk diffusion assay. The trypan blue dye exclusion assay also confirmed the antibacterial propensity of as-prepared bAgNPs. Furthermore, both Aq@bAgNPs and Et@bAgNPs oxidize bacterial membrane fatty acids and generate lipid peroxides which eventually form complexes with thiobarbituric acid (i.e., malondialdehyde-thiobarbituric acid adduct) to bring about bacterial death. Both the nanoparticles demonstrated good hemocompatibility against human as well as rat red blood cells (RBCs). In addition, they exhibited excellent biocompatibility in vivo in terms of rat liver (i.e., serum ALT, AST, and γ-GT) and kidneys (i.e., serum creatinine) function biomarkers.

Suppression of the Proliferation of Huh7 Hepatoma Cells Involving the Downregulation of Mutant p53 Protein and Inactivation of the STAT 3 Pathway with Ailanthoidol.

Ailanthoidol (ATD) has been isolated from the barks of Zanthoxylum ailanthoides and displays anti-inflammatory, antioxidant, antiadipogenic, and antitumor promotion activities. Recently, we found that ATD suppressed TGF-β1-induced migration and invasion of HepG2 cells. In this report, we found that ATD exhibited more potent cytotoxicity in Huh7 hepatoma cells (mutant p53: Y220C) than in HepG2 cells (wild-type p53). A trypan blue dye exclusion assay and colony assay showed ATD inhibited the growth of Huh7 cells. ATD also induced G1 arrest and reduced the expression of cyclin D1 and CDK2. Flow cytometry analysis with Annexin-V/PI staining demonstrated that ATD induced significant apoptosis in Huh7 cells. Moreover, ATD increased the expression of cleaved PARP and Bax and decreased the expression of procaspase 3/8 and Bcl-xL/Bcl-2. In addition, ATD decreased the expression of mutant p53 protein (mutp53), which is associated with cell proliferation with the exploration of p53 siRNA transfection. Furthermore, ATD suppressed the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and the expression of mevalonate kinase (MVK). Consistent with ATD, the administration of S3I201 (STAT 3 inhibitor) reduced the expression of Bcl-2/Bcl-xL, cyclin D1, mutp53, and MVK. These results demonstrated ATD’s selectivity against mutp53 hepatoma cells involving the downregulation of mutp53 and inactivation of STAT3.

Synergistic effect of folic acid and galantamine against experimentally induced oxidative stress in IMR 32 cells

Galantamine is an active constituent obtained from Galanthus nivalis L., a traditional herb known for its pharmacological properties, particularly nootropic effect. Folic acid is a dietary supplement that enhances neuronal activity. Effect of galantamine and folic acid on human neuronal cells is well known. In the present study, we explored the protective effect of galantamine and folic acid, both independently as well as in combination, over antioxidant defence system and nootropic effects on human neuroblastoma cells IMR-32. The treatment galantamine, folic acid and their combination was given for 24 h and cytotoxicity study was carried out by trypan blue dye exclusion assay. Apoptosis and necrosis were observed using Propidium iodide (PI) and Hoechst double staining method. Biochemical assays viz. total protein, protein carbonyl, lipid peroxidation and glutathione were analyzed along with super oxide dismutase and catalase. Result of cytotoxicity showed dose dependent increase in percent viability and significant decrease was observed in apoptosis and necrosis. Moreover, exposure to Galantamine, Folic acid and their combination significantly decreased lipid peroxidation and protein carbonyl formation along with the enhancement in antioxidant defence mechanism. Findings of these dose reliant toxicity study of Galantamine , Folic acid and their combination suggest that these has higher potency when given together and shows synergistic effect. They also causes repair of human neuronal cells IMR-32 cells enhancing the cell viability and consumption of Galantamine and Folic acid together will help in prevention of CNS disorders and neurodegeneration.

Cytotoxic Potential of Denture Adhesives on Human Fibroblasts-In Vitro Study.

(1) In recent years, there has been a significant increase in the availability of denture adhesives for stabilizing removable dentures. The aim of the present study was to assess the cytotoxicity of three denture adhesives on human fibroblasts. (2) Methods: Three denture adhesives were analyzed. Fibroblast cultures were established for the study and control groups in order to assess the incidence of necrosis and to evaluate the microscopic intracellular alterations induced. Following incubation with (study groups) or without adhesives (control group), trypan blue dye exclusion assay was used to determine the number of viable and/or dead cells. Microscopic specimens were stained with haematoxylin and eosin, scanned, digitally processed and then analyzed by a histopathologist. (3) Results: All three denture adhesives analyzed demonstrated various toxic effects in vitro on human fibroblast: quantitative evaluation—45.87–61.13% reduction of cell viability (p = 0.0001) and slight to moderate cytotoxicity in qualitative evaluation. (4) Conclusions: Denture adhesive creams demonstrated a toxic effect on human fibroblasts in vitro in quantitative and qualitative evaluation. In vivo observations are needed to find out if denture adhesives present a cytotoxic effect in patients.

Mechanism of polyphyllin Ⅰ targeting EGFR to affect proliferation and apoptosis of human breast cancer cells

This study aims to investigate the molecular mechanism of polyphyllin Ⅰ(PPⅠ) inhibiting proliferation of human breast cancer cells. Human breast cancer BT474 and MDA-MB-436 cells were treated with different concentrations of PPⅠ, and then the effect of PPⅠ on cell proliferation was detected by MTT assay, trypan blue dye exclusion assay, real-time cell analysis, and clone forming assay, respectively. The apoptosis was detected by Annexin V-FITC/PI staining and then analyzed by flow cytometry. The change of mitochondrial membrane potential was detected by flow cytometry after fluorescent probe JC-1 staining. Western blot was used to detect protein expression and phosphorylation. Molecular docking was performed to detect the binding between PPⅠ and EGFR. The affinity between PPⅠ and EGFR was determined by drug affinity responsive target stability assay. The results indicated that PPⅠ inhibited the proliferation and colony formation of BT474 and MDA-MB-436 cells in a time-and concentration-dependent manner. The PPⅠ treatment group showed significantly increased apoptosis rate and significantly decreased mitochondrial membrane potential. PPⅠ down-regulated the expression of pro-caspase-3 protein, promoted the cleavage of PARP, and significantly reduced the phosphorylation levels of EGFR, Akt, and ERK. Molecular docking showed that PPⅠ bound to the extracellular domain of EGFR and formed hydrogen bond with Gln366 residue. Drug affinity responsive target stability assay confirmed that PPⅠ significantly prevented pronase from hydrolyzing EGFR, indicating that PPⅠ and EGFR have a direct binding effect. In conclusion, PPⅠ inhibited the proliferation and induced apoptosis of breast cancer cells by targeting EGFR to block its downstream signaling pathway. This study lays a foundation for the further development of PPⅠ-targeted drugs against breast cancer.

Mitochondrial dysfunction is involved in the cellular activity inhibition by eleutheroside B in SMMC-7721 and HeLa cells.

Eleutheroside B, also known as syringin, has been shown to have various pharmacological activities such as anti-inflammatory, anti-irradiation and antidepressant, but there are few studies on its anti-cancer activity. Its anti-tumor effect on SMMC-7721 cells has not been revealed. Moreover, whether it induces autophagy is still unclear. Thus, the present study investigated whether Eleutheroside B induces apoptosis, autophagy and cellular motility in SMMC-7721 cells and HeLa cells, and explored the underlying molecular mechanisms. SMMC-7721 cells and HeLa cells treated with Eleutheroside B and cell viability measured by MTT assay and trypan blue dye exclusion assay. Apoptosis checked by flow cytometry combined, fluorescent staining. Apoptotic signal proteins and autophagy proteins were checked by Western blot. This study showed that Eleutheroside B inhibited the cell proliferation and blocked cell cycle, migration and invasion as well. Moreover, Eleutheroside B induced apoptosis in SMMC-7721 cells and HeLa cells. It upregulated Bax expression, while simultaneously decreasing Bcl-2 expression. Further elucidation of the mechanism revealed that Eleutheroside B induced mitochondrial dysfunction, with mitochondrial membrane potential collapse and cytochrome c release, suggesting that Eleutheroside B induced apoptosis by triggering mitochondrial pathway. Most importantly, Eleutheroside B could induce autophagy in SMMC-7721 cells and HeLa cells. Taken together, these results suggested Eleutheroside B is a potential therapeutic candidate for HCC and Human cervical cancer.

In vitro biocompatibility testing of different base materials used for elevation of proximal subgingival margins using human gingival epithelial cells.

To analyze the biological effects of four base materials used for elevation of proximal subgingival margins on gingival epithelial cells. Twenty-eight specimens for each of the four base materials (total 112 specimens) were used: resin-modified glass ionomer (RMGI), glass hybrid (HV-GIC), flowable bulk fill resin composite (Bulk Flow) and bioactive ionic resin (Activa). Proximal enamel and root dentin were used as controls. Gingival epithelial cell viability was calculated after direct incubation on all four types of material for either 24 h or 72 h using both the methyl tetrazolium and trypan blue dye exclusion assays. Data were analyzed statistically using one-way analysis of variance, Tukey post hoc test and independent sample t-test (P < 0.05). Cell viability values in both assays showed significant differences among the study groups. Bulk Flow showed the highest values, followed in order by Activa and the control groups. Both HV-GIC and RMGI had the lowest values. Cell viability in all of the study groups was higher after incubation for 72 h than after 24 h. In terms of biocompatibility with epithelial tissues, bulk fill resin composite appears to be most suitable, followed by bioactive composite, for subgingival placement than glass ionomer-based materials, especially that containing 2-hydroxy-ethyl methacrylate.

Extracts of Tagetes erecta exhibit potential cytotoxic and antitumor activity that could be employed as a promising therapeutic agent against cancer: A study involving in vitro and in vivo approach

Background and purposeGlobally, the burden of cancer is increasing consistently. Modern cancer therapies include lots of toxicity in the non-targeted organs reducing the life expectancy of the patients. Therefore, the development of safer alternative medicines with less toxicity and high efficacy is of immense importance. The present study was designed to evaluate the anticancer activity of a medicinal plant, “Tagetes erecta L.” (TE), in established cancer cell lines in vitro and in animal models in vivo. MethodsGC–MS analysis was performed to reveal the bioactive compounds present in the leaves of TE. To evaluate the antitumor potential of aqueous extract of TE (AETE), EAC (Ehrlich ascites carcinoma) induced solid tumor model was prepared in Swiss albino mice. The effect of AETE on body weight (BW), tumor weight (TW), and tumor volume (TV) was assessed in control and tumor-bearing animals. Further, the survival of the experimental animals was also monitored to assess the effect of AETE on life span. To determine the cytotoxicity induced by AETE, trypan blue dye exclusion assay, MTT assay, and LDH release assay was performed in EAC and HEp-2 cell lines by in vitro methods. Further, cell cycle assay and apoptosis assay was employed to ascertain the AETE-induced cytotoxicity is due to apoptosis or not. In addition, we have done a mitochondrial membrane potential assay using a flow cytometer to understand the mechanism of apoptosis. ResultsGC–MS analysis revealed the presence of hexadecanoic acid, Linolenic acid, Quinic acid, 2,3- dihydro benzofuran (Coumaran), and β-stigmasterol as major bioactive compounds in TE leaves. Results in animal studies showed that AETE treatment potentially reduced the TW and TV and increased the life span in EAC-induced tumor-bearing animals. Side effect analysis was also done in normal Swiss albino mice that confirmed the lack of toxicity of AETE. Studies in cancer cell lines indicated dose and time-dependent cytotoxicity in HEp-2 and EAC cells. Flow cytometric analysis established significant induction of apoptosis in EAC cells without arresting the cell cycle. In addition, AETE treatment led to a significant increase in cells with depolarised mitochondrial membrane potential. ConclusionThe present study indicated that AETE potentially inhibits tumor progression without disturbing normal body physiology. Thus, we conclude that AETE can be used as a potential therapeutic agent against cancer.

Anti-Tumor and Anti-Oxidant Activity of Ethanolic Extract of Epipremnum Aureum Linn. Leaves against DAL Induced Tumor in Swiss Albino Mice

Neoplasia literally means the process of “new growth,” and a new growth is called a neoplasm. The term tumor was originally applied to the swelling caused by inflammation. Neoplasms also may induce swellings, but by long precedent, the non-euplastic usage of tumor has passed into limbo; thus, the term is now equated with neoplasm. Oncology (Greek oncos = tumor) is the study of tumors or neoplasm’s. Cancer is the common term for all malignant tumors. The present study was designed to investigate the anti-tumor activity of ethanolic leaves extract of Epipremnum aureum Linn. and evaluated by in-vitro and in-vivo experimental models. To achieve objectives, EEEA was subjected to phytochemical screening and tested for oral toxicity test. The in-vitro study was carried out by means of MTT assay and Trypan blue dye exclusion assay using DAL cell lines. The in-vivo anti-tumor activity was evaluated against DAL tumour bearing mice by liquid tumour models. Preliminary phytochemical screening was confirmed the presence of flavonoids, glycosides, tannins, phenolic, steroids, and triterpeniods etc. EEEA showed good cytotoxic effect on DAL cell line in MTT assay and Trypan blue dye exclusion assay. Oral administration of EEEA in tumour bearing mice for 14 days, showed significant reduction in the percent increase in body weight, tumour volume, tumour weight, viable cell count when compared to the untreated mice of the DAL control group. The restoration of the haematological parameters towards the normal control was also observed. The results suggested that the EEEA exhibits significant anti-tumor activity towards both methods. The DAL-bearing mice orally administered leaves of Epipremnum aureum Linn., at 200 and 400 mg/kg body weight showed significant change in the average life span compared to animals of the tumor control group. The percentage increase in body weight, tumor cell volume, and number of viable tumor cells were found to be significantly less than the tumor control animals, indicating the anti-tumor nature of the extract.