scholarly journals Extraction Optimization, Structural Characterization, and Anti-Hepatoma Activity of Polysaccharides from Scutellaria Barbata D. Don

2021 ◽  
Author(s):  
Wenwen Su ◽  
Leilei Wu ◽  
Qichao Liang ◽  
Xiaoyue Lin ◽  
Xiaoyi Xu ◽  
...  
Keyword(s):  
Colony Formation ◽  
Hepg2 Cells ◽  
Flow Cytometric Analysis ◽  
G1 Phase ◽  
Sugar Residue ◽  
Extraction Temperature ◽  
Anticancer Activities ◽  
Scutellaria Barbata ◽  
Acidic Polysaccharide

Abstract Background The above experimental results show that SBP-2A isolated and purified from Scutellaria barbata may be a candidate drug for further evaluation in cancer prevention, which provides a clue for further studies on the molecular mechanism of its anticancer activity against human liver cancer cells.Methods The crude polysaccharide of Scutellaria barbata (SBP) was extracted with water and precipitated with alcohol. Optimal extraction conditions were determined by response surface methodology: the solid-liquid ratio was 1:25, the extraction time was 2 h, and the extraction temperature was 90 °C. With these conditions, the average extraction efficiency was 3.85 ± 0.13%. SBP was purified with a DEAE-52 cellulose column and Sephadex G-100 dextran gel column to obtain SBP-1A and SBP-2A fractions. The polysaccharide content, molecular weight, monosaccharide composition and basic structure were preliminarily identified. Then, a MTT assay was used to identify the polysaccharide components with anti-hepatoma effects. The antitumor activity of SBP-2A was evaluated by colony formation tests, morphological observations, apoptosis and cell cycle analyses.Results Structural analysis showed that SBP-1A and SBP-2A were mainly composed of arabinose and galactose, but the molar ratios were different; these were homogeneous acidic polysaccharide components with high purity, and the average molecular weights were 1.15 × 105 Da and 1.4 × 105 Da, respectively. FT-IR spectra showed that SBP-1A and SBP-2A contained uronic acid β-glucan, and the sugar residue of the polysaccharide was mainly pyranose. MTT and colony formation assays showed that SBP-2A significantly inhibited the proliferation of HepG2 cells. The cell distribution at different apoptotic stages was determined by the Hoechst 33258 test and Annexin V-FITC/PI staining. Flow cytometric analysis showed that SBP-2A induced HepG2 cell apoptosis by blocking the G1 phase.Conclusions Two polysaccharides (SBP-1A and SBP-2A) had been isolated from Scutellaria barbata. Preliminary characterization of the SBP-1A and SBP-2A was investigated. The anticancer activities were studied in vitro. SBP-2A significantly inhibited the proliferation of HepG2 cells and induced cells apoptosis by blocking the G1 phase.

scholarly journals Evaluation of 2-Thioxoimadazolidin-4-One Derivatives as Potent Anti-Cancer Agents through Apoptosis Induction and Antioxidant Activation: In Vitro and In Vivo Approaches

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 83
Author(s):  
Mohamed S. Nafie ◽  
Ahmed I. Khodair ◽  
Hebat Allah Y. Hassan ◽  
Noha M. Abd El-Fadeal ◽  
Hanin A. Bogari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
In Silico ◽  
Hepg2 Cells ◽  
Flow Cytometric Analysis ◽  
Apoptosis Induction ◽  
Rt Pcr ◽  
Anti Cancer ◽  
Ic50 Values

Background: Hepatocellular carcinoma (HCC) is one of the most widespread malignancies and is reported as the fourth most prevalent cause of cancer deaths worldwide. Therefore, we aimed to investigate the probable mechanistic cytotoxic effect of the promising 2-thioxoimidazolidin-4-one derivative on liver cancer cells using in vitro and in vivo approaches. The compounds were tested for the in vitro cytotoxic activity using MTT assay, and the promising compound was tested in colony forming unit assay, flow cytometric analysis, RT-PCR, Western blotting, in vivo using SEC-carcinoma and in silico to highlight the virtual mechanism of action. Both compounds 4 and 2 performed cytotoxic effects against HepG2 cells with IC50 values of 0.017 and 0.18 μM, respectively, compared to Staurosporine and 5-Fu as reference drugs with IC50 values of 5.07 and 5.18 µM, respectively. Compound 4 treatment revealed apoptosis induction by 19.35-fold (11.42% compared to 0.59% in control), arresting the cell cycle at G2/M phase. Moreover, studying gene expression that plays critical roles in cell cycle and apoptosis by RT-PCR demonstrated that compound 4 enhances the expression of the pro-apoptotic genes p53, PUMA, and Caspase 3, 8, and 9, and impedes the anti-apoptotic Bcl-2 gene in the HepG2 cells. It can also inhibit the PI3K/AKT pathway at both gene and protein levels, which was reinforced by the in silico predictions of the molecular docking simulations towards the PI3K/AKT proteins. Finally, in vivo study verified that compound 4 has a promising anti-cancer activity through activating antioxidant levels (CAT, SOD and GSH) and ameliorating hematological, biochemical, and histopathological findings.

Inhibition of the ATPase Domain of Human Topoisomerase IIa on HepG2 Cells by 1, 2-benzenedicarboxylic Acid, Mono (2-ethylhexyl) Ester: Molecular Docking and Dynamics Simulations

2019 ◽  
Vol 19 (6) ◽  
pp. 495-503 ◽  
Author(s):  
Jemimah Naine Selvakumar ◽  
Subathra Devi Chandrasekaran ◽  
George Priya C. Doss ◽  
Thirumal D. Kumar
Keyword(s):  
Molecular Docking ◽  
Hepg2 Cells ◽  
Flow Cytometric Analysis ◽  
Dynamics Simulation ◽  
Benzenedicarboxylic Acid ◽  
Bioassay Guided Fractionation ◽  
Marine Streptomyces ◽  
Dynamics Simulations ◽  
Molecular Docking And Dynamics

Background: The major attention has been received by the natural products in the prevention of diseases due to their pharmacological role. Objective: The major focus of the study was to search for highly potential anti-cancer compounds from marine Streptomyces sp. VITJS4 (NCIM No. 5574). Methods: Cytotoxic assay was examined by MTT assay on HepG2 cells. Bioassay-guided fractionation of the ethyl acetate extract from the fermented broth led to the isolation of the compound. The lead compound structure was elucidated by combined NMR and MS analysis, and the absolute configuration was assigned by extensive spectroscopic analysis. Results: On the basis of spectroscopic data, the compound was identified as 1, 2 benzenedicarboxylic acid, mono 2-ethylhexyl (BMEH). The compound exhibited in vitro anticancer potential against liver (HepG2) cancer cells. Based on the flow cytometric analysis, it was evident that the BMEH was also effective in arresting the cell cycle at G1 phase. Further, the Western blotting analysis confirmed the down-regulation of Bcl-2 family proteins, and activation of caspase-9 and 3. The molecular docking and dynamics simulation were performed to reveal the activity of the compound over a time period of 10ns. From the molecular dynamics studies, it was found that the stability and compactness were attained by the protein by means of the compound interaction. Conclusion: This study highlights our collaborative efforts to ascertain lead molecules from marine actinomycete. This is the first and foremost report to prove the mechanistic studies of the purified compound 1, 2-benzene dicarboxylic acid, mono(2-ethylhexyl) ester isolated from marine Streptomyces sp.VITJS4 against HepG2 cells.

scholarly journals Optimization of Extraction Conditions for Antioxidant Activity of Acer tegmentosum Using Response Surface Methodology

2021 ◽  
Vol 11 (3) ◽  
pp. 1134
Author(s):  
Inyong Kim ◽  
Jung-Heun Ha ◽  
Yoonhwa Jeong
Keyword(s):  
Hydrogen Peroxide ◽  
Response Surface Methodology ◽  
Response Surface ◽  
Hepg2 Cells ◽  
Extraction Methods ◽  
Total Phenolic ◽  
Extraction Temperature ◽  
Freeze Dried ◽  
Hydrothermal Extraction

Extraction from edible plants is a highly important process that has various biological functions. To maximize biological activity, extraction methods should facilitate optimal extraction of functional phytochemicals. In this study, the optimal hydrothermal extraction conditions of Acer tegmentosum were determined using response surface methodology (RSM), and HepG2 cells were treated with optimized extract and hydrogen peroxide. In a central composition design, the independent variables were extraction temperature (X1: 70–90 °C), extraction time (X2: 2–6 h), and solvent-to-solid ratio (X3: 50–150). The maximum total phenolic contents (276.70 ± 10.11 mg GAE/g) and DPPH (2,2-diphenyl-1-pictylhydrazyl) activity (33.45 ± 2.20%) of A. tegmentosum were estimated at optimized extraction conditions, as follows: X1: 89.34 °C, X2: 7.36 h, X3: 184.09. Using the calculated extraction conditions, functional phytochemicals were extracted by hydrothermal extraction and freeze-dried. A. tegmentosum treatment (>10 μg/mL) of HepG2 cells remarkably attenuated hydrogen-peroxide-inducible hepatic cellular death and reactive oxygen species production in vitro.

scholarly journals In vitro toxicological evaluation of ethyl carbamate in human HepG2 cells

2016 ◽  
Vol 5 (2) ◽  
pp. 697-702 ◽  
Author(s):  
Xia Cui ◽  
Jiayi Wang ◽  
Nannan Qiu ◽  
Yongning Wu
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Hepg2 Cells ◽  
Ethyl Carbamate ◽  
G1 Phase ◽  
Toxicological Evaluation ◽  
Cytotoxic Effects ◽  
Apoptosis And Necrosis

Ethyl carbamate could decrease the viability of HepG2 cells by arresting the cell cycle in the G1 phase, as well as apoptosis and necrosis. Moreover, an oxidative stress mechanism also contributed to the cytotoxic effects of EC.

Novel Dihydropyrimidinone-Derived Selenoesters as Potential Cytotoxic Agents to Human Hepatocellular Carcinoma: Molecular docking and DNA Fragmentation

2020 ◽  
Vol 20 ◽  
Author(s):  
Jean C. Benassi ◽  
Flavio A.R. Barbosa ◽  
Valdelúcia M.A.S. Grinevicius ◽  
Fabiana Ourique ◽  
Daniela Coelho ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Fragmentation ◽  
Hepg2 Cells ◽  
Molecular Mechanisms ◽  
Hydrophobic Interactions ◽  
Cyclin A ◽  
Cytotoxic Agents ◽  
Anticancer Activities ◽  
Organoselenium Compounds

Background and Objective: Evidences point out promising anticancer activities of Dihydropyrimidinones (DHPM) and organoselenium compounds. The aim of this study was to evaluate the cytotoxic and antiproliferative potential of DHPM-derived selenoesters (Se-DHPM), as well as their molecular mechanisms of action. Methods: Se-DHPM cytotoxicity was evaluated against cancer lines (HeLa, HepG2, HRT-18 and MCF-7) and normal cells (McCoy). HepG2 clonogenic assay allowed verifying antiproliferative effect. The propidium iodide/orange acridine fluorescence readings showed the type of cell death induced after treatments (72h). Molecular simulations with B-DNA and 49H showed docked positions (AutoDock Vina) and trajectories/energies (GROMACS). In vitro molecular interactions used CTDNA and 49H applying UV-Vis absorbance and fluorescence. Comet assay evaluated DNA fragmentation of HepG2 cells. Flow cytometry analysis verified HepG2 cell cycle effects. Levels of proteins (β-actin, p53, BAX, HIF-1α, γH2AX, PARP1, cyclin A, CDK-2 and pRB) were quantified by immunoblotting. Results: Among Se-DHPM, 49H was selectively cytotoxic to HepG2 cells, reduced cell proliferation and increased BAX (80%) and p53 (66%) causing apoptosis. Molecular assays revealed 49H inserted in CT-DNA molecule causing hypochromic effect. Docking simulations showed H-bonds and hydrophobic interactions, which kept the ligand partially inserted into DNA minor groove. 49H increased the DNA damage (1.5 fold) and γH2AX level (153%). Besides, treatments reduced PARP-1 (60%) and reduced pRB phosphorylation (21%) as well decreased cyclin A (46%) arresting cell cycle at G1 phase. Conclusions: Together all data obtained confirmed the hypothesis of disruptive interactions between Se-DHPM and DNA thereby highlighting its potential as a new anticancer drug.

scholarly journals C1QTNF6 regulates cell proliferation and apoptosis of NSCLC in vitro and in vivo

2020 ◽  
Author(s):  
Wei Zhang ◽  
Ganzhu Feng
Keyword(s):  
Cell Proliferation ◽  
Cancer Progression ◽  
Colony Formation ◽  
Cell Function ◽  
Flow Cytometric Analysis ◽  
A549 Cells ◽  
Migration And Invasion ◽  
Flow Cytometric

Objectives: Lung cancer has been reported as the leading cause of cancer-associated death in humans, and its incidence continues to increase in the world. A growing number of studies have shown that dysregulated genes are associated with the occurrence and poor prognosis of a variety of tumors, including NSCLC. C1q/tumor necrosis factor-related protein 6 (C1QTNF6), a member of the CTRP family, has been revealed to play a role in carcinogenesis and cancer progression. Nevertheless, the effects and mechanisms of C1QTNF6 in NSCLC remain unrevealed. Materials and methods: MTT and colony formation, flow cytometric and transwell assays were performed to explore the cell function. RT-PCR and western blot were used to analyze the mRNA and protein expression. Results: In this study, we found that C1QTNF6 significantly promoted the proliferation of SPCA1 and A549 cells by MTT and colony formation assays. In addition, downregulation of C1QTNF6 weakened the tumor growth in vivo. Besides, C1QTNF6 remarkably reduced apoptosis by flow cytometric analysis and TUNEL assay. Furthermore, the capability of migration and invasion was obviously enhanced when C1QTNF6 overexpression. Conclusion: Overall, our results demonstrated that inhibition of C1QTNF6 attenuated cell proliferation, migration, invasion and promoted apoptosis in vitro and in vivo of NSCLC. Based on the above results, our study provided us with a new and key perspective in understanding and treating NSCLC.

Identification of CCND2 As a RUNX1/ETO Target Required for Leukaemic Propagation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 835-835 ◽  
Author(s):  
Lynsey McKenzie ◽  
Natalia Martinez-Soria ◽  
Julia Draper ◽  
Sirintra Nakjang ◽  
Helen J Blair ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Fusion Protein ◽  
Cell Cycle Arrest ◽  
Colony Formation ◽  
G1 Phase ◽  
Cycle Arrest ◽  
Gene Sets

Abstract The translocation t(8;21) is the most common chromosomal rearrangement in AML and generates the RUNX1/ETO fusion protein. RUNX1/ETO is required for maintaining the leukaemic phenotype influencing both leukaemic clonogenicity and proliferation and is traditionally thought of as a transcriptional repressor through its actions with histone deacetylases and transcriptional co-repressors. Intriguingly our RNAi experiments have also identified many RUNX1/ETO target genes that are transcriptionally upregulated by this fusion protein. To delve deeper into the involvement of these upregulated genes in RUNX1/ETO driven leukaemia, targeted RNAi screens were performed both in vitro and in vivo, directed at genes found directly bound by RUNX1/ETO and that were found differentially expressed upon RUNX1/ETO knockdown. For the RNAi screens, we used a doxycycline (dox)-inducible lentiviral RNAi library covering each gene with 3 shRNAs. We transduced two t(8;21)-positive AML cell lines, Kasumi-1 and SKNO-1, with this library and performed parallel screens employing colony formation and long-term suspension culture assays in the in vitro arm, and intrafemoral xenotransplantation of immunodeficient NSG mice for the in vivo arm. For comparative purposes, both arms of the RNAi screen were performed in the presence and absence of dox. DNA was isolated throughout both screens and was analysed by Next Generation Sequencing (NGS). Comparison of the changes in level of integrated shRNA coding sequences between dox and no dox groups were made using DESEQ and those genes that are required for RUNX1/ETO driven leukaemia identified. As expected, RUNX1/ETO shRNA constructs diminished upon induction of shRUNX1/ETO expression by dox, therefore implicating RUNX1/ETO dependency in cell survival. Furthermore non-targeting control shRNA (shNTC) levels were unaffected. Out of the many hits identified (which include previously reported hits such as Pontin (RUBVL1), SKP2 and KIT, Cyclin D2 (CCND2) stood out significantly, since levels of shCCND2 were depleted in all dox samples from both arms of the screen. CCND2 is a cell cycle regulator whose activity is dependent on its binding to CDK4/6 in G1 phase. Phosphorylation of Rb (Retinoblastoma), by CDK4/6-CCND2, uncouples Rb from E2F allowing transcription of essential S phase genes. Here we show CCND2 knockdown downregulates both cell proliferation and colony formation in t(8;21) positive cells by causing G1 phase cell cycle arrest via a reduction in Rb phosphorylation, which is a phenotype copied by our RUNX1/ETO knockdown. Moreover, inhibition of CDK4/6-CCND2 by palbociclib (PD-0332991) in t(8;21) positive cells similarly reduces cell proliferation and colony formation via a G1 cell cycle arrest. Interestingly, primary CD34+ human stem/progenitor cells (HSPCs) containing an activated form of KIT (N822K) and the RUNX1/ETO 9a isoform are likewise responsive to palbociclib treatment. In addition to in vitro studies, palbociclib was tested in vivo in two separate experimental models: firstly, mice were intravenously injected with murine CD34+ cKit+ HSPCs expressing the RUNX1/ETO9a isoform and secondly, immunodeficient mice were intrahepatically injected with Kasumi-1. Mice were dosed with palbociclib upon confirmation of engraftment. Engraftment was detected using bioluminescence and survival rates recorded. Both studies showed significant increased survival with palbociclib treatment with the first giving an increased median survival of 59 versus 29 days (p<0.005) and the second giving a median survival of 87 and 67 days for palbociclib treated and control mice (p<0.005), respectively. Finally, Gene Set Enrichment Analysis (GSEA) shows the correlations between palbociclib treatment, CCND2 knockdown and RUNX1/ETO knockdown and shows common gene sets shared between them and other gene sets which include those targeted by MYC and E2F. Interestingly, gene sets that only correlate with knockdown of CCND2 and RUNX1/ETO and evidentially unaffected by CDK4/6 activity will be of interest, in order to target other RUNX1/ETO dependent proteins and pathways alongside of CDK4/6 inhibition. We are currently investigating the use of palbociclib with other drugs in order to develop new drug combinations with reduced toxicity and minimal risk of developing resistance. Disclosures No relevant conflicts of interest to declare.

scholarly journals Peiminine Inhibits Glioblastoma in Vitro and in Vivo Through Cell Cycle Arrest and Autophagic Flux Blocking

2018 ◽  
Vol 51 (4) ◽  
pp. 1566-1583 ◽  
Author(s):  
Boxian Zhao ◽  
Chen Shen ◽  
Zhixing Zheng ◽  
Xiaoxiong Wang ◽  
Wenyang Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Cycle Arrest ◽  
Colony Formation ◽  
Morphological Changes ◽  
Autophagic Flux ◽  
Anticancer Activities ◽  
Cycle Arrest

Background/Aims: Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates. Existing treatments do not provide substantial benefits to patients; therefore, novel treatment strategies are required. Peiminine, a natural bioactive compound extracted from the traditional Chinese medicine Fritillaria thunbergii, has many pharmacological effects, especially anticancer activities. However, its anticancer effects on GBM and the underlying mechanism have not been demonstrated. This study was conducted to investigate the potential antitumour effects of peiminine in human GBM cells and to explore the related molecular signalling mechanisms in vitro and in vivo Methods: Cell viability and proliferation were detected with MTT and colony formation assays. Morphological changes associated with autophagy were assessed by transmission electron microscopy (TEM). The cell cycle rate was measured by flow cytometry. To detect changes in related genes and signalling pathways in vitro and in vivo, RNA-seq, Western blotting and immunohistochemical analyses were employed. Results: Peiminine significantly inhibited the proliferation and colony formation of GBM cells and resulted in changes in many tumour-related genes and transcriptional products. The potential anti-GBM role of peiminine might involve cell cycle arrest and autophagic flux blocking via changes in expression of the cyclin D1/CDK network, p62 and LC3. Changes in Changes in flow cytometry results and TEM findings were also observed. Molecular alterations included downregulation of the expression of not only phospho-Akt and phospho-GSK3β but also phospho-AMPK and phospho-ULK1. Furthermore, overexpression of AKT and inhibition of AKT reversed and augmented peiminine-induced cell cycle arrest in GBM cells, respectively. The cellular activation of AMPK reversed the changes in the levels of protein markers of autophagic flux. These results demonstrated that peiminine mediates cell cycle arrest by suppressing AktGSk3β signalling and blocks autophagic flux by depressing AMPK-ULK1 signalling in GBM cells. Finally, peiminine inhibited the growth of U251 gliomas in vivo. Conclusion: Peiminine inhibits glioblastoma in vitro and in vivo via arresting the cell cycle and blocking autophagic flux, suggesting new avenues for GBM therapy.

Nanocurcumin: A Double-Edged Sword for Microcancers

2020 ◽  
Vol 26 (45) ◽  
pp. 5783-5792
Author(s):  
Kholood Abid Janjua ◽  
Adeeb Shehzad ◽  
Raheem Shahzad ◽  
Salman Ul Islam ◽  
Mazhar Ul Islam
Keyword(s):  
Intracellular Signaling ◽  
Dosage Forms ◽  
The Body ◽  
In Vivo Studies ◽  
Mrna Levels ◽  
Anticancer Activities ◽  
Road Map ◽  
Anticancer Effects

There is compelling evidence that drug molecules isolated from natural sources are hindered by low systemic bioavailability, poor absorption, and rapid elimination from the human body. Novel approaches are urgently needed that could enhance the retention time as well as the efficacy of natural products in the body. Among the various adopted approaches to meet this ever-increasing demand, nanoformulations show the most fascinating way of improving the bioavailability of dietary phytochemicals through modifying their pharmacokinetics and pharmacodynamics. Curcumin, a yellowish pigment isolated from dried ground rhizomes of turmeric, exhibits tremendous pharmacological effects, including anticancer activities. Several in vitro and in vivo studies have shown that curcumin mediates anticancer effects through the modulation (upregulation and/or downregulations) of several intracellular signaling pathways both at protein and mRNA levels. Scientists have introduced multiple modern techniques and novel dosage forms for enhancing the delivery, bioavailability, and efficacy of curcumin in the treatment of various malignancies. These novel dosage forms include nanoparticles, liposomes, micelles, phospholipids, and curcumin-encapsulated polymer nanoparticles. Nanocurcumin has shown improved anticancer effects compared to conventional curcumin formulations. This review discusses the underlying molecular mechanism of various nanoformulations of curcumin for the treatment of different cancers. We hope that this study will make a road map for preclinical and clinical investigations of cancer and recommend nano curcumin as a drug of choice for cancer therapy.

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