Expression and Functionality of Connexin-Based Channels in Human Liver Cancer Cell Lines

Liver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis. Among such mechanisms is cellular communication mediated by connexin-based gap junctions. The present study investigated changes in connexin expression and gap junction functionality in liver cancer in vitro. For this purpose, seven human liver cancer cell lines, as well as primary human hepatocytes, were subjected to connexin and gap junction analysis at the transcriptional, translational and activity level. Real-time quantitative reverse transcription polymerase chain reaction analysis showed enhanced expression of connexin43 in the majority of liver cancer cell lines at the expense of connexin32 and connexin26. Some of these changes were paralleled at the protein level, as evidenced by immunoblot analysis and in situ immunocytochemistry. Gap junctional intercellular communication, assessed by the scrape loading/dye transfer assay, was generally low in all liver cancer cell lines. Collectively, these results provide a full scenario of modifications in hepatocyte connexin production and gap junction activity in cultured liver cancer cell lines. The findings may be valuable for the selection of neoplastic hepatocytes for future mechanistic investigation and testing of anti-cancer drugs that target connexins and their channels.

Assessment of the Cytotoxic Activity of Triphala: A Semisolid Traditional Formulation on HepG2 Cancer Cell Line

Cancer chemotherapies may result in resistance, and therefore, contemporary treatments including natural products may find an increasing consideration. As per Persian medicine (PM), many natural products have been used for malignant and chronic diseases. Triphala, with a combination of Terminalia chebula Retz., Terminalia bellirica Retz., Phyllanthus emblica L., and honey, is a multi-ingredient traditional formulation attributed to anticancer activities in PM. This study is aimed at evaluating the cytotoxic activity of this preparation on HepG2, the human liver cancer cell line. Hydroalcoholic extracts were prepared from the formulation and its components. Compared with the control and Cisplatin, the extracts were tested using MTT assay at different concentrations. All concentrations of the preparation, as well as Cisplatin, were effective significantly against HepG2 cells. All extract preparations at multiple concentrations were significantly effective as evidenced by MTT assay when compared to the control group. The IC50 level for Triphala extract was 77.63 ± 4.3 μ g / ml . Based on the results, Triphala and its components have cytotoxic activity on the HepG2 cancer cell line and they can reduce the survival rate significantly.

Bioinorganic Synthesis of Polyrhodanine Stabilized Fe3O4/Graphene Oxide in Microbial Supernatant Media for Anticancer and Antibacterial Applications

Polyrhodanines have been broadly utilized in diverse fields due to their attractive features. The effect of polyrhodanine- (PR-) based materials on human cells can be considered a controversial matter, while many contradictions exist. In this study, we focused on the synthesis of polyrhodanine/Fe3O4 modified by graphene oxide and the effect of kombucha (Ko) supernatant on results. The general structure of synthetic compounds was determined in detail through Fourier-transform infrared spectroscopy (FT-IR). Also, obtained compounds were morphologically, magnetically, and chemically characterized using scanning electron microscopy (SEM) and vibrating sample magnetometer (VSM), energy dispersive X-ray (EDX) analysis. The antibacterial effects of all synthesized nanomaterials were done according to CLSI against four infamous pathogens. Also, the cytotoxic effects of the synthesized compounds on the human liver cancer cell line (Hep-G2) were assessed by MTT assay. Our results showed that Go/Fe has the highest average inhibitory effect against Escherichia coli and Pseudomonas aeruginosa, and this compound possesses the least antimicrobial effect on Staphylococcus aureus. Considering the viability percent of cells in the PR/GO/Fe3O4 compound and comparing it with GO/Fe3O4, it can be understood that the toxic effects of polyrhodanine can diminish the metabolic activity of cells at higher concentrations (mostly more than 50 µg/mL), and PR/Fe3O4/Ko exhibited some promotive effects on cell growth, which enhanced the viability percent to more than 100%. Similarly, the cell viability percent of PR/GO/Fe3O4/KO compared to PR/GO/Fe3O4 is much higher, which can be attributed to the presence of kombucha in the compound. Consequently, based on the results, it can be concluded that this novel polyrhodanine-based nanocompound can act as drug carriers due to their low toxic effects and may open a new window on the antibacterial agents.

Antitumor Activity of Turnera subulata Sm. (Turneraceae) in Hep G2 Cancer Cell Line

Turnera subulata are bright common garden flowers that are grown in Asian regions. Mostly flowers of several kinds have medicinal properties and applications. This is as one of a kind that contains compounds that has medicinal application. The present studies are targeted to investigate the phytochemical composition through GC-MS technique, antimicrobial activity and antitumor activity via MTT assay against Hep G2 (or HepG2), a human liver cancer cell line. Ethanol (EtOH) extracts has reasonable antitumor activity against Hep G2 for period of 24 h and 48 h and the aqueous part was non-reactive. From gas chromatography‑mass spectrometry (GC-MS), a sum of 27 identified natural compounds exhibiting against cancer cell. Some traces of flavouring agents and antifungal agent with very low GC-MS peak lengths are too observed furaneol (2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one), benzeneacetaldehyde, benzoic acid and undecanoic acid. Hence, the result that indicates the flavouring agents including flavonoids along with phenolic and other acidic compounds have important characteristic property in reducing and treating against cancer cells.

Experimental Application of High-content Screening in Evaluating the Induction of Cell-cycle Arrest and Apoptosis on Human Liver Cancer Cell Line Hep-G2

This study discusses the results of the experimental application of high-content screening (HCS) techniques in evaluating the induction of cell-cycle arrest and apoptosis on human liver cancer cell line, Hep-G2. Accordingly, the bisbenzimide-stained cells (Hoechst 33342; 350 to 500 nM) were analyzed by using an Olympus scanˆR HCS-system to determine the cell-cycle phases (G1, S, and G2/M) and apoptosis as well. As a result, the cell-cycle arrest could be indicated by an increase in G2/M population of Hep-G2 cells after 24h exposure to zerumbone (Zer4; 9 µg/mL) and a similar observation could be made for paclitaxel (Pac; 4 µg/mL) as a reference substance. Keywords Apoptosis, cell-cycle arrest, high-content screening, human liver cancer cell line Hep-G2. References [1] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144 (2011) 646–674.[2] M. Malumbres, M. Barbacid, Cell cycle, CDKs and cancer: a changing paradigm, Nat. Rev. Cancer 9 (2009) 153–166.[3] S. Diermeier-Daucher, et al., Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry, Cytometry A 75 (2009) 535-546.[4] J. Essers, et al., Nuclear dynamics of PCNA in DNA replication and repair, Mol. Cell Biol 25 (2005) 9350- 9359. [5] V. Roukos, et al., Dynamic recruitment of licensing factor Cdt1 to sites of DNA damage. J. Cell Sci. 124 (2011) 422-434.[6] M. Hesse, et al., Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle, Nat. Commun 3 (2012) 1076. doi: 10.1038/ncomms2089.[7] P. Cappella, F. Gasparri, M. Pulici, J. Moll, A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining, Cytometry A 73 (2008) 626–636. [8] S. Diermeier-Daucher, et al., Cell type specific applicability of 5-ethynyl-2’-deoxyundine (EdU) for dynamic proliferation assessment in flow cytometry, Cytometry A 75 (2009) 535–546.[9] T. Yokochi, D.M. Gilbert, Replication labeling with halogenated thymidine analogs, Curr. Protoc. Cell Biol, 35 (2007) 22.10.1–22.10.14. [10] T.J. McGarry, M.W. Kirschner, Geminin, an inhibitor of DNA replication, is degraded during mitosis, Cell 93 (1998) 1043–1053. [11] H. Nishitani, S. Taraviras, Z. Lygerou, T. Nishimoto, The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase. J. Biol. Chem 276 (2001) 44905–44911.[12] J. Pines, T. Hunter, Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B, Nature 346 (1990) 760–763. [13] A. Stathopoulou, et al., Cdt1 is differentially targeted for degradation by anticancer chemotherapeutic drugs. PLoS ONE 7, e34621 (2012). [14] M. Hesse, A. Raulf, G.A. Pilz, C. Haberlandt, A.M. Klein, R. Jabs, H. Zaehres, C.J. Fügemann, K. Zimmermann, J. Trebicka, A. Welz, A. Pfeifer, W. Röll, M.I. Kotlikoff, C. Steinhäuser, M. Götz, H.R. Schöler, B.K. Fleischmann, Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle, Nat. Commun 3 (2012): 1076.[15] D.A. Ridenour, M.C. McKinney, C.M. Bailey, P.M. Kulesa, CycleTrak: a novel system for the semiautomated analysis of cell cycle dynamics. Dev. Biol 365 (2012) 189–195. [16] A. Roukos, et al., Cell cycle staging of individual cells by fluorescence microscopy, Nat. Protoc 10 (2015) 334-348.[17] E. Harlow, D. Lane, Fixing attached cells in paraformaldehyde, CSH Protoc 3 (2006) doi: 10.1101/pdb.prot4294.[18] G. Mazzini, M. Danova, Fluorochromes for DNA staining and quantitation, Method. Mol. Biol 1560 (2017) 239-259.[19] A. Gottfried, E. Weinhold, Sequence-specific covalent labelling of DNA, Biochem. Soc. Trans 39 (2011) 623-628.[20] J. Bucevičius, G. Lukinavičius, R. Gerasimaitė, The use of Hoechst dyes for DNA staining and beyond, Chemosensor 6 (2018) 1-18.[21] V. Kumar, A.K. Abbas, J.C. Aster, Robbins and Cotran Pathologic Basis of Disease, Ninth ed., Elsevier/Saunders, Philadelphia (2015).[22] N.A. Jensen et al., Establishment of a high content assay for the identification and characterisation of bioactivities in crude bacterial extracts that interfere with the eukaryotic cell cycle, J. Biotechnol 140 (2009) 124-134.[23] H.S. Rahman, et al., Zerumbone induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in Jurkat cell line, Nat. Prod. Commun 9 (2014) 1237-1242.[24] S.I. Abdelwahab, et al., Zerumbone inhibits interleukin-6 and induces apoptosis and cell cycle arrest in ovarian and cervical cancer cells, Intern. Immunopharm 12 (2012) 594-602.[25] M. Xian, et al., Zerumbone, A bioactive sesquiterpene, induces G2/M cell cycle arrest and apoptosis in leukemia cells via a Fas- and mitochondria-mediated pathway, Cancer Sci 98 (2007) 118-126.[26] A. Sehrawat, et al., Zerumbone causes Bax-and Bak-mediated apoptosis in human breast cancer cells and inhibits orthotopic xenograft growth in vivo, Breast Cancer Res. Treat. 136 (2012) 429-441.[27] Y.Z. Zhou, et al., Zerumbone induces G1 cell cycle arrest and apoptosis in cervical carcinoma cells, Int. J. Clin. Exp. Med. 10 (2017) 6640-6647.

Inhibition of human liver cancer cell growth by evodiamine involves apoptosis and deactivation of PI3K/AKT pathway

Evodiamine is an active alkaloid member found in Traditional Chinese Herb (TCH) Evodia rutaecarpa. It has been reported to exhibit remarkable biological and medicinal activities including anticancer and anti-inflammatory. This study was designed to investigate the anticancer effects of evodiamine against human liver cancer and evaluate its effects on cell migration, cell invasion, cellular apoptosis and PI3K/AKT pathway. The results showed that evodiamine exhibits potent antiproliferative effects against two human liver cancer cell lines (HepG2 and PLHC-1) with an IC50 of 20 µM. Nonetheless, the cytotoxic effects of evodiamine were comparatively low against the normal cells as evident from the IC50 of 100 μM. The growth inhibitory effects of evodiamine were found to be due to the induction of apoptosis as revealed by the DAPI, AO/EB and annexin V/PI staining assays. The induction of apoptosis was also associated with upregulation of Bax and downregulation of Bcl-2 expression in a concentration dependent manner. The wound healing and transwell assay revealed that evodiamine caused a significant decline in the migration and invasion of the HepG2 and PLHC-1 cells. Investigation of the effects of evodiamine on the PI3K/AKT signalling revealed that evodiamine inhibited the phosphorylation of PI3K and AKT proteins. Taken together, the results showed that evodiamine inhibits the growth of human liver cancer via induction of apoptosis and deactivation of PI3K/AKT pathway. The results point towards the therapeutic potential of evodiamine in the treatment of liver cancer.

Cytotoxic effects of individual and binary combinations of zearalenone and ochratoxin a on liver

Co-contamination with mycotoxin represents a serious concern for human and animal health. In this in vitro study, we investigated the combined effects of ZEA and OTA, mycotoxins which frequently contaminate cereals, in binary mixtures on the viability of human liver cancer cell line (HepG2). Cell viability was assessed after 24 h using a neutral red assay. An antagonistic effect was observed for binary toxins combinations affecting 25% of cell viability (CI=4.18), which turn into a synergistic effect as followed: slight at IL50 (CI=1.51), moderate at IL75 (CI=0.554) and strong at IL90 (CI=0.203). In conclusion, our results show an important additive and even synergistic cytotoxic effect of two commonly occurred mycotoxins: zearalenone and ochratoxin when they are present simultaneously in food or feed. The co-exposure to mycotoxins lead to a higher toxicity than the exposure to single toxin. Our study provides important data for mycotoxins risk assessment. In this context, a re-evaluation of the guidance levels for mycotoxins will be required in the future, in order to reduce the health risk associated with the possible consumption of mycotoxin co-contaminated food or feed.

Multivariate Data Analysis Guided Identification of Anticancer Constituents of Spina Gleditsiae for Human Hepatoma Cells

Spina Gleditsia has been traditionally used as a lead ingredient of many formulas for cancer treatment in Traditional Chinese Medicine. This work aimed to reveal the phytochemical foundation of Spina Gleditsiae and underpin a fundamental understanding of its anticancer activities. The growth inhibition activities were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of 7 solvent extracts of Spina Gleditsia in the human liver cancer cell line HepG2. The activities were then mapped onto the secondary metabolite profile of crude extracts by principal components analysis (PCA) of High Performance Liquid Chromatography-Ultraviolet (HPLC-UV) data. The top 5 PCA components of the map discriminated extract activities, mainly based on the differential content of 3 compounds. The identified compounds were then tested individually using cytotoxic assay on cancer cells in vitro. Principal components analysis constructed secondary metabolite mapping quickly identified functional compounds from the crude herbal extracts. (2 R,3 R)-7,3′,4′-Trihydroxy-5-methoxy-flavanonol, (2 R,3 R)-5,7,3′-trihydroxy-4′-methoxy-flavanonol, and isovitexin inhibited the growth of human liver cancer cell line growth with activities of 38.6 ± 1.9, 40.7 ± 0.9, and 41.5 ± 3.1 μg/mL, respectively, comparable with that of cisplatin (IC50: 41.6 ± 2.9 μg/mL). (2 R,3 R)-7,3′,4′-Trihydroxy-5-methoxy-flavanonol, (2 R,3 R)-5,7,3′-trihydroxy-4′-methoxy-flavanonol, and isovitexin are, for the first time, identified as the anticancer constituents of Spina Gleditsia. It thus strongly underpins a fundamental understanding of the anticancer activities of Spina Gleditsia and supports its ongoing clinical uses in China.