Cytotoxic effect of hydroalcoholic extract of Cota tinctoria (L.) J.Gay on AGS and Hep-G2 cancer cell lines

Cota tinctoria is a medicinal plant which has been used for management of cancer in folk medicine of various regions. The aim of present study is to investigate cytotoxic activity of different concentrations of hydroalcoholic extract of C. tinctoria flowers on gastric (AGS) and liver (Hep-G2) cancer cell lines as well as Human Natural GUM fibroblast (HUGU) cells. Cell mortality rates were examined after 24, 48 and 72 h incubations using the MTT assay. IC50 of extract on AGS cells after 24, 48 and 72h was 1.46, 1.29 and 1.14 µg/mL respectively. The extract demonstrated IC50 of 5.15, 3.92 and 2.89 µg/mL on Hep-G2 cells after 24, 48 and 72 h respectively. No cytotoxic effect was detected on HUGU (Human Natural GUM fibroblast) cells. C. tinctoria seems to have a promising potential to be considered as a source for anticancer drug discovery. However, more experimental and clinical studies are required.

Impact of circ-0000221 in the Pathogenesis of Hepatocellular via Modulation of miR-661–PTPN11 mRNA Axis

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death in Egypt. A deep understanding of the molecular events occurring in HCC can facilitate the development of novel diagnostic and/or therapeutic approaches. In the present study, we describe a novel axis of hsa-circ-0000221–miR-661–PTPN11 mRNA proposed by in silico and in vitro analysis and its role in HCC pathogenesis. We observe a reduction in the expression levels of hsa-circ-0000221 and PTPN11 mRNA in HCC patients’ sera tested compared with control subjects. The reduction occurs with a concomitant increase in the expression of miR-661. Furthermore, the introduction of exogenous hsa-circ-0000221 into Hep-G2 or SNU449 cell lines results in detectable decrease in cellular viability and an increase in apoptotic manifestations that is associated with G1 accumulation and CCDN1 overexpression. Altogether, these findings indicate the tumor-suppressive role of hsa-circ-0000221 in HCC, which acts through miR-661 inhibition, along with a subsequent PTPN11 mRNA increase, where PTPN11 is known to inhibit cell proliferation in many forms of cancer. Our study encourages further investigation of the role of circRNAs in cancer and their potential use as molecular biomarkers.

Bioactive PKS–NRPS Alkaloids from the Plant-Derived Endophytic Fungus Xylaria arbuscula

A novel hybrid PKS–NRPS alkaloid, xylarialoid A (1), containing a 13-membered macrocyclic moiety and [5,5,6] fused tricarbocyclic rings, together with ten known cytochalasins (2–11), was isolated from a plant-derived endophytic fungus, Xylaria arbuscula. The chemical structures of all compounds were elucidated using 1D and 2D NMR, HR ESIMS spectroscopic analyses, and electronic circular dichroism (ECD) calculation. Compounds 1–3 and 10 exhibited significant antitumor activities against A549 and Hep G2 cell lines, with IC50 values of 3.6–19.6 μM. In addition, compound 1 showed potent anti-inflammatory activity against LPS-induced nitric oxide (NO) production in macrophage RAW 264.7 cells (IC50, 6.6 μM).

Development of Droplet Microfluidics Capable of Quantitative Estimation of Single-Cell Multiplex Proteins

This study presented constriction microchannel based droplet microfluidics realizing quantitative measurements of multiplex types of single-cell proteins with high throughput. Cell encapsulation with evenly distributed fluorescence labelled antibodies stripped from targeted proteins by proteinase K was injected into the constriction microchannel with the generated fluorescence signals captured and translated into protein numbers leveraging the equivalent detection volume formed by constriction microchannels in both droplet measurements and fluorescence calibration. In order to form the even distribution of fluorescence molecules within each droplet, the stripping effect of proteinase K to decouple binding forces between targeted proteins and fluorescence labelled antibodies was investigated and optimized. Using this microfluidic system, binding sites for beta-actin, alpha-tubulin, and beta-tubulin were measured as 1.15±0.59×106, 2.49±1.44×105, and 2.16±1.01×105 per cell of CAL 27 (N cell=15486), 0.98±0.58×106, 1.76±1.34×105 and 0.74±0.74×105 per cell of Hep G2 (N cell=18266). Neural net pattern recognition was used to differentiate CAL 27 and Hep G2 cells, producing successful rates of 59.4% (beta-actin), 64.9% (alpha-tubulin), 88.8% (beta-tubulin), and 93.0% in combination, validating the importance of quantifying multiple types of proteins. As a quantitative tool, this approach could provide a new perspective for single-cell proteomic analysis.

Studies on the effect of Celastrus orbiculatus (Celastraceae) extract on chemosensitivity of liver cancer cells via Wnt/β-catenin pathway

Purpose: To examine the efficacy of Celastrus orbiculatus extract (COE) on the chemosensitivity of liver cancer (LC) cells and its mechanism of action.Methods: Hep G2/ADM cells in the logarithmic growth phase were assigned to a control group (no treatment for cell culture medium only) and a study group (120 μg/ml COE added to the culture medium). After 48 h of incubation, the biological responses were compared. The study group wasdivided into groups A and B, while control group was divided into groups C and D, with 1 μmol/L XAV939 added in groups A and C. Cell proliferation, cell invasion, cell apoptosis rate, and apoptosis protein in the four groups were evaluated.Results: The study group showed significantly lower values in terms of cell proliferation and cell invasiveness (p < 0.05) and a higher apoptotic rate than the control group (p < 0.05)). The study group also demonstrated an elevated pro-apoptotic protein Bax level and a declined anti-apoptotic protein Bcl-2 level. In contrast to group B, the proliferation and invasiveness of Hep G2/ADM cells in group A treated with the inhibitor, XAV939, were significantly lower (p < 0.05), while the apoptotic rate exhibited a significant increase (p < 0.05). There was a rise in the level of pro-apoptotic protein, Bax, and a fall in the anti-apoptotic protein Bcl-2 level in group A. Lower levels of β-catenin, c-Myc, and cyclin D1 protein were observed in the study group compared with the control group (p < 0.05). Compared with other groups, the multiplication capacity and invasiveness of cells in group A treated with COE and inhibitor XAV939 significantly declined, while the apoptotic rate increased (p < 0.05).Conclusion: COE reverses drug resistance in chemotherapy by inhibiting the expression of Wnt/β-catenin pathway in LC cells. Therefore, COE has potentials for use along with chemotherapeutic agents in the management of liver cancer.

p-STAT3 is a PDC-E2 interacting partner in human cholangiocytes and hepatocytes with potential pathobiological implications

The E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC) is the key autoantigen in primary biliary cholangitis (PBC) and STAT3 is an inflammatory modulator that participates in the pathogenesis of many liver diseases. This study investigated whether PDC-E2 interacts with STAT3 in human cholangiocytes (NHC) and hepatocytes (Hep-G2) under cholestatic conditions induced by glyco-chenodeoxycholic acid (GCDC). GCDC induced PDC-E2 expression in the cytoplasmic and nuclear fraction of NHC, whereas in Hep-G2 cells PDC-E2 expression was induced only in the cytoplasmic fraction. GCDC-treatment stimulated phosphorylation of STAT3 in the cytoplasmic fraction of NHC. siRNA-mediated gene silencing of PDC-E2 reduced the expression of pY-STAT3 in NHC but not in HepG2 cells. Immunoprecipitation and a proximity ligation assay clearly demonstrated that GCDC enhanced pY-STAT3 binding to PDC-E2 in the nuclear and cytoplasmic fraction of NHC cells. Staining with Mitotracker revealed mitochondrial co-localization of PDC-E2/pS-STAT3 complexes in NHC and Hep-G2 cells. In cirrhotic PBC livers the higher expression of both PDC-E2 and pY-STAT3 was observed. The immunoblot analysis demonstrated the occurrence of double bands of PDC-E2 protein in control livers, which was associated with a lower expression of pY-STAT3. Our data indicate the interaction between PDC-E2 and phosphorylated STAT3 under cholestatic conditions, which may play a role in the development of PBC.

Cytotoxic and Anti-Inflammatory Activities of Dihydroisocoumarin and Xanthone Derivatives from Garcinia picrorhiza

Garcinia picrorhiza, a woody plant native to Sulawesi and Maluku Islands, Indonesia, has been traditionally used as a wound healing ointment. In our continuous search for bioactive compounds from this plant, 15 phenolic compounds were isolated from its stem bark, including a previously undescribed dihydroisocoumarin, 2′-hydroxyannulatomarin, and two undescribed furanoxanthones, gerontoxanthone C hydrate and 3′-hydroxycalothorexanthone. The structures of the new metabolites were elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR and HRESIMS. Gerontoxanthone C hydrate possessed cytotoxicity against four cancer cells (KB, HeLa S3, MCF-7, and Hep G2) with IC50 values ranging from 5.6 to 7.5 µM. Investigation on the anti-inflammatory activities showed that 3′-hydroxycalothorexanthone inhibited NO production in RAW 264.7 and BV-2 cell lines with IC50 values of 16.4 and 13.8 µM, respectively, whereas only (−)-annulatomarin possessed inhibition activity on COX-2 enzyme over 10% at 20 µM. This work describes the presence of 3,4-dihydroisocoumarin structures with a phenyl ring substituent at C-3, which are reported the first time in genus Garcinia. These findings also suggest the potential of furanxanthone derivatives as cytotoxic and anti-inflammatory agents for further pharmacological studies.