Tannic Acid and Ethyl Gallate Potentialize Paclitaxel Effect on Microtubule Dynamics in Hep3B Cells.

Among broad-spectrum anticancer agents, paclitaxel (PTX) has proven to be one of the most effective against solid tumors for which more specific treatments are lacking. However, drawbacks such as neurotoxicity and the development of resistance reduce its therapeutic efficacy. Therefore, there is a need for compounds able to improve its activity by synergizing with it or potentiating its effect, thus reducing the doses required. We investigated the interaction between PTX and tannins, other compounds with anticancer activity known to act as repressors of several proteins involved in oncological pathways. We found that both tannic acid (TA) and ethyl gallate (EG) strongly potentiate the toxicity of PTX in Hep3B cells, suggesting their utility in combination therapy. We also found that AT and EG promote tubulin polymerization and enhance the effect of PTX on tubulin, suggesting a direct interaction with tubulin. Biochemical experiments confirmed that TA, but not EG, binds tubulin and potentiates the apparent binding affinity of PTX for the tubulin binding site. Furthermore, the molecular docking of TA to tubulin suggests that TA can bind to two different sites on tubulin, one at the PTX site and the second at the interface of α and ß-tubulin (cluster 2). The binding of TA to cluster 2 could explain the overstabilization in the tubulin + PTX combinatorial assay. Finally, we found that EG can inhibit PTX-induced expression of pAkt and pERK defensive protein kinases, which are involved in resistance to PXT, by limiting cell death (apoptosis) and favoring cell proliferation and cell cycle progression. Our results support that tannic acid and ethyl gallate are potential chemotherapeutic agents due to their potentiating effect on paclitaxel.

Achillin Increases Chemosensitivity to Paclitaxel, Overcoming Resistance and Enhancing Apoptosis in Human Hepatocellular Carcinoma Cell Line Resistant to Paclitaxel (Hep3B/PTX).

Multidrug resistance (MDR) has become a major obstacle in the treatment of cancer, and is associated with mechanisms such as increased drug outflow, reduction of apoptosis, and/or altered drug metabolism. These problems can be mitigated by the coadministration of agents known as chemosensitizers, as they can reverse resistance to anticancer drugs and eventually resensitize cancer cells. We explore the chemosensitizing effect of Achillin, a guaianolide-type sesquiterpene lactone isolated from the Mexican medicinal plant Artemisia ludovisiana, to reverse MDR in Hep3B/PTX cells of hepatocellular carcinoma, which present resistance to paclitaxel (PTX). Achillin showed an important effect as chemosensitizer; indeed, the cytotoxic effect of PTX (25 nM) was enhanced, and the induction of G2/M phase cell cycle arrest and apoptosis were potentiated when combining with Achillin (100 µM). In addition, we observed that Achillin decreases P-gp levels and increases the intracellular retention of doxorubicin in Hep3B/PTX cells; in addition, homology structural modeling and molecular docking calculations predicted that Achillin interacts in two regions (M-site and R-site) of transporter drug efflux P-glycoprotein (P-gp). Our results suggest that the chemosensitizer effect demonstrated for Achillin could be associated with P-gp modulation. This work also provides useful information for the development of new therapeutic agents from guaianolide-type sesquiterpene lactones like Achillin.

Quercetin induces G2 phase arrest and apoptosis with the activation of p53 in an E6 expression­independent manner in HPV­positive human cervical cancer­derived cells.

Cervical cancer is the second most common cancer in women worldwide. Human papillomavirus (HPV) infection appears to be a necessary factor in the development of almost all cases (>95%) of cervical cancer. HPV E6 induces a change of control of p53 stabilization from Hdm2 to E6/E6AP in HPV­infected cells. It is well known that the LxxLL motif of cellular ubiquitin ligase E6AP binds to the pocket of E6 and causes a conformational change to enable E6 to bind p53 competently. In the ternary complex E6/E6AP/p53, p53 is polyubiquitinated by E6AP and subsequently degraded by a proteasome. Therefore, these cells are deficient in the processes regulated by p53, including apoptosis, damaged DNA repair, and the cell cycle. In the present study, it was demonstrated that quercetin induced G2 phase cell cycle arrest and apoptosis in both HeLa and SiHa cells, accompanied by an increase of p53 and its nuclear signal. It was also observed that quercetin increased the level of the p21 transcript and the pro­apoptotic Bax protein, which are two p53­downstream effectors. However, quercetin did not alter the expression of the HPV E6 protein in cervical cancer cells; therefore, the increase in p53 occurred in an E6 expression­independent manner. Furthermore, molecular docking demonstrated that quercetin binds stably in the central pocket of E6, the binding site of E6AP. These data suggest that quercetin increases the nuclear localization of p53 by interrupting E6/E6AP complex formation in cervical cancer cells.

Lignans from Bursera fagaroides Affect In Vivo Cell Behavior by Disturbing the Tubulin Cytoskeleton in Zebrafish Embryos.

By using a zebrafish embryo model to guide the chromatographic fractionation of antimitotic secondary metabolites, seven podophyllotoxin-type lignans were isolated from a hydroalcoholic extract obtained from the steam bark of Bursera fagaroides. The compounds were identified as podophyllotoxin (1), ß-peltatin-A-methylether (2), 5'-desmethoxy-ß-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7). The biological effects on mitosis, cell migration, and microtubule cytoskeleton remodeling of lignans 1⁻7 were further evaluated in zebrafish embryos by whole-mount immunolocalization of the mitotic marker phospho-histone H3 and by a tubulin antibody. We found that lignans 1, 2, 4, and 7 induced mitotic arrest, delayed cell migration, and disrupted the microtubule cytoskeleton in zebrafish embryos. Furthermore, microtubule cytoskeleton destabilization was observed also in PC3 cells, except for 7. Therefore, these results demonstrate that the cytotoxic activity of 1, 2, and 4 is mediated by their microtubule-destabilizing activity. In general, the in vivo and in vitro models here used displayed equivalent mitotic effects, which allows us to conclude that the zebrafish model can be a fast and cheap in vivo model that can be used to identify antimitotic natural products through bioassay-guided fractionation.

Phenolic Compounds Isolated from Caesalpinia coriaria Induce S and G2/M Phase Cell Cycle Arrest Differentially and Trigger Cell Death by Interfering with Microtubule Dynamics in Cancer Cell Lines.

Caesalpinia coriaria (C. coriaria), also named cascalote, has been known traditionally in México for having cicatrizing and inflammatory properties. Phytochemical reports on Caesalpinia species have identified a high content of phenolic compounds and shown antineoplastic effects against cancer cells. The aim of this study was to isolate and identify the active compounds of a water:acetone:ethanol (WAE) extract of C. coriaria pods and characterize their cytotoxic effect and cell death induction in different cancer cell lines. The compounds isolated and identified by chromatography and spectroscopic analysis were stigmasterol, ethyl gallate and gallic acid. Cytotoxic assays on cancer cells showed different ranges of activities. A differential effect on cell cycle progression was observed by flow cytometry. In particular, ethyl gallate and tannic acid induced G2/M phase cell cycle arrest and showed interesting effect on microtubule stabilization in Hep3B cells observed by immunofluorescence. The induction of apoptosis was characterized by morphological characteristic changes, and was supported by increases in the ratio of Bax/Bcl-2 expression and activation of caspase 3/7. This work constitutes the first phytochemical and cytotoxic study of C. coriaria and showed the action of its phenolic constituents on cell cycle, cell death and microtubules organization.

RhoA/ROCK pathway activity is essential for the correct localization of the germ plasm mRNAs in zebrafish embryos.

Zebrafish germ plasm is composed of mRNAs such as vasa and nanos and of proteins such as Bucky ball, all of which localize symmetrically in four aggregates at the distal region of the first two cleavage furrows. The coordination of actin microfilaments, microtubules and kinesin is essential for the correct localization of the germ plasm. Rho-GTPases, through their effectors, coordinate cytoskeletal dynamics. We address the participation of RhoA and its effector ROCK in germ plasm localization during the transition from two- to eight-cell embryos. We found that active RhoA is enriched along the cleavage furrow during the first two division cycles, whereas ROCK localizes at the distal region of the cleavage furrows in a similar pattern as the germ plasm mRNAs. Specific inhibition of RhoA and ROCK affected microtubules organization at the cleavage furrow; these caused the incorrect localization of the germ plasm mRNAs. The incorrect localization of the germ plasm led to a dramatic change in the number of germ cells during the blastula and 24hpf embryo stages without affecting any other developmental processes. We demonstrate that the Rho/ROCK pathway is intimately related to the determination of germ cells in zebrafish embryos.

Cytotoxic Activity and Chemical Composition of the Root Extract from the Mexican Species Linum scabrellum: Mechanism of Action of the Active Compound 6-Methoxypodophyllotoxin.

The cytotoxic activity and the chemical composition of the dichloromethane/methanol root extract of Linum scabrellum Planchon (Linaceae) were analyzed. Using NMR spectra and mass spectrometry analyses of the extract we identified eight main constituents: oleic acid (1), octadecenoic acid (2), stigmasterol (3), α-amyrin (4), pinoresinol (5), 6 methoxypodophyllotoxin (6), coniferin (7), and 6-methoxypodophyllotoxin-7-O-ß-D-glucopyranoside (8). By using the sulforhodamine B assay, an important cytotoxic activity against four human cancer cell lines, HF6 colon (IC50 = 0.57 µg/mL), MCF7 breast (IC50 = 0.56 µg/mL), PC3 prostate (IC50 = 1.60 µg/mL), and SiHa cervical (IC50 = 1.54 µg/mL), as well as toward the normal fibroblasts line HFS-30 IC50 = 1.02 µg/mL was demonstrated. Compound 6 (6-methoxypodophyllotoxin) was responsible for the cytotoxic activity exhibiting an IC50 value range of 0.0632 to 2.7433 µg/mL against the tested cell lines. Cell cycle studies with compound 6 exhibited a cell arrest in G2/M of the prostate PC3 cancer cell line. Microtubule disruption studies demonstrated that compound 6 inhibited the polymerization of tubulin through its binding to the colchicine site (binding constant K b = 7.6 × 10(6) M(-1)). A dose-response apoptotic effect was also observed. This work constitutes the first investigation reporting the chemical composition of L. scabrellum and the first study determining the mechanism of action of compound 6.

Cytotoxic podophyllotoxin type-lignans from the steam bark of Bursera fagaroides var. fagaroides.

The hydroalcoholic extract of the steam bark of B. fagaroides var. fagaroides displayed potent cytotoxic activity against four cancer cell lines, namely KB (ED50 = 9.6 × 10(-2) µg/mL), PC-3 (ED50 = 2.5 × 10(-1) µg/mL), MCF-7 (ED50 = 6.6 µg/mL), and HF-6 (ED50 = 7.1 × 10(-3) µg/mL). This extract also showed anti-tumour activity when assayed on mice inoculated with L5178Y lymphoma cells. Bioactivity-directed isolation of this extract, afforded seven podophyllotoxin-type lignans identified as podophyllotoxin (1), ß-peltatin-A-methylether (2), 5'-desmethoxy-ß-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7) by 1D and 2DNMR and FAB-MS analyses, and comparison with reported values. All the isolated compounds showed potent cytotoxic activity in the cell lines tested, especially compound 3, which exhibited greater activity than camptothecin and podophyllotoxin against PC-3 (ED50= 1.0 × 10(-5) µg/mL), and KB (ED50 = 1.0 × 10(-5) µg/mL). This is the first report of the isolation of podophyllotoxin and its acetate in a Bursera species.

Genetic and transcriptome characterization of model zebrafish cell lines.

Compared with the increasing use of zebrafish as a model organism in many laboratories, zebrafish cell lines are still unexploited and limited in application, partly due to their unknown genetic and physiological properties. We characterize two zebrafish embryonic fibroblast cell lines, ZF4 and PAC2. We demonstrate the genetic stability of these two zebrafish cell lines and achieved genetic manipulation by either lipid-mediated transfection or an electroporation- based nucleofection method. Data from zebrafish chip analysis (Affymetrix) demonstrate unique characteristics of these two cell lines in gene expression levels, showing that different zebrafish cell lines can be classified by their transcriptome profile. Their transcriptional responses to serum growth factor exposure suggest that zebrafish fibroblast cell lines may be used to study processes related to wound-healing or cancer.

The CD43 coreceptor molecule recruits the zeta-chain as part of its signaling pathway.

CD43 is an abundant cell surface sialoglycoprotein implicated in hemopoietic cell adhesion and activation. Cell stimulation through CD43 results in recruitment of different signaling proteins, including members of the Src family kinases, Syk, phospholipase Cgamma2, the adapter protein Shc, the guanine nucleotide exchange factor Vav, and activation of protein kinase C. In this study, we report that in human T lymphocytes, the zeta-chain is part of the CD43 signaling pathway. Upon CD43 engagement, the zeta-chain was tyrosine-phosphorylated, generating docking sites for tyrosine-phosphorylated zeta-associated protein of 70 kDa and Vav. In vitro kinase assays suggested that zeta-associated protein of 70 kDa could account for the kinase activity associated with the zeta-chain following CD43 engagement. Cross-linking CD43 on the surface of the Lck-deficient JCaM.1 cells failed to phosphorylate the zeta-chain and associated proteins, suggesting that Lck is a key element in the CD43 signaling pathway leading to zeta phosphorylation. CD43 engagement with beads coated with anti-CD43 mAb resulted in concentration of the zeta-chain toward the bead attachment site, but interestingly, the distribution of the T cell Ag receptor complex remained unaffected. Recruitment of the zeta-chain through CD43-mediated signals was not restricted to T lymphocytes because phosphorylation and redistribution of the zeta-chain was also observed in NK cells. Our results provide evidence that the zeta-chain functions as a scaffold molecule in the CD43 signaling pathway, favoring the recruitment and formation of downstream signaling complexes involved in the CD43-mediated cell activation of T lymphocytes and other leukocytes such as NK cells.