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.

Finding a Novel Chalcone-Cinnamic Acid Chimeric Compound with Antiproliferative Activity against MCF-7 Cell Line Using a Free-Wilson Type Approach.

In this work, we carried out the design and synthesis of new chimeric compounds from the natural cytotoxic chalcone 2',4'-dihydroxychalcone (2',4'-DHC, A) in combination with cinnamic acids. For this purpose, a descriptive and predictive quantitative structure-activity relationship (QSAR) model was developed to study the chimeric compounds' anti-cancer activities against human breast cancer MCF-7, relying on the presence or absence of structural motifs in the chalcone structure, like in a Free-Wilson approach. For this, we used 207 chalcone derivatives with a great variety of structural modifications over the α and ß rings, such as halogens (F, Cl, and Br), heterocyclic rings (piperazine, piperidine, pyridine, etc.), and hydroxyl and methoxy groups. The multilinear equation was obtained by the genetic algorithm technique, using logIC50 as a dependent variable and molecular descriptors (constitutional, topological, functional group count, atom-centered fragments, and molecular properties) as independent variables, with acceptable statistical parameter values (R2 = 86.93, Q2LMO = 82.578, Q2BOOT = 80.436, and Q2EXT = 80.226), which supports the predictive ability of the model. Considering the aromatic and planar nature of the chalcone and cinnamic acid cores, a structural-specific QSAR model was developed by incorporating geometrical descriptors into the previous general QSAR model, again, with acceptable parameters (R2 = 85.554, Q2LMO = 80.534, Q2BOOT = 78.186, and Q2EXT = 79.41). Employing this new QSAR model over the natural parent chalcone 2',4'-DHC (A) and the chimeric compound 2'-hydroxy,4'-cinnamate chalcone (B), the predicted cytotoxic activity was achieved with values of 55.95 and 17.86 µM, respectively. Therefore, to corroborate the predicted cytotoxic activity compounds A and B were synthesized by two- and three-step reactions. The structures were confirmed by 1H and 13C NMR and ESI+MS analysis and further evaluated in vitro against HepG2, Hep3B (liver), A-549 (lung), MCF-7 (breast), and CasKi (cervical) human cancer cell lines. The results showed IC50 values of 11.89, 10.27, 56.75, 14.86, and 29.72 µM, respectively, for the chimeric cinnamate chalcone B. Finally, we employed B as a molecular scaffold for the generation of cinnamate candidates (C-K), which incorporated structural motifs that enhance the cytotoxic activity (pyridine ring, halogens, and methoxy groups) according to our QSAR model. ADME/tox in silico analysis showed that the synthesized compounds A and B, as well as the proposed chalcones C and G, are the best candidates with adequate drug-likeness properties. From all these results, we propose B (as a molecular scaffold) and our two QSAR models as reliable tools for the generation of anti-cancer compounds over the MCF-7 cell line.

Identification of a cytotoxic factor from a non-pigmented entomopathogenic Serratia marcescens isolate toxic towards human carcinoma cell lines.

It has been reported that cell-free culture broths and some proteins from pigmented and non-pigmented Serratia spp. are cytotoxic towards cancerous and non-cancerous human cell lines. Looking for new molecules toxic against human cancerous cells but harmless towards normal human cells, the aim of this work was (a) to determine whether cell-free broths from the entomopathogenic non-pigmented S. marcescens 81 (Sm81), S. marcescens 89 (Sm89) and S. entomophila (SeMor4.1) presented cytotoxic activity towards human carcinoma cell lines; (b) to identify and purify the associated cytotoxic factor(s) and (c) to evaluate whether the cytotoxic factor(s) was cytotoxic towards non-cancerous human cells. This research was focussed on the observed morphology changes and the proportion of remaining viable cells after incubation in the presence of cell-free culture broths from the Serratia spp isolates to evaluate cytotoxic activity. The results showed that broths from both S. marcescens isolates presented cytotoxic activity and induced cytopathic-like effects on the human neuroblastoma CHP-212 and the breast cancer MDA-MB-231 cells. Slight cytotoxicity was observed in the SeMor4.1 broth. A serralysin-like protein of 50 kDa was identified in Sm81 broth as responsible for cytotoxic activity after purification by ammonium sulphate precipitation and ion-exchange chromatography followed by tandem-mass spectrometry (LC-MS/MS). The serralysin-like protein was toxic against CHP-212 (neuroblastoma), SiHa (human cervical carcinoma) and D-54 (human glioblastoma) cell lines in a dose-dependent manner and showed no cytotoxic activity in primary cultures of normal non-cancerous human keratinocytes and fibroblasts. Therefore, this protein should be evaluated for a potential use as an anticancer agent.

Exploring the Gallic and Cinnamic Acids Chimeric Derivatives as Anticancer Agents over HeLa Cell Line: An in silico and in†vitro Study.

Cervical cancer is one of the most aggressive and important cancer types in the female population, due to its low survival rate. Actually, the search for new bioactive compounds, like gallic and cinnamic acid, is one of the most employed options to finding a treatment. In the present study, 134 phenolic compounds with cytotoxic activity over HeLa cell line were used to generate a descriptive ( R 2 ${{R}^{2}}$ =0.76) and predictive ( Q 2 ${{Q}^{2}}$ =0.69 and Q e x t 2 ${{Q}_{{\rm e}{\rm x}{\rm t}}^{2}}$ =0.62) QSAR model. Structural, electronic, steric, and hydrophobic features are represented as different molecular descriptors in our QSAR model. From this model, nine gallate-cinnamate ester derivatives (N1-N9) were designed and synthesized. Furthermore, in vitro cytotoxic activity was evaluated against HeLa and non-tumorigenic cells. Derivatives N6, N5, N1, and N9 were the most active molecules with IC50ExpHeLa values from 7.26 to 11.95 µM. Finally, the binding of the synthesized compounds to the colchicine binding site on tubulin was evaluated by molecular docking as a possible action mechanism. N1, N5 and N6 can be considered as templates for the design of new cervical anticancer compounds.

Marine Cyanobacteria as Sources of Lead Anticancer Compounds: A Review of Families of Metabolites with Cytotoxic, Antiproliferative, and Antineoplastic Effects.

The marine environment is highly diverse, each living creature fighting to establish and proliferate. Among marine organisms, cyanobacteria are astounding secondary metabolite producers representing a wonderful source of biologically active molecules aimed to communicate, defend from predators, or compete. Studies on these molecules' origins and activities have been systematic, although much is still to be discovered. Their broad chemical diversity results from integrating peptide and polyketide synthetases and synthases, along with cascades of biosynthetic transformations resulting in new chemical structures. Cyanobacteria are glycolipid, macrolide, peptide, and polyketide producers, and to date, hundreds of these molecules have been isolated and tested. Many of these compounds have demonstrated important bioactivities such as cytotoxicity, antineoplastic, and antiproliferative activity with potential pharmacological uses. Some are currently under clinical investigation. Additionally, conventional chemotherapeutic treatments include drugs with a well-known range of side effects, making anticancer drug research from new sources, such as marine cyanobacteria, necessary. This review is focused on the anticancer bioactivities of metabolites produced by marine cyanobacteria, emphasizing the identification of each variant of the metabolite family, their chemical structures, and the mechanisms of action underlying their biological and pharmacological activities.

Methyl benzoate and cinnamate analogs as modulators of DNA methylation in hepatocellular carcinoma.

Phenolic acids represent a large collection of phytochemical molecules present in the plant kingdom; they have an important role as epigenetic regulators, particularly as inhibitors of DNA methylation. In the present study, 14 methyl benzoate and cinnamate analogs were synthesized (11-24). Their cytotoxic activity on hepatocellular carcinoma cells (Hep3B) and immortalized human hepatocyte cells was then evaluated. In addition, its effect on the inhibition of global DNA methylation in Hep3B was also determined. Our results showed that the cinnamic derivatives 11-14 and 20-22 were more potent than the free caffeic acid (IC50 109.7-364.2 µM), being methyl 3,4-dihydroxycinammate (12) the most active with an IC50  = 109.7 ± 0.8 µM. Furthermore, 11-14, 20-23 compounds decreased overall DNA methylation levels by 63% to 97%. The analogs methyl 4-hydroxycinnamate (11), methyl 3,4,5-trimethoxycinnamate (14), methyl 4-methoxycinnamate (21), and methyl 3,4-dimethoxycinnamate (22) showed relevant activities of both cytotoxicity and global DNA methylation inhibition. The molecular docking of 21 and 14 suggested that they partly bind to the SAH-binding pocket of DNA methyltransferase 1. These results emphasize the importance of natural products and their analogs as potential sources of DNA methylation modulating agents.

UPLC-QTOF-MS analysis of cytotoxic and antibacterial extracts of Hechtia glomerata Zucc.

Hechtia glomerata, a Mexican medicinal plant employed against bacterial infections and as food, is taxonomically related to the genus Tillandsia which has anticancer activity. Organic and aqueous extracts of H. glomerata leaves were prepared and tested for cytotoxic and antibacterial activity. UPLC-QTOF-MS analysis determined the chemical composition of active extracts to find cytotoxic and antibacterial compounds. Hexane extract was cytotoxic against HepG2, Hep3B and MCF7 (IC50: 24-28 µg/mL), whereas CHCl3/MeOH extract against PC3 and MCF7 (IC50: 25 and 32 µg/mL). CHCl3/MeOH extract showed antibacterial activity against Staphylococcus aureus and Enterococcus faecium (MIC: 125 and 62.5 µg/mL). Hexane extract cytotoxic compounds were ß-sitosterol, stigmasterol, phytol and ursolic acid. CHCl3/MeOH extract antibacterial and/or cytotoxic compounds were daucosterol, oleanolic acid, resveratrol, quercetin, kaempferol, apigenin, cyanidin, p-coumaric acid and caffeic acid. This plant could be useful against bacterial infections and cancer. However, in vivo studies are needed to determine its toxicity and therapeutic efficacy.

Metabolomic Profile and Cytotoxic Activity of Cissus incisa Leaves Extracts.

Cissus incisa leaves have been traditionally used in Mexican traditional medicine to treat certain cancerous illness. This study explored the metabolomic profile of this species using untargeted technique. Likewise, it determined the cytotoxic activity and interpreted all data by computational tools. The metabolomic profile was developed through UHPLC-QTOF-MS/MS for dereplication purposes. MetaboAnalyst database was used in metabolic pathway analysis and the network topological analysis. Hexane, chloroform/methanol, and aqueous extracts were evaluated on HepG2, Hep3B, HeLa, PC3, A549, and MCF7 cancer cell lines and IHH immortalized hepatic cells, using Cell Titer proliferation assay kit. Hexane extract was the most active against Hep3B (IC50 = 27 ± 3 µg/mL), while CHCl3/MeOH extract was the most selective (SI = 2.77) on the same cell line. A Principal Component Analysis (PCA) showed similar profiles between the extracts, while a Venn diagram revealed 80 coincident metabolites between the bioactive extracts. The sesquiterpenoid and triterpenoid biosynthesis pathway was the most significant identified. The Network Pharmacology (NP) approach revealed several targets for presqualene diphosphate, phytol, stearic acid, δ-tocopherol, ursolic acid and γ-linolenic acid, involved in cellular processes such as apoptosis. This work highlights the integration of untargeted metabolomic profile and cytotoxic activity to explore plant extracts, and the NP approach to interpreting the experimental results.

Cytotoxic Fractions from Hechtia glomerata Extracts and p-Coumaric Acid as MAPK Inhibitors.

Preliminary bioassay-guided fractionation was performed to identify cytotoxic compounds from Hechtia glomerata, a plant that is used in Mexican ethnomedicine. Organic and aqueous extracts were prepared from H. glomerata's leaves and evaluated against two cancer cell lines. The CHCl3/MeOH (1:1) active extract was fractionated, and the resulting fractions were assayed against prostate adenocarcinoma PC3 and breast adenocarcinoma MCF7 cell lines. Active fraction 4 was further analyzed by high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry analysis to identify its active constituents. Among the compounds that were responsible for the cytotoxic effects of this fraction were flavonoids, phenolic acids, and aromatic compounds, of which p-coumaric acid (p-CA) and its derivatives were abundant. To understand the mechanisms that underlie p-CA cytotoxicity, a microarray assay was performed on PC3 cells that were treated or not with this compound. The results showed that mitogen-activated protein kinases (MAPKs) that regulate many cancer-related pathways were targeted by p-CA, which could be related to the reported effects of reactive oxygen species (ROS). A molecular docking study of p-CA showed that this phenolic acid targeted these protein active sites (MAPK8 and Serine/Threonine protein kinase 3) at the same binding site as their inhibitors. Thus, we hypothesize that p-CA produces ROS, directly affects the MAPK signaling pathway, and consequently causes apoptosis, among other effects. Additionally, p-CA could be used as a platform for the design of new MAPK inhibitors and re-sensitizing agents for resistant cancers.

Antibacterial and cytotoxic activities of new sphingolipids and other constituents isolated from Cissus incisa leaves.

Cissus incisa is used in traditional Mexican medicine to treat certain ailments, infectious or cancerous diseases. Excepting for our previous research, this species had no scientific reports validating its traditional use. In this study, we evaluated the antibacterial and cytotoxic properties of the sphingolipids and others phytocompounds isolated from C. incisa leaves to increase the scientific knowledge of the Mexican flora. The antibacterial activity was evaluated against Gram-positive and Gram-negative bacteria by the Microdilution method. Meanwhile, the cytotoxic potential was determined on six human cancer cells: PC3, Hep3B, HepG2, MCF7, A549, and HeLa; using an aqueous solution cell proliferation assay kit. A cell line of immortalized human hepatocytes (IHH) was included as a control of non-cancerous cells. Selectivity index (SI) was determined only against the hepatocellular carcinoma cell lines. The phytochemical investigation of C. incisa leaves resulted in the isolation and characterization of five compounds: 2-(2'-hydroxydecanoyl amino)-1,3,4-hexadecanotriol-8-ene (1), 2,3-dihydroxypropyl tetracosanoate (2), ß-sitosterol-D-glucopyranoside (3), α-amyrin-3-O-ß-D-glucopyranoside (4), and a mixture of cerebrosides (5). Until now, this is the first report of the sphingolipids (1), (5-IV) and (5-V). Only the compound (4) and cerebrosides (5) exhibited antibacterial activity reaching a MIC value of 100 µg/mL against Pseudomonas aeruginosa resistant to carbapenems. While, the acetylated derivate of (3), compound (3Ac) showed the best cytotoxic result against PC3 (IC50 = 43 ± 4 µg/mL) and Hep3B (IC50 = 49.0 ± 4 µg/mL) cancer cell lines. Likewise, (3Ac) achieved better SI values on HepG2 and Hep3B cell lines. This research reveals the importance of study medicinal plants, to identify bioactive molecules as sources of potential drugs. The presence of these compounds allows us to justify the use of this plant in traditional Mexican medicine.

Metabolic Profile and Evaluation of Biological Activities of Extracts from the Stems of Cissus trifoliata.

Cissus trifoliata (L.) L belongs to the Vitaceae family and is an important medicinal plant used in Mexico for the management of infectious diseases and tumors. The present study aimed to evaluate the metabolic profile of the stems of C. trifoliata and to correlate the results with their antibacterial and cytotoxic activities. The hexane extract was analyzed using gas chromatography coupled with mass spectrometry (GC-MS) and the CHCl3-MeOH and aqueous extracts by ultraperformance liquid chromatography quadrupole time of fly mass spectrometry (UPLC-QTOF-MS). The antibacterial activity was determined by broth microdilution and the cytotoxicity was evaluated using MTS cell proliferation assay. Forty-six metabolites were putatively identified from the three extracts. Overall, terpenes, flavonoids and stilbenes characterize the metabolic profile. No antibacterial activity was found in any extract against the fifteen bacteria strains tested (MIC >500 µg/mL). However, high cytotoxic activity (IC50 ≤ 30 µg/mL) was found in the hexane and aqueous extracts against hepatocarcinoma and breast cancer cells (Hep3B, HepG2 and MCF7). This is the first report of the bioactive compounds of C. trifoliata stems and their antibacterial and cytotoxic properties. The metabolic profile rich in anticancer compounds correlate with the cytotoxic activity of the extracts from the stems of C. trifoliata. This study shows the antitumor effects of this plant used in the traditional medicine and justifies further research of its anticancer activity.

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.

16-Hydroxy-Lycopersene, a Polyisoprenoid Alcohol Isolated from Tournefortia hirsutissima, Inhibits Nitric Oxide Production in RAW 264.7 Cells and Induces Apoptosis in Hep3B Cells.

Three polyisoprenoid alcohols were isolated from the leaves of Tournefortia hirsutissima by a bioassay-guided phytochemical investigation. The compounds were identified as 16-hydroxy-lycopersene (Compound 1), (Z8,E3,ω)-dodecaprenol (Compound 2) and (Z9,E3,ω)-tridecaprenol (Compound 3). Compound 1, an unusual polyisoprenoid, was characterized by 1D and 2D NMR. We also determined the absolute configuration at C-16 by the modified Mosher's method. The in vitro antiproliferative and anti-inflammatory activities of the isolated compounds were evaluated. Among isolates, Compound 1 moderately inhibited the nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. On the other hand, Compound 1 displayed selective antiproliferative activity against HeLa, PC3, HepG2 and Hep3B cancer cells and was less potent against IHH non-cancerous cells. Compound 1 in Hep3B cells showed significant inhibition of cell cycle progression increasing the sub-G1 phase, suggesting cell death. Acridine orange/ethidium bromide staining and Annexin V-FITC/PI staining demonstrated that cell death induced by Compound 1 in cells Hep3B was by apoptosis. Further study showed that apoptosis induced by Compound 1 in Hep3b cells is associated with the increase of the ratio of Bax/Bcl-2, and caspase 3/7 activation. These results suggest that Compound 1 induce apoptotic cell death by the mitochondrial pathway. To our knowledge, this is the first report about the presence of polyprenol Compounds 1-3 in T. hirsutissima, and the apoptotic and anti-inflammatory action of Compound 1.

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.

Design, synthesis and QSAR study of 2'-hydroxy-4'-alkoxy chalcone derivatives that exert cytotoxic activity by the mitochondrial apoptotic pathway.

Eleven 4'-alkoxy chalcones were synthesized and biologically evaluated for their antiproliferative activity against four human tumor cell lines (PC-3, MCF-7, HF-6, and CaSki). Compounds 3a-3d and 3f were selective against PC-3, with IC50 values ranging from 8.08 to 13.75 µM. In addition, chalcones 3a-3c did not affect the normal fibroblasts BJ cells. The most active and selective compounds were further evaluated for their effect on the progression of cell cycle in PC-3 cells, and chalcones 3a and 3c induced a G2/M phase arrest. Furthermore, it was found that these three chalcones induced the mitochondrial apoptotic pathway by regulating Bax and Bcl-2 transcripts and by increasing caspase 3/7 activation. Otherwise, the QSAR model indicates that the double bond of the α,ß-unsaturated carbonyl, as well as the planar structure geometry, are important to the biological activity of the synthetized chalcones. Based on these studies, it was concluded that withdrawing substituents in ring A, decrease the antiproliferative activity. This is related to the possible mechanism of action of these compounds, where a Michael addition needs to take place in order to be a potent anticancer agent.

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.

Gallic acid sensitizes paclitaxel-resistant human ovarian carcinoma cells through an increase in reactive oxygen species and subsequent downregulation of ERK activation.

Paclitaxel (PTX) is currently used as a front-line chemotherapeutic agent for several types of cancer, including ovarian carcinoma; however, PTX-resistance frequently arises through multiple mechanisms. The development of new strategies using natural compounds and PTX in combination has been the aim of several prior studies, in order to enhance the efficacy of chemotherapy. In this study, we found the following: (i) gallic acid (GA), a phenolic compound, potentiated the capacity of PTX to decrease proliferation and to cause G2/M cycle arrest in the PTX-resistant A2780AD ovarian cancer cell line; (ii) GA exerted a pro-oxidant action by increasing the production of reactive oxygen species (ROS), and co-treatment with the antioxidant agent N­acetyl-L­cysteine (NAC) prevented GA+PTX-induced cell proliferation inhibition and G2/M phase arrest; (iii) PTX stimulated ERK phosphorylation/activation, and co-treatment with the MEK/ERK inhibitor PD98049 potentiated the proliferation inhibition and G2/M phase arrest; (iv) and finally, GA abrogated the PTX-induced stimulation of ERK phosphorylation, a response that was prevented by co-treatment with NAC. Taken together, these results indicate that GA sensitizes PTX-resistant ovarian carcinoma cells via the ROS­mediated inactivation of ERK, and suggest that GA could represent a useful co-adjuvant to PTX in ovarian carcinoma treatment.

Wnt/ß­catenin pathway activation and silencing of the APC gene in HPV­positive human cervical cancer­derived cells.

Although persistent infections with high­risk human papilloma virus (HPV) constitute the most significant cofactor for the development of cervical cancer, they are insufficient on their own. Mutations or epigenetic inactivation of the tumor suppressor adenomatous polyposis coli (APC), the two acting as prominent oncogenic mechanisms in a number of types of cancer, are frequently associated with aberrant activation of the Wnt/ß­catenin pathway. According to these observations, it was hypothesized that APC alteration may lead to ß­catenin deregulation and the abnormal expression of direct targets of the Wnt pathway in HPV­infected cervical cancer cells. The present study confirmed that the stabilization of ß­catenin correlates with enhanced transcriptional activity of the ß­catenin/T­cell factor complex in cervical cancer cell lines. Sequence analysis of the 'hot­spot' in the mutation cluster region did not exhibit genetic alterations that may be associated with APC gene inactivation. In addition, it was identified that there was a good correlation with the hypermethylation status of the APC promoter 1A and the abnormal accumulation of endogenous ß­catenin in cell lines and biopsies infected with HPV16, although not HPV18. Removal of the epigenetic markers led to an increase in APC levels and a reduction of ß­catenin expression in two transcriptional targets of the Wnt pathway: Matrix metalloproteinase­7 and vascular endothelial growth factor. The present study suggested that the increase in Wnt activity in certain cervical cancer­derived cells may be associated with an alteration in the methylation status of the APC gene promoter 1A.

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.

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.