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Antiproliferative activity of chloroformic fractions from leaves and inflorescences of Ageratina gracilis
Abstract and Figures
To obtain a scientific basis and justification of plant domestication in the use of Ageratina gracilis, we did an in vitro study of the anticancer potential of extracts and fractions from its leaves and inflorescences. Firstly, cytotoxicity was evaluated against five human tumorigenic cell lines by MTT assay. Subsequently, the chloroformic fractions, considered the most cytotoxic were tested for genotoxicity by comet assay, morphological effects were analyzed by fluorescent microscopy, cell cycle arrest by flow cytometry and early apoptosis induction through fluorescein-5-isothiocyanate (FITC) labeled Annexin-V assay. Non-polar extracts with IC50 values of <53µg/ml showed a high cytotoxicity. The highest cytotoxicity was achieved by chloroformic fraction from petroleum ether extract of leaves and inflorescences and chloroformic fraction from ethanolic extract of leaves, displaying a significant inhibition of cell viability particularly on A549 cells with an IC50 value of 25.9 µg/mL. Chloroformic fractions caused a high percent of DNA damage above 60 percent on A549 and MDAMB-231.The fractions also induced G1/S phase arrest of the cell cycle in A549 cells, furthermore it was confirmed the apoptotic activity chloroformic fraction from petroleum ether extract of inflorescences and chloroformic fraction from ethanolic extract of leaves on those cells by Annexin-V assay. These preliminary results indicate that A. gracilis has an antiproliferative activity against cancer cells, being a starting
point for forthcoming studies about the antineoplastic activity and its domestication conditions
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... Formazan crystals were dissolved with 100 ml of DMSO. The results were determined by the optical density (OD) determined by the absorbance at 570 nm [33]. Estimation of the MTAs concentrations required to reduce the 50% of cell viability (IC 50 ) was done using nonlinear regression from plotting cell survival (%) versus drug concentration [mg/mL]. ...
... The microtubules were stained with anti-atubulin monoclonal antibody DM1A (SigmaeAldrich) and goat anti-mouse Alexa Fluor 488 (Molecular Probes), both diluted in 5% BSA/TTBS (w/v) blocking solution. DNA staining was done with 1.0 mg/mL of DAPI (Invitrogen) [22,33]. Fluorescence was monitored using an epifluorescence microscope (Motic AE31), and the images were captured with the MoticCamPro 282A and analyzed with Motic Image plus 2.0 software. ...
Background:
Microtubule-targeting agents (MTAs) disrupt microtubule dynamics, thereby inducing apoptosis via mitochondrial pathway activation through the modulation in the expression of the Bcl-2 family.
Methods:
To describe topological features of the MTAs networks associated to intrinsic apoptosis induction in p53-null prostate cancer cells, we predicted and compared the interactomes and topological properties of Paclitaxel and Vincristine, and thus, the essential nodes corresponding with the pro- and anti-apoptotic proteins and their kinetics were subjected to experimental analysis in PC-3 cell line.
Results:
The essential nodes of the apoptotic pathways, TP53, and CASP3, were identified in both, Paclitaxel and Vincristine networks, but the intrinsic pathway markers BCL2, BAX, and BCL2L1 were identified as hub nodes only in the Paclitaxel network. An in vitro analysis demonstrated an increase in BimEL and the cleaved-caspase-3 proteins in PC-3 cells exposed to both treatments. Immunoprecipitation analysis showed that treatments induced the releasing of Bax from the anti-apoptotic complex with Bcl-2 protein and the role of BimEL as a de-repressor from sequestering complexes, in addition, new protein complexes were identified between BimEL or Bcl-2 and cleaved-caspase-3, contributing data to the Vincristine network for p53-null cells in response to MTAs.
Conclusion:
The differences in sensitivities, protein profiles, and protein complex kinetics observed between the drugs confirmed that the selectivity and stimulation of the apoptotic system vary depending on the cell's genotype, the drug used and its exposure period.
... Immunofluorescence microscopy analysis: Immunofluorescent assays were performed on A549 cells in order to examine possible microtubule and nuclear damage 24 hours after treatment with the flavanone, according to the methodology suggested previously 7 . One hundred thousand cells per well were seeded on a 24-well plate and grown in 5 % CO2 at 37 °C. ...
Background: Flavonoids isolated from plants have demonstrated an important role in cancer chemoprevention and chemotherapy. The genus Cromolaena has been shown to have active principles against this disease and found in species such as C. odorata, and C. laevigata in a concentration lower than 100 mg/L, however, flavonoids from C. leivensis has not been studied completely as an alternative in cancer treatment. Materials and Methods: The (2R)-5,7-dihydroxy flavanone or (R) Pinocembrin was isolated from leaves of Chromolaena leivensis (Hieron) using chromatography methods. Its structure and relative configuration were determined by NMR spectroscopy, gas chromatography coupled to mass spectrometry and X-ray diffraction. We evaluated the (R) Pinocembrin effects on cell proliferation, morphology, DNA damage, and cell cycle progression of cancer cell lines Results: The compound showed a decreasing cell proliferation rate against HT29, PC-3, A549, MDA-MB-231, and SiHa cancer cell lines with an IC50 values between 58.9 mg/L, and 30.9 mg/L, causing alterations in the stability of the cytoskeleton and G1-phase cell cycle arrest without affecting significantly the DNA integrity. Conclusion: The (R)-Pinocembrin is a potential molecule to be used in the treatment of cancer with an action on the cytoskeleton. Our study indicate that the medium polarity fraction obtained from C. leivensis is a promising fraction which could be used as in the treatment of cancer, especially as a coadjuvant.
This contribution is a completely updated and expanded version of the four prior analogous reviews that were published in this journal in 1997, 2003, 2007, and 2012. In the case of all approved therapeutic agents, the time frame has been extended to cover the 34 years from January 1, 1981, to December 31, 2014, for all diseases worldwide, and from 1950 (earliest so far identified) to December 2014 for all approved antitumor drugs worldwide. As mentioned in the 2012 review, we have continued to utilize our secondary subdivision of a "natural product mimic", or "NM", to join the original primary divisions and the designation "natural product botanical", or "NB", to cover those botanical "defined mixtures" now recognized as drug entities by the U.S. FDA (and similar organizations). From the data presented in this review, the utilization of natural products and/or their novel structures, in order to discover and develop the final drug entity, is still alive and well. For example, in the area of cancer, over the time frame from around the 1940s to the end of 2014, of the 175 small molecules approved, 131, or 75%, are other than "S" (synthetic), with 85, or 49%, actually being either natural products or directly derived therefrom. In other areas, the influence of natural product structures is quite marked, with, as expected from prior information, the anti-infective area being dependent on natural products and their structures. We wish to draw the attention of readers to the rapidly evolving recognition that a significant number of natural product drugs/leads are actually produced by microbes and/or microbial interactions with the "host from whence it was isolated", and therefore it is considered that this area of natural product research should be expanded significantly.
The following study is designed to determine the anticancer and cytotoxic potential of hydroalcoholic extract from rhizomes of Pa ris polyphylla to produce any cytotoxic effect on human A549 lung cancer cell lines. The hydroalcoholic analysed for spectral analysis through NMR, HPLC - DAD and LC - ESI - MS/MS methods and found to have Diosgenyl and pennogenyl saponins. The test conducted us ing MTT method using hum an lung cancer A549 cell lines as part of the in vitro preclinical characterization of compound and compared against Doxorubicin . More than 97 % increment in cell k illing at a concentration of 500 µ g/ml recorded in the cell line . Th e EC50 for the extract was calculated to be 52.34 ug/ml and at the concentration of 1.8559 ug/ml, doxorubicin exhibited approximately 98 % killing of the cells. The EC50 for the Doxorubicin was calculated to be 0.579 ug/ml . From the study it is concluded t he hydroalcoholic rhizome extracts Paris polyphylla has potential to exhibit anticancer activity.
9-Oxo-10, 11-dehydroageraphorone (euptox A), a cadenine sesquiterpene, is the main toxin from Eupatorium adenophorum. The aim of the present study was to examine the induction and mechanism of HeLa cell apoptosis by euptox A. The apoptosis‑inducing effect of the euptox A on HeLa cells was examined by MTT assay. The underlying mechanism was analyzed by flow cytometry and quantitative PCR. Flow cytometry results suggested that euptox A effectively inhibited the proliferation of HeLa cells, arrested the cell cycle transition from S to G2/M phase, did not continue to complete the cell cycle activity (mainly from 4 times and mitosis), and induced cell proliferation. The RT-qPCR detection results showed that euptox A induced apoptosis by improving the gene expression level of apoptotic proteases such as caspase-10 in HeLa cells. Its mechanism of action was associated with the upregulation of apoptotic gene expression and arresting of the cell cycle.
There is an increasing interest to identify plant-derived natural products with antitumor activities. In this work, we have studied the effects of aqueous leaf extracts from Amazonian Vismia and Piper species on human hepatocarcinoma cell toxicity. Results showed that, depending on the cell type, the plants displayed differential effects; thus, Vismia baccifera induced the selective killing of HepG2, while increasing cell growth of PLC-PRF and SK-HEP-1. In contrast, these two last cell lines were sensitive to the toxicity by Piper krukoffii and Piper putumayoense, while the Piperaceae did not affect HepG2 growth. All the extracts induced cytotoxicity to rat hepatoma McA-RH7777, but were innocuous (V. baccifera at concentrations < 75 µg/mL) or even protected cells from basal death (P. putumayoense) in primary cultures of rat hepatocytes. In every case, cytotoxicity was accompanied by an intracellular accumulation of reactive oxygen species (ROS). These results provide evidence for the anticancer activities of the studied plants on specific cell lines and suggest that cell killing could be mediated by ROS, thus involving mechanisms independent of the plants free radical scavenging activities. Results also support the use of these extracts of the Vismia and Piper genera with opposite effects as a model system to study the mechanisms of the antitumoral activity against different types of hepatocarcinoma.
Although apoptosis and necrosis have distinct features, the identification and discrimination of apoptotic and necrotic cell death in vitro is challenging. Immunocytological and biochemical assays represent the current gold standard for monitoring cell death pathways; however, these standard assays are invasive, render large numbers of cells and impede continuous monitoring experiments. In this study, both room temperature (RT)-induced apoptosis and heat-triggered necrosis were analyzed in individual Saos-2 and SW-1353 cells by utilizing Raman microspectroscopy. A targeted analysis of defined cell death modalities, including early and late apoptosis as well as necrosis, was facilitated based on the combination of Raman spectroscopy with fluorescence microscopy. Spectral shifts were identified in the two cell lines that reflect biochemical changes specific for either RT-induced apoptosis or heat-mediated necrosis. A supervised classification model specified apoptotic and necrotic cell death based on single cell Raman spectra. To conclude, Raman spectroscopy allows a non-invasive, continuous monitoring of cell death, which may help shedding new light on complex pathophysiological or drug-induced cell death processes.
Combretastatin A-4 (CA-4) is one of the most effective agents used in chemotherapy. Nevertheless, the contribution of p53 and Bim proteins in the CA-4-induced apoptosis in non-small lung cancer cells (NSCLC) remains unresolved, specifically on involving of p53 in the mitochondrial pathway activation by a transcription-independent mechanism. In this context, the p53-null H1299 and wt-p53 H460 NSCLC cells, in the absence and presence of pifithrin-µ (PFTµ), an inhibitor of p53 mitochondrial-translocation, were treated with CA-4 and different cellular endpoints were analyzed. In contrast to previous observations in H460 cells, CA-4 failed in the activation of an apoptotic response in H1299 cells, thus indicating an involvement of p53 in the cell death induced by the drug. We found that CA-4 led to p53 cellular re-localization in H460 cells; in particular, p53 was released from the microtubular network and accumulated at mitochondria where in it interact with Bim protein and other proteins of the Bcl-2 (B-cell leukaemia-2) family, leading to cytochrome c release, alteration in the mitochondrial membrane polarization, cell cycle arrest at the G2/M-phase, and cell death. Interestingly, the cytosolic and the mitochondrial accumulation of protein Bim was strictly dependent on p53 status. The extent of cell death was not reduced in H460 after combined treatment of PFTµ with CA-4. Overall, the data support a model of CA-4-induced apoptosis in NSCLC, for which the expression of p53 protein is essential, but its mitochondrial function, linked to p53-transcription independent apoptosis pathway, is negligible.
Anticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.
Significance
Drugs targeting microtubules are among the most active anticancer agents. In vitro and in preclinical models, these agents are said to interfere with mitosis. However human tumors divide too slowly for this paradigm to apply, evidenced by the failure of over a dozen well-designed antimitotic agents targeting the aurora kinases and kinesin spindle protein that had minimal antitumor activity but caused severe bone marrow suppression. We have proposed that microtubule-targeting agents interfere with the trafficking of critical proteins in interphase microtubules. If true, then one must identify critical proteins whose traffic on microtubules is impacted. We identify nine DNA repair proteins that traffic on microtubules, explaining why combinations of a microtubule-targeting agent and a DNA-damaging agent are frequently used in cancer therapy.
The cardiotoxicity induced by the anti-cancer Doxorubicin (DOX) involves increased oxidative stress, disruption of calcium homeostasis and activation of cardiomyocyte death. Nevertheless, antioxidants and caspase inhibitors often show little efficacy in preventing cell death. We hypothesize that a caspase-independent cell death mechanism with the release of the Apoptosis Inducing Factor (AIF) from mitochondria is involved in DOX toxicity. To test the hypothesis, H9c2 cardiomyoblasts were used as model for cardiac cells. Our results demonstrate that z-VAD-fmk, a pan-caspase inhibitor, does not prevent DOX toxicity in this cell line. DOX treatment results in AIF translocation to the nuclei, as confirmed by Western Blotting of cell fractions and confocal microscopy. Also, DOX treatment of H9c2 cardiomyoblasts resulted in the appearance of 50 kbp DNA fragments, a hallmark of AIF nuclear effects. AIF knockdown using a siRNA approach in H9c2 cells resulted in a reduction of DOX toxicity, including decreased p53 activation and poly-ADP-ribose-polymerase (PARP) cleavage. Among the proteases that could be responsible for AIF cleavage, DOX decreased calpain activity but increased cathepsin B activation, with inhibition of the latter partly decreasing DOX toxicity. Altogether, the results support that AIF release is involved in DOX-induced H9c2 cell death, which explains the limited ability of caspase inhibitors to prevent toxicity.
Chromatin has been successfully used as a tool for the study of genome function in cancers. Vincristine as a vinca alkaloid anticancer drug exerts its action by binding to tubulins. In this study the effect of vincristine on DNA and chromatin was investigated employing various spectroscopy techniques as well as thermal denaturation, equilibrium dialysis and DNA-cellulose affinity. The results showed that the binding of vincristine to DNA and chromatin reduced absorbance at both 260 and 210 nm with different extent. Chromopheres of chromatin quenched with the drug and fluorescence emission intensity decreased in a dose-dependent manner. Chromatin exhibited higher emission intensity changes compared to DNA. Upon addition of vincristine, Tm of DNA and chromatin exhibited hypochromicity without any shift in Tm. The binding of the drug induced structural changes in both positive and negative extremes of circular dichroism spectra and exhibited a cooperative binding pattern as illustrated by a positive slope observed in low r values of the binding isotherm. Vincristine showed higher binding affinity to double stranded DNA compared to single stranded one. The results suggest that vincristine binds with higher affinity to chromatin compared to DNA. The interaction is through intercalation along with binding to phosphate sugar backbone and histone proteins play fundamental role in this process. The binding of the drug to chromatin opens a new insight into vincristine action in the cell nucleus.