A549 cells distribution in the phases of the cell cycle after treatments with DMSO 1% (negative control), VCR (positive control for arresting in G2/M phase), and Chloroformic fractions of A. gracilis. Cells were incubated by 24, 48 and 72 h, and stained with PI. Cells distribution in the Sub-G1 (possible apoptosis), G1, S and G2/M phases of the cell cycle, was analyzed by using of FlowJo_V10 software. 

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... analysis performed on cells exposed to those fractions, by immunofluorescence microscopy was realized in order to confirm inhibitions of cell growth and estimate a possible apoptosis induction; changes in the microtubule integrity, nucleus, mitochondria, cell shape and size were detected. As it is known currently, the mode of death and morphological changes are dependent on the cell type, and the stimuli applied with other cell types ( Ziegler et al., 2004). In the inhibition of microtubule dynamics, a persistent alteration of biological processes is induced which eventually leads to apoptosis ( Mollinedo et al., 2003). In this study it was found a loss of the integrity of microtubules forming the cytoskeleton on all cells exposed to chloroformic fractions derived from petroleum ether extracts, and the most affected were SiHa and A549 cell lines in response to the chloroformic fraction from petroleum ether extract of inflorescences, chloroformic fraction from petroleum ether extract of leaves affected and A549 cells, the less effect on microtubule organization was observed in the cells exposed to chloroformic fraction from ethanolic extract of leaves (Fig. 2). Simultaneously with the microtubule destabilization, different nuclear phenotypes were induced by the treatments, finding in some cells as A549 and MDA-MB231, an initial increase in size thereof relative to the negative control, even with the positive control VCR, after 24 hours (Figs. 2 and 3). Previously, it was reported that VCR binds to DNA and chromatina on cancer cells (Mohammadgholi et al., 2013), according to this, the increase in the size of the nucleus could be related with the nuclear envelope disruption which allows vincristine, and perhaps components of chloroformic fractions, enter the nucleus. Another hypothesis is the induction of mitotic catastrophe, because it is known that different classes of cytotoxic agents can induce an abnormal mitosis that results in cell death ( Mansilla et al., 2006), given by factors such as abnormal nuclei, nucleus enlarging, multipolar mitoses, or multiple nuclei, characteristic of mitotic catastrophe (Maskey et al., 2013), as it was observed in this study. On the other hand, the mitochondrial material was affected by chloroformic fractions, showing the shuttling of complex IV subunit (COX IV) from cytoplasm to the nucleus, as was observed in A549 and PC3 cells treated with chloroformic fraction from petroleum ether extract of leaves and in MDA-MB-231 cells exposed to chloroformic fraction from petroleum ether extract of inflorescences.These results could support an evidence of programmed cell death induction in A549 cells, according previous reports where mitochondrial proteins were translocated from mitochondria to the nucleus after apoptotic stimuli ( Moreira et al., 2014). In addition, a decrease in the number of mitochondria was observed in A549 and MDA-MB-231 cells treated with chloroformic fraction from petroleum ether extract of leaves; A549 treated with chloroformic fraction from petroleum ether extract of inflorescences and MDA-MB-231 treated with chloroformic fraction from ethanolic extract of leaves (Fig. 3). All cell lines analyzed showed alterations by exposure to chloroformic fractions; however, according with the results, the most affected cells by treatments were A549. The size and complexity changes of that cell line was analyzed performing a basic flow cytometric analysis of the parameters forward-scatter area (FSC-A) and side- scatter area (SSC-A) data after 6 h of 50.000 events of treated or untreated cells. Fig. 4 shows the dot plot of sub- populations of A549 cells stained with PI. Synchronized and negative control cells were presented as normal in size and complexity; nevertheless, upon treatment with VCR, cells presented an increasing of FSC-A and SSC-A with the 87 % of cells located in the Q2 subpopulation, between chloroformic fractions the chloroformic fraction from ethanolic extract of leaves caused the most significant alteration on light scatter properties: 77 % of cells in Q2 (Fig. 4). The IC 50 values of petroleum ether, chloroformic, ethyl acetate and ethanolic fractions from petroleum ether and ethanolic extracts of A. gracilis inflorescences and leaves, on SiHa, HT29, A549, MDA MB-231 and PC3 cancer cell lines after 48 hours of incubation Fractions IC 50 µg/ml SiHa HT29 A549 MDAMB231 PC-3 VCR-control 0.960** 0.003** 0.059** 0.008** 0.054** PI-Petrol >200 >200 >200 >200 >200 PI-CHCl 3 29 On the other hand, a normal distribution in the cell cycle phases was observed with the negative controls of A549 cells. The synchronized cells showed a high population in G1 phase > 80% which was decreasing with the progress of incubation time with DMSO vehicle, but without affection in the normal development. The number of cells in Sub-G1 not exceed 2% during 72 hours of incubation. Vincristine sulfate, as expected, produced an arrest in G2/M phase of the cell cycle of A549 cells, avoiding cell proliferation and hence causing cell death ( Poruchynsky et al., 2015), as was evidenced in a time dependent manner of drug treatment. Instead, the chloroformic fraction from petroleum ether extract of inflorescences, chloroformic fraction from petroleum ether extract of leaves and chloroformic fraction from ethanolic extract of leaves, caused cell growth inhibition of A549 cells by blocking the G1 phase to S phase in the cell cycle (Fig. 5). However, cells treated with chloroformic fraction from petroleum ether extract of leaves decay aggressively at 48h indicating that cell cycle arrest induced by this fraction, occurred during a short time before imminent cell death. A similar behavior was evidenced on cells exposed to chloroformic fraction from petroleum ether extract of inflorescences but at 72 h, indicating that these fractions could be a highly active compound that causes rapid decrease in cell ...

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... in a time dependent manner of drug treatment. Instead, the chloroformic fraction from petroleum ether extract of inflorescences, chloroformic fraction from petroleum ether extract of leaves and chloroformic fraction from ethanolic extract of leaves, caused cell growth inhibition of A549 cells by blocking the G1 phase to S phase in the cell cycle (Fig. 5). However, cells treated with chloroformic fraction from petroleum ether extract of leaves decay aggressively at 48h indicating that cell cycle arrest induced by this fraction, occurred during a short time before imminent cell death. A similar behavior was evidenced on cells exposed to chloroformic fraction from petroleum ether extract ...