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Sun Yifu! Min Lowest price since launch. Deal of the Day. Page 10 of 2, Previous 6 7 8 9 10 11 12 13 14 15 Next. Did you find what you were looking for? Yes No. Works perfectly fine I tried on my self Flipkart Customer Certified Buyer 3months ago. Nice product really satisfied. JNK is susceptible to ROS response and is dragged into various cellular processes, including autophagy and apoptosis Apple fruits are one of the most important sources of polyphenolic compounds in the Western diet Apple polyphenols have shown high antioxidant capacity in vitro 15 , 16 and it has been described that the consumption of apple increases the antioxidant status of blood 16 , exhibits an anti-arteriosclerosis activity by reducing low-density lipoprotein oxidation 17 , and decreases cellular glucose levels and lipid uptake Furthermore, it has been reported that apple polyphenols inhibit the proliferation of cancer cells 15 and that phenolic phytochemicals present in the apple skin are able to prevent tumor formation in different types of cancer, including breast Malus pumila Miller cv.
Previous results from our laboratory have described the antiproliferative effect of polyphenol extract from Annurca apple APE in human HaCaT keratinocytes and in human breast carcinoma MCF-7 cells 22 , In the current study, we reported the anticancer effect of APE on triple negative MDA-MB human breast carcinoma cells and we explored the underlying molecular mechanism.
Cell viability was then assessed by MTT assay and expressed as a percentage of untreated cells. The distribution of cell cycle was assessed by flow cytometry. The full-length blots are included in the supplementary information Fig. As shown in Fig. We then examined the levels of phospho-cdc25C and cyclin-dependent inhibitors p27 and p Results showed a substantial dose-dependent increase of p27 and phospho-cdc25C levels.
As showed in Fig. Flow cytometry analysis was then performed. The percentage of cells is reported in the quadrants. Viable cells, lower left; non-viable necrotic cells, upper left; early apoptotic cells, lower right; late apoptotic cells, upper right. Analysis was carried out by triplicate determination on at least three separate experiments. Cell lysates were prepared and analyzed by western blotting assay for the expression of apoptosis-related proteins. The intensities of signals were expressed as arbitrary units.
The graph shows the densitometric intensity of caspase 3 band under the different experimental conditions. The full-length blots are included in the supplementary information Figs S2 , S3.
To explore the mechanism of apoptosis-mediated cell death, we next analyzed the levels of cleaved caspases and PARP. Activation of procaspase 3 was further evidenced from the cleavage of PARP. These results indicated that APE promoted apoptosis via a caspase-dependent mechanism.
To confirm the induction of intrinsic apoptotic pathway, we evaluated the levels of Bax and Bcl-2 proteins, two mitochondria-associated modulators of apoptosis. The balance of these pro- and anti-apoptotic members of Bcl-2 gene family has been thought to determine the functional integrity of the mitochondrial outer membrane and the commitment to apoptotic cell death in mammalian cells It has to be noted that Bcl-2 serves as the mitochondrial gate-keeper and is frequently overexpressed in tumors where it plays a critical role in chemo- and radio-resistance Findings indicated that the content of Bax slightly decreased after APE treatment while Bcl-2 levels were intensely reduced.
This prompted us to examine whether death receptor Fas played any role in our model. All the data above indicated that APE could efficiently induce apoptosis in MDA-MB triple negative breast cancer cells by activating both the extrinsic and intrinsic apoptotic pathways. Persistent autophagy in response to oxidative stress serves as a potent death signal To examine whether APE was able to induce autophagic cell death in MDA-MB cells, we analyzed autophagosome formation after staining with LysoTracker Red LTR , an acidotropic dye for staining and tracking cellular acidic compartments including autophagosome and autolysosome structures in living cells We found that APE induced a dose-dependent upregulation of p62, in agreement with reports showing that an increase in p62 expression is required for autophagy induction We then evaluated the protein levels of beclin-1, an important autophagic player involved in the autophagosome formation 28 , that resulted significantly decreased highlighting that APE induced-autophagy in MDA-MB cell is a beclinindependent process.
Chloroquine is used as positive control. The protein content of LC3B, p62, and beclin-1 was analyzed by western blotting. Overproduction of ROS caused damage to the cells and was involved in the regulation of a variety of cellular processes including autophagy, apoptosis, and cell cycle arrest Next, we analyzed ROS production by flow cytometry Fig.
NAC treatment inhibited the fluorescent signal induced by menadione, thus confirming that the signal was specifically produced by ROS increase. MFI values are reported. Menadione is used as positive control, MFI values are reported. Data were expressed as a percentage of untreated control. The results shown in Fig. The morphology of cells is closely related to cellular physiological status and functions.
Compared with the corresponding untreated cells, APE-treated MDA-MB cells displayed significant morphological changes, characteristic of apoptotic cells, such as loss of cell adhesion, membrane shrinkage, destruction and collapse of the cytoskeleton and the expulsion of the cell nucleus, cell rounding, condensed chromatin and nuclear fragmentation, and reduced cell density Fig.
All results were obtained from at least three independent experiments. The full-length blots are included in the supplementary information Figs S6 , S7. Western blotting analysis evidenced that APE remarkably decreased the level of Dusp-1 in concentration-dependent manner Fig. The full-length blots are included in the supplementary information Figs S8 — S APE downregulates the main cell growth and survival pathways. The full-length blots are included in the supplementary information Figs S12 , S New advances in anticancer drug discovery using natural compounds have been made in the last years and polyphenols have emerged as promising molecules being able to act as selective cytotoxic agents leading to tumor cell death through generation of toxic levels of ROS.
The present study showed that APE exhibited anticancer activities in MDA-MB triple negative breast cancer cells and provided experimental evidences on the underlying mechanisms. We found that APE selectively and efficiently causes a time- and dose-dependent inhibition of MDA-MB cell viability by a pro-oxidant cytotoxic effect while in non-tumorigenic MCF10A cells it exerted an antioxidant protective effect against the H 2 O 2 -induced oxidative stress. These results are in agreement with recent reports indicating that natural products such as, isoliensinine 32 , physagulide P 33 and Ziyuglycoside II 34 are able to selectively increase ROS levels in MDA-MB cells thus allowing to take APE into consideration as a promising novel natural agent for the selective targeting of these TNBC cells.
JNK plays a critical role in natural compounds-induced apoptosis suggesting a tumor-suppressive role of JNK signaling in cancer 13 , The duration of JNK activation is thought to be a critical factor in determining cell proliferation or apoptosis, with transient activation driving essentially prosurviving responses and prolonged activation promoting cell death by mechanisms that are cell type- and stimuli-dependent 13 , 36 , ROS are able to maintain a prolonged JNK activation by inhibiting Dusp-1 through reversible oxidation of a catalytic-site cysteine 38 , Interestingly, we found that also JNK, once activated, was able to downregulate Dusp-1 thus providing a positive feedback loop that contributed to potentiate its activation.
Cdc25 phosphatases regulate cell cycle progression in S phase and mitosis by removing inhibitory phosphate groups from cyclin-dependent kinases It has to be noted that this cell cycle regulatory mechanism is pindependent and could therefore be particularly relevant for the DNA damage response in cancer cells characterized by high levels of mutant p53, such as MDA-MB cells 36 , APE simultaneously triggered both extrinsic and intrinsic apoptosis.
Although autophagy acts primarily as a pro-survival mechanism during nutrient starvation, under some situations it can stimulate a non-apoptotic form of programmed cell death. Strikingly we found that APE-induced autophagy is beclinindependent. This evidence as well as the finding that autophagy inhibition attenuated cell growth inhibition strongly suggested that APE-induced autophagy could function as a pro-death mechanism in MDA-MB cells.
This hypothesis is supported by several considerations: i beclin-independent autophagy is invariably associated with cell death 27 , 42 ; ii autophagic cell death is considered a major non-apoptotic cell death mechanism regulated by JNK 43 , 44 ; iii in cancer cells, under certain conditions, a sustained JNK activation is crucial to induce autophagic cell death 43 , 44 ; iiii during apoptosis, caspase-mediated cleavage of beclin-1 generates fragments that translocate to the mitochondria and induce apoptosis, resulting in an amplified apoptotic cell death In addition to acting as a powerful cell death inducer, APE also displayed potent inhibitory effects on the expression and activity of several oncoproteins related to cell survival and proliferation.
In recent years evidence has been accumulated showing that p21 behaves like a two-faced regulator. Indeed, depending on cell type, cellular localization, p53 status, and the type and level of genotoxic stress, p21 can acquire either oncosuppressive or oncopromoting properties Regulation of breast cancer greatly depends on the subcellular localization of p Nuclear p21, indeed, has a tumor-suppressor activity inducing temporary arrest of cell cycle or leading to a chronic state of senescence or apoptosis.
On the other hand, cytosolic p21 displays oncogenic activities by downregulating apoptotic response and promoting cell proliferation 47 , 48 , The phosphorylation of p21 by AKT is crucial to localize p21 at cytosolic level where p21 can directly bind to JNK and function as a non-competitive inhibitor preventing the activation of JNK and inhibiting its catalytic activity Second, NF-kB is one of the major chemoresistance-related anti-apoptotic factors.
High NF-kB activity has been identified in drug-resistant cancer cell lines Moreover, the anti-cancer activities of many anti-inflammatory drugs are, at least in part, related to the inhibition of NF-kB Interestingly, we found that APE downregulated.
In this scenario ROS are the initial players and JNK, like a capable conductor, orchestrates and modulates numerous cellular events that ultimately resulted in cell death. The timing and duration of JNK activation determine whether cells proliferate or undergo programmed cell death.
Consistent with this view, we furnished evidences strongly suggesting that the sustained JNK activation, induced by the pro-oxidant effect of APE, could represent the key mechanism to trigger cell death in the highly resistant and invasive MDA-MB breast cancer cells. In conclusion, our study for the first time highlighted APE as a multi-faceted agent able to modulate the growth of triple negative MDA-MB breast cancer cells through the simultaneous regulation of multiple cell signaling pathways involved either in cell death as well as in cell proliferation and survival.
The ability of APE to affect cancer cells at multiple levels and, thus, to potentially circumvent the development of resistance, together with its capability to selectively kill tumor cells while exerting a protective antioxidant effect in normal cells, stimulate great interest in future investigations and make APE an attractive candidate for drug development against TNBC. Louis, MO. Raleigh, NC. Ultra-high quality water was used to prepare all buffers and solutions.
All reagents were of the purest commercial grade. Annurca Malus pumila Miller cv. Annurca apple undergoes a peculiar postharvest storage. The amounts of total pholyphenols in apple extracts were determined by Folin-Ciocalteu phenol reagent as described by Singleton et al.
The determination of the polyphenolic profile of APE was performed by HPLC analysis and confirmed the results already described in the literature 21 , The cell viabilities were normalized to the control. The IC 50 values were calculated by using the linear-regression analysis. All MTT assays were executed in quadruplicate.
According to Mele et al. For DAPI nuclear staining the cells were incubated with 2. Subsequently, following the procedure reported in Delle Cave et al. Cells were then washed with PBS and observed by fluorescence microscopy. Thereafter, the cells were harvested and washed twice with PBS.
Cells were processed for western blotting as described previousy The protein concentration was determined by Bradford method P values less than 0. DeSantis, C.
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