الفهرس 
Introduction and Aim of the WorkOnly 14 pages are availabe for public view
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Abstract
Breast cancer is the second most common type of cancer worldwide and displays the highest cancer-related mortality among women worldwide. It represents 35.1% of all female cancers in Egypt.
Autophagy, an evolutionally conserved stress response and degradative mechanism, is induced mainly by stresses, such as starvation and leads to the capture of intracellular proteins and organelles by double-membrane structures called autophagosomes. Cargos (protein aggregates and damaged mitochondria) are then delivered for degradation upon fusion of autophagosomes with lysosomes, which facilitates the recycling of biomolecules, and sustains survival. Despite its role in energy homeostasis, autophagy also regulates many cellular processes, such as quality control and degradation of misfolded proteins and damaged organelles. Therefore, dysfunctional autophagy can contribute to the pathogenesis of many diseases, including cancer, neurodegenerative disorders, and immunologic ailments.
Autophagy is a tightly controlled process, influenced by multiple signalling pathways. Autophagy-related genes (ATG genes) orchestrate the formation of the autolysosome, through a series of sequential steps: initiation, nucleation, elongation and maturation. Beclin1 (human homolog of Atg6) is important to initiate the phases of autophagy, and microtubule-associated protein 1A/1B-light chain 3B (also referred to as LC3B), a member of the Atg8 family, is one of the most studied autophagosome markers. As for the autophagosome maturation, LC3 lipidation is very essential and acts as a second ubiquitin-like conjugation in autophagy process. p62 (also known as SQSTM1/sequestome 1) serves as an adaptor protein that links ubiquitinated proteins to the autophagic machinery and enables their clearance in the lysosome, and can also be used as an evidence of the autophagic flux. Degradation of p62 is another widely used marker to monitor autophagic activity because p62 directly binds to LC3, and is selectively degraded by autophagy.
Several signaling pathways have been shown to promote the cellular growth in response to growth factors and nutrient availability. Inhibition of PI3K/AKT/m-TOR signalling pathway can inhibit the cell growth, invasion and metastasis of various tumors, such as breast cancer. m-TOR, which is a potent inhibitor of autophagy induction, is activated by the oncogenic PI3K/AKT pathway.
Aloin, a natural bioactive anthracycline extracted from the leaf exudates of Aloe vera, has been reported to possess a potential antitumor activity against different cancer cell lines. The cytotoxicity of aloin was mediated by different modes of action, including the increase in the percentage of S phase fraction and the proportion of cells cycling at a higher ploidy level (>G2M). The polypolidization induces apoptosis. In addition, inhibition of topoisomerase IIα and cyclin B1 proteins expression were reported in breast cancer cell lines.
The present study was proposed to elucidate the involvement of autophagy process in the mechanistic role of aloin cytotoxicity. To fulfill the main goal of this study, the cytotoxic effect of aloin was assessed in two different types of breast cancer cell lines, estrogen receptor positive (ER+) (T47D) and estrogen receptor negative (ER-) (MDA-MB231), compared to the reference drug, doxorubicin, an anthraquinone analog, to determine the concentrations that kill 50% of tumor cells (IC50 values).
T47D and MDA-MB231 breast cancer cell lines were cultured in complete culture media containing multiple increments of aloin (20-100μg/ml and 20-400 μg/ml, respectively) or doxorubicin (0.05-0.2 μg/ml and 0.1-1 μg/ml, respectively) for 24 and 72h. The cytotoxic effects of aloin and doxorubicin were evaluated by MTT assay and confirmed by the clonogenic assay. Formation of the autophagic ultrastructures in the control and treated tumor cells was initially examined by transmission electron microscopy (TEM). Confocal fluorescence microscope was used to detect the acidic vesicular organelles (AVOs) formation in the control and treated tumor cells. Quantification of the tumor cells containing AVOs was done by flowcytometery (FACS). The proceeding of autophagy process was emphasized by determination of the protein expression levels of some autophagy-related genes, such as p-AKT, p-m-TOR, beclin1, LC3B and p62 by Western blotting.
Results obtained from this study are summarized as follows:
• Exposure of T47D cells to aloin for 24 and 72h showed a significantly dose-dependent reduction in the percentage of cell viability, attaining a minimum value of 45.81% at the highest dose level (100µg/ml) in 72h exposure assay.
• Doxorubicin showed a significantly dose-dependent reduction in the percentage cell viability of T47D cells, attaining a minimum of 38.38% at the highest dose level (0.2 µg/ml) in 72h exposure assay.
• Likewise, treatment with multiple doses of aloin and doxorubicin significantly produced a dose-dependent inhibition of the colony formation of T47D cells, with a more pronounced effect in the continuous (72h) exposure regimen.
• MTT cytotoxicity assay in MDA-MB23 cells revealed that aloin gave rise to a dose-dependent reduction in the percent of cell viability of tumor cells, reaching a nadir of 55.6 and 42.35% at the highest dose (400µg/ml) in the 24 and 72h exposure assay, respectively.
• On the other hand, 24h exposure of MDA-MB231 to various increments of doxorubicin produced non-significant change in the percent of cell viability, while in the continuous exposure a significantly dose-dependent reduction in the percentage of cell viability was recorded, reaching a minimum of 30.87% at the highest dose (10µg/ml).
• Exposure of MDA-MB231 tumor cells to increasing concentrations of aloin and doxorubicin showed a significantly gradual increase in the percent of inhibition of colony formation in both time-dependent exposure assays.
• from the results of MTT assay, the IC50 values of aloin and doxorubicin were calculated for both cancer cell lines, and were found to be 75 and 0.1 μg/ml, respectively, for T47D cells, and 295 and 1μg/ml, respectively, for MDA-MB231 cells.
• Transmission electron micrograph of cultured T47D and MDA-MB231 cells after 24 and 72h exposure to IC50 values of aloin and doxorubicin revealed the presence of time-dependent double membrane autophagosomes and autophagolysosomes, as early and late autophagic features.
• Continuous exposure to aloin induced the formation of AVOs in both breast cancer cell lines. Augmented formation of AVOs was monitored in doxorubicin-treated cells at the same time endpoint.
• Quantification of the number of cells containing AVOs by acridine staining using the flow cytometry showed that aloin significantly increased the AVOs-positive cells recording 43.7% in T47D tumor cells, compared to the respective control (2.1%), and amounting 70.6% in MDA-MB231 cancer cells, compared to the respective control (48.69%). On the other hand, doxorubicin treatment increased the accumulation of acidic vesicles in T47D and MDA-MB231 tumor cells recording 92.93 and 61.23%, respectively.
• T47D and MDA-MB231 tumor cells cultured in complete growth media containing IC50 value of aloin for 24h showed an increase in the protein expression levels of p-AKT, beclin 1 and LC3B II gene, which was augmented in the continuous exposure assay.
• In contrast, repressed protein expression of p-mTOR kinase and p62 genes was detected in T47D and MDA-MB231 tumor cells briefly exposed to aloin (24h), which was more notable in the continuous exposure assay (72h).