Good Essay On Results: Role Of Xiap In Breast Cancer

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Figure 1: Down regulation of XIAP by specific inhibitor Embelin inhibits cell viability in a dose dependent manner. Investigation was conducted to determine the role of XIAP in the pathogenesis of BC and the impact of its down regulation in the inhibition of BC cell viability. Fig. 1 showed that all cell lines (CAL120, EVSAT, MCF7, and MDAMB231) experienced enormous drop to <40% in mean cell viability at 50μm, with least outcome variability found in EVSAT cell line, indicating dose-dependent vulnerability to Embelin action. Although the MCF7 cell line averaged <40% cell viability, certain samples recorded slightly higher viability levels than 40%. At 25μm, MDAMB231 cell line exhibited a mean cell viability level of 40 percent, indicating its high relative vulnerability to Embelin inhibition than the other cell lines. Conversely, the MCF7 cell line showed cell viability levels as high as ~65%, making it the least susceptible to Embelin influence in this dose. At 1μM, 5μM, and 10μM doses, MCF7 showed the strongest mean cell vulnerability at 10μM dose (~60%) with CAL120 as the least vulnerable (~90%) to Embelin inhibition. The EVSAT cell line showed most vulnerability at 5μM (~70%) while CAL120 the least inhibition (>90% with samples even showing growth). At 1μM, MDAMB231, MCF7, and EVSAT cell lines showed mean cell viability above 90% while CAL120 close to the negative control level. Overall, CAL120 showed least vulnerability to Embelin inhibition at doses 10μM or less; while EVSAT indicated strongest resistance against it at 50μM. Pronounced inhibitory effect of Embelin to the cell lines can be observed in the 25μM and 50μM doses after 24h. This result determines the working doses for Embelin when down regulation of XIAP inhibits cell viability in a dose-dependent manner.
Figure 2: Embelin induces apoptosis in BC cell lines. In order to understand the apoptotic behavior of Embelin at doses 25M and 50M after 24h exposure, the BC cell lines (CAL120, MDAMB231, MCF7, and EVSAT) were analyzed using flow cytometry after staining the samples with Annexin V (Ann) and propidium iodide (PI). Fig. 2A showed the results for MCF7 and MDAMB231 cell lines in four quadrants. The top left quadrant shows necrotic cells (-Ann, +PI); the top right quadrant, apoptotic cells (+Ann, -PI); the bottom left quadrant, live cells (-Ann, -PI), and; the bottom right quadrant, early apoptotic cells (+Ann, -PI). Moreover, Fig. 2B shows the apoptotic activity of Embelin, which is highest in CAL120 cell line (25μM: ~50%, 50μM: >85%) and lowest in MCF7 cell line (25μM: ~15%, 50μM: <40%). Both EVSAT and MDAMB231 cell lines, however, exhibited high apoptotic reaction at >60% in 50μM and ~40% in 25μM. The CAL120 cell line outcome indicates a pronounced resistance against down regulation of XIAP in both doses within a 24-hour exposure period. Conversely, MCF7 cell lines expressed the most pronounced vulnerability to inhibition and XIAP down regulation in both doses.
Figure 3: Embelin treatment down regulates XIAP and activates and cleaves caspase-9, caspase-3, and PARP in BC cell lines. There is a potential involvement of the caspases pathway in the down regulation of XIAP. To verify that, three BC cell lines (CAL120, EVSAT, and MDAMB231) were exposed to Embelin 25μM and 50μM for 24h, after which the cells were lysed and treated with anti-caspases antibodies. Fig. 3 showed clear down regulation of XIAP through the inhibition of C-9 and C-3 in both doses with relatively stronger effect in 50μM dose. Strongest down regulation can be observed in the CAL120 cell line, though. In addition, PARP cleavage can be observed also in all cell lines. These indicate that Embelin induced apoptosis in caspase-dependent cells. Furthermore, cell lines CAL120 and MDAMB231 showed down regulation in XIAP and Survivin in both doses, comparable to the C-9 and C-3 profile, with stronger effect in 50μM dose. However, Survivin resistance to down regulation can be observed in CAL120 at 25μM, indicating the working dose at 50μM or more.
Figure 4: Embelin induced C-3 down regulation activates mitochondrial apoptotic pathway. The question still remained on whether caspases pathway down regulation has any effect on the mitochondrial pathway in effecting apoptosis to BC cell lines. To trigger the reaction, three BC lines were treated with 25μM and 50μM Embelin doses to activate caspases 3 (C-3) and the extracted proteins treated with specific antibodies for Bid, Bcl-XI, and Bcl-2. Fig. 4 showed the down regulated expression Bid, Bcl-XI, and Bcl-2 in both doses, indicating that XIAP down regulation activates C-3, which effect apoptosis of the BC cell lines through the mitochondrial pathway.
XIAP induced apoptosis is caspase dependent. The down regulatory effect of 25μM and 50μM Embelin doses had been shown to activate the caspases pathway, which in turn activates the mitochondrial apoptotic pathway that caused the apoptosis of BC cell lines (CAL120, EVSAT, and MDAM231).