Elucidating the Potential of E-cadherin Re-expression along with Trichostatin A and Zebularine in Enhancing Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis in Human Breast Adenocarcinoma Cells

Background: Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an emerging anti-cancer agent that targets and eliminates cancer cells without harming normal healthy cells. However, TRAIL therapy is limited by resistance. Notably, the depletion of the E-cadherin (CDH1) gene has been significantly associated with TRAIL resistance, particularly in triple-negative breast cancer (TNBC) cells like MDA-MB-231. However, its re-expression has the potential to improve TRAIL-induced apoptosis. Objectives: This study focused on evaluating the potential of E-cadherin re-expression as the biomarker in sensitizing TRAIL-induced apoptosis and exploring the potential effects when E-cadherin is in combination with Trichostatin A (TSA), Zebularine (Zeb), and TRAIL, which is a triple combinational treatment (TZT). 

Methods: This study utilized three cell models: E-MDA-MB-231, which represents MDA-MB-231 that re-expressed E-cadherin, the parental MDA-MB-231 that inherently lacks E-cadherin, and MCF-7 that naturally expresses E-cadherin. Following 48 hours of drug treatment, the cells were stained with Haematoxylin and Eosin (H&E), followed by flow cytometric analysis of fluorescein isothiocyanate (FITC)-Annexin V/Propidium Iodide, reverse transcription-polymerase chain reaction (RT-PCR) for Bax/Bcl-2 ratio, and Western blot to determine the cleavage of poly (ADP-ribose) polymerase (PARP). 

Results: All treatments, both single and combinational, induced significant apoptotic morphology changes, with TZT-treated more evident in all cell lines. Moreover, based on flow cytometric analysis, TZ caused the highest early apoptosis in E-MDA-MB-231 (7.43%) and MDA-MB-231 (10.67%), while TZT was shown effectiveness in MCF-7 (42.23%). Consistent with flow cytometry results, further RT-PCR analysis indicated that E-cadherin in combination with TSA and Zeb (TZ) has the potential to improve the intrinsic apoptotic pathway, likely by increasing 1.5-fold of Bax/Bcl-2 ratio compared to the control, suggesting a shift toward a pro-apoptotic state. Finally, Western blot revealed that TZ, TRAIL, and TZT induced cleaved PARP in E-MDA-MB-231 and MDA-MB-231, suggesting apoptosis induction. Meanwhile in MCF-7 cells, cleaved PARP was observed only with TZ and TZT treatments. Notably, TRAIL treatment led to the highest cleaved PARP in E-MDA-MB-231 cells, while in MDA-MB-231, TZ treatment resulted in the most pronounced cleavage. This suggests that E-cadherin re-expression enhances TRAIL sensitivity in E-MDA-MB-231 cells, whereas apoptosis is more effectively induced by TZ treatment in MDA-MB-231 cells. 

Conclusion: In summary, H&E staining showed the positive effect of E-cadherin in sustaining apoptosis induced by TRAIL, especially in combination with TSA and Zeb. However, based on flow cytometry, RT-PCR, and Western blot results, TZ and TRAIL could potentially offer a more effective treatment option for E-MDA-MB-231. These findings suggest that TZ induced intrinsic apoptotic pathway via epigenetic modulation of CDH1 promoter while TRAIL-induced extrinsic apoptotic pathway via improved TRAIL signaling with E-cadherin re-expression, indicating the potentiality of E-cadherin as a biomarker for TRAIL treatment in TNBC.