Theaflavins target Fas/caspase-8 and Akt/pBad pathways to induce apoptosis in p53-mutated human breast cancer cells

Abstract

The most common alterations found in breast cancer are inactivation or mutation of tumor suppressor gene p53. The present study revealed that theaflavins induced p53-mutated human breast cancer cell apoptosis. Pharmacological inhibition of caspase-8 or expression of dominant-negative (Dn)-caspase-8/Fas-associated death domain (FADD) partially inhibited apoptosis, whereas caspase-9 inhibitor completely blocked the killing indicating involvement of parallel pathways that converged to mitochondria. Further studies demonstrated theaflavin-induced Fas upregulation through the activation of c-jun N-terminal kinase, Fas-FADD interaction in a Fas ligand-independent manner, caspase-8 activation and t-Bid formation. A search for the parallel pathway revealed theaflavin-induced inhibition of survival pathway, mediated by Akt deactivation and Bcl-xL/Bcl-2-associated death promoter dephosphorylation. These well-defined routes of growth control converged to a common process of mitochondrial transmembrane potential loss, cytochrome c release and activation of the executioner caspase-9 and -3. Overexpression of either constitutively active myristylated-Akt (Myr-Akt) or Dn-caspase-8 partially blocked theaflavin-induced mitochondrial permeability transition and apoptosis of p53-mutated cells, whereas cotransfection of Myr-Akt and Dn-caspase-8 completely abolished theaflavin effect thereby negating the possibility of existence of third pathways. These results and other biochemical correlates established the concept that two distinct signaling pathways were regulated by theaflavins to induce mitochondrial death cascade, eventually culminating to apoptosis of p53-mutated human breast cancer cells that are strongly resistant to conventional therapies.

Curcumin Enhances the Efficacy of Chemotherapy by Tailoring p65NFκB-p300 Cross-talk in Favor of p53-p300 in Breast Cancer

“…More than 98% of the non-adherent population was found to be CD16-negative, among which Ͼ92% were characterized by Wright staining (34). The cells were routinely maintained in complete DMEM or RPMI 1640 at 37°C in a humidified incubator containing 5% CO 2 (36). Cells were allowed to reach confluence before use.…”

Restoration of tumor suppressor p53 by differentially regulating pro- and anti-p53 networks in HPV-18-infected cervical cancer cells

“…For whole cell lysates, cells were resuspended and homogenized in buffer (100 mM Tris-Cl, pH 7.4, 300 mM NaCl, 1% NP-40 and 0.25% sodium-deoxycholate). All the buffers were supplemented with protease and phosphatase inhibitor mixtures (Bhattacharyya et al, 2007;Lahiry et al, 2010). For direct western blot analysis, the cell lysates or the particular fractions were separated by SDSpolyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and probed with specific antibodies, for example, anti-p53, -p63, -p73, -E6, -E7, -p21, -Bax, -PUMA, -ATM, -p-p38MAPK, -p38MAPK, -p-Ser-15-p53, -p-Ser-46-p53, -Cox-2, -Ub, -caspase-3, produced from Santa Cruz (Santa Cruz, CA, USA), thereafter the immunoblots were visualized by chemiluminescence.…”

“…Equal protein loading was confirmed with a-actin/ Histone-H1/MnSOD antibody (Santa Cruz). For the determination of direct interaction between two proteins, co-immunoprecipitation technique was employed (Bhattacharyya et al, 2007;Lahiry et al, 2010). Cox-2 and p53 reciprocal co-IP and p53-ubiquitin interaction were performed using cell lysates prepared in Nonidet P-40 (1%) lysis buffer containing protease inhibitors.…”

Proteomic approaches to study epigallocatechin gallate-provoked apoptosis of TSGH-8301 human urinary bladder carcinoma cells: Roles of AKT and heat shock protein 27-modulated intrinsic apoptotic pathways

“…Thus, these results suggest that EGCG-triggered apoptosis is involved in the decrease of the protein levels in HSP27 and p-AKT in TSGH-8301 cells. It has been reported that the pro-apoptotic protein BAD, a member of the Bcl-2 family, is rendered inactive when the phosphorylated serine/threonine protein kinase p-AKT converts p-BAD and BAD (52,53). To investigate the downstream effectors, we traced the phosphorylation status of BAD and also assessed the protein levels of Bcl-2 family, including Bcl-2, Bax and BAD in EGCG-treated TSGH-8301 cells with 0, 50, 75 and 100 µM for 24-h exposure.…”