Cancer patients often exhibit loss of proper cell-mediated immunity and reduced effector T-cell population in the circulation. Thymus is a major site of T-cell maturation, and tumors induce thymic atrophy to evade cellular immune response. Here, we report severe thymic hypocellularity along with decreased thymic integrity in tumor bearer. In an effort to delineate the mechanisms behind such thymic atrophy, we observed that tumor-induced oxidative stress played a critical role, as it perturbed nuclear factor-KB (NF-KB) activity. Tumor-induced oxidative stress increased cytosolic IKBA retention and inhibited NF-KB nuclear translocation in thymic T cells. These NF-KB-perturbed cells became vulnerable to tumor-secreted tumor necrosis factor (TNF)-A (TNF-A)-mediated apoptosis through the activation of TNF receptorassociated protein death domain-associated Fas-associated protein death domain and caspase-8. Interestingly, TNF-Adepleted tumor supernatants, either by antibody neutralization or by TNF-A-small interfering RNA transfection of tumor cells, were unable to kill T cell effectively. When T cells were overexpressed with NF-KB, the cells became resistant to tumor-induced apoptosis. In contrast, when degradationdefective IKBA (IKBA super-repressor) was introduced into T cells, the cells became more vulnerable, indicating that inhibition of NF-KB is the reason behind such tumor/TNF-Amediated apoptosis. Curcumin could prevent tumor-induced thymic atrophy by restoring the activity of NF-KB. Further investigations suggest that neutralization of tumor-induced oxidative stress and restoration of NF-KB activity along with the reeducation of the TNF-A signaling pathway can be the mechanism behind curcumin-mediated thymic protection. Thus, our results suggest that unlike many other anticancer agents, curcumin is not only devoid of immunosuppressive effects but also acts as immunorestorer in tumor-bearing host.

Theaflavins, the bioactive flavonoids of black tea, have been demonstrated to inhibit proliferation and induce apoptosis in a variety of cancer cells. However, the contribution of p53 in mammary epithelial carcinoma cell apoptosis by theaflavins remains unclear. It has been reported that p53 triggers apoptosis by inducing mitochondrial outer membrane permeabilization through transcription-dependent and -independent mechanisms. Using wild-type and mutant p53-expressing as well as p53-null cells we found a strong correlation between p53 status and theaflavin-induced breast cancer cell apoptosis. Apoptogenic effect was more pronounced in functional p53-expressing cells in which theaflavins raised p53 protein levels that harmonized with Bax up-regulation and migration to mitochondria. However, in the same cells, when p53-mediated transactivation was inhibited by pifithrin-alpha, theaflavins not only failed to increase transcription but also to induce apoptosis although p53 up-regulation was not altered. In contrast, Bax over-expression restored back theaflavin-induced apoptosis in pifithrin-alpha-inhibited/dominant-negative p53-expressing cells. Inhibition of Bax by RNA-interference also reduced theaflavin-induced apoptosis. These results not only indicated the requirement of p53-mediated transcriptional activation of Bax but also its role as down-stream effecter in theaflavin-induced apoptosis. Bax up-regulation resulted in mitochondrial transmembrane potential loss and cytochrome c release followed by activation of caspase cascade. In contrast, mitochondrial translocation of p53 and its interaction with Bcl-2 family proteins or activation of caspase-8 could not be traced thereby excluding the involvement of p53-mediated transcription-independent pathways. Together these findings suggest that in breast cancer cells, p53 promotes theaflavin-induced apoptosis in a transcription-dependent manner through mitochondrial death cascade.

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.

Edited by Quan Chen Keywords:Breast cancer Migration NF-jB p53 Reactive oxygen species Theaflavins a b s t r a c tThe present study demonstrates that theaflavins exploit p53 to impede metastasis in human breast cancer cells. Our data suggest that p53-dependent reactive oxygen species (ROS) induce p53-phosphorylation via p38MAPK in a feedback loop to inhibit IjBa-phosphorylation and NF-jB/p65 nuclear translocation, thereby down-regulating the metastatic proteins metalloproteinase (MMP)-2 and MMP-9. When wild-type p53-expressing MCF-7 cells are transfected with p53 short-interfering RNA, or treated with a pharmacological inhibitor of ROS, theaflavins fail to inhibit NF-jB-mediated cell migration. On the other hand, NF-jB over-expression bestows MCF-7 cells with resistance to the anti-migratory effect of theaflavins. These results indicate that inhibition of NF-jB via p53-ROS crosstalk is a pre-requisite for theaflavins to accomplish the anti-migratory effect in breast cancer cells. Structured summary:MINT-7295816: p53 (uniprotkb:P04637) physically interacts (MI:0915) with IKK beta (uniprotkb:O14920) by anti bait coimmunoprecipitation (MI:0006)

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