Inhibition of RelB by 1,25-dihydroxyvitamin D3 promotes sensitivity of breast cancer cells to radiation.

Aberrant constitutive expression of the NF-kappaB c-Rel and RelA subunits in breast cancer cells was shown to promote their survival. Recently, we demonstrated that aggressive breast cancers constitutively express high levels of the RelB subunit, which promotes their more invasive phenotype via induction of the BCL2 gene. As these cancers are frequently resistant to therapy, here we tested the hypothesis that RelB promotes their survival. High RelB expressing Hs578T and MDA-MB-231 breast cancer cells were more resistant to gamma-radiation than MCF7 and ZR-75 cells, which express lower RelB levels. Knockdown of RelB in Hs578T led to decreased survival in response to gamma-irradiation, while conversely ectopic expression of RelB in MCF7 cells protected these cells from radiation. Similar data were obtained upon treatment of Hs578T or MCF7 cells with the chemotherapeutic agent doxorubicin. High serum levels of 25-hydroxyvitamin D are associated with decreased breast cancer risk and mortality, although, the mechanisms of its protective actions have not been fully elucidated. Treatment of Hs578T and Her-2/neu-driven NF639 cells with 1,25-dihydroxyvitamin D3 decreased RelB/RELB gene expression and levels of pro-survival targets Survivin, MnSOD and Bcl-2, while increasing their sensitivity to gamma-irradiation. Thus, RelB, which promotes survival and a more highly invasive phenotype of breast cancer cells, is a target of 1,25-dihydroxyvitamin D3, providing one mechanism for the observed protective role of 25-hydroxyvitamin D in patients with breast cancer.

Differential sensitization of different prostate cancer cells to apoptosis.

Although protein kinase C (PKC) plays an important role in sensitizing prostate cancer cells to apoptosis, and suppression of PKC is able to trigger an apoptotic crisis in cells harboring oncogenic ras, little is known about whether dyregulation of Ras effectors in prostate cancer cells, together with loss of PKC, is synthetically lethal. The current study aims at investigating whether prostate cancer cells with aberrant Ras effector signaling are sensitive to treatment with HMG (a PKC inhibitor) for the induction of apoptosis. We show that prostate cancer DU145 cells expressing a high level of JNK1 become susceptible to apoptosis after treatment with HMG, in which caspase 8 is activated and cytochrome c is released to the cytosol. In contrast, the addition of HMG sensitizes LNCaP or PC3 prostate cancer cells harboring an active Akt to apoptosis, in which ROS is upregulated to induce the UPR and GADD153 expression. The concurrent activation of JNK1 and Akt has an additive effect on apoptosis following PKC suppression. Thus, the data identify Akt and JNK1 as potential targets in prostate cancer cells for PKC inhibition-induced apoptosis.

Pro-IL-16 recruits histone deacetylase 3 to the Skp2 core promoter through interaction with transcription factor GABP.

Pro-IL-16 is a PDZ domain-containing protein expressed in T cells. Our previous work showed that upon activation of normal T cells, pro-IL-16 mRNA and protein are diminished in close correlation to the down-regulation of p27KIP1 protein. In addition, we showed that pro-IL-16 regulates the transcription of Skp2, the mechanism of which, however, remains elusive. In this study, we identified GA binding protein beta1 subunit (GABPbeta1) and histone deacetylase 3 (HDAC3) as binding partners of pro-IL-16. Interestingly, both GABPbeta1 and HDAC3 have canonical PDZ-binding motifs and specifically bind to the first and second PDZ domain of pro-IL-16, respectively. Heat shock cognate protein 70 (HSC70) also copurified with the GST-PDZ1-containing fragment but lacks a C-terminal PDZ binding motif, suggesting that it binds through a different mechanism. We further showed that pro-IL-16 is located in a GABP transcriptional complex bound to the Skp2 promoter. In addition, we demonstrated that HDAC activity is critical for pro-IL-16-induced cell cycle arrest. Taken altogether, these data suggest that pro-IL-16 forms a complex with GABPbeta1 and HDAC3 in suppressing the transcription of Skp2. Thus, this study has revealed a novel mechanism with which pro-IL-16 regulates T cell growth through the Skp2-p27KIP1 pathway.

Modulation of lung cancer growth arrest and apoptosis by Phellinus Linteus.

The Phellinus Linteus (PL) mushroom has been shown to possess anti-tumor properties. Through influencing lymphocytes, PL indirectly augments the host's immune system against cancer cells. PL has also been demonstrated to reduce tumor proliferation. However, the mechanisms of PL against malignant growth have not yet been fully explored. In this study, we report that PL mediates the following two activities in mouse and human lung cancer cells: cell-cycle arrest at a low concentration of PL and apoptosis in response to a high dose of PL. After exposure to a low dose of PL, G(1) growth arrest occurred in the lung cancer cells. The negative growth control mediated by PL is evidenced by the decrease of the activities of cyclin-dependent kinases CDK2, 4, and 6. In contrast, at high doses, PL-induced lung cancer cells to undergo apoptosis in a dose-dependent fashion. This was evidenced by DNA fragmentation, caspase activation, and loss of clonogenecity in the lung cancer cells, all of which were lacking in the lung cancer cells treated with low concentrations of PL as well as the normal mouse lung epithelial cells exposed to either low or high concentrations of PL. The addition of the caspase inhibitor Z-VADfmk completely suppressed PL-induced apoptosis. Furthermore, the low dose of PL was able to synergize with doxorubicin to induce apoptosis in the lung cancer cells. Thus, our findings suggest that PL regulates two responses in the lung cancer cells: cell-cycle arrest and apoptosis.

Modulation of intracellular signaling pathways to induce apoptosis in prostate cancer cells.

An understanding of the molecular pathways defining the susceptibility of prostate cancer, especially refractory prostate cancer, to apoptosis is the key for developing a cure for this disease. We previously demonstrated that up-regulating Ras signaling, together with suppression of protein kinase C (PKC), induces apoptosis. Dysregulation of various intracellular signaling pathways, including those governed by Ras, is the important element in the development of prostate cancer. In this study, we tested whether it is possible to modulate the activities of these pathways and induce an apoptotic crash among them in prostate cancer cells. Our data showed that DU145 cells express a high amount of JNK1 that is phosphorylated after endogenous PKC is suppressed, which initiates caspase 8 cleavage and cytochrome c release, leading to apoptosis. PC3 and LNCaP cells contain an activated Akt. The inhibition of PKC further augments Akt activity, which in turn induces ROS production and the accumulation of unfolded proteins in the endoplasmic reticulum, resulting in cell death. However, the concurrent activation of JNK1 and Akt, under the condition of PKC abrogation, dramatically augment the magnitude of apoptosis in the cells. Thus, our study suggests that Akt, JNK1, and PKC act in concert to signal the intracellular apoptotic machinery for a full execution of apoptosis in prostate cancer cells.

Targeting retinoblastoma protein for degradation by proteasomes.

Inactivation of retinoblastoma protein (Rb) plays a key role in human tumorigenesis. Although the regulation of Rb by phosphorylation has been extensively studied, the regulation of proteasome-mediated Rb protein degradation is largely unknown. Viral oncoprotein E7, Epstein-Barr virus nuclear antigen 3C (EBNA3C), human cytomegalovirus pp71 and cellular oncoprotein gankyrin all contain the L-x-C-x-E Rb-binding motif and target Rb protein for degradation in either ubiquitin-dependent or ubiquitin-independent proteasome pathways. The molecular mechanisms, however, remain elusive. The MDM2 oncoprotein is overexpressed in a variety of human cancers. MDM2 functions as an ubiquitin E3 ligase and induces p53 protein degradation through ubiquitination-proteasome pathway. Both MDM2 central acidic domain and the C-terminal RING domain are critical for p53 degradation. MDM2 also interacts with Rb through its central acidic domain and inhibits Rb function in part by blocking Rb-E2F-DNA complex formation. Recently, we showed that MDM2 binds to C8 subunit of 20S proteasome and promotes Rb-C8 interaction, leading to a proteasome-dependent ubiquitin-independent degradation of Rb. Knockdown of MDM2 results in accumulation of hypophosphorylated Rb and inhibition of DNA synthesis. Taken together, we suggest that targeting Rb protein for degradation by proteasomes may represent a common neoplastic strategy during human cancer development.