The proteasome inhibitor PS-341 (bortezomib or Velcade), an approved drug for treatment of patients with multiple myeloma, is currently being tested in clinical trials against various malignancies, including lung cancer. Preclinical studies have shown that PS-341 induces apoptosis and enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human cancer cells with undefined mechanisms. In the present study, we show that PS-341 induced caspase-8-dependent apoptosis, cooperated with TRAIL to induce apoptosis, and up-regulated death receptor 5 (DR5) expression in human non-small cell lung cancer (NSCLC) cells. DR5 induction correlated with the ability of PS-341 to induce apoptosis. Blockage of PS-341-induced DR5 up-regulation using DR5 small interfering RNA (siRNA) rendered cells less sensitive to apoptosis induced by either PS-341 or its combination with TRAIL, indicating that DR5 upregulation mediates PS-341-induced apoptosis and enhancement of TRAIL-induced apoptosis in human NSCLC cells. We exclude the involvement of c-FLIP and survivin in mediating these events because c-FLIP (i.e., FLIP S ) and survivin protein levels were actually elevated on exposure to PS-341. Reduction of c-FLIP with c-FLIP siRNA sensitized cells to PS-341-induced apoptosis, suggesting that c-FLIP elevation protects cells from PS-341-induced apoptosis. Thus, the present study highlights the important role of DR5 up-regulation in PS-341-induced apoptosis and enhancement of TRAIL-induced apoptosis in human NSCLC cells. [Cancer Res 2007;67(10):4981-8]

The proteasome, a key component of the ubiquitin-proteasome pathway, has emerged as an important cancer therapeutic target. PS-341 (also called Bortezomib or Velcade) is the first proteasome inhibitor approved for newly diagnosed and relapsed multiple myeloma and is currently being tested in many clinical trials against other types of cancers. One proposed mechanism by which PS-341 exerts its anticancer effect is inactivation of nuclear factor-B (NF-B) through prevention of IB␣ degradation. In this study, we show that PS-341 at concentrations that effectively inhibited the growth of human cancer cells, instead of increasing IB␣ stability, paradoxically induced IB␣ degradation. As a result, PS-341 facilitated p65 nuclear translocation and increased NF-B activity. Moreover, IB␣ degradation by PS-341 occurred early before induction of apoptosis and could not be inhibited by a pan-caspase inhibitor or caspase-8 silencing; however, it could be prevented with calpain inhibitors, calcium-chelating agents, calpain knockdown, or calpastatin overexpression. In agreement, PS-341 increased calpain activity. These data together indicate that PS-341 induces a calpain-mediated IB␣ degradation independent of caspases. In the presence of a calpain inhibitor, the apoptosis-inducing activity of PS-341 was dramatically enhanced. Collectively, these unexpected findings suggest not only a novel paradigm regarding the relationship between proteasome inhibition and NF-B activity but also a strategy to enhance the anticancer efficacy of PS-341.

HERG expression levels and chemosensitivity were positively correlated for vincristine, paclitaxel, and hydroxy-camptothecin. Erythromycin was active as a modulator. These results suggest that HERG may serve as a molecular marker and modulating target for individualized cancer therapy.

2,5-Dimethyl-celecoxib (DMC) is a derivative of celecoxib, a cyclooxygenase-2 (COX-2) inhibitor with anticancer activity in both preclinical studies and clinical practice, and lacks COX-2-inhibitory activity. Several preclinical studies have demonstrated that DMC has better apoptosis-inducing activity than celecoxib, albeit with undefined mechanisms, and exhibits anticancer activity in animal models. In this study, we primarily investigated DMC's cooperative effect with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the induction of apoptosis and the underlying mechanisms in human nonsmall-cell lung cancer (NSCLC) cells. We found that DMC was more potent than celecoxib in decreasing the survival and inducing apoptosis of NSCLC cells. When combined with TRAIL, DMC exerted enhanced or synergistic effects on the induction of apoptosis, indicating that DMC cooperates with TRAIL to augment the induction of apoptosis. To determine the underlying mechanism of the synergy between DMC and TRAIL, we have demonstrated that DMC induces a CCAAT/ enhancer binding protein homologous protein-dependent expression of DR5, a major TRAIL receptor, and reduces the levels of cellular FLICE-inhibitory protein (c-FLIP) (both the long and short forms), key inhibitors of death receptor-mediated apoptosis, by facilitating c-FLIP degradation through a ubiquitin/proteasome-dependent mechanism. It is noteworthy that enforced expression of c-FLIP or silencing of DR5 expression using DR5 small interfering RNA abrogated the enhanced effects on induction of apoptosis by the combination of DMC and TRAIL, indicating that both DR5 up-regulation and c-FLIP reduction contribute to cooperative induction of apoptosis by the combination of DMC and TRAIL. Together, we conclude that DMC sensitizes human NSCLC cells to TRAIL-induced apoptosis via induction of DR5 and down-regulation of c-FLIP.

Celecoxib is a COX2 inhibitor that reduces the risk of colon cancer. However, the basis for its cancer chemopreventive activity is not fully understood. In this study, we defined a mechanism of celecoxib action based on degradation of c-FLIP, a major regulator of the death receptor pathway of apoptosis. c-FLIP protein levels are regulated by ubiquitination and proteasome-mediated degradation. We found that celecoxib controlled c-FLIP ubiquitination through Akt-independent inhibition of GSK3 kinase, itself a candidate therapeutic target of interest in colon cancer. Celecoxib increased the levels of phosphorylated GSK3 (p-GSK3), including the α and β forms, even in cell lines where p-Akt levels were not increased. PI3K inhibitors abrogated Akt phosphorylation as expected but had no effect on celecoxib-induced GSK3 phosphorylation. In contrast, PKC inhibitors abolished celecoxib-induced GSK3 phosphorylation, implying that celecoxib influenced GSK3 phosphorylation through a mechanism relied upon PKC but not Akt. GSK3 blockade either by siRNA or kinase inhibitors was sufficient to attenuate c-FLIP levels. Combining celecoxib with GSK3 inhibition enhanced attenuation of c-FLIP and increased apoptosis. Proteasome inhibitor MG132 reversed the effects of GSK3 inhibition and increased c-FLIP ubiquitination, confirming that c-FLIP attenuation was mediated by proteasomal turnover as expected. Our findings reveal a novel mechanism through which the regulatory effects of c-FLIP on death receptor signaling are controlled by GSK3, which celecoxib acts at an upstream level to control independently of Akt.