Downregulation of survivin expression and concomitant induction of apoptosis by celecoxib and its non-cyclooxygenase-2-inhibitory analog, dimethyl-celecoxib (DMC), in tumor cells in vitro and in vivo

BACKGROUND: 2,5-Dimethyl-celecoxib (DMC) is a close structural analog of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib (Celebrex(®)) that lacks COX-2-inhibitory function. However, despite its inability to block COX-2 activity, DMC is able to potently mimic the anti-tumor effects of celecoxib in vitro and in vivo, indicating that both of these drugs are able to involve targets other than COX-2 to exert their recognized cytotoxic effects. However, the molecular components that are involved in mediating these drugs' apoptosis-stimulatory consequences are incompletely understood. RESULTS: We present evidence that celecoxib and DMC are able to down-regulate the expression of survivin, an anti-apoptotic protein that is highly expressed in tumor cells and known to confer resistance of such cells to anti-cancer treatments. Suppression of survivin is specific to these two drugs, as other coxibs (valdecoxib, rofecoxib) or traditional NSAIDs (flurbiprofen, indomethacin, sulindac) do not affect survivin expression at similar concentrations. The extent of survivin down-regulation by celecoxib and DMC in different tumor cell lines is somewhat variable, but closely correlates with the degree of drug-induced growth inhibition and apoptosis. When combined with irinotecan, a widely used anticancer drug, celecoxib and DMC greatly enhance the cytotoxic effects of this drug, in keeping with a model that suppression of survivin may be beneficial to sensitize cancer cells to chemotherapy. Remarkably, these effects are not restricted to in vitro conditions, but also take place in tumors from drug-treated animals, where both drugs similarly repress survivin, induce apoptosis, and inhibit tumor growth in vivo. CONCLUSION: In consideration of survivin's recognized role as a custodian of tumor cell survival, our results suggest that celecoxib and DMC might exert their cytotoxic anti-tumor effects at least in part via the down-regulation of survivin – in a manner that does not require the inhibition of cyclooxygenase-2. Because inhibition of COX-2 appears to be negligible, it might be worthwhile to further evaluate DMC's potential as a non-coxib alternative to celecoxib for anti-cancer purposes

Regulation of PERK Signaling and Leukemic Cell Survival by a Novel Cytosolic Isoform of the UPR Regulator GRP78/BiP

The unfolded protein response (UPR) is an evolutionarily conserved mechanism to allow cells to adapt to stress targeting the endoplasmic reticulum (ER). Induction of ER chaperone GRP78/BiP increases protein folding capacity; as such it represents a major survival arm of UPR. Considering the central importance of the UPR in regulating cell survival and death, evidence is emerging that cells evolve feedback regulatory pathways to modulate the key UPR executors, however, the precise mechanisms remain to be elucidated. Here, we report the fortuitous discovery of GRP78va, a novel isoform of GRP78 generated by alternative splicing (retention of intron 1) and alternative translation initiation. Bioinformatic and biochemical analyses revealed that expression of GRP78va is enhanced by ER stress and is notably elevated in human leukemic cells and leukemia patients. In contrast to the canonical GRP78 which is primarily an ER lumenal protein, GRP78va is devoid of the ER signaling peptide and is cytosolic. Through specific knockdown of endogenous GRP78va by siRNA without affecting canonical GRP78, we showed that GRP78va promotes cell survival under ER stress. We further demonstrated that GRP78va has the ability to regulate PERK signaling and that GRP78va is able to interact with and antagonize PERK inhibitor P58IPK. Our study describes the discovery of GRP78va, a novel cytosolic isoform of GRP78/BiP, and the first characterization of the modulation of UPR signaling via alternative splicing of nuclear pre-mRNA. Our study further reveals a novel survival mechanism in leukemic cells and other cell types where GRP78va is expressed

Glucose-regulated protein 78 antagonizes cisplatin and adriamycin in human melanoma cells

Resistance of melanoma cells to chemotherapeutics remains a major obstacle to successful treatment of melanoma once it has spread beyond locoregional sites. We report in this study that activation of the unfolded protein response (UPR) is involved in resistance of melanoma cells to two chemotherapeutic drugs, cisplatin (CDDP) and adriamycin, and this is associated with glucose-regulated protein 78 (GRP78)-mediated inhibition of activation of caspase-4 and -7. The UPR was constitutively activated in cultured melanoma cell lines and fresh melanoma isolates as evidenced by elevated expression levels of the GRP78 protein and the active form of x-box-binding protein 1 messenger RNA. Treatment with CDDP or adriamycin further increased the levels, indicative of induction of endoplasmic reticulum stress and activation of the UPR by the drugs. Inhibition of GRP78 by small-interference RNA (siRNA)-sensitized melanoma cells to CDDP- and adriamycin-induced apoptosis. This was associated with enhanced caspase-4 and -7 activation as siRNA knockdown of the caspases blocked induction of apoptosis. In contrast, overexpression of GRP78 attenuated activation of caspase-4 and -7 and induction of apoptosis by the drugs. CDDP- and adriamycin-induced activation of caspase-4 and -7 appeared to be mediated by calpain activity in that it was blocked by the calpain inhibitors calpeptin and PD150606 even when GRP78 was inhibited by siRNA. These results provide new insights into resistance mechanisms of melanoma cells to CDDP and adriamycin and identify GRP78 as a potential target for enhancing chemosensitivity in melanoma