Sanguinarine-Dependent Induction of Apoptosis in Primary Effusion Lymphoma Cells

Abstract

Primary effusion lymphoma (PEL) is an incurable, aggressive B-cell malignancy that develops rapid resistance to conventional chemotherapy. In efforts to identify novel approaches to block proliferation of PEL cells, we found that sanguinarine, a natural compound isolated from the root plant Sanguinaria canadendid, inhibits cell proliferation and induces apoptosis in a dose-dependent manner in several PEL cell lines. Our data show that sanguinarine treatment of PEL cells results in upregulation of death receptor 5 (DR5) expression via generation of reactive oxygen species (ROS) and causes activation of caspase-8 and truncation of Bid (tBid). Subsequently, tBid translocates to the mitochondria causing conformational changes in Bax, leading to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. Sanguinarine-induced release of cytochrome c results in activation of caspase-9 and caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, leading to induction of caspasedependent apoptosis. In addition, we show that pretreatment of PEL cells with carbobenzoxy-Val-Ala-Asp-fluoromethylketone, a universal inhibitor of caspases, abrogates caspase and PARP activation and prevents cell death induced by sanguinarine. Moreover, treatment of PEL cells with sanguinarine down-regulates expression of inhibitor of apoptosis proteins (IAP). Finally, N-acetylcysteine, an inhibitor of ROS, inhibits sanguinarine-induced generation of ROS, up-regulation of DR5, Bax conformational changes, activation of caspase-3, and down-regulation of IAPs. Taken together, our findings suggest that sanguinarine is a potent inducer of apoptosis of PEL cells via up-regulation of DR5 and raise the possibility that this agent may be of value in the development of novel therapeutic approaches for the treatment of PEL. [Cancer Res 2007;67(8):3888-97]

Curcumin suppresses constitutive activation of nuclear factor-κB and requires functional Bax to induce apoptosis in Burkitt's lymphoma cell lines

“…3C, NAC pretreatment blocked conformational changes of Bax protein, whereas zVAD-fmk pretreatment was unable to inhibit such changes, indicating a role for ROS release but not caspase activation in these changes. The oligomerization of Bax has been shown previously to only occur in apoptotic cells, possibly playing a role in mediating cytochrome c release (32,38). To test this possibility, we determined whether curcumin treatment of Burkitt's lymphoma cells could trigger Bax oligomerization.…”

“…Detection of Bax Conformational Changes Cells were treated with indicated doses of curcumin and lysed with CHAPS lysis buffer [10 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, and 1% CHAPS] and immunoprecipitated with anti-Bax 6A7 monoclonal antibody and Bax conformation was detected as described earlier (32).…”

“…Cross-linked and oligomerized proteins were detected as described earlier. Cross-linking experiments were done as described earlier (32).…”

“…Amplification of glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. Quantification analysis of DR5 using real-time reverse-transcription PCR was done by Pfaffle methodology as described earlier (32).…”

“…To test this possibility, we determined whether curcumin treatment of Burkitt's lymphoma cells could trigger Bax oligomerization. PA682PB cells were treated with curcumin; the cross-linked proteins were separated on SDS-PAGE and immunoblotted for the analysis of Bax oligomerization as described (32). As shown in Fig.…”