ALBERT

Description: This report compares in detail, the role of curcumin treatment in a panel of Burkitt’s lymphoma (BL) cell lines that are either expressing full length Bax with a group of cell lines that are either completely deficient or have decreased expression of Bax. Multiple apoptotic stimuli induce conformational changes in Bax, a pro-apoptotic protein from the Bcl-2 family and its deficiency is a frequent cause of chemo-resistance in a variety of malignancies including Burkitt’s lymphoma. We extent our previous studies on Burkitt’s lymphoma to determine the role of curcumin treatment on BLs. Curcumin (diferuloymethane) is a naturally occurring yellow pigment isolated from the rhizomes of the plant curcuma longa. The medicinal value of curcumin has been well recognized with its anti-oxidant, anti-inflammatory and anti-tumor activities. Curcumin is also known to induce apoptosis in a variety of cancer cells including multiple myeloma and primary effusion lymphomas. To understand the role of Bax in curcumin-induced apoptosis, we used two groups of Burkitt’s lymphoma cell lines, one that expressed the Bax protein (AS283A, KK124 and Pa682PB) and the other group either did not or had decreased expression of Bax (BML895 CA46, and LW878). Cell viability decreased in a dose-dependent manner in AS283A, PA682PB and KK124 with curcumin treatment ranging between 0–40mM whereas only minimal changes in viability was observed in BML895, CA46 and LW878 after treatment. Curcumin induced a dose-dependent apoptosis in the Bax expressing group of cell lines while the cell lines that were either completely deficient or had decreased expression of Bax did not respond to curcumin treatment and remained refractory. In AS283A, KK124, and PA682PB, curcumin induced apoptosis through truncation of BID, loss of mitochondrial potential as determined by JC1 staining with subsequent activation of caspase3 followed by cleavage of PARP. However, in the curcumin resistant cell lines, there was no change in the mitochondrial potential after curcumin treatment and therefore apoptosis did not occur. In addition, zVAD-fmk, a universal inhibitor of caspases prevented caspase3 cleavage as well as cell death in the sensitive cell lines after curcumin treatment suggesting that curcumin-induced apoptosis is caspase dependent. Our findings suggest that Bax integrity is necessary for curcumin to induce apoptosis in Burkitt’s lymphoma cells. These results provide the molecular basis and preliminary data for new treatment strategies that may incorporate curcumin in regimens for Burkitt’s lymphoma treatment.

Description: Phosphatidylinositol 3′-kinase (PI3K) is a key player in cell-growth signaling in a number of lymphoid malignancies, but its role in diffuse large B-cell lymphoma (DLBCL) has not been fully elucidated. Therefore, we investigated the role of the PI3K/AKT pathway in a panel of 5 DLBCL cell lines and 100 clinical samples. Inhibition of PI3K by a specific inhibitor, LY294002, induced apoptosis in SUDHL4, SUDHL5, and SUDHL10 (LY-sensitive) cells, whereas SUDHL8 and OCI-LY19 (LY-resistant) cells were refractory to LY294002-induced apoptosis. AKT was phosphorylated in 5 of 5 DLBCL cell lines and inhibition of PI3K caused dephosphorylation/inactivation of constitutively active AKT, FOXO transcription factor, and GSK3 in LY-sensitive cell lines. In addition, there was a decrease in the expression level of inhibitory apoptotic protein, XIAP, in the DLBCL cell lines sensitive to LY294002 after treatment. However, no effect was observed in XIAP protein levels in the resistant DLBCL cell lines following LY294002 treatment. Finally, using immunohistochemistry, p-AKT was detected in 52% of DLBCL tumors tested. Furthermore, in univariate analysis, high p-AKT expression was associated with short survival. In multivariate analysis, this correlation was no longer significant. Altogether, these results suggest that the PI3K/AKT pathway may be a potential target for therapeutic intervention in DLBCL.

Description: Phosphatidylinositol 3′-kinase (PI3′-kinase) is a key player in cell growth signaling and has been shown to be activated by the K1 protein of Kaposi sarcoma associated herpes virus (KSHV/HHV8). However, the exact role of PI3′-kinase activation in KSHV-associated PEL has not been elucidated. Therefore, we have studied the PI3′-kinase pathway and apoptosis in five PEL cell lines (BC1, BC3, BCBL1, BCP1 and HBL6). Our data show that inhibition of PI3′-kinase by a specific inhibitor, LY294002, induced apoptosis as detected by Annexin V/Propidium Iodide dual staining in the majority of PEL cell lines, including BC1 (43.5+9%), BC3 (62.7+2.4%), BCBL1 (75+5.2%) and HBL6 (36+4.7%). In contrast, BCP1 was resistant to LY294002-induced apoptosis (2%+0.5). We then dissected the PI3′-kinase pathway by analyses of downstream targets of phosphorylation by Western blot. We found that AKT/PKB was constitutively phosphorylated, and thus activated, in all PEL cell lines including BCP1. Interestingly, 24 hours after LY294002 treatment, AKT was completely de-phosphorylated in all cell lines except BCP1, in which a residual phosphorylation level was detected. The downstream elements of AKT, ForkHead (FKHR) and GSK3 were also constitutively phosphorylated in all PEL cell lines. Similarly, treatment with LY294002 prevented this phenomenon in all the cell lines regardless of their final apoptotic endpoint. To confirm specificity of LY294002 treatment on the PI3′-kinase pathway, we tested an unrelated signaling cascade (p38/MAPK) and no changes were observed. Since FKHR was previously shown to upregulate Fas-L in a variety of cells, we analyzed the Fas/Fas-L system in sensitive PEL cell lines following treatment with LY294002. We have previously shown surface expression of CD95 in these cell lines. We now observed that neutralization of Fas/CD95 by the ZB4 antibody did not influence LY294002 apoptosis. Furthermore, co-treatment with LY294002 and CH11 had an additive apoptotic effect. Inhibition of PI3′-kinase activity further downstream induced cleavage of Bid in all PEL cells. However, cytochrome C was only released from mitochondria in LY294002- sensitive BC1 cells and not in the resistant BCP1 cells. The release of cytochrome C in the sensitive BC1 cell line led to activation of Caspase-9 and 3 and cleavage of PARP, none of which occured in the LY294002 resistant BCP1 cell line. Similarly, the expression of the inhibitor of apoptosis, XIAP, which is also a downstream target of AKT, was compromised in the sensitive cell lines following LY294002 treatment. Our data demonstrate that the PI3′-kinase pathway plays a major role in growth and survival of PEL cells since blocking PI3′-kinase activity induces apoptosis. Although this LY294002 induced apoptosis does not appear to involve Fas/Fas-L, it is caspase dependent and compromises XIAP expression. The residual AKT activity in the LY294002 resistant BCP1 cell line may be protecting this cell line from apoptosis. Altogether, these results suggest that blocking the PI3′-kinase pathway may be a potential target for therapeutic intervention in most primary effusion lymphomas.

Description: Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood resulting from the clonal proliferation of lymphoid precursors with arrested maturation. Chemotherapy can induce complete remission in more than 95% of cases of childhood ALL and achieve long-term survival in 70–80% of cases. However, ALL with the t(9:22) BCR-ABL translocation or Philadelphia chromosome (Ph1) are still highly resistant to chemotherapy from the onset. Thus, new therapeutic approaches are required to improve their prognosis. Characterization of the growth requirement of ALL cells suggest that these cancers are dependent on various cytokines via paracrine and/or autocrine mechanism in which the JAK family of proteins are closely implicated. Accordingly, tyrosine kinase inhibitors against JAKs are expected to become a new class of anti-tumor agents against these cancers. Curcumin has been shown to inhibit JAK-STAT pathway in a variety of hematological malignancies including multiple myeloma and primary effusion lymphomas. We therefore sought to determine whether curcumin suppresses the growth of acute lymphoblastic leukemia. We tested a panel of preB-ALL cell lines with various translocations after treatment with different doses of curcumin. The cell lines included REH (t12:21), RS4:11 (t4:11), 697 (t1:19) and SupB15(t9:22). Cell viability decreased in a concentration-dependent manner in 697, REH and RS4:11 with curcumin (0–40mM) whereas only minimal changes in viability was detected in SupB15. Curcumin induced apoptosis in all preB-ALL cell lines except SupB15 that was found to be refractory to curcumin treatment. Curcumin induced apoptosis via truncation of BID, loss of mitochondrial potential as determined by JC1 staining with subsequent release of cytochrome c from the mitochondria, and activation of caspase 3 and PARP. Curcumin treatment also caused the down-regulation of the IAPs, cIAP1 and XIAP. All these events occured in the sensitive cell lines 697, REH and RS4:11, however, in SupB15, curcumin failed to inhibit the expression of cIAP1 and XIAP and remained refractory to treatment. These results suggest that the IAPs may play an important role in curcumin induced apoptosis in preB-ALL cells. Altogether, our findings suggests a novel function for curcumin, acting as a growth suppressor of most preB-ALL cells and inducing apoptosis via down-regulation of IAPs. Therefore, curcumin may have a future therapeutic role in preB-ALL and possibly other malignancies.