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: Kaposi ‘s sarcoma-associated herpesvirus (KSHV or HHV8) has also been implicated in primary effusion lymphoma (PEL). KSHV/HHV8 has been shown to activate a number of growth and survival signaling pathways, including PI3-kinase and JAK/STAT, and protect cells from programmed cell death. Indeed Stat-3 is constitutively phosphorylated/activated in PEL cells and plays a role in their survival and proliferation. Curcumin (diferuloylmethane), a natural compound isolated from the plant Curcuma Ionga had very little or no toxicity in human cells and has been shown to have anti-tumor activity in vitro. We describe here that curcumin at 20 μM concentration inhibits cell proliferation in several PEL cell lines (BC1 64 + 1%, BC3 67 + 2%, BCBL1 62 +3% inhibition of growth) and induces apoptosis (BC1 51+ 5%, BC3 47 + 6%, BCBL1 48 + 4 % apoptosis cells). To characterize better the mechanism of action of curcumin, we studied the JAK/STAT pathway and the apoptotic cascade. Curcumin suppressed the constitutively active Stat3 transcription factor in a dose dependent manner. Curcumin also induced loss of mitochondrial membrane potential, as determined by JC1 staining with subsequent activation of capase-3 followed by polyadenosin-5′-diphosphate-ribose polymerase (PARP) cleavage. In addition, zVAD-fmk, a universal inhibitor of caspases prevented caspase-3 activation as well as PARP cleavage induced by curcumin treatment. IL-10 is a major autocrine growth factor for PEL cell survival. We quantified by ELISA the secretion of IL-10 in PEL cells and found that curcumin abrogated IL-10 secretion. Taken together, our findings suggest that curcumin suppresses constitutively active Stat-3 in PEL cells, leading to inhibition of proliferation and induction of caspase-dependent apoptosis. These results provide the molecular basis and preliminary data for new treatment strategies that may incorporate curcumin in regimens for PEL.

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: Primary effusion lymphoma (PEL) is an aggressive and fatal type of cancer. PEL cells produce a variety of autocrine cytokines and growth factors, which provides cyto-protection against conventional chemotherapeutic agents. In efforts to identify novel approaches to block the proliferation of PEL cells, we found that Sanguinarine, a natural compound isolated from the root plant Sanguinaria canadendid, that is being used as an anti-microbial agent, inhibited cell proliferation and induced apoptosis in a dose dependent manner in several PEL cell lines through a bax-dependent signaling pathway. Five PEL cell lines used in this study were treated with various doses of Sanguinarine ranging between 0.5–4μM inhibited cell proliferation in all the cell lines in a dose dependent manner (BC1 40–97%, BC3 46–93%, BCBL1 11–94%, BCP1 20–97% and HBL6 7–95%). Treatment with varying doses of Sanguinarine also induced apoptosis in all cell lines as determined by cell cycle analysis, annexinV/PI dual staining, TUNEL assay and DNA laddering. Sanguinarine treatment resulted in up-regulation of death receptor 5 (DR5) expression, activation of caspase-8 and Bid leading to Bax conformational changes and translocation to the mitochondrial causing loss of mitochondrial membrane potential as measured by JC1 staining and release of cytochrome c to the cytosole. Sanguinarine induced release of cytochrome c resulted in activation of caspase-3, followed by polyadenosin-5′-diphosphate-ribose polymerase (PARP) cleavage leading to inhibition of proliferation and induction of caspase-dependent apoptosis. Furthermore, pre-treatment of PEL cells with z-VAD-fmk, a universal inhibitor of caspases, abrogated caspase-3 and PARP activation and prevented cell death induced by Sanguinarine. Inhibitor of apoptosis proteins (IAPs), play an important role in protecting cells against apoptosis through their direct action on caspases-9 and -3. Treatment of PEL cells with Sanguinarine down-regulated the expression of IAPs; XIAP, cIAP1 and cIAP2. Taken altogether, our findings suggest that Sanguinarine induces apoptosis via up-regulation of DR5, activation of Bax in a caspase-dependent pathway and down-regulation of IAPs. These results provide the molecular basis and preliminary data for new treatment strategies that may incorporate Sanguinarine in regimens for primary effusion lymphoma treatment.

Description: Proteosome inhibition is a novel approach for treating malignancy and has been approved for clinical use. The proteosome is the primary proteolytic mechanism in eukaryotic cells and inhibition of its catalytic activity initiates a cascade of events affecting cell cycle and apoptotic activities. These activities ultimately lead to cell cycle arrest and apoptosis in malignant cells however, the normal counterpart of these cells are spared. In this study, we used a panel of primary effusion lymphoma cell lines (BC1, BC3, BCBL1 and HBL6) to study the effects of proteosome inhibitor, MG132 on cell proliferation and apoptosis. Our data showed that proteosome inhibitor MG132 decreased cell viability as well as induced apoptosis in a dose dependent manner ranging from 0.5–10μM. Furthermore, treatment with 2.5μM MG132 for 24hours induced 41% apoptosis in BC1, 51% in BC3, 41% in BCBL1 and 48% in HBL6 cell lines as detected by annexinV/PI dual staining. S-phase kinase-associated protein 2 (skp-2) is a proto-oncogene and over expressed in various types of tumors. We sought to determine the role of Skp-2 following proteosome inhibition in PELs. MG132 treatment of PEL cell lines resulted in down-regulation of SKP-2 protein in a dose dependent manner whereas the expression of p-27 was up-regulated demonstrating an inverse relationship between these two proteins. Furthermore, MG132 treatment of PELs led to conformational changes in Bax protein and translocation to the mitochondria leading to the loss of mitochondrial membrane potential with subsequent release of cytochrome c from mitochondria into cytosol. Cytochrome c release caused activation of caspase-3 followed by polyadenosin-5′-diphosphate-ribose polymerase (PARP) cleavage. In addition, proteosome inhibitor treatment also caused down-regulation of inhibitor of apoptosis protein, XIAP. Taken together, our findings show that proteosome inhibition causes down-regulation of skp-2, up-regulation of p-27, inhibition of proliferation as well as caspase-dependent apoptosis in primary effusion lymphoma cells suggesting a role of proteosome inhibitors in the treatment of these aggressive cancers.

Description: Phosphatidylinositol 3-kinase (PI3-kinase) is a key player in cell growth signaling in a number of lymphoid malignancies including myeloma and primary effusion lymphoma. However, its role in diffuse large B-cell lymphoma (DLBCL) has not been elucidated. Therefore, we have studied the PI3-kinase pathway and apoptosis in a panel of DLBCL cell lines (SUDHL4, SUDHL8, SUDHL10 and OCI-LY19). 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 DLBCL cell lines. We then dissected the PI3-kinase pathway by analyzing the downstream targets of phosphorylation by Western blot. We found that AKT/PKB was constitutively phosphorylated, and thus activated, in all DLBCL cell lines. The downstream elements of AKT, ForkHead (FKHR) and GSK3 were also constitutively phosphorylated in all DLBCL cell lines. Similarly, treatment with LY294002 prevented this phenomenon in all the cell lines regardless of their final apoptotic endpoint. Inhibition of PI3-kinase activity further downstream induced cleavage of Bid in all DLBCL cells and subsequently loss of mitochondrial membrane potential and release of cytochrome c from mitochondria in all DLBCL cell lines. The release of cytochrome C led to activation of Caspases 9 and 3 and cleavage of PARP. Finally expression of the inhibitor of apoptosis, XIAP, which is also a downstream target of AKT, was compromised in the all cell lines following LY294002 treatment. Our data demonstrate that the PI3-kinase pathway plays a major role in the survival and growth of DLBCL cells. Altogether, these results suggest that blocking the PI3-kinase pathway may be a potential target for therapeutic intervention in diffuse large B-cell lymphoma.

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.

Description: S-phase kinase-associated protein 2 (SKP-2) is a proto-oncogene that has been shown to be expressed in a number of tumors. A number of studies have shown that SKP-2 plays a role in the degradation of tumor suppressor genes by increased proteosome-dependent degradation. SKP-2 overexpression is highly representative of intrinsic biological aggressiveness of certain cancers including breast, non-small cell lung cancer and gastric carcinoma, how ever its role in hematological malignancies have not yet been explored. Therefore, in this study we examined 100 clinical samples of diffuse large B-cell lymphoma (DLBCL) to study the expression pattern of ubiquitin ligase subunit SKP-2 proto-oncogenes and its relation to proliferative index marker protein Ki67 to correlate tumor aggressiveness. The expression of SKP-2 and Ki67 were examined by immunohistochemistry using specific antibodies on formalin-embedded tissue sections of DLBCL patients. Our data showed that SKP-2 was over expressed in majority of DLBCL and was associated with expression pattern of the proliferating index marker Ki67 protein. Since increased proteosome-dependent degradation of tumor suppressor genes play a critical role in the etiology of various tumors and proteosome inhibition is a novel approach for treating malignancies and has been approved for clinical use, we sought to determine whether inhibition of proteosome by MG132, a specific proteosome inhibitor induces apoptosis in a panel of DLBCL cell lines. Our data showed that treatment of DLBCL cell lines by MG132 induced apoptosis in a dose dependent manner. Inhibition of proteosome also decreased the expression of SKP-2 leading to subsequent disruption of mitochondrial membrane potential causing release of cytochrome c into the cytosol. Release of cytochrome c resulted in activation of caspase-3 and cleavage of PARP ultimately leading to apoptosis. These data suggests that SKP-2 expression plays a major role in the oncogenesis of DLBCL and overexpression of SKP-2 can be used as useful prognostic marker. Furthermore, proteosome inhibitors can be used as future therapeutic modality in treating DLBCL.