ALBERT

Description: Molecular features that can classify diffuse large B cell lymphoma (DLBCL) in clinically relevant subgroups have recently been demonstrated in studies investigating Western Patients. Different etiologic factors and potentially also varying genetic predispositions have been demonstrated for lymphoma patients from developing countries. To investigate whether proposed molecular classifiers are equally important for Middle East as for Western populations, we analyzed coexpression of CD10/bcl6 (by immunohistochemistry), IgH/Cyclin D1 translocations (by fluorescence in situ hybridization) and methylation of the gene encoding the DNA repair enzyme O6 methyguanine -DNA methytransferase gene (MGMT) in a series of 236 DLBCL patients from Saudi Arabia. Clinical follow up data were available from 190 patients. Coexpression of CD10/bcl6 was found in 21%, IgH/Cyclin D1 translocations in 18.3%, and MGMT methylation in 70% of cases. With the exception of MGMT methylation which was reported in only 20–35% of European patients, these numbers were in the range expected from the Western literature. Despite the markedly higher number of MGMT methylation in Saudi cases, its proposed positive effect on DLBCL prognosis could be confirmed (p

Description: Age adjusted incidence of Hodgkin’s Lymphoma (HL) is markedly higher in Saudi Arabia than in the United States. For example, HL accounts for 10.5% of all neoplasia in children 15 years and younger in Saudi Arabia. EBV virus infection, which can induce HL transformation has been suspected to cause high HL incidence in developing countries. To investigate the role of EBV for the high frequency of HL in Saudi Arabia, we analyzed 265 HL from Saudi Arabia and 58 HL from Europe for EBV infection by in situ hybridization with fluoresce in-conjugated Epstein-Barr virus (EBER) on tissue microarray (TMA) sections. All Saudi and European HL were analyzed in one staining run under identical conditions. Unexpectedly, our data show only minor, statistically insignificant differences in EBV infection rates between Saudi (64 out of 150 informative cases; 42.6%) and European HL (11 out of 30 informative cases; 33%; p=0.5). Within the Arabian population, EBV positivity was more frequent in 79 children (53%) than among 133 adults (36%; p=0.015). EBV positivity was also linked to high stage with EBV positivity in 36% of 69 stage I/II and 64% of 73 stage III/IV tumors (p=0.009). EBV infection was most frequently seen in mixed cellularity HL (63% of 27 cases). This was significantly more frequent than in nodular sclerosing HL (39% of 136; p=0.02). Interestingly, EBV positivity was associated with good prognosis in Saudi childhood HL (p=0.016) but with poor prognosis in Saudi adulthood HL (p=0.0048). In conclusion, our data strongly suggest that EBV is not the main cause for the high prevalence of HL in Middle East countries. Among others, this would be consistent with a major role of genetic susceptibility genes for HL in these populations. Saudi Arabia with high consanguinity and large families would be ideal to search for HL susceptibility genes.

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: The mechanisms that regulate induction of the antiapoptotic state and mitogenic signals in primary effusion lymphoma (PEL) are not well known. In efforts to identify novel approaches to block the proliferation of PEL cells, we found that apigenin (4′,5,7,-trihydroxyflavone), a flavonoid, induces apoptosis in a dose dependent manner in several PEL cell lines. Such effects of apigenin appear to result from suppression of the constitutively active AKT, FOXO transcription factor and GSK3. Our data also demonstrate that apigenin induces loss of mitochondrial membrane potential with subsequent release of cytochrome c and activation of caspase-3, followed by polyadenosin-5′-diphosphate-ribose polymerase (PARP) cleavage. Altogether, our findings suggest a novel function for apigenin, acting as a suppressor of AKT/PKB pathway in PEL cells, leading to the induction of caspase-dependent apoptosis. Therefore, apigenin may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of AKT/PKB pathway.

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: Phosphotidylinositol 3′-kinases are lipid kinases that regulate signaling pathways involved in cell proliferation, adhesion, survival, motility, and are implicated to play an important oncogenic role in many cancer types. The PIK3CA gene encodes the p110α catalytic subunit of PI3K-like gene. Recently, high frequencies of PIK3CA somatic mutations have been detected in various kinds of human cancers notably colorectal, gastric, and breast cancers. It has also been shown that somatic mutations in the PIK3CA gene have been associated with a higher expression of p-AKT in certain cancers. However, the role of PIK3CA mutations in hematological malignancies has not been elucidated in detail. Therefore, we tested whether PIK3CA is subject to mutations in diffuse large B-cell lymphoma (DLBCL). Exons 9 and 20, encoding the highly conserved helical and kinase domains of PIK3CA, which account for 85% of somatic mutation in PIK3CA gene, were subjected to direct sequencing analysis in 219 cases of DLBCL. Somatic missense mutations were observed in 17 of 212 (8%) DLBCL cases. Out of which, 10 mutations (60%) occurred at codon 1047 in the kinase domain, which is one of the previously reported hot spot within the PIK3CA gene. Additionally, a silent polymorphism (T1025T) was found in one of the DLBCL sample. Moreover, activation of AKT was observed in 14 out of 17 (82%) of the DLBCL harboring PIK3CA mutations. Further, 9 out of 17 cases showed high level of pAKT. Additionally, 12 out of 17 DLBCL patients with PIK3CA mutations showed over-expression of the proliferative marker Ki-67. Expression of high level of pAKT was significantly correlated with over-expression of Ki-67 (p=0.0097). Overall survival in DLBCL cases harboring PIK3CA mutation showed poor survival as compared to the DLBCL cases lacking mutations though it was not statistically significant. These data indicate that mutations of PIK3CA play an oncogenic role in DLBCL tumors and argue for a role of PIK3CA targeting in treatment of DLBCL patients.

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.