Treatment Of Ramos Cells Research Articles

Abstract 3617: Inhibition of KLF4 expression in resistant B-NHL cell lines inhibited cell growth and sensitized the cells to drug-induced apoptosis

Abstract Kruppel-like factor 4 (KLF4) is a member of the KLF4 zinc-finger containing transcription factor family. Reported studies have indicated the involvement of KLF4 in the regulation of proliferation, apoptosis, and differentiation of B-cells and B-cell malignancies. We have recently reported that KLF4 is overexpressed in pediatric Burkitt lymphomas and is a predictive biomarker for survival. In addition, overexpression of KLF4 predicted unresponsiveness to CHOP treatment (Valencia-Hipolito A et al Leuk Lymphoma. 2014;55:1806-14). Preliminary findings demonstrated that the transcription factor Yin Yang 1 (YY1) plays, in part, a role in the regulation of KLF4 expression in B-NHL. We hypothesized that (1) the chemical inhibition of KLF4 may result in the inhibition of proliferation, induction of apoptosis, and sensitization to drug-induced apoptosis in B-NHL cell lines and (2) the inhibition of YY1 would mimic the chemical inhibition of KLF4. Analysis of two B-NHL cell lines revealed that the expression of KLF4 was high in Ramos and low Raji as compared to normal B-cells. Treatment with increasing concentrations (0.5-10 μM) of Kenpaullone (a KLF4 inhibitor) induced in Ramos cells both the inhibition of proliferation and cell survival and the induction of apoptosis; however, there was no effect on the treatment of Raji cells. Treatment of Ramos cells with the combination of Kenpaullone and CDDP potentiated apoptosis as compared to treatment with either the chemical inhibitor or CDDP used alone. The finding with the chemical inhibitor was validated with the transfection of Ramos cells with siRNA-KLF4. In addition, treatment with Kenpaullone inhibited the transcriptional expression of KLF4 (using a reporter assay system with pKLF4-GFP). Additional studies demonstrated that the transfection of Ramos cells with siRNA-YY1 resulted in significant inhibition of KLF4 expression and correlated with the inhibition of proliferation and the induction of apoptosis. The latter is due, in part, as KLF4 suppresses the extrinsic apoptotic pathway by inhibiting the activation and cleavage of caspases 7, 9, and 3.. Our previous analysis in TMA of pediatric lymphomas demonstrated, in all of the tumor tissues, the presence of a positive correlation between the expression of KLF4 and YY1 and that this correlation was markedly significant in the Burkitt subtype. In conclusion, the overexpression of KLF4 may be responsible, in part, in the pathogenesis, malignancy, and drug resistance of B-NHL lymphomas. In addition, the present findings suggest that the chemical inhibition of KLF4 by Kenpaullone treatment or the inhibition of YY1 may be considered as targets for therapeutic intervention in the treatment of B-lymphoma overexpressing KLF4, when used alone or in combination with sub-toxic chemo/immune-drugs. Current studies are evaluating the role of KLF4 inhibition in vivo using B-NHL tumor xenografts models. Citation Format: Mayra R. Montecillo-Aguado, Gabriel G. Vega, Hector Mayani, Sara Huerta-Yepez, Rogelio Hernández-Pando, Otoniel Martinez-Maza, Benjamin Bonavida, Mario I. Vega. Inhibition of KLF4 expression in resistant B-NHL cell lines inhibited cell growth and sensitized the cells to drug-induced apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3617. doi:10.1158/1538-7445.AM2015-3617

HIF-1α Induces a Chemoresistance Phenotype in a Non-Hodgkin‘s Lymphoma Cell Line Via up-Regulation of Bcl-xL

AbstractAbstract 5091Non-Hodgkin’s Lymphoma (NHL) is one of the most common cancers in childhood. The development of chemoresistance in tumor cells is one of the principal causes of treatment failure. This resistance has been associated to different mechanisms, being the overexpression of anti-apoptotic proteins such as Bcl-xL. It has been shown that this protein is regulated by the transcription factor HIF-1α, which is overexpressed in several tumors, including NHL, and the overexpression of both proteins may result in resistance to chemotherapy. We investigated the role of HIF-1α in resistance to chemotherapy via the induction of the Bcl-xL expression in NHL cell lines, using a pharmacological modulation of HIF-1α. Our data showed that treating the Ramos cell with ethyl 3, 4-dihydroxybenzoate, the inhibitor of prolylhydroxylase, induces accumulation of HIF-1α and this correlates with an increase of Bcl-xL as well as resistance to apoptosis after the exposition to chemotherapeutics drugs. In contrast, the treatment of Ramos cells with 2-methoxyestradiol, the inhibitor of HIF-1α activity, induced downregulation of the Bcl-xL expression, and this correlates with the sensitization of tumor cells to chemotherapeutic drugs. This data demonstrated that the up-regulation of the anti-apoptotic protein Bcl-xL in NHL cells correlates with HIF-1α expression and activity, through which induces a phenotype of chemoresistance. Disclosures:No relevant conflicts of interest to declare.

Hypoxia inducible factor-1α induces chemoresistance phenotype in non-Hodgkin lymphoma cell line via up-regulation of Bcl-xL

Non-Hodgkin lymphoma (NHL) is one of the most common cancers in childhood. The development of chemoresistance in tumor cells is one of the principal causes of treatment failure. This resistance has been associated with different mechanisms, one being the overexpression of anti-apoptotic proteins such as Bcl-xL. It has been shown that this protein is regulated by the transcription factor hypoxia inducible factor-1α (HIF-1α), which is overexpressed in several tumors including NHL, and the overexpression of both proteins may result in resistance to chemotherapy. We investigated the role of HIF-1α in resistance to chemotherapy via the induction of Bcl-xL expression in NHL cell lines, using a pharmacological modulation of HIF-1α. Our data showed that treating Ramos cells with ethyl 3,4-dihydroxybenzoate, an inhibitor of prolyl hydroxylase, induces accumulation of HIF-1α and this correlates with an increase of Bcl-xL as well as resistance to apoptosis after exposure to chemotherapeutics drugs. In contrast, the treatment of Ramos cells with 2-methoxyestradiol, an inhibitor of HIF-1α activity, induced down-regulation of Bcl-xL expression, and this correlated with the sensitization of tumor cells to chemotherapeutic drugs. These data demonstrate that up-regulation of the anti-apoptotic protein Bcl-xL in NHL cells correlates with HIF-1α expression and activity, through which a phenotype of chemoresistance is induced.

Selective Cytotoxicity of Amidinopiperidine Based Compounds Towards Burkitt’s Lymphoma Cells Involves Proteasome Inhibition

Serine proteases have proven to be promising pharmacological targets in contemporary drug discovery for cancer treatment. Since azaphenylalanine-based compounds manifest cytotoxic activity, we have selected serine protease inhibitors designed and synthesized in-house with large hydrophobic naphthalene moiety for screening. The cytotoxic potential of screened molecules was correlated to modifications of R1 residues. The most cytotoxic were compounds with greater basicity; amidinopiperidines, piperidines and benzamidines. Amidinopiperidine-based compounds exert cytotoxicity in low µM range, with IC50 18 µM and 22 µM for inhibitors 15 and 16 respectively. These compounds exhibited selective cytotoxicity towards the Burkitt’s lymphoma cells Ramos and Daudi, and proved nontoxic to PMBC, Jurkat and U937. They induce caspase-dependent apoptotic cell death, as demonstrated by the use of a pan-caspase inihibitor, zVADfmk, which was able to rescue Ramos cells from compound(s)-induced apoptosis. We confirm a disruption of the pro-survival pathway in Burkitt’s lymphoma through NFκB inhibition. The accumulation of phosphorylated precursor (p105) and inhibitory (IκB) molecules with no subsequent release of active NFκB implicated the involvement of proteasome. Indeed, we show that the amidinopiperidine-based compounds inhibit all three proteolytical activities of the human 20S proteasome, with the most prominent effect being on the trypsin-like activity. Consistently, treatment of Ramos cells with these compounds led to an increase in ubiquitinated proteins. The amidinopiperidine-based serine protease inhibitors presented are, as selective inducers of apoptosis in Burkitt’s lymphoma cells, promising leads for the development of novel chemotherapeutics.

Mcl-1 and YY1 inhibition and induction of DR5 by the BH3-mimetic Obatoclax (GX15-070) contribute in the sensitization of B-NHL cells to TRAIL apoptosis

The pan Bcl-2 family antagonist Obatoclax (GX15-070), currently in clinical trials, was shown to sensitize TRAIL-resistant tumors to TRAIL-mediated apoptosis via the release of Bak and Bim from Mcl-1 or Bcl-2/Bcl-XL complexes or by the activation of Bax, though other mechanisms were not examined. Herein, we hypothesize that Obatoclax-mediated sensitization to TRAIL apoptosis may also result from alterations of the apoptotic pathways. The TRAIL-resistant B-cell line Ramos was used as a model for investigation. Treatment of Ramos cells with Obatoclax significantly inhibited the expression of several members of the Bcl-2 family, dissociated Bak from Mcl-1 and inhibited the NFκB activity. Cells treated with Mcl-1 siRNA were sensitized to TRAIL apoptosis. We examined whether the sensitization of Ramos to TRAIL by Obatoclax resulted from signaling of the DR4 and/or DR5. Transfection with DR5 siRNA, but not with DR4 siRNA, sensitized the cells to apoptosis following treatment with Obatoclax and TRAIL. The signaling via DR5 correlated with Obatoclax-induced inhibition of the DR5 repressor Yin Yang 1 (YY1). Transfection with YY1 siRNA sensitized the cells to TRAIL apoptosis following treatment with Obatoclax and TRAIL. Overall, the present findings reveal a new mechanism of Obatoclax-induced sensitization to TRAIL apoptosis and the involvement of the inhibition of NFκB activity and downstream Mcl-1 and YY1 expressions and activities.

Abstract 3499: Identification of a novel mechanism by which the BH3 mimetic obatoclax sensitizes non-Hodgkin's lymphoma tumor cells to TRAIL-mediated apoptosis

Abstract The BH3 mimetic chemical antagonist obatoclax (GX15-070, Gemin X Pharmaceuticals) has been used in vitro and nowadays also in clinical trials for its potential therapeutic effects in both solid and lymph node malignancies. The effects of obatoclax treatment alone or in combination with cytotoxic drugs results in the reversal of tumor cell resistance. Studies by us and others have reported that inhibition of over expressed anti-apoptotic gene products such as Bcl-2 and Bcl-XL can reverse resistance to TRAIL. Obatoclax was shown to sensitize TRAIL-resistant solid tumors to TRAIL-apoptosis, inhibiting diverse anti-apoptotic molecules like Bcl-2, Bcl-XL and Mcl-1 through direct binding. We hypothesized that treatment of B-NHL cell lines with Obatoclax will result in tumor cell sensitization to TRAIL apoptosis by a different mechanism. B-NHL Ramos cell line were treated with different concentrations of Obatoclax (7-28 nM) and TRAIL (2.5-20 ng/ml) resulting in significant potentiation of apoptosis and synergy. Western analysis revealed that treatment with obatoclax resulted in significant inhibition of several members of the Bcl-2 family such as inhibition of Mcl-1, Bcl-XL, XIAP, and cIAP 1/2. In addition, obatoclax dissociated Bak from Mcl-1 as determined by immunoprecipitation. We found that, obatoclax inhibited the expression/activity of several members of the NF-κB pathway including phospho-p65, IKK2, and phosphor-IκB-α. The NF-κB activity inhibition by obatoclax was assessed by EMSA. We examined whether obatoclax-induced sensitization to TRAIL resulted from both signaling of DR4 and DR5. Treatment of Ramos cells with DR4 siRNA or DR5 siRNA and then treated with obatoclax and TRAIL resulted in the sensitization of cells treated with DR4 siRNA but not DR5 siRNA. We have previously reported that DR5 is negatively regulated by the transcription repressor YY1, upstream of NF-κB. We found that obatoclax inhibited YY1 transcription and expression. The direct role of YY1 inhibition by obatoclax in both DR5 upregulation and sensitization to TRAIL was determined in transfected cells with YY1 siRNA. Such cells showed upregulation of DR5 and sensitivity to TRAIL apoptosis. The inhibitory effect of obatoclax on Mcl-1 activity and the Mcl-1 direct role in sensitization was examined by treatment of cells with Mcl-1 siRNA. Such cells reversed their sensitivity to TRAIL-mediated apoptosis. Overall, the present findings reveal a new mechanism of obatoclax-induced sensitization to TRAIL by inhibiting NF-κB and downstream Mcl-1 and YY1. Both Mcl-1 and YY1 play a direct role in tumor cells sensitization to TRAIL mediated apoptosis. The present findings identify several new therapeutic targets modified by obatoclax whose modifications can reverse tumor cell resistance to TRAIL mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3499. doi:10.1158/1538-7445.AM2011-3499

The anti-CD20 mAb LFB-R603 interrupts the dysregulated NF-κB/Snail/RKIP/PTEN resistance loop in B-NHL cells: Role in sensitization to TRAIL apoptosis

LFB-R603 is a chimeric anti-CD20 mouse/human monoclonal antibody with a human IgG1 constant (Fc) region. In comparison to rituximab, LFB-R603 exhibits a high affinity to the Fcγ RIII (CD16) and a potent in vitro antibody-dependent cellular cytotoxicity (ADCC). We examined several experimental designs for the biological anti-tumor activity of LFB-R603 as well as its sensitizing activity to apoptosis in resistant non-Hodgin's B-cell lymphoma (B-NHL) in comparison to rituximab. Treatment of the B-NHL cell line Ramos with LFB-R603 was not toxic at the concentrations used and induced cell aggregation following culture at 24 and 48 h similarly to rituximab. Hence, we hypothesized that LFB-R603 may signal the tumor cells and modify intracellular survival/anti-apoptotic pathways. Treatment of Ramos cells with LFB-R603 inhibited the constitutively active NF-κB survival pathway, followed by significant potentiation of TRAIL-mediated apoptosis. We examined the underlying molecular mechanism by which the LFB-R603-mediated NF-κB inhibition results in the reversal of tumor cell resistance to TRAIL. We hypothesized that downstream gene products regulated by NF-κB that are involved in the resistance may be involved in LFB-R603-mediated sensitization. We found that LFB-R603 inhibited NF-κB activation and the anti-apoptotic factor Snail and induced the pro-apoptotic factor RKIP. The direct roles of Snail and RKIP modulation by LFB-R603 in the reversal of B-NHL resistance to TRAIL were examined by knocking down Snail and overexpressing RKIP in Ramos cells, respectively. Both approaches increased significantly the cell sensitivity to TRAIL apoptosis. In addition to changes observed in the expression levels of Snail and RKIP, Ramos cells treated with LFB-R603 induced the expression of PTEN along with inhibition of the PI3K-AKT activated pathway. PTEN silencing in Ramos cells pretreated with LFB-R603 and TRAIL inhibited TRAIL apoptosis; thus, demonstrating that PTEN induction by LFB-R603 has a direct role in tumor cell sensitization to TRAIL apoptosis. Several of the findings obtained in the experimental designs with LFB-R603 were superior to those obtained with rituximab. Overall, the findings demonstrate that LFB-R603 interferes with the dysregulated NF-κB/Snail/RKIP/PTEN/AKT resistance circuitry in B-NHL cells. Further, the findings suggest that LFB-R603 may sensitize tumor cells to various apoptotic stimuli including cytotoxic ligands such as TRAIL and chemotherapeutic drugs.

Inhibition of the RKIP and PTEN-Transcription Repressor Snail in B-NHL by the Proteasome Inhibitor NPI-0052: Roles of RKIP and PTEN Induction in the Reversal of Resistance to TRAIL Apoptosis.

Abstract 3938Poster Board III-874Several strategies have been examined to reverse tumor cell resistance to immunotherapy. For instance, proteasome inhibitors such as Bortezomib, MG-132 and NPI-0052 have been reported to sensitize hematopoietic tumor cells to TRAIL-mediated apoptosis. NPI-0052 mediates its cytotoxic and sensitizing effects in lymphoma cells through the inhibition of the NF-κB and PI3K/Akt survival pathways. The molecular mechanisms and associated gene products involved in NPI-0052-induced inhibition of these pathways are not known. We have reported that NPI-0052 induced the expression of Raf-1 kinase inhibitor protein (RKIP) via inhibition of NF-κB activity and downstream the RKIP transcription repressor Snail (Baritaki et al., Oncogene. 2009, PMID: 19633685). Also, Snail has been reported to repress phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and allowing the Akt pathway to remain activated. Based on these findings, we hypothesized that treatment of B-NHL cells with NPI-0052 will result in the induction of both RKIP and PTEN through inhibition of Snail and, consequently, will result in the inhibition of the NF-κB and Akt pathways, respectively. Treatment of Ramos cells with NPI-0052 resulted in the inhibition of NF-κB activity and Snail expression along with the induction of both RKIP and PTEN expression, as assessed by western. The direct roles of RKIP and PTEN in B-NHL cell sensitization to TRAIL were demonstrated by the use of cells transfected with siRNA RKIP and siRNA PTEN, whereby reversal of sensitivity occurred. In contrast, Snail knockdown by siRNA Snail resensitized the cells to TRAIL apoptosis. The inhibition by NPI-0052 of both the NF-κB and PI3K/Akt pathways resulted downstream in the inhibition of transcription and/or activity of anti-apoptotic gene products such as Bcl-2 family members. The direct role of Bcl-2 family anti-apoptotic gene products in the reversal of resistance by NPI-0052 was demonstrated by the use of the Bcl-2 family chemical inhibitor 2MAM3. Altogether, these findings demonstrate that NPI-0052 modifies a tightly regulated loop in the resistance of B-NHL cells, namely, the constitutive activation of NF-κB and Akt survival pathways that crosstalk and induce Snail. Snail in turn, represses RKIP and PTEN and, thus, the cells continue to survive and proliferate. However, upon treatment with NPI-0052, the inhibition of PI3K/Akt and NF-κB activities results downstream in the inhibition of Snail and the inhibition of Snail, in turn, derepresses the transcription of both RKIP and PTEN. In a feedback loop, the induction of RKIP represses the NF-κB pathway and the induction of PTEN represses the PI3K/Akt pathway. Hence, the inhibition by NPI-0052 of the NF-κB/Akt/Snail/RKIP/PTEN regulatory resistance loop in B-NHL cells results in the reversal of resistance. Further, these findings identify several new targets for potential therapeutic intervention in the reversal of resistance. Disclosures:Berenson:Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau. Palladino:Nereus Pharmaceuticals: Employment, Equity Ownership.

Mechanism of Obatoclax (GX15-070; Bcl-2 Family Inhibitor)-Induced-Sensitization of B-NHL Cells to TRAIL-Mediated Apoptosis: Inhibition of the NF-κB Survival Pathway and the DR5 Transcription Repressor Yin Yang1 (YY1)

Patients with B-NHL respond initially to treatment with rituximab (chimeric anti- CD20 monoclonal antibody) in combination with CHOP. However, a subset of patients does not respond or develop refractoriness to further treatments. Therefore, there is an urgent need to develop novel therapeutic strategies to treat unresponsive patients. We have explored the potential therapeutic efficacy of TRAIL though, most tumors and cell lines are resistant to TRAIL-induced apoptosis. Our previous findings and those of others have demonstrated that the overexpression of anti-apoptotic gene products such as Bcl-2, BclXL, and Mcl-1 regulates resistance to TRAIL and thus, inhibition of these gene products reverses resistance. Hence, we hypothesized that treatment of B-NHL cell lines with the Bcl-2 family inhibitor, Obatoclax (GX15-070; Gemin X Pharmaceuticals, Malvern, PA) will result in tumor cell sensitization to TRAIL apoptosis. We have used the B-NHL Ramos cell line as model. Treatment of Ramos cells with various concentrations of Obatoclax (7–28 nM) and TRAIL (2.5–20 ng/ml) resulted in significant potentiation of apoptosis and the combination treatment was synergistic. We then explored the mechanism of Obatoclax-induced sensitization to TRAIL. Treatment of Ramos cells with Obatoclax inhibited NF-κB activity and downstream anti-apoptotic gene products regulated by NF- κB (example Bcl-xl, Mcl-1 and XIAP) as assessed by western. Since Obatoclax inhibited NF-κB activity, we explored its effect on the transcription repressor YY1 and DR5 expression. Treatment of Ramos with Obatoclax significantly inhibited YY1 expression concomitantly with upregulation of total and surface DR5 expression that are regulated by NF-κB. The direct role of YY1 in the regulation of resistance to TRAIL was demonstrated by treatment of Ramos with siRNA YY1. Such treated cells showed upregulation of DR5 expression and sensitization to TRAIL apoptosis. The sensitization by Obatoclax resulted in activation of both Type I and Type II apoptotic pathways when used in combination with TRAIL. These findings establish a novel mechanism of Obatoclax-induced gene modification aside from its direct inhibition of Bcl-2 family. Further, our findings with Obatoclax are different from those recently reported by Song et al., [JBC 2008; July 3 (Epub ahead of print)] demonstrating that ABT-737, a small molecule Bcl-2 inhibitor, potentiated TRAIL-induced apoptosis via activation of NF-κB and NF-κB-induced upregulation of DR5 transcription via NF-κB DNA binding site on the DR5 promoter. It is possible that Obatoclax and ABT-737 mediate their sensitization to TRAIL via distinct mechanisms. In summary, our findings demonstrate the potential therapeutic application of Obatoclax in combination with TRAIL or agonist DR4/DR5 antibodies in the reversal of tumor cell resistance to TRAIL.

The Novel Proteasome Inhibitor NPI-0052 Induces the Expression of Raf-1 Kinase Inhibitor Protein RKIP) in B-NHL via Inhibition of the Transcription Repressor Snail: Roles of Snail and RKIP in Sensitization to TRAIL Apoptosis

A subset of patients with B cell malignancies does not respond to conventional current cytotoxic therapies. Novel immunotherapeutic approaches have been considered such as TRAIL and agonist DR4/DR5 mAbs that are currently in phase I/II clinical trials in patients with a variety of tumor types. However, most tumors are not sensitive to TRAIL/agonist antibodies due to the development of a generalized resistance to apoptotic stimuli. Therefore, for these therapies to be effective it is a requisite that resistance is modified by the use of sensitizing agents that target cell survival anti-apoptotic pathways or, more selectively, specific gene products in the apoptotic pathways. Proteasome inhibitors such as bortezomib and the novel inhibitor NPI-0052 have shown both direct cytotoxicity and also sensitizing ability when used in combination with biologics and cytotoxic drugs. We have recently reported that treatment of TRAIL-resistance B-NHL cell lines with NPI-0052 (not toxic by itself) in combination with TRAIL resulted in significant potentiation of apoptosis and synergy was achieved. Sensitization was due, in part, to inhibition of NF-kB activity and induction of Raf-1 kinase inhibitor protein (RKIP) [Baritaki et al., J Immunol 80:6199, 2008]. The role of RKIP in TRAIL apoptosis by NPI-0052 in Ramos cells was examined as well as the mechanism of NPI-0052-induced expression of RKIP. Like NPI-0052, overexpression of RKIP in Ramos cells sensitized the cells to TRAIL apoptosis and, in contrast, treatment with siRNA RKIP reversed this sensitizing effect. A recent report by our collaborators [Beach et al., Oncogene 27:2243,2008] demonstrated that RKIP transcription is negatively regulated, in part, by the metastasis-inducer transcription factor Snail. Thus, we hypothesized that NPI-0052-induced upregulation of RKIP may be due to inhibition of Snail. Indeed, treatment of Ramos cells with NPI-0052 significantly downregulated Snail expression concomitantly with upregulation of RKIP. Treatment of Ramos cells with siRNA Snail resulted in upregulation of RKIP and sensitization to TRAIL apoptosis. In contrast, ectopic expression of Snail repressed RKIP expression. Further, ectopic expression of RKIP inhibited Snail expression while siRNA RKIP upregulated Snail. These findings revealed the existence of a regulatory RKIP-NF-kB-Snail-RKIP loop. NPI-0052-induced inhibition of Snail also resulted in inhibition of Snail-induced gene products involved in metastasis. These findings demonstrate that NPI-0052 regulates B-NHL sensitivity to TRAIL apoptosis via the inhibition of Snail and induction of RKIP. These findings support the potential therapeutic application of the combination of NPI-0052 and TRAIL in the treatment of patients with resistant B-NHL and other malignancies.

Synergistic cytotoxicity of Bcl-xL inhibitor, gossypol and chemotherapeutic agents in non-Hodgkin’s lymphoma cells

Anti-apoptotic proteins Bcl-2 and Bcl-xL are overexpressed in 80% of non-Hodgkin’s lymphoma cells and are thought to play an important role in the resistance of lymphoma cells to current chemotherapeutic agents. Gossypol, an orally-active polyphenolic aldehyde derived from the cotton plant, has been known to have potential anti-neoplastic activity. Recently, gossypol was found to bind to the BH3 binding groove of Bcl-xL and with lesser affinity to Bcl-2. The present study was conducted to determine whether gossypol increases the sensitivity of non-Hodgkin’s lymphoma cells to the actions of chemotherapeutic agents by potentiating treatment-induced apoptosis. The interactions observed between gossypol and chemotherapeutic drugs were analyzed using the median effect principle (CalcuSyn analysis). Our data showed that treatment of Ramos cells with gossypol not only induced cell arrest on the G0/G1 phase, but also augmented apoptosis and growth inhibition induced by etoposide (VP-16), doxorubicin hydrochloride (ADM), vincristine (VCR), and paclitaxel (taxol). However, when gossypol was combined with cisplatin (DDP) an antagonistic effect was observed. Gossypol-induced cell cycle arrest was accompanied by decreased expression of cyclin D1 in Ramos cells. In addition, the peroxisome proliferator-activated receptor (PARP) pathway is, at least in part, involved in the gossypol-induced apoptosis when combined with VP-16. These data indicate that single-agent gossypol is effective in inhibiting growth of non-Hodgkin’s lymphoma cells in vitro and combination studies with certain secondary chemotherapeutic agents further demonstrate it’s synergistic cytotoxicity. These findings support future preclinical and clinical studies of gossypol in the treatment of non-Hodgkin’s lymphoma.

Nitric Oxide Sensitizes B-NHL Cells to TRAIL-Mediated Apoptosis through Induction of RKIP, Inhibition of YY1 and Upregulation of DR5.

A better knowledge of the mechanisms of survival and escape from apoptosis of B-cell lymphoma cells has led to the proposal of new drugs that selectively interfere at the different steps of these cascades. Among the novel agents that have recently emerged for the sensitization of several resistant tumor cells to chemo- or immuno-mediated cell death are agents that induce nitric oxide (NO) (Bonavida B et al., Drug Resistance Update, 2006). The molecular mechanisms which are implicated in the NO-induced sensitization of tumor cells to apoptosis are not clearly elucidated. Recent findings in our laboratory have reported that treatment of various cancer cell lines with the NO donor, DETA/NONOate, sensitizes tumor cells to FasL- and TRAIL-mediated apoptosis. Sensitization is mediated through inhibition of NF-κB and YY1 activities and upregulation of death receptors. The objective of the present study was to examine whether DETA/NONOate is also able to sensitize B-NHL cells to TRAIL-mediated apoptosis and to identify additional molecular targets which could be involved in the NO-mediated chemo- and immuno-sensitization of tumor cells. Contrary to YY1, RKIP (Raf kinase inhibitor protein) has been shown to play a pivotal role in suppression of mitogenesis through inhibition of Raf/MEK/ERK and NF-κB survival signaling pathways (Yeung et al., Molecular Cellular Biology ; 21:7207, 2001). Since both NO and RKIP inhibit NF-κB, we hypothesized that NO may induce RKIP expression and may participate in NO-mediated effects. The TRAIL-resistant B-NHL cell line, Ramos, was treated with DETA/NONOate and examined for changes in YY1 and RKIP protein and m-RNA levels, as well as for sensitization to TRAIL-mediated apoptosis. The findings demonstrate that treatment of Ramos cells with DETA/NONOate results in slight induction of RKIP m-RNA levels followed by significantly elevated RKIP protein expression. In contrast, both YY1 transcript and protein levels were found remarkably reduced after DETA/NONOate treatment. Since YY1 has been recently shown to negatively regulate DR5 transcription, NO-induced suppression of YY1 was able to sensitize Ramos cells to TRAIL-mediated apoptosis through significant DR5 upregulation, as assessed by flow cytometry. However, DR4 surface protein expression remained unchanged. Noteworthy, the rituximab resistant Ramos clone (RR1) was not sensitized by NO, while no changes were observed in both DR4 and DR5 levels, suggesting different regulation of cell sensitivity to TRAIL. The above findings support the role of NO as an immuno-sensitizing agent via inhibition of YY1 and suggest, for the first time, the pivotal role of RKIP as one potential NO target for apoptosis induction in tumor cells. Further experimental studies will clarify the interlink between RKIP and YY1 involved in NO-based B-cell lymphoma sensitization to apoptosis and validation with patient derived tissues.

Induction of RKIP and Inhibition of the Transcription Repressor YY1 in B-NHL by the Novel Proteasome Inhibitor NPI-0052 and Sensitization to TRAIL-Induced Apoptosis.

Proteasome inhibitors have been shown to exert multiple effects including direct cytotoxic activity in sensitive cancer cells. In addition, proteasome inhibitors have recently been used therapeutically for certain cancers such as multiple myeloma and mantle cell lymphoma. We have recently reported that the novel proteasome inhibitor, NPI-0052 (Nereus Pharmaceuticals), can sensitize drug-resistant B-NHL tumor cells to apoptosis by various chemotherapeutic drugs. Also, NPI-0052 inhibits the NF-κB survival pathway and inhibition of NF-κB sensitizes tumor cells to TRAIL-induced apoptosis. We hypothesized that inhibition of NF-κB by NPI-0052 may also lead to sensitization of TRAIL-resistant B-NHL cells to TRAIL-mediated apoptosis. Human Burkitt's lymphoma,Ramos cells, were treated with various concentrations of NPI-0052 (0–10ng/ml for 24h) and were then treated with recombinant human TRAIL (0–100ng/ml for 24h). The cells were examined for apoptosis by Annexin V/PI. The combination treatment resulted in significant potentiation of cytotoxicity and synergy in apoptosis was achieved. We have then examined a potential underlying mechanism of NPI-0052-mediated sensitization. The transcription repressor YY1 was recently shown to negatively regulate the transcription of the TRAIL death receptor DR5 and thus overexpression of YY1 is a resistant factor for TRAIL-induced apoptosis. Hence, treatment of Ramos cells with NPI-0052 resulted in significant inhibition of YY1 expression and upregulation of surface DR5 expression. We have also examined the effect of NPI-0052 on RKIP (Raf-kinase inhibitor protein) (Yeung et al., Molecular Cellular Biology; 21:7207, 2001) expression. Since both NPI-0052 and RKIP inhibit the NF-κB pathway, we hypothesized that treatment of Ramos cells with NPI-0052 may result in significant upregulation of RKIP expression. The findings, indeed, demonstrate that NPI-0052 significantly induced RKIP expression in Ramos cells. Overall, the findings show, for the first time, that NPI-0052 sensitizes B-NHL tumor cells to TRAIL-mediated apoptosis. Sensitization by NPI-0052 was correlated with both the induction of RKIP expression and inhibition of YY1 and upregulation of DR5 expression. Currently, TRAIL or agonist monoclonal antibodies against DR4 or DR5 are being examined clinically for anti-tumor activity. Therefore, our findings suggest the potential application of the combination of NPI-0052 and TRAIL or agonistic monoclonal antibodies against DR4 or DR5 in the treatment of tumor cells that are resistant to drugs and TRAIL.

Rituximab-Mediated Upregulation of PTEN Expression and Inhibition of Both the PI3K/AKT Pathway and Pleitrophin (PTN) Expression in B-NHL: Roles in Cell Growth Inhibition and Chemosensitization.

We have recently reported that rituximab treatment of B-NHL cell lines, like Ramos, inhibited the PI3K/AKT signaling pathway and downregulated Bcl-xL expression. The role of the AKT pathway in chemosensitization was corroborated by treating Ramos cells with the AKT inhibitor, LY294002, and resulted in sensitization of the cells to drug-induced apoptosis. We have investigated a potential underlying mechanism responsible for the rituximab-mediated inhibition of the AKT pathway. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor phosphatase and functions as a negative regulator of the PI3K pathway through its phosphatase activity. We hypothesized that rituximab may upregulate PTEN expression and thus, inhibiting the AKT pathway. Treatment of Ramos cells with rituximab (20μg/ml for 20h) resulted in significant upregulation of PTEN expression (as assessed by both Western and RT-PCR). Time kinetic analysis showed that PTEN is upregulated as early as 6–9h post rituximab treatment. In addition, since rituximab inhibits cell proliferation and cell growth, we have examined the effect of rituximab on the expression of the growth factor pleitrophin (PTN). PTN is a heparin-binding and secreted growth differentiation factor that mediates various functions such as cell motility and migration, survival, growth and differentiation. Treatment of Ramos cells with rituximab inhibited PTN expression as early as 12h post treatment. The present findings suggested that rituximab-mediated induction of PTEN expression and inhibition of both the AKT pathway and PTN epxression may be interrelated and play an important role in rituximab-mediated cell growth inhibition and chemosensitization. A recent report by Li et al., (JBC, 281:10663,2006) demonstrated that PTEN null cells exhibited upregulation of PTN expression and activation of the PI3K/AKT pathway. Further, inhibition of PTN resulted in inhibition of the AKT pathway, thus establishing a feedback mechanism. Our findings with rituximab are consistent with the Li's findings'. The mechanism by which rituximab inhibits PTN expression is not clear. Reported studies have implicated the role of AP-1 in PTN transcription and in agreement, our findings have also demonstrated that rituximab inhibits AP-1. Overall, the present studies suggest novel targets modified by rituximab namely, PTN and PTEN, which can be considered for therapeutic intervention in the treatment of both rituximab-sensitive and rituximab-resistant tumors.

Tumor localization and antitumor efficacy of novel sapphyrin compounds

Sapphyrins are pentapyrrolic metal-free expanded porphyrins with potential medical use as anticancer agents. The novel sapphyrin derivative, PCI-2050, functionalized with 2-[2-(2-methoxyethoxy)ethoxy]ethoxy groups to enhance solubility and a modified bipyrrole moiety was found to be more potent in inducing apoptosis than the previously described sapphyrin PCI-2000. Because some sapphyrins may localize to tumors, we took advantage of the intrinsic fluorescence of these compounds to develop a flow cytometry-based assay to track sapphyrin biodistribution in tumor-bearing mice. Ex vivo analysis of sapphyrin-injected animals revealed that PCI-2050 preferentially localized to tumor, whereas PCI-2000 distributed into normal tissues rather than tumor. PCI-2050 uptake in xenograft tumor cells and resultant tumor cell cytotoxicity was dose dependent. To investigate structure-activity relationships, we focused on PCI-2050 and three derivatives that differ by their alkyl substituents on the bipyrrole moiety: PCI-2051, PCI-2052, and PCI-2053. Treatment of Ramos cells in culture or treatment of Ramos xenograft-bearing animals with each of the sapphyrins followed by ex vivo growth of tumor cells revealed the same pattern of cytotoxicity: PCI-2050 > PCI-2052 > PCI-2051 > PCI-2053. Thus, subtle changes in the alkyl substituents on the bipyrrole moiety result in significant changes in antitumor activity. PCI-2050 displayed significant antitumor efficacy in both Ramos and RKO xenograft models without hematologic, hepatic, or renal abnormalities as assessed by complete blood counts and serum chemistries. On the basis of these findings, it is concluded that the sapphyrin PCI-2050 warrants further evaluation as a potential anticancer agent due to its intrinsic proapoptotic activity and tumor localization ability.

Tumor necrosis factor-α and CD40L modulate cell surface morphology and induce aggregation in Ramos Burkitt's lymphoma cells

Interaction of CD40L and its cognate receptor is an essential component of B-lymphocyte signaling, affecting various aspectsof B-cell differentiation pathways and immunoglobulin gene expression. However, much less is known about the biologicalconsequences of B-cell signaling through tumor necrosis factor (TNF)-α and its cognate receptors TNF-R1 and 2. We usedRamos Burkitt's lymphoma cell line as a model system to study the direct effects of these cytokines on B cells. Treatment ofRamos cells with either TNF-α or CD40L, but not with interleukin (IL)- 4, interferon (IFN)-γ and transforming growthfactor (TGF)-β, resulted in enhanced cell aggregation and enhancement of adherence to glass cover-slips. Scanning electronmicroscopy showed that Ramos cells have a polarized cell surface morphology and exhibit at least 3 cell surfacemorphological domains: microvilli, filopodia and ruffled membranes. The cells adhered to the glass matrix through multiplefilopodia/podopodia-like cell processes and demonstrated distinct ruffled-like membrane projections on their opposite pole.Induction by TNF-α or CD40L, but not with IL-4, IFN-γ and TGF-β, resulted in increased number and complexity of bothtypes of membrane projections. TNF-α and CD40L upregulated the expression of the adhesion molecule intercellularadhesion molecule-1 and the Fas receptor on Ramos cells, without affecting the expression levels of membraneimmunoglobulin M or its secretion rate. Reverse transcriptase-polymerase chain reaction, and flow cytometry demonstratedthat Ramos cells expressed TNF-R1 but very little if any TNF-R2, indicating that TNF-α exerted its effects on Ramos cellsthrough the former receptor.

Rituximab-Mediated Inhibition of the PI3K-Akt Survival Signaling Pathway in B-NHL Cell Lines Leading to Downregulation of Bclxl Expression and Chemosensitization.

Rituximab (chimeric anti-CD20 monoclonal antibody) has been used in the treatment of B-NHL. We have reported in vitro that rituximab treatment signals B-NHL cell lines Ramos and Daudi and inhibits both the ERK 1/2 MAPK and NF-κB signaling pathways leading to selective inhibition of Bclxl expression and sensitization to drug-induced apoptosis. The inhibition of the NF-κB signaling pathway by rituximab was shown to be due, in part, to the induction of the Raf Kinase Inhibitor Protein (RKIP) (Jazirehi, et al., 2005 Cancer Research 65:264–276). The PI3K-Akt signaling pathway is a key regulator of cell survival and aberrant activation of the PI3K-Akt pathway has been implicated in both drug resistance and resistance to apoptosis-inducing stimuli. Akt can promote cell survival by indirectly activating the proximal transcription factor NF-κB through the phosphorylation of I-kappa B kinase (I-κB) (Ozes et. al. Nature 401:82–85, 1999). This study investigated whether NF-κB inhibition by rituximab and downregulation of Bclxl expression was also the result of rituximab-mediated inhibition of the PI3K-Akt pathway. Ramos and Daudi B-NHL cell lines were treated with rituximab (20 ug/ml) and cell lysates were prepared and both Akt and phospho-Akt (p-Akt) expression were examined by western blot. The findings demonstrate that both cell lines show constitutively activated p-Akt and treatment with rituximab significantly inhibited p-Akt but not Akt. Time kinetics analysis demonstrated that inhibition of p-Akt was first detected at 3–6 hours following rituximab treatment and inhibition was maintained up to 24 hours. Concomitantlly, a similar time kinetics revealed inhibition of NF-κB activity as assessed by EMSA. Since the inhibition of NF-kB activity resulted in significant downregulation of Bclxl expression, we also examined the role of the Akt pathway in the regulation of Bclxl expression. Tumor cells were treated with the Akt inhibitor LY294002 and analysis of cell lysates showed significant downregulation of Bclxl expression. Rituximab was previously shown to sensitize B-NHL cells to drug-induced apoptosis via inhibition of NF-κB activity and Bclxl expression. We examined if inhibition of the Akt pathway also chemosensitized the cells. Treatment of Ramos cells with the Akt inhibitor LY294002 significantly sensitized the cells to CDDP-induced apoptosis and synergy was achieved. Altogether, these findings demonstrate, for the first time, that rituximab inhibits the Akt pathway and that this pathway is involved in the regulation of tumor- cell resistance to chemotherapeutic drugs. This study also proposes that the Akt pathway is a potential targeting pathway for therapeutic intervention in the treatment of rituximab and drug-resistant B-NHL.

P72syk tyrosine kinase is activated by oxidizing conditions that induce lymphocyte tyrosine phosphorylation and Ca2+ signals

We have used H2O2 as a pharmacologic agent to examine the effects of oxidizing conditions on lymphocyte signal pathways. Treatment of Ramos cells with 5-10 mM H2O2 gave rapid and strong tyrosine phosphorylation of multiple cellular proteins and activation of p72syk to levels equal to or greater than that observed upon surface Ig cross-linking. Strong Ca2+ signals that could be blocked by the tyrosine kinase inhibitor herbimycin A were also observed under these conditions. However, there was no increase in activity for the Src family kinases p56lck, p59fyn, or p56/p53lyn. Our findings that the p72syk tyrosine kinase responds to H2O2 treatment of cells suggest that this kinase is likely to contribute to cellular tyrosine phosphorylation and calcium signaling induced by oxidizing conditions. Furthermore, H2O2 may be useful as a pharmacologic agent to distinguish the effects of p72syk-related kinases from those of Src family kinases.

Structure-function analysis of the human integrin VLA-4 (α4/β1): Correlation of proteolytic α4 peptides with α4 epitopes and sites of ligand interaction

The structure-function relationship of the human integrin VLA-4 (α4/β1; CD49d/CD29), has been studied in the human B-cell line Ramos by immunochemical and functional analysis. Ramos cells expressed the 150-kDa non-proteolyzed form of the α4 chain, which could be digested upon mild trypsin treatment to generate the 80- and 65-kDa proteolyzed forms, as well as α4 polypeptides of 55 and 50 kDa. In addition, treatment of Ramos cells with high doses of pronase predominantly yielded the 55- and 50-kDa α4 peptides. The trypsin-generated 80- and 65-kDa α4 polypeptides, but not the 55- and 50-kDa fragments, were able to associate with the β1 chain. Distinct anti-VLA-4 mAb against four different α4 epitopes, referred to as epitopes A, B1, B2, and C, recognized the 150-kDa α4 chain both associated or non-associated with the β1 chain. The α4 proteolytic forms of 80, 65 and 50 kDa were precipitated by the anti-α4 mAb directed against the four different α4 epitopes. On the other hand, the 55-kDa α4 peptide was present in precipitates from anti-α4 mAb specific for epitopes A, B1 and C, but absent in precipitates from the anti-α4 mAb specific for epitope B2. The different adhesive capacities of the VLA-4 integrin, namely the interaction with a 38-kDa fibronectin fragment containing the CS-1 region of plasma fibronectin (Fn-38), the binding to the vascular cell adhesion molecule-1 (VCAM-1), or the ability to mediate the anti-α4-induced cell aggregation, were not altered on VLA-4 from cells upon mild trypsin treatment, when compared to non-treated cells. However, the 55- and 50-kDa α4 forms generated by high-dose pronase cell treatment, failed to mediate cell interaction with Fn-38 or VCAM-1 ligands, and cell aggregation could not be triggered through VLA-4 under these conditions.

Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells.

Blot hybridization with labeled tubulin cDNA showed that treatment of Ramos cells, a human cell line of lymphoblastoid origin, with either alpha or beta interferon (IFN) induced a marked increase in the amount of tubulin mRNA sequences. The level of tubulin mRNA sequences increased rapidly after exposure of cells to IFN-alpha and reached a maximum after 1 h of treatment, which was four times the control level. Treatment with IFN-beta induced a maximal increase after 4 h; the amount of tubulin mRNA sequences was seven times higher than the control level. The mRNA extracted from IFN-treated and nontreated cells was translated in vitro in a reticulocyte lysate cell-free system containing [35S]methionine. Electrophoretic analysis of the labeled cell-free products showed an increase in the amount of translatable tubulin mRNA that parallels the time course of induction of tubulin mRNA sequences. Two-dimensional gel electrophoresis of the labeled protein products directed by mRNA indicates that IFN caused a more pronounced increase in the level of alpha-tubulin than beta-tubulin mRNA. Treatment with colchicine, which disrupts the cell microtubules, caused a marked decrease in the tubulin mRNA content. Concomitant treatment of the cells with colchicine and IFN abolished the interferon-dependent induction of tubulin mRNA.