Caffeic Acid Phenethyl Ester Induces Apoptosis in Acute Myeloid Leukemia Cells - Possible Role of Chemokine/G-Protein Signaling.

Abstract Abstract 4802 Caffeic acid phenethyl ester (CAPE) is an active phenolic compound present in propolis obtained from honeybee hives. It is reported to present a spectrum of biological activities including antioxidant, anti-inflammatory and antitumoral. The antitumoral activity of CAPE as evaluated by several studies in vitro and in vivo seems to be related to distinct effects like inhibition of angiogenesis, invasion and metastasis and induction of apoptosis or differentiation of cancer cells. In the scenario of AML the demonstration of CAPE-induced apoptosis or cellular differentiation is restricted to the HL-60 cell line. Our aim was to evaluate the effects of CAPE treatment on primary AML samples as well as APL cell lines NB4 and NB4-R2 (a cell resistant to ATRA-induced differentiation) and on AML cell line Kasumi-1 (representative of core binding factor leukemia with AML1-ETO rearrangement). Proliferation and viability was evaluated by cell count with tripan blue in Neubauer chamber at fixed time intervals. Differentiation was evaluated by flow cytometer determination of CD11b expression. Apoptotic cells were defined as sub-G0 fraction and were evaluated by flow cytometer determination of propidium iodide- DNA fluorescence. Also apoptosis was detected by the annexin-V method. Leishman stained cytospins were used to confirm apoptosis or differentiation. CAPE did not induce differentiation in the cell lines NB4, NB4-R2 or Kasumi-1 and did not alter the differentiation induced by ATRA in NB4 cells. CAPE inhibited the proliferation of AML cell lines in a time and dose dependent fashion. The ED50 in 24h treatment for NB4 cell line (tripan blue) was 32.1 mcg/ml. ED50 (at 24h) for induction of apoptosis in the more sensitive assay using annexin-V in NB4 cells after 24h was 7.5mcg/ml and for Kasumi-1 was 10.2mcg/ml. CAPE (32 mcg/ml) significantly induced apoptosis after 24h in cells from AML patients (n=10), mean (IC95%) of 40.5% (29.26 – 51.76) versus control treated cells 18.16% (12.27 – 24.05); p=0.0004 In order to evaluate the mechanisms of CAPE-induced apoptosis in NB4 cells we performed a microarray analysis after 12 hours treatment (32mcg/ml). The majority of downregulated genes fall into two categories: positive cell cycle regulators and ribosomal genesis / protein traduction. In the other hand, upregulated genes fall into several categories, we point out chemokines and G- protein signalization genes. (Table 1 and 2) The role of IL-8 and Gro chemokines, that signaling by G-protein coupled receptors, has been determined in tumor progression and invasiveness. We are currently investigating the possibility that CAPE exerts an inhibitory effect in chemokine signaling in APL. In conclusion, CAPE-induced apoptosis in AML is associated with the regulation of specific genes. These properties are interesting and need further investigation. Disclosures: No relevant conflicts of interest to declare.

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Role of the G-Protein-Coupled Receptor Signaling Pathway in Insecticide Resistance

International Journal of Molecular Sciences ◽

10.3390/ijms20174300 ◽

2019 ◽

Vol 20 (17) ◽

pp. 4300 ◽

Cited By ~ 2

Author(s):

Ting Li ◽

Nannan Liu

Keyword(s):

Insecticide Resistance ◽

Cell Line ◽

Signaling Pathway ◽

G Protein ◽

Cell Lines ◽

Sf9 Cells ◽

Camp Production ◽

G Protein Coupled Receptor ◽

G Protein Coupled

The G-protein-coupled receptor (GPCR) regulated intracellular signaling pathway is known to be involved in the development of insecticide resistance in the mosquito, Culex quinquefasciatus. To elucidate the specific role of each effector in the GPCR regulating pathway, we initially expressed a GPCR, G-protein alpha subunit (Gαs), adenylate cyclase (AC), and protein kinase A (PKA) in insect Spodoptera frugiperda (Sf9) cells and investigated their regulation function on cyclic AMP (cAMP) production and PKA activity. GPCR, Gαs, and AC individually expressed Sf9 cells showed higher cAMP production as the expression of each effector increased. All the effector-expressed cell lines showed increased PKA activity however. Moreover, Sf9 cytochrome P450 gene expression and cell tolerance to permethrin were examined. The relative expression of CYP9A32gene in Sf9 cells tested was significantly increased in all effector-expressed cell lines compared to a control cell line; these effector-expressed cell lines also showed significantly higher tolerance to permethrin. Inhibitor treatments on each effector-expressed cell line revealed that Bupivacaine HCl and H89 2HCl robustly inhibited cAMP production and PKA activity, respectively, resulting in decreased tolerance to permethrin in all cell lines. The synergistic functions of Bupivacaine HCl and H89 2HCl with permethrin were further examined in Culex mosquito larvae, providing a valuable new information for mosquito control strategies.

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Targeting xIAP Is More Important Than Bcl-Xl in Lymphomas with Constitutive Activation of NF-kB.

Blood ◽

10.1182/blood.v108.11.2390.2390 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2390-2390

Author(s):

Yanjuan He ◽

Joan Cain ◽

Lee Ratner ◽

Leon Bernal-Mizrachi

Keyword(s):

Cell Lines ◽

Annexin V ◽

Fold Increase ◽

Daudi Cell ◽

Xiap Expression ◽

Apoptotic Effect ◽

Effective Doses ◽

Induction Of Apoptosis ◽

Apoptotic Pathways

Abstract Pathways resulting in resistance to apoptosis are essential to the process of lymphomagenesis. One such pathway, the nuclear factor-kB (NFkB), has been shown to be a key element in coordinating the anti-apoptotic effect of these malignancies. However the mechanisms used by which NFkB prevents apoptosis are not well understood. It has been suggested that NFkB inhibits activation of the intrinsic, extrinsic and common apoptotic pathways. Previous work in our lab using two different virally mediated lymphoma models (Tax/HTLV1 and LMP1/EBV driven tumors) has identified two candidates that could explain these results: X chromosome-linked inhibitor of apoptosis (xIAP) and BCL-xL. Although the current literature extensively demonstrates the role of BCL-xL in lymphomas, little is known about the importance of xIAP in these malignancies. To answer this question we tested the apoptotic effect of etoposide or tumor necrosis factor (TNF) after knocking down bcl-xL and xIAP expression in our lymphoma models (SC and Daudi cell lines) using a lentivirus expressing siRNAs. After 24 hours of treatment with etoposide and TNF, we measured apoptosis by flow cytometry using double staining with Annexin V-Alexa Fluorescense and propidium iodide. Interestingly, xIAP siRNA-expressing cell lines demonstrated 2–4 fold increase in the induction of apoptosis after treatment with etoposide as compared to a nearly 2 fold increase in those expressing Bcl-xL siRNA (see Table below). No synergism was seen after treatment with TNF. Based on this finding, we then tested a novel small molecule, homolog smac, (SHC, kindly provided by Dr. PG Harren) to determine the possible therapeutic effect of xIAP inhibitors. After titration, the two most effective doses were selected (25 μM and 50 μM) to treat Daudi cell lines for 24hrs, with either etoposide or TNF. At doses of 25 μM , we observed a 2 fold increase in the induction of apoptosis produced by etoposide compared to that seen in control (DMSO + etoposide) or SHC alone and no synergism with TNF confirming the siRNA data. More importantly, at doses of 50 μM, SHC alone demonstrated activity with a 5 fold increase in apoptosis and a nearly 10 fold increase as compared to control (DMSO) when etoposide was added. Overall, we have demonstrated that xIAP and bcl-xL are important in mediating NFkB-resistance to apoptosis. However, our findings suggested that xIAP is a more potent anti-apoptotic signal and opens the door for further drug development aimed at testing xIAP-inhibitors in lymphomas. Induction of Apoptosis in xIAP or Bcl-xL siRNA expressing cell lines siRNA/Compound Etoposide TNF Untreated xIAP 43.1 ± 17.6 17.04 ± 1.4 14.3 ± 2 SC Bcl-xL 18.39± 3.7 9.4 ± 0.22 12.5 ± 2.7 Luc/DMSO 14.9 ± 1.8 14.4 ± 5.6 14.03 ± 1.25 xIAP 9.2 ± 3.2 4.7 ± 0.48 4.6 ± 0.44 Bcl-xL 8.9 ± 0.5 5.3 ± 1.7 4.16 ± 0.4 Daudi Luc/DMSO 5.49 ± 1.71 4.28 ± 0.5 6.2 ± 0.9 SHC 25 μM 20.07 ± 4.8 12.8 ± 3.9 12.1 ± 3.2 SHC 50 μM 47.7 ± 14.55 38.3 ± 0.99 32.7 ± 8.99

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Voruciclib, an Oral, Selective CDK9 Inhibitor, Enhances Cell Death Induced By the Bcl-2 Selective Inhibitor Venetoclax in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-118372 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1361-1361 ◽

Cited By ~ 2

Author(s):

Daniel A Luedtke ◽

Yongwei Su ◽

Holly Edwards ◽

Lisa Polin ◽

Juiwanna Kushner ◽

...

Keyword(s):

Cell Lines ◽

Myeloid Leukemia ◽

Caspase 3 ◽

Combination Index ◽

Survival Rates ◽

Annexin V ◽

Antileukemic Activity ◽

Induction Of Apoptosis ◽

Induced Apoptosis

Abstract Introduction: Patients with acute myeloid leukemia (AML) face overall 5-year survival rates of 65% and 27% for children and adults, respectively, leaving significant room for improvement. Relapse remains a major contributor to such low overall survival rates, and leukemic stem cells (LSCs) that survive treatment are believed to be responsible for AML relapse. The anti-apoptotic protein Bcl-2 is overexpressed in bulk AML cells and LSCs and is associated with poor clinical outcomes. Thus, Bcl-2 represents a promising therapeutic target for the treatment of AML. Venetoclax (ABT-199) is a selective Bcl-2 inhibitor that has shown great potential for treating a number of malignancies, including AML. Venetoclax inhibits Bcl-2, preventing it from sequestering pro-apoptotic Bcl-2 family protein Bim, leading to Bim activated Bax/Bak, resulting in apoptosis. However, Mcl-1 can also sequester Bim and prevent apoptosis. We previously showed that directly targeting Mcl-1 can enhance the antileukemic activity of venetoclax (Luedtke DA, et al. Signal Transduct Target Ther. Apr 2017). Alternatively, we proposed that indirect targeting of Mcl-1 may preserve or enhance the antileukemic activity of venetoclax, and prevent resistance resulting from Mcl-1. It has been reported that inhibition of CDK9 can downregulate cell survival genes regulated by superenhancers, including Mcl-1, MYC, and Cyclin D1. One CDK9 inhibitor in clinical development, flavopiridol (alvocidib), has progressed to phase II clinical trials in AML. However, off target effects and dose-limiting toxicities remain a concern. Voruciclib is an oral, selective CDK inhibitor differentiated by its potent inhibition of CDK9 as compared to other CDK inhibitors. This selectivity may potentially circumvent toxicities resulting from inhibition of non-CDK targets like MAK and ICK that are inhibited by flavopiridol. Voruciclib has been shown in vitro to promote apoptosis and decrease Mcl-1 expression levels in chronic lymphocytic leukemia (CLL) cells (Paiva C, et al. PLOS One. Nov 2015) and inhibit tumor growth in mouse xenograft models of diffuse large B-cell lymphoma (DLBCL) in combination with venetoclax (Dey J. et al Scientific Reports. Dec 2017). Based on these data, voruciclib may downregulate Mcl-1 in AML cells and therefore synergistically enhance the antileukemic activity of venetoclax. Methods/Results: Culturing AML cell lines (THP-1, U937, MOLM-13, MV4-11, and OCI-AML3) and primary patient samples with various concentrations of voruciclib resulted in a concentration-dependent increase in Annexin V+ cells (2 μM voruciclib induced 13.8-55.8% Annexin V+ cells) along with increased levels of cleaved caspase 3 and PARP, demonstrating that voruciclib induces apoptosis in AML cells. Next, we tested the combination of voruciclib and venetoclax in AML cell lines and primary AML patient samples at clinically achievable concentrations. Annexin V/PI staining, flow cytometry analysis, and combination index calculation (using CalcuSyn software) revealed synergistic induction of apoptosis by voruciclib and venetoclax combination (combination index values for MV4-11, U937, THP-1, and MOLM-13 cells were <0.73; treatment with 2 µM voruciclib and venetoclax for 24 h resulted in >80% apoptosis). Importantly, synergy was observed in both venetoclax sensitive and resistant cell lines. This was accompanied by increased cleavage of caspase 3 and PARP. Lentiviral shRNA knockdown of Bak and Bax partially rescued AML cells from voruciclib-induced apoptosis, showing that voruciclib induces apoptosis at least partially through the intrinsic apoptosis pathway. However, Bak and Bax knockdown had little to no effect on induction of apoptosis by the combination treatment, indicating that there might be other molecular mechanisms underlying the synergistic interaction between the two agents. Treatment with the pan-caspase inhibitor Z-VAD-FMK partially rescued cells from combination treatment induced-apoptosis. Discussion: Collectively, these results demonstrate that voruciclib and venetoclax synergistically induce apoptosis in AML cells in vitro and reverse venetoclax resistance. Further studies to determine the mechanism of action and in vivo efficacy of this promising combination in AML xenografts and PDX models are underway. Disclosures Ge: MEI Pharma: Research Funding.

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Bim Promoter Is Highly Methylated in Malignant Lymphoid Cell Lines, Leading to Downregulation of Bim Expression and Protection from Apoptosis.

Blood ◽

10.1182/blood.v106.11.2625.2625 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2625-2625 ◽

Cited By ~ 1

Author(s):

Rocco G. Piazza ◽

Vera Magistroni ◽

Federica Andreoni ◽

Anna Franceschino ◽

Carlo Gambacorti

Keyword(s):

Cell Line ◽

Cell Lines ◽

Lymphoid Cell ◽

Protein Level ◽

Methylation Status ◽

Annexin V ◽

Cpg Sites ◽

Induction Of Apoptosis ◽

High Level ◽

Lymphoid Cell Lines

Abstract Bim, a proapoptotic, BH3-only, Bcl-2 family member, is the major physiological antagonist of the antiapoptotic Bcl-2 proteins in B and T lymphocytes. It is essential for the induction of apoptosis of activated T cells following an immune response and for the homeostasis of B cells; it also plays a key role in the induction of apoptosis of early hematopoietic progenitors following cytokine-deprivation. We performed a CpG Islands prediction analysis on Bim promoter, identifying a putative CpG Island. Using a Bisulfite Modification-Clonal Sequencing Analysis (BMCSA), we investigated the methylation status of 19 CpG sites (from nucleotide −504 to +64 from the ATG start site) in the Bim promoter in 12 malignant hematological cell lines: 7 of lymphoid and 5 of myeloid origin. A minimum of 6 clones were analysed. An homogeneous, very high level of methylation was present in all the lymphoid cell lines (Average Level of Methylation (ALM) 93.4 ± 4.4% Standard Deviation [SD]) and a variable level of methylation in the myeloid cell lines (ALM 37.1 ± 32.4%). The lowest ALM was found in lymphocytes from healthy donors (15.5 ± 2.1%). Evidence of Bim promoter methylation was also found in frozen tumor samples from patients affected by NPM/ALK+ lymphomas. We treated the 12 cell lines with the demethylating agent 5-azacytidine (AZA). The changes in the methylation status of Bim promoter were evaluated by BMCSA and the corresponding induction of Bim by Real-Time PCR (TaqMan) and by Western Blot. The demethylation of Bim promoter led to a potent induction of Bim at the mRNA and protein level. In the lymphoid, NPM/ALK positive, SUDHL-1 cell line, in which a complete demethylation (from 100% to 0%) was achieved, the increase in the expression of Bim was 7.7-fold and this correlated with a potent induction of apoptosis, as assessed by TUNEL and Annexin V assays. Similar results were obtained using a different demethylating agent: 5-aza-2′deoxycytidine (DAC). To assess whether the methylation of Bim promoter is an active process, a wash-out experiment was performed on the SUDHL-1 (high level of methylation, 100%), on the PML/RAR alpha positive myeloid NB4 (intermediate level of methylation, 33%) and on the BCR/ABL positive LAMA-R cell lines (unmethylated) previously treated with AZA or DAC. This experiment showed that the demethylation is reversible and that, following remethylation, the expression of Bim at mRNA and protein level is reduced to the initial value. In the NB4 cell line, in which methylation is clustered on the last 6 CpG sites, remethylation occurs following the same pattern. No de novo methylation was seen in LAMA-R after the wash-out. To address the biological role for the methylation of Bim promoter, we generated a TET-ON inducible system for BimS (the most potent proapoptotic isoform of Bim) in the highly methylated NPM/ALK+ Karpas-299 cell line, showing that, following an induction of Bim expression, the cells are potently induced to apoptosis, as assessed by FACS using TUNEL and Annexin V assays. We conclude that Bim promoter is actively methylated in several leukemias/lymphomas of T and B origin and that its methylation is associated with the downregulation of Bim expression and with protection from apoptosis.

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Role of Phosphatidylinositol 3′- Kinase Pathway in Primary Effusion Lymphoma Survival.

Blood ◽

10.1182/blood.v104.11.2282.2282 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2282-2282

Author(s):

Azhar R. Hussain ◽

Shahab Uddin ◽

Khalid Al-Hussein ◽

Pulicat S. Manogaran ◽

Marina I. Gutierrez ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Kinase Activity ◽

Pi3 Kinase ◽

Phosphatidylinositol 3 Kinase ◽

Fas L ◽

Induced Apoptosis ◽

Pi3 Kinase Pathway ◽

Kinase Pathway

Abstract 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.

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Synergy in Apoptosis Is Achieved in B-NHL by the Combination of Rituximab and the Novel Proteasome Inhibitor NPI-0052: Pivotal Role of Induction of the Immune Surveillance Cancer Gene Product RKIP

Blood ◽

10.1182/blood.v112.11.4973.4973 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4973-4973

Author(s):

Mario I. Vega ◽

Melisa Martinez- Paniagua ◽

Sara Huerta-Yepez ◽

Eriko Suzuki ◽

Kazuo Umezawa ◽

...

Keyword(s):

Cell Lines ◽

Combination Treatment ◽

Proteasome Inhibitor ◽

Single Agent ◽

Gene Products ◽

Apoptotic Gene ◽

Minimal Toxicity ◽

Induction Of Apoptosis ◽

Induced Apoptosis

Abstract Treatment of patients with B-NHL with a combination of rituximab and CHOP resulted in significant clinical response in greater than 90% of patients. The underlying mechanism of synergy achieved in-vivo is not clear; however, our recent studies with B-NHL cell lines revealed rituximab-induced inhibition of intracellular survival pathways that were responsible for reversal of resistance. The combination of rituximab and CHOP is associated with drug-induced toxicity, and thus, it is desirable to have a nontoxic agent that can replace CHOP with similar and improved clinical responses. The proteasome inhibitor, NPI-0052, has been shown to exert minimal toxicity, and induce cytotoxic activity against certain tumor cell lines and is currently in Phase I/II clinical trials as single agent and in combination with Zolinza against various cancers. We have reported that rituximab inhibits the NF-κB pathway concomitantly with the induction of Raf-1 kinase inhibitor protein (RKIP) and inhibition downstream of anti-apoptotic gene products (e.g. Bcl-2, Bc-lXL, Mcl-1, etc.). Likewise, NPI-0052 has also recently been shown to induce the expression of RKIP and inhibits downstream anti-apoptotic gene products. Based on the above findings, we hypothesized that treatment of resistant B-NHL cells with the combination of rituximab and NPI-0052 may result in the complementary induction of apoptosis through additive and/or synergistic effects as a result of inhibiting several survival and anti-apoptotic gene products regulated by NF-κB and induction of RKIP. This study was designed to test this hypothesis. Treatment of Ramos B-NHL cells with rituximab (20 μg/ml for 24 h) or NPI-0052 (20–40 nM) did not yield any significant apoptosis; however, the combination treatment resulted in significant potentiation of apoptosis and synergy was achieved. Treatment with rituximab or NPI-0052 alone resulted in inhibition of the NF-κB pathway, namely, IκBα and downstream BclXL and Mcl-1 and there was no activation of caspases. There was, however, significant induction of RKIP expression by each agent alone. The combination treatment resulted in additive effects with the activation of caspases 8, 9 and 3 and induction of apoptosis. The role of NF-κB inhibition by rituximab in synergy was corroborated with the use of the NF-κB inhibitor, DHMEQ, which sensitized the cells to apoptosis by NPI-0052. The role of RKIP induction in the regulation of apoptosis by NPI-0052 was demonstrated in cells over-expressing RKIP, which were sensitized to NPI-0052-induced apoptosis. In contrast, treatment with si-RNA RKIP reversed rituximab-induced sensitization to NPI-0052-induced apoptosis. Altogether, these findings reveal one mechanism by which rituximab sensitizes B-NHL cells to NPI-0052 apoptosis as the result of the concomitant induction of RKIP and inhibition of the NF-κB survival pathway. The findings also suggest the potential clinical application of rituximab and NPI-0052 in the treatment of patients with B-NHL with minimal toxicity. Furthermore, the findings suggest that agents that can induce RKIP may mimic rituximab in the sensitization to NPI-0052-induced apoptosis and their therapeutic application in patients who are not responsive to rituximab.

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Intact Rac Signaling Is Important for Leukemia Cell Survival

Blood ◽

10.1182/blood.v116.21.2885.2885 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2885-2885

Author(s):

Anja Troeger ◽

Pascal-David Johann ◽

Mumine Senturk ◽

Michael D. Milsom ◽

David A. Williams

Keyword(s):

Western Blot ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Hematopoietic Cells ◽

Leukemia Cell ◽

Jurkat Cells ◽

Knock Down ◽

Induced Apoptosis

Abstract Abstract 2885 Rho GTPases, Ras-related small G proteins, regulate multiple cell processes in hematopoietic cells. There is growing evidence that acute myeloid leukemia (AML) blasts and particularly MLL-rearranged AML blasts, rely on Rac activity (Mulloy JC et al, Blood, 2010). However, little is known about the role of these GTPases in acute lymphoblastic leukemia (ALL) and particularly precursor B cell ALL. To investigate the role of Rac and potential compensation by other GTPases in ALL, we first assessed the protein expression and activation of Rac in a number of B-ALL cell lines (SEM; RS4,11; REH; Nalm 6; Raji), compared with a T-ALL cell line (Jurkat) and several AML cell lines (ML2; MV4,11). Of these cell lines SEM; RS4,11; ML2 and MV4,11 are characterized by MLL-fusion genes. Jurkat and MLL-rearranged AML cell lines show higher expression of Rac proteins compared to B cell leukemia lines (Table 1). Overall, B-ALL cell lines exhibit highly variable levels of Rac expression and activity with no obvious correlation to the presence of MLL-fusion proteins. We then investigated proliferation and apoptosis in cell lines treated with the small molecule inhibitor NSC23766 (NSC), which blocks interaction of a subset of guanine exchange factors (GEFs) with Rac and thus inhibits its activation. Treatment with NSC led to ∼2-fold increase in cells arrested at G0/G1 and induced apoptosis in a dose-dependent fashion at NSC concentrations previously demonstrated to be non-toxic in normal hematopoietic cells (Muller LUW et al., Leukemia, 2008) (Table 2). The lymphoid cell lines Jurkat, Raji and SEM appeared less responsive to NSC with no increased apoptosis at 40μM NSC. There was no correlation between NSC response and baseline expression or activation status of Rac. However, cell lines resistant to NSC exhibited a paradoxical and transient early increase in Rac activation, suggesting the existence of compensatory activation mechanisms. To determine if the relative resistance observed in some cell lines was related to dependence on GEFs not targeted by NSC and to validate that the inhibitory effect of NSC was specifically due to Rac inhibition in sensitive cells, shRNAs were utilized to knock-down different members of the Rac subfamily. Effective shRNA-mediated knockdown was validated by western blot. Knockdown of Rac1 or Rac2 consistently induced apoptosis compared to non-targeting vector controls in NSC sensitive cell lines ML2 and Nalm6, with ML2 cells appearing slightly more sensitive to knock-down of Rac2 (Table 3). Knock-down of either Rac1 or Rac2 had little effect upon Jurkat cells which are resistant to NSC treatment. These data suggest that Jurkat cells are not dependent upon Rac signaling for survival; however we cannot discount the possibility that some compensation may occur between Rac1 and Rac2. These experiments demonstrate the importance of intact Rac signaling pathways for the survival of the majority of leukemia cell lines tested and demonstrate that dependence on Rac signaling is not restricted to leukemias characterized by MLL-rearrangements. Our observations also suggest that activation of different Rac isoforms may influence sensitivity towards pharmacological Rac inhibition. Table 1: Baseline Expression of Rac assessed by Western blot Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji Rac/b-actin expression* 1.6 2.5 1.7 0.5 0.7 0.8 1.0 1.0 (*arbitrary units, italics indicate cell lines carrying MLL-rearrangements) Table 2: % AnnexinV+ cells after treatment of the different cell lines with increasing doses the Rac-specific inhibitor NSC Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji control 6%+1.4 6%+1.3 9%+0.3 12%+3.6 9%+1.9 7%+1.5 9%+2 13%+2.3 20uM NSC 6%+1.4 9%+1.3 15%+0.3** 21%+8.5 8%+1.5 6%+1.9 25%+6.4 16%+3 40uM NSC 7%+1.8 24%+9.1 60%+4** 52%+11* 10%+1.3 10%+3.4 39%+11 16%+1.9 80uM NSC 15%+3.5* 73%+14.7** 97%+0.4** 80%+4** 17%+1.2* 46%+10.5** 62%+12.3* 22%+4 (Mean±SEM; n=5; * p<0.05; ** p<=0.01 versus control, bolded columns indicate increased NSC sensitivity) Table 3: % AnnexinV+ cells 7 days after lentiviral transduction of the different cell lines with Rac1 and Rac2-specific shRNA Cell line Jurkat ML-2 Nalm 6 non targeting control 4.3%+0.3 14.2%+8 11.4%+2.2 Rac1 shRNA* 8.0%+3.5 26.3%+7.9 36.8%+8.5 non targeting control 9.6%+4.2 8.1%+4.0 16.2%+3.1 Rac2 shRNA* 18.7%+4.5 35.5%+12.9 43.7%+7.1 (Mean±SEM; n=6; * second set of Rac1 and Rac2 shRNAs gave comparable results) Disclosures: No relevant conflicts of interest to declare.

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CD45RO regulates the HIV-1 gp120-mediated apoptosis of T cells by activating Lck

Biological Chemistry ◽

10.1515/hsz-2017-0211 ◽

2018 ◽

Vol 399 (6) ◽

pp. 583-591

Author(s):

Kelei Li ◽

Zhe Cong ◽

Zhuoying Peng ◽

Ting Chen ◽

Jing Xue ◽

...

Keyword(s):

T Cells ◽

Cell Line ◽

Cell Lines ◽

Tyrosine Phosphatase ◽

Extracellular Domain ◽

Jurkat Cells ◽

Transfected Cells ◽

Induced Apoptosis ◽

Hiv 1

Abstract CD45 has been reported to regulate the HIV-1 gp120-induced apoptosis of Jurkat cells. Here, we demonstrate that the extracellular domain of CD45 plays an important role in this function. We observed that CD45RO-transfected cells, but not cells transfected with other CD45 isoforms, underwent significant apoptosis induced by gp120. However, a CD45RA-transfected cell line treated with an O-glycan inhibitor was able to undergo apoptosis. The role of the extracellular domain of CD45 was further confirmed using CD45 isoform-transfected cell lines by analyzing the phosphorylation of Lck, which is a direct substrate of CD45 tyrosine phosphatase, and by using an Lck inhibitor. These results suggest that CD45RO modulates HIV-1 gp120-induced apoptosis by regulating the activity of Lck.

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Zoledronate Can Overcome Drug Resistance in a Dexamethasone Resistant Myeloma Cell Line.

Blood ◽

10.1182/blood.v104.11.4894.4894 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4894-4894

Author(s):

Masayuki Koizumi ◽

Chiaki Nakaseko ◽

Chikako Ohwada ◽

Ryuko Cho ◽

Miki Nishimura ◽

...

Keyword(s):

Dna Synthesis ◽

Cell Line ◽

Cell Lines ◽

Antitumor Effect ◽

Annexin V ◽

Myeloma Cell ◽

Myeloma Cell Line ◽

Human Myeloma Cell Line ◽

Induced Apoptosis

Abstract <Introduction> Multiple myeloma (MM) is almost invariably fatal despite all available chemotherapeutic and supportive treatment. Initial treatment of MM with dexamethasone (Dex), a key drug for MM chemotherapy, effectively induces myeloma cell death. However, prolonged drug exposure results in the development of Dex-resistance and clinical failures. Therefore, one of the most important issues in myeloma therapy is to overcome resistance to Dex at the stage of clinical refractoriness. Several recent in-vitro studies have demonstrated an antitumor effect of nitrogen-containing amino-bisphosphonates (N-BPs) in some tumor cell lines, including myeloma cell lines. However, few data are available concerning the effects of N-BPs on Dex resistant myeloma cell lines. In this study, we have established a Dex-resistant human myeloma cell line and investigated the antitumor effect of the third generation bisphosphonate Zoledronate (ZOL) on the Dex-resistant subline. <Methods> 1) Development of de novo resistance through continuous exposure to Dex. A Dex-resistant human myeloma cell line (Dex-R) was selected from the Dex-sensitive myeloma cell line RPMI8226 by continuously exposing cells to gradually increasing doses of Dex. Resistance was validated by flow cytometry. 2) Apoptosis assay. Dual-color FACS with propidium iodide (PI) and Annexin V was used to detect Dex-induced apoptosis in RPMI8226 and Dex-R. 3) Surface markers. Phenotypes of sensitive and resistant cells were compared by FACS analysis. 4) Cell Proliferation and DNA synthesis assay. RPMI8226 and Dex-R cells were cultured in 96 wells plates for 72 hours in the absence or presence of Dex (1 μM) or increasing concentrations of ZOL (2.5 to 50 μM). Cell growth was assessed by WST-8 assay kit. DNA synthesis was measured according to thymidine uptake. 5) Actin staining. RPMI8226 and Dex-R cells were cultured for 48 hours in the absence or presence of Dex (2 μM) or ZOL (40 μM). The distribution of f-actin stained by fluorescent phalloidin was investigated by confocal fluorescence microscopy. <Results> The developed subline Dex-R showed reduced apoptotic and antiproliferative responses to Dex treatment. Flow cytometry on 24 hours Dex exposure revealed significantly low percentage of apoptotic (Annexin V positive and PI negative) population in Dex-R cells compared with RPMI8226 cells (6.7% vs. 29.9%). Our data demonstrate that Dex-R cells showed increased CD38, CD44, CD49d (VLA-4) expression, and decreased CD45, CD95 (Fas), CD138 (Syndecan-I) expression as compared to native RPMI8226 cells. ZOL induced apoptosis and inhibited DNA synthesis and cell proliferation in both RPMI8226 and Dex-R cell lines in a dose-dependant manner. Interestingly, some modifications of the cellular morphology were observed in ZOL-treated cells. Rhodamine-phalloidin staining of the f-actin cytoskeleton showed disruption of the cytoskeleton in these deformed cells. This fibroblast-like cellular morphology was common to Dex-R and RPMI8226 cells treated with ZOL, but was not detected under Dex treatment. This result might reflect different mechanism of antitumor effect between Dex and ZOL.. <Conclusion> Our results suggest that ZOL can induce myeloma cell death in vitro in a different mechanistic way than Dex. ZOL might be effective as an antitumor drug for Dex resistant myeloma.

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Chemotherapy Sensitizing Effects of Methadone in Glioblastoma Cells Are Drug- and Cell Line-dependent

10.21203/rs.3.rs-62227/v1 ◽

2020 ◽

Author(s):

Bodo Haas ◽

Janine Ciftcioglu ◽

Sanja Jermar ◽

Sandra Weickhardt ◽

Niels Eckstein ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Pain Treatment ◽

Annexin V ◽

Response Curves ◽

Drug Concentrations ◽

Data Points ◽

Induction Of Apoptosis ◽

Apoptosis And Necrosis ◽

Mtt Assays

Abstract Background D,L-methadone (MET), an analgesic drug used for pain treatment and opiate addiction has achieved attention from oncologist and social media as possible chemosensitizing agent in glioblastoma multiforme (GBM) treatment. MET has been reported to enhance doxorubicin-induced cytotoxicity in GBM cells via activation of the µ-opioid receptor (MOR) and subsequent apoptosis induction. Here, we further aimed at quantifying MET effects in comparison to other opioids alone and in combination with doxorubicin and clinically more relevant temozolomide (TMZ) in a set of GBM cell lines and primary GBM cells. Methods MOR expression in GBM cells was investigated by immunofluorescence and immunoblotting. Resistance to drugs alone or in combination was assessed by MTT assays. Concentration effect curves were fitted to data points by nonlinear regression analysis and IC50 values were calculated. Apoptotic rates were determined by Annexin V staining. Results We found that MET alone was cytotoxic to GBM cells at high micromolar concentrations in MTT assays by induction of apoptosis and necrosis while morphine and oxycodone were hardly cytotoxic. Naloxone was not able to block MET-induced cytotoxicity, indicating that cell death inducing effects of MET are not MOR dependent. We recorded doxorubicin and TMZ concentration response curves by MTT assays in combination with fixed MET concentrations. MET only enhanced doxorubicin cytotoxicity in one cell line and in part in primary cells at certain MET concentrations. MET was not effective in sensitizing cells towards TMZ. Contrarily, in two cell lines MET even decreased sensitivity towards TMZ. Conclusions MET can be considered cytotoxic to GBM cells only at clinically not relevant concentrations by induction of apoptosis and necrosis. Sensitizing effects are only observed in combination with doxorubicin but not with TMZ and are highly dependent on cell line and applied drug concentrations.

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