Resveratrol Plus Simvastatin Are Effective in Decreasing IgM Secretion in Asymptomatic Waldenstrom Macroglobulinemia. One Year Follow-up.

Abstract Abstract 4401 Background Waldenström macroglobulinemia (WM) is a distinct B-cell lymphoproliferative disorder characterized by lymphoplasmacytic bone marrow infiltration along with an immunoglobulin M (IgM) monoclonal gammopathy. Asymptomatic patients with monoclonal IgM and without morphologic evidence of bone marrow infiltration < 10% clonal marrow cells) are classified as having IgM-MGUS.Therapy is postponed for asymptomatic patients, and progressive anemia is the most common indication for initiation of treatment. Resveratrol (3,4',5-tri-hydroxy-trans-stilbene) is an antioxidant constituent of a wide variety of plant species including grapes. It has gained considerable attention because of its anticancer properties, as shown in solid and hematologic malignancies. Published data show that resveratrol has significant antitumor activity in WM cells line. Moreover, simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor, induced inhibition of proliferation, cytotoxic effect and apoptosis in IgM secreting cell lines as well as in primary CD19(+) WM cells. With this background we have treated 4 patients with asymptomatic WM with an association of simvastatin and resveratrol to test the efficacy of such of drugs in asymptomatic Waldenstrom macroglobulinemia Methods 4 pts (3 males and 1 female), median age 42,3 yrs (range, 42–73) and asymptomatic WM were treated with a schedule containing resveratrol 40 mg/die and simvastatin 20 mg/die for at least 90 days. At enrollment patients characteristic were hemoglobin level median, 12.1 g/dL,serum beta(2)-microglobulin level median, 2.4 mg/L, and IgM peaks median, 1.8 g/dL. All patients have taken regularly the drugs and there have been no adverse events.CK and LDH serum levels were kept in the normal range. Results In all IgM-MGUS patients a reduction of more than 50% of the IgM peak was observed after 3 months of therapy and it was still maintained at 12 months of follow-up. In SWM patient the reduction was about 25% and it was manteined over time. Striking, another patient with Waldentrom disease resistant to the previous therapy with EDX and anti-CD20 MoAb achieved a CR only after adding resveratrol and simvastatin. Conclusions Our data demonstrate clearly that the association between resveratrol with simvastatin decreases IgM secretion in Waldenstrom macroglobulinaemia and can be useful in asymptomatic or low risk patients not having any adverse effects. Disclosures: Off Label Use: Simvastatin showed in vitro activity on waldentrom cell lines Resveratrol showed in vitro activity on waldenstro cell lines.

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Flavopiridol Induces Apoptosis in Chronic Lymphocytic Leukemia Cells Via Activation of Caspase-3 Without Evidence of bcl-2 Modulation or Dependence on Functional p53

Blood ◽

10.1182/blood.v92.10.3804 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3804-3816 ◽

Cited By ~ 197

Author(s):

John C. Byrd ◽

Charlotte Shinn ◽

Jamie K. Waselenko ◽

Ephraim J. Fuchs ◽

Teresa A. Lehman ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Caspase 3 ◽

Mononuclear Cells ◽

P53 Protein ◽

Lymphocytic Leukemia ◽

Vitro Activity ◽

Interleukin 4 ◽

In Vitro Activity

Abstract Flavopiridol has been reported to induce apoptosis in lymphoid cell lines via downregulation of bcl-2. The in vitro activity of flavopiridol against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing cytotoxicity were studied. The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at a flavopiridol concentration of 1.15 μmol/L (95% confidence interval [CI] ±0.31), 0.18 μmol/L (95% CI ±0.04), and 0.16 μmol/L (95% CI ±0.04), respectively. Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.18 μmol/L of flavopiridol resulted in both decreased expression of p53 protein and cleavage of the caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changes in bcl-2 protein expression alterations. We evaluated flavopiridol’s dependence on intact p53 by exposing splenocytes from wild-type (p53+/+) and p53 null (p53−/−) mice that demonstrated no preferential cytotoxicity as compared with a marked differential with F-ara-a and radiation. Incubation of CLL cells with antiapoptotic cytokine interleukin-4 (IL-4) did not alter the LC50 of flavopiridol, as compared with a marked elevation noted with F-ara-a in the majority of patients tested. These data demonstrate that flavopiridol has significant in vitro activity against human CLL cells through activation of caspase-3, which appears to occur independently of bcl-2 modulation, the presence of IL-4, or p53 status. Such findings strongly support the early introduction of flavopiridol into clinical trials for patients with B-CLL.

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MYD88 Negative Waldenstrom Macroglobulinemia Has Distinct Clinical & Biological Features As Compared To Its MYD88 Mutant Counterpart

Blood ◽

10.1182/blood.v122.21.4299.4299 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4299-4299

Author(s):

Nikhil V Patkar ◽

Prashant Deshpande ◽

Russel Mascarenhas ◽

PG Subramanian ◽

Prashant Tembhare ◽

...

Keyword(s):

Bone Marrow ◽

Partial Response ◽

Conflicts Of Interest ◽

International Prognostic Index ◽

Post Treatment ◽

Waldenström Macroglobulinemia ◽

Waldenstrom Macroglobulinemia ◽

Myd88 L265p Mutation ◽

Myd88 L265p

Abstract Introduction Waldenstrom Macroglobulinemia (WM) harbors a mutation in MYD88 gene (MYD88L265P) with frequencies varying from 67% to 91%. Although of diagnostic use its clinical significance in terms of prognosis and treatment response is unclear. We retrospectively analyzed WM for MYD88 L265P mutation, immunogenetic profile (presence of somatic hypermutations and biased gene usage) & correlated these with standard clinical variables including prognosis and patient outcome. Patients & Methods 32 cases WM (diagnosed as per WHO 2008/2001 criteria) were retrospectively accrued from 2007-2013. Genomic DNA extracted from bone marrow aspirate smears was subjected to an allele specific oligonucleotide PCR to detect the MYD88L265P mutation using fluorescently labeled primers followed by capillary electrophoresis. Immunogenetics was assessed in 29 patients. Clonal FR1/FR2 regions of the VH gene were amplified & sequenced. Sequence data was compared to the closest germline sequences on NCBI & IMGT databases. Laboratory variables (Hb, WBC, platelet, M Protein concentration, S. IgM, b2M level, S. Globulin, LDH, %of lymphoplasmacytic lymphocytes) were evaluated at baseline along with the International Prognostic Index (ISSWM). Response evaluation was done as per VIth International Consensus guidelines after treatment as well as at last follow up. 2-tailed Student's t-Test & Chi squared test were applied for statistical analysis. Results Median age was 60 years (range: 46-77), male predominant (87.5%).Majority of patients had cytopenia (90.6%) of one or more blood lineages. Median IWSSM was 3 (n=26). The median follow up was 21.5 months (range: 1 week to 82 months). Majority of patients were treated with cyclophosphamide/vincristine/prednisone ± rituximab (55.1%), followed in others by bendamustine/rituximab(13.8%) or fludarabine/cyclophosphamide/rituximab,(13.8%) or cyclophosphamide/thalidomide/dexamethasone (10.3%). MYD88 L265P mutation was found in 84.3% (27/32) of patients. The immunogenetic results here pertain only to samples with productive IGHV gene rearrangement [22/29 (∼76%) cases]. 96% of cases revealed somatic hypermutations. 59% of cases showed a biased use for the VH3 gene followed by VH4 (22.7%) and VH1 (18.18%). The commonest gene used was IGHV3-7 (27.3%) followed by IGHV1-18 (18.2%). Clinical features separating MYD88 negative from MYD88 mutated WM are seen in Table 1. MYD88 negative WM presented with lower number of infiltrating tumor cells in the bone marrow (p=0.05), older age (p=0.02) and had a lower IWSSM score at presentation (p=0.03) as compared to mutated WM. Majority of the MYD88 negative group were in VGPR,(very good partial response) or CR (complete response) (75%:VGPR/CR) post treatment as compared to MYD88 mutated patients [21%: VGPR/CR, 31.6%: PR (partial response): 26.3%, SD (stable disease):15.8%, PD (progressive disease):6.3%]. At the last follow up 44.4% of MYD88 mutated WM had PD where as no patient in MYD88 WT had changed their initial post treatment status. Two patients with MYD88 mutation died due to disease related complications. Conclusion Our data indicates that WM is a biologically heterogeneous subset dichotomized by MYD88 mutations. WM patients with MYD88 mutations present at younger age with high tumor burden in the bone marrow, high risk of progression and poor therapeutic response. Although limited in number, MYD88 negative WM patients were not associated with PD as compared to the mutated group. Overall MYD88mutation may be considered as an adverse prognostic factor in WM. Disclosures: No relevant conflicts of interest to declare.

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Evaluation of essential oils from 22 Guatemalan medicinal plants for in vitro activity against cancer and established cell lines

Journal of Medicinal Plants Research ◽

10.5897/jmpr2017.6528 ◽

2018 ◽

Vol 12 (3) ◽

pp. 42-49 ◽

Cited By ~ 1

Author(s):

Byron Miller Andrew ◽

Gordon Cates Rex ◽

O’Neill Kim ◽

Alfonso Fuentes Soria Juan ◽

Vicente Espinoza Luis ◽

...

Keyword(s):

Medicinal Plants ◽

Essential Oils ◽

Cell Lines ◽

Vitro Activity ◽

In Vitro Activity ◽

Established Cell Lines ◽

Established Cell

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In vitro activity of sorafenib against imatinib- and sunitinib-resistant kinase mutations associated with drug-resistant GI stromal tumors

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.10500 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 10500-10500

Author(s):

M. C. Heinrich ◽

R. Carden ◽

D. Griffith ◽

C. Liang ◽

A. Marino-Enriquez ◽

...

Keyword(s):

Cell Lines ◽

Binding Pocket ◽

Vitro Activity ◽

Activation Loop ◽

Second Line ◽

In Vitro Activity ◽

Stromal Tumors ◽

Line Therapy ◽

Exon 11

10500 Background: Most gastrointestinal stromal tumors (GISTs) harbor mutant KIT or platelet-derived growth factor receptor alpha (PDGFRA) kinases, which are targets of imatinib (front-line therapy) or sunitinib (second-line therapy). Resistance to imatinib (IM) or sunitinib (SU) is commonly associated with the acquisition of secondary kinase mutations. In phase II studies, sorafenib (SOR), which targets KIT, PDGFRs, and several other kinases, has demonstrated efficacy in GIST patients after failure of IM and SU. We evaluated the in vitro activity of SOR against IM and/or SU resistant kinases. Methods: Kinase mutants were biochemically profiled for SOR, SU, and IM sensitivity by measuring inhibition of kinase phosphorylation after drug treatment in mutant-expressing CHO cells. We also tested the biochemical and cellular activity of SOR and IM against GIST cells derived from IM-resistant tumor clones. Results: SOR had superior potency to IM against IM-resistant KIT secondary mutations involving the ATP binding pocket (V654A, T670I) and the activation loop (D816H, D820A/G, N822K, Y823D). SOR and SU had similar potency against IM-resistant KIT secondary mutations involving the ATP binding pocket. In contrast, SOR had markedly increased potency, compared to SU, against IM-resistant KIT activation loop mutations. Both SOR and SU were more potent than IM against the isolated KIT exon 9 mutation. To confirm these observations in a GIST cellular context, we tested the relative potency of IM and SU against two previously described IM-resistant GIST cell lines (GIST430: exon 11 + V654A, GIST48: exon 11 + D820A). SOR was significantly more potent than IM against KIT in these cell lines. This also correlated with enhanced inhibition of downstream signaling pathways and cellular proliferation. Conclusions: SOR has superior in vitro potency compared with IM or SU against a panel of GIST-relevant mutant kinases. Notably, SOR, unlike SU, is active against most IM-resistant secondary mutations involving the KIT activation loop. Based on these results, we hypothesize that SOR might have superior clinical activity to SU in the setting of imatinib-resistant GIST and should be further evaluated for clinical efficacy in the second-line setting. [Table: see text]

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Cyclin-Dependent Kinase Inhibitor Seliciclib Shows In Vitro Activity in Diffuse Large B Cell Lymphomas.

Blood ◽

10.1182/blood.v108.11.4738.4738 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4738-4738

Author(s):

Francesco Bertoni ◽

Katia Lacrima ◽

Andrea Rinaldi ◽

Sara Vignati ◽

Vittoria Martin ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Active Agent ◽

B Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Vitro Activity ◽

In Vitro Activity ◽

Large B Cell

Abstract Background. Despite recent improvements in treatment, a significant fraction of patients with diffuse large B cell lymphoma (DLBCL) still fail therapy. Therefore, new therapeutic modalities are needed to advance the cure rate. Seliciclib (CYC202, R-roscovitine) is a purine analogue developed as an inhibitor of CDK2/cyclin E CDK7/cyclin H and CDK9/cyclin T. Seliciclib has been shown to be active in B cell neoplasms, such as mantle cell lymphoma, chronic lymphocytic leukemia and in multiple myeloma in vitro. The aim of this study was to assess the in vitro activity of seliciclib in DLBCL. Materials and methods. The anti-proliferative activity of seliciclib was tested in nine human DLBCL cell lines and six DLBCL primary cell cultures. The effects of seliciclib on the cell cycle and on apoptosis, as well as on transcription-related proteins were assessed. Results. The cell viability of all DLBCL cell lines and primary cells was reduced by seliciclib treatment. The IC50 for the cell lines ranged from 13 to 36 μM. The effect of seliciclib was independent of the genetic aberrations characterizing the cell lines. After seliciclib exposure cells accumulated in G2/M or in G1 phase, with most of the cells showing signs of apoptosis. Despite the clear cytotoxic effect and induction of apoptosis, we could not identify a unique mechanism of action. Conclusions. Our in vitro data suggest that seliciclib is an active agent in DLBCL. Its efficacy is apparently independent of the underlying chromosomal translocations characteristic of DLBCL. The drug might represent a new therapeutic agent in this lymphoma subtype.

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Selective Inhibition of the Chymotrypsin-Like Activity of the Immunoproteasome and Constitutive Proteasome Represents a Valid Anti-Tumor Strategy in Waldenstrom Macroglobulinemia.

Blood ◽

10.1182/blood.v114.22.4911.4911 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4911-4911

Author(s):

Aldo M. Roccaro ◽

Antonio Sacco ◽

Monette Aujay ◽

Hai Ngo ◽

Feda Azab ◽

...

Keyword(s):

Bone Marrow ◽

Dna Synthesis ◽

Cell Lines ◽

Parp Cleavage ◽

Low Grade ◽

Employment Equity ◽

Waldenström Macroglobulinemia ◽

Waldenstrom Macroglobulinemia ◽

Synergistic Cytotoxicity ◽

Equity Ownership

Abstract Abstract 4911 Introduction Proteasome inhibition represents a valid therapeutical approach in several tumors and its use has been validated in Waldenstrom macroglobulinemia (WM), where single-agent Bortezomib has been tested in phase 2 clinical trials, achieving 40% to 80% responses. Nevertheless, a significant fraction of patients relapse, or develop significant neuropathy. Therefore preclinical evaluation of new proteasome inhibitor is needed in order to improve patient outcome. We tested PR-047, a new orally bioavailable analog of carfilzomib which selectively inhibits the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome, in WM. Methods WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Expression of imunoproteasome and constitutive proteasome subunits (beta1, beta2, beta5; LMP2, MECL1, LMP7) were detected primary WM cells and cell lines by an ELISA-based assay. Cytotoxicity, DNA synthesis, cell cycle and apoptosis were measured by thymidine uptake, MTT, PI staining/flow cytometry analysis and DNA fragmentation, respectively. NF-kB activity has been evaluated on nuclear proteins using a DNA-binding ELISA-based assay. Cell signaling and apoptotic pathways were determined by Western Blot. Determination of the additive or synergistic effect of drugs combination was calculated using the CalcuSyn software based on the Chou-Talalay method. Results Primary bone-marrow derived WM cells are characterized by higher expression of the immunopreoteasome as compared to the constitutive proteasome. PR-047 inhibited the chymotrypsin-like activity of both the immunoproteasome (LMP7) and the constitutive proteasome (beta5) and in WM cells, leading to induction of cytotoxicity in primary WM cells; as well as to programmed cell death in a caspase-dependent and -independent manner, as shown by activation of c-jun-N-terminal kinase; inhibition of NF-kB; and initiation of the unfolded protein response. PR-047 induced cytotoxicity and inhibited DNA synthesis in primary WM cells (IC50: 50-80nM), as well as in IgM secreting low grade lymphoma cells (IC50: 30-50nM). Importantly, PR-047 exerted cytotoxicity in WM cells, even in the context of bone marrow milieu, by inhibiting IL-6- and IGF1-BMSC secretion and BMSCs-induced phosphorylation of Akt and ERK in WM cells. Moreover, combination of PR-047 and bortezomib induced synergistic cytotoxicity in WM cells, as shown by enhanced caspases-, PARP-cleavage; NF-kB inhibition; and synergy in inhibiting the chymotrypsin- and caspase-like activities of the immunoproteasome and constitutive proteasome. Conclusion These preclinical findings demonstrate that PR-047 targets WM cells, due to its anti-CT-L activity of both immunoproteasome and constitutive proteasome, providing the framework for testing PR-047 in this disease. Disclosures Aujay: Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

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Significant in-Vitro Activity of a Novel JAK2 Inhibitor in Multiple Myeloma Associated with Preferential Cytotoxicity for CD45+ Myeloma Cells.

Blood ◽

10.1182/blood.v114.22.2848.2848 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2848-2848

Author(s):

Vijay Ramakrishnan ◽

Jessica Haug ◽

Teresa Kimlinger ◽

Timothy Halling ◽

Linda Wellik ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Lines ◽

Research Funding ◽

Myeloma Cell ◽

Vitro Activity ◽

In Vitro Activity ◽

Myeloma Cells ◽

Stat Pathway

Abstract Abstract 2848 Poster Board II-824 Background: Multiple myeloma remains incurable with current therapies and novel approaches based on disease biology are needed. IL-6 is a critical cytokine involved in myeloma cell proliferation and survival and exerts its activity primarily through the JAK/STAT pathway. In addition to IL6, other cytokines are also believed to cross talk with the JAK/STAT pathway, making it a crucial interface for survival signals. It has been implicated in myeloma cell interaction with the microenvironment and resistance to apoptotic stimuli from different drugs, and represents a potential therapeutic target. We examined the pre-clinical activity of a novel JAK2 tyrosine kinase inhibitor TG101209. Methods: TG101209 (N-tert-butyl-3-(5-methyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-ylamino)-benzenesulfonamide) was synthesized by TargeGen Inc. (San Diego, CA, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and using Apo2.7 in primary patient cells. CD45 expression was estimated using flow cytometry and cells were gated by their CD45 expression to assess differential effects of the drug. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways. Results: TG101209 resulted in a dose and time dependent inhibition of cell growth in the MM cell lines tested. Most of the cytotoxicity was evident by 48 hours, with minimal increase seen up to 96 hours of incubation. At 48 hours of incubation, the median inhibitory concentration was between 2 and 4uM with similar IC50 seen for myeloma cell lines sensitive or resistant to conventional therapies. The IC50s were maintained when the cells were treated in co-culture with stromal cells or in the presence of IL6, IGF or VEGF. Increasing doses of IL6 was not able to rescue the cells from the drug. Dose dependent decrease in proliferation of the cell lines was evidenced by decreased thymidine incorporation. Apoptotic changes in cells following drug treatment was confirmed by flow cytometry for Annexin and PI. Cleavage of caspases 3, 8 and 9 were confirmed on flow cytometry. Addition of the pan-caspase inhibitor zvad-fmk did not prevent drug-induced apoptosis confirming non-caspase mediated mechanisms of cell death as well. Primary myeloma cells from several patients were treated with increasing doses of the drug and IC50 similar to cell lines were seen in 8/10 patient samples tested. Interestingly, evaluation of U266 cell lines, which have a mix of CD45+ and negative cells as well as primary patient cells demonstrated more profound cytotoxicity and anti-proliferative activity of the drug on the CD45+ population relative to the CD45- cells. Immunoblotting studies demonstrated significant down regulation of IL-6 induced pSTAT3 with minor effects on the pERK and pAkt. The effect on pSAT3 was sustained compared to that on pERK and pAkt. This was accompanied by significant down regulation of Bcl-xL. Studies in a mouse model of myeloma are planned. Conclusion: These studies demonstrate significant in-vitro activity of JAK2 inhibition in multiple myeloma. In particular, the preferential targeting of CD45 cells, considered to reflect the proliferative compartment in myeloma holds out the promise for more sustained impact on the disease from a therapeutic standpoint. This is likely explained by the increased sensitivity of the CD45 cells to cytokines as a result of higher expression of different cytokine receptors as has been previously shown. This leads to increased activity of and dependence of the cells on the JAK-STAT pathway and likely explains the increased effect of the pathway inhibition. These studies form the framework for clinical evaluation of the drug in the setting of myeloma. Disclosures: Kumar: CELGENE: Research Funding; MILLENNIUM: Research Funding; BAYER: Research Funding; GENZYME: Research Funding; NOVARTIS: Research Funding.

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Flavopiridol Induces Apoptosis in Chronic Lymphocytic Leukemia Cells Via Activation of Caspase-3 Without Evidence of bcl-2 Modulation or Dependence on Functional p53

Blood ◽

10.1182/blood.v92.10.3804.422k36_3804_3816 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3804-3816 ◽

Cited By ~ 7

Author(s):

John C. Byrd ◽

Charlotte Shinn ◽

Jamie K. Waselenko ◽

Ephraim J. Fuchs ◽

Teresa A. Lehman ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Caspase 3 ◽

Mononuclear Cells ◽

P53 Protein ◽

Lymphocytic Leukemia ◽

Vitro Activity ◽

Interleukin 4 ◽

In Vitro Activity

Flavopiridol has been reported to induce apoptosis in lymphoid cell lines via downregulation of bcl-2. The in vitro activity of flavopiridol against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing cytotoxicity were studied. The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at a flavopiridol concentration of 1.15 μmol/L (95% confidence interval [CI] ±0.31), 0.18 μmol/L (95% CI ±0.04), and 0.16 μmol/L (95% CI ±0.04), respectively. Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.18 μmol/L of flavopiridol resulted in both decreased expression of p53 protein and cleavage of the caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changes in bcl-2 protein expression alterations. We evaluated flavopiridol’s dependence on intact p53 by exposing splenocytes from wild-type (p53+/+) and p53 null (p53−/−) mice that demonstrated no preferential cytotoxicity as compared with a marked differential with F-ara-a and radiation. Incubation of CLL cells with antiapoptotic cytokine interleukin-4 (IL-4) did not alter the LC50 of flavopiridol, as compared with a marked elevation noted with F-ara-a in the majority of patients tested. These data demonstrate that flavopiridol has significant in vitro activity against human CLL cells through activation of caspase-3, which appears to occur independently of bcl-2 modulation, the presence of IL-4, or p53 status. Such findings strongly support the early introduction of flavopiridol into clinical trials for patients with B-CLL.

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