Rapamycin inhibits the growth of cholangiocarcinoma cells in vitro

15153 Background: In the present study, anti-neoplastic effect of rapamycin against cholangiocarcinoma was studied in vitro. Methods: Expression of mTOR in 4 cholangiocarcinoma cell lines, TFK1, HuCCT1, NOZW, and OZ was evaluated by real-time PCR. Then, the four cholangiocarcinoma cell lines were cultured with rapamycin (0, 25, 50, 100, 200 nM), gemcitabine (0, 0.5, 1, 2 μM), or both, and anti-proliferative effect was evaluated by MTT assay. Results: All the four cholangiocarcinoma cell lines expressed endogenous mTOR- mRNA. Level of expression was the highest in HuCCT1 (65.8), and the lowest in TFK1 (17.6). Then, rapamycin significantly inhibited the growth of all the four cholangiocarcinoma cell lines, in dose-dependent manner. Gemcitabine inhibited the growth of NOZW (48.4%) and HuCCT1 (48.9%), but less efficiently in TFK1 (5.9%) and OZ (27.4%). Furthermore, synergistic anti-proliferative effect of rapamycin and gemcitabine was observed in TFK1 (39.1%), NOZW (38.9%), and OZ (47.1%), not in HuCCT1 (18.9%). Conclusion: Rapamycin effectively inhibited the growth of the cholangiocarcinoma cell lines, and synergistic effect with gemcitabine was observed in three of the four cell lines. No significant financial relationships to disclose.

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Hif-1α Inhibitors Could Successfully Inhibit the Progression of Differentiated Thyroid Cancer in Vitro

Pharmaceuticals ◽

10.3390/ph13090208 ◽

2020 ◽

Vol 13 (9) ◽

pp. 208

Author(s):

Min-Hee Kim ◽

Tae Hyeong Lee ◽

Jin Soo Lee ◽

Dong-Jun Lim ◽

Peter Chang-Whan Lee

Keyword(s):

Cell Proliferation ◽

Thyroid Cancer ◽

Cell Lines ◽

Cancer Progression ◽

Hypoxia Inducible Factor ◽

Soft Agar ◽

Dependent Manner ◽

Thyroid Cancer Cell Lines ◽

Dose Dependent

Hypoxia-inducible factor (HIF)-1α plays an important role in cancer progression. In various cancers, including thyroid cancer, overexpression of HIF-1α is related to poor prognosis or treatment response. However, few studies have investigated the role of HIF-1α inhibition in thyroid cancer progression. We evaluated the utility of the HIF-1α inhibitor IDF-11774 in vitro utilizing two thyroid cancer cell lines, K1 and BCPAP. Both cell lines were tested to elucidate the effects of IDF-11774 on cell proliferation and migration using soft agar and invasion assays. Here, we found that a reduction of HIF-1α expression in BCPAP cells was observed after treatment with IDF-11774 in a dose-dependent manner. Moreover, cell proliferation, migration, and anchorage-independent growth were effectively inhibited by IDF-11774 in BCPAP cells but not in K1 cells. Additionally, invasion of BCPAP but not K1 cells was controlled with IDF-11774 in a dose-dependent manner. Our findings suggest that promoting the degradation of HIF-1α could be a strategy to manage progression and that HIF-1α inhibitors are potent drugs for thyroid cancer treatment.

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Lenalidomide Strongly Enhances Natural Killer (NK) Cell Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) of Rituximab Treated Non-Hodkin’s Lymphoma Cell Lines In Vitro.

Blood ◽

10.1182/blood.v108.11.3714.3714 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3714-3714 ◽

Cited By ~ 2

Author(s):

Lei Wu ◽

Peter Schafer ◽

George Muller ◽

David Stirling ◽

J. Blake Bartlett

Keyword(s):

Nk Cells ◽

Tumor Cells ◽

Cell Lines ◽

Nk Cell ◽

Dependent Manner ◽

Activation Markers ◽

Early Results ◽

Ifn Γ ◽

Dose Dependent

Abstract Lenalidomide (Revlimid® is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone is for the treatment of multiple myeloma patients who have received at least one prior therapy. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL). Potential mechanisms of action include anti-angiogenic, anti-proliferative and immunomodulatory activities. Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Furthermore, lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo. We have utilized an in vitro ADCC system to assess the ability of lenalidomide to directly enhance human NK cell function in response to therapeutic antibodies, such as rituximab (chimeric anti-CD20 mAb). Isolated NK cells produced little or no IFN-γ in response to IgG and/or IL-2 or IL-12. However, pre-treatment of NK cells with lenalidomide greatly enhanced IFN-γ production by NK cells in a dose-dependent manner. In a functional ADCC assay, NHL cell lines (Namalwa, Farage & Raji) were pre-coated with rituximab and exposed to NK cells pre-treated with lenalidomide in the presence of either exogenous IL-2 or IL-12. After 4 hours in culture the viability of the tumor cells was assessed. Lenalidomide consistently and synergistically increased the killing of tumor cells in a dose-dependent manner and up to >4-fold compared to rituximab alone. Rituximab alone had only a small effect in this model and there was no killing of cells in the absence of rituximab. The presence of either exogenous IL-2 or IL-12 was required to see enhanced killing by lenalidomide. In cancer patients lenalidomide has been shown to increase serum IL-12 levels and is also known to induce IL-2 production by T cells in vitro. Potential mechanisms for enhanced ADCC include increased signaling through NK FCγ receptors and/or IL-2 or IL-12 receptors. However, we found that these receptors are unaffected by lenalidomide, although downstream effects on NK signaling pathways are likely and are being actively investigated. In conclusion, we have shown that lenalidomide strongly enhances the ability of rituximab to induce ADCC mediated killing of NHL cells in vitro. This provides a strong rationale for combination of these drugs in patients with NHL and CLL.

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Effect of the green tea polyphenol epigallocatechin gallate (EGCG) on growth and IL-6 signalling pathways in malignant plasma cells

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.8114 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 8114-8114

Author(s):

R. Burger ◽

H. Czekalla ◽

K. Richter ◽

T. Ahrens ◽

A. Guenther ◽

...

Keyword(s):

Cell Lines ◽

Green Tea ◽

Epigallocatechin Gallate ◽

Myeloma Cell ◽

Signalling Pathways ◽

Dependent Manner ◽

Green Tea Polyphenol ◽

Dose Dependent ◽

Human Myeloma Cell

8114 Background: Epigallocatechin gallate (EGCG) is the predominant polyphenolic constituent of green tea leaves that possesses antitumor, antiinflammatory, and antioxidant activity. EGCG exerts its effects through potentially multiple mechanisms including inhibition of growth factor receptor signalling. The compound is currently under investigation in a phase I/II clinical trial for treatment of patients with early stage chronic lymphocytic leukemia at Mayo Clinic. The goal of our study was to examine the in vitro effects of EGCG in multiple myeloma (MM). Methods: A panel of human myeloma cell lines (n=6) including the IL-6 dependent INA-6 cell line was used to evaluate the sensitivity to EGCG. Cells were cultured for three days in the absence or presence of EGCG at concentrations between 6.25 μM and 100 μM. Cell viability was determined in a colorimetric tetrazolium (MTS) based assay and by trypanblue exclusion. For signalling experiments, INA-6 cells were IL-6 and serum starved and then treated with EGCG for two hours before IL-6 was added. Whole cell lysates were prepared and subjected to SDS-PAGE and Western blot analysis. Results: EGCG inhibited the in vitro growth of human myeloma cell lines by inducing cell death in a time and dose-dependent manner. IC50 concentrations were between 12,5 μM and 50 μM. IL-6 mediated growth of INA-6 cells was inhibited at similar doses. The addition of excess amounts of IL-6 could not protect from EGCG induced cytotoxicity. Pretreatment of INA-6 cells with EGCG resulted in a dose-dependent inhibition of IL-6 induced STAT3 tyrosine phosphorylation. In these cells, stimulation with IL-6 leads to upregulation of Mcl-1 expression. In contrast, phosphorylation of p44/p42 MAPK, which is constitutively activated in INA-6 cells, was not affected. Conclusion: EGCG has growth inhibitory activity on myeloma cells. Specific inhibition of signalling pathways that regulate expression of anti-apoptotic proteins could be one mechanism how EGCG exerts its activity. Our work provides the rationale for further studies to evaluate the effect of EGCG not only in B-CLL, but also in plasma cell tumors. No significant financial relationships to disclose.

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Inhibition of Tumor Cell Proliferation In Vitro by Benzamide Derivatives

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.997.225 ◽

2014 ◽

Vol 997 ◽

pp. 225-228 ◽

Cited By ~ 1

Author(s):

Yan Ling Wu ◽

Li Wen Shen ◽

Yan Ping Ding ◽

Yoshimasa Tanaka ◽

Wen Zhang

Keyword(s):

Tumor Cell ◽

Cell Lines ◽

Malignant Tumors ◽

Dependent Manner ◽

Tumor Cell Lines ◽

Lead Compounds ◽

Proliferative Effect ◽

Benzamide Derivatives

Benzamide derivatives have been shown to have antitumor activity in various tumor cell lines in vitro as well as in vivo. In this study, we examined the anti-proliferative effect of four benzamide derivativeson Hela, H7402, and SK-RC-42 tumor cell lines in vitro by means of Real-Time cell assay (RTCA), and found that four benzamide derivatives suppressed proliferation of tumor cells in a time-and dose-dependent manner. The anti-proliferative activity of benzamide derivatives demonstrated that theycould be promising lead compounds for developing therapeutic agents for malignant tumors.

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An Immune Based, Anti-CD138 Targeting Antibody for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-119112 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5617-5617 ◽

Cited By ~ 3

Author(s):

Tengteng Yu ◽

Lijie Xing ◽

Liang Lin ◽

Jiye Liu ◽

Kenneth Wen ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Myeloma Cell ◽

Dependent Manner ◽

Cellular Cytotoxicity ◽

Adcc Activity ◽

Effector Cells ◽

Equity Ownership ◽

Dose Dependent

Abstract CD138 (Syndecan-1), a member of integral membrane family of heparan sulfate proteoglycans (HSPGS), is highly expressed on differentiated plasma cells (PC) and is both a primary diagnostic biomarker of multiple myeloma (MM) as well as an indicator of poor clinical prognosis. This surface antigen is an attractive candidate for targeted immunotherapy for MM, given its constitutive expression during disease progression, including smoldering myeloma, a relatively early asymptomatic phase of disease that is potentially amenable to early treatment. We here investigated the targeted use of chimeric anti-CD138 monoclonal antibody (mAb) 1610 and confirm its in vitro anti-tumor potency based on an immune directed cellular cytotoxicity against a diverse panel of CD138 positive MM cell lines, both resistant or sensitive to conventional and current MM therapies and varying levels of CD138 expression as measured by cell immunostaining and quantitative RT-PCR. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using a calcein-AM based release assay in the presence of human natural killer (NK) effector cells purified from four different healthy donors. MAb 1610 lysed CD138-expressing MM cell lines in a dose dependent manner. This ADCC activity was mAb 1610 specific (in comparison to isotype control), CD138 target dependent, and mediated in the presence of human NK effector cells (co-cultured at an effector:target cell ratio of 20:1). MAb 1610 dependent-cytotoxicity was observed at concentrations as low as 0.01 µg/ml with maximal lysis occurring at approximately 1 µg/ml and extrapolated sub-nanomolar ED50 potencies (Table 1) based on these data. All MM cell lines were subject to mAb 1610-mediated lysis, albeit with slightly different sensitivities that modestly correlated with their relative CD138 cell surface expression levels. This anti CD138 mAb-dependent cellular toxicity included MM1SR and H929R cell lines, both of which are resistant to lenalidomide. MAb 1610 induced specific cell lysis of JJN3 cells, but not of CD138 knock out JJN3 cells or CD138-negative B lymphocytes, further confirming that mAb 1610 specifically induced ADCC against-CD138 expressing MM cells in a target specific manner. Using an orthogonal cytometric based assay, the ability of mAb 1610, in a dose-dependent manner, to activate NK cells was also shown in the presence of CD138 target cells, as evidenced by increased expression of CD107 (a marker for NK cell degranulation) and cytokine production in NK cells. Importantly, the CD138 targeting cytotoxic activities of mAb 1610 translationally extend to MM cells autologously derived directly from MM patients with newly diagnosed and relapsed/refractory diseases. The concomitant use of autologously derived effector cells from these patients to mediate antibody dependent myeloma cell killing further suggests the relevance of anti-CD138 directed immune-based therapeutic strategy in humans. In further replication of human disease, we also co-cultured MM1.S or MM1.R cells with human bone marrow stromal cells (BMSCs) which support myeloma cell growth by promoting an immunosuppressive microenvironment within the BM. Importantly, mAb 1610-dependent cytotoxicity against MM1.S or MM1.R cells was not attenuated by the co-presence of BMSCs. Similarly, IL-6 (10 ng/ml) did not significantly affect mAb 1610-induced ADCC activity, indicating a mechanism of action that can overcome growth promotion, immune suppression, and drug resistance conferred by the tumor promoting BM microenvironment. Taken together, these in vitro studies further demonstrate as a proof-of-concept the use of an antibody CD138 targeting strategy mediated through an immune based mechanism of myeloma plasma cell killing. Based on these results, optimization and further biological characterization of chimeric mAb 1610 in advance of pre-clinical studies is anticipated. Disclosures Myette: Visterra Inc.: Employment. Chaganty:Visterra Inc.: Employment. Adari:Visterra Inc.: Employment. Tissire:Visterra Inc.: Employment. Deotale:Visterra Inc.: Employment. Shriver:Visterra Inc.: Employment. Munshi:OncoPep: Other: Board of director. Anderson:Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; Bristol Myers Squibb: Consultancy.

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Cytotoxic effect of Montivipera bornmuelleri’s venom on cancer cell lines: in vitro and in vivo studies

PeerJ ◽

10.7717/peerj.9909 ◽

2020 ◽

Vol 8 ◽

pp. e9909

Author(s):

Carol Haddoub ◽

Mohamad Rima ◽

Sandrine Heurtebise ◽

Myriam Lawand ◽

Dania Jundi ◽

...

Keyword(s):

Tumor Growth ◽

Cell Lines ◽

Cytotoxic Effect ◽

In Vivo Studies ◽

Dependent Manner ◽

In Vivo Testing ◽

Murine Fibrosarcoma ◽

Dose Dependent

Background Montivipera bornmuelleri’s venom has shown immunomodulation of cytokines release in mice and selective cytotoxicity on cancer cells in a dose-dependent manner, highlighting an anticancer potential. Here, we extend these findings by elucidating the sensitivity of murine B16 skin melanoma and 3-MCA-induced murine fibrosarcoma cell lines to M. bornmuelleri’s venom and its effect on tumor growth in vivo. Methods The toxicity of the venom on B16 and MCA cells was assessed using flow cytometry and xCELLigence assays. For in vivo testing, tumor growth was followed in mice after intratumoral venom injection. Results The venom toxicity showed a dose-dependent cell death on both B16 and MCA cells. Interestingly, overexpression of ovalbumin increased the sensitivity of the cells to the venom. However, the venom was not able to eradicate induced-tumor growth when injected at 100 µg/kg. Our study demonstrates a cytotoxic effect of M. bornmuelleri’s venom in vitro which, however, does not translate to an anticancer action in vivo.

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Regulation of c-ret expression by retinoic acid in rat metanephros: implication in nephron mass control

AJP Renal Physiology ◽

10.1152/ajprenal.1998.275.6.f938 ◽

1998 ◽

Vol 275 (6) ◽

pp. F938-F945 ◽

Cited By ~ 4

Author(s):

Evelyne Moreau ◽

José Vilar ◽

Martine Lelièvre-Pégorier ◽

Claudie Merlet-Bénichou ◽

Thierry Gilbert

Keyword(s):

Retinoic Acid ◽

Molecular Mechanisms ◽

Kidney Development ◽

Mrna Level ◽

Cell Surface Receptor ◽

Dependent Manner ◽

All Trans Retinoic Acid ◽

Surface Receptor ◽

Dose Dependent

Vitamin A and its derivatives have been shown to promote kidney development in vitro in a dose-dependent fashion. To address the molecular mechanisms by which all- trans-retinoic acid (RA) may regulate the nephron mass, rat kidneys were removed on embryonic day 14( E14) and grown in organ culture under standard or RA-stimulated conditions. By using RT-PCR, we studied the expression of the glial cell line-derived neurotrophic factor (GDNF), its cell surface receptor-α (GDNFR-α), and the receptor tyrosine kinase c-ret, known to play a major role in renal organogenesis. Expression of GDNF and GDNFR-α transcripts was high at the time of explantation and remained unaffected in culture with or without RA. In contrast, c-ret mRNA level, which was low in E14 metanephros and dropped rapidly in vitro, was increased by RA in a dose-dependent manner. The same is true at the protein level. Exogenous GDNF barely promotes additional nephron formation in vitro. Thus the present data establish c-ret as a key target of retinoids during kidney organogenesis.

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Lestaurtinib (CEP701) Inhibits Proliferation and Activates Apoptosis In Hodgkin Lymphoma Cells through Inhibition of the JAK/STAT Signaling Pathway

Blood ◽

10.1182/blood.v116.21.3919.3919 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3919-3919

Author(s):

Alfons Navarro ◽

Tania Díaz ◽

Antonio Martinez ◽

Anna Gaya ◽

Mariano Monzó

Keyword(s):

Hodgkin Lymphoma ◽

Cell Lines ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Dependent Manner ◽

Constitutive Activation ◽

Downstream Target ◽

Dose Dependent ◽

Stat Pathway

Abstract Abstract 3919 Background: The constitutive activation of the JAK/STAT pathway plays an important role in the pathogenesis and proliferation of Hodgkin Lymphoma (HL). Although somatic activating point mutations in the JAK2 gene have been reported in myeloproliferative disorders (MPD), they are rarely described in HL, where JAK2 amplification is associated with mutations of regulator genes such as SOCS-1, constitutive activation of STAT proteins or miRNA deregulation. Recently, many JAK2 inhibitors, including Lestaurtinib (CEP701), have been reported to have clinical efficacy in MPD. CEP701 is a multitargeted tyrosine kinase inhibitor that potently inhibits FLT3 at nanomolar concentrations. Recent studies in MPD have further shown that CEP701 inhibitory activity is not limited to FLT3 and can suppress JAK2/STAT5 signaling through JAK2 inhibition. As a first step towards elucidating the potential role of CEP701 in HL therapy, we have analyzed its efficacy in vitro. Methods: Four HL cell lines, L-428, L-1236, HDMYZ and L-540, were assayed for proliferation, apoptosis and levels of proteins in the JAK2/STAT pathway (pJAK2, JAK2, pSTAT5, STAT5, Bcl-xL) after CEP701 treatment. 100,000 cells were plated in a 96-well plate in 100 ml culture medium with CEP701 or DMSO (vehicle control) at concentrations of 30–300 nM. After 1 or 24 hours of incubation with CEP701, the levels of the proteins and of FLT3 were analyzed by Western blot. Proliferation was analyzed with CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) and apoptosis by CaspaseGlo 3/7 after 48 hours of treatment. Results: The proliferation analysis showed an effective dose-dependent inhibition of cell growth in the 4 HL cell lines after treatment with increasing concentrations of CEP701. At 48h, in comparison to cells treated with DMSO alone (normalized to 100%), in cells treated with 100nM of CEP701, we observed a marked inhibition of 35% in L-428, 55% in L-1236, 15% in HDMYZ and 77% in L-540. Moreover, apoptosis increased by 38%, 31%, 21% and 25%, respectively. The protein analysis showed that after one hour, CEP701 inhibited phosphorylation of JAK2 (pJAK2) and its downstream target STAT5 (pSTAT5) in a dose-dependent manner, with no changes in the non-phosphorylated proteins. The downstream target Bcl-xL also decreased. Conclusions: Taken together, these data demonstrate that growth inhibition and apoptosis activation by CEP701 in HL cells correlates with the inhibition of the JAK2/STAT5-dependent signal transduction pathway. Here we present the first biological evidence that Lestaurtinib could be a promising new agent in the treatment of patients with HL. Supported by a FIS grant (PS09/00547). Disclosures: No relevant conflicts of interest to declare.

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PEG10 is associated with treatment-induced neuroendocrine prostate cancer

Journal of Molecular Endocrinology ◽

10.1530/jme-18-0226 ◽

2019 ◽

Vol 63 (1) ◽

pp. 39-49 ◽

Cited By ~ 6

Author(s):

Soojin Kim ◽

Daksh Thaper ◽

Samir Bidnur ◽

Paul Toren ◽

Shusuke Akamatsu ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Lines ◽

Resistant Cell ◽

Dependent Manner ◽

Castration Resistant ◽

Neuroendocrine Prostate Cancer ◽

Marker Status ◽

Dose Dependent

Neuroendocrine (NE) differentiation of advanced prostate adenocarcinoma following androgen receptor (AR) axis-directed therapy is becoming increasingly recognized. Several models of this transdifferentiation provide insight into its molecular pathogenesis and have highlighted the placental gene PEG10 for further study. Using our unique model of enzalutamide resistance (ENZR) and NE differentiation, we studied PEG10/AR interplay in enzalutamide treatment-resistant cell lines 42DENZR and 42FENZR compared to LNCaP and castration-resistant 16DCRPC cells. ENZR cell lines with positive terminal NE marker status also displayed higher baseline expression of PEG10 compared to LNCaP and 16DCRPC. Antagonism of AR activity increased PEG10 expression followed by an increase in terminal NE markers. Conversely, stimulating AR activity via androgen supplementation reversed PEG10 and NE marker expression in a time and dose-dependent manner. These results were supported by human data showing that PEG10 expression is highest in NEPC and that AR-dependent gene, PSA, is negatively correlated with PEG10 in adenocarcinoma. Further, ChIP assay confirmed binding of activated AR to the PEG10 enhancer, decreasing PEG10 expression. While PEG10 did not drive NEPC, its knockdown reduced NE markers in our cell lines. Moreover, PEG10 knockdown in vitro- and in vivo-attenuated tumor growth. Overall, these observations indicate that PEG10 is an AR-repressed gene which modulates NE markers in ENZR cells and targeting PEG10 in advanced prostate cancer with NE features is a rational and viable option.

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Regulation of Histone Deacetylase in Waldenström Macroglobulinemia

Blood ◽

10.1182/blood.v112.11.2617.2617 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2617-2617

Author(s):

Xiaoying Jia ◽

Aldo M. Roccaro ◽

Abdel Kareem Azab ◽

Hai T. Ngo ◽

Antonio Sacco ◽

...

Keyword(s):

Bone Marrow ◽

Western Blot ◽

Histone Deacetylase ◽

Cell Lines ◽

Parp Cleavage ◽

Low Grade ◽

Dependent Manner ◽

Apoptotic Pathways ◽

Dose Dependent

Abstract Background: Waldenström’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Histone deacetylase (HDAC) inhibitors represent promising new treatment strategy in B cell malignancies. We therefore investigated the in vitro effect of the novel hydroxamic acid derivative HDAC inhibitor LBH589 in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTS assay and thymidine uptake assay. Cell signaling and apoptotic pathways were determined by Western Blot and immunofluorescence. Results: LBH589 induced a significant decrease of proliferation and triggered cytotoxicity in all cell lines tested and primary CD19+ WM cells (IC50 of 20–40nM), even in the presence of BMSC, IL-6 and IGF-1, which induce resistance to conventional therapies. Importantly, LBH589 did not induce cytotoxicity in healthy donor peripheral blood mononuclear cells. LBH589 induced both intrinsic and extrinsic apoptotic pathways, with caspase-9, caspase-8, caspase-3, and PARP cleavage in a dose-dependent manner. We also demonstrated significant upregulation of the proapoptotic transcription factor p53 and down-regulation of the anti-apoptotic proteins BclxL, Mcl-1 and c-myc. We then demonstrated that LBH589 induced apoptosis in WM cells in a caspase-independent manner through induction of autophagy, as shown by upregulation of LC3B and Rab7 expression. We further determined the mechanism of action of LBH589 in WM, investigating the effect of LBH589 on histone acetylation and NF-kB pathways. We found that LBH589 induced a dose-dependent increase in histone H3-H4 acetylation; and inhibited both canonical and non-canonical pathways of NF-κB, as shown by western blot and immunofluorescence. In addition, LBH589 augmented rituximab, fludarabine, bortezomib, and perifosine-induced cyotoxicity in WM cells. Conclusion: LBH589 has significant antitumor activity in WM in vitro, providing the framework for clinical trials evaluating LBH589 as a new therapeutic agent in patients with WM.

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