Protein kinase C inhibitor enzastaurin induces in vitro and in vivo antitumor activity in Waldenström macroglobulinemia

Blood ◽  
2007 ◽  
Vol 109 (11) ◽  
pp. 4964-4972 ◽  
Author(s):  
Anne-Sophie Moreau ◽  
Xiaoying Jia ◽  
Hai T. Ngo ◽  
Xavier Leleu ◽  
Garrett O'Sullivan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Antitumor Activity ◽  
Mononuclear Cells ◽  
Xenograft Model ◽  
Parp Cleavage ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Synergistic Cytotoxicity

AbstractWaldenström macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Protein kinase Cβ (PKCβ) regulates cell survival and growth in many B-cell malignancies. In this study, we demonstrate up-regulation of PKCβ protein in WM using protein array techniques and immunohistochemistry. Enzastaurin, a PKCβ inhibitor, blocked PKCβ activity and induced a significant decrease of proliferation at 48 hours in WM cell lines (IC50, 2.5-10 μM). Similar effects were demonstrated in primary CD19+ WM cells, without cytotoxicity on peripheral blood mononuclear cells. In addition, enzastaurin overcame tumor cell growth induced by coculture of WM cells with bone marrow stromal cells. Enzastaurin induced dose-dependent apoptosis at 48 hours mediated via induction of caspase-3, caspase-8, caspase-9, and PARP cleavage. Enzastaurin inhibited Akt phosphorylation and Akt kinase activity, as well as downstream p-MARCKS and ribosomal p-S6. Furthermore, enzastaurin demonstrated additive cytotoxicity in combination with bortezomib, and synergistic cytotoxicity in combination with fludarabine. Finally, in an in vivo xenograft model of human WM, significant inhibition of tumor growth was observed in the enzastaurin-treated mice (P = .028). Our studies therefore show that enzastaurin has significant antitumor activity in WM both in vitro and in vivo, providing the framework for clinical trials to improve patient outcome in WM.

scholarly journals Dual targeting of the proteasome regulates survival and homing in Waldenström macroglobulinemia

Blood ◽  
2008 ◽  
Vol 111 (9) ◽  
pp. 4752-4763 ◽  
Author(s):  
Aldo M. Roccaro ◽  
Xavier Leleu ◽  
Antonio Sacco ◽  
Xiaoying Jia ◽  
Molly Melhem ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Proteasome Inhibitors ◽  
B Cell Lymphoma ◽  
Parp Cleavage ◽  
Low Grade ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Synergistic Cytotoxicity

AbstractWaldenström macroglobulinemia (WM) is an incurable low-grade B-cell lymphoma characterized by high protein turnover. We dissected the biologic role of the proteasome in WM using 2 proteasome inhibitors, NPI-0052 and bortezomib. We found that NPI-0052 inhibited proliferation and induced apoptosis in WM cells, and that the combination of NPI-0052 and bortezomib induced synergistic cytotoxicity in WM cells, leading to inhibition of nuclear translocation of p65NF-κB and synergistic induction of caspases-3, -8, and -9 and PARP cleavage. These 2 agents inhibited the canonical and noncanonical NF-κB pathways and acted synergistically through their differential effect on Akt activity and on chymotrypsin-like, caspaselike, and trypsinlike activities of the proteasome. We demonstrated that NPI-0052–induced cytotoxicity was completely abrogated in an Akt knockdown cell line, indicating that its major activity is mediated through the Akt pathway. Moreover, we demonstrated that NPI-0052 and bortezomib inhibited migration and adhesion in vitro and homing of WM cells in vivo, and overcame resistance induced by mesenchymal cells or by the addition of interleukin-6 in a coculture in vitro system. Theses studies enhance our understanding of the biologic role of the proteasome pathway in WM, and provide the preclinical basis for clinical trials of combinations of proteasome inhibitors in WM.

scholarly journals Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1654-1664 ◽  
Author(s):  
Dharminder Chauhan ◽  
Ajita Singh ◽  
Mohan Brahmandam ◽  
Klaus Podar ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitors ◽  
Xenograft Model ◽  
Er Stress Response ◽  
Synergistic Cytotoxicity ◽  
Proteolytic Activities ◽  
Dose Combination ◽  
Inhibition Of Tumor Growth

AbstractOur recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in multiple myeloma (MM) cells, and importantly, that is distinct from bortezomib (Velcade) in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 plus bortezomib–induced synergistic apoptosis is associated with: (1) activation of caspase-8, caspase-9, caspase-3, and PARP; (2) induction of endoplasmic reticulum (ER) stress response and JNK; (3) inhibition of migration of MM cells and angiogenesis; (4) suppression of chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) proteolytic activities; and (5) blockade of NF-κB signaling. Studies in a xenograft model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth and CT-L, C-L, and T-L proteasome activities in tumor cells. Immununostaining of MM tumors from NPI-0052 plus bortezomib–treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Taken together, our study provides the preclinical rationale for clinical protocols evaluating bortezomib together with NPI-0052 to improve patient outcome in MM.

scholarly journals Dual targeting of the PI3K/Akt/mTOR pathway as an antitumor strategy in Waldenstrom macroglobulinemia

Blood ◽  
2010 ◽  
Vol 115 (3) ◽  
pp. 559-569 ◽  
Author(s):  
Aldo M. Roccaro ◽  
Antonio Sacco ◽  
Emanuel N. Husu ◽  
Costas Pitsillides ◽  
Steven Vesole ◽  
...  
Keyword(s):  
Significant Inhibition ◽  
Mtor Pathway ◽  
Clinical Activity ◽  
Independent Manner ◽  
Waldenström Macroglobulinemia ◽  
Constitutive Activation ◽  
Dual Targeting ◽  
Waldenstrom Macroglobulinemia

Abstract We have previously shown clinical activity of a mammalian target of rapamycin (mTOR) complex 1 inhibitor in Waldenstrom macroglobulinemia (WM). However, 50% of patients did not respond to therapy. We therefore examined mechanisms of activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR in WM, and mechanisms of overcoming resistance to therapy. We first demonstrated that primary WM cells show constitutive activation of the PI3K/Akt pathway, supported by decreased expression of phosphate and tensin homolog tumor suppressor gene (PTEN) at the gene and protein levels, together with constitutive activation of Akt and mTOR. We illustrated that dual targeting of the PI3K/mTOR pathway by the novel inhibitor NVP-BEZ235 showed higher cytotoxicity on WM cells compared with inhibition of the PI3K or mTOR pathways alone. In addition, NVP-BEZ235 inhibited both rictor and raptor, thus abrogating the rictor-induced Akt phosphorylation. NVP-BEZ235 also induced significant cytotoxicity in WM cells in a caspase-dependent and -independent manner, through targeting the Forkhead box transcription factors. In addition, NVP-BEZ235 targeted WM cells in the context of bone marrow microenvironment, leading to significant inhibition of migration, adhesion in vitro, and homing in vivo. These studies therefore show that dual targeting of the PI3K/mTOR pathway is a better modality of targeted therapy for tumors that harbor activation of the PI3K/mTOR signaling cascade, such as WM.

scholarly journals SHC014748M, a Novel Selective Inhibitor of PI3Kδ, Demonstrates Promising Pre-Clinical Antitumor Activity in B Cell Lymphomas and CLL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5306-5306
Author(s):  
Lei Fan ◽  
Chao Wang ◽  
Zhiqiang Wang ◽  
Xian Zhang ◽  
Lei Cao ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
B Cell ◽  
Cell Lines ◽  
Xenograft Model ◽  
Selective Inhibitor ◽  
Lymphoma Cell ◽  
Class I ◽  
B Cell Lymphomas

Introduction : PI3Kδ, one of the class I PI3Ks, is found expressed primarily in leukocytes and plays an essential role in B-cell development and function. Here, we comprehensively evaluated the in vitro and in vivo antitumor activity and the underlying mechanism of SHC014748M, an oral selective inhibitor of PI3Kδ under Phase I clinical evaluation. Methods : Biochemical and cell-based assays were used to measure compound potency and selectivity in lymphoma cell lines as well as primary CLL cells, and PI3K/AKT pathway was measured by Western blot assay, Alphalisa and Elisa. Xenograft model was carried out to validate in-vivo antitumor potency of the compound. Besides, chemokines and cytokines derived from blood samples of patients were also detected. Results: SHC014748M was 125- to 306-fold more selective for PI3Kδ inhibition relative to other class I PI3K enzymes and showed in vitro activity in most of 23 B lymphoma cell lines. We identified that SHC014748M treatment resulted in a 3.1- to 5.5-fold increase in annexin V/7-ADD staining, indicating a significant apoptosis induction. SHC014748M inhibited phosphorylation of AKT, targets downstream of PI3Kδ, in lymphoma cells. Among the 15 primary CLL cells, the 50% inhibitory concentration (IC50) of SHC014748M varies from 850 nM to 37040 nM respectively and expression of phosphorylation AKT decreased to the normal levels in the presence of SHC014748M or positive control, Idelalisib. In-vivo study revealed that SHC014748M significantly reduced lymphoma cell growth in the treatment group compared with control mice. CCL4, CCL17, CCL22 and CXCL13 derived from patients decreased sharply after SHC014748M treatment. Conclusion: According to the results, SHC014748M appeared to be a novel promising compound in the treatment of B cell lymphomas and CLL. Disclosures Wang: Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment. Wang:Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment. Zhang:Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment.

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

Blood ◽  
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.

Lidocaine Induces Apoptosis and Suppresses Tumor Growth in Human Hepatocellular Carcinoma Cells In Vitro and in a Xenograft Model In Vivo

2017 ◽  
Vol 126 (5) ◽  
pp. 868-881 ◽  
Author(s):  
Wei Xing ◽  
Dong-Tai Chen ◽  
Jia-Hao Pan ◽  
Yong-Hua Chen ◽  
Yan Yan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Antitumor Activity ◽  
Hepg2 Cells ◽  
Xenograft Model ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Bax Protein

Abstract Background Recent epidemiologic studies have focused on the potential beneficial effects of regional anesthetics, and the differences in cancer prognosis may be the result of anesthetics on cancer biologic behavior. However, the function and underlying mechanisms of lidocaine in hepatocellular carcinoma both in vitro and in vivo have been poorly studied. Methods Human HepG2 cells were treated with lidocaine. Cell viability, colony formation, cell cycle, and apoptosis were assessed. The effects of lidocaine on apoptosis-related and mitogen-activated protein kinase protein expression were evaluated by Western blot analysis. The antitumor activity of lidocaine in hepatocellular carcinoma with or without cisplatin was investigated with in vitro experiments and also with animal experiments. Results Lidocaine inhibited the growth of HepG2 cells in a dose- and time-dependent manner. The authors also found that lidocaine arrested cells in the G0/G1 phase of the cell cycle (63.7 ± 1.7% vs. 72.4 ± 3.2%; P = 0.0143) and induced apoptosis (1.7 ± 0.3% vs. 5.0 ± 0.7%; P = 0.0009). Lidocaine may exert these functions by causing an increase in Bax protein and activated caspase-3 and a corresponding decrease in Bcl-2 protein through the extracellular signal-regulated kinase 1/2 and p38 pathways. More importantly, for the first time, xenograft experiments (n = 8 per group) indicated that lidocaine suppressed tumor development (P < 0.0001; lidocaine vs. control) and enhanced the sensitivity of cisplatin (P = 0.0008; lidocaine plus cisplatin vs. cisplatin). Conclusions The authors’ findings suggest that lidocaine may exert potent antitumor activity in hepatocellular carcinoma. Furthermore, combining lidocaine with cisplatin may be a novel treatment option for hepatocellular carcinoma.

scholarly journals microRNA expression in the biology, prognosis, and therapy of Waldenström macroglobulinemia

Blood ◽  
2009 ◽  
Vol 113 (18) ◽  
pp. 4391-4402 ◽  
Author(s):  
Aldo M. Roccaro ◽  
Antonio Sacco ◽  
Changzhong Chen ◽  
Judith Runnels ◽  
Xavier Leleu ◽  
...  
Keyword(s):  
Expression Profiling ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Staging System ◽  
Microrna Expression ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
And Migration

AbstractMultilevel genetic characterization of Waldenström macroglobulinemia (WM) is required to improve our understanding of the underlying molecular changes that lead to the initiation and progression of this disease. We performed microRNA-expression profiling of bone marrow–derived CD19+ WM cells, compared with their normal cellular counterparts and validated data by quantitative reverse-transcription–polymerase chain reaction (qRT-PCR). We identified a WM-specific microRNA signature characterized by increased expression of microRNA-363*/-206/-494/-155/-184/-542-3p, and decreased expression of microRNA-9* (ANOVA; P < .01). We found that microRNA-155 regulates proliferation and growth of WM cells in vitro and in vivo, by inhibiting MAPK/ERK, PI3/AKT, and NF-κB pathways. Potential microRNA-155 target genes were identified using gene-expression profiling and included genes involved in cell-cycle progression, adhesion, and migration. Importantly, increased expression of the 6 miRNAs significantly correlated with a poorer outcome predicted by the International Prognostic Staging System for WM. We further demonstrated that therapeutic agents commonly used in WM alter the levels of the major miRNAs identified, by inducing downmodulation of 5 increased miRNAs and up-modulation of patient-downexpressed miRNA-9*. These data indicate that microRNAs play a pivotal role in the biology of WM; represent important prognostic marker; and provide the basis for the development of new microRNA-based targeted therapies in WM.

scholarly journals Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl)

Blood ◽  
2006 ◽  
Vol 109 (4) ◽  
pp. 1669-1677 ◽  
Author(s):  
Klaus Podar ◽  
Marc S. Raab ◽  
Jing Zhang ◽  
Douglas McMillin ◽  
Iris Breitkreutz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Xenograft Model ◽  
Multidrug Resistant ◽  
The Novel ◽  
Pkc Inhibitor ◽  
Downstream Signaling ◽  
Synergistic Cytotoxicity ◽  
And Migration

Abstract In multiple myeloma (MM) protein kinase C (PKC) signaling pathways have been implicated in cell proliferation, survival, and migration. Here we investigated the novel, orally available PKC-inhibitor enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits phorbol ester–induced activation of PKC isoforms, as well as phosphorylation of downstream signaling molecules MARCKS and PKCμ. Importantly, it also inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs), costimulation with fibronectin, vascular endothelial growth factor (VEGF), or interleukin-6 (IL-6), as well as MM patient serum. Consequently, enzastaurin inhibits proliferation, survival, and migration of MM cell lines and MM cells isolated from multidrug-resistant patients and overcomes MM-cell growth triggered by binding to BMSCs and endothelial cells. Importantly, strong synergistic cytotoxicity is observed when enzastaurin is combined with bortezomib and moderate synergistic or additive effects when combined with melphalan or lenalidomide. Finally, tumor growth, survival, and angiogenesis are abrogated by enzastaurin in an in vivo xenograft model of human MM. Our results therefore demonstrate in vitro and in vivo efficacy of the orally available PKC inhibitor enzastaurin in MM and strongly support its clinical evaluation, alone or in combination therapies, to improve outcome in patients with MM.

scholarly journals PI3K inhibitor significantly enhances the antitumor activity of oncolytic virus in osteosarcoma

2020 ◽  
Author(s):  
Ye Wang ◽  
Xin Yang ◽  
Hong Li ◽  
Haiyang Xie
Keyword(s):  
Clinical Trials ◽  
Antitumor Activity ◽  
Oncolytic Virus ◽  
Xenograft Model ◽  
In Vitro Cytotoxicity ◽  
Pi3k Inhibitor ◽  
Oncolytic Viruses ◽  
Osteosarcoma Cell

Abstract Background: Osteosarcoma (OS) is a highly aggressive malignancy with less than 30% 5-year survival rate among patients with metastatic or recurrent cancer. However, the treatment for osteosarcoma has not been modified in the last three decades. Oncolytic viruses have shown encouraging results in pre-clinical trials, but have failed to translate into high therapeutic efficacy in clinical trials. In this study, we will determine the therapeutic effect of combining PI3K inhibitor with an oncolytic virus against osteosarcoma. Material and Methods: Osteosarcoma cell lines and xenograft model were treated with ZSTK474 and/or VSVΔ51, the tumor suppressive ability was verified by in vitro cytotoxicity experiments and in vivo antitumor activity experiments, and the antitumor mechanism was explored through the study of apoptosis-related signaling pathways. Results: ZSTK474 sensitized the osteosarcoma cells to VSVΔ51, and augmented apoptosis via endoplasmic reticulum stress. The combination treatment also showed greater in vivo tumor inhibition compared to either ZSTK474 or VSVΔ51 alone, and significantly enhanced the tumor infiltration of immune cells. Conclusion: PI3K inhibitors combined with oncolytic virus is a promising strategy against osteosarcoma.

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