Single Agent and Synergistic Activity of Maritoclax with ABT-263 in Nasopharyngeal Carcinoma (NPC) Cell Lines

The BCL-2 anti-apoptotic proteins are over-expressed in many cancers and hence are attractive therapeutic targets. In this study, we tested the sensitivity of two Nasopharyngeal Carcinoma (NPC) cell lines HK1 and C666-1 to Maritoclax, which is reported to repress anti-apoptotic protein MCL-1 and BH3 mimetic ABT-263, which selectively inhibits anti-apoptotic proteins BCL-2, BCL-XL and BCL-w. We investigated the sensitisation of the NPC cell lines to these drugs using the SYBR Green I assay and 3D NPC spheroids. We report that Maritoclax repressed anti-apoptotic proteins MCL-1, BCL-2, and BCL-XL in a dose- and time-dependent manner and displayed a single agent activity in inhibiting cell proliferation of the NPC cell lines. Moreover, combination of Maritoclax and ABT-263 exhibited synergistic antiproliferative effect in the HK1 cells. Similar results were obtained in the 3D spheroids generated from the HK1 cells. More notably, 3D HK1 spheroids either treated with single agent Maritoclax or combination with ABT-263, over 10 days, did not develop resistance to the treatment rapidly. Collectively, the findings illustrate that Maritoclax as a single agent or combination with BH3 mimetics could be potentially useful as treatment strategies for the management of NPC.

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Single Agent and Synergistic Activity of Maritoclax with ABT-263 in Nasopharyngeal Carcinoma (NPC) Cell Lines

10.1101/471888 ◽

2018 ◽

Author(s):

Benedict Shi Xiang Lian ◽

Kwok-Wai Lo ◽

Alan Soo-Beng Khoo ◽

Nethia Mohana-Kumaran

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Single Agent ◽

Synergistic Activity ◽

Dependent Manner ◽

Preclinical Models ◽

Apoptotic Protein ◽

Apoptotic Proteins ◽

Bh3 Mimetic ◽

3D Spheroids

AbstractObjectiveThe BCL-2 anti-apoptotic proteins are over-expressed in many cancers and hence are attractive therapeutic targets. In this study we tested the sensitivity of two NPC cell lines HK1 and C6661-1 to Maritoclax which is reported to repress anti-apoptotic protein MCL-1 and BH3 mimetic ABT-263 which selectively inhibits anti-apoptotic proteins BCL-2, BCL-XL and BCL-w. We investigated the sensitization of the NPC cell lines to these drugs using the SYBR Green I assay and 3D NPC spheroids.ResultsWe report that Maritoclax repressed MCL-1, BCL-2, and BCL-XL in a dose- and time-dependent manner and displayed a single agent activity in inhibiting cell proliferation of the NPC cell lines. Moreover, combination of Maritoclax and ABT-263 exhibited synergistic cell proliferation effect in the HK1 cell line. Similar results were obtained in the 3D spheroids. More notably, 3D spheroids either treated with single agent Maritoclax or combination with ABT-263 over 10 days did not develop resistance to the treatment rapidly. Collectively, the findings illustrate that Maritoclax as a single agent or combination with BH3 mimetics could be a potential treatment strategy for NPC but further studies in preclinical models are warranted to fully unravel the prospects of these drugs.

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Synergistic Activity of the Aurora Kinase Inhibitor MK-0457 (VX-680) with Idarubicin, Ara-C, and Inhibitors of BCR-ABL.

Blood ◽

10.1182/blood.v108.11.1384.1384 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1384-1384 ◽

Cited By ~ 3

Author(s):

Russell R. Hoover ◽

Matthew W. Harding

Keyword(s):

Clinical Trials ◽

Cell Viability ◽

Cell Lines ◽

Kinase Inhibitor ◽

Single Agent ◽

Leukemia Cell ◽

Parp Cleavage ◽

Synergistic Activity ◽

Dependent Manner ◽

Wild Type

Abstract MK-0457 (VX-680) is a reversible small molecule kinase inhibitor that targets Aurora A, B, and C with Ki values of 0.7, 18, and 4.6 nM, respectively. MK-0457 also inhibits Flt3 (Ki = 30 nM), and both the wild type and the T315I mutant of BCR-ABL (Ki = 30 and 40 nM, respectively). Clinical trials are ongoing in patients with solid tumors and hematological malignancies. Recent data show that MK-0457 is active in patients against subtypes of AML, BCR-ABL T315I mutant CML, and Philadelphia positive (Ph+) ALL. To support multi-agent clinical trials, the activity of MK-0457 in combination with idarubicin, Ara-C, and BCR-ABL inhibitors was investigated. The viability of a panel of AML, ALL, and CML cell lines was assessed following single agent and either simultaneous or sequential combinations of agents. Combination effects were evaluated using the Bliss Independence Model. MK-0457 as a single agent markedly inhibited leukemia cell viability (at 72 hrs) with an IC50 range of 20–300 nM for MV4-11, Molt-4, Molm-13, K562, LAMA-84, MEG-01, and KU812F cells. Additionally, MK-0457 inhibited the viability of BaF3 cells transformed by wild type, T315I, or Y253F mutants of BCR-ABL with similar IC50s (approximately 300 nM). The sequential combination of MK-0457 followed by either idarubicin or Ara-C showed greater synergy than simultaneous combinations in a cell line dependent manner. MK-0457 displayed strong synergy in simultaneous combination with Gleevec (imatinib mesylate) in a panel of human CML-derived cell lines and BaF3 cells expressing wild type BCR-ABL. MK-0457 enhanced the Gleevec-mediated cell death of K562 leukemia cells as evidenced by increased caspase activity, PARP cleavage, and induction of the sub-G1 population. At concentrations where synergy was observed by cell viability analysis, the MK-0457/Gleevec combination resulted predominantly in aneuploidy and G2/M arrest, consistent with inhibition of Aurora kinases by MK-0457. These results support the clinical evaluation of MK-0457 combined with idarubicin and Ara-C in AML and with BCR-ABL inhibitors in CML and Ph+ ALL.

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Dual Inhibition of Anti-apoptotic Proteins BCL-XL and MCL-1 Enhances Cytotoxicity of Nasopharyngeal Carcinoma Cells

10.21203/rs.3.rs-1085342/v1 ◽

2021 ◽

Author(s):

Siti Fairus Abdul Rahman ◽

Azali Azlan ◽

Kwok-Wai Lo ◽

Ghows Azzam ◽

Nethia Mohana-Kumaran

Keyword(s):

Nasopharyngeal Carcinoma ◽

Cell Survival ◽

Cell Lines ◽

Treatment Strategies ◽

Selective Inhibitor ◽

Carcinoma Cells ◽

Gene Expressions ◽

Apoptotic Genes ◽

Apoptotic Proteins ◽

The Many

Abstract One of the many strategies that cancer cells evade death is through up-regulation of the BCL-2 anti-apoptotic proteins. Hence, these proteins have become attractive therapeutic targets. Given that different cell population rely on different anti-apoptotic proteins for survival, it is crucial to determine which proteins are important for Nasopharyngeal carcinoma (NPC) cell survival. Here we determined the survival requirements for the NPC cells using combination of the CRISPR/Cas9 technique and selective BH3-mimetics. A human apoptosis RT2 Profiler PCR Array was first employed to profile the anti-apoptotic gene expressions in NPC cell lines HK-1 and C666-1. The HK-1 cells expressed all the anti-apoptotic genes (MCL-1, BFL-1, BCL-2, BCL-XL, and BCL-w). Similarly, the C666-1 cells expressed all the anti-apoptotic genes except BFL-1 (undetectable level). Notably, both cell lines highly expressed MCL-1. Deletion of MCL-1 sensitized the NPC cells to BCL-XL selective inhibitor A-1331852, suggesting that MCL-1 and BCL-XL may be important for NPC cell survival. Co-inhibition of MCL-1 and BCL-2 with MCL-1 selective inhibitor S63845 and BCL-2 selective inhibitor ABT-199 inhibited NPC cell proliferation but the effect on cell viability was more profound with co-inhibition of MCL-1 and BCL-XL with S63845 and A-1331852, implying that MCL-1 and BCL-XL are crucial for NPC cell survival. Furthermore, co-inhibition of MCL-1 and BCL-XL inhibited the growth and invasion of NPC spheroids. Deletion of BFL-1 sensitized NPC cells to A-1331852 suggesting that BFL-1 may play a role in NPC cell survival. Taken together co-inhibition of BCL-XL and MCL-1/BFL-1 could be potential treatment strategies for NPC.

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MCL-1 Inhibition By the Selective MCL-1 Inhibitor AMG-176 Induces in Vitro Activity Against Burkitt Lymphoma Cell Lines and Synergistically Enhances the Cytotoxic Effect of Chemotherapy and BH3 Mimetics

Blood ◽

10.1182/blood-2019-129052 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5303-5303 ◽

Cited By ~ 2

Author(s):

Tara Daly ◽

Thomas Ippolito ◽

Juan J. Gu ◽

Cory Mavis ◽

Pallawi Torka ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Lines ◽

Western Blotting ◽

Single Agent ◽

Cytotoxic Chemotherapy ◽

Time Dependent ◽

Synergistic Activity ◽

Minimal Effect ◽

Bh3 Mimetic

Background: Although >90% of Burkitt Lymphoma (BL) patients will be cured by intensive multi-agent immunochemotherapy, relapsed and refractory BL patients have poor prognoses demonstrating the need to identify novel therapies. Myeloid cell leukemia-1 (MCL-1) is a pro-survival protein within the BCL-2 family that has been investigated as a therapeutic target in several leukemia and lymphoma pre-clinical models and clinical studies. Analysis of pediatric BL tumors has implicated MCL-1 in BL pathogenesis (Giulino-Roth et al., Blood 2012) and targeting MCL-1 may be effective in MYC-driven lymphomas (Kelly et al., Genes Dev 2014). High MCL-1 expression has also been associated with therapy resistance and can confer resistance to BH3 mimetic agents (Carrington et al., Cell Death Differ 2017). AMG176, a selective MCL-1 inhibitor, is an effective promoter of apoptosis and is currently under investigation in clinical trials for relapsed and refractory multiple myeloma, lymphoma, and acute myeloid leukemia. Hypothesis: We hypothesized that MCL-1 inhibition with AMG176 will promote apoptosis and increase sensitivity of BL therapy-sensitive (TSCL) and therapy-resistant (TRCL) cell lines to other chemotherapy drugs. Methods: A panel of TSCLs (Raji, Ramos and Daudi) and one TRCL (Raji 4RH) BL cell lines were exposed to increasing concentrations of AMG176 as a single agent and in combination with Doxorubicin or Venetoclax at different time points. Cell viability was analyzed using PrestoBlue. Synergy in drug combination exposures was determined by Calcusyn software and reported as a combination index (CI) value with values increasingly <1 indicating increasing synergistic activity. Apoptosis induction was analyzed by flow cytometry for Annexin V (AV) and Sytox-7AAD and by western blotting for PARP. Effects of MCL-1 inhibition on the expression of other pro- and anti-apoptotic BCL-2 family member proteins was assessed by western blotting. AMG176 mechanism(s) of action was evaluated by co-immunoprecipitation followed by western blotting for pro-apoptotic BCL-2 family members known to be bound by MCL-1. Results: Single agent AMG176 exposure of BL cell lines exhibited a dose and time-dependent decrease in viable cells. 48hr IC50 values of Ramos (0.3097uM) and Raji (1.652uM) showed an increased sensitivity to AMG176 as compared to Daudi (9.616uM) and Raji 4RH (12.45uM). Exposure of cells to AMG176 also resulted in altered expression levels of pro-survival and pro-apoptotic proteins of the BCL-2 family proteins. Co-immunoprecipitation performed after 48hrs of AMG176 exposure exhibited a decrease in MCL-1:BIM and MCL-1:BAK complexes in Raji and Ramos cells promoting a pro-apoptotic environment. After 48hr exposure to AMG176, induction of apoptosis was observed in a dose-dependent manner in all cell lines as evidenced by increased PARP cleavage on western blot and AV-Sytox positivity on flow cytometry. To investigate the ability of MCL-1 inhibition to enhance the activity of cytotoxic chemotherapy, BL cells were exposed to AMG176 in combination with Doxorubicin. Daudi and Raji exhibited strong synergy with peak synergistic activity observed at 72hrs (Daudi CI values <0.1 at all AGM176 concentrations with 0.1-0.2uM Doxorubicin; Raji CI ranged from 0.233-0.295 at multiple AMG176 concentrations with <0.4uM Doxorubicin) while a minimal effect was observed in Ramos and Raji 4RH. As high MCL-1 is associated with resistance to Venetoclax by sequestering free BIM following Venetoclax exposure, the combination of AMG176 and Venetoclax was investigated and also showed significant synergy in BL cells. A minimal effect was observed in Daudi and Raji 4RH in Venetoclax combination but Ramos and Raji, the more AMG176 sensitive cell lines, showed peak synergy at 24hrs at high doses of Venetoclax (Ramos CI values ranged from 0.3-0.478; Raji CI ranged from 0.511 to 0.67). Conclusion: MCL-1 inhibition with AMG176 demonstrates dose- and time-dependent anti-lymphoma activity impairing in vitro proliferation and inducing apoptosis in BL cells. AMG176 exhibits synergistic activity in combination with cytotoxic chemotherapy in both therapy-sensitive and therapy-resistant BL cell lines and synergistically enhances response to the BH3 mimetic agent Venetoclax. These findings highlight the potential of MCL-1 inhibition as a therapeutic option in BL. This work was supported by T35 NIH training grant (T35AI089693). Disclosures No relevant conflicts of interest to declare.

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7β-(3-Ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z Notonipetranone Attenuates Neuropathic Pain by Suppressing Oxidative Stress, Inflammatory and Pro-Apoptotic Protein Expressions

Molecules ◽

10.3390/molecules26010181 ◽

2021 ◽

Vol 26 (1) ◽

pp. 181

Author(s):

Amna Khan ◽

Adnan Khan ◽

Sidra Khalid ◽

Bushra Shal ◽

Eunwoo Kang ◽

...

Keyword(s):

Oxidative Stress ◽

Neuropathic Pain ◽

Interleukin 1 ◽

Related Factor ◽

Dependent Manner ◽

Glutathione S Transferase ◽

Quinone Oxidoreductase ◽

Apoptotic Protein ◽

Apoptotic Proteins ◽

Apoptosis And Inflammation

7β-(3-Ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (ECN), a sesquiterpenoid obtained from a natural source has proved to be effective in minimizing various side effects associated with opioids and nonsteroidal anti-inflammatory drugs. The current study focused on investigating the effects of ECN on neuropathic pain induced by partial sciatic nerve ligation (PSNL) by mainly focusing on oxidative stress, inflammatory and apoptotic proteins expression in mice. ECN (1 and 10 mg/kg, i.p.), was administered once daily for 11 days, starting from the third day after surgery. ECN post-treatment was found to reduce hyperalgesia and allodynia in a dose-dependent manner. ECN remarkably reversed the histopathological abnormalities associated with oxidative stress, apoptosis and inflammation. Furthermore, ECN prevented the suppression of antioxidants (glutathione, glutathione-S-transferase, catalase, superoxide dismutase, NF-E2-related factor-2 (Nrf2), hemeoxygenase-1 and NAD(P)H: quinone oxidoreductase) by PSNL. Moreover, pro-inflammatory cytokines (tumor necrotic factor-alpha, interleukin 1 beta, interleukin 6, cyclooxygenase-2 and inducible nitric oxide synthase) expression was reduced by ECN administration. Treatment with ECN was successful in reducing the caspase-3 level consistent with the observed modulation of pro-apoptotic proteins. Additionally, ECN showed a protective effect on the lipid content of myelin sheath as evident from FTIR spectroscopy which showed the shift of lipid component bands to higher values. Thus, the anti-neuropathic potential of ECN might be due to the inhibition of oxidative stress, inflammatory mediators and pro-apoptotic proteins.

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BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance

ESMO Open ◽

10.1136/esmoopen-2018-000387 ◽

2018 ◽

Vol 3 (6) ◽

pp. e000387 ◽

Cited By ~ 10

Author(s):

Chiara Tarantelli ◽

Elena Bernasconi ◽

Eugenio Gaudio ◽

Luciano Cascione ◽

Valentina Restelli ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Antitumour Activity ◽

Single Agent ◽

Treatment Strategies ◽

Bet Inhibitor ◽

Mantle Cell

BackgroundThe outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials.Materials and methodsThe activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines.ResultsBirabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines.ConclusionOur data provide the rationale to evaluate birabresib in patients affected by MCL.

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Hesperidin Induces Mitochondria Mediated Intrinsic Apoptosis in HPV-positive Cervical Cancer Cells via Regulation of E6/p53 Expression

10.21203/rs.3.rs-105340/v1 ◽

2020 ◽

Author(s):

Fang Ren ◽

Gong Zhang ◽

Caiyu Li ◽

Gailing Li ◽

Yuan Cao ◽

...

Keyword(s):

Cervical Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Protein Level ◽

Potential Candidate ◽

Dependent Manner ◽

Antitumor Effects ◽

P53 Expression ◽

Cervical Cancer Cells ◽

Apoptotic Proteins

Abstract Background: Hesperetin, an active compound found in citrus fruits, possesses antiproliferative effects toward several types of cancer cell lines, including cervical cancer. In this study, we explore the antitumor effects of Hesperetin on the human cervical cancer human papilloma virus (HPV)-positive (CaSki and HeLa) and HPV-negative (C-33A) cell lines and further elucidated the underlying mechanisms of this action. Methods: Cell viability and proliferation was measured by the MTT assay and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, respectively. dUTP-fluorescein nick end-labeling (TUNEL) staining, Annexin V‑fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining and flow cytometry was used to assess the degree of apoptosis. JC-1 staining assay was used to evaluate the change of mitochondrial membrane potential (ΔΨm) and Western blot assays were used to determine apoptosis-related factors at protein level. Results: Hesperetin (100, 200 and 400 μM) exhibited a significant exclusive inhibitory effect against the growth of HPV-infected CaSki and HeLa cancer cells via induction of apoptosis in a concentration-dependent manner, while it was almost not active in HPV-negative C-33A cancer cells and normal cervix epithelial H8 cells. Moreover, this antitumor effect executed by Hesperetin was associated with disruption of ΔΨm, the release of cytochrome c from mitochondria, activation of pro-apoptotic proteins (Bax, cleaved caspase-3 and caspase-9) and inhibition of anti-apoptotic proteins (Bcl-2). During this process, cleaved caspase-8 remained unchanged. In addition, Hesperetin led to a downregulation of E6 oncoprotein expression and upregulation of tumor suppressor protein p53 level. Conclusions: Collectively, these results implicated that Hesperetin can induce apoptosis of HPV‑positive cervical cancer cells via a mitochondria-mediated intrinsic signaling pathway, together with the repression of E6 and enhancement of p53 protein level, indicating Hesperetin may be considered as a potential candidate for the development of innovative anti-HPV cervical cancer agents.

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Essential Role of the INK4/CDK4,6/Rb/E2F Signaling Pathway in AML with the Flt3 Internal Tandem Duplication.

Blood ◽

10.1182/blood.v106.11.2752.2752 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2752-2752

Author(s):

Lisheng Wang ◽

Jie Wang ◽

Blaser W. Bradley ◽

Caligiuri A. Michael ◽

Briesewitz Roger

Keyword(s):

Cell Lines ◽

Essential Role ◽

Tandem Duplication ◽

Dependent Manner ◽

U937 Cells ◽

Phase I Clinical Trials ◽

Internal Tandem Duplication ◽

Survival Signal ◽

Apoptotic Protein

Abstract Mutationally activated tyrosine kinases provide a critical survival signal to cancer cells, thus, making such kinases and their downstream effectors attractive targets for cancer therapy. To study signaling of mutated kinases we have chosen the receptor tyrosine kinase Flt3 that harbors an activating internal tandem duplication (ITD) in about 25% of AML patients. The use of a Flt3 inhibitor (THRX-165724, Theravance, Inc.) in two Flt3 ITD AML cell lines (MOLM13 and MV4-11) led to the inhibition of the INK4/CDK4,6/Rb/E2F pathway within three hours as reflected by the downregulation of D-cyclin gene expression followed by a decrease in D-cyclin protein. As a result of reduced D-cyclin levels, CDK4,6 activity was downregulated as revealed by the hypophosphorylation of the main substrate of CDK4,6, the Rb protein. THRX-165724 had no effect on D-cyclin levels or Rb hyperphosphorylation in THP-1 and U937 cells, two AML cell lines that express wildtype Flt3. Furthermore, THRX-165724 did not affect the proliferation or survival of these two cell lines. To investigate the role of the INK4/CDK4,6/Rb/E2F pathway as part of the proliferation and survival signal provided by the Flt3 ITD, we used PD-0332991, a highly selective CDK4,6 kinase inhibitor from Pfizer currently in phase I clinical trials for solid tumors. A dose-response experiment revealed that in cells PD-0332991 inhibits CDK4,6 activity with an IC50 of 30–40 nM as assayed by following the dephosphorylation of Rb. The compound does not inhibit Flt3 kinase activity even at the highest concentrations tested (500 nM). In MV4-11 and MOLM13 cells, PD-0332991 induced G1 specific cell cycle arrest within 24 hours and apoptosis after 3 days. Hence, in MV4-11 and MOLM13, PD-0332991 is cytostatic and cytotoxic. In contrast, PD-0332991 was neither cytotoxic nor cytostatic in THP-1 and U937 cells. In a clonogenic assay PD-0332991 reduced in a dose dependent manner the colony formation of MV4-11 and MOLM13 cells as well as primary patient blasts with Flt3 ITD. In contrast, THP-1 and U937 cells as well as CD34+ primary hematopoietic progenitor cells were not affected in their ability to form colonies. In MV4-11 and MOLM13 cells PD-0332991 induced the downregulation of the anti-apoptotic protein Bcl-2 and in MOLM13 it also induced the upregulation of the pro-apoptotic protein Bak. The deregulation of Bcl-2 and Bak may represent the underlying mechanism for the observed cytotoxicity of PD-0332991 in MV4-11 and MOLM13. In a mouse model of AML, NOD/SCID mice were inoculated with MOLM13 cells which caused leukemia within eight days. Mice treated with PD-0332991 starting at day seven after inoculation had a significant survival advantage demonstrating that PD-0332991 has anti-leukemic activity in vivo (median survival time 15.0 days versus 11.5 days, P=.0003). Our data suggest that the activation of the INK4/CDK4,6/Rb/E2F pathway by Flt3 ITD has an essential role for proliferation and survival in AML cells. Targeting the INK4/CDK4,6/Rb/E2F pathway in Flt3 ITD AML using CDK4,6 inhibitors like PD-0332991 should be explored for clinical efficacy in FLT3 ITD AML.

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Akt Inhibitor Perifosine-Induced Cytotoxicity Is Associated with Significant Downregulation of Survivin in Human Multiple Myeloma (MM) Cells.

Blood ◽

10.1182/blood.v108.11.3410.3410 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3410-3410

Author(s):

Teru Hideshima ◽

Hiroshi Yasui ◽

Laurence Catley ◽

Noopur Raje ◽

Dharminder Chauhan ◽

...

Keyword(s):

Cell Lines ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Single Agent ◽

Survivin Expression ◽

Mek Inhibitor ◽

Parp Cleavage ◽

Caspase 8 ◽

Akt Inhibitor ◽

Apoptotic Proteins

Abstract Perifosine (NSC 639966; Keryx Biopharmaceuticals, New York, NY) is a synthetic novel alkylphospholipid, a new class of anti-tumor agents which potently inhibits Akt (PKB) activity. Our previous studies have shown that Perifosine induces significant cytotoxicity in MM cells triggered by c-Jun NH2-terminal kinase (JNK) activation followed by caspase-8, caspase-9, and PARP cleavage even in the presence of cytokines (ie, IL-6 and IGF-1) or bone marrow stromal cell (BMSCs). Importantly, MEK inhibitor and bortezomib enhance Perifosine-induced cytotoxicity. It has also shown significant anti-tumor activity in a human MM cell xenograft mouse model (Hideshima et al. Blood2006, 107:4053–4062). In this study, we further delineated molecular mechanisms whereby Perifosine triggers cytotoxicity as a single agent and in combination with bortezomib in MM cells. In most MM cell lines, the IC50 for Perifosine-induced cytotoxicity is 5–10 μM range assessed by MTT assay at 24h; however, apoptosis assessed by APO2.7 staining, varied in each cell line. Moreover, neither the degree of JNK phosphorylation nor caspase-8/9/PARP cleavage correlated with Perifosine-induced cytotoxicity. Therefore we further examined expression level of anti-apoptotic proteins in MM cell lines and found that survivin, which has a crucial role in regulation of caspase-3 activity, was markedly downregulated by Perifosine treatment in a time- and dose-dependent fashion, without affecting expression of other anti-apoptotic proteins (ie, cIAP, XIAP, Bcl-2, Bcl-xL). Since survivin is a known downstream protein of β-catenin/TCF-4 cascade, we next hypothesized that Perifosine may inhibit β-catenin activity. As expected, Perifosine significantly downregulated both phosphorylation and protein expression of β-catenin, associated with downregulation of survivin and enhanced caspase-3 cleavage. Real-time PCR confirmed that gene expression of survivin was suppressed 35% and 55% after 3h and 6h Perifosine treatment, respectively. Since β-catenin is a substrate of proteasomes, we further examined whether bortezomib could augment survivin expression by blocking its degradation. Importantly, bortezomib significantly upregulated β-catenin and survivin, which was blocked in the presence of Perifosine. These results suggest that inhibition of bortezomib-induced survivin expression, at least in part, accounts for enhanced bortezomib-induced cytotoxicity by Perifosine. Based upon these preclinical studies, a rational combination trial of bortezomib with Perifosine to treat relapsed refractory MM is currently ongoing.

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The BH3 Mimetic, ABT-737, Is Effective Against Bcl-2 Overexpressing Lymphoid Tumors.

Blood ◽

10.1182/blood.v108.11.826.826 ◽

2006 ◽

Vol 108 (11) ◽

pp. 826-826 ◽

Cited By ~ 1

Author(s):

Kylie D. Mason ◽

Cassandra J. Vandenberg ◽

Mark F. van Delft ◽

Andrew H. Wei ◽

Suzanne Cory ◽

...

Keyword(s):

Cell Lines ◽

Genetic Manipulation ◽

Single Agent ◽

B Cell Lymphoma ◽

Cell Types ◽

Clinical Samples ◽

Bh3 Mimetic ◽

Mouse Lymphoma

Abstract Lymphoid tumors often respond poorly to conventional cytotoxics, a common cause being their impaired sensitivity to apoptosis, such as that caused by Bcl-2 overexpression. A strategy to overcoming this is to use mimics of the natural antagonists of pro-survival Bcl-2, the BH3 only proteins. A promising BH3 mimetic is ABT-737, which targets Bcl-2 and closely related pro-survival proteins. We evaluated its potential utility by testing it on cell lines, clinical samples and on a relevant mouse lymphoma model. We assessed the sensitivity of B cell lymphoma cell lines and primary CLL samples to ABT-737, either alone or in combination. To ascertain its efficacy in vivo, we utilized a mouse model based on the Eμ-myc tumor that is readily transplantable and amenable to genetic manipulation. When syngeneic recipient mice were inoculated with tumors, they develop widespread lymphoma, fatal unless treated by agents such as cyclophosphamide. We found that ABT-737, on its own, was cytotoxic only to a subset of cell lines and primary CLL samples. However, it can synergize potently with agents such as dexamethasone, suggesting that this agent might be useful in combination with currently used chemotherapeutics. In the Eμ myc mouse lymphoma model, treatment with ABT-737 alone did not control the disease as multiple independently derived tumors proved refractory to treatment with this agent. However, ABT-737 was partially effective as a single agent for treating bitransgenic tumors derived from crosses of the Eμmyc and Eμ-bcl-2 transgenic mice. ABT-737 therapy prolonged the survival of recipient mice transplanted with tumors from 30 to 60 days. When combined with a low dose of cyclophosphamide (50mg/kg), long term stable remissions were achieved, which were sustained even longer than control mice treated with much higher doses of cyclophosphamide (300mg/kg). We found that ABT-737 was well tolerated as a single agent and when combined with low doses of cytotoxics such as cyclophosphamide. Thus, ABT-737 may prove to be efficacious for those tumors highly dependent on Bcl-2 for their survival. We found that despite its high affinity for Bcl-2, Bcl-xL and Bcl-w, many cell types proved refractory to ABT-737 as a single agent. We show that this resistance reflects its inability to target another pro-survival relative Mcl-1. Down-regulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in the Eμmyc/bcl-2 bitransgenic mouse lymphoma model conferred marked resistance as mice transplanted with such tumors died as rapidly as the untreated counterparts. However, enhanced Bcl-2 overexpression on these tumors had little impact on the in vivo response, suggesting that ABT-737 can be utilized even when Bcl-2 is markedly overexpressed. ABT-737 appears to be a promising agent for the clinic. It potently sensitizes certain lymphoid tumors to conventional cytotxics in vitro. The synergy observed between dexamethasone and ABT-737 on some lymphoid lines in culture suggests that it is attractive for clinical testing. Encouragingly, ABT-737 appeared efficacious in vivo against Bcl-2 overexpressing tumors when combined with a reduced dose of cyclophosphamide, suggesting that it will be useful for treating even those Bcl-2-overexpressing tumors that are normally highly chemoresistant.

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