HSP70 Inhibitor, YK5, Synergizes with Chemotherapeutic Agents and Prevents Chemoresistance in Acute Myelogenous Leukemia (AML).

Abstract Abstract 2476 Stress-inducible heat shock protein 70 (HSP70) is a major cytoprotective factor and a molecular chaperone that interacts with HSP90 to form a multi-chaperone complex. Cancer cells are highly dependent on this complex due to their increased demand for protein synthesis. HSP70 overexpression inhibits apoptosis and has been associated with drug resistance and poor prognosis. YK5, a novel inhibitor of tumor-HSP70, has been shown to induce potent cell death in AML blast, progenitor, and stem cell populations with minimal effects in normal hematopoietic cells. Due to the role of HSP70 in drug resistance, we examined the effect of combining YK5 with other chemotherapeutic agents, including arsenic trioxide, cytarabine, suberoylanilide hydroxamic acid (SAHA) and PU-H71, a novel tumor-specific HSP90 inhibitor. We tested the ability of YK5 to synergize with either AsO3, AraC, SAHA, or PU-H71 in primary AML samples. Using multiparameter flow cytometry to measure viability after 48 hours of treatment, we found that combining 1μM YK5 with either 500nM AsO3 or PU-H71 resulted in a significant increase in cell death when compared to either agent alone (n=9, mean viability: 51.8, 67.2, and 13.4% for AsO3, YK5, and AsO3/YK5, respectively, P = 0.0018; mean viability: 57.1 and 20.8% for PU-H71 and PU-H71/YK5, respectively, P = 0.0029). A synergistic relationship between YK5 and both AsO3 and PU-H71 was found in all nine primary samples (combination indexes 0.29 – 0.76 with YK5/AsO3, 0.33 – 0.83 with YK5/PU-H71). In contrast, the combination of YK5 with either AsO3 or PU-H71 in CD34+ cord blood mononuclear cells did not result in a significant increase in cell death when compared to either agent alone (mean viability: 42.4, 72.4, and 37.2% for AsO3, YK5, and AsO3/YK5, respectively; mean viability: 61.1 and 51.1% for PU-H71 and PU-H71/YK5, respectively). YK5 in combination with either AraC or SAHA, however, did not result in a significant increase in cell death when compared to either drug alone, with an additive effect being demonstrated with a 1:1 YK5 to AraC/SAHA drug ratio (Mean CI = 0.9918). To determine the mechanism of the observed synergistic activity, intracellular HSP70 and active caspase-3, a client of HSP70, were measured using flow cytometry. Both AsO3 and PU-H71 significantly increased intracellular HSP70 and caspase-3 (Mean fold change = 18.3, 21.0 of HSP70 and 9.9, 8.3 of Caspase-3 for AsO3 and PU-H71 treatment, respectively), while treatment with AraC or SAHA resulted in no change in HSP70 levels. Furthermore, quantitative PCR revealed that treatment with either AsO3 or PU-H71 strongly upregulated HSPA1A and HSPA6, the main stress-inducible isoforms of HSP70 (Mean fold change = 15.9, 14.1 of HSPA1A, and 20.8, 23.4 of HSPA6 for AsO3 and PU-H71 treatment, respectively). AraC and SAHA had no significant upregulation of these genes. We have previously shown that increased levels of HSPA1A correlate with sensitivity to HSP70 inhibition via YK5. To further explore the mechanism of this observed synergy, flow cytometry was used to measure the levels of reactive oxygen species (ROS). Treatment with AsO3, PU-H71, AraC, or SAHA resulted in a significant increase in ROS (Mean fold change = 2.75, 1.92, 2.89, 1.67, respectively). Quantitative PCR also confirmed the activation of the oxidative stress response by the upregulation of heme oxygenase 1 (HMOX1) by treatment with these drugs (Mean fold change = 10.9, 8.7, 11.2, 7.7, respectively). YK5, however, did not induce ROS or upregulate HMOX1. Interestingly, pretreatment with NAC in primary AML samples (n=4) resulted in no protection from YK5 synergistic effect when combined with either AsO3 or PU-H71. These results suggest that YK5 synergizes with AsO3 and PU-H71 due to the increase in intracellular HSP70 caused by these drugs. This synergy is most likely due to the activation of the heat shock response and independent of the production of ROS due to drug treatment. In summary, we have found that the novel tumor-HSP70 inhibitor YK5 can synergize with AsO3 and PU-H71 in primary human AML, and that the basis of this synergism is due to the increase in intracellular HSP70 caused by these chemotherapeutic agents. HSP70 inhibition represents a novel approach in AML treatment and can be particularly significant to drug-resistant patients when combined with other chemotherapy. Disclosures: Roboz: Astex Pharmaceuticals: Research Funding.

Download Full-text

ARTEMISININ MODULATING EFFECT ON HUMAN BREAST CANCER CELL LINES WITH DIFFERENT SENSITIVITY TO CYTOSTATICS

Experimental Oncology ◽

10.31768/2312-8852.2017.39(1):25-29 ◽

2017 ◽

Vol 39 (1) ◽

pp. 25-29 ◽

Cited By ~ 4

Author(s):

V F Chekhun ◽

N Yu Lukianova ◽

T Borikun ◽

T Zadvornyi ◽

A Mokhir

Keyword(s):

Breast Cancer ◽

Drug Resistance ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Iron Homeostasis ◽

Chemotherapeutic Agents ◽

Fold Change ◽

Breast Cancer Cell Lines ◽

Cellular Iron ◽

Mcf 7

Aim: To explore effects of Artemisinin on a series of breast cancer cells with different sensitivity to typical cytotoxic drugs (doxorubicin — Dox; cisplatin — DDP) and to investigate possible artemisinin-induced modification of the mechanisms of drug resistance. Materials and Methods: The study was performed on wild-type breast cancer MCF-7 cell line (MCF-7/S) and its two sublines MCF-7/Dox and MCF-7/DDP resistant to Dox and DDP, respectively. The cells were treated with artemisinin and iron-containing magnetic fluid. The latter was added to modulate iron levels in the cells and explore its role in artemisinin-induced effects. The MTT assay was used to monitor cell viability, whereas changes of expression of selected proteins participating in regulation of cellular iron homeostasis were estimated using immunocytochemical methods. Finally, relative expression levels of miRNA-200b, -320a, and -34a were examined by using qRT-PCR. Results: Artemisinin affects mechanisms of the resistance of breast cancer cells towards both Dox and DDP at sub-toxic doses. The former drug induces changes of expression of iron-regulating proteins via different mechanisms, including epigenetic regulation. Particularly, the disturbances in ferritin heavy chain 1, lactoferrin, hepcidin (decrease) and ferroportin (increase) expression (р ≤ 0.05) were established. The most enhanced increase of miRNA expression under artemisinin influence were found for miRNA-200b in MCF-7/DDP cells (7.1 ± 0.98 fold change), miRNA-320a in MCF-7/Dox cells (2.9 ± 0.45 fold change) and miRNA-34a (1.7 ± 0.15 fold change) in MCF-7/S cells. It was observed that the sensitivity to artemisinin can be influenced by changing iron levels in cells. Conclusions: Artemisinin can modify iron metabolism of breast cancer cells by its cytotoxic effect, but also by inducing changes in expression of iron-regulating proteins and microRNAs (miRNAs), involved in their regulation. This modification affects the mechanisms that are implicated in drug-resistance, that makes artemisinin a perspective modulator of cell sensitivity towards chemotherapeutic agents in cancer treatment.

Download Full-text

BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death [published erratum appears in Blood 1994 Jun 15;83(12):3835]

Blood ◽

10.1182/blood.v83.5.1179.1179 ◽

1994 ◽

Vol 83 (5) ◽

pp. 1179-1187 ◽

Cited By ~ 330

Author(s):

A McGahon ◽

R Bissonnette ◽

M Schmitt ◽

KM Cotter ◽

DR Green ◽

...

Keyword(s):

Cell Death ◽

Chronic Myelogenous Leukemia ◽

Apoptotic Cell ◽

Chimeric Protein ◽

Chemotherapeutic Agents ◽

Myelogenous Leukemia ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Expression Of Genes ◽

Induction Of Apoptosis

Abstract Apoptosis is the major form of cell death associated with the action of chemotherapeutic agents on tumor cells, and therefore the expression of genes that interfere with apoptosis can have important consequences for the efficacy of therapeutic approaches. Here we show that K562, a chronic myelogenous leukemia (CML) cell line expressing the BCR-ABL fusion protein, are resistant to the induction of apoptosis by a number of agents and conditions. Antisense oligodeoxynucleotides corresponding to the translation start of bcr downregulate bcr-abl protein in these cells and render them susceptible to induction of apoptosis by chemotherapeutic agents or serum deprivation. Expression of a temperature sensitive v-Abl protein reverses the effects of the antisense oligonucleotides, such that the cells remain resistant to apoptosis at the permissive temperature. These data indicate that bcr- abl acts as an anti-apoptosis gene in CML cells and suggests that the effect is dependent on the abl kinase activity in this chimeric protein. Inhibition of bcr-abl to render CML cells susceptible to apoptosis can be combined with therapeutic drugs and/or treatment capable of inducing apoptosis to provide an effective strategy for elimination of these cells.

Download Full-text

Tris DBA Palladium Overcomes Hypoxia Mediated Drug Resistance in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.2978.2978 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2978-2978

Author(s):

Pilar De La Puente ◽

Barbara Muz ◽

Feda Azab ◽

Micah John Luderer ◽

Jack L. Arbiser ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Proliferation ◽

Drug Resistance ◽

Cell Lines ◽

Research Funding ◽

Caspase 3 ◽

Cellular Processes ◽

Hypoxic Conditions

Abstract Introduction: Despite recent progress in novel and targeted therapies, multiple myeloma (MM) remains a therapeutically challenging incurable disease. The regulation of important cellular processes and its link to cancer presented Src as an attractive target for MM. Src is a non-receptor protein tyrosine kinase which regulates multiple fundamental cellular processes including cell growth, migration, survival and differentiation. Activated Src in cancer lead to studies with Src as a target for anti-cancer drugs, and numerous Src inhibitors have become available to test the importance of Src in tumor initiation and progression. In MM, it has been described that in cell lines and MM patient-derived tumors, c-Src is constitutively activated, which plays an important role in drug resistance mechanisms. Tris dibenzylideneacetone dipalladium (Tris DBA), a small-molecule palladium complex, was shown to reduce Src/NMT-1 complex in melanoma cells, as well as inhibit downstream signaling including mitogen-activated protein kinase (MAPK kinase) and phosphoinositol-3-kinase (PI3K). We suggest a novel strategy to improve the treatment of MM and overcome the drug resistance for the current therapeutic agents by specific inhibition of Src in MM cells by an organopalladium compound, Tris DBA. Methods: Tris DBA was prepared by Dr. Arbiser. MM cell lines (MM.1S, MM.1R, H929, RPMI-8826, and OPM2) and PBMCs were cultured with Tris DBA (0-10 µM) for 24h. MM cells were analyzed for cell proliferation by MTT assay; cell cycle by DNA staining with PI and analyzed by flow cytometry; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with proliferation, cell cycle, and apoptosis was analyzed by western blotting. In addition, cell proliferation assay of Tris DBA with or without combination of proteasome inhibitors (PIs) bortezomib or carfilzomib for 24h was analyzed on the proliferation of MM cells in normoxic or hypoxic conditions. Moreover, we tested the effect of combination treatment on cell cycle and apoptosis signaling under normoxic conditions. We then evaluated the effect of Tris DBA on HIF1α expression, migration and drug resistance under normoxic or hypoxic conditions. Results: The Src inhibitor Tris DBA reduced the proliferation of MM cell lines with an IC50 of about 1.5 - 3 µM after 24h treatment as a single agent, while none of the normal PBMC controls showed effect on their proliferation in the same dose range. These results were consistent with the decreased expression of proliferation signaling proteins from MAPK pathways (pERK), as well as PI3K (pS6R). Src inhibition led to the induction of a sub-G1 peak, which indicated accumulating apoptotic cells shown by DNA staining with PI. Apoptosis was then analyzed by Annexin/PI and confirmed by cleavage of caspase-3 and PARP. We found that Tris DBA synergized with bortezomib and carfilzomib by inhibiting proliferation of MM cells and reducing cell cycle protein signaling more than either of the drugs alone. Moreover, the Tris DBA/Bortezomib or Tris DBA/Carfilzomib combination therapies significantly increased apoptosis by caspase-3 cleavage more than treatment with either proteasome inhibitor individually. Tris DBA inhibited HIF1α expression in both normoxic and hypoxic conditions. HIF1α is an important target for hypoxia-driven drug resistance. Our studies confirmed hypoxia promoted faster chemotaxis of MM cells towards the chemo-attractants found in stromal cell conditioned media, and that Tris DBA treatment could overcome this hypoxia-induced effect. In addition, the development of hypoxia-induced drug resistance to individual bortezomib or carfilzomib treatment was overcome with combination treatment of Tris DBA under hypoxic conditions. Conclusions: Tris DBA reduces proliferation and induces G1 arrest and apoptosis in MM cells. Tris DBA synergized with PIs reducing proliferation and cell cycle signaling, as well as increasing apoptosis more than each drug alone. Tris DBA overcame hypoxia-induced effects such as enhanced chemotaxis or drug resistance to PIs by inhibition of HIF1α expression. Moreover, we found that Tris DBA is an effective anti-myeloma agent alone or in combination with other targeted drugs and that it reverses hypoxia-induced drug resistance in myeloma. These results suggest the use of Tris DBA as a new therapeutic agent in relapsed refractory myeloma. Disclosures Arbiser: ABBY Therapeutics: Other: Jack L Arbiser is listed as inventor on a US Patent for imipramine blue. He is cofounder of ABBY Therapeutics, which has licensed imipramine blue from Emory University.. Azab:Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner ; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding.

Download Full-text

Correlation of P-glycoprotein expression and function in childhood acute leukemia: a children's cancer group study

Blood ◽

10.1182/blood.v88.1.309.bloodjournal881309 ◽

1996 ◽

Vol 88 (1) ◽

pp. 309-318

Author(s):

SP Ivy ◽

RS Olshefski ◽

BJ Taylor ◽

KM Patel ◽

GH Reaman

Keyword(s):

Drug Resistance ◽

Cell Lines ◽

Childhood Leukemia ◽

Alkylating Agents ◽

Chemotherapeutic Agents ◽

Myelogenous Leukemia ◽

Response To Treatment ◽

Acute Leukemias ◽

P Glycoprotein ◽

And Function

Clinical drug resistance may be attributed to the simultaneous selection and expression of genes modulating the uptake and metabolism of chemotherapeutic agents. P-glycoprotein (P-gp) functions as a membrane-associated drug efflux pump whose increased expression results in resistance to anthracyclines, epipodophyllotoxins, vinca alkaloids, and some alkylating agents. This type of resistance occurs as both de novo and acquired resistance to therapy for leukemia. We have studied P- gp expression and function in childhood acute leukemias by developing a series of doxorubicin- and vincristine-selected CEM, T-cell lymphoblastoid cell lines that recapitulate the low levels of expression and resistance seen clinically. These cell lines have been used to develop flow cytometric assays for the semiquantitative measurements of P-gp expression with the MRK16 monoclonal antibody and P-gp function using the enhanced retention of rhodamine 123 in the presence of verapamil, a resistance modulator. Kolmogorov-Smirnov statistics, represented by the D measurement, are used to determine the difference in level of P-gp expression by comparing MRK16 staining to an IgG2a isotype control. When D is > 0.09, there is an excellent correlation (R = 0.82) between P-gp expression and function. The evaluation of 107 bone marrow specimens from 84 children with lymphoblastic or myelogenous leukemia showed a statistically significant (P = .004) increase in P-gp function at relapse. P-gp expression at relapse, however, approached but did not reach a significant level (P = .097). Using this methodology, we can identify patients with levels of P-gp expression and function that we can define clinically, as well as children with discordant multidrug resistance phenotypes. This study supports the role of P-gp-mediated drug resistance in childhood leukemia and confirms that P-gp expression and function are measurable in their leukemic blasts. These assays provide the means for the in vitro testing of resistance modulators and the monitoring of in vivo response to treatment with these agents.

Download Full-text

The Gain of the Short Arm of Chromosome 2 (2p+) Induces XPO1 Overexpression and Drug Resistance in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.492.492 ◽

2015 ◽

Vol 126 (23) ◽

pp. 492-492 ◽

Cited By ~ 1

Author(s):

Adrien Cosson ◽

Elise Chapiro ◽

Jérome Lambert ◽

Hong-Anh Cung ◽

Caroline Algrin ◽

...

Keyword(s):

Flow Cytometry ◽

Drug Resistance ◽

Cell Death ◽

Prognostic Factors ◽

Functional Characterization ◽

Snp Array ◽

Genetic Alterations ◽

Lymphocytic Leukemia ◽

Bax Protein

Abstract Introduction: CLL is a heterogeneous disease in terms of response to treatment, with some patients reaching complete and prolonged remissions, while others relapsing early and requiring several lines of treatments. This highly variable course is partly explained by the existence of a heterogenic panel of genetic alterations (mutations, chromosomal abnormalities) that allow the development of drug-resistant aggressive CLL subclones. Therefore, a functional characterization of the cytogenetic alterations associated to CLL drug resistance may provide new means of improving the current therapeutic strategies. We and others have already reported that the gain of 2p (2p+) is recurrent in CLL. However, the candidate gained gene(s) on the 2p remain to be identified. Previously data: we have observed that the 2p gain is frequent in previously untreated CLL Binet stages B/C (21/132, 15.9%), and is associated with bad prognostic factors, such as 11q deletion (p=0.0008) and unmutated IGHV (p=0.02). Using a SNP-array approach, we have identified a minimally gained region of 1.28Mb on 2p16.1-15. This region included the gene CRM1/XPO1 (Chromosome Region Maintenance 1/Exportin-1), a gene also recurrently mutated in CLL. A qPCR assessment confirmed that XPO1 was overexpressed in the 2p+/CLL patients (1.4-fold increase compared to 2p-/CLL; p=0.02). The objective of our work was to identify the potential role of XPO1 in CLL drug resistance by using the selective XPO1 inhibitor Selinexor (KPT-330, provided by Karyopharm Therapeutics), which is currently in Phase II human clinical trials in hematological and solid cancers. Methods: We have analyzed 36 2p+/CLL and we have searched for XPO1 mutations in 436 CLL samples. CLL drug resistance associated to XPO1 overexpression/mutation was assessed by measuring the rate of programmed cell death (PCD) on cells from 2p- and wildtype (wt) XPO1/CLL (n=20), 2p+/XPO1 wt/CLL (n=8) and on XPO1 mut/CLL (n=6). After 24 hours treatment with Fludarabin + Cyclophosphamid + Rituximab (FCR), Ibrutinib (Ibru), Idelalisib + Rituximab (Ide+R) and Selinexor, cells were stained with Annexin-V and propidium iodide and PCD was assessed by flow cytometry. KPT-301 was used as a negative control. For the inhibition assay, the inhibitor Q-VD-Oph was added 30 min before inducing cell death. Mitochondrial membrane depolarisation was assessed using tetramethyllrhodamine ethyl ester probe and flow cytometry analysis. Results: (i) Using a FISH approach, we fully confirmed the gain of XPO1 in 2p+/CLL samples. Additionally, we found that the XPO1 gain was often subclonal, suggesting that it tends to arise late in leukemic development. Longitudinal FISH analyses, performed on 8 2p+/CLL-treated patients, showed a similar or increasing percentage of cells carrying XPO1 gain at relapse, when compared to diagnosis; (ii) XPO1 was mutated in 23/436 (5.3%) CLL and in 2/30 (6.7%) 2p+/CLL; (iii) Selinexor induced PCD in 2p-/XPO1 wt/CLL (35% of PCD). The results were similar in all tested CLL, independently of prognostic factors (del13q, tri12, del11q, del17p, IGHV status), while sparing the non leukemic cells from patients or B cells from healthy donors; (iv) Selinexor induced CLL PCD through a caspase-dependant apoptotic pathway, as evidenced by inhibition of cell death by Q-VD-Oph, and cleavage of the caspase-3. Selinexor also induced mitochondrial depolarization and was associated with upregulation and activation of the pro-apopototic Bax protein; (v) XPO1 mut/CLL were significantly resistant to PCD induced by Selinexor (p=0.003). In contrast, the mutations in XPO1 had no effect in FCR and Ibru PCD induction; (vi) 2p+/CLL cells were resistant to PCD induced by all tested drugs: FCR (p=0.01), Ibru (p=0.003), Ide+R (p=0.004) and Selinexor (p=0.0001). Conclusion: Our data show that 2p+/CLL is associated to FCR, Ibru and Ide+R drug resistance. Strikingly, Selinexor, a new XPO1 inhibitor, is unable to induce PCD in 2p+ and/or XPO1 mut CLL, which strongly suggests a key role for XPO1 in the CLL drug resistance associated to the 2p gain. Altogether, our work provide substantial progress in the understanding of the role of XPO1 in CLL drug resistance and suggests that the assessment of the 2p gain and the mutations in XPO1 will be considered before to decide a CLL therapy. As 2p gain could be observed in other B malignancies, it is tempting to extend these recommendations to all Selinexor treatments. Disclosures Choquet: Janssen: Consultancy; Roche: Consultancy. Leblond:Janssen: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Mundipharma: Honoraria.

Download Full-text

FTY720 Has Potent Anti-Leukemic Effects on Acute Lymphoblastic Leukemia Cells and Results In Caspase Independent Cell Death

Blood ◽

10.1182/blood.v116.21.3260.3260 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3260-3260

Author(s):

Craig T. Wallington-Beddoe ◽

John Hewson ◽

Kenneth Francis Bradstock ◽

Linda J. Bendall

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Cell Lines ◽

Western Blotting ◽

Caspase 3 ◽

Lymphoblastic Leukemia ◽

Parp Cleavage ◽

Oxygen Species ◽

Ic50 Values

Abstract Abstract 3260 Introduction: Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, which usually responds to chemotherapy. Long-term survival in adults is poor with most developing disease relapse, whilst Ph+ ALL has a particularly poor prognosis. FTY720 is an immunosuppressive drug that has recently demonstrated efficacy in phase 3 trials of relapsing/remitting multiple sclerosis. FTY720 also appears promising in a number of malignancies with the proposed mechanism being the reactivation of PP2A, a protein serine/threonine phosphatase whose activity may be reduced in malignant cells. Here we report findings of in vitro testing of FTY720 on Ph+ and negative ALL cell lines and primary patient samples, describing mechanisms of cell death. Methods: ALL cell lines and primary patient samples were treated with 1 nM - 100 μM FTY720 for 24 hours. Viability was measured by flow cytometry using propidium iodide and annexin V staining. Cellular proliferation was measured by 3H-thymidine incorporation. Flow cytometry and western blotting were used to measure caspase 3 activation whilst western blotting was used to assess caspase 3, PARP cleavage and LC3II formation. Electron microscopy permitted a detailed examination of cell ultra-structure and confocal microscopy with lysosensor blue staining enabled visualisation of acidic vacuoles. Reactive oxygen species generation was assessed by flow cytometry using the cell permeable dye carboxy-H2DCFDA. Results: FTY720 produced a profound reduction in proliferation and viability of Ph+ (ALL1 cells) and Ph− (REH, NALM6 and LK63 cells) cell lines and patient samples (n=7) in the low micromolar range. IC50 values for loss of viability at 24 hours ranged from 5.3 μM for ALL1 to 7.9 μM for LK63. The IC50 values for proliferation at 24 hours were 1.4 μM for ALL1 and 3.5 μM for REH. Caspase 3 activation was observed only at very low levels by flow cytometry whilst both caspase 3 and PARP cleavage were not detected by western blotting. Inhibition of caspases by ZVAD-FMK failed to rescue ALL cells from FTY720 induced cell death, demonstrating a caspase independent cell death mechanism. Light microscopy revealed prominent cytoplasmic vacuolation, and electron microscopy showed features consistent with autophagy and necrosis. Western blotting demonstrated strong LC3II bands and confocal microscopy, using lysosensor blue, revealed prominent acidic vacuolation, all confirming the induction of autophagy. Reactive oxygen species were generated in response to FTY720 treatment and partial reversal of this by N-acetyl-cysteine produced a concomitant increase in cell viability. PP2A inhibition with okadaic acid failed to rescue cells from FTY720-induced cell death. Conclusion: FTY720 is a highly active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase independent mechanism of cell death with the occurrence of autophagy and necrosis. PP2A activation is not solely responsible for leukemic cell death. Data on the in vivo effects of FTY720 on ALL cells in NOD-SCID mice will be presented. Disclosures: Bendall: Genzyme: Honoraria.

Download Full-text

Hypoxia Induces Drug Resistance In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.1852.1852 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1852-1852

Author(s):

Barbara Muz ◽

Feda Azab ◽

Pilar De La Puente ◽

Irene M. Ghobrial ◽

Ravi Vij ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Flow Cytometry ◽

Drug Resistance ◽

Mtt Assay ◽

Research Funding ◽

Caspase 3 ◽

Advisory Board ◽

Associated Proteins ◽

Apoptosis Proteins

Abstract Introduction Multiple Myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) and overproduction of immunoglobulin. The interaction of MM cells with the BM microenvironment was shown to play an important role in the drug resistance in MM. Hypoxia was shown to develop in the BM niche during progression of MM and to play a major role in the dissemination of MM cells to the new BM niches. In this study, we tested the effect of hypoxia on the proliferation, cells cycle, apoptosis and induction of drug resistance in MM cells. Methods MM cell lines were exposed to normoxia (21% O2) or hypoxia (1% O2) and the effect of hypoxia on cell survival (by MTT assay), apoptosis (by annexin-PI staining and analysis with flow cytometry) and cell cycle (by cell fixation and permeabilization, RNA degradation and DNA staining with PI, and analysis with flow cytometry) was tested. Moreover, the effect of hypoxia on the expression of PI3K pathway-associated proteins (p-PI3K, p-AKT and p-mTOR), cell cycle proteins (cyclin-D, cyclin-E, p-Rb and p27) and apoptosis proteins (cleaved PARP, caspase-3, Bcl-2, Bcl-Xl and Mcl-1) were studied. We tested the effect of hypoxia on the sensitivity of MM cells to different therapeutic agents using MTT assay after the MM cells were treated with increasing concentrations of bortezomib, carfilzomib and melphalan. Finally, we examined the effect of a HIF inhibitor on the sensitivity of MM cells to therapy. Results Hypoxia decreased the proliferation of MM cells and reduced phosphorylation of p-PI3K, p-AKT and p-mTOR. Similarly, MM cells exhibited G1 cell cycle arrest, decreased expression of cell cycle-associated proteins including cyclin-D3, cyclin-E, p-Rb, and increase in cell cycle inhibitory protein p27. However, hypoxia did not alter the apoptosis of MM cells, where neither apoptosis was detected in MM cells due to hypoxia using annexin-PI staining, nor pro-apoptosis proteins were activated due to hypoxia as shown by the unchanged levels of cleaved PARP, caspase-3, Bcl-2, Bcl-Xl, and Mcl-1 proteins. Moreover, we found that hypoxic cells displayed less sensitivity to bortezomib and carfilzomib, but it did not induce any change in melphalan activity. Treatment with carfilzomib or bortezomib (5nM for 48hrs) showed decreased survival of about 50% of normoxic cells, while no effect of the drugs was observed on survival in hypoxia. The same trends where observed in higher and lower concentrations of the drugs. Moreover, we found that treatment with a HIF inhibitor could partially rescue the sensitivity of MM cells to bortezomib and carfilzomib. Conclusions We report that hypoxia decreases the proliferation and cell cycle of MM cells without signs of apoptosis, and that hypoxia induces drug resistance to bortezomib and carfilzomib, an effect which was partially reversed by a HIF inhibitor. This data suggests the hypoxia signaling as a therapeutic target for sensitization of MM cells to therapy, and suggests the use of HIF inhibitors in combination with other drugs as a novel therapeutic strategy for treatment of MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Download Full-text

Dual targeting of heat shock proteins 90 and 70 promotes cell death and enhances the anticancer effect of chemotherapeutic agents in bladder cancer

Oncology Reports ◽

10.3892/or.2014.3132 ◽

2014 ◽

Vol 31 (6) ◽

pp. 2482-2492 ◽

Cited By ~ 34

Author(s):

LIANG MA ◽

FUMINORI SATO ◽

RYUTA SATO ◽

TAKANORI MATSUBARA ◽

KENICHI HIRAI ◽

...

Keyword(s):

Bladder Cancer ◽

Cell Death ◽

Heat Shock ◽

Heat Shock Proteins ◽

Chemotherapeutic Agents ◽

Anticancer Effect ◽

Dual Targeting ◽

Shock Proteins

Download Full-text

Haeamtang Induces Apoptosis of Colon Cancer HT-29 Cells through Activation of Caspase-3

The American Journal of Chinese Medicine ◽

10.1142/s0192415x07005363 ◽

2007 ◽

Vol 35 (05) ◽

pp. 897-909 ◽

Cited By ~ 5

Author(s):

Phil-Dong Moon ◽

Hyun-Na Koo ◽

Hyun-Ja Jeong ◽

Ho-Jeong Na ◽

Su-Jin Kim ◽

...

Keyword(s):

Colon Cancer ◽

Flow Cytometry ◽

Cell Death ◽

Mtt Assay ◽

Caspase 3 ◽

Water Extract ◽

Time Dependent ◽

Dependent Manner ◽

Caspase Inhibitor ◽

Ht 29

The effect of Haeamtang (HAT) on the colon cancer HT-29 cells was investigated in this study. A water extract of HAT significantly decreased the number of HT-29 cells in a dose-and time-dependent manner as determined by a MTT assay. Flow cytometry results revealed a dose- and time-dependent increase of dead cells in HT-29 cells treated with HAT extract. The anticancer activity of the H AT extract is attributed to apoptosis induced in HT-29 cells, which was demonstrated by increased caspase-3 activity and poly-ADP-ribose polymerase fragmentation. A selective caspase inhibitor, z-VAD-fmk, inhibited the HAT-induced cell death. Taken together, these results demonstrate that HAT extract induces apoptosis in HT-29 cells.

Download Full-text

Targeting the p62-ZZ/N-End Rule Pathway in Multiple Myeloma Overcomes Proteasome Inhibitor-Resistance Via Induction of Necroptosis and Enhances the Bone Anabolic Effects of Proteasome Inhibitors

Blood ◽

10.1182/blood-2019-131865 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4391-4391

Author(s):

Silvia Marino ◽

Daniela Nicoleta Petrusca ◽

Edward Simpson ◽

Judith L Anderson ◽

Xiang-Qun Xie ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Drug Resistance ◽

Cell Death ◽

Bone Formation ◽

Cell Viability ◽

Caspase 3 ◽

Critical Role ◽

Proteasome Inhibitors ◽

Regulation Of Transcription

Multiple myeloma (MM) is incurable and 80% of MM patients develop MM bone disease (MMBD). MMBD lesions do not heal due to the persistent suppression of bone formation, which markedly increases mortality and contributes to MM drug resistance. Current treatments for MMBD, such as bisphosphonates and denosumab, target bone destruction but do not result in new bone formation. Although proteasome inhibitors (PIs) have greatly improved survival of MM patients, they only have transient bone anabolic effects. Further, development of drug resistance to PIs remains a major clinical problem. We previously showed that the ZZ domain of p62 (sequestosome-1),plays an important role in both MM growth and suppression of osteoblast (OB) differentiation in the MM microenvironment, by regulating multiple signaling pathways and acting as a cargo-receptor for autophagy. Recently, our collaborators showed that the p62-ZZ is a high-affinity N-recognin of the N-end rule pathway (NERP). p62-ZZ also serves as the molecular switch for necroptotic versus apoptotic cell death pathways. We previously reported that MM cells or TNFα prevent OB differentiation by inducing persistent epigenetic repression of the Runx2-P1 promoter in MM patient bone marrow stromal cells (BMSCs), via the transcriptional repressor Gfi1. We found that blocking p62-ZZ by saturating it with a novel synthetic p62-ZZ/NERP competing ligand, XRK3F2, prevented and reversed MM-induced Gfi1 occupancy at the Runx2-P1 promoter, allowing BMSCs to increase OB marker gene expression and to mineralize. These results suggest that targeting the p62-ZZ/N-end rule pathway would enhance the bone anabolic effects of PIs. To test this hypothesis, we first exposed normal OBs to different doses of bortezomib (Btz) or XRK3F2 or their combination. The combination significantly increased OB differentiation markers Runx2 (60%), Osterix (20%) and ATF4 (60%), and induced mineral deposition compared with either drug alone. The combination also blocked TNFα up-regulation of Gfi1 and suppression of OB differentiation. Interestingly, none of the concentrations tested decreased OB viability. Studies with MM patient-BMSCs showed that XRK3F2 reversed suppression of OB differentiation induced by MM cells, allowing them to mineralize. Importantly, our preliminary in vivo data showed that administration of XRK3F2 to mice with established MM induced dramatic cortical bone formation in MM-containing bones but had no effect on tumor burden. We and others previously showed that MM cells subjected to sustained proteasomal inhibition, rely on p62-mediated autophagic degradation to reduce the proteotoxic load caused by excessive immunoglobulin synthesis. We recently found that targeting the p62-ZZ domain in human MM cells, increases Btz-induced MM cell death, independently of their p53 status. The combination also significantly reduced cell viability in Btz resistant cells although no caspase 3 activation was observed, suggesting a caspase-3 independent cell death. To determine the mechanism(s) responsible for MM cell death induced by the combination, we pretreated MM cell lines and primary CD138+ MM cells with Z-DEVD (20μM), bafilomycin (Baf, 40nM), or necrostatin1 (NEC1, 50μM). The anti-MM effects of XRK3F2 or Btz+XRK3F2 were fully blocked by NEC1, an inhibitor of necroptosis, but not by inhibitors of caspase-dependent apoptosis (Z-DEVD) or autophagy (Baf), supporting that p62-ZZ regulates necroptosis in MM cells. RNAseq analysis of the additive effect of Btz+XRK3F2 on gene expression showed a total of 583 differentially regulated genes, including 374 significantly down-regulated and 209 significantly upregulated. GO term analysis of up-regulated DEGs identified an enrichment in the endoplasmic reticulum (ER) stress and ER unfolded protein response, and regulation of transcription in response to stress and autophagy. In summary, our results demonstrate that targeting the p62-ZZ/N-end rule pathway in combination with PIs in MM significantly reduces MM cell viability by activating multiple death pathway and overcomes PI-resistance of MM cells. In addition, targeting the p62-ZZ in OBs potentiates the bone anabolic action of PIs and reverses the persistent OB suppression induced by MM cells to allow bone formation. Thus, p62-ZZ plays a critical role in MM and bone cells and identifies p62-ZZ as an important molecular target for the treatment of MMBD. Disclosures Xie: Oxis Biotech: Consultancy; ID4Pharma: Other: Founder. Roodman:Amgen: Membership on an entity's Board of Directors or advisory committees.

Download Full-text