The Combination of a Histone Deacetylase (HDAC) Inhibitor with the BCL-2 Inhibitor GX15-070 Has Synergistic Antileukemia Effect by Inducing Both Apoptotic and Autophagic Pathways.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1633-1633
Author(s):  
Yue Wei ◽  
Tapan Kadia ◽  
Weigang Tong ◽  
Susan O’Brien ◽  
Jean Viallet ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Western Blot ◽  
Blot Analysis ◽  
Hdac Inhibitor ◽  
Caspase 3 ◽  
Single Agent ◽  
Apoptotic Cells ◽  
Caspase Activation ◽  
Apoptotic Protein ◽  
Induction Of Apoptosis

Abstract HDAC inhibitors have limited single agent clinical activity in leukemia, a probable effect of dysregulated proapoptotic pathways. We hypothesized that the combination of a HDAC inhibitor with a Bcl-2 inhibitor may result in synergistic antileukemia activity. To test this concept, we modeled in vitro the combination of MGCD0103, a class 1 specific HDAC inhibitor, with GX15-070 (Obatoclax), a novel Bcl-2 homology domain-3 (BH3) mimetic The cytotoxic potential of the combination was first assessed measuring cell viability using trypan blue extraction assays in leukemia cell lines HL60, U937, THP1, and Molt4. Mathematical modeling analysis indicated a strong synergistic effect of the combination of MGCD0103 and GX15-070 in terms of growth inhibition. Inhibitory effect of the combination at concentrations of MGCD0103 600 nM and GX15-070 200 nM was 64 fold the effect of single agent MGCD0103 and 16 fold that of GX15-070. Analysis of sequence of treatment indicated that the optimal sequence was addition of GX15-070 first followed by MGCD0103. We then analyzed the effect of the combination on the induction of apoptosis using Annexin V flow assays. The combination also showed a marked synergistic effect in terms of apoptosis induction (the combination was 217% more potent than single agent MGCD0103 and 79% more than GX15-070). Western blot analysis indicated that the use of the combination substantially enhanced the cleavage/activation of caspase-3 and the subsequent cleavage of caspase-3 substrate PARP. As demonstrated in AML and other cell types previously, we showed that GX15-070 dissociated the pro-apoptotic protein BAK from MCL-1. The release of BAK has been demonstrated to play an important role in caspase activation. Western blot analysis also demonstrated that while anti-apoptotic protein XIAP, the major inhibitor of caspase 3, was reduced in MGCD0103 treated cells, the reduction was enhanced with the combination. These results suggest that GX15-070 may sensitize XIAP to the down-regulation effect of MGCD0103, which in turn further activates caspase 3. The combinatory effect of BAK release by GX-15-070, with the suppression of XIAP by MGCD0103, that is potentiated by GX15-070, may play a critical role in the synergistic effect of these two agents on caspase activation, which then results in increased induction of apoptosis. Because in initial experiments we evidenced a discrepancy between cell viability and apoptosis, we also studied the effect of the combination on the induction of autophagy using electron microscopy: non-apoptotic cells after MGCD0103 treatment did not show increase autophagic characteristics compared to control cells, while more non-apoptotic cells under GX15-070 evidenced swollen endoreticulum, a feature of early autophagy. Most non-apoptotic cells treated with MGCD0103 and GX15-070 combination showed accumulation of macro-autophagosomes, a marker of late autophagy, indicating that more severe autophagy was induced by the combination. Furthermore, induction of LC3-II, a marker of autophagy, was observed after combination. We confirmed these results (both in terms of apoptosis and autophagy) ex vivo in primary cells (n=8) obtained from patients with relapsed/refractory AML. In conclusion, the combination of an HDAC inhibitor with GX15-070 has synergistic antileukemia activity and should be studied in human clinical trials.

Pan-BCL-2 Family Small Molecule Inhibitor GX015-070 (GeminX, Inc.) Exhibits Single Agent Cytotoxicity, Is Synergistic with Select Anti-Leukemia Cytotoxic Agents, and Induces Apoptosis in Cell Lines with t(4;11)(q21;q23) Translocations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3368-3368 ◽  
Author(s):  
Jessicca M. Rege ◽  
Blaine W. Robinson ◽  
Manish Gupta ◽  
Jeffrey S. Barrett ◽  
Peter C. Adamson ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Family Member ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Single Agent ◽  
Cytotoxic Agents ◽  
Response Surface Modeling

Abstract Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

scholarly journals DIAPH1 Is Upregulated and Inhibits Cell Apoptosis through ATR/p53/Caspase-3 Signaling Pathway in Laryngeal Squamous Cell Carcinoma

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Jiechao Yang ◽  
Liang Zhou ◽  
Yanping Zhang ◽  
Juan Zheng ◽  
Jian Zhou ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Laryngeal Squamous Cell Carcinoma ◽  
Poor Overall Survival

Cancer bioinformatics has been used to screen possible key cancer genes and pathways. Here, through bioinformatics analysis, we found that high expression of diaphanous related formin 1 (DIAPH1) was associated with poor overall survival in head and neck squamous cell carcinoma and laryngeal squamous cell carcinoma (LSCC). The effect of DIAPH1 in LSCC has not been previously investigated. Therefore, we evaluated the expression, function, and molecular mechanisms of DIAPH1 in LSCC. Immunohistochemistry and western blot analysis confirmed the significant upregulation of DIAPH1 in LSCC. We used DIAPH1 RNA interference to construct two DIAPH1-knockdown LSCC cell lines, AMC-HN-8 and FD-LSC-1, and validated the knockdown efficiency. Flow cytometry data showed that DIAPH1 inhibited apoptosis. Further, western blot analysis revealed that DIAPH1 knockdown increased the protein levels of ATR, p-p53, Bax, and cleaved caspase-3, -8, and -9. Thus, DIAPH1 is upregulated in LSCC and may act as an oncogene by inhibiting apoptosis through the ATR/p53/caspase-3 pathway in LSCC cells.

4-Acetyl-12,13-Epoxyl-9-Trichothecene-3,15-Diol from Isaria japonica Mediates Apoptosis of Rat Bladder Carcinoma NBT-II Cells by Decreasing Anti-apoptotic Bcl-2 Expression and Increasing Pro-apoptotic Bax Expression

2004 ◽  
Vol 32 (03) ◽  
pp. 377-387 ◽  
Author(s):  
Hyung-Jin Kim ◽  
Seon Il Jang ◽  
Young-Jun Kim ◽  
Hyun-Ock Pae ◽  
Hae-Young Won ◽  
...  
Keyword(s):  
Cell Death ◽  
Bladder Carcinoma ◽  
Cytotoxic Effect ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Rat Bladder ◽  
Apoptotic Protein ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

We studied the effect of 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol (AETD) isolated from Isaria japonica, one of the most popular Chinese fungal medicines, on the induction of apoptosis in rat bladder carcinoma NBT-II cells. AETD was cytotoxic to NBT-II cells, and this cytotoxic effect appears to be attributed to its induction of apoptotic cell death, as AETD induced nuclear morphological changes and internucleosomal DNA fragmentation, and increased the proportion of hypodiploid cells and activity of caspase-3. AETD treatment also decreased the expression of the anti-apoptotic protein Bcl-2 and increased the expression of the pro-apoptotic protein Bax. These results provide important information in understanding the mechanism(s) of AETD-induced apoptosis.

Abstract 255: Cytoprotective Effects of Erythropoietin and Erythropoietin-derivatives in Ischaemic Human Myotubes and the Role of Pi3k/akt Signalling Pathway

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Rebekah Sian Hwee Yu ◽  
Daryll Baker ◽  
David Abraham ◽  
Janice Tsui
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Signalling Pathway ◽  
Critical Limb Ischaemia ◽  
Human Myotubes ◽  
Muscle Biopsies

Objectives Erythropoietin (Epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously demonstrated the presence and interaction of the EpoR and βcR in human skeletal muscle. Here we aim to investigate the potential cytoprotective effects of Epo and an Epo-derivative (ARA-290) in a human in vitro model of skeletal muscle and establish a potential downstream signalling pathway utilised in protecting cells from apoptosis (including Jak-2, PI3k/Akt, NFkB). Methods Gastrocnemius muscle biopsies were obtained from patients with critical limb ischaemia and control samples were obtained from non-ischaemic patients. Human myoblasts were isolated from muscle biopsies, cultured, and allowed to differentiate into myotubes in order to investigate the cytoprotective effects of Epo and ARA-290 on myotubes subjected to simulated ischaemia. The PI3k inhibitors, LY294002 and wortmannin, were then used to determine the role of PI3k/Akt pathway in mediating cytoprotection. Following this, inhibitors against the upstreatm (Jak-2) and downstream (NFkB) molecules were also investigated. Western blot analysis, using the pro-apoptotic marker cleaved caspase-3 was performed and compared with levels of Akt and phosphorylated-Akt, using western blot analysis. Results Exogenous administration of Epo and ARA-290 were able to ameliorate the ischaemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. Addition of all inhibitors, to ARA-290 or Epo pre-treated cells, abolished the reduction in apoptosis. Conclusion The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic insult suggests a potential role in tissue protection in skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.

Starving Tumors: Inhibition of Glycolysis Reduces Viability of Human Endometrial and Ovarian Cancer Cells and Enhances Antitumor Efficacy of GnRH Receptor-Targeted Therapies

2013 ◽  
Vol 23 (1) ◽  
pp. 34-40 ◽  
Author(s):  
Madita Reutter ◽  
Günter Emons ◽  
Carsten Gründker
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Targeted Therapies ◽  
Caspase 3 ◽  
Single Agent ◽  
Gnrh Receptor ◽  
Ovarian Cancer Cells ◽  
Inhibition Of Glycolysis ◽  
Induction Of Apoptosis ◽  
Glycolysis Inhibitor

ObjectiveIncreased glycolysis for energy production is necessary for survival of tumor cells and thus represents a selective therapeutic target. We have analyzed in vitro whether inhibition of glycolysis can reduce the viability of human endometrial and ovarian cancer cells and whether it can enhance the antitumor efficacy of GnRH receptor-targeted therapies.Materials and MethodsCell viability of ovarian and endometrial cancer cells treated without or with glycolysis inhibitor 2-Deoxy-D-Glucose (2DG) alone or in combination with GnRH-II antagonist [Ac-D2Nal1, D-4Cpa2, D-3Pal3,6,Leu8, D-Ala10]GnRH-II or with cytotoxic GnRH-I agonist AEZS-108 (AN-152) was measured using alamar blue assay. Induction of apoptosis was analyzed using TUNEL assay and quantified by measurement of loss of mitochondrial membrane potential. Apoptotic signaling was measured by quantification of activated caspase-3 by using the Western blot technique.ResultsTreatment of endometrial and ovarian cancer cells with glycolysis inhibitor 2DG resulted in a significant decrease of cell viability and a significant increase of apoptosis. Treatment with 2DG in combination with the GnRH-II antagonist or with AEZS-108 resulted in a significant reduced viability compared with single-agent treatments. The observed reduction in viability was due to induction of apoptosis. Also for apoptosis induction, a significant stronger effect in the case of cotreatments compared with single-agent treatments could be observed. These additive effects could be correlated to increased activation of caspase-3.ConclusionsThe glycolytic phenotype of human endometrial and ovarian cancer cells can be targeted for therapeutic intervention. In addition, cotreatment of a glycolysis inhibitor with GnRH receptor-targeted therapies might be a suitable therapy for GnRH receptor-positive human endometrial and ovarian cancers.

scholarly journals Ovarian Cancer HO-8910 Cell Apoptosis Induced by Crocin in vitro

2015 ◽  
Vol 10 (2) ◽  
pp. 1934578X1501000 ◽  
Author(s):  
Dan Xia
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
Western Blot ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Cell Cycle Distribution ◽  
Apoptotic Pathway ◽  
Apoptosis Rate

The effect and mechanism of ovarian cancer HO-8910 cell apoptosis induced by crocin. MTT assay was performed to detect the inhibitory action of crocin on the proliferation of HO-8910 cells. Flow cytometry was used to test the cell cycle distribution and apoptosis rate of ovarian cancer HO-8910 cells. Western blot analysis was utilized to measure the levels of apoptotic proteins such as p53, Fas/APO-1, and Caspase-3. MTT analysis revealed that crocin significantly inhibited the growth of HO-8910 cells. Additionally, flow cytometry illustrated that crocin raised the proportion of HO-8910 cells in the G0/G1 phase and increased their apoptosis rate. Furthermore, Western blot analysis revealed that crocin up-regulated the expression of p53, Fas/APO-1, and Caspase-3. The results of this study showed that crocin can significantly inhibit the growth of HO-8910 cells and arrest them in the G0/G1 phase. Crocin can also promote ovarian cancer HO-8910 cell apoptosis, most likely by increasing p53 and Fas/APO-1 expression, and then activating the apoptotic pathway regulated by Caspase-3.

scholarly journals Mytoxin B and Myrothecine A Induce Apoptosis in Human Hepatocarcinoma Cell Line SMMC-7721 via PI3K/Akt Signaling Pathway

Molecules ◽  
2019 ◽  
Vol 24 (12) ◽  
pp. 2291 ◽  
Author(s):  
Huiliang Song ◽  
Yi Fu ◽  
Dan Wan ◽  
Wenjing Xia ◽  
Fengwei Lyu ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Akt Signaling ◽  
Akt Signaling Pathway ◽  
Hepatocarcinoma Cell ◽  
Apoptotic Protein ◽  
Human Hepatocarcinoma Cell Line ◽  
Hepatocarcinoma Cell Line

Trichothecene macrolides comprise a class of valuable leading compounds in developing anticancer drugs, however, there are few reports concerning their anticancer mechanisms, especially the anticancer mechanism of the 10,13-cyclotrichothecane derivatives that are found mainly in symbiotic fungi. In vitro anticancer activity of two trichothecene macrolides mytoxin B and myrothecine A against the human hepatocarcinoma cell line SMMC-7721 was investigated in the present study. MTT assay showed that mytoxin B and myrothecine A inhibited the proliferation of SMMC-7721 cells in dose- and time-dependent manners. Annexin V-FITC/PI dual staining assay revealed that mytoxin B and myrothecine A both could induce SMMC-7721 cells apoptosis in a dose-dependent manner. The decreased expression level of anti-apoptotic protein Bcl-2 and the increased expression level of pro-apoptotic protein Bax were observed apparently in Western blot analysis. The reduced ratio of Bcl-2/Bax further confirmed the apoptosis-inducing effect of mytoxin B and myrothecine A on SMMC-7721 cells. Moreover, the expression levels of caspases-3, -8, and -9, and cleaved caspases-3, -8, and -9 were all upregulated in both mytoxin B and myrothecine A-treated cells in Western blot analysis, which indicated that both compounds might induce SMMC-7721 cells apoptosis through not only the death receptor pathway but also the mitochondrial pathway. Finally, mytoxin B and myrothecine A were found to reduce the activity of PI3K/Akt signaling pathway that was similar to the effect of LY294002 (a potent and specific PI3K inhibitor), suggesting that both mytoxin B and myrothecine A might induce SMMC-7721 cells apoptosis via PI3K/Akt pathway.

Effects of riluzole (RZ) and ionizing radiation (IR) in metabotropic glutamate receptor-1 (GRM1) positive human melanoma

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 9083-9083
Author(s):  
C. Green ◽  
D. Schiff ◽  
A. Khan ◽  
S. Goyal ◽  
J. Goydos ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cell Survival ◽  
Glutamate Receptor ◽  
Blot Analysis ◽  
Caspase 3 ◽  
Metabotropic Glutamate Receptor ◽  
Human Melanoma ◽  
Metabotropic Glutamate

9083 Background: Melanoma has long been known to be relatively radio-resistant. GRM1 is a metabotropic glutamate receptor that has been detected in human melanoma cell lines and biopsies. Riluzole (RZ), a glutamate release inhibitor, has been shown to arrest GRM1 positive human melanoma cells in G2/M and sub-G1 phases of the cell cycle. The purpose of this study was to determine if RZ enhances the lethal effects of IR in human melanoma. Methods: ATP luminescence assays were performed. Clonogenic assays were performed and cell survival curves generated. Cell cycle analysis was performed utilizing flow cytometry. Western blot analysis was performed utilizing cleaved PARP and caspase-3 antibodies as markers of apoptosis. Results: Luminescence assays revealed 25uM Riluzole to be the necessary concentration for clonogenic assays. At 2Gy, there was a 48% reduction (p≤0.05) in cell survival in RZ-treated cells. At 4 Gy, there was a 19% reduction (p≤0.05) in cell survival in RZ-treated cells. No differences were seen at 6 and 8 Gy. Cell cycle analysis showed that the combination of IR and RZ was superior to IR alone in increasing the number of cells in sub-G1, which represents apoptotic death. Western blot analysis showed that the combination of IR and RZ showed yielded increased cleaved PARP and caspase-3 activity when compared to IR alone. Conclusions: Riluzole is a FDA approved drug that has long been used in ALS. It is relatively non-toxic and crosses the blood brain barrier. Our data shows that Riluzole in combination with radiation eliminates the radio-resistant shoulder of the C8161 survival curve. RZ and IR, as combination therapy are more lethal than IR or RZ alone in human melanoma, as demonstrated by flow cytometry and WB analysis. This data has promising implications for melanoma patients with brain metastases. No significant financial relationships to disclose.

Proteasome Inhibitors Can Induce Caspase-3 Activation and Apoptosis in Human CML Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4681-4681
Author(s):  
Byung-Su Kim ◽  
Chang Up Kim ◽  
Young-Ju Kim ◽  
Eun Kyung Bae ◽  
Jinhee Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Proteasome Inhibitor ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Expression Patterns ◽  
Proteasome Inhibitors ◽  
Acridine Orange Staining

Abstract The proteasome is a multi-enzyme complex that provides the ubiquitin-dependent degradation of many cytoplasmic and nuclear proteins involved in cell cycle progression and apoptosis. Inhibition of the proteasome represents a promising approach for the treatment of cancer because it can lead to cell cycle arrest and activation of caspases in tumor cells. There are several proteasome inhibitors that have been reported to induce apoptosis in various tumors. However, the effect of proteasome inhibition in human myeloid leukemia has not been reported so far. In this study, we tested two peptide-aldehyde proteasome inhibitors (MG115, MG132) on two human CML cell lines (K562, KCL22). At first, we treated both cell lines for 24, 48 and 72 hours with different doses of MG115 and MG132 and cell viability was tested by MTT assay. It showed substantial time and dose dependent cytotoxicity in both CML cell lines. Acridine orange staining also revealed DNA fragmentation. We then performed caspase-3 colorimetric assay after treating both cell lines for 6, 12 and 24 hours with 0.78μM of MG115, MG132. K562 showed the continuous rising of caspase-3 activity, while KCL22 exhibited the initial increase and subsequent mild decrease of caspase-3 activity. In addition, western blot analysis showed the reduction of procaspase-3 expression. The expression of Bcl-2 and Bcl-XL was reduced by western blot. p21 expression was slightly increased and that of cyclin D1 was decreased. Additionally, the treatment of proteasome inhibitor in CML cell lines initially induced phosphorylation of Jun kinase. We next examined the expression of heat shock proteins (Hsp70, Hsp90) after treating for 6, 12, 24 hours with the same proteasome inhibitors. Western blot analysis results indicated that expression patterns were different between MG115 and MG132. MG115 induced the slight increase of Hsp70 and Hsp90 in K562, but the reduction of both in KCL22. Meanwhile, MG132 produced the decrease of Hsp70 and Hsp90 in both K562, KCL22. In summary, our work supports that a proteasome inhibitor can induce apoptosis in human CML cell lines. We are currently focusing on the combined effect of proteasome inhibitor and Hsp90 inhibitor on CML. IC50 of Proteasome Inhibitors Cell line Proteasome Inhibitor 24hr 48hr 72hr K562 MG115 3.01 μM 1.14 μM 0.59 μM K562 MG132 μ 2.13 M 1.03 μM 0.57 μM KCL22 MG115 156.92 μM 1.36 μM 0.73 μM KCL22 MG132 1.56 μM 0.93 μM μ 0.75 M

scholarly journals DIPG-71. SELECTIVE HDAC INHIBITOR RG2833 INDUCES DIPG CELL DEATH VIA DOWNREGULATION OF THE NFĸB PATHWAY

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii300-iii301
Author(s):  
Katherine Barnett ◽  
Orlandi Novak ◽  
Charles Eberhart ◽  
Eric Raabe
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Hdac Inhibitor ◽  
Single Agent ◽  
Diffuse Intrinsic Pontine Glioma ◽  
T Test ◽  
Growth And Survival ◽  
Histone 3

Abstract Histone deacetylase (HDAC) inhibitor panobinostat demonstrated activity against diffuse intrinsic pontine glioma (DIPG) in vitro, but its efficacy in vivo was limited by toxicity and poor blood brain barrier penetration. RG2833 (RGFP109) is a selective HDAC1/3 inhibitor that has established brain penetration. In clinical trials, the Cmax (plasma) of RG2833 was 32uM. RG2833 demonstrated cytotoxicity against temozolomide-resistant glioblastoma and downregulated the NFĸB pathway. Because this pathway is overexpressed in DIPG and may play a role in DIPG cell growth and survival, we hypothesized that RG2833 would kill DIPG cells. Treatment of DIPG cell lines with RG2833 as a single agent suppresses cell proliferation in the 5–10μM range (MTS assay for HSJD007 p=0.0004 10μM vs DMSO, JHH-DIPG1 p=0.001 10μM vs DMSO, SF-7761 p=0.04 10μM vs DMSO, SU-DIPG13 p=0.01 10μM vs DMSO by t-test). RG2833 induces apoptosis by 48 hours as measured by Western blot for cPARP and cleaved caspase 3 immunofluorescence (HSJD007 p<0.003 8μM vs DMSO, JHH-DIPG1 p=0.0026 10μM vs DMSO by t-test). RG2833 also slows cell proliferation as measured by Western blot for pRb and immunofluorescence for BrdU (HSJD007 p=0.008 8μM vs DMSO, JHH-DIPG1 p=0.0002 10μM vs DMSO by t-test). Western blot confirmed a dose-dependent increase in histone 3 acetylation with RG2833 treatment at 5 hours. We detected increased acetylated p65 and decreased expression of the NFĸB regulated pro-survival genes BCL2, BCL-xL, and XIAP with RG2833 treatment. Together, this data shows that HDAC inhibitor RG2833 may be a promising therapeutic candidate for DIPG via downregulation of the NFĸB pathway.

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