The Potential Role of STAT's in Anti-Leukemic Therapy with Different Drugs

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5120-5120
Author(s):  
Hatice Demet Kiper ◽  
Burcin Tezcanli Kaymaz ◽  
Ozlem Purclutepe ◽  
Ceyda Tunakan Dalgic ◽  
Nur Selvi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Drug Exposure ◽  
Conflicts Of Interest ◽  
Time Dependent ◽  
Blue Dye ◽  
Dependent Manner ◽  
Down Regulation ◽  
Expression Levels ◽  
Induced Apoptosis

Abstract Abstract 5120 STAT pathways play a pivotal role in oncogenesis and leukemogenesis, thus targeting STAT signalling appears to be an effective anticancer treatment strategy. It has been described that constitutive activation of STAT3 and STAT5 plays a pro-oncogenic role both in acute and chronic myeloid neoplasms. In this study, we aimed to clarify the potential relationship between drug-induced apoptosis with different agents and STAT pathway. A third-generation bisphosphonate; zoledronate, an angiotensin-converting enzyme inhibitor (ACE-I); enalapril, a proteasome inhibitor which is used for treatment of multiple myeloma; bortezomib and a second-generation tyrosine kinase inhibitor; dasatinib were examined in this goal. Cell viability and cytotoxicity tests were conducted by using Trypan blue dye exclusion and XTT assays, respectively. Apoptotic analyses were performed by AnnexinV-EGFP staining method and fluorescence microscopy. Expression levels of STAT3, −5A and −5B genes were analysed in myeloid cell lines by qRT-PCR. The results showed that zoledronate; bortezomib and dasatinib decreased viability and proliferation and induced apoptosis in CML cell line K562 in a dose- and time-dependent manner which is associated by prominent decrease of STAT3, STAT5A and STAT5B mRNA expressions. Enalapril was also found to be cytotoxic and induced apoptosis in APL cell line HL60 in a dose- and time-dependent manner and the expression levels of STAT5A gene have significantly reduced in enalapril-treated HL60 cells as compared to untreated controls. Treatments of cell lines with other drugs were also associated with significant apoptosis in certain time points. The results and changes in expression of STAT's in mRNA level at 72nd hours are summarized in table. Taken together all these data showed that targeting STAT pathways by different drugs may be an appropriate approach in anti-leukemic therapy. This finding is important to propose that discovery or identification of novel agents targeted STATs may open new windows to the other hematological and solid malignancies which are associated with aberrant STAT expression. Table: The changes in STAT expressions after drug exposure in time-dependent manner with the dose of IC50. DRUGS CELL LINE IC50 APOPTOSIS (%) STAT3 mRNA Down Regulation (%) STAT5A mRNA Down Regulation (%) STAT5B mRNA Down Regulation (%) ENALAPRIL HL-60 7 μM 20 20* 76 5* ZOLEDRONATE K562 60 μM 34 63 31 57 BORTEZOMIB K562 177 μM 37 98 100 99 DASATINIB K562 3,314 nM 75 NA 33 78 * : Not significant NA: not applied Disclosures: No relevant conflicts of interest to declare.

Proteomic Evaluation of Pathways Associated with Dexamethasone-Induced Apoptosis and Resistance in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 642-642
Author(s):  
Karen S. Rees-Unwin ◽  
Rachel A. Craven ◽  
Sarah Hanrahan ◽  
Nicholas F. Totty ◽  
Ann M. Dring ◽  
...  
Keyword(s):  
Cell Lines ◽  
Resting State ◽  
Drug Exposure ◽  
Fold Change ◽  
Protein Profiling ◽  
Dependent Manner ◽  
Receptor Complex ◽  
Resistant Line ◽  
2D Page ◽  
Induced Apoptosis

Abstract Glucocorticoids are the mainstay of treatment for patients with myeloma (MM). Unfortunately many patients become resistant to therapy and the median survival is approximately 4.5 years. This study has used a global protein-expression approach to further characterise the pathways of dexamethasone (dex)-induced apoptosis and resistance in the sensitive and resistant MM.1 sub-line’s (MM.1S and MM.1R respectively). Following dex treatment, protein from MM.1S was separated by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using a pH range of 4–7 in the first dimension. Changes in expression pattern were identified between dex-treated and untreated cells with 24 spots downregulated and 3 spots upregulated. Identification by mass spectrometry (4700 Proteomics analyser: MALDI TOF-TOF, Applied Biosystems) demonstrated that 10/24 of the downregulated proteins were involved in cell survival and proliferation whereas the three upregulated proteins were involved in post translational modification, protein folding and trafficking. A comparison with gene expression studies identified a number of corresponding genes, as well as a number of proteins/genes highlighted in one study but not the other. Interestingly the upregulation of FKBP51 a key regulatory component of the Hsp90 steroid receptor complex was observed in both gene and protein analysis. Importantly 2D-PAGE of the dex-resistant line MM.1R demonstrated no increase in FKBP51, confirming its role in mediating dex-induced apoptosis. The Hsp90 receptor complex is important in maintaining the glucocorticoid receptor (GR) in a state receptive to dex and comprises of a number of molecules including the GR, Hsp90, and the immunophilin’s FKBP51 and FKBP52. Western and immunoblotting analysis of the complex in the MM.1S cell line identified FKBP51/52 and GR expression to increase in a time dependent manner whilst Hsp90 expression remained constant. No changes were observed in the resistant line post drug exposure. Overexpression of FKBP51 at resting state or the inability to induce expression following treatment with dex has been associated with glucocorticoid resistance in other diseases. Gene array analysis of 29 myeloma cases showed no statistical difference in FKBP51 expression between presenting myeloma cases sensitive to dex and relapsed refractory cases resistant to dex, suggesting in myeloma resistance is not mediated by a resting state upregulation of FKBP51. RQ-PCR of MM.1S and a number of other cell lines sensitive to dex showed between 5 and 24-fold change in FKBP51 expression post drug exposure, whereas cell lines resistant to dex showed <2 -fold change. Similar results were observed in primary pateint cells, suggesting that an increase in FKBP51 following dex exposure may be a surrogate marker for dex sensitivity. In conclusion these protein profiling studies have identified a number of novel proteins involved in dex-induced apoptosis and resistance, many of which warrant further investigation.

MYC Responsible for down-Regulation of CD33 Expression in Human Myeloma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5116-5116
Author(s):  
Karim Shamsasenjan ◽  
Ken-ichiro Otsuyama ◽  
Mohd S. Iqbal ◽  
Maged S. Mahmoud ◽  
Michio M. Kawano
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Down Regulation ◽  
Myeloma Cells ◽  
Expression Levels ◽  
Stat3 Phosphorylation ◽  
Serum Crp ◽  
Human Myeloma Cells

Abstract Human myeloma cells from about 10% of cases with multiple myeloma expressed CD33 and have monocytoid morphology with convoluted nuclei, and all these patients had no increase in serum CRP values. In CD33(+) myeloma cells as well as myeloma cell lines, CD33 expression levels were correlated with the increased expression levels of CEBPA (C/EBPα) gene. This correlation was confirmed by the finding that transfection with the CEBPA gene induced CD33 expression in a CD33(−) myeloma cell line. As suggested by the lack of an increase in serum CRP values in CD33(+) myelomas, IL-6 down-regulated the expression of CD33 in CD33(+) myeloma cell lines along with the down-regulation of CEBPA gene expression. Cucurbitacin I (STAT3 inhibitor) but not U0126 (MAPK inhibitor) could abolish the effect of IL-6. Furthermore, IL-6 up-regulated the expression of MYC via STAT3 phosphorylation and MYC bound to the promoter region of CEBPA gene followed by the down-regulation of the CEBPA expression. It was confirmed that introduction of shRNA for MYC into a CD33(+) myeloma cell line blocked the IL6-induced down-regulation of CD33 and CEBPA expression. Therefore, these results indicate that IL-6 can reverse the expression level of CD33 by up-regulating MYC followed by the down-regulation of CEBPA expression.

Bcl-2 Antagonism Potentiates MEK1/2/Chk1 Inhibitor Lethality in Multiple Myeloma Cells Overexpressing Bcl-2 through a Stat3-Dependent Mechanism

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4763-4763
Author(s):  
Xin-Yan Pei ◽  
Michael W Sanderson ◽  
Leena E Youssefian ◽  
Jessica Felthousen ◽  
Lora B Kramer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Down Regulation ◽  
Bh3 Mimetics ◽  
Survival Factor ◽  
Survival Signaling ◽  
Induced Apoptosis

Abstract Introduction: Multiple myeloma (MM) is characterized by deregulation of members of the Bcl-2 family of apoptotic regulatory proteins. This has led to the development of BH3-mimetics such as ABT-737 which inhibits Bcl-2/xL but not Mcl-1. Previously, we reported that simultaneous inhibition of Chk1 and MEK1/2 dramatically induced apoptosis in cultured and primary MM cells, including cells resistant to conventional agents, while sparing their normal counterparts (Pei et al., Blood 2007, 2011). Recently, we reported that this strategy circumvented MM cell resistance conferred by overexpression of Mcl-1, an important survival factor in this disease (Pei et al., PLoS One 2014). However, Bcl-2 overexpression confers significant resistance to the Chk1/MEK1/2 inhibition strategy. This raised the possibility that BH3-mimetics targeting Bcl-2 might circumvent this resistance mechanism. The purpose of the present studies was to determine whether BH3-mimetics could overcome such resistance while preserving anti-myeloma selectivity. An additional goal was to employ a new mathematical model to characterize interactions combining three novel agents that coordinately inhibit multiple survival signaling pathways. Methods: Various parental and Bcl-2 or Bcl-xL-over-expressing MM cell lines, as well as primary CD138+ MM cells were employed. ABT-737, the MEK1/2 inhibitor PD184352 (PD), and the Chk1/Wee1 inhibitor (Chk1i) were obtained from Abbott, Millipore and Calbiochem, respectively. Cells were exposed to agents alone or in various combinations for 4 -72 h, after which effects on apoptosis and signaling pathways were determined. Results: Co-administration of ABT-737 potentiated PD/Chk1i-mediated lethality in multiple parental MM cell lines, in association with Mcl-1 down-regulation, Bim up-regulation, and increased DNA damage (ΥH2A.X). Consistent with earlier findings, ectopic expression of Bcl-2 or Bcl-xL protected MM cells from the PD/Chk1i regimen. However, co-administration of ABT-737 significantly restored sensitivity towards PD/Chk1i lethality. Mathematical modeling indicated 3-agent synergistic interactions, particularly in Bcl-2 overexpressing cells. PD/Chk1i exposure inhibited phosphorylation (T705 and S727) of Stat3, another important survival factor for MM cells, while cells expressing constitutively active Stat3 (CA-STAT3) exhibited resistance to this regimen. However, the latter event was reversed by co-exposure to ABT-737. Moreover, combining ABT-737 with PD/Chk1i resulted in release of Bim from anti-apoptotic proteins including Bcl-2, Bcl-xL, and Mcl-1, accompanied by Bak and Bax conformational change (activation). Knock-down of Bim by shRNA significantly protected cells from apoptosis induced by the 3-agent combination, indicating a functional role for Bim in anti-MM activity of this regimen. Furthermore, similar interactions, together with down-regulation of pStat3, were also observed in bortezomib-resistant MM cells, as well as in patient-derived CD138+ MM cells. In contrast, the regimen was minimally toxic to normal cord blood CD34+ cells or CD138- bone marrow cells. Finally, co-culture of parental or bortezomib-resistant MM cells with HS-5 stromal cells induced up-regulation of pStat3, while treatment with ABT-737 in combination with PD/Chk1i prevented Stat3 activation and robustly induced apoptosis despite the presence of stromal cells. Conclusion: ABT-737 co-administration synergistically potentiates the lethality of the PD/Chk1i regimen in MM cells, including bortezomib-resistant and primary MM cells, but not in normal hematopoietic progenitors. It also overcomes PD/Chk1i resistance conferred by overexpression of Bcl-2 or Bcl-xL, as well as by microenvironmental factors. Mechanisms responsible for these interactions are likely to be multi-factorial, including inactivation of Stat3, up-regulation of Bim, release of Bim from Bcl-2, Bcl-xL, and Mcl-1, and activation of Bak and Bax. Collectively, these findings demonstrate that combining BH3-mimetics with Chk1/MEK1/2 inhibition circumvents multiple forms of drug resistance in MM cells while exhibiting minimal toxicit toward normal hematopoietic cells. They also argue that a strategy targeting three coordinate survival signaling pathways may be highly effective in killing MM cells, particularly those resistant to current anti-MM therapies. Disclosures No relevant conflicts of interest to declare.

Novel Small Molecule MDM2 Inhibitor MI-63 Induces p53-Dependent Apoptosis in AML Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2596-2596
Author(s):  
Ismael Samudio ◽  
Martin Dietrich ◽  
Paul Corn ◽  
Dajun Yang ◽  
Gautam Borthakur
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Small Molecule ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Wild Type ◽  
Wild Type P53 ◽  
Induced Apoptosis ◽  
Related Proteins ◽  
Mdm2 Inhibitor

Abstract Although TP53 mutations are rare in acute myeloid leukemia (AML), inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulator MDM2 (murine double minute 2). We investigated the effects of MI-63, a small molecule that activates p53 by inhibition of MDM2-p53 interaction [ Ki value of 3 nM (J Med Chem.2006;49(12):3432–5)] in AML cell lines. Treatment with MI-63 triggered apoptosis (evidenced by loss of membrane potential and externalization of phosphatidylserine) in AML cell lines with wild-type p53 (OCI-AML-3 and MOLM13) in a time and concentration-dependent manner (IC50 at 72 hrs.= 2.5 μM for OCI-AML-3 and 1 μM for MOLM-13), while a p53-null AML cell line (HL-60) was resistant (IC50 not reached at 10 μM). Moreover, knockdown of p53 in OCI-AML3 cells rendered this cell line resistant to MI-63 induced apoptosis while control vector infected OCI-AML-3 cells remained as sensitive to MI-63 similar to the parental cells. Mechanistic studies showed that MI-63 blocks G1/S phase transition in AML cells with wild-type p53 resulting in accumulation of cells in G1 phase (percentage cells inG1 phase at 24 hrs. = 88.66% vs 43.49% in cultures with DMSO control) while MI-61, a skeletally related but inactive control compound failed to do so (41.63%). Treatment with MI-63 increased cellular levels of p53 and p53 dependent proteins in OCI-AML-3 cells that include p21 and BH3-only pro-apoptotic protein Puma and pro-apoptotic multi-domain Bcl-2 family member Bax. Additionally, MI-63 induced a profound decrease in the levels of MDM4, an MDM2 homolog that has been reported to mediate resistance to the effects of nutlin-3a, suggesting that MI-63 may offer a therapeutic advantage in cells expressing high levels of MDM4. Finally, supporting the concept that increased levels of p53 modulate the apoptotic rheostat both directly, by behaving as a BH3-only protein, and indirectly by increasing the levels of sensitizer BH3-only proteins, MI-63 potently synergized with AT-101, an orally available pan inhibitor of Bcl-2, Bcl-xL and Mcl-1 (currently being evaluated as an antitumor agent in Phase I/II trials by Ascenta Therapeutics), to induce mitochondrial dysfunction and apoptosis in OCI-AML-3 cells (average combination index = 0.055±0.019). Taken together our results support preclinical evaluation of novel small molecule MI-63 alone and in combination with Bcl-2 inhibitors for the therapy of AML. The studies in primary AML samples are ongoing. Fig.1: MI-63 Induced Apoptosis Requires Intact p53 Fig.1:. MI-63 Induced Apoptosis Requires Intact p53 Fig.2: Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included) Fig.2:. Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included)

The BH3 Mimetic, ABT-737, Overcomes Stromal-Mediated Pro-Survival Signals and Synergizes with PHA-767491, a Dual Cdc7/CDK9 Inhibitor, In Acute Myeloid Leukaemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1841-1841
Author(s):  
Ruth C Morrell ◽  
Eva Szegezdi ◽  
Anna Halpin-McCormick ◽  
Karen Cawley ◽  
Afshin Samali ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
In Vivo Studies ◽  
Relative Reduction ◽  
Induced Apoptosis ◽  
Cdk9 Inhibitor ◽  
Acute Myeloid

Abstract Abstract 1841 ABT-737 is a small molecule inhibitor of Bcl-2 and Bcl-xL with reported activity in pre-clinical in-vitro and in-vivo studies of acute myeloid leukaemia(AML) but to date no data has been reported on its activity in an AML co-culture model. To address this, we examined the effects of co-culture of AML cell lines (MOLM-13, ML-1, KG-1, OCI-AML2) with HS5 cells, a human stromal cell line, on sensitivity to Ara-C and ABT-737. All cell lines cultured in the presence of HS-5 stroma demonstrated a significant reduction in Ara-C-induced apoptosis (% relative reduction - OCI-AML2:80%; ML1:65%; MOLM-13:53%; KG-1:55%) as compared to cells cultured in suspension in normal complete media, with the effect on expression of Bcl-2 family members being currently under evaluation. In contrast, in the presence of ABT-737, HS-5 co-culture did not provide any protective effect whatsoever to AML cells, with IC50 ranging from 0.1 to 0.3μM in the cell lines noted above, regardless of the presence of stroma. OCI-AML3, an AML cell line known to express high levels of Mcl-1 was resistant to ABT-737 in both normal suspension cultures and co-culture. Indeed Mcl-1, an important pro-survival protein in haematopoietic cells is thought to be a key factor promoting resistance to ABT-737 and it has recently been reported that transcriptional upregulation of Mcl-1 may follow exposure to ABT-737. Thus, the combination of ABT-737 with strategies to deplete Mcl-1 is particularly attractive. Cdk9 inhibition is such a strategy. Since Cdk9 phosphorylates RNA polymerase II affecting the rate of transcription, inhibition leads to a depletion of proteins with short half-lives, such as Mcl-1. Here we report that resistance of OCI-AML3 cells to ABT-737-induced apoptosis can be overcome by combination with PHA-767491, a novel dual Cdc7/CDK9 inhibitor. OCI-AML3 cells were treated with increasing concentrations of ABT-737, PHA-767491 or both. Co-administration resulted in a strong synergistic apoptosis-inducing effect as assessed by AnnexinV staining, with combination indices, as calculated by Chou et Talalay, for a range of doses of both drugs of <1 (range 0.3–0.9). Importantly, the sensitising effect of PHA-767491 was seen only at concentrations (≥ 2μM) that resulted in significant downregulation of Mcl-1 protein expression, implicating Mcl-1 downregulation as a possible cause of synergy. We are currently investigating the precise role of Mcl-1 in this regard. In conclusion, taken together, these studies support that ABT-737, possibly in combination with agents to deplete Mcl-1, represents a promising therapeutic strategy for AML and warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mir-155 Expression Raises the Apoptotic Threshold in Waldenström Macroglobulinemia By Inhibition of FOXO3a and Bim

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1671-1671 ◽  
Author(s):  
Brian T Gaudette ◽  
Lawrence H. Boise
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Therapeutic Index ◽  
Fold Increase ◽  
Malignant Cell ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Apoptotic Proteins ◽  
Induced Apoptosis

Abstract Waldenström Macroglobulinemia (WM) is a proliferative disorder of lymphoplasmacytic cells in the lymph nodes and bone marrow. Phenotypically, WM cells are CD19+, CD20+, CD22+, CD38+, CD138+/- and are lymphoid or plasmacytic in morphology. The disease is characterized by abundant secretion of monoclonal, IgM which causes much of morbidity associated with WM. The disease carries a high prevalence of activating somatic mutations in MyD88 (91%) and CXCR4 (28%), which have been shown to contribute to poor prognosis. These mutations involve signaling cascades that activate pathways known to enhance survival signaling including Bcl-xL. Generally, upregulation of pro-apoptotic Bcl-2 family proteins is observed as cancer cells break differentiation and proliferation checkpoints. To counter this, it becomes necessary for the cell to increase expression of anti-apoptotic Bcl-2 proteins making it dependent on a particular protein or set of proteins for survival. However, we have previously shown data that Bcl-2 family expression in WM is characterized by low expression of both pro- and anti-apoptotic proteins. To investigate a mechanism for this regulation, we examined the Bcl-2 family expression in three WM cell lines and observed that in two lines, BCWM.1 and MWCL-1, the pro-apoptotic BH3-only protein Bim was expressed at very low levels or absent, respectively, which corresponded with low sensitivity to inducers of Bim-dependent intrinsic apoptosis including ABT-737 and dexamethasone. These cell lines were sensitive to bortezomib which can induce apoptosis independent of Bim via a tBid-dependent mechanism. In the third WM cell line, RPCI-WM1, Bim was expressed at moderate levels but the pro-apoptotic proteins Bak and Bax were underexpressed and absent, respectively, which rendered the cell line completely apoptosis-deficient. Having ruled out genomic copy number variation at the loci corresponding to these genes and finding no evidence of epigenetic silencing by methylation, we examined the expression of microRNAs targeting these genes. We first examined the predicted targets of seven commonly dysregulated microRNAs in WM. Of these only one, miR-494, was found to have a moderately conserved target site in the 3’ UTR of Bim. However, the expression pattern of miR-494 did not correlate with the pattern of Bim expression in the WM cell lines. None of these microRNAs were predicted to target Bax or Bak. Therefore, we examined the expression of the remaining commonly dysregulated microRNAs and found that miR-155 was expressed at much higher levels in BCWM.1 and MWCL-1 than in RPCI-WM1 or the multiple myeloma (MM) cell line MM1.s. miR-155 is known to both directly and indirectly regulate FOXO3a, a transcription factor important in the induction of Bim. Confirming this, we observed low protein expression of FOXO3a in both BCWM.1 and MWCL-1 cells. To test this mechanism we stably expressed an anti-miR that targets miR-155 or a control anti-miR in all three WM cell lines and observed an increase in mRNA for FOXO3a and Bim as well as an increase in Bim protein in BWCM.1 and MWCL-1 cells expressing anti-miR-155, while no effect on Bim was observed in the RPCI-WM1 line that does not express miR-155 at high levels. This corresponded with a two-fold increase in ABT-737-induced apoptosis in both BWCM.1 and MWCL-1 in the absence of any additional death signal. As expected, miR-155 antagonism did not significantly increase bortezomib-induced apoptosis. These data indicate that miR-155 expression raises the apoptotic threshold in WM by limiting FOXO3a-mediated Bim expression. Cancer therapy relies on the ability to kill malignant cells at a lower dose than would kill healthy cells. This therapeutic index relies heavily on what is termed mitochondrial priming which is a measure of the expression of pro-apoptotic proteins in a cell. The malignant cell remains alive due to sequestration of these proteins by anti-apoptotic proteins, yet requires less death signaling to cause release of sufficient quantities of pro-apoptotic proteins to activate apoptosis. The data presented here indicate that increased expression of miR-155 raises the apoptotic threshold of WM cells by inhibiting Bim expression and thereby compromises the therapeutic index of many agents. Therefore, the sensitivity to a variety of apoptosis-inducing therapies would be increased by targeting miR-155 in combination as part of the treatment modality. Disclosures No relevant conflicts of interest to declare.

scholarly journals Enhancing the Therapeutic Efficacy of Daunorubicin and Mitoxantrone with Bavachinin, Candidone, and Tephrosin

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Sina Darzi ◽  
Seyed Abbas Mirzaei ◽  
Fatemeh Elahian ◽  
Sadegh Shirian ◽  
Amir Peymani ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Cancer Treatment ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell Line ◽  
Multidrug Resistant ◽  
Resistant Cell ◽  
Dependent Manner ◽  
Expression Levels ◽  
Treatment Research

The capability of flavonoids in sensitizing cancer cells was demonstrated in numerous works to chemotherapy and converse multidrug resistance by modulating efflux pumps and apoptosis mechanisms. Three flavonoids, namely, bavachinin, tephrosin, and candidone, have been recently introduced to cancer treatment research presenting various activities, such as antibacterial, immunomodulatory, cell death, and anticancer. Less information exists regarding the therapeutic significance of these flavonoids in cancer treatment, especially in overcoming multidrug resistance (MDR). Here, we tempted to investigate the potency of these agents in reversing MDR by analyzing their effects as chemosensitizers on cell cytotoxicity, P-gp and ABCG2 protein expression levels, and their function on two multidrug-resistant cell lines, P-gp-overexpressing human gastric adenocarcinoma cell line (EPG85.257RDB) and ABCG2-overexpressing human epithelial breast cancer cell line (MCF7/MX). The inhibitory concentration of 10% (IC10) of bavachinin, tephrosin, and candidone in EPG85.257RDB cells was 1588.7 ± 202.2, 264.8 ± 86.15, and 1338.6 ± 114.11 nM, respectively. Moreover, these values in MCF7/MX cell were 2406.4 ± 257.63, 38.8 ± 4.28, and 27.9 ± 5.59 nM, respectively. Expression levels of ABCG2 and P-gp were not significantly downregulated by these flavonoids. Maximum levels of daunorubicin and mitoxantrone accumulations and minimum rates of drug efflux in both cell lines were detected 48 hrs posttreatment with tephrosin and bavachinin, respectively. Chemosensitization to mitoxantrone and daunorubicin treatments was, respectively, achieved in MCF7/MX and EPG85.257RDB cells in response to IC10 of bavachinin and tephrosin, independently. These effects did not follow time-dependent manner, and each flavonoid had its cell-dependent patterns. Overall, bavachinin, tephrosin, and candidone showed potency to sensitize MDR cells to daunorubicin and mitoxantrone and could be considered as attractive MDR modulators for cancer treatment. However, their action was time and cell specific.

Targeting Myeloma Stem Cells through Simultaneous Inhibition of Wnt and Hedgehog (Hh) Signaling Pathways

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 615-615
Author(s):  
Guido J. Tricot ◽  
Ye Yang ◽  
Chujiao Xu ◽  
Hongwei Xu ◽  
Ming Zeng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Signaling Pathways ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cyclin D3 ◽  
Expression Levels ◽  
Gli1 Expression

Abstract Abstract 615 Cancer stem cells (CSC) have been identified in a variety of tumor types, including multiple myeloma (MM). Different signaling pathways, such as Wnt, Hedgehog (Hh), Notch and Bmi, are up-regulated in CSC. MM stem cells (MMSC) are enriched in the side population and have increased aldehyde dehydrogenase 1 A1 (ALDH1A1) activity. They are non-cycling and resistant to drugs typically used to treat MM. In this study, MMSC were isolated from 10 MM cell lines (CD138− fraction) and 4 primary MM patients by selecting the CD19+ CD138− cells from bone marrows heavily involved with MM. We confirmed that the CD19+/CD138− cells were part of the malignant clone by comparing expression levels of spiked genes, such as MAF-B and Cyclin D3, in MMSC and CD138+ MM cells. We also verified that CD138− from MM cell lines and the primary CD138−CD19+ had stem cell characteristics by clonogenic in vitro assays and tumor formation in NOD/SCID mice. GEP and real-time PCR analyses indicated that RARa2 levels were significantly higher levels in MM cell line-derived and primary MMSC than in CD138+ bulk MM cells. We also showed that over-expression of RARα2 in low-expressing MM cell lines resulted in increased Wnt and Hh activity, as evidenced by higher levels of nuclear β-catenin, Cyclin D1, TCF4, LEF1, Smo and GLI1. Expression levels of these proteins were also higher in MMSC than in CD138+ MM cells. We subsequently evaluated the efficacy of inhibition of Wnt (CAY10404, a COX-2 inhibitor) and Hh (cyclopamine and itraconazole) signaling on MMSC in vitro and also in vivo using the 5TGM1 mouse model. These drugs given separately induced potent cell death and growth inhibition in MMSC. Quantitative PCR and western blot revealed that CAY10404 decreased nuclear β-catenin and Gli1 expression, while cyclopamine and itraconazole inhibited nuclear GLI1 expression. The efficacy of CAY10404 and cyclopamine was further evaluated in the 5TGM1 mouse model. The CD138− fraction of this cell line has many of the same characteristics as human CD138− MMSC. After injection of 0.5 M cells into the C57BL/KaLwRij mice through the tail vein and allowing growth for 1 week, CAY10404 (20mg/kg, I.P.) and cyclopamine (20mg/kg, I.P) were given separately three times per week. Interestingly, we observed that the mice injected with untreated CD138+ cells survived longer than untreated mice receiving CD138− cells (P < 0.05). Treatment with CAY10404 and cyclopamine significantly reduced the tumor burden measured by idiotype IgG2 protein levels and dramatically prolonged survival (P < 0.05). We conclude that activation of Wnt and Hh pathway maintains the “stemness” features of MMSC. In MMSC, these pathways are activated by increased RARα2 expression, which we have shown to be related to drug resistance. MMSC can be eradicated by a combination of Wnt and Hh inhibitors. Disclosures: No relevant conflicts of interest to declare.

Low-dose Triptolide could enhance the Idarubicin-induced apoptosis in KG1α cells through down-regulation of MDR1 and NF-κb

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4922-4922
Author(s):  
Bing Xu ◽  
Jiahong Tang ◽  
Feili Chen ◽  
Yanyan Li ◽  
Huijuan Dong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Low Dose ◽  
Conflicts Of Interest ◽  
Therapeutic Window ◽  
Severe Toxicity ◽  
Flow Cytometry Analysis ◽  
Down Regulation ◽  
Hoechst 33342 ◽  
Induced Apoptosis

Abstract Background Triptolide (TPL), a diterpenoid triepoxide derived from Tripterygium wilfordii, has been used in Traditional Chinese Medicine (TCM) for centuries. Recently, TPL has also been shown to have strong anti-cancer ability both in vitro and in vivo. It could induce apoptosis in a variety of cancer cell lines. However, the clinical applications of TPL are limited by its narrow therapeutic window and severe toxicity. Thus, the focus of study about TPL has been switched to the ability of low dose of TPL in enhancing the drug-induced apoptosis of cancer cells. KG1a cell line is a kind of cell line which has been demonstrated to have a certain proportion of leukemia stem cells which are responsible for the relapse of leukemia. Usage of this kind of cell line could resemble the relapse situation of patients. MDR1 and NF-ΚB are two important factors closely related to drug-resistance. Dow-regulation of these two genes could lead to the sensitization of cells to drugs. Aims This study aims to explore whether low-dose TPL could enhance the Idarubicin-induced apoptosis in KG1a cells through down-regulation of MDR1 and NF-κB. Methods Hoechst 33342 staining and flow cytometry analysis were used to determine the ability of TPL in enhancing the Idarubicin-induced apoptosis in KG1a cells. RT-PCR as well as western blotting analysis were used to determine whether TPL combined with Idarubicin could down-regulate MDR1 and NF-κB. Results Hoechst 33342 staining and flow cytometry analysis showed that low-dose of TPL((IC20F5.0nM)) could significantly enhance the Idarubicin-induced apoptosis in KG1a cells(Idarubicin alone vs Idarubicn combined with TPL: 15.13%: 60.17%; P<0.05). TPL combined with Idarubicin could down-regulate expression of MDR1 both at protein and mRNA level with the down-regulation of NF-κB at protein level. Conclusion Low-dose Triptolide could enhance the Idarubicin-induced apoptosis IN KG1a cells through down-regulation of MDR1 and NF-κB Disclosures: No relevant conflicts of interest to declare.

Curcumin Induces Apoptosis by Inhibiting the Expression of IAPs in Human Precursor B-Acute Lymphoblastic Leukemic (PreB-ALL) Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 883-883 ◽  
Author(s):  
Azhar R. Hussain ◽  
Abdul K. Siraj ◽  
Pulicat S. Manogaran ◽  
Khawla S. Al-Kuraya ◽  
Shahab Uddin
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Dependent Manner ◽  
Curcumin Treatment ◽  
Down Regulation ◽  
Term Survival ◽  
Childhood All ◽  
Induced Apoptosis

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood resulting from the clonal proliferation of lymphoid precursors with arrested maturation. Chemotherapy can induce complete remission in more than 95% of cases of childhood ALL and achieve long-term survival in 70–80% of cases. However, ALL with the t(9:22) BCR-ABL translocation or Philadelphia chromosome (Ph1) are still highly resistant to chemotherapy from the onset. Thus, new therapeutic approaches are required to improve their prognosis. Characterization of the growth requirement of ALL cells suggest that these cancers are dependent on various cytokines via paracrine and/or autocrine mechanism in which the JAK family of proteins are closely implicated. Accordingly, tyrosine kinase inhibitors against JAKs are expected to become a new class of anti-tumor agents against these cancers. Curcumin has been shown to inhibit JAK-STAT pathway in a variety of hematological malignancies including multiple myeloma and primary effusion lymphomas. We therefore sought to determine whether curcumin suppresses the growth of acute lymphoblastic leukemia. We tested a panel of preB-ALL cell lines with various translocations after treatment with different doses of curcumin. The cell lines included REH (t12:21), RS4:11 (t4:11), 697 (t1:19) and SupB15(t9:22). Cell viability decreased in a concentration-dependent manner in 697, REH and RS4:11 with curcumin (0–40mM) whereas only minimal changes in viability was detected in SupB15. Curcumin induced apoptosis in all preB-ALL cell lines except SupB15 that was found to be refractory to curcumin treatment. Curcumin induced apoptosis via truncation of BID, loss of mitochondrial potential as determined by JC1 staining with subsequent release of cytochrome c from the mitochondria, and activation of caspase 3 and PARP. Curcumin treatment also caused the down-regulation of the IAPs, cIAP1 and XIAP. All these events occured in the sensitive cell lines 697, REH and RS4:11, however, in SupB15, curcumin failed to inhibit the expression of cIAP1 and XIAP and remained refractory to treatment. These results suggest that the IAPs may play an important role in curcumin induced apoptosis in preB-ALL cells. Altogether, our findings suggests a novel function for curcumin, acting as a growth suppressor of most preB-ALL cells and inducing apoptosis via down-regulation of IAPs. Therefore, curcumin may have a future therapeutic role in preB-ALL and possibly other malignancies.

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