RAD001, An Inhibitor of mTOR, Enhances Monocytic Differentiation Induced by ATRA through Phosphorylation of C/EBPα In AML Cell Lines

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2152-2152
Author(s):  
Hideki Yoshida ◽  
Toshihiko Imamura ◽  
Atsushi Fujiki ◽  
Yoshifumi Hirashima ◽  
Mitsuru Miyachi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Microarray Analysis ◽  
Combination Treatment ◽  
Annexin V ◽  
Resistant Cell ◽  
Pcr Analysis ◽  
Monocytic Differentiation ◽  
Cd11b Expression ◽  
Qrt Pcr ◽  
Induced Apoptosis

Abstract Abstract 2152 Background: CCAAT/ enhancer binding protein alpha (C/EBPα) is a critical transcription factor that controls monocytic and granulocytic differentiation. Several recent studies have reported that C/EBPα expression is down-regulated in acute myeloid leukemia (AML), leading to suppression of monocytic differentiation. All-trans-retinoic acid (ATRA) induces numerous transcriptional factors, including C/EBPα; however, ATRA alone is not sufficient to induce monocytic differentiation in AML. The purpose of this study was to identify agents that increase the efficacy of ATRA. RAD001 (Everolimus; provided by Novartis), a rapamycin analog, is a relatively new drug that inhibits the Akt/ PI3K/ mTOR pathway. To assess the utility of differentiation therapy as a treatment for types of AML other than acute promyelocytic leukemia, we evaluated the effects of RAD001 and ATRA combination treatment in several AML cell lines. Methods: Three AML cell lines (U-937, THP-1, and KOCL48) and two primary AML samples were treated with 2.5–5.0 nM RAD001 and 1 μM ATRA for five days. Cell growth was analyzed by counting nuclei using a Coulter counter. Monocytic differentiation was assessed by morphological analysis and flow cytometric analysis (FCM) of CD11b expression. An Annexin V assay was carried out to measure apoptosis. Microarray analysis using an Agilent expression array was employed to determine changes in gene expression associated with ATRA and RAD001 combination treatment. Quantitative RT-PCR (qRT-PCR) analysis was performed to validate the microarray results. Western blotting was carried out to measure the phosphorylation of C/EBPα at Ser 21. Results: We determined that ATRA and RAD001 treatment induced morphological changes characteristic of monocytic differentiation. Microarray analysis of THP-1 revealed that ATRA and RAD001 induced expression of a set of genes associated with monocytic differentiation, including MPEG1, CD11b, CD115 and CD14. FCM analysis confirmed that ATRA and RAD001 intensified CD11b expression in the three cell lines tested, especially in the two ATRA-resistant cell lines (KOCL48 and U937). qRT-PCR analysis also revealed that ATRA and RAD001 treatment increased expression of C/EBPα and C/EBPε, which is involved in the terminal stages of monocytic differentiation, in all three cell lines and two primary samples compared to treatment with ATRA only. Expression of PU.1 was also increased by combination treatment in all cells tested except the U937 cell line. Western blot analysis revealed that ATRA and RAD001 decreased phosphorylation of C/EBPα at serine 21. ATRA and RAD001 combination treatment also suppressed cell growth in two ATRA-resistant cell lines (growth inhibition rate: 70–80%). The Annexin V assay demonstrated that ATRA and RAD001 combination treatment strongly induced apoptosis in the three cell lines tested. Microarray analysis revealed that FasL,FADD, and caspase 8, which are associated with apoptotic pathways, showed the greatest degree of up-regulation in THP-1 cells treated with ATRA and RAD001. qRT-PCR analysis confirmed up-regulation of these genes in all three cell lines and in both primary AML samples, indicating that ATRA and RAD001 induce apoptosis in AML cells through the extrinsic cell death signaling pathway. Conclusions: RAD001 induced monocytic differentiation through induction of a set of genes associated with monocytic differentiation and phosphorylation of C/EBPα at Ser 21 when combined with ATRA. This combination therapy also induced apoptosis in AML cells through activation of the extrinsic cell death signaling pathway. Disclosures: No relevant conflicts of interest to declare.

In Vitro Evaluation of Apoptosis and Cell Death of Neuroblastoma Cell Lines Exposed to Busulfan.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4459-4459
Author(s):  
Morris Kletzel ◽  
Sarah C. Tallman ◽  
Marie Olszewski ◽  
Wei Huang
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Annexin V ◽  
Resistant Cell ◽  
Primary Tumors ◽  
Induced Apoptosis ◽  
Neuroblastoma Cell Lines

Abstract Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan. Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control. Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p<0.005). 24 Hour % Cell Viability for Varying Doses of Busulfan (mg/ml) Dose 0 Dose 0.001 Dose 0.005 Dose 0.01 Dose 0.05 Dose 0.1 Mean 66.1 44.4 40.3 40.7 37.7 39 SEM 5.56 5.17 5.96 6.17 6.03 5.60 Median 65 33.5 38 39 37 31 Range 39 to 97 14 to 87 4 to 89 6 to 93 4 to 77 5 to 88 The overall mean decrease in cell viability when compared to the control was 25.7%. However, there were only modest differences in effectiveness when comparing the doses, with an average of only 5–7% difference between doses. Further, there was much variability between the different cell lines, some with changes in apoptosis and cell death of over 50%, while other lines showed no changes at all. Limited differences were seen after 6 hours, and after 72 hours any effect of busulfan was masked by cell death due to other factors, as seen through increased cell death in untreated cells. Conclusion: Busulfan induced apoptosis and cell death in vitro in neuroblastoma cell lines at a mean of 76.43% for non-resistant lines, 59.33% for primary tumors and 35% for resistant cell lines (at middle dose 0.01mg/ml). The resistance of certain cell lines confirms the difficulties of treating multi-drug resistant cells in often heterogeneous neuroblastoma tumors. That some cell lines were responsive shows the potential of using busulfan to treat neuroblastoma in the future.

LSD1 Inhibitor Activates Retinoic Acid Pathway in MLL Fusion Positive Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1345-1345
Author(s):  
Mio Yano ◽  
Toshihiko Imamura ◽  
Kenichi Sakamoto ◽  
Hideki Yoshida ◽  
Atsushi Fujiki ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Morphological Changes ◽  
Pcr Analysis ◽  
Monocytic Differentiation ◽  
Differentiation Therapy ◽  
Cd11b Expression ◽  
Nbt Reduction ◽  
Acid Pathway

Abstract Abstract 1345 Background: Among the subtypes of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) responds dramatically to differentiation therapy with all-trans retinoic acid (ATRA). However, ATRA is not sufficient to induce differentiation in non-APL AML. Although the molecular basis for the poor response of non-APL AML to ATRA was poorly understood, Lysine-specific demethylase 1 (LSD1), the histone demetylase, was found to inhibit the retinoic acid pathway by chromatin modification through H3K4 demethylation, resulting in silencing of gene expression targeted by retinoic acid. Herein, we first evaluated whether MLL fusion partners, such as MLL-AF9 and MLL-AF4/AF5q31, affect the sensitivity of ATRA in human and murine MLL fusion positive AML cells, which is one of the most aggressive pediatric AML. In addition, we also assess whether the LSD1 inhibitor affects the ATRA sensitivity in MLL fusion positive AML cells. Methods: Three human AML cell lines with MLL fusion (THP-1 and MOLM-13 expressing MLL-AF9, and KOCL48 expressing MLL-AF4) and two murine leukemic cell lines derived from murine Lin- hematopoietic progenitors transduced by retroviral vector expressing MLL fusion genes, such as MLL-AF9 and MLL-AF5q31 were used in this study. To test the sensitivity of ATRA, all cell lines were treated with 1 μM ATRA for three days. Cell growth was analyzed by counting nuclei using a Coulter counter. Monocytic differentiation was assessed by morphological analysis, NBT reduction test and flow cytometric analysis (FCM) of CD11b expression. FCM analysis was also carried out to evaluate cell cycle and annexin V assay. Quantitative RT-PCR (qRT-PCR) analysis and western blotting was carried out to measure the RARα, C/EBPα, C/EBPε, and PU.1 expressions. To determine whether Tranylcypromine (TCP), which is a nonreversible LSD1 inhibitor, could decrease the IC50 of ATRA in MLL-AF4/AF5q31 positive cells, KOCL48 and murine MLL-AF5q31 expressing cells were treated with 0μM or 10μM TCP and titrating doses of ATRA (ranging from 0μM to 10μM). After three days, cell count was analyzed by counting nuclei using a Coulter counter to evaluate IC50 of ATRA in each cell lines. Results: We first determined that morphological changes characteristic of monocytic differentiation, CD11b expression and NBT reduction are more readily induced by ATRA in human and murine MLL-AF9 positive cells than human and murine MLL-AF4/AF5q31 positive cells The NBT reduction percentage was 17.6±1.69 in THP-1, but 2.7±1.2 in KOCL48 cells (p<0.01). The ATRA treatment also induced growth inhibition accompanied with G0/G1 arrest and apoptosis more efficiency in MLL-AF9 positive cells than MLL-AF4/AF5q31 cells. The IC50 of ATRA for THP-1 cells was 0.21±0.04 μM, but 5.31±1.50 μM for KOCL48 cells (p<0.01) The percentage of cells arrested in G0/G1 phase and Annexin/PI positive cells were 84% and 17.8% in THP-1 but 40% and 4.8% in KOCL48, respectively. Furthermore, qRT-PCR analysis and western blot analysis revealed that ATRA increased expression level of RARα, C/EBPα, C/EBPε, and PU.1, which is involved in monocytic differentiation through retinoic acid pathway, in MLL-AF9 positive cells, but not in MLL-AF4/AF5q31 positive cells. Collectively, retinoic acid pathway is more impaired in MLL-AF4/AF5q31 positive cells than MLL-AF9 positive cells. Next, we also determined that ATRA and TCP combination treatment suppressed cell growth and decreased the IC50 of ATRA in KOCL48 and murine MLL-AF5q31 expressing cells (IC50 of ATRA: 0.20±0.10 μM and 0.20±0.09 μM with TCP, vs 5.5±3.2 μM and over 10 μM without TCP, p<0.05), accompanied with morphological changes and CD11b expression, suggesting that inhibition of LSD1 restores ATRA sensitivity in both cell lines. Conclusions: Our data demonstrate that retinoic acid pathway was more profoundly impaired in MLL-AF4/AF5q31 positive cell than MLL-AF9 positive cells, suggesting MLL-AF4/AF5q31 contributes inactivation of retinoic acid pathway. Our data also demonstrate TCP restore the sensitivity of ATRA in ATRA-resistant MLL-AF4/AF5q31 positive cell lines, suggesting LSD1 plays a major role in inactivation of retinoic acid pathway in MLL-AF4/AF5q31 positive cells. Therefore, LSD1 inhibitor might be important novel therapeutic option for differentiation therapy of MLL-fusion positive AML, especially for ATRA resistant MLL-AF4/AF5q31 positive cells. Disclosures: No relevant conflicts of interest to declare.

MLL-AF9 Positive Acute Myeloid Leukemia Cells Are Sensitive To All-Trans Retinoic Acid

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3827-3827
Author(s):  
Kenichi Sakamoto ◽  
Toshihiko Imamura ◽  
Mio Yano ◽  
Hideki Yoshida ◽  
Atsushi Fujiki ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Morphological Changes ◽  
Monocytic Differentiation ◽  
Cd11b Expression ◽  
Qrt Pcr ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Nbt Reduction

Abstract Background Among the subtypes of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) responds dramatically to differentiation therapy with all-trans retinoic acid (ATRA). But, ATRA is not sufficient to induce differentiation in non-APL AML. Herein, we first evaluated whether MLL fusion partners, such as MLL-AF9 and MLL-AF4/AF5q31, affect the sensitivity of ATRA in human and murine MLL fusion positive AML cell lines. In addition, we also assessed the level of H3K4me2 modification for the RARα gene in human AML cell lines, and whether the LSD1 inhibitor affected the ATRA-resistant MLLfusion positive AML cell lines. Methods Three human AML cell lines with MLL fusion (THP1 and MOLM13 expressing MLL-AF9, and KOCL48 expressing MLL-AF4) and two murine leukemic cell lines derived from murine Lin- hematopoietic progenitors transduced by retroviral vector expressing MLL fusion genes, such as MLL-AF9 and MLL-AF5q31 were used in this study. To test the sensitivity of ATRA, all cell lines were treated with 1 μM ATRA for three days. Monocytic differentiation was assessed by morphological analysis, NBT reduction test and flow cytometric analysis (FCM) of CD11b expression. Quantitative RT-PCR (qRT-PCR) analysis and western blotting was carried out to measure the RARα, C/EBPα, C/EBPε, and PU.1 expressions. Cytotoxicity assay was performed to determine the IC50 of ATRA in these cell lines and whether ATRA could decrease the IC50 of cytarabine in MLL-AF9positive cells by using WST assays. Chromatin immunoprecipitation (ChIP) assay was performed to determine the value of H3K4me2 status using RARα-specific primers. To determine whether tranylcypromine (TCP), which is a nonreversible LSD1 inhibitor, could reactivate ATRA sensitivity, we treated KOCL48 with 10 μM TCP and 1μM ATRA. Results We first determined that morphological changes characteristic of monocytic differentiation, CD11b expression and NBT reduction are more readily induced by ATRA in human and murine MLL-AF9 positive cells than MLL-AF4/AF5q31 positive cells. The NBT reduction percentage was 12.5±3.77 in THP1, 13.1±2.02 in MOLM13, but 7.00±2.64 in KOCL48 cells (p<0.05). The ATRA treatment also induced growth inhibition and increased gene expression related to monocytic differentiation through retinoic acid (RA) pathway, more efficiency in MLL-AF9 positive cells than MLL-AF4/AF5q31 cells. The IC50 of ATRA for THP1, MOLM13 and murine MLL-AF9 cells was 3.85, 1.24 and 1.95 μM, but over 10 μM for KOCL48 and murine MLL-AF5q31 cells. Furthermore, qRT-PCR and western blot revealed that ATRA increased expression level of RARα, C/EBPα, C/EBPε, and PU.1 in MLL-AF9 positive cells, but not in MLL-AF4/AF5q31 positive cells. Collectively, RA pathway is more impaired in MLL-AF4/AF5q31 positive cells than MLL-AF9 positive cells. In addition, the increase in RARα, C/EBPα, C/EBPε, and PU.1 mRNA expressions were observed in two primary MLL-AF9 positive AML cells treated with ATRA. Next, we also carried out ChIP assay and the H3K4me2/ H3 on the RARα promoter in MLL-AF9 positive cells were higher than MLL-AF4 positive cell. Furthermore, ATRA and TCP combination treatment in KOCL48 induced morphological changes, CD11b expression, and increased the expression level of RARα, C/EBPα, C/EBPε, and PU.1, suggesting that inhibition of LSD1 restores ATRA sensitivity. Finally, ATRA in combination with cytarabine treatment in MLL-AF9 positive cells enhanced cytarabine sensitivity: the IC50 of cytarabine in THP1, MOLM13, and murine MLL-AF9cells was 4.18, 0.04, and0.065 μM without ATRA and 0.13, 0.0005, and 0.015 μM with ATRA, respectively. Conclusions Our data demonstrated that RA pathway was more profoundly impaired in MLL-AF4/AF5q31 positive cells than MLL-AF9 positive cells, suggesting type of MLL fusion protein contributes inactivation of RA pathway. Our data also identified the sensitivity of ATRA was correlated with the ratio of H3K4me2/ H3 on the RARα promoter, and TCP restore the sensitivity of ATRA in KOCL48, suggesting the decrease of the H3K4me2/H3 plays a role in inactivation of RA pathway. Additionally, we revealed that synergistic antileukemic activity of ATRA in combination with cytarabine in MLL-AF9 positive cells. Therefore, ATRA in combination with cytarabine might be novel therapeutic option for the ATRA sensitive AML cells, especially for MLL-AF9 positive cells. Disclosures: No relevant conflicts of interest to declare.

Specific MLL Fusion Partners Affect the Sensitivity of Human and Murine Leukemic Cell Lines to Demethylating Agents

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2158-2158
Author(s):  
Atsushi Fujiki ◽  
Toshihiko Imamura ◽  
Hideki Yoshida ◽  
Yoshifumi Hirashima ◽  
Mitsuru Miyachi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Fusion Proteins ◽  
Leukemic Cells ◽  
Pcr Analysis ◽  
Inhibition Rate ◽  
Monocytic Differentiation ◽  
Murine Cell ◽  
Qrt Pcr

Abstract Abstract 2158 Background and Introduction: The CCAAT/enhancer binding protein (C/EBP)α is a transcriptional factor that plays a key role in granulocytic/monocytic differentiation. We have demonstrated that ATRA and 5-Aza-2′-deoxycytidine (5-Aza) efficiently induce over-expression of C/EBPα, leading to monocytic differentiation, cell cycle arrest and apoptosis in various AML cell lines (Fujiki A, et al. The 51st Annual ASH Meeting). In addition, we also found that THP-1 cells, which express MLL-AF9, are more sensitive to treatment with a combination of ATRA and 5-Aza than KOCL48 cells, which express MLL-AF4. To explore whether the expression of MLL fusion partners affects the degree of monocytic differentiation induced by combination treatment with ATRA and 5-Aza, we compared the expression levels of various transcriptional factors and cell surface markers in human and murine leukemic cells expressing variousMLL fusion proteins. Materials and Methods: THP-1 and KOCL48, two human AML cell lines containing MLL rearrangements, and MLL-AF9, MLL-ENL and MLL-AF5q31, three murine leukemic cell lines expressing MLL fusion proteins, were used in this study. Thee murine cell lines were derived from murine Lin-hematopoietic progenitors transduced by retroviral vectors expressing MLL fusion proteins. To test the effect of Aza and ATRA on cell growth, all of the cell lines were treated with 50 nM 5-Aza for two days, followed by 1 μM ATRA for an additional three or five days. Cell growth was then analyzed by nuclei counting using a Coulter counter. Cell cycle analysis was also performed by flow cytometry (FCM). In addition, an Annexin V assay was performed to measure the level of apoptosis. To assess whether monocytic differentiation was induced, the level of CD11b/Mac 1 expression was evaluated by FCM. In addition, the level of expression of different transcription factors, including C/EBPa, C/EBPe and PU.1/ Sfpi1, was analyzed by quantitative PCR (qRT-PCR) analysis. Results: Although both THP-1 and KOCL-48 cells were moderately resistant to ATRA (growth inhibition rate of 40–50%), the addition of 5-Aza efficiently suppressed the growth in these two cell lines (growth inhibition rate of 80%). Cell cycle analysis revealed that G1 arrest occurred at almost the same level in both cell lines. However, an Annexin V assay revealed that treatment with ATRA and 5-Aza induced 1.5 times more Annexin positive THP-1 cells than KOCL-48 cells. Morphological studies of treated THP-1 and KOCL-48 cells revealed characteristic features of apoptosis, such as an extended cytoplasm containing vacuoles, the presence of fine granules, and irregular shaped nuclei. However, FCM analysis revealed that, following treatment, CD11b was expressed at higher levels in THP-1 cells than in KOCL-48 cells. Similarly, qRT-PCR analysis demonstrated that PU.1 expression was induced to higher levels in the THP-1 cell line than in KOCL-48 cells following ATRA/5-Aza treatment (p<0.05), suggesting that these two cell lines are differentially sensitive to combination therapy. In addition, the murine cell lines expressing either MLL-AF5q31 or MLL-ENL were resistant to treatment with ATRA /5-Aza (growth inhibition rate: 10–30%). However, the murine cell line expressing MLL-AF9 was sensitive to ATRA/5-Aza treatment (growth inhibition rate: 90%). Using qRT-PCR analysis, it was found that C/EBPα and Sfpi1 expression increased in MLL-AF9-expressing murine leukemic cells but not in MLL-ENL or MLL-AF5q31-expressing murine leukemic cells. Conclusions: Monocytic differentiation is more readily induced by combination treatment with ATRA and 5-Aza in MLL-AF9-expressing cells than in MLL-ENL- or MLL-AF4/AF5q31-expressing cells. The presence of specific MLL fusion partners might affect the sensitivity of cell lines to demethylating agents through inducing different degrees of DNA methylation. Disclosures: No relevant conflicts of interest to declare.

Absence of temporal ordering of apoptotic features in heat-shock treated leukemia and lymphoma cell lines

1996 ◽  
Vol 54 ◽  
pp. 758-759
Author(s):  
D. W. Fairbain ◽  
M.D. Standing ◽  
K.L. O'Neill
Keyword(s):  
Heat Shock ◽  
Cell Lines ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Resistant Cell ◽  
Membrane Blebbing ◽  
Late Loss ◽  
Induced Apoptosis ◽  
Dying Cells ◽  
In Cells

Apoptosis is a genetically defined response to physiological stimuli that results in cellular suicide. Features common to apoptotic cells include chromatin condensation, oligonucleosomal DNA fragmentation, membrane blebbing, nuclear destruction, and late loss of ability to exclude vital dyes. These characteristics contrast markedly from pathological necrosis, in which membrane integrity loss is demonstrated early, and other features of apoptosis, which allow a non-inflammatory removal of dead and dying cells, are absent. Using heat shock-induced apoptosis as a model for examining stress response in cells, we undertook to categorize a variety of human leukemias and lymphomas with regard to their response to heat shock. We were also interested in determining whether a common temporal order was followed in cells dying by apoptosis. In addition, based on our previous results, we investigated whether increasing heat load resulted in increased apoptosis, with particular interest in relatively resistant cell lines, or whether the mode of death changed from apoptosis to necrosis.

Bortezomib blocks Bax degradation in malignant B cells during treatment with TRAIL

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2797-2805 ◽  
Author(s):  
Feng-Ting Liu ◽  
Samir G. Agrawal ◽  
John G. Gribben ◽  
Hongtao Ye ◽  
Ming-Qing Du ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Resistant Cell ◽  
Protein Levels ◽  
Bax Protein ◽  
Degradation Activity ◽  
Potent Drug ◽  
Induced Apoptosis

Proapoptotic Bcl-2 family member Bax is a crucial protein in the induction of apoptosis, and its activation is required for this process. Here we report that Bax is a short-lived protein in malignant B cells and Bax protein levels decreased rapidly when protein synthesis was blocked. Malignant B cells were relatively resistant to tumor necrosis factor–related apoptosis inducing ligand (TRAIL)–induced apoptosis, and this correlated with low basal Bax protein levels. Furthermore, during treatment with TRAIL, the resistant cell lines showed prominent Bax degradation activity. This degradation activity was localized to mitochondrial Bax and could be prevented by truncated Bid, a BH3-only protein; in contrast, cytosolic Bax was relatively stable. The proteasome inhibitor bortezomib is a potent drug in inducing apoptosis in vitro in malignant B-cell lines and primary chronic lymphocytic leukemic (CLL) cells. In CLL cells, bortezomib induced Bax accumulation, translocation to mitochondria, conformational change, and oligomerization. Accumulation and stabilization of Bax protein by bortezomib-sensitized malignant B cells to TRAIL-induced apoptosis. This study reveals that Bax instability confers resistance to TRAIL, which can be reversed by Bax stabilization with a proteasome inhibitor.

scholarly journals Activation of MYC, a bona fide client of HSP90, contributes to intrinsic ibrutinib resistance in mantle cell lymphoma

2018 ◽  
Vol 2 (16) ◽  
pp. 2039-2051 ◽  
Author(s):  
Jimmy Lee ◽  
Liang Leo Zhang ◽  
Wenjun Wu ◽  
Hui Guo ◽  
Yan Li ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Resistant Cell ◽  
Intrinsic Resistance ◽  
Mantle Cell ◽  
Myc Gene ◽  
Bona Fide ◽  
Ibrutinib Resistance ◽  
Induced Apoptosis

Abstract The BTK inhibitor ibrutinib has demonstrated a remarkable therapeutic effect in mantle cell lymphoma (MCL). However, approximately one-third of patients do not respond to the drug initially. To identify the mechanisms underlying primary ibrutinib resistance in MCL, we analyzed the transcriptome changes in ibrutinib-sensitive and ibrutinib-resistant cell lines on ibrutinib treatment. We found that MYC gene signature was suppressed by ibrutinib in sensitive but not resistant cell lines. We demonstrated that MYC gene was structurally abnormal and MYC protein was overexpressed in MCL cells. Further, MYC knockdown with RNA interference inhibited cell growth in ibrutinib-sensitive as well as ibrutinib-resistant cells. We explored the possibility of inhibiting MYC through HSP90 inhibition. The chaperon protein is overexpressed in both cell lines and primary MCL cells from the patients. We demonstrated that MYC is a bona fide client of HSP90 in the context of MCL by both immunoprecipitation and chemical precipitation. Furthermore, inhibition of HSP90 using PU-H71 induced apoptosis and caused cell cycle arrest. PU-H71 also demonstrates strong and relatively specific inhibition of the MYC transcriptional program compared with other oncogenic pathways. In a MCL patient-derived xenograft model, the HSP90 inhibitor retards tumor growth and prolongs survival. Last, we showed that PU-H71 induced apoptosis and downregulated MYC protein in MCL cells derived from patients who were clinically resistant to ibrutinib. In conclusion, MYC activity underlies intrinsic resistance to ibrutinib in MCL. As a client protein of HSP90, MYC can be inhibited via PU-H71 to overcome primary ibrutinib resistance.

scholarly journals Vulpinic Acid as a Natural Compound Inhibits the Proliferation of Metastatic Prostate Cancer Cell by Inducing Apoptosis

2021 ◽  
Author(s):  
Demet Cansaran Duman ◽  
Gamze Guney Eskiler ◽  
Betül Çolak ◽  
Elif Sozen Kucukkara
Keyword(s):  
Prostate Cancer ◽  
Metastatic Prostate Cancer ◽  
Molecular Mechanisms ◽  
Treatment Options ◽  
Annexin V ◽  
Current Treatment ◽  
Pcr Analysis ◽  
G1 Arrest ◽  
Advanced Analysis ◽  
Induced Apoptosis

Abstract Lichen secondary metabolites have drawn considerable attention in recent years due to limitations of current treatment options. Vulpinic acid (VA) obtained from Letharia vulpina lichen species exerts a remarkable cytotoxic effect on different cancer types. However, the therapeutic efficacy of VA in metastatic prostate cancer (mPC) cells has not been investigated. In the present study, we aimed to identify VA-mediated cytotoxicity in PC-3 mPC cells compared with control cells. After identification of the cytotoxic concentrations of VA, VA induced apoptosis was analyzed by Annexin V, cell cycle, acridine orange and propidium iodide staining and RT-PCR analysis. Our findings showed that VA significantly decreased the viability of PC-3 cells (p < 0.01) and caused a considerable early apoptotic effect through G0/G1 arrest, nuclear bleebing and the activation of particularly initiator caspases. Therefore, VA may be a potential treatment option for mPC patients. However, the underlying molecular mechanisms of VA-induced apoptosis with advanced analysis should be further performed.

scholarly journals Mutual Expression of ALDH1A1, LOX, and Collagens in Ovarian Cancer Cell Lines as Combined CSCs- and ECM-Related Models of Drug Resistance Development

2018 ◽  
Vol 20 (1) ◽  
pp. 54 ◽  
Author(s):  
Karolina Sterzyńska ◽  
Andrzej Klejewski ◽  
Karolina Wojtowicz ◽  
Monika Świerczewska ◽  
Marta Nowacka ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Extracellular Matrix ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Lysyl Oxidase ◽  
Resistant Cell ◽  
Pcr Analysis ◽  
Ovarian Cancer Cells

A major contributor leading to treatment failure of ovarian cancer patients is the drug resistance of cancer cell. CSCs- (cancer stem cells) and ECM (extracellular matrix)-related models of drug resistance are described as independently occurring in cancer cells. Lysyl oxidase (LOX) is another extracellular protein involved in collagen cross-linking and remodeling of extracellular matrix and has been correlated with tumor progression. The expression of LOX, COL1A2, COL3A1, and ALDH1A1 was performed in sensitive (A2780, W1) and resistant to paclitaxel (PAC) (A2780PR1 and W1PR2) and topotecan (TOP) (W1TR) cell lines at the mRNA (real-time PCR analysis) and protein level (Western blot and immunofluorescence analysis). The ALDH1A1 activity was measured with the ALDEFLUOR test and flow cytometry analysis. The protein expression in ovarian cancer tissues was determined by immunohistochemistry. We observed an increased expression of LOX and collagens in PAC and TOP resistant cell lines. Subpopulations of ALDH1A1 positive and negative cells were also noted for examined cell lines. Additionally, the coexpression of LOX with ALDH1A1 and COL1A2 with ALDH1A1 was observed. The expression of LOX, collagens, and ALDH1A1 was also detected in ovarian cancer lesions. In our study LOX, ALDH1A1 and collagens were found to be coordinately expressed by cells resistant to PAC (LOX, ALDH1A1, and COL1A2) or to TOP (LOX and ALDH1A1). This represents the study where molecules related with CSCs (ALDH1A1) and ECM (LOX, collagens) models of drug resistance are described as occurring simultaneously in ovarian cancer cells treated with PAC and TOP.

scholarly journals Dihydrochalcone Derivative Induces Breast Cancer Cell Apoptosis via Intrinsic, Extrinsic, and ER Stress Pathways but Abolishes EGFR/MAPK Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-18 ◽  
Author(s):  
Wasitta Rachakhom ◽  
Patompong Khaw-on ◽  
Wilart Pompimon ◽  
Ratana Banjerdpongchai
Keyword(s):  
Breast Cancer ◽  
Er Stress ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Mapk Pathway ◽  
Annexin V ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Mcf 7

Dihydrochalcone derivatives are active compounds that have been purified from the Thai medicinal plant Cyathostemma argenteum. The objectives of this study were to investigate the effects of two dihydrochalcone derivatives on human breast cancer MDA-MB-231 and MCF-7 cell proliferation and to study the relevant mechanisms involved. The two dihydrochalcone derivatives are 4′,6′-dihydroxy-2′,4-dimethoxy-5′-(2″-hydroxybenzyl)dihydrochalcone (compound 1) and calomelanone (2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone, compound 2), both of which induced cytotoxicity toward both cell lines in a dose-dependent manner by using MTT assay. Treatment with both derivatives induced apoptosis as determined by annexin V-FITC/propidium iodide employing flow cytometry. The reduction of mitochondrial transmembrane potential (staining with 3,3′-dihexyloxacarbocyanine iodide, DiOC6, employing a flow cytometer) was established in the compound 1-treated cells. Compound 1 induced caspase-3, caspase-8, and caspase-9 activities in both cell lines, as has been determined by specific colorimetric substrates and a spectrophotometric microplate reader which indicated the involvement of both the extrinsic and intrinsic pathways. Calcium ion levels in mitochondrial and cytosolic compartments increased in compound 1-treated cells as detected by Rhod-2AM and Fluo-3AM intensity, respectively, indicating the involvement of the endoplasmic reticulum (ER) stress pathway. Compound 1 induced cell cycle arrest via enhanced atm and atr expressions and by upregulating proapoptotic proteins, namely, Bim, Bad, and tBid. Moreover, compound 1 significantly inhibited the EGFR/MAPK signaling pathway. In conclusion, compound 1 induced MDA-MB-231 and MCF-7 cell apoptosis via intrinsic, extrinsic, and ER stress pathways, whereas it ameliorated the EGFR/MAPK pathway in the MCF-7 cell line. Consequently, it is believed that compound 1 could be effectively developed for cancer treatments.

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