Apoptosis In Chronic Myelogenous Leukemia Cells Research Articles

Methyl-Thiol-Bridged Oxadiazole and Triazole Heterocycles as Inhibitors of NF-κB in Chronic Myelogenous Leukemia Cells.

Nuclear factor kappa beta (NF-κB) is a transcriptional factor that plays a crucial role in regulating cancer cell proliferation. Therefore, the inhibition of NF-κB activity by small molecules may be beneficial in cancer therapy. In this report, methyl-thiol-bridged oxadiazole and triazole heterocycles were synthesized via click chemistry and it was observed that the lead structure, 2-(((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-5-(4-methoxybenzyl)-1,3,4-oxadiazole (4c), reduced the viability of MCF-7 cells with an IC50 value of 7.4 µM. Compound 4c also caused concentration-dependent loss of cell viability in chronic myelogenous leukemia (CML) cells. Furthermore, compound 4c inhibited the activation of NF-κB in human CML cells as observed by nuclear translocation and DNA binding assays. Functionally, compound 4c produced PARP cleavage and also suppressed expression of Bcl-2/xl, MMP-9, COX-2, survivin, as well as VEGF, resulting in apoptosis of CML cells. Moreover, ChIP assay showed that compound 4c decreased the binding of COX-2 to the p65 gene promoter. Detailed in silico analysis also indicated that compound 4c targeted NF-κB in CML cells. In conclusion, a novel structure bearing both triazole and oxadiazole moieties has been identified that can target NF-κB in CML cells and may constitute a potential novel drug candidate.

Design, semi-synthesis and examination of new gypsogenin derivatives against leukemia via Abl tyrosine kinase inhibition and apoptosis induction

Chronic myelogenous leukemia (CML) is characterized by Philadelphia translocation arising from Bcr-Abl fusion gene, which encodes abnormal oncoprotein showing tyrosine kinase (TK) function. Certain mutations in kinase domain, off-target effects and resistance problems of current TK inhibitors require the discovery of novel Abl TK inhibitors. For this purpose, herein, we synthesized new gypsogenin derivatives (6a-l) and evaluated their anticancer effects towards CML cells along with healthy cell line and different leukemic cells. Among these compounds, compound 6l was found as the most active anti-leukemic agent against K562 CML cells compared to imatinib exerting less cytotoxicity towards PBMCs (healthy). This compound also revealed significant anti-leukemic effects against Jurkat cell line. Besides, compound 6l enhanced apoptosis in CML cells with 52.4 % when compared with imatinib (61.8 %) and inhibited Abl TK significantly with an IC50 value of 13.04 ± 2.48 μM in a large panel of kinases accentuating Abl TK-mediated apoptosis of compound 6l in CML cells. Molecular docking outcomes showed that compound 6l formed mainly crucial interactions in the ATP-binding cleft of Abl TK similar to that of imatinib. Ultimately, in silico pharmacokinetic evaluation of compound 6l indicated that this compound was endowed with anti-leukemic drug candidate features.

Farnesiferol C Induces Apoptosis in Chronic Myelogenous Leukemia Cells as an Imatinib Sensitizer via Caspase Activation and HDAC (Histone Deacetylase) Inactivation.

Herein the underlying apoptotic mechanism of Farnesiferol C (FC) derived from Ferula assafoetida was elucidated in chronic myelogenous leukemia (CML) K562 and KBM5 cells. FC showed significant cytotoxicity in K562 and KBM5 cells, more so than in U937 and UL-60 acute myeloid leukemia (AML) cells. Cleaved PARP and caspase 9/3 attenuated the expression of Bcl2 and induced G1 arrest in K562 and KBM5 cells. Also, FC effectively abrogated the expression of cell cycle related proteins, such as: Cyclin D1, Cyclin E, Cyclin B1 in K562, and KBM5 cells, but caspase 3 inhibitor Z-DEVD-FMK rescued the cleavages of caspase 3 and PARP induced by FC in K562 cells. Of note, FC decreased histone deacetylase 1 (HDAC1) and HDAC2, and enhanced histone H3 acetylation K18 (Ac-H3K18) in K562 and KBM5 cells. Furthermore, combination of FC and Imatinib enhanced the apoptotic effect of Imatinib as a potent Imatinib sensitizer in K562 cells. Overall, our findings provide scientific evidence that inactivation of HDAC and caspase activation mediate FC induced apoptosis in CML cells.

An ent-Kaurane Diterpenoid Isolated from Rabdosia excisa Suppresses Bcr-Abl Protein Expression in Vitro and in Vivo and Induces Apoptosis of CML Cells.

Chronic myelogenous leukemia (CML) is a disease of the blood stem cells that features the oncoprotein Bcr-Abl. Tyrosine kinase inhibitors (TKIs) are used to treat CML patients, but these have limited efficacy due to the emergence of resistance via genetic mutation. Kamebakaurin is an ent-kaurane diterpenoid that has been isolated from Rabdosia excisa (Maxim.) H.Hara. Herein, we investigate the potential of kamebakaurin as a chemotherapy reagent for the treatment of CML. We conducted in vitro and in vivo biological experiments and found that kamebakaurin potently inhibits cell proliferation, mainly by enhancing cell apoptosis and down-regulating Bcr-Abl protein levels. In addition, kamebakaurin was found to inhibit tumor growth and has no side effects on five internal organs for in vivo experiment. These results suggest that kamebakaurin is a potential anticancer agent and is a key compound for further investigations.

Long Noncoding RNA (lncRNA) Small Nucleolar RNA Host Gene 5 (SNHG5) Regulates Proliferation, Differentiation, and Apoptosis of K562 Cells in Chronic Myeliod Leukemia

BackgroundHuman chronic myelogenous leukemia (CML) is a hematopoietic stem cell disorder with high malignant and invasive activity. lncRNA SNHG5 has been reported to be upregulated in CML. However, whether it affects the proliferation, differentiation, and apoptosis in CML cells is still unknown. This study investigated the role of SNHG5 in CML and revealed the potential mechanism.Material/MethodsK562 cells were transfected with shRNA, and the expression level of SNHG5 was assessd by quantitative RT-PCR. The proliferation ability was determined by CCK-8 assay. Western blot analysis was performed to detect protein expressions related to cell proliferation, differentiation, and apoptosis. Cell apoptosis rate was analyzed by flow cytometry. The DNA methylation level was determined by methylation-specific PCR (MSP).ResultsOur results show that inhibition of SNHG5 induced by RNA interference significantly inhibits K562 cells proliferation and induces cell differentiation with the increased expression of CD42b, CD11b, CD14, GATA-1, and β-globin. Flow cytometry analysis indicated that inhibition of SNHG5 significantly induced cell apoptosis with decreased expression of Bcl-2 and increased expression of Bax and cleaved capase-3. Additionally, Western blot and MSP analyses confirmed that inhibition of SNHG5 increased the expression of DR4 gene through suppressing its methylation.ConclusionsInhibition of SNHG5 suppressed K562 cell proliferation through inducing the differentiation and apoptosis by inhibiting methylation of DR4. Therefore, downregulated SNHG5 could play a key role in CML progression, and might provide a new strategy for the treatment of CML.

PF‑114, a novel selective inhibitor of BCR‑ABL tyrosine kinase, is a potent inducer of apoptosis in chronic myelogenous leukemia cells.

A t(9;22) chromosomal translocation which forms the chimeric tyrosine kinase breakpoint cluster region (BCR)‑Abelson murine leukemia viral oncogene homolog1(ABL) is a key mechanism underlying the pathogenesis of chronic myelogenous leukemia(CML). Pharmacological inhibition of BCR‑ABL with imatinib (Gleevec) has been reported as an effective targeted therapy; however, mutations (including the kinase domain of ABL) suppress the efficacy of inhibitors. PF‑114, a derivative of the third generation BCR‑ABL inhibitor ponatinib, demonstrated a high inhibitory activity against wild-type and mutant BCR‑ABL forms, such as the clinically important T315I. Furthermore, PF‑114 exhibited preferential kinase selectivity, safety, notable pharmacokinetic properties and therapeutic efficacy in a murine model. Investigation into the mechanisms of CML cell death revealed an exceptional potency of PF‑114 (at low nanomolar concentrations) for the CML‑derived K562 cell line, whereas leukemia cell lines that lack the chimeric tyrosine kinase were markedly more refractory. The molecular ordering of events mechanistically associated with K562 cell death included the dephosphorylation of CrkL adaptor protein followed by inhibition of ERK1/2 and Akt, G1 arrest, a decrease of phosphorylated Bcl‑2‑associated death promoter, Bcl‑2‑like protein11, BH3 interacting‑domain death agonist, Bcl‑extra large and Bcl‑2 family apoptosis regulator, and reduced mitochondrial transmembrane potential. Increased AnnexinV reactivity, activation of caspases and poly(ADP‑ribose)polymerase cleavage were proposed to lead to internucleosomal DNA fragmentation. Thus, PF‑114 may be a potent inducer of apoptosis in CML cells. Nevertheless, activation of STAT3 phosphorylation in response to PF‑114 may permit cell rescue; thus, a combination of BCR‑ABL and STAT3 inhibitors should be considered for improved therapeutic outcome. Collectively, the targeted killing of BCR‑ABL‑positive cells, along with other beneficial properties, such as invivo characteristics, suggests PF‑114 as a potential candidate for analysis in clinical trials with CML patients.

Femtosecond laser pulse assisted photoporation for drug delivery in Chronic myelogenous leukemia cells

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder occurs in the pluripotent hematopoietic stem cell. Currently, first-generation tyrosine kinase inhibitor (TKI) imatinib is the mainstay for the treatment of CML. Second generation TKI's like ponatinib, dasatinib, nilotinib, and bafetinib were treated against resistant CML. However, several CML patients develop resistance towards all existing inhibitors. Curcumin (Curcuma longa) a plant-derived natural compound is an effective bioactive component against various cancers including CML. Many studies have shown that curcumin induces time- and dose-dependent apoptosis in CML cells by regulating various downstream molecular regulators. Despite curcumin's selective cytotoxicity towards cancer cells, it has very poor bioavailability both in in-vitro and in-vivo conditions. In this present study, we have used femtosecond laser (fs-laser) pulses to ablate the cell membrane and standardized the conditions required for creating a cell membrane pores with less lethality. Following fs-laser pulse irradiation, K562 cells were incubated along with curcumin 30 μM for 0 h, 6 h,12 h and 24 h. Interestingly irradiated cells have shown higher sensitivity towards curcumin than non-irradiated cells. Immunoblotting studies showed higher induction levels of cleaved caspase 3 and 9 in irradiated population than non-irradiated. In summary, the results prove that irradiation by fs-laser pulses enhanced the bioavailability of curcumin and shows caspase-mediated cell death in irradiated CML cells than other populations.

Inhibition of ER stress-related IRE1α/CREB/NLRP1 pathway promotes the apoptosis of human chronic myelogenous leukemia cell

Endoplasmic reticulum (ER) stress is induced in chronic myelogenous leukemia (CML) cells. As an important sensor of ER stress, inositol-requiring protein-1α (IRE1α) promotes the survival of acute myeloid leukemia. NLRP1 inflammasome activation promotes metastatic melanoma growth and that IRE1α can increase NLRP1 inflammasome gene expression. This study aimed to investigate the role and molecular mechanism of IRE1α in CML cell growth. We found that overexpression of IRE1α or NLRP1 significantly promoted the proliferation and decreased the apoptosis of CML cells, whereas downregulation of these two genes showed the opposite effects. 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, reduced the expression of IRE1α and NLRP1. IRE1α elevated NLRP1 expression via cAMP responsive element binding protein (CREB) phosphorylation. NLRP1 inflammasome was activated in CML cells and its activation partly reversed ER stress inhibitor-induced cell apoptosis. Furthermore, inhibition of IRE1α/NLRP1 pathway sensitized CML cells to imatinib-mediated apoptosis. Additionally, IRE1α expression was elevated and NLRP1 inflammasome was activated in primary cells from CML patients. Downregulation of IRE1α or NLRP1 suppressed the proliferation and elevated the apoptosis of primary CML cells. Collectively, this study demonstrated that the IRE1α/CREB/NLRP1 pathway contributes to the progression of CML and the development of imatinib resistance. Hence, targeting ER stress-related IRE1α expression or NLRP1 inflammasome activation may block CML development.

Effects of Pimozide Derivatives on pSTAT5 in K562 Cells.

STAT5 is a transcription factor, a member of the STAT family of signaling proteins. STAT5 is involved in many types of cancer, including chronic myelogenous leukemia (CML), in which this protein is found constitutively activated as a consequence of BCR-ABL expression. The neuroleptic drug pimozide was recently reported to act as an inhibitor of STAT5 phosphorylation and is capable of inducing apoptosis in CML cells in vitro. Our research group has synthesized simple derivatives of pimozide with cytotoxic activity and that are able to decrease the levels of phosphorylated STAT5. In this work we continued the search for novel STAT5 inhibitors, synthesizing compounds in which the benzoimidazolinone ring of pimozide is either maintained or modified, in order to obtain further structure-activity relationship information for this class of STAT5 inhibitors. Two compounds of the series showed potent cytotoxic activity against BCR-ABL-positive and pSTAT5-overexpressing K562 cells and were able to markedly decrease the levels of phosphorylated STAT5.

Nickel pyrithione induces apoptosis in chronic myeloid leukemia cells resistant to imatinib via both Bcr/Abl-dependent and Bcr/Abl-independent mechanisms.

BackgroundAcquired imatinib (IM) resistance is frequently characterized by Bcr-Abl mutations that affect IM binding and kinase inhibition in patients with chronic myelogenous leukemia (CML). Bcr-Abl-T315I mutation is the predominant mechanism of the acquired resistance to IM. Therefore, it is urgent to search for additional approaches and targeting strategies to overcome IM resistance. We recently reported that nickel pyrithione (NiPT) potently inhibits the ubiquitin proteasome system via targeting the 19S proteasome-associated deubiquitinases (UCHL5 and USP14), without effecting on the 20S proteasome. In this present study, we investigated the effect of NiPT, a novel proteasomal deubiquitinase inhibitor, on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl.MethodsCell viability was examined by MTS assay and trypan blue exclusion staining assay in KBM5, KBM5R, K562, BaF3-p210-WT, BaF3-p210-T315I cells, and CML patients’ bone marrow samples treated with NiPT. Cell apoptosis in CML cells was detected with Annexin V-FITC/PI and rhodamine-123 staining followed by fluorescence microscopy and flow cytometry and with western blot analyses for apoptosis-associated proteins. Expression levels of Bcr-Abl in CML cells were analyzed by using western blotting and real-time PCR. The 20S proteasome peptidase activity was measured using specific fluorogenic substrate. Active-site-directed labeling of proteasomal DUBs, as well as the phosphorylation of USP14 was used for evaluating the inhibition of the DUBs activity by NiPT. Mouse xenograft models of KBM5 and KBM5R cells were analyzed, and Bcr-Abl-related proteins and protein biomarkers related to proliferation, differentiation, and adhesion in tumor tissues were detected by western blots and/or immunohistological analyses.ResultsNiPT induced apoptosis in CML cells and inhibited the growth of IM-resistant Bcr-Abl-T315I xenografts in nude mice. Mechanistically, NiPT induced decreases in Bcr-Abl proteins, which were associated with downregulation of Bcr-Abl transcription and with the cleavage of Bcr-Abl protein by activated caspases. NiPT-induced ubiquitin proteasome system inhibition induced caspase activation in both IM-resistant and IM-sensitive CML cells, and the caspase activation was required for NiPT-induced Bcr-Abl downregulation and apoptotic cell death.ConclusionsThese findings support that NiPT can overcome IM resistance through both Bcr-Abl-dependent and Bcr-Abl-independent mechanisms, providing potentially a new option for CML treatment.

Oroxylin A reverses the drug resistance of chronic myelogenous leukemia cells to imatinib through CXCL12/CXCR7 axis in bone marrow microenvironment.

Imatinib (IM), a tyrosine-kinase inhibitor, is used in treatment of multiple cancers, most notably Philadelphia chromosome-positive (Ph+ ) chronic myelogenous leukemia (CML). However, the majority of patients continue to present with minimal residual disease occurred in the bone marrow (BM) microenvironment. One of the key factors that contribute to leukemia cell drug resistance is chemokine CXCL12. In the current study, co-culturing CML cell K562 and KU812 with BM stromal cell M2-10B4 attenuated IM-induced apoptosis. CXCL12/CXCR7 pathway was activated in co-culture models, which was further proved to be related to drug resistance by silencing CXCR7. ERK phosphorylation and downstream apoptosis related proteins' activation were also observed in co-culture group after the activation of CXCR7. Moreover, oroxylin A, a bioactive flavonoid isolated from the root of Scutellaria baicalensis Georgi, was found to be effective in reversing BM stroma induced CML resistance to IM. After cells were treated with weakly toxic concentration of oroxylin A, cell apoptosis induced by IM in co-culture model was enhanced. And the activated CXCL12/CXCR7 pathway, the expression of p-ERK and downstream apoptosis related proteins were suppressed. The in vivo study also showed that oroxylin A increased apoptosis of CML cells with low systemic toxicity, and the mechanism was consistent with the in vitro study. In conclusion, oroxylin A improved sensitivity of CML cells to IM treatment in BM microenvironment through regulating CXCL12/CXCR7 pathway. © 2016 Wiley Periodicals, Inc.

Interferon gamma modulates sensitivity of CML cells to tyrosine kinase inhibitors

Immune effector cells such as T and NK cells can efficiently eliminate tumor cells. However, when activating oncogenic signaling pathways or protective mechanisms against cell death are active, immune cells can also confer therapy resistance. Here, we analyzed the role of activated T and NK cells and released cytokines on tyrosine kinase inhibitors imatinib and nilotinib – mediated apoptosis induction and proliferation of chronic myelogenous leukemia (CML) cells. Incubation of CML cells with activated, but not with resting CD3+ T cells or with activated NK cells significantly inhibited TKI-induced apoptosis induction in CML cells as quantified by nuclear fragmentation assays. Transwell experiments revealed a critical role for T or NK cell-derived cytokines for CML cell protection. Accordingly, CML cells treated with IFNγ also showed a clearly reduced sensitivity to TKI-mediated cell death induction and inhibition of proliferation. In contrast, IFNα or other pro-inflammatory mediators and cytokines, such as TNFα and GM-CSF did not impair TKI-induced apoptosis in CML cells. On a molecular level, IFNγ-exposed CML cells showed a significantly reduced caspase-3 activation and PARP-1 cleavage as well as an increased expression of anti-apoptotic molecule xIAP. Finally, IFNγ diminished TKI-induced downregulation of Jak-2 and STAT-5 phosphorylation and increased nuclear expression of RUNX-1, which may at least in part contribute to the reduced sensitivity to TKI effects. Our results demonstrate that IFNγ released by activated T or NK cells may interfere with the therapeutic effects of TKI in CML. Our findings may have important implications for the understanding of inflammation-mediated BCR-ABL independent resistance mechanisms.

Dual induction of mitochondrial apoptosis and senescence in chronic myelogenous leukemia by myrtucommulone A.

Despite recent advances in the treatment of chronic myelogenous leukemia (CML), the development of drug resistance and minimal residual disease remain major challenges for the treatment of CML patients, thus highlighting the need to develop innovative new approaches to improve therapeutic outcome. Myrtucommulone A (MCA) is a nonprenylated acylphloroglucinol isolated from the leaves of myrtle, a plant traditionally used in folk medicine. To date, studies addressing bioactivities of myrtle and its specific components are rare. Here, we investigated the biological effects of MCA, focusing on its anti-leukemic activity. As evidenced by fragmented nuclei after Hoechst/propidium iodide staining and poly (ADP-ribose) polymerase cleavage, MCA induces apoptosis in CML cells through down-regulation of anti-apoptotic proteins. Interestingly, we showed that chronic treatment with MCA at low doses induced senescence in CML cells. Taken together, this study highlights the chemotherapeutical potential of this natural product in human leukemia.

Abstract 341: Imatinib induces expression of Bim and apoptosis in chronic myelogenous leukemia cells via p38/H2AX pathway

Abstract H2AX is a novel human tumor suppressor protein and plays an important role in apoptosis of cancer cells. Increasing published data indicate that H2AX phosphorylation (Ser139) contributes to its regulation of cancer cell apoptosis. Thus, charactering the mechanisms and signaling pathways involved in H2AX phosphorylation (Ser139) will certainly provide better understanding on H2AX function in cancer cells. Our previous report showed that caspase-3/Mst1 pathway regulates phosphorylation of H2AX at Ser139 in chronic myelogenous leukemia (CML) cells. Recently we found another pathway which regulates H2AX phosphorylation (Ser139) in CML cells. When the CML cells were subjected to imatinib treatment, MAPK family member ERK1/2 activity was inhibited, but p38 activity was increased, another member JNK1/2 had no response. Then we used p38 inhibitor SB202190 and siRNA against p38 to block p38 activation and observed that H2AX phosphorylation and apoptosis of CML cells was concurrently inhibited. Our results also showed that inhibition of MSK1/MSK2 did not affect H2AX phosphorylation (Ser139), although the p38 and ERK1/2 downstream MSK1/MSK2 could be activated by imatinib in CML cells. Overall, these data confirmed that p38 signaling pathway is also required for H2AX phosphorylation (Ser139) in CML cells. Our further evidences indicated that inhibition of H2AX phosphorylation (Ser139) regulated by p38 during apoptotic induction with imatinib suppressed the expression of apoptosis-related gene Bim. Taken together, these data demonstrated that p38/H2AX pathway regulates Bim expression and subsequent apoptosis in CML cells. Citation Format: Min Xiong, Tianhui Niu, Yaqiong Dong, Chengrong Lu. Imatinib induces expression of Bim and apoptosis in chronic myelogenous leukemia cells via p38/H2AX pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 341. doi:10.1158/1538-7445.AM2014-341

Abstract 1456: MicroRNA improves the efficacy of imatinib on the treatment of chronic myeloid leukemia

Abstract Human chronic myelogenous leukemia (CML) is a malignancy of pluripotent hematopoietic cells that is caused by the deregulated activity of the tyrosine kinase that is encoded by the chimeric bcr-abl oncogene. Given that CML cells are usually highly resistant to chemotherapy, novel and more efficacious strategies against this disease are urgently needed. MicroRNA (miRNA) is a set of newly discovered 20-22 nucleotides RNA molecules, and they broadly repress their cognate target genes at post-transcriptional/trans-lational level. To date, there are approximately 2,500 human miRNA transcripts have been characterized, which are estimated to modulate more than 30% human genes by being involved in many essential normal biological and pathological processes including differentiation, proliferation, apoptosis, and tumorigenesis as well. In this study, we studied let-7a, a miRNA with tumor suppressive signature, for its therapeutic potential in CML. When treating CML K562 and KU812 cell by imatinib, let-7a can be significantly induced. Overexpression of let-7a can sensitize K562 and KU812 cells to imatinib, while knock-down of let-7a can increase the resistance of these cells towards this drug. The functional studies found that let-7a was able to promote apoptosis of CML cells, inhibit the tumor cell proliferation, and lead to a cell cycle arrest in the G0/G1 phase. These results demonstrate that let-7a is a potential target for treating CML by imatinib and/or other anti-cancer drugs. The pro-apoptosis and anti-proliferation roles of let-7a in CML cells may provide novel insights into discovery and development of more efficacious drugs for treating this malignant disease. Citation Format: Bin Yi, Ruixia Ma, Xiangling Feng, Zhiwei Xing, Xiaoguo Zhang, Gary Piazza, Yaguang Xi. MicroRNA improves the efficacy of imatinib on the treatment of chronic myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1456. doi:10.1158/1538-7445.AM2014-1456

H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in chronic myelogenous leukemia cells induced by imatinib.

Increasing evidence suggests that histone H2AX plays a critical role in regulation of tumor cell apoptosis and acts as a novel human tumor suppressor protein. However, the action of H2AX in chronic myelogenous leukemia (CML) cells is unknown. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. Here, we report that H2AX was involved in apoptosis of CML cells. Overexpression of H2AX increased apoptotic sensitivity of CML cells (K562) induced by imatinib. However, overexpression of Ser139-mutated H2AX (blocking phosphorylation) decreased sensitivity of K562 cells to apoptosis. Similarly, knockdown of H2AX made K562 cells resistant to apoptotic induction. These results revealed that the function of H2AX involved in apoptosis is strictly related to its phosphorylation (Ser139). Our data further indicated that imatinib may stimulate mitogen-activated protein kinase (MAPK) family member p38, and H2AX phosphorylation followed a similar time course, suggesting a parallel response. H2AX phosphorylation can be blocked by p38 siRNA or its inhibitor. These data demonstrated that H2AX phosphorylation was regulated by p38 MAPK pathway in K562 cells. However, the p38 MAPK downstream, mitogen- and stress-activated protein kinase-1 and -2, which phosphorylated histone H3, were not required for H2AX phosphorylation during apoptosis. Finally, we provided epigenetic evidence that H2AX phosphorylation regulated apoptosis-related gene Bim expression. Blocking of H2AX phosphorylation inhibited Bim gene expression. Taken together, these data demonstrated that H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in CML cells induced by imatinib.

Gambogic Acid Induces Apoptosis in Imatinib-Resistant Chronic Myeloid Leukemia Cells via Inducing Proteasome Inhibition and Caspase-Dependent Bcr-Abl Downregulation

Chronic myelogenous leukemia (CML) is characterized by the constitutive activation of Bcr-Abl tyrosine kinase. Bcr-Abl-T315I is the predominant mutation that causes resistance to imatinib, cytotoxic drugs, and the second-generation tyrosine kinase inhibitors. The emergence of imatinib resistance in patients with CML leads to searching for novel approaches to the treatment of CML. Gambogic acid, a small molecule derived from Chinese herb gamboges, has been approved for phase II clinical trial for cancer therapy by the Chinese Food and Drug Administration (FDA). In this study, we investigated the effect of gambogic acid on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl. CML cell lines (KBM5, KBM5-T315I, and K562), primary cells from patients with CML with clinical resistance to imatinib, and normal monocytes from healthy volunteers were treated with gambogic acid, imatinib, or their combination, followed by measuring the effects on cell growth, apoptosis, and signal pathways. The in vivo antitumor activity of gambogic acid and its combination with imatinib was also assessed with nude xenografts. Gambogic acid induced apoptosis and cell proliferation inhibition in CML cells and inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Our data suggest that GA-induced proteasome inhibition is required for caspase activation in both imatinib-resistant and -sensitive CML cells, and caspase activation is required for gambogic acid-induced Bcr-Abl downregulation and apoptotic cell death. These findings suggest an alternative strategy to overcome imatinib resistance by enhancing Bcr-Abl downregulation with the medicinal compound gambogic acid, which may have great clinical significance in imatinib-resistant cancer therapy.

Abstract 1848: Let-7 brings new insights into chronic myeloid leukemia therapy.

Abstract Human chronic myelogenous leukemia (CML) is a malignancy of pluripotent hematopoietic cells that is caused by the deregulated activity of the tyrosine kinase that is encoded by the chimeric bcr-abl oncogene. Given that CML cells are usually highly resistant to chemotherapy, novel and more efficacious strategies against this disease are urgently needed. MicroRNA (miRNA) is a set of newly discovered 20-22 nucleotides RNA molecules, and they broadly repress their cognate target genes at post-transcriptional/translational level. Up to date, there are approximately 2,000 human miRNA transcripts have been characterized, which are estimated to modulate more than 30% human genes and are widely involved in many essential biological processes including differentiation, proliferation, apoptosis, and also tumorigenesis. In this study, we studied let-7, a miRNA with tumor suppressive signature, for its therapeutic potential in CML. When treating CML K562 and KU812 cell by imatinib, let-7 can be significantly induced. Overexpression of let-7 can sensitize K562 and KU812 cells to imatinib, while knock-down of let-7 can increase the drug resistance of these cells. The mechanistic studies found that let-7 was able to promote apoptosis of CML cells, inhibit the tumor cell proliferation, and lead to a cell cycle arrest in the G0/G1 phase. These results demonstrate that let-7 is a potential target for treating CML by imatinib and/or other anti-cancer druges. The pro-apoptosis and anti-proliferation roles of let-7 in CML cells may provide novel insights into discovery and development novel drugs for conquering this malignant disease. Citation Format: Bin Yi, Xiaobo Li, Wenjun Du, Gary A. Piazza, Yaguang Xi. Let-7 brings new insights into chronic myeloid leukemia therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1848. doi:10.1158/1538-7445.AM2013-1848 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 4963: Tanshinone IIA induces JNK-dependent intrinsic apoptosis via suppressing astrocyte-elevated gene 1 expression in chronic myelogenous leukemia KBM-5 cells

Abstract Tanshinone IIA (Tan IIA) is a phytochemical isolated from the root of Salviae miltiorrhizae (Danshen) and has been reported to have various biological activities such as anti-inflammation, anti-oxidation and anti-cancer. However, the underlying molecular mechanisms to control anti-cancer effect of Tan IIA in chronic myelogenous leukemia (CML) have not been fully understood yet. In the present study, we investigated whether Tan IIA has the anti-cancer potential by inducing apoptosis in CML cells. Tan IIA significantly decreased the viability of human CML KBM-5 cells, but not normal peripheral blood lymphocytes. Tan IIA induced apoptotic cell death by increasing the sub-G1 cell populations and stimulating atypical shrunken of nucleus and DNA fragmentation. Also, Tan IIA significantly reduced the mitochondrial membrane potential, stimulated the release of cytochrome C from mitochondria into the cytosol and activated intrinsic caspase cascade. Furthermore, Tan IIA enhanced phosphorylation of JNK and suppressed the expression of astrocyte-elevated gene-1 (AEG-1). In contrast, blocking JNK activity using JNK inhibitor SP600125 reversed Tan IIA-induced apoptosis and AEG-1 expression. Taken together, our findings suggest that Tan IIA induces JNK-dependent intrinsic apoptosis via suppressing AEG-1 expression in KBM-5 cells as a valuable candidate agent for CML therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4963. doi:1538-7445.AM2012-4963

Biological effects of decreasing RBM15 on chronic myelogenous leukemia cells

RNA binding motif protein 15 (RBM15) was originally described as a 5’ translocation partner of the MAL gene in t(1;22)(p13;q13)infant acute megakaryocytic leukemia. Although previous investigations have shown that Rbm15 has broad regulatory effects within murine hematopoiesis through modulating Notch-induced transcriptional activation, which plays key roles in leukemogenesis, it is not clear what the functions of RBM15 are in the regulation of hematological malignancies. In the present study, we show that RBM15 expression was significantly increased in patients with blast-crisis chronic myelogenous leukemia (CML) compared with chronic-phase or accelerated-phase disease by real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. To further elucidate the role of RBM15 in CML, we introduced RBM15 small interfering RNA (siRNA) using pSUPER into CML cells. Fluorescence activated cell sorting (FACS), real-time RT-PCR and Western blot were used to study changes in RBM15 expression levels in transduced cells by comparing with control cells. Decreasing RBM15 levels with RNA interference could inhibit the growth and proliferation, block the cell cycle and induce apoptosis in CML cells. Knockdown of RBM15 may also act to inhibit clonogenicity and induce differentiation of CML cells along the myeloid lineage. Our studies also show that the effects of RBM15 on CML cells may be mediated, at least in part, via its effect on Notch signaling. These findings demonstrate that RBM15 does indeed play a critical role in the survival of CML cells, which may have potential application in designing molecular therapies for CML treatment.