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PDF file - 927K, The effect of Aurora kinases inhibitors on HL-60 and KG1a cell lines.

PDF file - 1715K, Aurora kinases inhibitors and mTOR inhibitors had synergistic effect on the expression of cleaved caspase 3 and p62.

PDF file - 1113K, Aurora kinases inhibitors had no effect with mTOR inhibitors on PBMCs.

PDF file - 1100K, Combination of Aurora kinases inhibitors and 2DG had minimal effect on PBMCs.

PDF file - 1470K, Aurora kinases inhibitors had synergistic effect with mTOR inhibitors on the glycolytic metabolism of U937 cells.

PDF file - 2735K, Aurora kinases inhibitors had synergistic effect with mTOR inhibitors on the proliferation of U937 and NB4 cells.

PDF file - 1081K, Cell viability detection in Aurora kinase A knockdown-U937 cells.

This study firstly aimed to reveal the gene expression differences of CIAPIN1 between myelomas cells from bone marrow cells of multiple myeloma patients and normal human, and subsequently investigate the regulation role of this gene on tumorigenicity ability of multiple myeloma (MM) cell line U266 via in vitro colony formation and in vivo xenograft studies. RT-PCR results obtained from 18 MM patients and 10 health people showed that the expression of CIAPIN1 gene was 4 times higher in normal human compared to MM patients. Besides, CIAPIN1 siRNA (si-CIAPIN1) transfected U266 cells presented higher proliferation ratio and superior colony forming ability than U266 cells and U266 cells transfected with non-coding siRNA (controls) evaluated by CCK8 test and soft agar colony formation assay, respectively. In a mice MM xenograft model, the si-CIAPIN1 transfected U266 cells induced the biggest tumor compared to the controls. Furthermore, CIAPIN1 overexpressed U266 cells were developed and compared with the si-CIAPIN1 transfected U266 cells to study the role of CIAPIN1 in the production of apoptosis related proteins in U266 cells. Results indicated that CIAPIN1 facilitated apoptosis promoting proteins expression in U266 cells, such as upregulation of BAX, BAK, Bcl-xs and BIM, and downregulation of p38, PKC, Bcl-2 and Bcl-xl proteins. Therefore, CIAPIN1 can be a potential suppression target gene in multiple myeloma.

The present study aimed to characterize the function and hemorheology of red blood cells (RBCs) recovered during liver transplantation surgery in patients with hepatitis B and decompensation. A total of 15 hepatitis B patients with decompensation who underwent liver transplantation surgery were included in the present study. Blood samples were recovered during the liver transplantation surgery using an Autologous Blood Recovery System. The morphology and structure of RBCs were characterized and compared between pre-operative and recovered blood samples. In addition, the physiological functions of RBCs were measured and compared between pre-operative and recovered blood samples. No significant differences in the morphological score, 2,3-diphosphoglycerate, Na+K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, malondialdehyde and osmotic fragility were identified between RBCs in the pre-operative and recovered blood samples. The level of free hemoglobin in RBCs of the recovered blood samples was significantly higher than in the pre-operative blood samples (P0.05) in the low-shear blood viscosity, plasma viscosity, relative blood viscosity, erythrocyte aggregation index or Casson yield stress were identified between recovered and pre-operative blood samples. These findings suggested that autologous blood transfusion in liver transplantation surgery in patients with hepatitis B and decompensation had no significant influence on the morphology, structure, function and hemorheology of RBCs.

The interaction between bone marrow stromal cells and leukemia cells is critical for the persistence and progression of leukemia, and this interaction may account for residual disease. However, the link between leukemia cells and their environment is still poorly understood. In our study, runt‑related transcription factor 3 (RUNX3) was identified as a novel target gene affected by As2O3 and involved in mesenchymal stem cell (MSC)‑mediated protection of leukemia cells from As2O3‑induced apoptosis. We observed induction of RUNX3 expression and the translocation of RUNX3 into the nucleus after As2O3 treatment in leukemia cells. In K562 chronic myeloid leukemia cells, downregulation of endogenous RUNX3 compromised As2O3‑induced growth inhibition, cell cycle arrest, and apoptosis. In the presence of MSC, As2O3‑induced expression of RUNX3 was reduced significantly and this reduction was modulated by CXCL12/CXCR4 signaling. Furthermore, overexpression of RUNX3 restored, at least in part, the sensitivity of leukemic cells to As2O3. We conclude that RUNX3 plays an important role in As2O3‑induced cellular responses and allows cells to overcome MSC‑mediated drug resistance. Therefore, RUNX3 is a promising target for therapeutic approaches to overcome MSC‑mediated drug resistance.

Background/Aims: Resistance of leukemia stem cells (LSCs) to chemotherapy in patients with acute myeloid leukemia (AML) causes relapse of disease. Hedgehog (Hh) signaling plays a critical role in the maintenance and differentiation of cancer stem cells. Yet its role in AML remains controversial. The purpose of the present study is to investigate the role of GLI1, the transcriptional activator of Hh signaling, in AML progenitor cells and to explore the anti-AML effects of GLI small-molecule inhibitor GANT61. Methods: The expression of GLI1 mRNA and protein were examined in AML progenitor cells and normal cells. The proliferation, colony formation, apoptosis and differentiation of AML progenitor cells were also analyzed in the presence of GANT61. Results: Kasumi-1 and KG1a cells, containing more CD34+ cells, expressed higher level of GLI1 compared to U937 and NB4 cells with fewer CD34+ cells. Consistently, a positive correlation between the protein levels of GLI1 and CD34 was validated in the bone marrow mononuclear cells (BMMC) of AML patients tested. GANT61 inhibited the proliferation and colony formation in AML cell lines. Importantly, GANT61 induced apoptosis in CD34+ enriched Kasumi-1 and KG1a cells, whereas it induced differentiation in U937 and NB4 cells. Furthermore, GANT61 enhanced the cytotoxicity of cytarabine (Ara-c) in primary CD34+ AML cells, indicating that inhibition of GLI1 could be a promising strategy to enhance chemosensitivity. Conclusions: The present findings suggested that Hh signaling was activated in AML progenitor cells. GLI1 acted as a potential target for AML therapy.

Background Hemophagocytic lymphohistiocytosis (HLH) is a rare and potentially fatal condition that can be primary or secondary. Secondary HLH due to Mycobacterium tuberculosis (TB) is uncommon. We report a case of tuberculosis-associated HLH in an umbilical cord blood transplant (UCBT) recipient and discuss its clinical characteristics and challenges. Methods Hematologic investigations, bone marrow aspirates, Xpert MTB/RIF test of TB with peripheral blood were performed. Immune modulation with anti-TB therapy was initiated. Results Subsequent treatment with anti-TB treatment resulted rapid clinical response and disease remission. Conclusion It is important to consider TB as one of the underlying cause of HLH in high-risk patients, particularly those in immunodeficient states. Early diagnosis and treatment can improve the survival rates of patients with tuberculosis-associated HLH.

Background Individual patient data from two multicentre, randomised trials comparing atezolizumab with docetaxel in previously treated advanced non-small-cell lung cancer (NSCLC) were analyzed to identify any preferable therapeutic approaches for patient subsets stratified by EGFR mutation status. Methods We pooled 1,137 patients from the phase II POPLAR trial (NCT01903993) and the phase III OAK trial (NCT02008227). Patients were randomly assigned to receive either atezolizumab or docetaxel and were analyzed based on stratification of EGFR mutation status. We built a novel algorithm integrating the blood-based tumor mutation burden and the ctDNA maximum somatic allele frequency (bTMB-MSAF score) and developed a novel clinicopathologic-genomic nomogram to predict individual survival. Results In the whole intention-to-treat population, OS was significantly longer with atezolizumab than with docetaxel (hazard ratio [HR] 0.72, P Conclusions Atezolizumab showed better OS and PFS than docetaxel in previously treated EGFR wild-type NSCLC patients with a bTMB-MSAF score Clinical trial identification NCT01903993 and NCT02008227. Legal entity responsible for the study Herui Yao. Funding Herui Yao is funded by the National Science and Technology Major Project under Grant [2020ZX09201021]; National Natural Science Foundation of China under Grant [81372819, 81572596, U1601223]; Natural Science Foundation of Guangdong Province under Grant [2017A030313828]; and Guangzhou Science and Technology Program under Grant [201704020131]. Disclosure All authors have declared no conflicts of interest.

BACKGROUND Extramedullary blast crisis (EBC) of T315I BCR-ABL mutated chronic myelogenous leukemia (CML) is extremely rare. METHODS We report an unusual case characterized by fever, right shoulder swelling, pleural effusion, and multiple bone destruction as the first signs of EBC of T315I BCR-ABL mutated CML. RESULTS The patient did not respond to chemotherapy consisting of anthracyclines, cytarabine and etoposide. His condition improved after the treatment with ponatinib combined with allogenic stem cell transplantation (alloHCT), but soon worsened after ponatinib withdrawal. He got slight relief after he continued ponatinib. CONCLUSIONS Ponatinib is an important treatment to control EBC even after allo-HCT.

Drug resistance still represents a major obstacle to successful chronic myeloid leukemia (CML) treatment and novel compounds or strategies to override this challenging problem are urgently required. Here, we evaluated a novel compound AKI603 against oncogenic Aurora kinase A (Aur-A) in imatinib-resistant CML cells. We found that Aur-A was highly activated in imatinib-resistant KBM5-T315I cells. AKI603 significantly inhibited the phosphorylation of Aur-A kinase at Thr288, while had little inhibitory effect on BCR-ABL kinase in both KBM5 and KBM5-T315I cells. AKI603 inhibited cell viability, and induced cell cycle arrest with polyploidy accumulation in KBM5 and KBM5-T315I cells. Moreover, inhibition of Aur-A kinase by AKI603 suppressed colony formation capacity without promoting obvious apoptosis. Importantly, AKI603 promoted cell differentiation in both CML cell types. Thus, our study suggested the potential clinical use of small molecule Aurora kinase inhibitor AKI603 to overcome imatinib resistance in CML treatment.

We conducted a phase II, noncomparative, multicenter study to assess the efficacy and safety of allogeneic bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) expanded in vitro for patients with aplastic anemia (AA) refractory to immunosuppressive therapy. Seventy‐four patients from seven centers received allogeneic BM‐MSCs at a dose of 1–2 × 106 cells/kg per week for 4 weeks. Responses were assessed at 0.5, 1, 2, 3, 6, 9, and 12 months after the first cells infusion. Patients with response at 1 month continued to receive four infusions. All patients were evaluable. The overall response rate was 28.4% (95% confidence interval, 19%–40%), with 6.8% complete response and 21.6% partial response. The median times to response of leukocytic, erythrocytic, and megakaryocytic linages were 19 (range, 11–29), 17 (range, 12–25), and 31 (range, 26–84) days, respectively. After median follow‐up of 17 months, overall survival was 87.8%. Seven patients developed transitory and mild headache and fever, but no other adverse events were observed. Antithymocyte globulin used in previous treatment and no activated infection throughout treatment were predictors for response. Allogeneic BM‐MSCs infusion is a feasible and effective treatment option for refractory AA. The trial was registered at www.clinicaltrials.gov as NCT00195624. Stem Cells Translational Medicine 2017;6:1569–1575

Long-term utilization of immunosuppression in organ transplant recipients leads to decreased immune-mediated tumor surveillance and increased risk of developing malignant tumors. However, chronic myeloid leukemia (CML) following living donor liver transplantation (LDLT) is rarely reported. The current case report presents a 42-year-old male patient who developed CML 14 months following LDLT. The patient achieved complete hematologic remission and early molecular response at 3 months imatinib treatment and major molecular response at 12 months imatinib treatment. The pathogenesis, risk factors, treatment and prognosis for CML following liver transplantation are unclear. Therefore, further analysis through accumulation of cases will be of great importance to prevent and treat this rare complication following liver transplantation.

The development of targeted tyrosine kinase inhibitors (TKIs) has succeeded in altering the course of chronic myeloid leukemia (CML). However, a number of patients have failed to respond or experienced disease relapse following TKI treatment. Proviral integration site for moloney murine leukemia virus-1 (PIM-1) is a serine/threonine kinase that participates in regulating apoptosis, cell cycle, signal transduction and transcriptional pathways, which are associated with tumor progression, and poor prognosis. SMI-4a is a selective PIM-1 kinase inhibitor that inhibits PIM-1 kinase activity in vivo and in vitro. The present study aimed to explore the mechanism underlying the antitumor effect of SMI-4a in K562 and imatinib-resistant K562 (K562/G) cell lines. It was demonstrated that SMI-4a inhibited the proliferation of K562 and K562/G cells using a WST-8 assay. The Annexin V-propidium iodide assay demonstrated that SMI-4a induced apoptosis of K562 and K562/G cells in a dose-, and time-dependent manner. Furthermore, Hoechst 33342 staining was used to verify the apoptosis rate. The clone formation assay revealed that SMI-4a significantly inhibited the colony formation capacity of K562 and K562/G cells. Western blot analysis demonstrated that SMI-4a decreased phosphorylated (p)-Ser9-glycogen synthase kinase (GSK) 3β/pGSK3β and inhibited the translocation of β-catenin. In addition, the downstream gene expression of apoptosis regulator Bax and poly(ADP-ribose) polymerase-1 was upregulated, and apoptosis regulator Bcl-2 and Myc proto-oncogene protein expression levels were downregulated. Immunofluorescence results demonstrated changes in the expression level of β-catenin in the plasma and nucleus. The results of the present study suggest that SMI-4a is an effective drug to use in combination with current chemotherapeutics for the treatment of imatinib-resistant CML.

The emergence of resistance to imatinib mediated by mutations in the BCR-ABL has become a major challenge in the treatment of chronic myeloid leukemia (CML). Alternative therapeutic strategies to override imatinib-resistant CML are urgently needed. In this study, we investigated the effect of AKI603, a novel small molecule inhibitor of Aurora kinase A (AurA) to overcome resistance mediated by BCR-ABL-T315I mutation. Our results showed that AKI603 exhibited strong anti-proliferative activity in leukemic cells. AKI603 inhibited cell proliferation and colony formation capacities in imatinib-resistant CML cells by inducing cell cycle arrest with polyploidy accumulation. Surprisingly, inhibition of AurA by AKI603 induced leukemia cell senescence in both BCR-ABL wild type and T315I mutation cells. Furthermore, the induction of senescence was associated with enhancing reactive oxygen species (ROS) level. Moreover, the anti-tumor effect of AKI603 was proved in the BALB/c nude mice KBM5-T315I xenograft model. Taken together, our data demonstrate that the small molecule AurA inhibitor AKI603 may be used to overcome drug resistance induced by BCR-ABL-T315I mutation in CML.

Natural products possessing anticancer activity have been extensively studied because of their low toxicity and potential effect. α-Mangostin, a component of Garcinia mangostana Linn, is a xanthone derivative shown to have antioxidant and antitumor properties. This study was carried out to investigate how to improve the anticancer effects of α-mangostin in chronic myeloid leukemia (CML) cell lines bearing wild-type BCR-ABL or BCR-ABL-T315I mutation. We showed that α-mangostin inhibited cell proliferation of K562, KBM5 and KBM5-T315I cells in both a time- and dose-dependent manner. Significantly, α-mangostin increased the number of apoptotic cells and induced DNA fragmentation compared to control cells. Moreover, α-mangostin selectively inhibited proliferation in primary CML cells, while showing limited lethality in normal hematopoietic progenitors. Additionally, α-mangostin induced not only apoptosis but also autophagy in CML cells. α-Mangostin dramatically increased the expression levels of LC-3II, an autophagosome marker in mammals, and the accumulation of autophagic vacuoles (AVs). Inhibition of autophagy by chloroquine enhanced α-mangostin-mediated cytotoxicity through increasing apoptosis. Taken together, our data suggest that targeting the autophagy pathway is a promising therapeutic strategy to enhance α-mangostin-induced apoptosis. Our study provides an approach for future studies to explore this combination for the treatment of CML.

A previously healthy 34-year-old female presented with a 5-month history of progressive backache and weakness in the left fingers. Magnetic resonance imaging (MRI) showed soft tissue masses in the spinal canal distributed along the nerve course. The patient's baseline laboratory data were normal. Surgical intervention was performed and histological examination identified isolated spinal granulocytic sarcoma (GS). A bone marrow biopsy also presented normal findings. However, the patient developed numbness and pain in the right lower limb two months later. Fluorodeoxyglucose (FDG)-positron emission tomography (PET) showed FDG uptake in the left trapezius muscle, cervix uteri, iliac bone, lymphadenectasis of the pelvic wall and left axillary fossa. Cerebrospinal fluid (CSF) examination allowed a diagnosis of central nervous system leukemia (CNSL). The patient underwent chemotherapy and intrathecal injection, resulting in the elimination of the residual lesion. Correct diagnosis and adequate treatment are essential to achieve optimal results in patients with isolated spinal GS.

Background Chronic myelogenous leukemia (CML) is a hematological stem cell disorder. Tyrosine kinase inhibitors (TKIs) are the standard treatments for CML, but a number of patients fail to respond effectively due to gene mutations. Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, has been shown to have anti-tumor effect on solid tumor whereas the anti-CML effect and its underlying mechanism have not been completely elucidated. Methods The cytotoxic effects of celecoxib and/or imatinib were evaluated by MTT assay. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis or necrosis was analyzed by Annexin-V/PI, Hoechst 33342 staining and Western blot assays. Autophagy suppression effect of celecoxib was examined by Western blot and LysoTracker probe labelling. Lysosensor probe labelling was used to detect the effect of celecoxib on the lysosomal function. Results In this study, we found that celecoxib had therapy efficacy in KBM5 and imatinib-resistant KBM5-T315I CML cell lines. Celecoxib caused significant cytotoxic effect in both cell lines, especially in KBM5-T315I cells exposed to celecoxib for 72 h. Moreover, celecoxib induced necrosis and apoptosis while inhibited autophagy in CML cell lines and patient samples. Furthermore, this study demonstrated that celecoxib prevented the autophagic flux by inhibiting lysosome function. Celecoxib was tested in combination with imatinib, demonstrating that celecoxib could strengthen the cytotoxicity of imatinib in imatinib-resistant CML cells. Conclusions These findings showed that celecoxib had therapy efficacy on CML cells. And it is first time to demonstrate that celecoxib is an autophagy suppresser and a combination of celecoxib and imatinib might be a promising new therapeutic strategy for imatinib-resistant CML cells. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-1012-8) contains supplementary material, which is available to authorized users.

Recent studies have suggested that dysregulation of autophagy plays a pivotal role in tumorigenesis. Here, we determined the prognostic value of autophagy-related protein Beclin 1 in gastric cancer. A total of 153 primary gastric cancer patients were subjected to analysis of Beclin 1 expression and survival prognosis. Among them, 68 patients were assigned randomly and used as a training set to generate a cutoff score for Beclin 1 expression by receive operating characteristic (ROC) curve analysis. The ROC-generated cutoff score was subjected to analyze the association of Beclin 1 with clinical characteristics and patient outcome. In a testing set (n = 85) and overall patients (n = 153), both univariate and multivariate analysis found that low expression of Beclin 1 predicted adverse overall survival and progression-free survival for gastric cancer patients. Furthermore, in each stage of gastric cancer patients, Beclin 1 expression was a prognostic indicator in patients with stage II, III and IV. Importantly, a reverse relationship between Beclin 1 and Bcl-xL expression was demonstrated. In patients of elevated Bcl-xL expression, a subset with lower Beclin 1 expression displayed an inferior overall survival and progression-free survival than those with higher Beclin 1 expression. Thus, our data demonstrated that low expression of Beclin 1, associated with high Bcl-xL, played as an independent biomarker, contributing to a more aggressive cancer cell phenotype and poor prognosis for gastric tumor.

Epigenetic therapy has had a significant impact on the management of haematologic malignancies. The aim of this study was to assess whether 5-aza-CdR and TSA inhibit the growth of leukaemia cells and induce caspase-3-dependent apoptosis by upregulating RUNX3 expression.K562 and Reh cells were treated with 5-aza-CdR, TSA or both compounds. RT-PCR and Western blot analyses were used to examine the expression of RUNX3 at the mRNA and protein levels, respectively. Immunofluorescence microscopy was used to detect the cellular location of RUNX3. Additionally, after K562 cells were transfected with RUNX3, apoptosis and proliferation were studied using Annexin V staining and MTT assays.The expression of RUNX3 in leukaemia cell lines was markedly less than that in the controls. Demethylating drug 5-aza-CdR could induce RUNX3 expression, but the combination of TSA and 5-aza-CdR had a greater effect than did treatment with a single compound. The combination of 5-aza-CdR and TSA induced the translocation of RUNX3 from the cytoplasm into the nucleus. TSA enhanced apoptosis induced by 5-aza-CdR, and Annexin V and Hoechst 33258 staining showed that the combination induced apoptosis but not necrosis. Furthermore, apoptosis was dependent on the caspase-3 pathway. RUNX3 overexpression in K562 cells led to growth inhibition and apoptosis and potentiated the effects of 5-aza-CdR induction.RUNX3 plays an important role in leukaemia cellular functions, and the induction of RUNX3-mediated effects may contribute to the therapeutic value of combination TSA and 5-aza-CdR treatment.

Tanshinone I (Tan I), a diterpene quinone extracted from herbal medicine Salvia miltiorrhiza Bunge, has recently been reported to have antitumor effects. As the mechanism of its proapoptotic effects on human myeloid leukemia cells has not been extensively studied, we performed an in-depth evaluation of the effects of Tan I on apoptosis in human K562 and HL-60 cells. The results revealed that Tan I could inhibit the growth of leukemia cells and cause apoptosis in a time- and dose-dependent manner. Apoptosis was observed clearly by flow cytometry and Hoechst 33258 staining, as well as DNA fragmentation analysis. After treatment by Tan I for 48 h, the percentage of disruption of mitochondrial membrane potential (Δψm) was increased in a dose-dependent manner. Western blotting analysis demonstrated the cleavage of caspase-3 zymogen protein and a dose-dependent cleavage of poly-(ADP-ribose) polymerase. Tan I-induced apoptosis was accompanied by a significant decrease in survivin and an increase in Bax. Moreover, Tan I treatment remarkably downregulated the phosphorylation of both P85/PI3K and Akt in a time-dependent manner, and the PI3K/AKT-specific inhibitor (LY294002) mimicked the apoptosis-inducing effects of Tan I. We therefore conclude that the induction of apoptosis by Tan I in these leukemia cells is mainly related to the disruption of Δψm, the upregulation of Bax expression, and the activation of caspase-3. This process is highly correlated with the inactivation of PI3K/Akt/survivin signaling pathways. The results indicate that Tan I may serve as an effective adjunctive reagent in the treatment of leukemia.

Tanshinone I (Tan-I) is a diterpene quinone extracted from the traditional herbal medicine Salvia miltiorrhiza Bunge. Recently, Tan-I has been reported to have anti-tumor effects. In this study, we investigated the growth inhibition and apoptosis inducing effects of Tan-I on three kinds of monocytic leukemia cells (U937, THP-1 and SHI 1). Cell viability was measured by MTT assay. Cell apoptosis was assessed by flow cytometry (FCM) and AnnexinV/PI staining. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR-enzyme-linked immunosorbent assay (ELISA) were used to detect human telomerase reverse transcriptase (hTERT) expression and telomerase activity before and after apoptosis. The activity of caspase-3 was determined by Caspase colorimetric assay kit and Western blot analysis. Expression of the anti-apoptotic gene Survivin was assayed by Western blot and Real-time RT-PCR using the ABI PRISM 7500 Sequence Detection System. The results revealed that Tan-I could inhibit the growth of these three kinds of leukemia cells and cause apoptosis in a time- and dose-dependent manner. After treatment by Tan-I for 48 h, Western blotting showed cleavage of the caspase-3 zymogen protein with the appearance of its 17-kD subunit, and a 89-kD cleavage product of poly (ADP-ribose) polymerase (PARP), a known substrate of caspase-3, was also found clearly. The expression of hTERT mRNA as well as activity of telomerase were decreased concurrently in a dose-dependent manner. Moreover, Real-time RT-PCR and Western blot revealed a significant down-regulation of Survivin. We therefore conclude that the induction of apoptosis by Tan-I in monocytic leukemia U937 THP-1 and SHI 1 cells is highly correlated with activation of caspase-3 and decreasing of hTERT mRNA expression and telomerase activity as well as down-regulation of Survivin expression. To our knowledge, this is the first report about the effects of Tan-I on monocytic leukemia cells.

Objective To investigate the apoptotic effects of peroxisome proliferator-activated receptor γ (PPAR-γ) agonist rosiglitazone (RGZ) on leukemia HL-60 cells. Methods Cell apoptosis was measured by flow cytometry, Hoechst 33258 staining, and DNA fragmentation assay. The expression of caspase-3 and apoptosis-related gene Bax and Bcl-2 as well as survivin expression were analyzed by Western blotting. Results Rosiglitazone (over 40 μmol/L) could inhibit the growth of HL-60 cells and substantially cause apoptosis. Apoptotic cells were observed clearly by Hoechst staining, especially after the cells were treated with RGZ for 72 h, and a DNA fragmentation assay showed a typical DNA ladder. Western blotting showed cleavage of the caspase-3 zymogen protein (32-kD) with the appearance of its 17-kD cleavage and down-regulation of anti-apoptotic protein Bcl-2 and survivin as well as up-regulaton of pro-apoptotic protein Bax . Conclusion Rosiglitazone can inhibit HL-60 cell growth by the induction of apoptosis. Activation of caspase-3 and down-regulation of Bcl-2 and survivin as well as up-regulation of Bax expression may be one of its most important mechanisms.

This study investigates the ability of a synthetic PPAR-γ agonist, rosiglitazone (RGZ), to induce apoptosis in leukemia K562 cells. The results revealed that RGZ (>40 mmol/L) inhibits the growth of K562 cells and causes apoptosis in a time and dose-dependent manner. Apoptosis is observed clearly by Hoechst 33258 staining. Western blotting analysis demonstrates the cleavage of caspase-3 zymogen protein with the appearance of its 17-kD subunit and a dose-dependent cleavage of poly (ADP-ribose) polymerase. Furthermore, RGZ treatment down-regulates anti-apoptotic protein Bcl-2 and up-regulates pro-apoptotic protein Bax in a dosedependent manner after the cells are treated for 48 hours. Telomerase activity is decreased concurrently in a dosedependent manner. We therefore conclude that RGZ induces apoptosis in K562 cells in vitro, and that RGZ-induced apoptosis in K562 cells is highly correlated with activation of caspase-3, decreasing telomerase activity, down-regulation of the anti-apoptotic protein Bcl-2, and up-regulation of the pro-apoptotic protein Bax.

In this study two monocytic leukemia cell lines, U937 and THP-1 cells, were used to investigate the anti-proliferation effects caused by ponicidin. Cell viability was measured by an MTT assay. Cell apoptosis was assessed by flow cytometry as well as DNA fragmentation analysis. Cell morphology was observed using an inverted microscope and Hoechst 33258 staining. RT-PCR and Western blot analysis were used to detect survivin as well as Bax and Bcl-2 expressions after the cells were treated with different concentrations of ponicidin. The results revealed that ponicidin could inhibit the growth of U937 and THP-1 cells significantly by induction of apoptosis. The suppression was in both time- and dose-dependent manner. Marked morphological changes of cell apoptosis were observed clearly after the cells were treated with ponicidin for 48 approximately 72 h. RT-PCR and Western blot analysis demonstrated that both survivin and Bcl-2 expressions were down-regulated remarkably while Bax expression remained constant before and after apoptosis occurred. We therefore conclude that ponicidin has significant anti-proliferation effects by inducing apoptosis on leukemia cells in vitro, downregulation of survivin as well as Bcl-2 expressions may be the important apoptosis inducing mechanisms. The results suggest that ponicidin may serve as potential therapeutic agent for leukemia.

Zoledronic acid (ZOL), a nitrogen‑containing bisphosphonate, is widely used in metastatic bone disease. Previous studies indicate that ZOL has marked anti‑leukemia activity, however, the underlying mechanism of action remains to be elucidated. The present study aimed to explore the mechanism of the anti‑leukemia effect of ZOL in leukemia cells. It was observed that ZOL inhibited the proliferation of HL‑60 and adriamycin‑resistant HL‑60 (HL‑60/A) cells using a WST‑8 assay. An Annexin V‑propidium iodide indicated that ZOL induced apoptosis of the two cell types in a dose‑ and time‑dependent manner. Hoechst 33342 staining was also used to verify the levels of apoptosis. The colony formation assay demonstrated that ZOL significantly inhibited colony formation capacity in acute myeloid leukemia (AML) cells. This was achieved by the induction of S‑phase cell cycle arrest, downregulation of B‑cell lymphoma 2 (Bcl‑2) and upregulation of Bcl‑2 associated X protein and cleaved poly (ADP‑ribose) polymerase. The results indicate that ZOL inhibited cell proliferation by inducing apoptosis via the mitochondrial apoptotic pathway and this anti‑leukemic activity appeared notably enhanced in HL‑60/A cells. As ZOL is already available for clinical use, these results indicate that it may be an effective addition to the chemotherapeutic strategies for AML.

To explore the expression and clinical significance of Hedgehog signaling transcription factor Gli1 in acute lymphoblastic leukemia (ALL) patients.The clinical specimens were obtained from 32 newly diagnosed and 6 relapsed ALL patients. Normal bone marrow cells from 15 healthy donors were used as controls. Real-time qPCR and Western blot were applied to detect Gli1 mRNA and protein expression in bone marrow mononuclear cells (BMMNC) of these samples respectively. The relation of Gli1 mRNA levels with clinical parameter was also evaluated.The expression level of Gli1 mRNA in de novo and relapsed ALL patients was significantly higher than that in the normal controls (P0.05). There was no stalistically significant difference of the Gli1 mRNA expression between de novo and relapsed ALL cases (P0.05). In 24 de novo ALL patients with complete remission (CR) after induction chemotherapy, the levels of Gli1 mRNA were significantly reduced as compared with levels before treatment (P0.05). However, in 4 ALL patients without remission, no obvious difference of Gli1 mRNA levels were observed as compared with levels of Gli1 before treatment (P0.05). A positive correlation between the Gli1 mRNA expression level and white blood cell count (WBC) was found in the BMMNC of ALL patients (R = 0.725, P0.05). Similarly, Gli1 protein expression was significantly higher in the de novo and relapsed ALL cases compared with normal controls. The Gli1 protein level was down-regulated when the ALL patients was in CR.The expression of Gli1 mRNA and protein has been found to be high in de novo and relapsed ALL patients, and the change of Gli1 expression maybe relate to therapeutic efficacy and prognosis of ALL patients.

Tanshinone IIA, a diterpene quinone extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, is used widely and successfully in clinics in China for treating inflammatory diseases. Recently tanshinone IIA has been reported to have apoptosis inducing effects on a large variety of cancer cells. In this study, the anti-proliferation and apoptosis inducing effects of tanshinone IIA as well as its influence on cell adhesion to and invasion through the extracellular matrix (ECM) on acute promyelocytic leukemia (APL) NB4 cells in vitro were studied. Cell proliferation was assessed by MTT assay, cell apoptosis was observed by Hoechst 33258 staining and flow cytometry (FCM); The variation of caspase-3 and apoptotic related genes were assayed by Western blotting, cell mitochondrial membrane potential as well as cell adhesive and invasive effects were also investigated by using standard methods. The results showed that tanshinone IIA exhibited induction of apoptosis by activation of caspase-3, downregulation of anti-apoptotic protein bcl-2 and bcl-xl and upregulation of pro-apoptotic protein bax, as well as disruption of the mitochondrial membrane potential. Furthermore, treatment by tanshinone IIA could reduce cell adhesion to and invasion through ECM in leukemia NB4 cells. These data provide a potential mechanism for tanshinone IIA-induced apoptosis and cell growth inhibition in leukemia NB4 cells, suggesting that tanshinone IIA may serve as an effective adjunctive reagent for the treatment of APL.

The present study was undertaken to investigate the mechanisms of oridonin induced apoptosis on human hepatocellular carcinoma BEL-7402 cells. BEL-7402 cells in culture medium in vitro were given different concentrations of oridonin. Cell proliferation was measured by MTT assay and [(3)H]-thymidine uptake, cell apoptotic rate was detected by flow cytometry (FCM), morphology of cell apoptosis was observed by Hoechst 33258 staining and DNA fragmentation. Caspase-3 as well as Bcl-2 and Bax expressions were detected by Western blotting. The results revealed that oridonin could inhibit the growth of BEL-7402 cells and cause apoptosis significantly, the suppression was both in time- and dose-dependent manner. Marked morphological changes of cell apoptosis were observed very clearly by Hoechst 33258 staining as well as DNA fragmentation analysis after the cells exposed to oridonin for 60h; Western blotting showed cleavage of the caspase-3 zymogen protein (32-kDa) with the appearance of its 20-kDa subunit; Along with the apoptotic process Bcl-2 expression was down-regulated and Bax expression up-regulated concurrently observed by Western blotting. We therefore conclude that oridonin can inhibit cell growth by induction of apoptosis in BEL-7402 cells via activation of caspase-3 as well as down-regulation of Bcl-2 and up-regulation of Bax expression. The results indicate that oridonin may be an important potential anti-hepatocellular carcinoma reagent.

The present study was undertaken to investigate the mechanisms of peroxisome proliferator activated receptor-gamma (PPAR-gamma) ligand-induced apoptosis on human myeloid leukemia K562 and HL-60 cell lines. The results revealed that both 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ) have significant anti-proliferation- and apoptosis-inducing effects on these two kinds of leukemia cells. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly using Wright's and Hoechst 33258 staining. Reverse transcription-PCR and western blot analyses demonstrated that both survivin and bcl-2 expression were downregulated markedly, while bax expression was upregulated concurrently when apoptosis occurred. We therefore conclude that 15d-PGJ2 and TGZ have significant apoptosis effects on K562 and HL-60 cells in vitro, and that upregulation of bax as well as downregulation of survivin and bcl-2 expression may be the important apoptosis-inducing mechanisms. The results suggest that PPAR-gamma ligands may serve as potential therapeutic agents for both acute and chronic myeloid leukemia.

Oridonin, an extract from the Chinese herb Rabdosia rubescens, is currently one of the most important traditional Chinese herbal medicines. We investigated the anti-proliferative effect of oridonin on the lung cancer cell line SPC-A-1 and its mechanism of action. Growth inhibition was measured using a microculture tetrazolium assay and apoptosis was measured by several standard methods. Western blot analysis measured the expression of bcl-2 and bax proteins. Oridonin (> 28 μmol/l) inhibited the growth of SPC-A-1 cells and induced apoptosis. Marked morphological changes indicative of apoptosis were observed, especially in cells treated with oridonin for 48–60 h. Western blot analysis revealed downregulation of bcl-2 and upregulation of bax proteins following treatment with oridonin for 48 h. We conclude that oridonin demonstrated anti-proliferative and apoptosis-inducing effects on SPC-A-1 cells in vitro, and that changes in bcl-2 and bax protein levels may play an important role in its mechanism of action.

Jurkat cells in culture medium in vitro were exposed to different concentrations of oridonin. The proliferation rate of the cells was measured by MTT assay, cell apoptotic rate was detected by flow cytometry, morphology of cell apoptosis was observed by Hoechst 33258 fluorescence staining, DNA fragmentation was assayed by agarose gel electrophoresis, caspase-3 and poly(ADP-ribose) polymerase (PARP) expressions were detected by Western blotting using polyclonal anti-caspase-3 antibody and mouse anti-human PARP monoclonal antibodies, and caspase-3 activity was assayed with a colorimetric assay kit before and after apoptosis had occurred. Oridonin (over 32 μmol/l) inhibited the growth of Jurkat cells and caused significant apoptosis. The suppression was both time-dependent and dose-dependent. Marked morphological changes of cell apoptosis, including condensation of chromatin and nuclear fragmentation, were clearly observed by Hoechst 33258 fluorescence staining, as well as by agarose gel electrophoresis. Western blotting showed cleavage of the caspase-3 zymogen protein (32 kDa), with the appearance of its 17 kDa subunit, and a cleaved 89-kDa fragment of 116 kDa PARP was also found, together with a concurrent increase in caspase-3 apoptotic activity. Oridonin can induce apoptosis in Jurkat cells via activation of caspase-3; the results indicate that oridonin might be an important potential anti-leukaemia reagent.

Nowadays, drug resistance still represents a major obstacle to successful acute myeloid leukemia (AML) treatment and the underlying mechanism is not fully elucidated. Here, we found that high expression of c-Myc was one of the cytogenetic characteristics in the drug-resistant leukemic cells. c-Myc over-expression in leukemic cells induced resistance to chemotherapeutic drugs, enhanced colony formation capacity and inhibited cell differentiation induced by all-trans retinoic acid (ATRA). Meanwhile, inhibition of c-Myc by shRNA or specific c-Myc inhibitor 10058-F4 rescued the sensitivity to cytotoxic drugs, restrained the colony formation ability and promoted differentiation. RT-PCR and western blotting analysis showed that down-regulation of C/EBPβ contributed to the poor differentiation state of leukemic cells induced by c-Myc over-expression. Importantly, over-expression of C/EBPβ could reverse c-Myc induced drug resistance. In primary AML cells, the c-Myc expression was negatively correlated with C/EBPβ. 10058-F4, displayed anti-proliferative activity and increased cellular differentiation with up-regulation of C/EBPβ in primary AML cells. Thus, our study indicated that c-Myc could be a novel target to overcome drug resistance, providing a new approach in AML therapy.

Background Report of a rare and serious complication of chemotherapy with daunorubicin and cytarabine (DA regimen). Methods We report a special case of a patient diagnosed chronic myeloid leukemia (CML) with accelerated phase, who simultaneously suffered from acute pleural and pericardial effusion while receiving chemotherapeutic treatment with DA regimen. Results Following treatment with DA regimen, the patient had the symptoms of chest distress and shortness of breath, followed by respiratory failure and pericardial tamponade. The patient's condition was improved when treated with the puncturation through the pericardium and pleural cavity, coupled with glucocorticosteroid treatment. Conclusions Physicians should be made aware of the potential for emergency pleural and pericardial effusion caused by daunorubicin and cytarabine in order to accurately diagnose and treat these conditions and thereby decrease mortality related to chemotherapy.

Aurora kinases are overexpressed in large numbers of tumors and considered as potential therapeutic targets. In this study, we found that the Aurora kinases inhibitors MK-0457 (MK) and ZM447439 (ZM) induced polyploidization in acute myeloid leukemia (AML) cell lines. The level of glycolytic metabolism was significantly increased in the polyploidy cells, which were sensitive to glycolysis inhibitor 2-deoxy-D-glucose (2DG), suggesting that polyploidy cells might be eliminated by metabolism deprivation. Indeed, inhibition of mTOR pathway by mTOR inhibitors (rapamycin and PP242) or 2DG promoted not only apoptosis but also autophagy in the polyploidy cells induced by Aurora inhibitors. Mechanically, PP242 or2DGdecreased the level of glucose uptake and lactate production in polyploidy cells as well as the expression of p62/SQSTM1. Moreover, knockdown of p62/SQSTM1 sensitized cells to the Aurora inhibitor whereas overexpression of p62/SQSTM1 reduced drug efficacy. Thus, our results revealed that inhibition of mTOR pathway decreased the glycolytic metabolism of the polyploidy cells, and increased the efficacy of Aurora kinases inhibitors, providing a novel approach of combination treatment in AML. Mol Cancer Res; 11(11); 1326–36. ©2013 AACR.