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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.

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.

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.

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.

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.

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.

Our aim was to specifically transfer the cytosine deaminase (CD) and thymidine kinase (TK) genes into mucin 1 (MUC1)-positive leukemia cells by anti-MUC1 antibody directed infection of replication-defective lentivirus and to evaluate the targeted cytotoxicity of double suicide genes to leukemia. The target gene vector (containing CD and TK) and envelope (containing GFP and anti-MUC1) and packaging plasmids were cotransfected into 293T cells to produce the recombinant lentivirus. Suicide genes in virus-infected leukemia cells (U937, Jurkat, and K562) were detected by western blot. The cytotoxicity and bystander effect in vitro and the therapeutic effect in vivo were detected after treatment with the prodrugs. The results revealed that combined treatment with prodrug 5-fluorocytosine (5-FC) and ganciclovir (GCV) inhibited leukemia cell growth and caused significant bystander effect than treatment with either prodrug alone. TK/GCV treatment alone induced degeneration and cell death while the effect of CD/5-FC alone mainly caused vacuolar degeneration and necrosis. The addictive effects of combinatorial use of GCV and 5-FC mainly induced swelling of the mitochondria followed by necrosis of the leukemia cells. In vivo experiments revealed that both single and combinatorial prodrug treatments could prolong the survival time of leukemic mice. In summary, anti-MUC1 antibody directed lentiviral vector successfully transduced dual suicide genes and exerted targeted cytotoxicity against MUC1 positive leukemia cells. This targeted lentiviral dual suicide gene delivering system provides a promising approach for clinical treatment of leukemia in future.

The mitogen-activated protein kinase (MAPK) pathway has a protective function on the management of hematologic malignancies. The aim of this study was to assess whether the induction of MAPK-mediated effects contributes to the therapeutic value of combination sorafenib and daunorubicin (DNR) treatment. Herein, we found that DNR increased phosphorylation of extracellular signal-regulated kinases (ERK1/2) in K562 cells. ERK1/2 activity was blocked by either the mitogen-induced extracellular kinase (MEK) inhibitor U0126 or a multi-kinase inhibitor sorafenib. Of note, sorafenib sensitized K562 to DNR by inhibiting proliferation and inducing apoptosis in a dose-dependent manner which was through blocking the RAF/MEK/ERK pathway. Moreover, K562 cells transfected with a constitutively active MEK2DD plasmid showed increasing IC50 values following DNR treatment compared with control cells. Combination of DNR with MEK inhibitor U0126 synergistically inhibited K562 cell growth. In conclusion, our results indicated that sorafenib sensitized K562 cells to DNR-induced cytotoxicity by downregulating p-ERK1/2 expression. DNR in combination with sorafenib may represent a new and potential therapeutic strategy in treating acute leukemia with high p-ERK1/2 levels.

To explore the mechanism underlying antileukaemia effect of sodium valproate, the growth and survival of the K562 cell line were investigated. Global profiles of gene expression in K562 cells exposed to sodium valproate were assessed and validated. The differentially expressed genes identified were further used to query the connectivity map database to retrieve a ranked list of compounds that act on the same intracellular targets as sodium valproate. A significant increase in cell apoptosis and a change in gene expression profile were observed in valproate-exposed K562 cells. The significant enrichment analysis of gene ontology terms for the differentially expressed genes showed that these genes were involved in many important biological processes. Eight differentially expressed genes involved in apoptosis were verified by quantitative real-time PCR. The connectivity map analysis showed gene expression profile in K562 cells exposed to sodium valproate was most similar to that of HDACi and PI3K inhibitors, suggesting that sodium valproate might exert antileukaemic action by inhibiting HDAC as well as inhibiting PI3K pathway. In conclusion, our data might provide clues to elucidate the molecular and therapeutic potential of VPA in leukaemia treatment, and the connectivity map is a useful tool for exploring the molecular mechanism of drug action.

The aim of this study was to investigate the proliferation-inhibitory and inducing apoptotic effects of decitabine (DAC) on acute promyelocytic leukemia NB4-R2 cells. Cell inhibitory rate was determined by cell proliferation and cytotoxicity assay (WST-1 assay) after NB4-R2 cells were treated with 0.01 - 0.5 µmol/L DAC for 24, 48 and 72 h. Apoptosis of NB4-R2 cells treated with 0.05 - 5 µmol/L DAC for 48 h was detected by flow cytometry with PI staining and AnnexinV/PI staining. Reverse transcription-PCR (RT-PCR) was used to determine the mRNA expression level of MDR1 gene encoding P-glycoprotein (P-gp). The results indicated that DAC (0.01 - 0.5 µmol/L) inhibited the proliferation of NB4-R2 cells in both time- and concentration-dependent manners. The IC(50) of DAC on the viability of NB4-R2 cells after treatment for 48 and 72 h were 0.089 and 0.064 µmol/L respectively. DAC (0.05 - 5 µmol/L) induced NB4-R2 cell apoptosis in dose-dependent manner with down-regulation of MDR 1 gene expression. It is concluded that a low concentration of DAC (0.5 µmol/L) inhibits cell proliferation, while higher concentration of DAC (1 or 5 µmol/L) induces apoptosis on NB4-R2 cells, accompanied with reduction of MDR1 levels.

Peroxisome proliferator-activated receptor gamma (PPARγ) plays an important role in regulating energy balance, glucose and lipid metabolisms and inflammation. PPARγ also exerts multiple anti-cancer effects including tumor growth and angiogenesis inhibition, induction of cell differentiation, and apoptosis. Perturbed Wnt/β-catenin signaling likely plays a key role in tumorigenesis and the interaction between PPARγ and the transcriptional regulator β-catenin maybe important in this process. Phosphorylation of β-catenin by GSK-3β inactivates it and suppresses tumor cell proliferation and self-renewal of tumor stem cells. In combination with Frizzled, Wnt suppresses GSK-3β and causes degradation of β-catenin and activation of many tumor proliferation factors. In the present study, we investigated the effects of PPARγ agonist rosiglitazone (RGZ) and PPARγ antagonist GW9662 on the growth, mitotic cycle, and apoptosis of human lymphoma cell line, Raji cells. We also studied the influence of PPARγ ligands on the expression of β-catenin and GSK-3β in Raji cells to reveal whether Wnt/GSK-3β/β-catenin signaling pathways are involved in PPARγ ligands triggered Raji cell apoptosis. Results showed that both RGZ and GW9662 can inhibit the growth of Raji cells by inducing apoptosis and arresting cell cycle; however, there was no correlation between these effects and expression of PPARγ. Both the PPARγ ligands, RGZ and GW9662, appear to reciprocally regulate the mRNA and protein expressions of GSK-3β, which promotes apoptosis, and of β-catenin, which blocks apoptosis. These results suggest that PPARγ ligands mediate their effects via Wnt/GSK-3β/β-catenin signaling on Raji cell proliferation and survival.

Background Aurora kinase ensures accurate chromosome segregation during cell cycle, maintaining genetic integrity in cell division. VX-680, a small-molecule Aurora kinase inhibitor, interferes with mitotic entry and formation of bipolar spindles. Here, we evaluated VX-680 as a potential agent for treatment of all-trans retinoid acid (ATRA)-resistant acute promyelocytic leukemia (APL) in vitro. Methods CD11b expression was utilized to assess cell differentiation by flow cytometry. Immunofluorescence staining was conducted to analyze formation of cell monopolar spindle. Cell proliferation was evaluated by MTT assay. Sub-G1 population and Annexin V/PI staining were used to measure cell apoptosis. Hoechst 33342 staining was applied for identifying morphological changes in nucleus of apoptotic cell. Aurora-A (Aur-A) activation and the signaling pathways involved in apoptosis were detected by Western blot. JC-1 probe was employed to measure mitochondrial depolarization. Results VX-680 inhibited Aur-A by reducing autophosphorylation at the activation site, Thr288, accompanied by producing monopolar mitotic spindles in APL cell line NB4-R2 that was resistant to ATRA. In addition, we found that VX-680 inhibited cell proliferation as assessed by MTT assay. Flow cytometry showed that VX-680 led to apoptotic cell death in both dose- and time-dependent manners by either Sub-G1 or Annexin V/PI analysis. Hoechst 33342 staining represented typical apoptotic cells with nuclear fragmentation in VX-680 treated cells. Importantly, VX-680 inhibition of Aurora kinase suppressed Akt-1 activation and induced mitochondrial depolarization, which eventually resulted in apoptosis by activation of caspase pathway, as indicated by increasing proteolytic cleavage of procaspase-3 and poly ADP ribose polymerase (PARP) in NB4-R2 cells. Conclusions Our study suggested potential clinical use of mitotic Aurora kinase inhibitor in targeting ATRA-resistant leukemic cells.

This study was aimed to investigate the effect of multikinase inhibitor sorafenib on the proliferation and apoptosis of U937 cells and its possible mechanism. U937 cells were treated with different concentrations of sorafenib for 48 hours. Cell viability was determined by Cell Counting Kit-8; cell apoptosis and cell ratio in cell cycle were detected by flow cytometry with Annexin V/PI staining and PI staining respectively; expressions of GSK-3β, β-catenin and cyclin-D1 were assayed by Western blot. The results showed that the proliferation of U937 cells was inhibited by sorafenib in a dose-dependent manner (p0.05). Sorafenib induced cell apoptosis and cell cycle G(1)/G(0) arrest. Compared with results of Western blot before treatment, expression of inactivated GSK-3β, β-catenin and Cyclin-D1 down-regulated in a dose-dependent manner after treatment with sorafenib, this same changes were observed after up-regulation of inactivated GSK-3β by LiCl (p0.05). It is concluded that sorafenib inhibits the proliferation of U937 cells and induces cell apoptosis through reducing negative regulation of WNT signal pathway on inactivated GSK-3β and down-regulating β-catenin and cyclin-D1 level, which result in U937 cell cycle G(1)/G(0) arrest.

To investigate the apoptosis-inducing effect of peroxisome proliferator-activated receptor γ (PPARγ) agonist ciglitazone (CGZ) on leukemic HL-60 cells and its mechanisms of action.HL-60 cells in vitro culture medium were subject to different concentrations of CGZ (10-50 µmol/L) for 24, 48 and 72 h. MTT assay was used to detect the cell inhibitory rate and agarose gel electrophoresis to observe DNA fragmentation. Flow cytometry (FCM) and Annexin V/PI staining were used to detect CGZ and/or GW9662 (PPARγ antagonist)-induced cell apoptosis. The expression of PPARγ was examined by RT-PCR and Western blotting. The caspase-3 and protein levels in mitogen-activated protein kinase signaling pathways (MAPKs, p-P38, p-ERK and p-JNK) were also detected.CGZ (over 30 µmol/L) could inhibit the growth of HL-60 cells in both time- and dose-dependent manner. After treatment for 72 h, the cell growth inhibitory rate in 50 µmol/L CGZ (84% ± 11%) treated cells was found more higher than that in both 40 µmol/L and 30 µmol/L CGZ treated cells (72% ± 13%, 59% ± 13%, P0.01) and a typical DNA ladder was also observed by agarose gel electrophoresis. The expression of PPARγ was gradually up-regulated by CGZ treatment and could be down-regulated partially by PPARγ antagonist GW9662. The results also revealed that CGZ-induced cell apoptosis (49.7%, 72 h) could not be inhibited thoroughly by GW9662 (36.2%, control:3.2%). It indicated that the CGZ-induced cell apoptosis was partially PPARγ-independent. Western blotting showed a cleavage of caspase-3 zymogen protein and up-regulation of p-P38 protein. Thus it indicated that the activations of caspase-3 and P38 MAPK were involved in CGZ-induced cell apoptosis.CGZ inhibits cell growth by induction of cell apoptosis in HL-60 cells via PPARγ dependent and independent signaling pathways. The activations of caspase-3 and P38 MAPK may be one of the important mechanisms in CGZ in treated HL-60 cells.

OBJECTIVE To investigate the apoptosis inducing effects of ponicidin (PON) on leukemic K562 cells and its mechanisms of action. METHOD K562 cells in culture medium in vitro were given different concentrations of PON (10-50 micromol x L(-1)) for 24, 48 and 72 h. The inhibitory rate of the cells was measured by MTT assay, cell apoptotic rates were detected by flow cytometry (FCM) using Annexin V staining after K562 cells were treated with different concentrations of PON for 72 hours, and cell morphology was observed by Wright-Giemsa staining. Western blot was used to detect caspase-3 and poly(ADP-ribose) polymerase (PARP) expression, and the protein levels in mitogen-activated protein kinase signaling pathways (MAPKs, p-P38, p-ERK and p-JNK) as well as p-AKT and p-P85 in PI3K/AKT signaling pathways were also detected. RESULT PON (over 30 micromol x L(-1)) could inhibit the growth of K562 cells in both time- and dose-dependent manner. FCM analysis revealed that apoptotic cells were gradually increased in a dose-dependent manner after treatment for 72 hours, and that marked morphological changes of cell apoptosis such as condensation of chromatin was clearly observed by Wright-Giemsa staining after treatment by 50 micromol x L(-1) PON. Western blot showed cleavage of the caspase-3 zymogen protein (32 kD), with the appearance of its 17 kD subunit, and a cleaved 89 kD fragment of 116 kD PARP was also found. Furthermore, Western blotting also showed that expression of p-AKT and p-P85 in PI3K/AKT signaling pathways was downregulated dramatically whereas the expression of p-P38 as well as p-ERK and p-JNK remained unchanged after the cells were treated by PON for 48 h. CONCLUSION The results demonstrate that PON exhibits in vitro anti-leukemia effect by induction of apoptosis in K562 cells, and that PON induced apoptosis in K562 cells mainly related to activation of caspase-3 as well as inactivation of PI3K/AKT signaling pathway via down regulation of the expression of p-AKT and p-P85 protein levels. These results provide strong laboratory evidence for further anti-leukemia trials of PON.

The aim of this study was to investigate the effect of sorafenib combined with daunorubicin on leukemic k562 cell line. The inhibitory effect of sorafenib alone and its combination with daunorubicin on K562 cell proliferation was detected by MTT method; the synergistic effect was measured by CDI (coefficient of drug interaction); the apoptosis of K562 cells was observed by flow cytometry with Hoechst 33258 staining. The results showed that the sorafenib alone or its combination with daunorubicin could significantly inhibit K562 cell proliferation and the combination of both drugs displayed synergistic effect on K562 cells, meanwhile the apoptotic cells increased. It is concluded that the combination of sorafenib and daunorubicin has a obviously synergistic inhibitory effect on leukemic cell line K562.

Background The mitotic Aurora-A kinase exerts crucial functions in maintaining mitotic fidelity. As a bona fide oncoprotein, Aurora-A aberrant overexpression leads to oncogenic transformation. Yet, the mechanisms by which Aurora-A enhances cancer cell survival remain to be elucidated. Results Here, we found that Aurora-A overexpression was closely correlated with clinic stage and lymph node metastasis in tongue carcinoma. Aurora-A inhibitory VX-680 suppressed proliferation, induced apoptosis and markedly reduced migration in cancer cells. We further showed that insulin-like growth factor-1, a PI3K physiological activator, reversed VX-680-decreased cell survival and motility. Conversely, wortmannin, a PI3K inhibitor, combined with VX-680 showed a synergistic effect on inducing apoptosis and suppressing migration. In addition, Aurora-A inhibition suppressed Akt activation, and VX-680-induced apoptosis was attenuated by Myr-Akt overexpression, revealing a cross-talk between Aurora-A and PI3K pathway interacting at Akt activation. Significantly, we showed that suppression of Aurora-A decreased phosphorylated Akt and was associated with increased IkappaBα expression. By contrast, Aurora-A overexpression upregulated Akt activity and downregulated IkappaBα, these changes were accompanied by nuclear translocation of nuclear factor-κB and increased expression of its target gene Bcl-xL. Lastly, Aurora-A overexpression induced IkappaBα reduction was abrogated by suppression of Akt either chemically or genetically. Conclusion Taken together, our data established that Aurora-A, via activating Akt, stimulated nuclear factor-κB signaling pathway to promote cancer cell survival, and promised a novel combined chemotherapy targeting both Aurora-A and PI3K in cancer treatment.

In the present study we investigated the in vitro apoptosis inducing effects of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand ciglitazone (CGZ) on acute promyelocytic leukemia (APL) NB4 cells and its mechanisms of action. The results revealed that CGZ (10-50 micromol/l) inhibited the growth of leukemia NB4 cells and caused apoptosis in a time- and dose-dependent manner. Apoptosis was observed clearly by flow cytometry (FCM) and DNA fragmentation analysis. After treatment by CGZ for 48 h, the percentage of disruption of mitochondrial membrane potential (Deltapsim) was increased in a dose-dependent manner. Western blotting demonstrated the cleavage of caspase-3 zymogen protein and a time-dependent cleavage of poly (ADP-ribose) polymerase (PARP). The results also demonstrated that PPAR-gamma expression was increased concomitantly when apoptosis occurred, and that CGZ-induced apoptosis was inhibited by the PPAR-gamma antagonist GW9662, suggesting a PPAR-gamma dependent signaling pathway in CZG-induced cell death. Moreover, CGZ treatment remarkably downregulated the expression of the X-linked inhibitor of apoptosis protein (XIAP), which was inhibited by GW9662. Of note, a small-molecule XIAP antagonist (1396-12) mimicked the effect of CGZ-induced apoptosis via activation of caspase-3, 7 and 9. The apoptosis-inducing effects by CGZ on fresh APL cells were also found to be remarkable by using FCM and Wright's staining observation. Taken together, our results suggest that downregulation of XIAP and activation of capase-3 play an important role in mediating the PPAR-gamma-dependent cell death induced by CGZ in APL cells. These data provide a novel insight into potential therapeutic strategies for treatment of leukemia.

Tanshinone IIA, a diterpene quinone extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, has been reported to have anti-tumor effects on a large variety of cancer cells. The present study was undertaken to investigate the in vitro antiproliferation and apoptosis inducing effects of Tanshinone IIA on leukemia THP-1 cell lines and its mechanisms of action. MTT assay was used to detect the cell growth inhibitory rate; cell apoptotic rate and the mitochondrial membrane potential (Deltapsim) were investigated by flow cytometry (FCM), apoptotic morphology was observed by Hoechst 33258 staining and DNA fragmentation analysis. The expression of caspase-3 and different apoptosis modulators were analyzed by Western blotting. The results revealed that Tanshinone IIA inhibited the growth of THP-1 cells and caused significant apoptosis, the suppression was both in time- and dose-dependent manner. After treatment by Tanshinone IIA for 48 h, the percentage of disruption of Deltapsim gradually increased in a dose-dependent manner along with marked changes of cell apoptosis. Western blotting showed cleavage of the caspase-3 zymogen protein (32-kDa) with the appearance of its 20-kDa subunit and a dose-dependent cleavage of PARP, with the appearance of 89-kDa fragment; The expression of Bcl-2 and survivin was down-regulated remarkably while Bax expression was up-regulated concurrently after the cells were treated with Tanshinone IIA for 48 h. We therefore conclude that Tanshinone IIA has significant growth inhibition effects on THP-1 cells by induction of apoptosis, and that Tanshinone IIA-induced apoptosis on THP-1 cells is mainly related to the disruption of Deltapsim and activation of caspase-3 as well as down-regulation of anti-apoptotic protein Bcl-2, survivin and up-regulation of pro-apoptotic protein Bax. The results indicate that Tanshinone IIA may serve as a potential anti-leukemia reagent.

Ponicidin, an ent-kaurane diterpenoid derived from a constituent of the herbal supplement PC-SPES, Rabdosia rubescens, is recently reported to have anti-tumor effects on a large variety of cancers. In this study, we demonstrate that ponicidin exhibits cytotoxicity, induces apoptosis, disrupts the mitochondrial membrane potential, and triggers the activation of caspase-3, -8 and -9 in lung cancer A549 and GLC-82 cells. Ponicidin treatment of lung cancer cells caused downregulation of anti-apoptotic protein Bcl-2 and survivin as well as upregulaton of pro-apoptotic protein Bax in a time dependent manner when apoptosis ocurred. Ponicidin induced activation of caspase-3 can be blocked by a caspase-3-specific inhibitor z-DEVD-FMK Furthermore, the caspase-8-specific inhibitor z-IETD-FMK could block the ponicidin-induced activation of caspase-3, PARP cleavage, and prevented the release of cytochrome c from mitochondria into the cytoplasm. This indicate that activated caspase-8 initiates the release of cytochrome c during ponicidin-induced apoptosis. We therefore conclude that ponicidin has significant apoptosis-inducing effects by activation of caspase-3 -8, and -9 as well as downregulation of anti-apoptotic protein Bcl-2, survivin and upregulation of pro-apoptotic protein Bax, with caspase-8 acting as an upstream activator. The data offer a potential mechanism for ponicidin-induced apoptosis in lung cancer cells, suggesting that ponicidin may severve as an effective reagent for the treatment of lung cancer, and that in vivo anti-cancer effects as well as its potential clinical effectiveness need further investigation.

Oridonin, an ent-kaurane diterpenoid derived from the herbal Rabdosia rubescens, has been recently reported to have antitumor effects on a large variety of cancer cells. The present study was undertaken to investigate the in vitro antiproliferation and apoptosis inducing effects of oridonin on HPB-ALL cell lines and its mechanisms of action. HPB-ALL cells in culture medium in vitro were treated with different concentrations of oridonin (16-56 micromol/L). MTT assay was used to detect the cell growth inhibitory rate, and the cell viability was assessed by the trypan blue dye-exclusion method. Cell apoptosis and the mitochondrial membrane potential (delta psi m) were investigated by flow cytometry (FCM), Hoechst 33258 staining, and DNA fragmentation analysis. The expression of caspase-3 and different apoptosis modulators, including Fas and Bcl-2 family members, was analyzed by Western blotting. The results revealed that oridonin could significantly inhibit the growth of HPB-ALL cells and cause apoptosis, and the suppression was both time- and dose-dependent. After treatment with oridonin for 48 hr, the percentage of disruption of delta psi m gradually increased in a dose-dependent manner along with marked changes of cell apoptosis, and necrotic cells increased remarkably after the cells were treated with oridonin for 72 hr; Western blotting showed cleavage of the caspase-3 zymogen protein (32 kDa) with the appearance of its 20-kDa subunit when apoptosis occurred; expression of Bcl-2 and Bcl-XL was downregulated remarkably while expression of Bax and Bid was upregulated concurrently after the cells were treated with oridonin for 24 hr. Of note, the expressions of Fas and other Bcl-2 family members including Bak and Bad remained constant before and after apoptosis occurred. We therefore conclude that oridonin has significant antiproliferation effects on HPB-ALL cells by induction of apoptosis as well as directly causing cell necrosis and that oridonin-induced apoptosis on HPB-ALL cells is mainly related to the disruption of delta psi m and activation of caspase-3 as well as downregulation of anti-apoptotic protein Bcl-2, Bcl-XL, and upregulation of pro-apoptotic proteins Bax and Bid. The results indicate that oridonin may serve as a potential antileukemia reagent.

The anti-proliferation effects of oridonin on acute promyelocytic leukemia (APL) cells and its mechanisms were studied in vitro. NB4 cells as well as fresh leukemia cells obtained from APL patients in culture medium were treated with different concentrations of oridonin. Cell growth inhibition, apoptosis and related pathways were assessed by MTT assay as well as flow cytometry (FCM) and western blot analysis. The data revealed that oridonin (over 16 micromol/L) could inhibit the growth of NB4 cells by induction of apoptosis. Marked changes of cell apoptosis were observed very clearly by using electron microscopy and DNA fragmentation analysis after the cells exposed to oridonin for 48 h; Western blotting showed cleavage of the caspase-3 zymogen protein (32-kDa) with the appearance of its 20-kDa subunit as well as a cleaved 89-kDa fragment of 116-kDa PARP when apoptosis occurred. The expression of Bcl-2 was down-regulated remarkably accompanied by the disruption of the mitochondrial membrane potential (delta(psi)m). The anti-proliferative and apoptosis-inducing effects by oridonin in fresh APL cells were also found remarkably using Trypan Blue dye exclusion method and Wright's staining. We concluded that oridoning has significant anti-proliferative and apoptosis-inducing effects on NB4 cells by activation of caspase-3 and cleavage of PARP as well as by down regulation of Bcl-2 and disruption of the delta(psi)m. Furthermore, oridonin demonstrated apparent cell growth inhibition effects on fresh APL cells in vitro. The results indicated that oridonin may serve as a potential anti-leukemia reagent.

Ponicidin, an extract from the Chinese herb Rabdosia rubescens, is currently one of the most important traditional Chinese herbal medicines. Ponicidin has been reported to have anti-tumor effects on a large variety of malignant diseases. In this study, we investigated the anti-proliferation effects of ponicidin on human myeloid K562 and HL-60 cells. Cell viability was measured by MTT assay; cell apoptosis was assessed by flow cytometry, DNA fragmentation analysis and Hoechst 33258 staining. Caspase-3 and poly(ADP-ribose) polymerase (PARP) activation and Bax and Bcl-2 expression were detected by Western blot analysis. The results revealed that ponicidin could significantly inhibit the growth of K562 and HL-60 cells by induction of apoptosis. The suppression was both time- and dose-dependent. Cell apoptosis was observed clearly after the cells were treated with ponicidin for 48-72 h. Western blotting showed cleavage of the caspase-3 zymogen protein (32 kDa) with the appearance of its 17 kDa subunit, together with a cleaved 89-kDa fragment of 116 kDa PARP when apoptosis occurred. Bcl-2 expression was down-regulated while Bax expression up-regulated concurrently when the cells were treated with ponicidin for 24-48 h. Therefore, we conclude that ponicidin has significant anti-proliferation effects by induction of apoptosis on myeloid leukemia cells in vitro, down-regulation of Bcl-2, and up-regulation of Bax, and that activation of caspase-3 and PARP may be an important apoptosis-inducing mechanism. The results suggest that ponicidin may serve as a potential therapeutic agent for leukemia.