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Chronic hepatitis B virus (HBV) infection can lead to fibrosis, liver cirrhosis, and primary hepatocellular carcinoma. Investigating host factors that regulate HBV replication helps to identify antiviral targets. In the current study, we identified Nicotinamide N-Methyltransferase gene (NNMT) as a novel factor that regulates HBV transcription. NNMT is up-regulated at both the mRNA and protein levels in HepG2.2.15 cells compared to HepG2 cells. Overexpression of NNMT reduces HBV replication in several cell models, while knockdown of NNMT enhances HBV DNA levels. Mechanistically, NNMT suppresses HBV DNA replication by inhibiting HBV RNA transcription. The region required for the inhibitory effect of NNMT was narrowed to nt 1672-1708 in enhancer II by luciferase assays. On the other hand, ChIP assays and EMSA results showed that NNMT does not bind to this region substantially, either directly or indirectly. Next, a collection of hepatic nuclear receptor transcription factors was screened to determine whether they were affected by NNMT overexpression. NR5A1, a positive regulator of HBV replication, decreased significantly after NNMT overexpression. Collectively, the findings of this study shed light on the regulation of HBV transcription.

Glioblastoma (GBM) is one of the most common malignant and incurable brain tumors. The identification of a gene signature for GBM may be helpful for its diagnosis, treatment, prediction of prognosis and even the development of treatments. In this study, we used the GSE108474 database to perform GSEA and machine learning analysis, and identified a 33-gene signature of GBM by examining astrocytoma or non-GBM glioma differential gene expression. The 33 identified signature genes included the overexpressed genes COL6A2, ABCC3, COL8A1, FAM20A, ADM, CTHRC1, PDPN, IBSP, MIR210HG, GPX8, MYL9 and PDLIM4, as well as the underexpressed genes CHST9, CSDC2, ENHO, FERMT1, IGFN1, LINC00836, MGAT4C, SHANK2 and VIPR2. Protein functional analysis by CELLO2GO implied that these signature genes might be involved in regulating various aspects of biological function, including anatomical structure development, cell proliferation and adhesion, signaling transduction and many of the genes were annotated in response to stress. Of these 33 signature genes, 23 have previously been reported to be functionally correlated with GBM; the roles of the remaining 10 genes in glioma development remain unknown. Our results were the first to reveal that GBM exhibited the overexpressed GPX8 gene and underexpressed signature genes including CHST9, CSDC2, ENHO, FERMT1, IGFN1, LINC00836, MGAT4C and SHANK2, which might play crucial roles in the tumorigenesis of different gliomas.

Motivation MIB2 (metal ion-binding) attempts to overcome the limitation of structure-based prediction approaches, with many proteins lacking a solved structure. MIB2 also offers more accurate prediction performance and more metal ion types. Results MIB2 utilizes both the (PS)2 method and the AlphaFold Protein Structure Database to acquire predicted structures to perform metal ion docking and predict binding residues. MIB2 offers marked improvements over MIB by collecting more MIB residue templates and using the metal ion type-specific scoring function. It offers a total of 18 types of metal ions for binding site predictions. Availability and implementation Freely available on the web at http://bioinfo.cmu.edu.tw/MIB2/. Supplementary information Supplementary data are available at Bioinformatics online.

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.

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.

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.

The antiproliferation effects and inhibition of telomerase activity of oridonin on leukemic HL-60 cells were studied. HL-60 cells in culture medium were treated with different concentrations of oridonin. The inhibitory rate of the cells was measured by MTT assay. Cell apoptotic rate was detected by flow cytometry (FCM). Morphology of cell apoptosis was observed by Wright's stain. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR-enzyme-linked immunosorbent assay (ELISA) were used to detect hTERT mRNA expression and telomerase activity before and after apoptosis. Oridonin (over 8 micromol/l) could inhibit the growth of HL-60 cells and cause apoptosis significantly. The suppression was in both time-dependent and dose-dependent manner. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly by Wright's stain especially after the cells were treated 48-60 h by oridonin. The expression of hTERT mRNA as well as activity of telomerase were decreased concurrently by treatment with oridonin in HL-60 cells. Oridonin can downregulate the hTERT mRNA expression and decrease the telomerase activity of HL-60 cells; it has apparent antiproliferation and apoptosis-inducing effects on HL-60 cells in vitro.

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.

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.

Background Rapamycin, isolated from Streptomyces hygroscopicus , is recently reported to have immunosuppressant and anti-tumor effects on a large variety of cancers. To date, no detailed data are available about the effects of rapamycin on hepatocellular carcinoma cells. Objective In this study, the anti-proliferation effects of rapamycin on hepatocellular carcinoma cells BEL-7402 and HepG-2 in vitro were studied. Methods Cell viability was assessed by MTT assay and [ 3 H]-thymidine uptake, cell apoptosis was observed by Hoechst 33258 staining and flow cytometry (FCM). The variation of caspase-3 and apoptotic related genes was assayed by Western blotting, cell mitochondrial membrane potential was also investigated by using standard methods. Results Rapamycin could inhibit the growth of hepatocellular carcinoma 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. Rapamycin exhibits induction apoptosis by activation of caspase-3 and disruption of the mitochondrial membrane potential on hepatocellular carcinoma cells in vitro. Western blotting analysis demonstrated that anti-apoptotic protein Bcl-2 was down-regulated while pro-apoptotic protein Bcl-xl up-regulated remarkably in a time-dependent manner when apoptosis occurred. Conclusion Rapamycin has significant anti-proliferation effect by induction of apoptosis via activation of caspase-3 and disruption of mitochondrial membrane potential, as well as by down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bcl-xl on hepatocellular carcinoma cells. The data provide a potential mechanism for rapamycin-induced apoptosis in hepatocellular carcinoma cells, suggesting that rapamycin may serve as both an effective adjunctive reagent for the treatment of residual cancer cells and immunosuppressant after liver transplantation of hepatocellular carcinoma, 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.