Proliferation Of NB4 Cells Research Articles

Molecular mechanism of different sensitivities of PML-RARα to apoptosis induced by apoptosis inducing agents

Objective To study the molecular mechanism of different sensitivities to apoptosis induced by low concentration of As2O3 in PML-RARα negative HL-60 cells and PML-RARα positive NB4 cells. Meth-ods NB4 and HL-60 cells were cultured with As2O3 for 1 to 4 days; cell proliferation were detected by MTT method; the apoptosis was detected by flow cytometry,Bcl-2,Bax and Fas mRNA were determined by RT-PCR. Results The proliferation of NB4 cells was inhibited obviously by As2O3(1.0 μmol/L)with the induction of apoptosis( P 0.05). Inhibition of proliferation and apoptosis were not obvious in PML-RARα negative HL-60 cells induced by low concentration As2O3 ( P >0.05), and there was no obvious variation of Bcl-2, Bax, Fas mRNA expres-sion or Bcl/Bax ratio( P >0.05). Conclusion The ratio of Bcl-2/Bax is contributed to the different sensitiv-ities of PML-RARα negative HL-60 cells and positive NB4 cells induced by low concentration of As2O3. Key words: Apoptosis; Arsenicals

Preliminary Study on Effects of Marsdenia Tenacissima Injection on NB4 Cells.

Purpose To study the effect of Marsdenia tenacissima injection on NB4 leukemia cells and its mechanisms.Methods After treated with Marsdenia tenacissima, viability of NB4 leukemia cells were determined by trypan blue assay. Cell cycle was assessed by FACS. Cell differentiation were determined by cell morphology, cell surface differentiation antigen and NBT reduction test. Cell cycle and apoptosis associated proteins were measured by western blot.Results Marsdenia tenacissima inhibits the growth of leukemia cells in a dose and time dependent manner. Marsdenia tenacissima induces G0/G1 phase arrest of NB4 cells, accompanied by P21 up-regulation. Marsdenia tenacissima did not change the protein level of PARP and p27. Marsdenia tenacissima could increase the expression of CD11b and percent of NBT positive cells, induces differentiation of NB4 cells.Conclusion Marsdenia tenacissima could inhibit the proliferation of NB4 cells through inducing cell cycle arrest and cell differentiation.

Berbamine selectively induces apoptosis of human acute promyelocytic leukemia cells via survivin-mediated pathway

Currently, resistance and relapse are still major problems in acute promyelocytic leukemia (APL) cases. Thus, new agents that override the resistance are crucial to the development of curative therapies for APL. In this study, we investigated the effects of berbamine on the proliferation of APL cell line NB4 and its possible mechanisms. NB4 cells were treated with berbamine at different concentrations (0-64 microg/ml) for 72 hours. MTT assay was used to determine proliferation inhibition of NB4 cells. Cell apoptosis was evaluated by both flow cytometry (FCM) and morphological examination. PML/RAR-alpha and survivin mRNAs were measured by nested-RT-PCR and RT-PCR, respectively. Activated-caspase 3 was determined by FCM. Berbamine greatly inhibited the proliferation of NB4 cells in dose- and time-dependent manners, and its IC50 value was 3.86 microg/ml at 48 hours. Both morphological observations and FCM results showed that berbamine induced apoptosis of NB4 cells with concomitant increase of activated caspase-3 and decrease of survivin mRNA. After treatment with berbamine at 8 microg/ml for 48 hours, the percentage of apoptotic cells increased from 2.83% to 58.44% (P<0.01), and the percentage of cells with activated-caspase 3 elevated from 2.06% to 70.89% (P<0.01), whereas, level of survivin mRNA was reduced to 38.24% of control (P<0.01). However, no significant change was observed in PML/RAR-alpha mRNA. Berbamine induces caspase-3-dependent apoptosis of leukemia NB4 cells via survivin-mediated pathway, suggesting that berbamine may be a novel potential agent against APL with a mechanism distinct from that of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO).

Gnidilatimonoein fromDaphne mucronata induces differentiation and apoptosis in leukemia cell lines

Gnidilatimonoein is a new diterpene ester, recently isolated from the leaves of Daphne macronata with potent anti-tumoral and anti-metastastic activities (Yazdanparast et al., 2004). Promyeloblastic (KG1), promyelocytic (NB4) and promonocytic (U937) cells were cultured in the presence of various concentrations of the drug (0.5-3.0 microM) for 3 days. Herein, we report that gnidilatimonoein induces differentiation and apoptosis in KG1, NB4 and U937 cells. The drug inhibited growth and proliferation of KG1, NB4 and U937 cells with IC50 values of 1.5, 1.5 and 1.0 microM, respectively, after 72 h of treatment. Cell viability was also decreased by 18%, 20% and 23%, respectively, after 72 h treatment with the drug. NBT reducing assay revealed that the inhibition of proliferation is associated with differentiation especially toward monocytes-like morphology. Indeed, the drug at 0.5-1.5 microM induced differentiation by 5-50% in the cells. Acridine orange/ethidium bromide (AO/EtBr) double staining and DNA fragmentation assays revealed that apoptosis occurred after differentiation of the cells. Based on the present data, it seems that the new compound is a good candidate for further evaluation as an effective chemotherapeutic agent acting through induction of differentiation and apoptosis.

Zerumbone, a bioactive sesquiterpene, induces G2/M cell cycle arrest and apoptosis in leukemia cells via a Fas‐ and mitochondria‐mediated pathway

We demonstrated here for the first time that zerumbone (ZER), a natural cyclic sesquiterpene, significantly suppressed the proliferation of promyelocytic leukemia NB4 cells among several leukemia cell lines, but not human umbilical vein endothelial cells (HUVECs), by inducing G2/M cell cycle arrest followed by apoptosis with 10 microM of IC50. Treatment of NB4 cells with growth-suppressive concentrations of ZER resulted in G2/M cell cycle arrest that was associated with a decline of Cyclin B1 protein, but with the phosphorylation of ATM/ Chk1/Chk2. In addition, ZER induced the phosphorylation of Cdc25C at the Thr48 residue and Cdc2 at the Thr14/Tyr15 residues. Furthermore, ZER-induced apoptosis in NB4 cells was initiated by the expression of Fas (CD95)/Fas Ligand (CD95L), concomitant with the activation of caspase-8. ZER was also found to induce the cleavage of Bid, a mediator that is known to connect the Fas/CD95 cell death receptor to the mitochondrial apoptosis pathway. ZER also induced the cleavage of Bax and Mcl-1 proteins, but not Bcl-2 or Bcl-XL. ZER-induced apoptosis took place in association with a loss of the mitochondrial transmembrane potential as well as the activation of caspase-3 and -9, resulting in the degradation of the proteolytic poly (ADP-ribose) polymerase (PARP). ZER also triggered a release of cytochrome c into the cytoplasm. Both antagonistic anti-Fas antibody ZB4 and pan-caspase inhibitor Z-VAD inhibited ZER-induced apoptosis in NB4 cells. Taken together, ZER is an inducer of apoptosis in leukemic cells that specifically triggers the Fas/CD95- and mitochondria-mediated apoptotic signaling pathway.

Distinct effects of different concentrations of sodium selenite on apoptosis, cell cycle, and gene expression profile in acute promyeloytic leukemia-derived NB4 cells

Selenium at a low concentration has a chemopreventive role against cancer, while at a high concentration, it exerts a direct antitumor effect. However, the mechanisms remain elusive. In this article, we discovered that Na(2)SeO(3) at 20 micromol/l concentration could significantly inhibit the proliferation of NB4 cells, affect the cell cycle distribution of cell population, and induce cellular changes characteristic of apoptotic cells, while this same compound at 2 micromol/l concentration had no such effects. The mechanisms underlying these overt differences caused by treatment of different concentrations of selenium were further investigated. cDNA microarray analysis showed that after treatment by 20 micromol/l Na(2)SeO(3), 34 genes were changed in expression, while treatment by 2 micromol/l Na(2)SeO(3) resulted in the changes of 29 genes. Nine genes were regulated in both groups, among which three showed opposite changes caused by 2 and 20 micromol/l Na(2)SeO(3). The majority of regulated genes did not coincide between the two experiment groups. In conclusion, 2 and 20 micromol/l Na(2)SeO(3) could have different effects on NB4 cells, and some genes might be involved in the underlying mechanisms. Our findings could provide basis for further uncovering the molecular mechanisms of the chemopreventive and antitumor effects of selenium and, in turn, for probing the rationality of treating leukemia with selenium.

Bcl-2 siRNA Induces Apoptosis and Enhances the Sensitivity to Arsenic Trioxide in NB4 Cells.

Bcl-2 is the prominent member of a family of proteins responsible for dysregulation of apoptosis, prevention of cancer-cell death, and resistance to chemotherapy and radiotherapy. Overexpression of Bcl-2 protein was shown to result in resistance to a variety of apoptosis-inducing signals in acute leukemia. Arsenic trioxide (As203)induces clinical remission in acute promyelocytic leukemia with minimal toxicity and apoptosis in APL-derived NB4 cells at low (0.5 to 2 μmol/L) concentration. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing a target protein by specifically digesting its mRNA. In this study, we studied the effect of transfection with siRNA targeting Bcl-2 in NB4 cells in terms of proliferation and induction of apoptosis. Further,we investigated whether the Bcl-2 siRNA may enhance the sensitivity of NB4 cells to arsenic trioxide. Synthetic siRNA was transferred against Bcl-2 into NB4 cells using Oligofectamine transfection protocol in the presence or absence of arsenic trioxide. RT-PCR and Western blotting analysis of Bcl-2 expression in NB4 cells was performed after transfection. The inhibition of cell growth was assessed by a modified MTT assay, counting cell number. Apoptosis was determined by morphological observation and flow cytometric analysis. When synthetic Bcl-2 siRNA was delivered into NB4 cells, no Bcl-2 transcripts were detected at 24h, and Bcl-2 protein levels were reduced by approximately 72% for 48h after siRNA transfer. Bcl-2 siRNA treatment for 72h led to 36% apoptosis of cells,as compared to control siRNA treatment. Control siRNA had no significant effect on growth and apoptosis of cells. Simultaneous administration of As203 and Bcl-2 siRNA was able to reduce the mRNA and protein expression of Bcl-2 significantly compared to Bcl-2 siRNA alone demonstrating a synergistic effect of combined As203 and siRNA treatment (P<0.05). Proliferation in NB4 cells was strongly inhibited (about 86%) following combination treatment for 72h as opposed to only 40% after administration of 0.5μmol/L As203 alone. When NB4 cells were incubated with 0.5μmol/L As203 for 72h after siRNA transfer, the percentage of apoptotic cells significantly increased (P<0.05), compared with combined As203 and control siRNA treatment or As203 treatment alone, respectively. No significant difference on apoptosis was seen in cells treated with As203 alone or treated with As203 and control siRNA. These results show that Bcl-2 siRNA could induce apoptosis and enhance the sensitivity to arsenic trioxide in NB4 cells. Our results suggest that the combination of arsenic trioxide and Bcl-2 siRNA might be an effective anti-leukemia protocol.

Effect on NB4 Cells Proliferation, Differentiation and Resistance to Chemotherapy Apoptosis by VEGF-C cDNA Transfection.

VEGF-C secreted by kinds of tumor cells play the key role in lymphangiogenesis via activating tyrosine kinase of VEGFR-3, which is one of most important pathway of infiltration and metastasis of tumor cells. In recent years, the effects of VEGF-C on hematological malignant cells are paid attention. In the current study, the recombinant eukaryotic expression plasmid (pcDNA3.1-VEGF-C) and the vacant pcDNA3.1 vector were introduced into the acute promyelocytic leukemia cell line- NB4 cells (VEGFR-3+/VEGFR-2−) by lipofectamine mediation and positive clones were screened by G418. The stable expression of VEGF-C was detected by reverse transcriptase-PCR and Western blotting. The proliferation ability of NB4/VEGF-C cells is analysed by MTT assay. After NB4/VEGF-C cells were induced by ATRA (1μmol/L), the expression levels of C/EBPα gene which can promote myelocytes differentiation and maturation, CD11b on cells surface and morphological variation were analysed by real-time quantitative PCR (RT-QPCR), flow cytometry analysis (FCM), Wright-Giemsa staining respectively. Furthermore, after the NB4/VEGF-C cells were cultured together with Etoposide (200ng/ml) for 48 hours, the numbers of AnnexinV+/PI−cells (apoptotic cells) were determined by FCM to investigate the effect of VEGF-C on the cells apoptosis and expression levels of antiapoptotic bcl-2 gene in these cells were analysed by RT-QPCR. The NB4/pcDNA3.1 cells was used as control during the experiments. From these experiments, NB4/VEGF-C cells can stably secret active protein VEGF-C in the cell supernatants. The cell growth curve shows that the proliferation ability of NB4/VEGF-C cells is stronger compared with NB4/pcDNA3.1cells. The expression levels of C/EBPα gene of NB4/VEGF-C cells after induced by ATRA is only 1/32 that of NB4/pcDNA3.1 cells. Morphological analysis shows that the degree of promyelocytes gradually maturing into myelocytes among NB4/VEGF-C cells is weaker compared with the controls after induced by ATRA. After induced by Etoposide, the percent of apoptotic cells of NB4/VEGF-C cells (7.20±2.52%) is significantly lower than that of NB4/pcDNA3.1 cells (16.07±3.58%)( P=0.005) and the bcl-2 gene expression level of NB4/VEGF-C cells is 2.28 times more than that of NB4/pcDNA3.1 cells. The results implied that the VEGF-C via VEGFR-3 signaling pathway could promote the proliferation of NB4 cells by autocrine pathway and inhibit the NB4cells differentiation and maturation and chemotherapy-induced apoptosis via upregulating bcl-2 gene expression. Thus, VEGF-C/VEGFR-3 signaling loops might play an important role in disease progression and be potential therapeutic target for the treatment of leukemias.

Studies on the Effects of Berbamine on the Human Leukemia Cell Line NB4.

Purpose: To study the effects of berbamine on the human leukemia cell line NB4Methods: Berbamine compound was dissolved to the concentration of 1mg/ml with 0.9% sodium chloride and diluted to desired concentrations (as indicated in figure ledends 0.5,1,2,4,8,12,16ug/mL)with culture medium. Growth of NB4 cells was determined by MTT assay based upon the reduction of tetrazolium dye 3–4,5-dimethylthiazol-2,5-diphenyltetrazolium(MTT). Morphological observation was used to detect the characteristic changes of apoptosis in NB4 cells and DNA agarose electrophoresis were used to detect the characteristic changes of apoptosis in NB4 cells, while apoptosis rate was measured by flow cytometry assay. The _expression of PML/RARα gene was determined by nested-PCR.Result: MTT results showed berbamine could inhibit the growth of NB4 cells significantly after treated with different concentration of berbamine for different time, IC50 was 3.86 ug•mL-1at 48 hours. Typical nuclear condensation and apotosis body were observed as early as 24 hours following a 8μgμg•mL-1 berbamine treatment;while the DNA agarose electrophoresis also show the typical DNA ladder,and the apoptosis rate of NB4 cell was increased from 2.83%to 58.84% after treated with 12ug•mL-1 berbamine for 48 hours. The _expression of PML/RARα gene wasn't changed obviously.Conclusion: Berbamin could inhibit the proliferation of NB4 cells and inducing apoptosis might be its one of mechanisms. The berbamine-induced apoptosis is not related to the _expression of PML/RARα gene.

Effects of curcumin on proliferation of NB4 cells and acetylation of histone H3 and P53

Objective: To investigate the effects of curcumin on the proliferation of NB4 cells and the acetylation of histone H3 and no-histone p53. Methods: NB4 cells were cultured and treated with or without curcumin at different concentration (50 µmol/L, 25µmol/L, 12.5µmol/L, 6.25µmol/L, 3.125µmol/L) at various time points (oh, 4h, 8h, 12h, 24h). Western blot analysis was performed to determine the level of acetylated histone H3, p53 and acetylated p53, MTT assay was performed to examine the proliferation of NB4 cells. Results: the proliferation of NB4 cells was inhibited by curcumin in a time- and dose-dependent manner, the IC50 at 24h and 36h were 40µmol/L and 25µmol/L respectively. The levels of acetylated histone H3 and acetylated p53 were increased obviously. Conclusion: curcumin possessed the foundation of deacetylases inhibitor, increased the level of acetylated histone H3 and the expression of tumor suppressor p53, enhanced later’s activity, and inhibited the proliferation of NB4 cell.

Signaling Mechanisms of Anti-Proliferation by Sphingosine 1-Phoshate(S1P) on Acute Promyelocytic Leukemia(APL) Cell Lines.

There is abundant evidence that S1P can function as a second messenger important for regulation of calcium homeostasis, cell growth, and suppression of apoptosis. In many cases, the intracellular level of S1P and ceramide, another important sphingolipid metabolite associated with cell death and cell growth arrest, coordinately determine cell fate. N, N-dimethylsphingosine(DMS) potentiates TNF- and FasL- induced apoptosis in the human acute leukemia jurkat, U937 and HL-60 cell line, which suggests that combining DMS with cytotoxic drugs might be a useful chemotherapeutic approach. In this study, we investigated the role of S1P on proliferation, survival and its signaling mechanisms on the NB4 cell, the human acute promyelocytic leukemia cell line, which has chromosomal abnormality, t(15:17). NB4 cells were exposed to S1P at various concentrations (1, 5, 10 μM) for 48 hours, and then the cell growth was determined by MTT assay. We studied whether NB4 cells have S1P receptors or not. To investigate the signaling mechanisms of S1P on the NB4 cell, we used MAP kinase inhibitors and a PI3K inhibitor including PD98059, U0126 (specific ERK pathway inhibitor), SB203580 (specific p38 pathway inhibitor), and LY294002 (specific PI3K pathway inhibitor). We also examined the effect of pertussis toxin (PTX), a Gi protein inhibitor, on the proliferation of NB4 cells after S1P treatment.RT-PCR analysis revealed a putative S1P receptor, Edg-3 mRNA expressed in NB4 cells. In the MTT assay, S1P inhibited cell proliferation in a dose-dependent manner. At 10 μM, NB4 cell proliferation was most inhibited. In addition, we found that the inhibitory effect of S1P on the NB4 cell proliferation was inhibited by PD98059, U0126, and PTX. However, SB203580 and LY294002 had not caused an inhibitory effect of S1P on the NB4 cell proliferation.In contrast to previous knowledge that S1P increases the survival of leukemic cells because apoptosis is prevented by caspase inactivation, our data suggests that S1P inhibits NB4 cell proliferation, through ERK pathway activation, not p38 nor JNK pathways. In addition, the pertussis toxin(PTX)-sensitive GTP-binding protein-coupled receptor, Edg-3, seems to be involved in the antiproliferative signaling of S1P to the NB4 cell.Understanding the signaling mechanisms of anti-proliferation by S1P can uncover new therapeutic targets for APL and the results of this study warrant further investigation on synergism of S1P with known therapeutic agent, ATRA and Arsenic trioxide on APL cells.

Sodium selenite induces apoptosis in acute promyelocytic leukemia-derived NB4 cells by a caspase-3-dependent mechanism and a redox pathway different from that of arsenic trioxide.

Two relatively recent discoveries stand behind our current effort to investigate the effects of the chemopreventive agent, selenium, on the proliferation and survival of NB4 cells. The first is that certain selenium compounds such as sodium selenite have pro-oxidant ability to catalyze the oxidation of thiols and simultaneously generate superoxide. The second lies in the exquisite susceptibility of NB4 cells to arsenic trioxide-induced, reactive oxygen species (ROS)-mediated apoptosis due to less efficiency of the cellular defense system. In this study, we demonstrated that sodium selenite could induce apoptosis in NB4 cells via the classic mitochondrial pathway involving caspase-3 activation and Bcl-2 cleavage. An increase in the basal cellular glutathione (GSH) content rendered NB4 cells resistant to arsenic trioxide, but could sensitize NB4 cells to sodium selenite. Moreover, combined treatment of NB4 cells with all- trans retinoic acid (ATRA) at low concentration and sodium selenite exhibited a synergistic effect on apoptosis induction. Together, our results suggest that selenite is a promising candidate for treatment of acute promyelocytic leukemia (APL) and the mechanism underlying its anticancer effects warrants further investigation.

Cytotoxic activities of Coriolus versicolor (Yunzhi) extract on human leukemia and lymphoma cells by induction of apoptosis

Coriolus versicolor (CV), also known as Yunzhi, is one of the commonly used Chinese medicinal herbs. Although recent studies have demonstrated its antitumour activities on cancer cells in vitro and in vivo, the exact mechanism is not fully elucidated. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized aqueous ethanol extract prepared from Coriolus versicolor on a B-cell lymphoma (Raji) and two human promyelocytic leukemia (HL-60, NB-4) cell lines using a MTT cytotoxicity assay, and to test whether the mechanism involves induction of apoptosis. Cell death ELISA was employed to quantify the nucleosome production resulting from nuclear DNA fragmentation during apoptosis. The present results demonstrated that CV extract at 50 to 800 μg/ml dose-dependently suppressed the proliferation of Raji, NB-4, and HL-60 cells by more than 90% (p < 0.01), with ascending order of IC 50 values: HL-60 (147.3 ± 15.2 μg/ml), Raji (253.8 ± 60.7 μg/ml) and NB-4 (269.3 ± 12.4 μg/ml). The extract however did not exert any significant cytotoxic effect on normal liver cell line WRL (IC 50 > 800 μg/ml) when compared with a chemotherapeutic anticancer drug, mitomycin C (MMC), confirming the tumour-selective cytotoxicity. Nucleosome productions in HL-60, NB-4 and Raji cells were significantly increased by 3.6-, 3.6- and 5.6-fold respectively upon the treatment of CV extract, while no significant nucleosome production was detected in extract-treated WRL cells. The CV extract was found to selectively and dose-dependently inhibit the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway.

Inhibitory Effect of Oridonin on the Proliferation of NB4 Cells and Its Mechanism

To investigate the anti-proliferation effect of oridonin on leukemic NB4 cells and its mechanism. NB4 cells in culture medium in vitro were given different concentrations of oridonin. The inhibitory rate of the cells were measured by MTT assay, cell apoptotic rate was detected by flow cytometry (FCM), morphology of cell apoptosis was observed by hoechst 33258 fluorescence staining, and the activity of telomerase was detected using TRAP-PCR-ELISA before and after apoptosis occurred. Oridonin (over 8 μmol/L) could decrease the telomerase activity, inhibit the growth of NB4 cells and induce apoptosis significantly in a time- and dose-dependent manner. Marked morphological changes of cell apoptosis were observed by hoechst 33258 fluorescence staining especially after the cells treated by oridonin for 48—60 h. Oridonin could inhibit the proliferation and induce the apoptosis of NB4 cells in vitro. One of the mechanisms may be the decrease of the telomerase activity of NB4 cells.

Targeting PML/RARalpha transcript with DNAzymes results in reduction of proliferation and induction of apoptosis in APL cells.

DNAzymes are nucleic acid enzymes that can recognise specific RNA substrate via Watson-Crick base pairing and cleave it with multiple turnovers. We have designed and examined the effects of DNAzymes targeting the PML/RARalpha fusion gene in acute promyelocytic leukaemia (APL). The DNAzymes (DZ1 and DZ3) were designed to cleave the PML/RARalpha transcript at the GC nucleotides at the fusion point and three nucleotides upstream of that respectively. Disabled DNAzymes were synthesised and used as controls. Cell-free cleavage reactions were performed on total RNA from NB4 cell line and PML/RARalpha and RARalpha-amplified RNA fragments (aRNA). Postcleavage examination showed that DZ1 and DZ3 cleave PML/RARalpha efficiently and specifically. NB4 APL cells transfected with DZ1 or DZ3 showed a significant suppression of PML/RARalpha protein expression. These DNAzymes also inhibited the proliferation of NB4 cells, reduced the viability rate, and induced apoptosis in these cells. The disabled DNAzymes showed no effect on NB4 cells. The two DNAzymes did not produce any significant effect on K562 cells, which were used as control cells. DNAzymes are more resistant to serum than ribozymes. These data show that targeting the PML/RARalpha fusion gene with DNAzymes can induce apoptosis in APL cells and may have a role in the treatment of APL. They also show DNAzymes are promising tools for targeting specific genes in leukaemia.

Vitamin D2 analog 19-nor-1,25-dihydroxyvitamin D2: antitumor activity against leukemia, myeloma, and colon cancer cells.

1,25-Dihydroxyvitamin D(3) inhibits growth of several types of human cancer cells in vitro, but its therapeutic use is hampered because it causes hypercalcemia. 19-nor-1,25-Dihydroxyvitamin D(2) (paricalcitol) is a noncalcemic vitamin D analog that is approved by the Food and Drug Administration for the treatment of secondary hyperparathyroidism. We investigated the antitumor activity and mechanism of action of paricalcitol in vitro and in vivo. Effects of paricalcitol on proliferation, the cell cycle, differentiation, and apoptosis were examined in cancer cell lines. Effects on tumor growth were examined with colon cancer cell xenografts in nude mice (five in the experimental group and five in the control group). The interaction of paricalcitol with the vitamin D receptor (VDR) in mononuclear spleen cells and myeloid stem cells from wild-type and VDR knockout mice was examined. All statistical tests were two-sided. Paricalcitol inhibited the proliferation of myeloid leukemia cell lines HL-60, NB-4, and THP-1 cells at an effective dose that inhibited growth 50% (ED(50)) of 2.4-5.8 x 10(-9) M by inducing cell cycle arrest and differentiation. Paricalcitol inhibited the proliferation of NCI-H929 myeloma cells at an ED(50) of 2.0 x 10(-10) M by inducing cell cycle arrest and apoptosis. Paricalcitol also inhibited the proliferation of colon cancer cell lines HT-29 (ED(50) = 1.7 x 10(-8) M) and SW837 (ED(50) = 3.2 x 10(-8) M). HT-29 colon cancer xenografts in paricalcitol-treated nude mice were smaller (1044 mm(3) and 1752 mm(3), difference = 708 mm(3), 95% confidence interval = 311 to 1104 mm(3); P =.03) and weighed less (1487 mg and 4162 mg, difference = 2675 mg, 95% confidence interval = 2103 to 3248 mg; P<.001) than those in vehicle-treated mice. Paricalcitol induced committed myeloid hematopoietic stem cells from wild-type but not from VDR knockout mice to differentiate as macrophages. Paricalcitol has anticancer activity against myeloid leukemia, myeloma, and colon cancer cells that may be mediated through the VDR. Because it has been approved by the Food and Drug Administration, clinical trials of this agent in certain cancers are reasonable.

Cytotoxicity of anti-CD64-ricin a chain immunotoxin against human acute myeloid leukemia cells in vitro and in SCID mice.

Blast cells from patients with acute myeloid leukemia (AML) commonly express CD64, the high-affinity receptor for immunoglobulin G (FcgammaRI). An immunotoxin (MDX-44) was constructed by coupling humanized anti-CD64 monoclonal antibody (mAb) H22 via a bivalent linker to deglycosylated ricin A-chain (RA). Human leukemia cell lines were incubated with MDX-44 or H22/free RA. The effect of MDX-44 on the proliferation of leukemia cells was assessed by [(3)H]thymidine incorporation. In the presence of interferon-gamma (IFN-gamma), MDX-44 significantly inhibited the proliferation of CD64(+) HL-60, NB4, and U937 cells in 72-h cultures in a dose-dependent manner. The mechanism of action appeared to be the induction of apoptosis, as measured by propidium iodide staining and flow cytometry analysis. However, CD64(-) KG-1a and Daudi cells were not affected by MDX-44/IFN-gamma. Incubating HL-60 cells with MDX-44/IFN-gamma resulted in a 99% decrease in colony-forming units, whereas colony-forming cells in normal bone marrow were not significantly suppressed by such treatment. Cells from 60% of AML patients (6/10) were inhibited by MDX-44/IFN-gamma, and the inhibition was correlated with CD64 expression on these cells (r = 0.65). In a human AML model in NOD/SCID mice, MDX-44/IFN-gamma inhibited 95-98% of peritoneal exudate AML cell proliferation and 85-90% of solid leukemia masses. The effect of MDX-44 on AML cells was dependent on activation of cells by IFN-gamma. MDX-44/IFN-gamma may have value in the therapy of AML cells expressing cell-surface CD64.

Effects of Novel RAR- and RXR-Selective Retinoids on Myeloid Leukemic Proliferation and Differentiation In Vitro

Retinoids such as all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA) have an important role in many aspects of proliferation and differentiation of hematopoietic cells. They exert their effects by binding to retinoic acid receptors (RARs) and/or retinoid X receptors (RXRs). We studied the effects of novel retinoids on proliferation and differentiation of HL-60 and NB4 myeloid leukemic cells, as well as acute promyelocytic leukemia (APL) cells from patients. RXR-selective SR11345 (Retinoid C) had little ability to inhibit the clonal growth and to induce the differentiation of either HL-60 or NB4 cells. However, SR11276 (Retinoid E), which activated both the RAR and RXR classes, and SR11278 (Retinoid D), which activated the RAR subtypes alpha, beta, and gamma, could inhibit clonal growth of both cell types, as well as leukemic cells from APL patients. The combination of ATRA and either SR11276 or SR11278 additively inhibited APL cell proliferation. SR11302 (Retinoid A), with reported anti-AP-1 activity and no activation of RARs and RXR and SR11363 (Retinoid B), which selectively activated RARbeta and gamma, were inactive. The clonal proliferation of both HL-60 and NB4 cells that were pulse-exposed to 10(-9) mol/L ATRA, SR11276, SR11278, or SR11345 for 3 days, washed, and plated in methylcellulose culture were inhibited by 0%, 51%, 21%, and 1% for HL-60 cells and 43%, 41%, 35%, and 1% for NB4, respectively, compared with nontreated control cells. When the HL-60 cells were pulse-exposed to 10(-9) mol/L of either SR11278 or SR11276, plus 10(-9) mol/L ATRA for 3 days, colony numbers were reduced by 46% and 64%, respectively. Induction of leukemic cell differentiation as determined by the nitroblue tetrazolium (NBT) assay showed that the combination of 10(-7) mol/L of either SR11278 or SR11276 with 10(-7) mol/L ATRA had additive effects on HL-60 cells, NB4 cells, and fresh APL cells. Induction of CD11b expression on both HL-60 and NB4 cells occurs during their differentiation. Expression of this antigen was synergistically augmented by the combination of either 10(-7) to 10(-8) mol/L SR11278 or 10(-7) to 10(-9) mol/L SR11276 with 10(-9) mol/L ATRA compared with either analog alone in HL-60 cells. Expression of the novel myeloid specific transcription factor C/EBPepsilon was increased by SR11278 and SR11276 in both the HL-60 and NB4 cell lines. We conclude that retinoids or combination of retinoids with specificities for both RAR and RXR may markedly enhance the ability of ATRA to inhibit clonal growth and induce differentiation of HL-60 and NB4 leukemic cells. This occurs in the absence of continuous contact with retinoids.

Bryostatin-1 and 1alpha,25-dihydroxyvitamin D3 synergistically stimulate the differentiation of NB4 acute promyelocytic leukemia cells.

One of the objectives of treatment for patients with acute promyelocytic leukemia (APL) is to induce tumor cell differentiation and block cell proliferation. Acute promyelocytic leukemia cells (NB4) responded to the combination treatment of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] plus phorbol 12-myristate 13-acetate (PMA) and differentiated into monocyte/macrophage-like cells, as well as expressed strong alkaline phosphatase (ALP) activities. Since PMA has limited clinical application due to its tumor-promoting effect, another protein kinase C activator, bryostatin-1, was currently tested for its interaction with 1alpha,25(OH)2D3 to induce NB4 cell differentiation and block cell proliferation. Bryostatin-1 alone, but not 1alpha,25(OH)2D3 alone, significantly inhibited cell proliferation and induced NB4 cell differentiation into monocyte/macrophages; however neither bryostatin-1 nor 1alpha,25(OH)2D3 alone induced ALP expression. Like PMA, bryostatin-1 synergistically interacted with 1alpha,25(OH)2D3 to stimulate ALP expression 30-fold over the control (P < 0.001) and further promote appearance of monocyte/macrophage-like cells. The ALP stimulation was both time- and dose-dependent. Thus, we demonstrate for the first time that the combination of bryostatin-1 and 1alpha,25(OH)2D3 strongly affect NB4 cell differentiation and proliferation. Therefore, this proposed combination treatment may be an alternatively potential therapeutic regimen for APL patients and assay of ALP may be a more sensitive and facile way to monitor the possible remission of APL patients.

The induction of apoptosis and cell cycle arrest by arsenic trioxide in lymphoid neoplasms.

Arsenic trioxide (As2O3) has recently been shown to induce complete remission in acute promyelocytic leukemia (APL). As2O3 reportedly has dose-dependent dual effects on APL cells, triggering apoptosis at relatively high concentrations and inducing differentiation at lower concentrations. However, its effect is still controversial for other AML cells and hematological neoplasms. We studied the in vitro effect of As2O3 on lymphoid lineage cells: lymphoma cell lines, NOL-3, Raji and Daudi, a myeloma cell line, NOP-1, normal peripheral blood lymphocytes (PBL), non-Hodgkin's lymphoma (NHL) cells and chronic lymphocytic leukemia (CLL) cells, and compared it with the effect on APL cell line, NB4, as well as other myeloid cell lines, HL-60 and NKM-1. As2O3 at a concentration of 1 micromol/l markedly inhibited both proliferation and viability of NB4, NOP-1, NOL-3 and NKM-1 cells, but it reduced only viability in normal PBL, CLL cells and NHL cells. As2O3 induced apoptosis and down-regulated bcl-2 expression in NB4, NOP-1 and NKM-1 cells. On the other hand, in HL-60, Raji and Daudi cells, 1 micromol/l As2O3 inhibited only the proliferation weakly, and neither induced apoptosis nor down-regulated bcl-2 expression, but arrested only cell cycle at G1 phase. As2O3 at a low concentration of 0.1 micromol/l had no effect on proliferation and viability of these cells except for NB4. These results showed that As2O3 exerted variable and definite effects on lymphoid cells and indicated that As2O3 might be clinically useful in lymphoid neoplasms such as malignant lymphoma and CLL.