Whole transcriptome sequencing and integrated network analysis elucidates the effects of 3,8-Di-O-methylellagic acid 2-O-glucoside derived from Sanguisorba offcinalis L., a novel differentiation inducer on erythroleukemia cells.

Acute erythroid leukemia (AEL) is a rare and aggressive hematologic malignancy with no specific treatment. Sanguisorba officinalis L. (S. officinalis), a well-known traditional Chinese medicine, possesses potent anticancer activity. However, the active components of S. officinalis against AEL and the associated molecular mechanisms remain unknown. In this study, we predicted the anti-AML effect of S. officinalis based on network pharmacology. Through the identification of active components of S. officinalis, we found that 3,8-Di-O-methylellagic acid 2-O-glucoside (DMAG) not only significantly inhibited the proliferation of erythroleukemic cell line HEL, but also induced their differentiation to megakaryocytes. Furthermore, we demonstrated that DMAG could prolong the survival of AEL mice model. Whole-transcriptome sequencing was performed to elucidate the underlying molecular mechanisms associated with anti-AEL effect of DMAG. The results showed that the total of 68 miRNAs, 595 lncRNAs, 4030 mRNAs and 35 circRNAs were significantly differentially expressed during DMAG induced proliferation inhibition and differentiation of HEL cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the differentially expressed miRNAs, lncRNAs, mRNAs and circRNAs were mainly involved in metabolic, HIF-1, MAPK, Notch pathway and apoptosis. The co-expression networks showed that miR-23a-5p, miR-92a-1-5p, miR-146b and miR-760 regulatory networks were crucial for megakaryocyte differentiation induced by DMAG. In conclusion, our results suggest that DMAG, derived from S. officinalis might be a potent differentiation inducer of AEL cells and provide important information on the underlying mechanisms associated with its anti-AEL activity.

Paris Saponin VII Induces Apoptosis and Cell Cycle Arrest in Erythroleukemia Cells by a Mitochondrial Membrane Signaling Pathway.

BACKGROUND AND PURPOSE: Leukemia is considered a top-listed ailment, according to WHO, which contributes to the death of a major population of the world every year. Paris Saponin VII (PS), a saponin which was isolated from the roots of Trillium kamtschaticum, from our group, was reported to provide hemostatic, cytotoxic and antimicrobial activities. However, its molecular mechanism underlying the anti-proliferative effects remains unclear. Thus, this study hypothesized to assess that mechanism in PS treated HEL cells. METHODS: The MTT assay was used to analyze the PS inhibited cell viability in the HEL cells. We further found that PS could induce S phase cell cycle arrest through flow cytometry as well as the western blot analysis of intrinsic and extrinsic apoptotic molecules. RESULTS: The MTT assay showed the IC50 concentration of PS as 0.667µM. The study revealed that PS treatment inhibits cell proliferation dose-dependently. It further caused mitochondrial membrane potential changes by PS treatment. Mechanistic protein expression revealed a dose-dependent upsurge for Bid and Bim molecules, while Bcl2 and PARP expression levels were significantly (P<0.05) down-regulated in PS treated HEL cells resulting in caspase -3 release and increased the Bim levels upon 24h of incubation. CONCLUSION: These findings indicate that PS possesses an excellent anti-leukemic activity via the regulation of the mitochondrial pathway, leading to S phase cell cycle arrest and caspase-dependent apoptosis, suggesting it as a potential alternative chemotherapeutic agent for leukemia patients.

Effect of Sargassum fusiforme polysaccharide on apoptosis and its possible mechanism in human erythroleukemia cells.

This study aimed to investigate the effects of Sargassum fusiforme polysaccharide (SFPS I, II, and III) on the apoptosis and regulation of human erythroleukemia (HEL) cells. The effect of different doses of SFPS on HEL cell growth was detected using the Cell Counting Kit-8 method, and apoptosis was detected by Hoechst staining. Cell cycle distribution and apoptosis were detected using flow cytometry. Expression of the cell cycle gene, p53, antiapoptotic genes, Bcl-xL and Bcl-2, and pro-apoptotic genes, Bax, Bad, and Caspase-3, as well as the expression of the corresponding proteins, were detected using real-time quantitative polymerase chain reaction (qPCR) and Western blot. The results showed that SFPS II and III decreased HEL cell viability and induced HEL cell apoptosis. Different concentrations of SFPS (I, II, and III) were detected that induced much less toxic effect in normal human embryonic lung (MRC-5) cells, and SFPS I increased cell proliferation, indicating its favorable selectivity towards cancer cells. The mechanism by which SFPS induced apoptosis was also found to be related to the induction of cell cycle arrest in the G0/G1 phase and the increased expression of apoptosis-related genes and proteins. We concluded that SFPS induces HEL cell apoptosis, possibly via activation of the Caspase pathway, providing the theoretical basis for the development of SFPS-based anti-tumor drug products.

Selective ERK1/2 agonists isolated from Melia azedarach with potent anti-leukemic activity.

BACKGROUND: MAPK/ERK kinases transmit signals from many growth factors/kinase receptors during normal cell growth/differentiation, and their dysregulation is a hallmark of diverse types of cancers. A plethora of drugs were developed to block this kinase pathway for clinical application. With the exception of a recently identified agent, EQW, most of these inhibitors target upstream factors but not ERK1/2; no activator of ERK1/2 is currently available. METHOD: A library of compounds isolated from medicinal plants of China was screened for anti-cancer activities. Three limonoid compounds, termed A1541-43, originally isolated from the plant Melia azedarach, exhibiting strong anti-leukemic activity. The anti-neoplastic activity and the biological target of these compounds were explored using various methods, including western blotting, flow cytometry, molecular docking and animal model for leukemia. RESULTS: Compounds A1541-43, exhibiting potent anti-leukemic activity, was shown to induce ERK1/2 phosphorylation. In contrast, the natural product Cedrelone, which shares structural similarities with A1541-43, functions as a potent inhibitor of ERK1/2. We provided evidence that A1541-43 and Cedrelone specifically target ERK1/2, but not the upstream MAPK/ERK pathway. Computational docking analysis predicts that compounds A1541-43 bind a region in ERK1/2 that is distinct from that to which Cedrelone and EQW bind. Interestingly, both A1541-43, which act as ERK1/2 agonists, and Cedrelone, which inhibit these kinases, exerted strong anti-proliferative activity against multiple leukemic cell lines, and induced robust apoptosis as well as erythroid and megakaryocytic differentiation in erythroleukemic cell lines. These compounds also suppressed tumor progression in a mouse model of erythroleukemia. CONCLUSIONS: This study identifies for the first time activators of ERK1/2 with therapeutic potential for the treatment of cancers driven by dysregulation of the MAPK/ERK pathway and possibly for other disorders.

Effects of bufalin on up-regulating methylation of Wilm's tumor 1 gene in human erythroid leukemic cells.

OBJECTIVE: To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells. METHODS: The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively. RESULTS: The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC50) of 0.046 µmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner. CONCLUSIONS: Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G0/G1 phase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.

A screen for Fli-1 transcriptional modulators identifies PKC agonists that induce erythroid to megakaryocytic differentiation and suppress leukemogenesis.

The ETS-related transcription factor Fli-1 affects many developmental programs including erythroid and megakaryocytic differentiation, and is frequently de-regulated in cancer. Fli-1 was initially isolated following retrovirus insertional mutagenesis screens for leukemic initiator genes, and accordingly, inhibition of this transcription factor can suppress leukemia through induction of erythroid differentiation. To search for modulators of Fli-1, we hereby performed repurposing drug screens with compounds isolated from Chinese medicinal plants. We identified agents that can transcriptionally activate or inhibit a Fli-1 reporter. Remarkably, agents that increased Fli-1 transcriptional activity conferred a strong anti-cancer activity upon Fli-1-expressing leukemic cells in culture. As opposed to drugs that suppress Fli1 activity and lead to erythroid differentiation, growth suppression by these new Fli-1 transactivating compounds involved erythroid to megakaryocytic conversion (EMC). The identified compounds are structurally related to diterpene family of small molecules, which are known agonists of protein kinase C (PKC). In accordance, these PKC agonists (PKCAs) induced PKC phosphorylation leading to activation of the mitogen-activated protein kinase (MAPK) pathway, increased cell attachment and EMC, whereas pharmacological inhibition of PKC or MAPK diminished the effect of our PKCAs. Moreover, in a mouse model of leukemia initiated by Fli-1 activation, the PKCA compounds exhibited strong anti-cancer activity, which was accompanied by increased presence of CD41/CD61 positive megakaryocytic cells in leukemic spleens. Thus, PKC agonists offer a novel approach to combat Fli-1-induced leukemia, and possibly other cancers,by inducing EMC in part through over-activation of the PKC-MAPK-Fli-1 pathway.

[Reversion Mechanism Study of PESV to Multidrug Resistance at Leukemia Stem Cell Level].

OBJECTIVE: To explore the effect of peptide extract from scorpion venom (PESV) to multidrug resistance (MDR) of leukemic stem cell (LSC) in vivo. METHODS: K562/A02 cells were cultured and collected in the logarithmic phase. K562/A02 stem cells were screened using immunomagnetic beads for reserve. K562/A02 LSC was injected to 5 of 40 BABL/c nude mice for preparing subcutaneous tumor. The rest 35 nude mice were then randomly divided into 7 groups, i.e., the normal control group, the model group, the Adriamycin (ADM) group, the PESV group, the ADM +high dose PESV group, the ADM + middle dose PESV group, the ADM +low dose PESV group, 5 in each group. Tumor tissue was embedded in all groups except the normal control group. One milliliter normal saline was peritoneally injected to mice in the model group after modeling, once per day. ADM 0. 05 mg was peritoneally injected to mice in the ADM group, once per other day. PESV 2 µg was peritoneally injected to mice in the PESV group, once per day. Mice in 3 ADM + PESV groups were peritoneally injected with ADM 0. 05 mg (once per other day) plus PESV (5, 2, and 1 µg respectively, once per day). All medication lasted for 14 days. P-glycoprotein (P-gp) was detected using flow cytometry. Breast cancer resistance protein (BCRP) and mRNA expression of multidrug resistance 1 (MDR1) were measured using RT-PCR. Aldehyde dehydrogenase 1 (ALDH1) was detected using immunohistochemistry. Phosphoinositide 3-kinase (PI3K) was detected using Western blot. NF-κB content was detected using ELISA. RESULTS: CD34 + CD38-ratio was 31.5% and IC50 was (60.33 ± 10. 68) µg/mL before K562/A02 cells were screened with immunomagnetic beads, while they were 92. 8% and (58. 33 ±9. 72) µg/mL after screen. The tumor formation rate was 100% in modeling mice. Compared with the model group, no statistical difference of each index occurred in the ADM group (P <0. 05). There was statistical difference in BCRP, MDR1 mRNA, or NF-κB factor between the model group and the PESV group (P <0. 05). The expression level of P-gp obviously decreased and the protein expression of P13K was down-regulated in 3 ADM + PESV groups (P <0. 05); mRNA expression of BCRP decreased and mRNA ex- pression of MDR1 obviously increased in the ADM + high dose PESV group and the ADM + middle dose PESV group, with statistical difference (P <0. 05). Protein expression of P13K was down-regulated in the ADM+ high dose PESV group, with statistical difference (P <0. 05). P-gp value, BCRP mRNA expression, MDR1 mRNA expression, PI3K, and NF-κB factor were all obviously down-regulated in the ADM +high dose PESV group, as compared with the ADM group and the PESV group respectively (P <0. 05). There was no statistical difference in ALDH1 positive rate among all groups (P >0. 05). Conclusion PESV combined ADM could down-regulate expression levels of P-gp, BCRP, MDR1, P13K, and NF-κB, strengthen the sensitivity of K562/A02 LSC to ADM in vivo, and reverse MDR of LSC.

[Regulatory effect of ginsenoside Rh2 on HDAC1/2 activity and cyclin in human erythroleukemia K562 cells].

OBJECTIVE: To investigate the effects of the 20(S)-ginsenoside Rh2 [Rh2(S)]on cell proliferation, histone deacetylase 1 (HDAC1) and HDAC2 activity, and expression of cyclin in human erythroleukemia K562 cells. METHODS: The K562 cells were treated with Rh2(S) at various concentrations (10-80 µmol/L). Cell proliferation activity was detected by CCK-8 assay. Flow cytometry (FCM) was used to detect cell cycle and apoptotic changes. The HDAC activity of cells was measured by chemical colorimetry. The protein expressions of HDAC1, HDAC2, cyclin D1, CDK4, p16INK4A and p21 after 48 hour-treatment of Rh2 (S) (10, 20, 40, 60 µmol/L) were examined by Western blotting. RESULTS: The proliferation of K562 cells was inhibited by Rh2 (S) (20-80 µmol/L) in dose-and time-dependent manner. FCM analyses revealed that the number of the K562 cells treated with 60 µmol/L Rh2(S) was arrested in G0/G1 phase. The apoptosis rates of K562 cells were respectively (8.09±0.86)%, (9.44±0.53)% and (22.80±2.16)% after induced by 20, 40, 60 µmol/L Rh2(S), which showed statistically significant difference (P<0.05) compared with the control group (2.63±0.14)%. HDAC activity of the cells treated with Rh2(S) (40, 60 µmol/L) was reduced. Western blotting showed that the expressions of HDAC1, HDAC2, cyclin D1 and CDK4 decreased after induced by Rh2(S), and p16INK4A, p21 proteins were enhanced significantly. CONCLUSION: The Rh2(S) can inhibit the proliferation of K562 cells and induce its cycle arrest and apoptosis through inhibiting HDAC1 and HDAC2 activity, down-regulating the expression of cyclin D1 and activating p16INK4A and p21.

In vitro antileukaemic activity of extracts from Daphne gnidium leaves against sensitive and multidrug resistant K562/R7 cells.

The antiproliferative potential of extracts of Daphne gnidium L. (Thymelaeaceae) on K562 cells was assessed, and the capacity of these extracts to disturb the cell cycle of K562 cells and to inhibit human P-glycoprotein was evaluated. The antiproliferative activity was evaluated using the MTT assay. The cell cycle analysis and the inhibition of P-glycoprotein were tested by flow cytometry. All the tested extracts exhibited significant anti-proliferative effects. Ethyl acetate extract has the strongest cytotoxic effect with an IC50 of 18.5 µg/ml. Furthermore, cell cycle analysis revealed that cells treated with chloroform, butanol and aqueous extracts were arrested predominantly in G2-M phase. Butanol extract was the most active extract. Percentage of cells arrested in G2-M was 34 %, 36.67 % and 42.63 % respectively, after treatment with 25, 75 and 100 µg/ml of the extract, versus 19 % in the cells treated with the vehicle solvent. In addition, chloroform extract had the ability to inhibit human P-glycoprotein-mediated daunorubicin in K562/R7 leukaemic cells in a dose-dependent manner compared to the positive control, cyclosporin A. These findings demonstrate that extracts from D. gnidium leaves have antileukaemic activity by perturbing the cell cycle of K562 and inhibiting human P-glycoprotein in K562/R7 cell line.

Effects of Danshen Injection () on inhibiting proliferation and inducing apoptosis through down-regulation of mutant JAK2 gene and its protein phosphorylation in human erythroid leukemic cells.

OBJECTIVE: To explore the effects of Danshen Injection () on inhibition proliferation, inducing apoptosis and its possible mechanisms on human erythroid leukemic (HEL) cells. METHODS: The commercial Chinese patent medicine of Danshen Injection was extracted and isolated from Chinese herb of Salvia miltiorrhiza bung. The inhibition effects of proliferation were assayed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method in HEL cells treated by Danshen Injection at various concentrations for 48 h. The cellular apoptosis was observed in morphology, analyzed by flow cytometry with annexin V and propidium iodide (PI) staining, and examined by DNA degradation ladder on agarose gel electrophoresis. Meanwhile, the expression levels of mutant Janus kinasez (JAK2) gene and phosphorylation-JAK2 (P-JAK2) protein were detected by allele specific-polymerase chain reaction and Western blot. RESULTS: The proliferation of HEL cells was effectively inhibited by Danshen Injection in a dose-dependent manner, with suppression rates from 19.46±2.31% to 50.20±5.21%. Typical apoptosis cells was observed in Danshen Injection treated HEL cells, the rates of annexin V positive cells increased obviously in a dose-dependent manner, as well as the DNA degradation ladder of apoptosis revealed on gel electrophoresis. The expression levels of mutant JAK2 gene and P-JAK2 protein reduced gradually with increasing dosage of Danshen injection. CONCLUSION: Danshen Injection could not only significantly inhibit the proliferation, but also induce apoptosis in HEL cells; down-regulation of the mutant JAK2 gene and P-JAK2 protein expressions are probably one of its molecular mechanisms.

Enhanced adhesion/migration and induction of Pyk2 expression in K562 cells following imatinib exposure.

Concern about extramedullary relapse (EMR) despite favorable response in the bone marrow has been raised with the use of imatinib for treatment of chronic myeloid leukemia (CML). In the present study we show an increase in adhesion, migration and invasion capabilities of the CML cell line K562 following imatinib administration. Imatinib induced upregulation of Pyk2 mRNA and protein levels. Pyk2 inhibition resulted in a reduction of K562 cells' adhesion and migration subsequent to imatinib treatment. This effect was similar to that shown by us previously with all trans retinoic acid (ATRA) in the acute promyelocytic leukemia (APL) cell line NB4. Our data pinpoint Pyk2 as a shared key mediator of targeted-therapy induced adhesion and migration and may implicate that targeting Pyk2 may serve as an effective therapeutic strategy to reduce EMR in APL and CML.

Antioxidants may protect cancer cells from apoptosis signals and enhance cell viability.

Quercetin is one of the most abundant dietary flavonoids widely present in many fruits and vegetables. Previous in vitro studies has shown that quercetin acts as an antioxidant and anti-inflammatory agent and it has potent anticarcinogenic properties as an apoptosis inducer. In this study we examined apoptotic effects of quercetin on the K562 erythroleukemia cell line. K562 cells were induced to undergo apoptosis by hydrogen peroxide. Cell viability and apoptosis level were assessed by annexin V and PI staining methods using flow cytometry. Viability of K562 cells was increased by low dose of quercetin (5-100 µM) for 3 hours. High doses of quercetin proved toxic (100-500 µM, 24 hours) and resulted in decrease of K562 cell viability as expected (P<0.01). As to results, 100 µM quercetin was defined as a protective dose. Also, K562 cell apoptosis due to hydrogen peroxide was decreased in a dose dependent manner. As indicated in previous studies, reduction of superoxides by free radical scavengers like quercetin could be beneficial for prevention of cancer but consumption of such flavonoids during cancer treatment may weaken effects of chemotherapeutics and radiotherapy. Especially cancer patients should be carefully considered for traditional phytotherapy during cancer treatment, which can lead to controversial results.

Anti-angiogenic quassinoid-rich fraction from Eurycoma longifolia modulates endothelial cell function.

Targeting angiogenesis could be an excellent strategy to combat angiogenesis-dependent pathophysiological conditions such as cancer, rheumatoid arthritis, obesity, systemic lupus erythematosus, psoriasis, proliferative retinopathy and atherosclerosis. Recently a number of clinical investigations are being undertaken to assess the potential therapeutic application of various anti-angiogenic agents. Many of these angiogenesis inhibitors are directed against the functions of endothelial cells, which are considered as the building blocks of blood vessels. Similarly, roots of a traditional medicinal plant, Eurycoma longifolia, can be used as an alternative treatment to prevent and treat the angiogenesis-related diseases. In the present study, antiangiogenic potential of partially purified quassinoid-rich fraction (TAF273) of E. longifolia root extract was evaluated using ex vivo and in vivo angiogenesis models and the anti-angiogenic efficacy of TAF273 was investigated in human umbilical vein endothelial cells (HUVEC). TAF273 caused significant suppression in sprouting of microvessels in rat aorta with IC50 11.5µg/ml. TAF273 (50µg/ml) showed remarkable inhibition (63.13%) of neovascularization in chorioallantoic membrane of chick embryo. Tumor histology also revealed marked reduction in extent of vascularization. In vitro, TAF273 significantly inhibited the major angiogenesis steps such as proliferation, migration and differentiation of HUVECs. Phytochemical analysis revealed high content of quassinoids in TAF273. Specially, HPLC characterization showed that TAF273 is enriched with eurycomanone, 13α(21)-epoxyeurycomanone and eurycomanol. These results demonstrated that the antiangiogenic activity of TAF273 may be due to its inhibitory effect on endothelial cell proliferation, differentiation and migration which could be attributed to the high content of quassinoids in E. longifolia.

JAK2-V617F-mediated signalling is dependent on lipid rafts and statins inhibit JAK2-V617F-dependent cell growth.

Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs.

The effect of silymarin on telomerase activity in the human leukemia cell line K562.

Telomerase has been proposed as a novel and potentially selective target in cancer therapy. Silymarin, which is a standardized mixture of flavonolignans from the medical plant Silybum marianum, has potent effects against various types of cancer cells, but its effect on telomerase activity in the human leukemia cell line K562 has not been investigated. The aim of this study was to examine the mechanism of silymarin-induced apoptosis in K562 cells, with particular emphasis on its effect on telomerase activity. The antiproliferation effect of silymarin on K562 cells was evaluated by the MTT assay. To measure apoptosis, Hoechst 33342 staining and flow cytometry were used. The telomerase activity was determined using the telomeric repeat amplification protocol (TRAP)-ELISA assay. The treatment of the K562 cells with silymarin resulted in a significant inhibition of cell growth and telomerase activity. Also, a positive correlation was found between telomerase inhibition and induction of apoptosis in silymarin-treated K562 cells. These results suggest a novel mechanism in the anticancer activity of silymarin in human leukemia K562 cells and may provide a basis for future development of anti-telomerase therapies.

The influence of the total flavonoids of Hedysarum polybotry on the proliferation, cell cycle, and expressions of p21Ras and proliferating cell nuclear antigen gene in erythroleukemia cell line K562.

OBJECTIVE: To investigate the effect of total flavonoids of Hedysarum polybotry on the proliferation, cell cycle, and expressions of p21(Ras) and proliferating cell nuclear antigen (PCNA) gene in erythroleukemia cell line K562. METHODS: The effect of total flavonoids of Hedysarum polybotry on K562 cell line survival was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay. The time- and dose-dependent manner was also observed. The cell cycle and apoptosis were analyzed with flow cytometry (FCM). The immunocytochemistry method was applied to quantitatively analyze the effects of flavonoids of Hedysarum polybotry on changes p21(Ras) and PCNA gene expressions. RESULTS: Flavonoids of Hedysarum polybotry (20-100 µg/mL) significantly inhibited the proliferation of K562 cells in a time- and dose-dependent manner. After K562 cells were cultured for 48 h, total flavonoids of Hedysarum polybotry had no significant effect on the apoptosis of K562 cells but showed significantly inhibition (P<0.01), indicating that total flavonoids of Hedysarum polybotry could induce K562 cells arrested at G(0)/G(1) and G(2)/M phases. Compared with the control group, p21(Ras) and PCNA gene expressions were decreased significantly in K562 cells treated with total flavonoids of Hedysarum polybotry (40 and 80 µg/mL, respectively) for 48 h. CONCLUSION: The inhibitory effect on proliferation of K562 cells was observed in the groups treated with flavonoids of Hedysarum polybotry, which might be related to cells arresting.

Δ12-prostaglandin J3, an omega-3 fatty acid-derived metabolite, selectively ablates leukemia stem cells in mice.

Targeting cancer stem cells is of paramount importance in successfully preventing cancer relapse. Recently, in silico screening of public gene-expression datasets identified cyclooxygenase-derived cyclopentenone prostaglandins (CyPGs) as likely agents to target malignant stem cells. We show here that Δ(12)-PGJ(3), a novel and naturally produced CyPG from the dietary fish-oil ω-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA; 20:5) alleviates the development of leukemia in 2 well-studied murine models of leukemia. IP administration of Δ(12)-PGJ(3) to mice infected with Friend erythroleukemia virus or those expressing the chronic myelogenous leukemia oncoprotein BCR-ABL in the hematopoietic stem cell pool completely restored normal hematologic parameters, splenic histology, and enhanced survival. More importantly, Δ(12)-PGJ(3) selectively targeted leukemia stem cells (LSCs) for apoptosis in the spleen and BM. This treatment completely eradicated LSCs in vivo, as demonstrated by the inability of donor cells from treated mice to cause leukemia in secondary transplantations. Given the potency of ω-3 polyunsaturated fatty acid-derived CyPGs and the well-known refractoriness of LSCs to currently used clinical agents, Δ(12)-PGJ(3) may represent a new chemotherapeutic for leukemia that targets LSCs.

Ethyl acetate extract and its major constituent, isorhamnetin 3-O-rutinoside, from Nitraria retusa leaves, promote apoptosis of human myelogenous erythroleukaemia cells.

OBJECTIVE: Fractionation of ethyl acetate extract (EA) obtained from Nitraria retusa leaves was assessed using different methods of chromatography, and isorhamnetin3-O-rutinoside (I3-O-R) was isolated from this extract. Its structure was determined using data obtained from (1) H and (13) C NMR spectra, as well as by various correlation experiments (COSY, HMQC and HMBC). Both EA extract and I3-O-R were investigated for their ability to induce apoptosis in human chronic myelogenous erythroleukaemia cells (K562). MATERIALS AND METHODS: Apoptosis of cells from the K562 line was detected by DNA fragmentation, PARP cleavage and by evaluating activities of caspases 3 and 8. RESULTS: Apoptosis, revealed by DNA fragmentation and PARP cleavage, was observed after 48-h incubation of these human myelogenous erythroleukaemia cells (K562), with the tested products. Likewise, caspase 3 and caspase 8 activities were induced in the presence of the EA extract and I3-O-R after 48 h of incubation. CONCLUSION: Our results strongly suggest the involvement of the extrinsic pathway of apoptosis in cells treated by both the original EA extract and its major component, I3-O-R.

Cytotoxic effect and apoptotic mechanism of tanshinone A, a novel tanshinone derivative, on human erythroleukemic K562 cells.

Tanshinone A is a novel derivative of phenanthrene-quinone extracted from Salvia miltiorrhiza BUNGE, a traditional herbal medicine. Cytotoxic effect of tanshinone A was observed in this study. Additionally its mechanism of promoting apoptosis was also investigated. MTT and SRB assays were applied to measure the effects of tanshinone A on the cell viability, the cell cycle distribution and cell apoptosis were measured by flow cytometry using PI staining and Annexin V/PI double staining method respectively. The changes of mitochondrial membrane potential were also detected by flow cytometry. Spectrophotometric method was used to detect the changes of caspase-3 activity. Western blotting assay was used to evaluate the expression of bcl-2, bax and c-Myc proteins. Results indicated that tanshinone A displayed a significant inhibitory effect on the growth of K562 cells in a dose- and time-dependent manner, and showed obvious minor damage to LO2 cells. Tanshinone A could arrest K562 cells in the G(0)/G(1) phase and induce apoptosis, decrease the mitochondrial transmembrane potential, decrease the expressions of bcl-2 and c-Myc proteins, increase the expression of bax protein and the activity of caspase-3. Accordingly, it was presumed that the apoptosis induction may be through the endogenous pathway. Subsequently, tanshinone A could be a promising candidate in the development of a novel antitumor agent.

Mortalin inhibitors sensitize K562 leukemia cells to complement-dependent cytotoxicity.

Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.