Synthesis of new pyrrolo[1,2-a]quinoxaline derivatives as potential inhibitors of Akt kinase

Akt kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel Akt kinase inhibitors, based on a quinoxaline or pyrazinone scaffold. A series of new substituted pyrrolo[1,2-a]quinoxaline derivatives, structural analogues of these active quinoxaline or pyrazinone pharmacophores, was synthesized from various substituted 2-nitroanilines or 1,2-phenylenediamine via multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC(50) of 4.5 microM, and 1h that inhibited U937 and MCF7 cell lines with IC(50) of 5 and 8 microM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730.     

Sensitization of Chemo-Resistant Human Chronic Myeloid Leukemia Stem-Like Cells to Hsp90 Inhibitor by SIRT1 Inhibition

Development of effective therapeutic strategies to eliminate cancer stem-like cells (CSCs), which play a major role in drug resistance and disease recurrence, is critical to improve cancer treatment outcomes. The current investigation was undertaken to examine the effectiveness of the combination treatment of Hsp90 inhibitor and SIRT1 inhibitor in inhibiting the growth of chemo-resistant stem-like cells isolated from human chronic myeloid leukemia K562 cells. Inhibition of SIRT1 by use of SIRT1 siRNA or SIRT1 inhibitors (amurensin G and EX527) effectively potentiated sensitivity of Hsp90 inhibitors (17-AAG and AUY922) in CD44^high K562 stem-like cells expressing high levels of CSC-related molecules including Oct4, CD34, β-catenin, c-Myc, mutant p53 (mut p53), BCRP and P-glycoprotein (P-gp) as well as CD44. SIRT1 depletion caused significant down-regulation of heat shock factor 1 (HSF1)/heat shock proteins (Hsps) as well as these CSC-related molecules, which led to the sensitization of CD44^high K562 cells to Hsp90 inhibitor by SIRT1 inhibitor. Moreover, 17-AAG-mediated activation of HSF1/Hsps and P-gp-mediated efflux, major causes of Hsp90 inhibitor resistance, was suppressed by SIRT1 inhibitor in K562-CD44^high cells. Our data suggest that combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be an effective therapeutic approach to target CSCs that are resistant to current therapies.     

Synthesis of New Pyrrolo[1,2-a]quinoxaline Derivatives as Potential Inhibitors of Akt Kinase

Akt kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel Akt kinase inhibitors, based on a quinoxaline or pyrazinone scaffold. A series of new substituted pyrrolo[1,2-a]quinoxaline derivatives, structural analogues of these active quinoxaline or pyrazinone pharmacophores, was synthesized from various substituted 2-nitroanilines or 1,2-phenylenediamine via multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC₅₀ of 4.5 μM, and 1h that inhibited U937 and MCF7 cell lines with IC₅₀ of 5 and 8 μM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730.     

Influence of culture medium pH on cytotoxicity of CD95L in vitro

CD95L belongs to the tumor necrosis factor-alpha (TNF-alpha) family, the members of which induce apoptosis by activation of their specific receptors. However, there are a few publications suggesting that two of these factors, TNF-alpha and TNF-beta, are able to reveal cytotoxic effect in pH-dependent manner. Therefore we investigated, whether CD95L may also reveal pH-dependent cytotoxicity. We analyzed influence of CD95L on U937 and K562 human cell lines at pH 5.1 and pH 7.4 using radioactive chromium release and tetrazolium salt (MTT) reduction assays. Expression of CD95 in both cell lines was estimated using RNase Protection Assay and FACS analysis. It has been found that short incubation of cells at pH 5.1 did not visibly affect their viability, as measured after 16 or 20 h. Incubation of U937 with CD95L at pH 7.4 resulted in a dose-dependent cell cytotoxicity. The effect was significantly augmented by incubation of cells with CD95L at pH 5.1. K562 cell line was resistant to CD95L at pH 7.4. This result correlated with the lack of CD95 expression in K562 cells. However, incubation at pH 5.1 resulted in a sensitization of K562 cells to CD95L. Our results suggest that CD95L, similarly to TNF-alpha, is able to reveal its cytotoxic activity in a receptor-independent manner and this activity strongly depends on pH of the environment.     

Calcium-independent phospholipase A2 mediates proliferation of human promonocytic U937 cells

We have investigated the possible involvement of two intracellular phospholipases A(2), namely group VIA calcium-independent phospholipase A(2) (iPLA(2)-VIA) and group IVA cytosolic phospholipase A(2) (cPLA(2)alpha), in the regulation of human promonocytic U937 cell proliferation. Inhibition of iPLA(2)-VIA activity by either pharmacological inhibitors such as bromoenol lactone or methyl arachidonyl fluorophosphonate or using specific antisense technology strongly blunted U937 cell proliferation. In contrast, inhibition of cPLA(2)alpha had no significant effect on U937 proliferation. Evaluation of iPLA(2)-VIA activity in cell cycle-synchronized cells revealed highest activity at G(2)/M and late S phases, and lowest at G(1). Phosphatidylcholine levels showed the opposite trend, peaking at G(1) and lowest at G(2)/M and late S phase. Reduction of U937 cell proliferation by inhibition of iPLA(2)-VIA activity was associated with arrest in G(2)/M and S phases. The iPLA(2)-VIA effects were found to be independent of the generation of free arachidonic acid or one of its oxygenated metabolites, and may work through regulation of the cellular level of phosphatidylcholine, a structural lipid that is required for cell growth/membrane expansion.     

Inducible p27(Kip1) expression inhibits proliferation of K562 cells and protects against apoptosis induction by proteasome inhibitors

Overexpression of the cyclin-dependent kinase inhibitor p27(Kip1) has been demonstrated to induce cell cycle arrest and apoptosis in various cancer cell lines, but has also been associated with the opposite effect of enhanced survival of tumor cells and increased resistance towards chemotherapeutic treatment. To address the question of how p27(Kip1) expression is related to apoptosis induction, we studied doxycycline-regulated p27(Kip1) expression in K562 erythroleukemia cells. p27(Kip1) expression effectively retards proliferation, but it is not sufficient to induce apoptosis in K562 cells. p27(Kip1)-expressing K562 cells, however, become resistant to apoptosis induction by the proteasome inhibitors PSI, MG132 and epoxomicin, in contrast to wild-type K562 cells that are efficiently killed. Cell cycle arrest in the S phase by aphidicolin, which is not associated with an accumulation of p27(Kip1) protein, did not protect K562 cells against the cytotoxic effect of the proteasome inhibitor PSI. The expression levels of p27(Kip1) thus constitute an important parameter, which decides on the overall sensitivity of cells against the cytotoxic effect of proteasome inhibitors.     

CD147对白血病细胞U937生长和肿瘤形成的影响

目的：研究CD147对白血病细胞U937生长和肿瘤形成的影响。方法：分别采用脂多糖（LPS）或CD147单克隆抗体处理U937细胞;用RT-PCR和流式细胞术分别在mRNA和蛋白水平检测各组中CD147的表达情况;用流式细胞术检测在LtX3和CD147单克隆抗体作用下U937细胞周期的变化;用MTT法对各组细胞的生长状况进行分析;将细胞经皮下接种于裸鼠体内,对各组间肿瘤生长速度、肿瘤体积及裸鼠存活时间进行统计分析。结果：LPS在体外能够诱导自血病细胞U937表面CD147的表达,同时细胞增殖旺盛,但细胞凋亡数增加;使用CD147抗体阻断CD147后,能够将细胞周期阻断在G0／G1期,细胞活力下降,并诱导细胞凋亡;CD147抗体体外预处理能够抑制U937细胞在裸鼠体内的生长,使小鼠存活时间延长。结论：LPS可诱导U937细胞表面CD147分子表达增加,从而促进U937细胞的生长和肿瘤形成。     

羟基脲（Hydroxyurea）对细胞周期与珠蛋白基因表达的影响（简报）

Although the hydroxyurea (HU) has been extensively studied, little is known of its molecular mechanism in controlling the expression of human globin gene and in modulating the progression of cell-cycle in K 562 cell. In the present study, the effect of hydroxyurea on proliferative kinetics of K 562 cells was examined by monitoring the number of cells during a period of 8 day's cell culture. Our results showed that there was a dose related decrease in cell growth when K562 cells were incubated with HU. Moreover, cell-cycle analysis demonstrated that HU had profound effect on cell-cycle distribution. In the case of the induced K 562 cells, there was an increased accumulation of cells in S phase and a decreased fraction of cells in G 1 and G 2 + M phase. Furthermore, HU could induce the expression of human beta-globin gene in the induced K 562 cells. Our results indicate that HU has a potential to inhibit the proliferation of K 562 cells and to stimulate the terminal differentiation of this cell.     

STAT5 in regulation of chronic leukemia K562 cell proliferation: Inhibitory effect of WHI-P131

In this study, we examined the role of JAK/STAT signaling in the regulation of chronic leukemia K562 cell proliferation. STAT3 and STAT5 tyrosine phosphorylation was used as a marker of the activation status of STAT proteins. We demonstrated that, in growing cultures of K562 cells, both STAT3 and STAT5 are constitutively activated. To determine the significance of STAT activity in maintaining the high level of K562 proliferation, we tested two JAK inhibitors, AG-490 (JAK2 and JAK3 inhibitor) and WHI-P131 (a new specific JAK3 inhibitor). We showed that, during the prolonged cultivation (48 h) of K562 cells with AG-490 or WHI-P131, the cells remained viable. It was found that treatment with WHI-P131 (30–100 μM) decreased tyrosine phosphorylation of STAT5 and did not affect the high level of STAT3 phosphorylation. In proliferating K562 cells, AG-490 (25–50 μM) did not influence on STAT3 and STAT5 phosphorylation. The flow cytometry analysis revealed a dose-dependent decrease in G1 and S phases and an increase in G2/M phases in WHI-P131-treated K562 cells and no changes in cell cycle structure in AG-490-treated cultures. Thus, our findings indicate the preferential role of STAT5 (not constitutively active STAT3) in the proliferation of leukemia K562 cells and demonstrate the specificity of WHI-P131 inhibitory effect; unlike other JAK drugs that stimulate apoptosis and decrease proliferation, WHI-P131 prevents K562 cells growth by arresting in G2/M phases of the cell cycle.     

Apoptosis induction in human leukemic cells by a novel protein Bengalin,isolated from Indian black scorpion venom: Through mitochondrial pathway and inhibition of heat shock proteins

Scorpion venom possesses protein toxins having numerous biological activities, some of which are potentially anticancerous. Previously we had reported antiproliferative activity of the venom of Indian black scorpion, Heterometrus bengalensis Koch. Here we have isolated and purified a novel protein named Bengalin (72 kDa) from the venom, responsible for antiproliferative and apoptogenic activities against human leukemic cells U937 (histiocytic lymphoma) and K562 (chronic myelogenous leukemia). N-terminal sequence of first 20 amino acids of Bengalin was G-P-L-T-I-L-H-I-N-D-V-H-A-A/R-F-E-Q/G-F/G-N-T. Bengalin induced cell growth inhibition at IC₅₀ values of 3.7 and 4.1 μg/ml for U937 and K562 cells respectively did not significantly affect normal human lymphocytes. Inhibition of U937 and K562 cell proliferation occurred by apoptosis as evidenced from damaged nuclei, cell cycle arrest at sub G1 phase, increase of early apoptotic cells, augmentation of DNA fragmentation and also a reduction of telomerase activity. Further insights revealed that Bax:Bcl2 ratio was elevated after Bengalin treatment. Moreover Bengalin elicited loss of mitochondrial membrane potential (MMP) which commenced cytochrome c release in cytosol, decreased heat shock protein (HSP) 70 and 90 expression, activated caspase-9, caspase-3 and induced poly(ADP-ribose) polymerase (PARP) cleavage. We have also determined that HSP70 and 90 inhibitions correlated with Bengalin induced antiproliferation, caspase-3 upregulation, apoptogenesis and increased DNA fragmentation. These results hypothesize that Bengalin might provide a putative molecular mechanism for their anticancer effect on human leukemic cells which might be mediated by mitochondrial death cascade. Inhibition of HSPs might also play a crucial role in induction of apoptosis.     

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDA, in chronic myeloid leukemia (K562) cells

We studied the effect of 2-(6-(2-thieanisyl)-3(Z)-hexen-1,5-diynyl)aniline(THDA), a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in K562 cells. THDA was found to inhibit the growth of K562 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in K562 cells following 24 h exposure to THDA. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a time-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that THDA did not change the steady-state levels of cyclin B1, cyclin D3 and Cdc25C, but decreased the protein levels of Cdk1, Cdk2 and cyclin A. THDA also caused a marked increase in apoptosis, which was associated with activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose) polymerase. These molecular alterations provide an insight into THDA-caused growth inhibition, G2/M arrest and apoptotic death of K562 cells.     

Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.     

Reversal of multidrug resistance in cancer cells by Rhizoma Alismatis extract

Prolonged chemotherapy may lead to the selective proliferation of multidrug resistant (MDR) cancer cells. In MDR HepG2-DR and K562-DR cells that over-expressed P-glycoprotein (Pgp), the extract of the rhizomes of Alisma orientalis (Sam) Juzep. showed a synergistic growth inhibitory effect with cancer drugs that are Pgp substrates including actinomycin D, puromycin, paclitaxel, vinblastine and doxorubicin. At the same toxicity levels the herbal extract was more effective than verapamil, a standard Pgp inhibitor, in enhancing cellular doxorubicin accumulation and preventing the efflux of rhodamin-123 from the MDR cells. The extract restored the effect of vinblastine on the induction of G(2)/M arrest in MDR cells. Our data suggest that A. orientalis may contain components that are effective inhibitors of Pgp.     

Bcr-abl translocation can occur during the induction of multidrug resistance and confers apoptosis resistance on myeloid leukemic cell lines

Apoptosis was studied in parental and mdr-1 expressing U937, HL60 and K562 myeloid leukemic cell lines using mdr unrelated inducers of apoptosis such as Ara-C, cycloheximide, serum deprivation, ceramide, monensin and UV irradiation. Apoptosis was efficiently induced by all these treatments in U937 and HL60 cells while K562 cells exhibited an apoptosis-resistant phenotype except with UV and monensin. The pattern of apoptosis resistance in mdr-1 expressing U937 (U937-DR) and HL60 (HL60-DR100) was similar to that presented by K562. This apoptosis-resistant phenotype of mdr cells was not overcome by concentrations of verapamil inhibiting the P-gp 170 pump. The acquisition of this phenotype was posterior to the mdr-1 expressing phenotype since a HL60-DR5 variant, selected at the beginning of the induction of resistance, presented a low level of mdr-1 expression without resistance to apoptosis. The variations observed in the Fas (CD95) expression between sensitive and resistant cells were not sufficient to account for apoptosis resistance. However, a high expression in Abl antigen was found in all the apoptosis-resistant cells. RT-PCR and Western blot analysis showed that this increase in Abl antigen content was accompanied by the expression in U937-DR and HL60-DR100 cells of a hybrid bcr/abl mRNA and a 210 kD Bcr/Abl protein which was constitutive in K562. This expression was due to the translocation of abl and the amplification of the bcr-abl translocated gene. These results are in agreement with the role of Bcr/Abl tyrosine protein kinase as an inhibitor of apoptosis independently of the mdr-1 expression. They also suggest that translocation of the abl gene in the bcr region is a highly probable rearrangement in the mdr-1 expressing myeloid cells and that Bcr/Abl tyrosine kinase effect on apoptosis needs the regulation of intracellular pH and is inactive against UV-induced apoptosis.     

敲低去泛素化酶USP13抑制K562细胞的增殖*

目的:研究去泛素化酶USP13对人慢性髓系白血病细胞系K562增殖和凋亡的影响,并进行初步的机制探究。方法:构建pLKO.1-shUSP13-GFP慢病毒干涉载体,慢病毒包装后感染并建立稳定敲低USP13的K562细胞株。免疫印迹检测K562细胞中USP13蛋白的敲低效率。流式细胞术分析敲低USP13对K562细胞增殖和凋亡的影响。免疫共沉淀和蛋白质泛素化实验探究USP13调控K562细胞的分子机制。结果:成功构建pLKO.1-shUSP13-GFP慢病毒干涉载体,同时利用慢病毒体系获得稳定敲低USP13的K562细胞株。流式细胞术结果显示,敲低USP13促进K562细胞凋亡、抑制细胞增殖。分子机制研究发现,敲低USP13通过增强c-Myc泛素化进而导致其蛋白质水平降低。结论:初步揭示了USP13调控K562细胞增殖和凋亡的分子机制,为治疗慢性髓系白血病提供了潜在的靶点。     

HFCL细胞对U937细胞的诱导分化作用

为了观察正常人骨髓成纤维样细胞系HFCL对急性单核细胞白血病U937细胞促分化作用,及其对经典诱导分化剂TPA诱导分化作用的影响,先建立U937细胞和HFCL细胞共培养体系,以细胞形态学改变、硝基四氦唑蓝(NBT)、流式细胞仪检测细胞周期和CD11b、CD13、CD14、CD33细胞表面抗原作为诱导分化指标;Western印迹检测P38蛋白的表达变化。结果发现,与HFCL细胞共培养后,U937细胞出现分化成熟的形态学改变,且与HFCL细胞直接接触组的诱导分化作用大于用transwell组。同时发现U937细胞与HFCL细胞共培养后,G1期细胞增高,S期细胞减少;CD11b、CD13、CD14和CD33表达增高;且NBT阳性细胞增高至46、3％。Western印迹检测结果显示,直接接触组总P38蛋白表达增加。而且HFCL细胞还能增强TPA对U937的诱导分化作用。