U937 Apoptosis Research Articles

Formononetin and Dihydroartemisinin Act Synergistically to Induce Apoptosis in Human Acute Myeloid Leukemia Cell Lines.

Enhanced cell survival and drug resistance in tumor cells have been linked to the overexpression of antiapoptotic members of the Bcl-2 family proteins, including Bcl-2 and Mcl-1. The aim of this study was to explore the impact of formononetin and dihydroartemisinin combination on the growth and apoptosis of acute myeloid leukemia (AML) cells. In this experimental study, the cell survival and cell proliferation were tested by MTT assay and trypan blue staining. The evaluation of cell apoptosis was conducted using Hoechst 33342 staining and a colorimetric assay to measure caspase-3 activity. To determine the mRNA levels of Mcl-1, Bcl-2, Bax, and Cyclin D1, a quantitative real-time polymerase chain reaction (qRT-PCR) was performed. We showed that treatment with either formononetin or dihydroartemisinin alone, led to significant decrease in the cell survival and growth, and triggered apoptosis in U937 and KG-1 AML cell lines. Moreover, treatment with each of the compounds alone significantly decreased the mRNA levels of Mcl-1, Bcl-2 and Cyclin D1 mRNA, while, the expression level of Bax mRNA was enhanced. Combination of two compounds showed a synergistic anti-cancer effect. The anti-leukemic potential of formononetin and dihydroartemisinin is exerted through the effect on cell cycle progression and intrinsic pathway of apoptosis. Therefore, they can be considered as a potential anti-leukemic agent alone or along with existing chemotherapeutic drugs.

The Effects of ABT-199 and Dihydroartemisinin Combination on Cell Growth and Apoptosis in Human U937 and KG-1 Cancer Cells.

Change in the balance of Bcl-2 family proteins is one of the main reasons for resistance of tumor cells to ABT-199. In this study, the effect of dihydroartemisinin on cell growth, apoptosis and sensitivity of the AML cells to ABT-199 was investigated. Cell proliferation and survival were assessed by trypan blue staining and MTT assay, respectively. Cell apoptosis was measured by Hoechst 33342 staining and caspase-3 activity assay. The expression levels of Bcl-2, Mcl-1 and Bax mRNA were tested by qRT-PCR. Our data showed that combination therapy significantly reduced the IC50 value and synergistically decreased the AML cell survival and growth compared with dihydroartemisinin or ABT-199 alone. Treatment with each of ABT-199 or dihydroartemisinin alone clearly enhanced the Bax mRNA expression and inhibited the expression of Mcl-1 and Bcl-2 mRNA. Inhibition of Mcl-1 mRNA by dihydroartemisinin was associated with enhancement of apoptosis induced by ABT-199 in AML cells. In conclusion, dihydroartemisinin not only triggers the intrinsic pathway of apoptosis, but also can increase the sensitivity of the AML cells to ABT-199 via suppression of Mcl-1 expression.

Study on the Synthetic Lethal Mechanism of Selective Killing of ALDH2 Deficient Acute Myeloid Leukemia Cells By Inhibiting BRIP1

Background and objective: Although targets related to DNA double strand damage repair have clear tumor therapeutic potential, few have identified DNA double strand damage repair dependence in AML cells through genetic screening to date. In our previous experiments, we found that ALDH2 showed low expression in most newly diagnosed AML, and their unique ALDH2 vulnerability may make them preferentially dependent on BRIP1 by the DNA double-strand damage repair pathway, and have more synthetic lethal effects than other normal cells. Therefore, this study explored whether blocking BRIP1-mediated DNA double-strand damage repair could selectively eliminate AlDH2-deficient AML cells while preserving AlDH2-expressing normal cells present in most human tissues. Methods: We examined the dependence of ALDH2-expressing cells on BRIP1 and methylation levels of ALDH2 promoters in different AML cell lines. The expressions of BRIP1 and ALDH2 in 6 AML cell lines and clinical samples were detected by Real-time PCR and Western blot, and the reverse expression pattern between the two was studied, and the correlation between the expression of ALDH2 and DNA base excision repair was analyzed. By targeting the inhibition of ALDH2 and BRIP1, in vitro and in vivo models were constructed to observe the proliferation and apoptosis of different AML cell lines and primary cells. Results: In the group of AML cells with low expression of ALDH2, we found that BRIP1 showed a certain high expression compared with other groups. Among the 46 newly diagnosed AML samples, we divided the clinical samples into high and low groups based on the difference in the expression level and methylation degree of ALDH2. Compared with the group with high expression of ALDH2, BRIP1 in the group with low expression of ALDH2 was significantly increased (P < 0.01). The proliferation level of AML cells was significantly decreased (P < 0.01). BRIP1 inhibition induced apoptosis of U937 and TPH1 cells (AML cell lines with low expression of ALDH2) in a dose - and time-dependent manner (P < 0.05). In addition, in AML cell lines with high expression of ALDH2, we inhibited the expression of both ALDH2 and BRIP1, and the growth of AML cells was significantly down-regulated (P < 0.01). Similarly, we observed the same results when constructing an in vivo AML infiltration model in mice using AML primary cells. Conclusion: The dependence of ALDH2-deficient cells on BRIP1 and the effect of targeting BRIP1 on the proliferation of AML cells could provide new ideas for finding new molecular targets for the treatment of AML.

Azacitidine Combined with Novel Flavonoid Derivative GL-V9 Demonstrated Synergistic Anti-Leukemia Effect in Acute Myeloid Leukemia By Targeting DDIT4/mTOR Signaling

Background Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of malignant progenitor cells and impaired cell differentiation. GL-V9, a nature compound derived from wogonin, has shown superior anti-tumor potential by inhibiting cancer cell growth. Azacitidine (AZA), a DNA hypomethylation agent, has demonstrated therapeutic efficacy against AML. In this study, we investigated the anti-leukemia effect and potential mechanism of AZA combined with GL-V9 in AML cells, aiming to provide evidence for future clinical treatment. Methods We employed Cell Counting Kit-8 (CCK-8) to assess cell viability, Annexin-V/PI staining followed by flow cytometry analysis to measure apoptosis. CalcuSyn analysis was used to evaluate the synergistic effect. RNA-seq was performed in U937 cells and differentially expressed genes (DEGs) were identified and subjected to KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. RT-qPCR and Western Blot were used to examine gene expression. Results Our results demonstrated significant cell proliferation arrest in U937 and MV4-11 cells treated with the combination of AZA and GL-V9, compared to the single drug ( Fig. 1A&B). CalcuSyn analysis showed a strong synergistic effect for the combination ( Fig. 1A&B). Similar results were also found in primary AML cells derived from 2 AML patients [patient 1 exhibited t(6;11) (q27;q23) chromosomal translocation, while Patient 2 presented with secondary AML, transformed from CMML (Chronic Myelomonocytic Leukemia) and featured ASXL1 and SRSF2 mutations] ( Fig. 1C&D). Moreover, AZA plus GL-V9 induced a substantial increase in cell apoptosis in U937 and MV4-11 cells compared to the single drug ( Fig. 1E&F). Consistently, the AZA+GL-V9 combination led to a notable increase in the protein level of Bax, BAD, BIM, and a decrease in BCL2 ( Fig. 1G). These data indicated the combination of AZA with GL-V9 has synergistic anti-leukemia effects in AML. To understand the underlying mechanisms of this synergy, we performed RNA-seq analysis in U937 cells, identifying 1385 and 586 DEGs (|log 2FC|≥2.0, P<0.05) upon AZA or GL-V9 treatment, respectively ( Fig. 2A). The mTOR signaling pathway exhibits prominent enrichment in the KEGG analysis of the overlapping DEGs between AZA and GL-V9 ( Fig. 2B). DDIT4, a negative regulator of mTOR emerged as one of the top DEGs in response to both AZA and GL-V9 treatment. Importantly, the AZA+GL-V9 treatment exhibited a higher expression level of DDIT4 compared to either drug alone and suppressed the phosphorylation of mTOR ( Fig. 2C). Additionally, the DDIT4 expression level was significantly decreased in AML patients, which derived from a public database (TCGA-AML, GSE13159)] ( Fig. 2D) and Zhongda Hospital (Nanjing, China) compared to the healthy controls ( Fig. 2E). To be noticed, high DDIT4 expression was associated with extended overall survival (P=0.021) ( Fig. 2F) and relapse-free survival (P=0.015) ( Fig. 2G), indicating DDIT4 may act as a tumor suppressor in AML. These findings suggested that the combination may exert the anti-leukemia effect by targeting the DDIT4/mTOR signaling pathway, as summarized in Fig. 2H. Conclusions Our study provides novel evidence of the synergistic effect on cell growth arrest and apoptosis treated with a new nature compound derived from wogonin combined with AZA in AML. Furthermore, we identified the mechanism underlying the synergy through targeting of DDIT4/mTOR signaling. These results offer preliminary support for the potential application of this combination therapy in treating AML patients.

Cytarabine-induced destabilization of MCL1 mRNA and protein triggers apoptosis in leukemia cells

Although cytarabine (Ara-C) is the mainstay of treatment for acute myeloid leukemia (AML), its cytotoxic mechanisms for inducing apoptosis are poorly understood. Therefore, we investigated the Ara-C-induced cell death pathway in human AML U937 cells. Ara-C-induced downregulation of MCL1 is associated with the induction of mitochondrial depolarization and apoptosis. Ara-C triggered NOX4-mediated ROS production, which in turn activated p38 MAPK but inactivated AKT. Ara-C-induced DNA damage modulates p38 MAPK activation without affecting AKT inactivation in U937 cells. Inactivated AKT promotes GSK3β-dependent CREB phosphorylation, which in turn increases NOXA transcription, thereby triggering the degradation of MCL1 protein. Activated p38 MAPK induces HuR downregulation, leading to accelerated MCL1 mRNA turnover. A similar pathway also explains the Ara-C-induced THP-1 cell death. Collectively, our data confirm that Ara-C-triggered apoptosis in the AML cell lines U937 and THP-1 is mediated through the destabilization of MCL1 mRNA and protein. Furthermore, Ara-C acts synergistically with the BCL2 inhibitor ABT-199 to induce cell death in ABT-199-resistant and parental U937 cells by inhibiting MCL1 expression.

Bone marrow‐derived mesenchymal stem cells inhibit NK cell function via Tim‐3/galectin‐9 in multiple myeloma patients

Bone marrow‐derived mesenchymal stem cells inhibit NK cell function via Tim‐3/galectin‐9 in multiple myeloma patients

Effects of Thiomersal on Apoptosis and Autophagy of Leukemia Cell Lines

To investigate the effects of different concentrations of thiomersal on apoptosis and autophagy regulation of human leukemia cell lines U937, CEM-C1 and BALL-1. The inhibitory effect of thiomersal on the proliferation of U937, CEM-C1 and BALL-1 cells was detected by CCK-8 assay. Annexin V-FITC/PI double staining flow cytometry was used to detect the apoptosis rate. Western blot was used to detect the effects of thiomersal on autophagy signaling pathway and the expression of PI3K, Akt, mTOR, p-mTOR, caspase-3 and LC3-II proteins. Within 24 and 48 hours, thiomersal inhibited the proliferation of U937, CEM-C1 and BALL-1 cell lines in a time and dose-dependent manner (r24 h=0.295, r24 h=0.452, r24 h=0.103; r48 h=0.821, r48 h=0.665, r48 h=0.821), but no significant time and dose-dependent effect was observed at 72 hours. After 48 hours treatment of thiomersal, the apoptosis rate of U937, CEM-C1 and BALL-1 cells increased in a dose-dependent manner (r=0.819, r=0.763, r=0.835). After 48 hours treatment of thiomersal, the expression levels of PI3K, Akt, mTOR and p-mTOR protein in U937, CEM-C1 and BALL-1 cells decreased in a concentration-dependent manner, the R value of U937 cells was -0.975, -0.899, -0.925 and -0.915, respectively, that of CEM-C1 cells was -0.960, -0.920, -0.861 and -0.927, and that of BALL-1 cells was -0.939, -0.911, -0.896 and -0.926,. which suggested that thiomersal-induced apoptosis of U937, CEM-C1 and BALL-1 cells might be due to the inhibition of PI3K/Akt/mTOR pathway. Thiomersal promoted the apoptosis of U937, CEM-C1 and BALL-1 cells via caspase-3 pathway, and the expressions of caspase-3 and LC3-II were up-regulated in a dose-dependent manner (r=0.976, r=0.914; r=0.976, r=0.986; r=0.961, r=0.974). Thiomersal can inhibit the proliferation and promote the apoptosis of U937, CEM-C1 and BALL-1 cells. A certain concentration of thiomersal can down-regulate the expression of PI3K/Akt/mTOR pathway related proteins PI3K, Akt, mTOR and p-mTOR in U937, CEM-C1 and BALL-1 cells, and activate autophagy and apoptosis by down-regulation of PI3K/Akt/mTOR pathway.

Daemonorops draco Blume Induces Apoptosis Against Acute Myeloid Leukemia Cells via Regulation of the miR-216b/c-Jun.

Daemonorops draco Blume (DD), also called dragon’s blood, has been used as a traditional Korean medicine, especially for relieving pain caused by wound infection. Recently, it has been described that DD has antibacterial and analgesic effects. In this study, the underlying anticancer effect of DD associated with apoptosis was investigated in acute myeloid leukemia cell lines U937 and THP-1. DD exhibited cytotoxic effects and induced apoptosis in U937 and THP-1 cells. Moreover, DD treatment significantly reduced mitochondrial membrane potential (ΔΨ). The protein expression of cleaved poly(ADP-ribose) polymerase, cleaved caspase-3, p-H2A.X, CCAAT/enhancer-binding protein (CHOP), and activating transcription factor 4 was upregulated by DD treatment. Consistently, DD-treated cells had increased reactive oxygen species (ROS) level in a concentration-dependent manner via miR-216b activation in association with c-Jun inhibition. N-acetyl-L-cysteine pretreatment reversed the cytotoxic effect of DD treatment as well as prevented ROS accumulation. Collectively, the results of this study suggest that the anticancer effect of DD in AML was mediated by CHOP-dependent apoptosis along with ROS accumulation and included upregulation of miR-216b followed by a decrease in c-Jun.

Effect of fresh soy milk and its compounds on apoptosis in human leukemic cells and peripheral blood mononuclear cells


 
 
 
 Purpose: To determine the effect of fresh soy milk and its compounds (coumestrol, daidzein and genistein) on apoptosis in human leukemic cells and peripheral blood mononuclear cells (PBMC).
 Methods: The apoptotic effect of fresh soy milk and its compounds on human leukemic cells (K562, Jurkat, U937) and PBMC was determined by flow cytometry. The PBMC from healthy donors were isolated by conducting density gradient centrifugation principle. Lymphoprep and cytotoxicity of the compounds was evaluated by 3-(4,5- ‘;1dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) MTT) assay.
 Results: PBMC treatment of daidzein and genistein had a significantly higher half-maximal inhibitory concentration (IC50) value (p < 0.01) and (p < 0.0001), compared to the leukemia cells. In addition, soy milk had a significantly higher IC50 value (p < 0.05) in PBMC than the leukemia cells. Coumestrol induced a significantly increased apoptotic cells in K562, U937 cells (p < 0.01) and Jurkat cells (p < 0.05). However, daidzein only increased apoptosis (p < 0.05) in K562 and U937. Genistein reported a significantly increased apoptotic cell in K562, Jurkat (p < 0.01) and U937 cells (p < 0.001). Nonetheless, in PBMC, significant apoptosis by the compounds did not occur. However, only soy milk induced a significantly increased apoptosis in PBMC (p < 0.05).
 Conclusion: The findings indicate that coumestrol, daidzein, genistein and soy milk inhibits the viability of leukemia cells by apoptosis induction, but they are non-toxic toward normal cells. Therefore, the results suggest that the compound alone, and thus, soy milk might have a therapeutic effect in leukemia.
 
 
 

The Effect of MicroRNA-143 on the Proliferation and Apoptosis of Leukemia Cell Line U-937

To investigate the expression of microRNA-143 (miR-143) in patients with acute myeloid leukemia (AML), and the effect of miR-143 over-expression on the proliferation and apoptosis of AML cells. And to verify the targeting relationship between miR-143 and long non-coding RNA MALAT-1. So as to explore the possible regulatory mechanism of miR-143 in the pathogenesis of AML. The expression of miR-143 in bone marrow cells of AML patients was detected by RT-qPCR. After miR-143 was over-expressed in U-937 cell lines, the proliferation of U-937 cell lines was detected by CCK-8, clone formation assay, and flow cytometry. In addition, cell apoptosis was detected by Hoechst 33258 fluorescence staining and flow cytometry. At the same time, bioinformatics, RT-qPCR and dual luciferase reporter gene assay were used to predicted and verified the targeting relationship between miR-143 and MALAT-1. Expression of miR-143 in AML patients was significantly lower than those in normal controls. Over-expressed miR-143 could inhibit the proliferation of U-937 cells and promote the apoptosis of the cell. The miR-143 binding site was located on the MALAT-1 RNA sequence, and MALAT-1 was down-regulated in U-937 cells after over-expressed miR-143. However, the expression of miR-143 showed no significantly changed after MALAT-1 silencing. Expression of miR-143 in AML patients is lower than that in normal controls. Over-expression of miR-143 can inhibit the proliferation of U-937 cells and promote its apoptosis. And its mechanism may be related to its targe regulation of MALAT-1.

Study of Bone Marrow Mesenchymal Stem Cells Regulating NK Cells Function through Tigit/ CD226 in Patients with Multiple Myeloma

Introduction：Bone marrow microenvironment plays a significant supporting role in the proliferation, differentiation, migration, survival and drug resistance of myeloma cells. To detect the expression of the co-inhibitory receptor TIGIT and the co-stimulatory receptor CD226 on the surface of natural killer cells (NK cells) in multiple myeloma (MM) patients, and changes in the immune phenotypes and killing function of NK cells. Furthermore, to explore the underlying mechanism of bone marrow mesenchymal stem cells (BMSCs) regulating NK cells function through TIGIT/CD226 in patients with MM. The expressions of TIGIT, CD226, activated molecules NKG2D and CD107a and functional molecules IFN-γ and Perforin on the NK cells in bone marrow or peripheral blood were detedted by FCM. BMSCs and NK cells were co-cultured at a ratio of 1:4 in vitro, which was grouped according to different monoclonal antibodies (mAbs) added. After co-cultured, the changes in the expression of NKG2D, NKp30, NKp44 and CD69 of NK cells and IFN-γ concentration were examined by FCM. NK cells and U266 were co-cultured with different E:T ratio and the apoptosis of U266 was detected to evaluate the killing function of NK cells by FCM. The expressions of CD155 on BMSCs in the bone marrow were detected by FCM. The correlations between CD155 expression on BMSCs and the production of IFN-γ and Perforin in bone marrow NK cells, as well as clinical characteristics were analyzed.Results: 1. In bone marrow, expression of TIGIT was significantly higher in patients with NDMM than those in patients with CR and HC (both P values <0.01). CD226 was significantly lower in NDMM patients than those in patients with CR and HC (both P values <0.01). NK cell surface activated molecules NKG2D and CD107a, and functional molecules IFN-γ and Perforin were significantly decreased in NDMM (Fig. A). In peripheral blood, TIGIT, CD226 and above immune phenotype expression levels were basically consistent with the trend in bone marrow. 2. In co-culture experiments of BMSCs and NK cells, only added anti-TIGIT mAbs, compared to both added anti-TIGIT and CD226 mAbs, the immune phenotypes of NK cells significantly increased (Fig. B). And the concentration of IFN-γ in the co-culture supernatants also increased (Fig. C). 3. After co-cultured with BMSCs, NK cells were co-cultured again with U266 in vitro, the apoptosis level of U266 in TIGIT mAbs group increased than those in TIGIT+ CD226 mAbs group at different E: T ratio (Fig. D). 4. The expression of CD155, the ligand of TIGIT and CD226, increased notably on the surface of BMSCs in MM patients(P<0.01) (Fig. E). 5. The expression of CD155 on BMSCs was negatively correlated with the production of IFN-γ and perforin in bone marrow NK cells with NDMM patients. And it has correlation with clinical characteristics including β2-MG, LDH, ALB, Hb, ISS stage and R-ISS stage in patients with MM.Summary: BMSCs may regulate NK cells through TIGIT/CD226. High expression of TIGIT on NK cells may mediate the inhibitory effect of BMSCs in MM patients. Blocking TIGIT could restore NK cells activation and killing function. CD155, a common ligand of TIGIT and CD226, was overexpressed on BMSCs in NDMM patients, but was low expressed on bone marrow MM cells. And it has correlation with NK cells function and clinical characteristics, indicating that CD155 may be involved in the regulation of NK cells function by BMSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Camellia sinensis and Phyllanthus amarus Ethanol Extracts Induced Apoptosis and Cell Cycle Arrest on Human Leukemic Cell Lines

Leukaemia is a heterogeneous hematologic malignancy characterized by unregulated proliferation of the early blood-forming cells which starts in bone marrow. The basic strategy of leukaemia therapy involves the induction of leukemic cells apoptosis. Research on natural products have shown that some plant derivatives have anticancer properties by inducing apoptosis of leukemic cells. Plants such as Camellia sinensis and Phyllanthus amarus are those that had gained a wide interest due to their anti-cancer effect. The aim of this study was to investigate the anti-cancer effects of C. sinensis and P. amarus extracts on human leukemic cell lines by analysing the cell cycle and determining the apoptotic state. The cell lines were treated with ethanolic plant extracts at the concentrations of 31.25 - 500 µg/mL for 24 h followed by MTT assay to determine the IC50. The IC50 of C. sinensis and P. amarus on the U937 cells were 170±10.39 and 210±6.78 µg/mL, respectively. Flow cytometric analysis of apoptosis using Annexin V/propidium iodide (PI) staining was also performed. C. sinensis extract at 170 µg/mL significantly increase apoptosis in U-937 (p&lt;0.001), Jurkat (p&lt;0.05) and K-562 cells (p&lt;0.01) when compared to untreated cells. Meanwhile, P. amarus extract at 210 µg/mL significantly induced apoptosis in both U937 and K562 cells (p&lt;0.05) but not Jurkat cells and caused cell cycle arrest at S phase in U-937 cells (p&lt;0.001) and at G0/G1 in K652 cells (p&lt;0.05) when compared to control. Based on the findings, both C. sinensis and P. amarus extracts showed potential in inducing apoptosis in human leukemic cell lines. In addition, P. amarus has the capability to disrupt cell cycle.

Curcumin in combination with homoharringtonine suppresses lymphoma cell growth by inhibiting the TGF-β/Smad3 signaling pathway.

Both homoharringtonine (HHT) and curcumin exhibit anti-proliferative effects on lymphoma cells, but the effects of combined HHT and curcumin treatment remain unclear. Here, we investigated the effects of HHT/curcumin combination on the proliferation, apoptosis, and invasion in lymphoma cells. CCK-8, flow cytometry, and transwell assays were used to assess proliferation, apoptosis, and invasion of U937 and Raji cells. p-Smad3, E-cadherin, and N-cadherin expression were also measured in Raji cells using Western blot assays. Combination of HHT and curcumin synergistically inhibited U937 and Raji cell proliferation and invasion. In addition, the combination treatment markedly increased apoptosis of Raji cells as evidenced by increased Bax, cleaved caspase 3, and cleaved caspase 9 expression. Meanwhile, the combination treatment promoted anti-tumor mechanisms in Raji cells as indicated by decreases in p-Smad3 and N-cadherin and increases in E-cadherin. In vivo experiments showed that the combination treatment suppressed tumor growth in a mouse Raji xenograft model. Our findings indicate that combination of HHT and curcumin inhibited lymphoma cell growth by downregulating the TGF-β/Smad3 pathway. These results suggest that HHT combined with curcumin might be a promising therapeutic approach for the treatment of lymphoma.

Discovery of novel tetrahydrobenzo[b]thiophene-3-carbonitriles as histone deacetylase inhibitors

The discovery and development of isoform-selective histone deacetylase (HDAC) inhibitor is a challenging task because of the sequence homology among HDAC enzymes. In the present work, novel tetrahydro benzo[b]thiophene-3-carbonitrile based benzamides were designed, synthesized, and evaluated as HDAC inhibitors. Pharmacophore modeling was our main design strategy, and two novel series of tetrahydro benzo[b]thiophene-3-carbonitrile derivatives with piperidine linker (series 1) and piperazine linker (series 2) were identified as HDAC inhibitors. Among all the synthesised compounds, 9h with 4-(aminomethyl) piperidine linker and 14n with piperazine linker demonstrated good activity against human HDAC1 and HDAC6, respectively. Both the compounds also exhibited good antiproliferative activity against several human cancer cell lines. Both these compounds (9h and 14n) also induced cell cycle arrest and apoptosis in U937 and MDA-MB-231 cancer cells. Overall, for the first time, this research discovered potent isoform-selective HDAC inhibitors using cyclic linker instead of the aliphatic chain and aromatic ring system, which were reported in known HDAC inhibitors.

Phosphonic Analogs of Alanine as Acylpeptide Hydrolase Inhibitors.

Acylpeptide hydrolase is a serine protease, which, together with prolyl oligopeptidase, dipeptidyl peptidase IV and oligopeptidase B, belongs to the prolyl oligopeptidase family. Its primary function is associated with the removal of N-acetylated amino acid residues from proteins and peptides. Although the N-acylation occurs in 50-90 % of eukaryotic proteins, the precise functions of this modification remains unclear. Recent findings have indicated that acylpeptide hydrolase participates in various events including oxidized proteins degradation, amyloid β-peptide cleavage, and response to DNA damage. Considering the protein degradation cycle cross-talk between acylpeptide hydrolase and proteasome, inhibition of the first enzyme resulted in down-regulation of the ubiquitin-proteasome system and induction of cancer cell apoptosis. Acylpeptide hydrolase has been proposed as an interesting target for the development of new potential anticancer agents. Here, we present the synthesis of simple derivatives of (1-aminoethyl)phosphonic acid diaryl esters, phosphonic analogs of alanine diversified at the N-terminus and ester rings, as inhibitors of acylpeptide hydrolase and discuss the ability of the title compounds to induce apoptosis of U937 and MV-4-11 tumor cell lines.

Effect of Calomelanone, a Dihydrochalcone Analogue, on Human Cancer Apoptosis/Regulated Cell Death in an In Vitro Model.

Calomelanone, 2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone, possesses anticancer activities. This study was conducted to investigate the cytotoxic effect of calomelanone, a dihydrochalcone analogue, on human cancer cells and its associated mechanisms. The cytotoxic effect of calomelanone was measured by MTT assay. Annexin V-FITC/propidium iodide and DiOC6 staining that employed flow cytometry were used to determine the mode of cell death and reduction of mitochondrial transmembrane potential (MTP), respectively. Caspase activities were measured using specific substrates and colorimetric analysis. The expression levels of Bcl-2 family proteins were determined by immunoblotting. Reactive oxygen species were also measured using 2′,7′-dihydrodichlorofluorescein diacetate and dihydroethidium (fluorescence dyes). Calomelanone was found to be toxic towards various human cancer cells, including acute promyelocytic HL-60 and monocytic leukemic U937 cells, in a dose-dependent manner at 24 h and human hepatocellular HepG2 cells at 48 h. However, the proliferation of HepG2 cells increased at 24 h. Calomelanone was found to induce apoptosis in HL-60 and U937 at 24 h and HepG2 apoptosis at 48 h via the intrinsic pathway by inducing MTP disruption. This compound also induced caspase-3, caspase-8, and caspase-9 activities. Calomelanone upregulated proapoptotic Bax and Bak and downregulated antiapoptotic Bcl-xL proteins in HepG2 cells. Moreover, signaling was also associated with oxidative stress in HepG2 cells. Calomelanone induced autophagy at 24 h of treatment, which was evidenced by staining with monodansylcadaverine (MDC) to represent autophagic flux. This was associated with a decrease of Akt (survival pathway) and an upregulation of Atg5 (the marker of autophagy). Thus, calomelanone induced apoptosis/regulated cell death in HL-60, U937, and HepG2 cells. However, it also induced autophagy in HepG2 depending on duration, dose, and type of cells. Thus, calomelanone could be used as a potential anticancer agent for cancer treatment. Nevertheless, acute and chronic toxicity should be further investigated in animals before conducting investigations in human patients.

Novel Small Molecular Compound AE-848 Potently Induces Human Multiple Myeloma Cell Apoptosis by Modulating the NF-κB and PI3K/Akt/mTOR Signaling Pathways.

BackgroundWe aimed to investigate the anti-multiple myeloma (MM) activity of the new small molecular compound AE-848 (5-bromo-2-hydroxyisophthalaldehyde bis[(1-methyl-1H-benzimidazol-2-yl)hydrazone]) and its underlying anti-MM mechanism.MethodsCell viability and apoptosis were detected and quantified by using MTT and flow cytometry, respectively. JC-1 dye-related techniques were used to assess mitochondrial membrane potential (MMP). Western blotting was applied to detect the expression of NF-κB and PI3K/Akt/mTOR pathway-associated proteins. The in vivo activity of AE-848 against MM was evaluated in a MM mouse model.ResultsApplication of AE-848 into the in vitro cell culture system significantly reduced the viability and induced apoptosis of the MM cell lines, RPMI-8226 and U266, in a dose- and time-dependent manner, respectively. JC-1 dye and Western blotting analysis revealed that AE-848 induced the cleavage of caspase-8, caspase-3, and poly ADP-ribose polymerase (PARP), resulting in loss of mitochondrial membrane potential (MMP). Both the NF-κB and PI3K/AKT/mTOR signaling pathways were involved in AE-848-induced apoptosis of U266 and RPMI8226 cells. Moreover, AE-848 leads to cell cycle arrest of MM cells. Its anti-MM efficacy was further confirmed in a xenograft model of MM. AE-848 administration significantly inhibited MM tumor progression and prolonged the survival of MM-bearing mice. More importantly, our results demonstrated that AE-848 markedly induced primary MM cell apoptosis.ConclusionOur results for the first time showed that the small compound AE-848 had potent in vitro and in vivo anti-myeloma activity, indicating that AE-848 may have great potential to be developed as a drug for MM treatment.

PFKFB4 is critical for the survival of acute monocytic leukemia cells

Acute myeloid leukemia (AML), which is characterized by an overproliferation of blood cells, is divided into several subtypes in adults and children. Of those subtypes, acute monocytic leukemia (M4/M5, AMoL) is reported to be associated with abnormal gene fusions that result in monocytic cell differentiation being blocked. However, few studies have shown a relationship between cellular metabolism and the initiation of AMoL. Here, we use the open-access database TCGA to analyze the expression of enzymes in the metabolic cycle and find that PFKFB4 is highly expressed in AMoL. Subsequently, knocking down PFKFB4 in THP-1 and U937 cells significantly inhibits cell growth and increases the sensitivity of cells to chemical drug-induced apoptosis. In line with the gene-editing alterations, treatment with a PFKFB4 inhibitor exhibits similar effects on THP-1 and U937 proliferation and apoptosis. In addition, we find that PFKFB4 functions as a reliable target of the epigenetic regulator MLL, which is a well-known modulator in AMoL. Mechanistically, MLL promotes PFKFB4 expression at the transcriptional level through the putative E2F6 binding site in the promoter of the pfkfb4 gene. Taken together, our results suggest PFKFB4 serves as a downstream target of MLL and functions as a potent therapeutic target in AMoL.

NUPR1 Silencing Induces Autophagy-Mediated Apoptosis in Multiple Myeloma Cells Through the PI3K/AKT/mTOR Pathway.

Nuclear protein 1 (NUPR1) is a stress-related small molecule and plays important roles in various tumors, including multiple myeloma (MM). Autophagy is essential for maintaining cellular homoeostasis in response to stress and, together with apoptosis, determines cell fate. Previous studies indicate that NUPR1 is involved in cancer progression of MM, but the underlying mechanisms have not been elucidated. In this study, we confirmed that NUPR1 and basal autophagy markers were highly expressed in the bone marrow of MM patients. The overexpression of NUPR1 was correlated with staging (both by Revised International Staging System [RISS] and Durie-Salmon [D-S] Staging System), levels of hemoglobin and calcium, and bone marrow plasma cell ratio in the MM patients. NUPR1 silencing reduced autophagy activities and induced apoptosis in U266 and RPMI 8226. We further observed a decrease in NUPR1 silencing-induced apoptosis in the presence of rapamycin, while an increase in apoptosis after chloroquine and 3-methyladenine treatment. Analysis of the mechanism indicated that PI3K/AKT/mTOR pathway was involved in autophagy-mediated apoptosis upon NUPR1 knockdown. In summary, our results demonstrate that NUPR1 silencing suppresses autophagy activities and induces autophagy-mediated apoptosis in MM cells through the PI3K/AKT/mTOR pathway, which exhibits potential as a treatment strategy for MM.

Prazosin inhibits the proliferation and survival of acute myeloid leukaemia cells through down-regulating TNS1

BackgroundPrazosin, a non-selective α1-adrenoceptor and a selective α2B-adrenoceptor antagonist, is reported to possess anti-cancer activity in some types of cancer. The aim of this study was to investigate the effect of prazosin on acute myeloid leukemia (AML) and the underlying relevant mechanisms. MethodsAML cell lines U937 and HL60 were treated with different concentration of prazosin (5, 10 and 15 μM), CCK8 and flow cytometry assays were performed to examine the effects of prazosin on cell viability, cell cycle distribution and apoptosis. Western blot assay was used to detect the expression of related proteins. ResultsWe observed that prazosin inhibited cell viability of U937 and HL60 cells and induced the rate of apoptosis in a dose-dependent manner, as well as induced cell cycle arrest at G1 phase. The activation of PI3K/Akt/mTOR signaling pathway was significantly suppressed by prazosin via reducing the phosphorylation of Akt and mTOR. Moreover, by RNA-seq analysis, we found that the expression of tensin 1 (TNS1) was down-regulated by prazosin, and down-regulation of TNS1 could inhibit cell viability of U937 and HL60 cells, as well as induced cell apoptosis. The PI3K/Akt/mTOR signaling pathway was also suppressed by depletion of TNS1. Furthermore, up-regulation of TNS1 could reverse the effects of prazosin on viability and apoptosis in U937 and HL-60 cells, as well as the PI3K/Akt/mTOR signaling pathway. ConclusionThese results highlight an anti-cancer activity of prazosin on AML by inhibiting the PI3K/Akt/mTOR pathway and targeting TNS1.