Knockdown of diacylglycerol kinase zeta (DGKZ) induces apoptosis and G2/M phase arrest in human acute myeloid leukemia HL-60 cells through MAPK/survivin/caspase pathway.

Diacylglycerol kinase zeta (DGKZ) is associated with the pathogenesis of a variety of malignant diseases, but its biological function on acute myeloid leukemia (AML) has not been explored. The aim of this study was to analyze apoptosis induced by knockdown of DGKZ and its mechanism in human acute myeloid leukemia HL-60 cells. qRT-PCR was carried out to detect the expression of DGKZ in HL-60, THP-1, Jurkat, K562, and CD34 cell lines. Additionally the expression of DGKZ in AML cells obtained from patients were detected by qRT-PCR. Cell Counting Kit-8 (CCK-8) assay was used to determine the viability of HL-60 cells DGKZ knocked down. Apoptosis and cell cycle phase of HL-60 cells after DGKZ knockdown were evaluated by flow cytometry. Western blot analysis was performed to investigate expressions of the proteins related to apoptosis and cell cycle. Results showed that expression of DGKZ was significantly higher in HL-60 and AML cells obtained from patients than those of Jurkat, THP-1, K562 and human CD34 cell. Compared with the shCtrl group, DGKZ was markedly knocked down in HL-60 cells transfected with lentivirus encoding shRNA. DGKZ knockdown significantly inhibited the proliferation and induced cycle arrest at the G2/M phase in HL-60 cells. The expressions of MAPK, caspase-3, caspase-8, cytochrome C markedly increased and p-MAPK and survivin decreased in HL-60 cells after DGKZ knockdown. The results suggest that knockdown of DGKZ can induce apoptosis and G2/M phase arrest in human acute myeloid leukemia HL-60 cells through the MAPK/survivin/caspase pathway.

Knockdown of IARS2 Inhibited Proliferation of Acute Myeloid Leukemia Cells by Regulating p53/p21/PCNA/eIF4E Pathway.

IARS2 encodes mitochondrial isoleucine-tRNA synthetase, which mutation may cause multiple diseases. However, the biological function of IARS2 on acute myeloid leukemia (AML) has not yet been identified. In the present study, qRT-PCR was used to determine the expression of IARS2 in K562, THP1, and HL-60 leukemia cells. Additionally the mRNA levels of IARS2 in CD34 cells and AML cells obtained from patients were detected by qRT-PCR. IARS2-shRNA lentiviral vector was established and used to infect acute myeloid leukemia HL-60 cells. qRT-PCR and Western blot analysis were employed to assess the knockdown effect of IARS2. The proliferation rate and cell cycle phase of HL-60 cells after IARS2 knockdown were evaluated by CCK-8 assay and flow cytometry. The PathScan Antibody Array was used to determine the expression of cell cycle-related proteins in HL-60 cells after IARS2 knockdown. The expression of proliferation-related proteins in HL-60 cells after IARS2 knockdown was determined by Western blot analysis. Results showed that IARS2 expression was stable and much higher in HL-60, THP-1, and K562 leukemia cells and AML cells obtained from patients than that of human CD34 cells. Compared with cells of the shCtrl group, IARS2 was markedly knocked down in cells that were transfected with lentivirus encoding shRNA of IARS2 in HL-60 cells (p < 0.05). IARS2 knockdown significantly inhibited the proliferation and induced cycle arrest at the G1 phase in HL-60 cells. Additionally IARS2 knockdown significantly increased the expression of p53 and p21, and decreased the expression of PCNA and eIF4E in HL-60 cells. In conclusion, IARS2 knockdown can inhibit acute myeloid leukemia HL-60 cell proliferation and cause cell cycle arrest at the G1 phase by regulating the p53/p21/PCNA/eIF4E pathways.

The effects and mechanism of peiminine-induced apoptosis in human hepatocellular carcinoma HepG2 cells.

Peiminine is a compound isolated from Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae family), which has demonstrated antitumor activities. But its precise molecular mechanism underlying antitumor activity remain elusive. In this study, peiminine-induced apoptosis towards human hepatocellular carcinoma and its molecular mechanism were investigated. MTT assay was employed to assess anticancer effects of peiminine upon Hela, HepG2, SW480 and MCF-7 cell lines. Nuclear staining and flow cytometry were carried out to detect apoptosis induced by peiminine. Mitochondrial membrane potential evaluation and Western blot analysis were performed to investigate the mechanism of peiminine-induced apoptosis. The results showed peiminine reduced the viability of HepG2 cells in a time- and dose-dependent manner and had an IC50 of 4.58 µg/mL at 24h. Peiminine significantly increased the percentage of apoptotic cells and the mitochondrial membrane potential dose-dependently in HepG2 cells. The results of Western blotting indicated the expressions of Bcl-2, procaspase-3, procaspase-8, procaspase-9, and PARP decreased in HepG2 cells treated with peiminine, while the expressions of Bax, caspase-3, caspase-8, caspase-9, and cleaved PARP1 increased. The result suggests that peiminine can induce apoptosis in human hepatocellular carcinoma HepG2 cells through both extrinsic and intrinsic apoptotic pathways.

A Familial 14q32.32q32.33 Duplication/17p13.3 Deletion Syndrome with Facial Anomalies and Moderate Intellectual Disability.

To our knowledge, a derivative chromosome 17 formed by a subtelomeric translocation involving chromosomes 17 and 14 has not been reported before. Here, we present the clinical and molecular cytogenetic characteristics of 2 family members with a subtelomeric rearrangement involving chromosome regions 14q32.32q32.33 and 17p13.3. The patients had moderate intellectual disability, a high forehead, a broad nasal root, downslanting palpebral fissures, epicanthal folds, retrognathia, hypertelorism, wrinkled skin over the glabella and metopic suture, and mild finger clubbing. Array CGH detected a 2.52-Mb duplication of 14q32.32q32.33 (103,805,680-106,396,479) and a 1.2-Mb deletion of 17p13.3 (87,009-1,298,869) confirmed to be pathogenic by quantitative PCR and loss of heterozygosity analysis of 17p13.3. The derivative chromosome 17 was inherited from a parental balanced translocation. To our knowledge, this cytogenetic aberration has not been described previously. The refinement of the genetic location will improve the knowledge of the genes responsible for this phenotype.

A novel mitochondrial DNA deletion in a patient with Pearson syndrome and neonatal diabetes mellitus provides insight into disease etiology, severity and progression.

Pearson syndrome (PS) is a rare, mitochondrial DNA (mtDNA) deletion disorder mainly affecting hematopoietic system and exocrine pancreas in early infancy, which is characterized by multi-organ involvement, variable manifestations and poor prognosis. Since the clinical complexity and uncertain outcome of PS, the ability to early diagnose and anticipate disease progression is of great clinical importance. We described a patient with severe anemia and hyperglycinemia at birth was diagnosed with neonatal diabetes mellitus, and later with PS. Genetic testing revealed that a novel mtDNA deletion existed in various non-invasive tissues from the patient. The disease course was monitored by mtDNA deletion heteroplasmy and mtDNA/nucleus DNA genome ratio in different tissues and at different time points, showing a potential genotype-phenotype correlation. Our findings suggest that for patient suspected for PS, it may be therapeutically important to first perform detailed mtDNA analysis on non-invasive tissues at the initial diagnosis and during disease progression.

Osthole induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

CONTEXT: Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong's Classic of Materia Medica in the Han Dynasty, had remarkable antiproliferative activity against human hepatocellular carcinoma HepG2 cells in culture. OBJECTIVES: This study evaluated the effects of osthole on cell growth, nuclear morphology, cell cycle distribution, and expression of apoptosis-related proteins in HepG2 cells. MATERIALS AND METHODS: Cytotoxic activity of osthole was determined by the MTT assay at various concentrations ranging from 0.004 to 1.0 µmol/ml in HepG2 cells. Cell morphology was assessed by Hoechst staining and fluorescence microscopy. Apoptosis and cell-cycle distribution was determined by annexin V staining and flow cytometry. Apoptotic protein levels were assessed by Western blot. RESULTS: Osthole exhibited significant inhibition of the survival of HepG2 cells and the half inhibitory concentration (IC50) values were 0.186, 0.158 and 0.123 µmol/ml at 24, 48 and 72 h, respectively. Cells treated with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 µmol/ml showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction. The increase of apoptosis induced by osthole was correlated with down-regulation expression of anti-apoptotic Bcl-2 protein and up-regulation expression of pro-apoptotic Bax and p53 proteins. CONCLUSION: Osthole had significant growth inhibitory activity and the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.