Hematologic malignancies, including leukemia, lymphoma, and multiple myeloma, are hazardous diseases characterized by the uncontrolled proliferation of cancer cells. Dysregulated cell cycle resulting from genetic and epigenetic abnormalities constitutes one of the central events. Importantly, cyclin-dependent kinases (CDKs), complexed with their functional partner cyclins, play dominating roles in cell cycle control. Yet, efforts in translating CDK inhibitors into clinical benefits have demonstrated disappointing outcomes. Recently, mounting evidence highlights the emerging significance of WEE1 G2 checkpoint kinase (WEE1) to modulate CDK activity, and correspondingly, a variety of therapeutic inhibitors have been developed to achieve clinical benefits. Thus, WEE1 may become a promising target to modulate the abnormal cell cycle. However, its function in hematologic diseases remains poorly elucidated. In this review, focusing on hematologic malignancies, we describe the biological structure of WEE1, emphasize the latest reported function of WEE1 in the carcinogenesis, progression, as well as prognosis, and finally summarize the therapeutic strategies by targeting WEE1.
Humans ; Protein-Tyrosine Kinases/physiology* ; Hematologic Neoplasms/drug therapy* ; Cell Cycle Proteins/antagonists & inhibitors* ; Nuclear Proteins/antagonists & inhibitors* ; Cyclin-Dependent Kinases ; Molecular Targeted Therapy ; Animals

OBJECTIVE: To investigate the interaction between interleukin-17 (IL-17) and interferon-γ (IFN-γ) and how their interaction affects the growth of mouse hepatoma Hepa1-6 cells. METHODS: Hepa1-6 cells treated with IL-17 and IFN-γ either alone or in combination were examined for changes in cell proliferation using MTT assay and in cell cycle distribution using flow cytometry. Western blotting was used to detect the protein expression levels of proliferating cell nuclear antigen (PCNA), cyclin D1, P21 and P16 and the phosphorylation of p38MAPK, ERK1/2 and Stat1 in the cells. RESULTS: Compared with control group, IFN-γ treatment obviously inhibited the growth and proliferation of Hepa1-6 cells, induced cell cycle arrest at G0/G1 phase, reduced the protein expression of PCNA and cyclin D1, and increased the protein expression of P21. IL-17 alone had no effect on the growth of Hepa1-6 cells. In the combined treatment, IL-17 significantly antagonized the effects of IFN-γ. Compared with those treated with IFN-γ alone, the cells with the combined treatment showed significantly decreased G0/G1 cell population, increased the protein expressions of PCNA and cyclin D1, and decreased the protein expression of P21. IL-17 significantly inhibited IFN-γ-induced phosphorylation of p38MAPK and ERK1/2 without affecting the phosphorylation of Stat1. CONCLUSIONS IL-17 obviously reverses the antitumor effects of IFN-γ to promote the proliferation of mouse hepatoma cells and accelerate the development of hepatocellular carcinoma.
Animals ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Interferon-gamma ; antagonists & inhibitors ; Interleukin-17 ; pharmacology ; Liver Neoplasms ; metabolism ; pathology ; Mice ; Neoplasm Proteins ; metabolism ; Proliferating Cell Nuclear Antigen ; metabolism

OBJECTIVE: To investigate the expression profile of miR-122-5p in melanoma tissues and the effect of miR-122-5p on the proliferation, cell cycle and apoptosis of human melanoma cell lines SK-MEL-110 and A375. METHODS: The expression profiles of miR-122-5p in melanoma and pigmented nevus tissues were detected using real-time fluorescence quantitative PCR (qRT-PCR). SK-MEL-110 and A375 cells transfected with miR-122-5p inhibitor or negative control inhibitor (NC) I were examined for miR-122- 5p expression using qRT-PCR and changes in cell proliferation, cell cycle and apoptosis using MTT assay or flow cytometry. NOP14 mRNA and protein expressions in the cells were detected using qRT- PCR and Western blotting, respectively. Luciferase reporter assay was used to confirm the identity of NOP14 as the direct target of miR-122-5p. RESULTS: The relative expression of miR-122-5p in human pigmented nevus tissues and melanoma tissues was 1.23±0.270 and 7.65 ± 1.37, respectively. The relative expression of miR-122-5p in SK-MEL-110 and A375 cells transfected with miR-122-5p inhibitor was 0.21 ± 0.08 and 0.17 ± 0.05, respectively. miR-122-5p inhibitor obviously inhibited the cell proliferation and increased the percentage of cells in G1 stage in both SK-MEL-110 and A-375 cells, but did not cause obvious changes in the apoptosis of the two cells. miR-122-5p inhibitor did not significantly affect the expression level of NOP14 mRNA, but obviously increased the expression level of NOP14 protein. Luciferase reporter assay revealed a significantly lower luciferase activity in cells co-transfected with miR-122-5p mimics and wild-type psi-CHECK2-3'UTR plasmid than in the cells cotransfected with NC and wild-type psi-CHECK2-3'UTR plasmid (0.21 ± 0.14 0.56 ± 0.1, < 0.01). CONCLUSIONS miR-122-5p expression is upregulated in melanoma tissues, indicating its involvement in the development of melanoma. miR-122-5p inhibits the proliferation of SK-MEL-110 and A-375 cells possibly by affecting the cycle through NOP14.
Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Luciferases ; metabolism ; Melanoma ; etiology ; metabolism ; pathology ; MicroRNAs ; antagonists & inhibitors ; metabolism ; Neoplasm Proteins ; metabolism ; Nevus, Pigmented ; etiology ; metabolism ; pathology ; Nuclear Proteins ; metabolism ; Skin Neoplasms ; etiology ; metabolism ; pathology ; Up-Regulation

OBJECTIVE: To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes. METHODS: The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for -SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting. RESULTS: Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein ( < 0.05) and increased expressions of CaN ( < 0.05) and NFAT3 proteins with also increased intracellular CaN and Ca concentrations ( < 0.01). Alda-1 treatment significantly lowered Ca concentration ( < 0.05), intracellular CaN content ( < 0.01), and CaN and NFAT3 protein expressions ( < 0.05), and increased ALDH2 protein expression ( < 0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca concentration ( < 0.01) and intracellular CaN content ( < 0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (>0.05) but significantly reduced the expression of NFAT3 protein ( < 0.05). CONCLUSIONS Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca-CaN-NFAT3 signaling pathway.
Aldehyde Dehydrogenase, Mitochondrial ; antagonists & inhibitors ; metabolism ; Animals ; Animals, Newborn ; Benzamides ; pharmacology ; Benzodioxoles ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Culture Media ; Enzyme Inhibitors ; pharmacology ; Glucose ; administration & dosage ; pharmacology ; Isoflavones ; pharmacology ; Mitochondria, Heart ; enzymology ; Myocytes, Cardiac ; drug effects ; metabolism ; NFATC Transcription Factors ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the molecular mechanism of action of BET bromodomain inhibitor JQ1 in treating airway remodeling in asthmatic mice.

METHODSA total of 24 mice were randomly divided into control group, ovalbumin (OVA)-induced asthma group (OVA group), and JQ1 intervention group (JQ1+OVA group), with 8 mice in each group. OVA sensitization/challenge was performed to establish a mouse model of asthma. At 1 hour before challenge, the mice in the JQ1+OVA group were given intraperitoneal injection of JQ1 solution (50 μg/g). Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected at 24 hours after the last challenge, and the total number of cells and percentage of eosinophils in BALF were calculated. Pathological staining was performed to observe histopathological changes in lung tissue. RT-PCR and Western blot were used to measure the mRNA and protein expression of E-cadherin and vimentin during epithelial-mesenchymal transition (EMT).

RESULTSCompared with the control group, the OVA group had marked infiltration of inflammatory cells in the airway, thickening of the airway wall, increased secretion of mucus, and increases in the total number of cells and percentage of eosinophils in BALF (P<0.01). Compared with the OVA group, the JQ1+OVA group had significantly alleviated airway inflammatory response and significant reductions in the total number of cells and percentage of eosinophils in BALF (P<0.01). Compared with the control group, the OVA group had significant reductions in the mRNA and protein expression of E-cadherin and significant increases in the mRNA and protein expression of vimentin (P<0.01); compared with the OVA group, the JQ1+OVA group had significant increases in the mRNA and protein expression of E-cadherin and significant reductions in the mRNA and protein expression of vimentin (P<0.01); there were no significant differences in these indices between the JQ1+OVA group and the control group (P>0.05).

CONCLUSIONSMice with OVA-induced asthma have airway remodeling during EMT. BET bromodomain inhibitor JQ1 can reduce airway inflammation, inhibit EMT, and alleviate airway remodeling, which provides a new direction for the treatment of asthma.

Airway Remodeling ; drug effects ; Animals ; Asthma ; drug therapy ; Azepines ; pharmacology ; Cadherins ; analysis ; genetics ; Epithelial-Mesenchymal Transition ; Female ; Mice ; Nuclear Proteins ; antagonists & inhibitors ; Ovalbumin ; immunology ; RNA, Messenger ; analysis ; Transcription Factors ; antagonists & inhibitors ; Triazoles ; pharmacology ; Vimentin ; analysis ; genetics

OBJECTIVETo observe the effects of estrogen on epidermis growth of mice and proliferation of keratinocytes (human epidermal cell line HaCaT), and to explore its mechanism.

METHODS(1) Five adult C57BL/6 mice in estrus cycle were identified by vaginal exfoliative cytology diagnosis and set as estrus group, while another 5 adult C57BL/6 mice with ovary resected before sexual development were set as ovariectomized group. The full-thickness skin from the tail root of mice in two groups were collected. The thickness of epidermis was observed and measured after HE staining. The distribution of proliferating cell nuclear antigen (PCNA)-positive cells in epidermis was observed by immunohistochemical staining, the number of which was counted. (2) HaCaT cells in logarithmic growth phase were cultured with RPMI 1640 nutrient solution containing 10% fetal bovine serum, and they were divided into negative control group (NC), pure estradiol group (PE), protein kinase B (Akt) inhibitor group (AI), and extracellular signal-regulated kinase (ERK) inhibitor group (EI) according to the random number table, with 20 wells in each group. To nutrient solution of each group, 1 μL dimethyl sulfoxide, 1 μL 17β-estradiol (100 nmol/L), 1 μL LY294002 (10 μmol/L), and 1 μL PD98059 (30 μmol/L) were added in group NC, group PE, group AI, and group EI respectively, and the last two groups were added with 1 μL 17β-estradiol (100 nmol/L) in addition. At post culture hour (PCH) 0 (immediately after culture), 24, 48, 72, 5 wells of cells from each group were collected to detect the proliferation activity of cells by cell counting kit 8 and microplate reader. (3) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 wells in each group. At PCH 72, cell cycle distribution was detected by flow cytometer to calculate proliferation index (PI) of cells. (4) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 dishes in each group. At PCH 72, the protein levels of phosphorylated Akt (p-Akt), phosphorylated ERK (p-ERK), and PCNA were determined with Western blotting. The cell experiments were repeated for 3 times. Data were processed with t test, one-way analysis of variance, analysis of variance of factorial design, and LSD test.

RESULTS(1) The epidermis thickness of mice in ovariectomized group was (33.5±3.0) μm, which was obviously thinner than that in estrus group [(51.4±3.1) μm, t=20.7, P<0.01]. The PCNA-positive cells mainly aggregated in the basal layer of epidermis of mice in two groups. The number of PCNA-positive cells in epidermis of mice in ovariectomized group was 37±12 per 200 fold visual field, obviously fewer than that in estrus group (96±15 per 200 fold visual field, t=15.3, P<0.01). (2) During PCH 0 to 48, there were no significant differences in the proliferation activity of cells between group PE and group NC (with P values above 0.05). At PCH 72, compared with that in group NC, the proliferation activity of cells in group PE was obviously increased (P<0.01). The proliferation activity of cells in groups AI and EI was obviously lower than that in the previous two groups (with P values below 0.01). (3) Compared with that in group NC [(51.6±1.1)%], the PI of cells in group PE was obviously increased [(58.5±0.8)%, P<0.05]. The PI values of cells in groups AI and EI were (34.9±0.8)% and (48.2±0.4)% respectively, both obviously lower than those in the previous two groups (with P values below 0.01). (4) Compared with that of group NC (0.566±0.034), the protein level of p-Akt in cells of group PE was significantly increased (1.048±0.077, P<0.01). Compared with that of group PE, the protein level of p-Akt was obviously decreased in cells of groups AI and EI (respectively 0.682±0.095 and 0.672±0.019, with P values below 0.01). Compared with that of group NC (0.469±0.013), the protein level of p-ERK obviously increased in cells of groups PE, AI, and EI (respectively 1.064±0.089, 1.010±0.038, 0.778±0.065, with P values below 0.01). The protein level of p-ERK in cells of group EI was obviously lower than that in group PE (P<0.01). Compared with that of group NC (0.386±0.053), the protein level of PCNA was obviously increased in cells of group PE (0.743±0.043, P<0.01). The protein levels of PCNA in cells of groups AI and EI were 0.264±0.019 and 0.223±0.065 respectively, both obviously lower than those in the previous two groups (with P values below 0.01).

CONCLUSIONSLack of estrogen damages the growth ability of epidermis of mice. Estrogen (17β-estradiol) can promote the proliferation of HaCaT cells by increasing the expression of PCNA via activating ERK/Akt signaling pathway.

Animals ; Cell Cycle ; Cell Line ; Cell Proliferation ; drug effects ; Epidermis ; cytology ; drug effects ; growth & development ; Estradiol ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Female ; Humans ; Keratinocytes ; cytology ; drug effects ; Mice ; Mice, Inbred C57BL ; Phosphorylation ; Proliferating Cell Nuclear Antigen ; metabolism ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; Signal Transduction

OBJECTIVETo investigate the effects of bromodomain-containing protein 4 (BRD4) inhibitor GSK525762A on the proliferation and apoptosis of primary common B-cell acute lymphoblastic leukemia (common B-ALL) cells from adult patients, then to further explore the possible mechanisms.

METHODSPurified leukemia cells from 14 common B-ALL adult patients (4 Ph⁺ and 10 Ph⁻ cases) were obtained by flow cytometry sorting, and maintained in a mimic bone marrow microenvironment culture system for short-term culture. Leukemia cells were treated with various concentrations of GSK525762A. The inhibitory effects of BRD4 inhibitor on common B-ALL leukemia cells were measured by CCK-8 assay and the apoptosis of those cells was determined by AnnexinⅤ/7-AAD staining using flow cytometry. The transcripts of c-MYC, CDK6 and Bcl-2 were detected by quantitative RT-PCR, and the expression of c-MYC, CDK6 and Bcl-2 proteins were detected via Western blot.

RESULTSGSK525762A could inhibit the proliferation of leukemia cells from all 14 common B-ALL patients in a dose-dependent manner, the median value of IC50 was 256.25 (90.64-1 378.39)nmol/L. GSK525762A could promote cells apoptosis of B-ALL leukemia cells in a dose-dependent manner, the median apoptosis rates respectively were 45.17%(9.38%-70.91%), 66.02% (24.36%-96.34%) and 89.29% (39.29%-99.37%) after treated by 500, 1 000 and 2 500 nmol/L GSK525762A. GSK525762A has a similar effect on Ph⁺ ALL and Ph⁻ B-ALL, but the effect of proliferation inhibition and apoptosis enhancement on Ph+ B-ALL is weaker than that on Ph⁻ B-ALL. Compared with vehicle control group, the levels of c-MYC, Bcl-2 and CDK6 transcripts in leukemic cells were reduced after treatment for 24 h and 48 h by 1 000 nmol/L GSK525762A, and there are no significant differences in the downregulation of c-MYC and CDK6 mRNA between Ph⁺ and Ph⁻ B-ALL; however, the inhibitory effect on Bcl-2 transcription was weaker in Ph⁺ B-ALL cells than that in Ph⁻ B-ALL cells. Moreover, c-MYC, Bcl-2 and CDK6 protein levels decreased in GSK525762A treated group.

CONCLUSIONGSK525762A could strongly inhibit the proliferation of common B-ALL and trigger apoptosis; meanwhile it has certain effects against Ph⁺ ALL in vitro. The effect may be achieved by down-regulation of c-MYC, CDK6 and Bcl-2 expression.

Apoptosis ; Benzodiazepines ; pharmacology ; Cell Line, Tumor ; drug effects ; Cyclin-Dependent Kinase 6 ; metabolism ; Down-Regulation ; Flow Cytometry ; Humans ; Nuclear Proteins ; antagonists & inhibitors ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Transcription Factors ; antagonists & inhibitors

This study was purposed to investigate the effect of bromodomain-containing protein 4 (BRD4) inhibitor GSK525762A on the proliferation and apoptosis of chronic myeloid leukemia blast crisis KU812 cells and its mechanism. KU812 cells were treated with different concentrations of GSK525762A (100, 250, 500, 1 000, 2 500 and 5000 nmol/L) and the inhibitory effects of drug on KU812 cell proliferation after 48 and 72 hours were detected by using CCK-8 assay. KU812 cells were treated with 3 different concentrations of GSK525762A (1.0, 2.5 and 5 µmol/L) and the cell apoptosis after 72 hours were assayed by using flow cytometry. KU812 cells were treated with DMSO and 2.5 µmol/L GSK525762A, and the mRNA levels of C-MYC, BCL-2, CDK6, BCL-xL, BAK and BAX were determined by using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results showed that GSK525762A could significantly inhibit the proliferation of KU812 cells and the inhibitory effect on KU812 cell proliferation was dependent on the dose-course and time-course of GSK525762A treatment. GSK525762A treatment could induce apoptosis of KU812 cells in a dose-dependent manner. After GSK525762A treatment, the mRNA levels of proliferation-promoting genes ( C-MYC and CDK6) and pro-survival genes ( BCL-2 and BCL-xL) decreased, while the transcription level of pro-apoptosis genes BAK and BAX increased, as compared to that of the control group. It is concluded that GSK525762A can inhibit the proliferation of KU812 cells and induce cell apoptosis possibly through depressing the transcription of C-MYC, BCL-2, CDK6 and BCL-xL gene, and down-regulating BAK and BAX transcription.
Apoptosis ; drug effects ; Benzodiazepines ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Nuclear Proteins ; antagonists & inhibitors ; Proto-Oncogene Proteins c-bcl-2 ; Transcription Factors ; antagonists & inhibitors

Estrogen has anti-colorectal cancer effects which are thought to be mediated by mismatch repair gene (MMR) activity. Estrogen receptor (ER) expression is associated with microRNA (miRNA) expression in ER-positive tumors. However, studies of direct link between estrogen (especially estradiol E2), miRNA expression, and MMR in colorectal cancer (CRC) have not been done. In this study, we first evaluated the effects of estradiol (E2) and its antagonist ICI182,780 on the expression of miRNAs (miR-31, miR-155 and miR-135b) using COLO205, SW480 and MCF-7 cell lines, followed by examining the association of tissue miRNA expression and serum E2 levels using samples collected from 18 colorectal cancer patients. E2 inhibited the expressions of miRNAs in COLO205 cells, which could be reversed by E2 antagonist ICI 182.780. The expression of miR-135b was inversely correlated with serum E2 level and ER-beta mRNA expression in CRC patients' cancer tissues. There were significant correlations between serum E2 level and expression of ER-beta, miR-135b, and MMR in colon cancer tissue. This study suggests that the effects of estrogen on MMR function may be related to regulating miRNA expression via ER-beta, which may be the basis for the anti-cancer effect in colorectal cells.
Adaptor Proteins, Signal Transducing/genetics/metabolism ; Adult ; Aged ; Cell Line, Tumor ; Colorectal Neoplasms/*genetics/metabolism ; DNA Mismatch Repair/*genetics ; Estradiol/analogs & derivatives/blood/*pharmacology ; Estrogen Antagonists/pharmacology ; Estrogen Receptor beta/genetics/*metabolism ; Female ; *Gene Expression Regulation, Neoplastic ; Humans ; Male ; MicroRNAs/genetics/*metabolism ; Middle Aged ; MutS Homolog 2 Protein/genetics/metabolism ; Nuclear Proteins/genetics/metabolism ; RNA, Messenger/biosynthesis

OBJECTIVETo investigate the effect of NSC348884, a nucleophosmin small molecular inhibitor, on the growth of hepatocellular carcinoma cell line HepG2 and its underlying mechanism.

METHODSAfter HepG2 cells were treated by NSC348884 for 4 days, the effect of HepG2 cells on proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay, the expression variation of nucleophosmin oligomer and monomer was measured using Western blotting, and cell apoptotic rate was detected by flow cytometry.

RESULTSThe proliferation of HepG2 cells was remarkably inhibited by NSC348884 treatment when the drug concentration ranged from 1 micromol/L to 10 micromol/L (P < 0.05), with a 50% inhibiting concentration of 1.4 micromol/L. After treatment for 24 hours, the expression level of nucleophosmin oligomer decreased obviously while that of nucleophosmin monomer increased (both P < 0.05). After treatment by 1 micromol/L and 2 micromol/L NSC348884, the 24-hour apoptotic rates of HepG2 cells were (13.770 +/- 0.335)% and (19.021 +/- 0.237)%, respectively, which were significantly higher than in the control group (6.950 +/- 0.207)% (P < 0. 05).

CONCLUSIONNSC348884 can promote the transformation of nucleophosmin oligomer to monomer and thus inhibit the growth of hepatic carcinoma cell line HepG2 in vitro.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Hep G2 Cells ; Humans ; Indoles ; pharmacology ; Nuclear Proteins ; antagonists & inhibitors ; metabolism