AZD1775 and anti-PD-1 antibody synergistically sensitize hepatoma to radiotherapy / 中华医学杂志(英文版)

BACKGROUND@#Radiation (IR)-induced DNA damage triggers cell cycle arrest and has a suppressive effect on the tumor microenvironment (TME). Wee1, a cell cycle regulator, can eliminate G2/M arrest by phosphorylating cyclin-dependent kinase 1 (CDK1). Meanwhile, programed death-1/programed death ligand-1 (PD-1/PDL-1) blockade is closely related to TME. This study aims to investigate the effects and mechanisms of Wee1 inhibitor AZD1775 and anti-PD-1 antibody (anti-PD-1 Ab) on radiosensitization of hepatoma.@*METHODS@#The anti-tumor activity of AZD1775 and IR was determined by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay on human and mouse hepatoma cells HepG2, Hepa1-6, and H22. The anti-hepatoma mechanism of AZD1775 and IR revealed by flow cytometry and Western blot in vitro . A hepatoma subcutaneous xenograft mice model was constructed on Balb/c mice, which were divided into control group, IR group, AZD1775 group, IR + AZD1775 group, IR + anti-PD-1 Ab group, and the IR + AZD1775 + anti-PD-1 Ab group. Cytotoxic CD8 + T cells in TME were analyzed by flow cytometry.@*RESULTS@#Combining IR with AZD1775 synergistically reduced the viability of hepatoma cells in vitro . AZD1775 exhibited antitumor effects by decreasing CDK1 phosphorylation to reverse the IR-induced G2/M arrest and increasing IR-induced DNA damage. AZD1775 treatment also reduced the proportion of PD-1 + /CD8 + T cells in the spleen of hepatoma subcutaneous xenograft mice. Further studies revealed that AZD1775 and anti-PD-1 Ab could enhance the radiosensitivity of hepatoma by enhancing the levels of interferon γ (IFNγ) + or Ki67 + CD8 T cells and decreasing the levels of CD8 + Tregs cells in the tumor and spleen of the hepatoma mice model, indicating that the improvement of TME was manifested by increasing the cytotoxic factor IFNγ expression, enhancing CD8 + T cells proliferation, and weakening CD8 + T cells depletion.@*CONCLUSIONS@#This work suggests that AZD1775 and anti-PD-1 Ab synergistically sensitize hepatoma to radiotherapy by enhancing IR-induced DNA damage and improving cytotoxic CD8 + T cells in TME.

CUDC-101 as a dual-target inhibitor of EGFR and HDAC enhances the anti-myeloma effects of bortezomib by regulating G2/M cell cycle arrest / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

CUDC-101, an effective and multi-target inhibitor of epidermal growth factor receptor (EGFR), histone deacetylase (HDAC), and human epidermal growth factor receptor 2 (HER2), has been reported to inhibit many kinds of cancers, such as acute promyelocytic leukemia and non-Hodgkin's lymphoma. However, no studies have yet investigated whether CUDC-101 is effective against myeloma. Herein, we proved that CUDC-101 effectively inhibits the proliferation of multiple myeloma (MM) cell lines and induces cell apoptosis in a time- and dose-dependent manner. Moreover, CUDC-101 markedly blocked the signaling pathway of EGFR/phosphoinositide-3-kinase (PI3K) and HDAC, and regulated the cell cycle G2/M arrest. Moreover, we revealed through in vivo experiment that CUDC-101 is a potent anti-myeloma drug. Bortezomib is one of the important drugs in MM treatment, and we investigated whether CUDC-101 has a synergistic or additive effect with bortezomib. The results showed that this drug combination had a synergistic anti-myeloma effect by inducing G2/M phase blockade. Collectively, our findings revealed that CUDC-101 could act on its own or in conjunction with bortezomib, which provides insights into exploring new strategies for MM treatment.

miR-18a enhances the radiosensitivity of nasopharyngeal carcinoma cells through inducing autophagy / 中华耳鼻咽喉头颈外科杂志

Objective: To explore the impacts of miR-18a overexpression or depression on the radiosensitivities of nasopharyngeal carcinoma cell line CNE1 and CNE2 and underlying mechanisms. Methods: CNE1 and CNE2 were transfected with miR-18a mimics, inhibitor and the corresponding control vectors. qRT-PCR and western blot were used to determine the ataxia telangiectasia mutated (ATM) expressions in CNE1 and CNE2. CNE1 and CNE2 with stably expressing miR-18a and miR-18a siRNA were constructed. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the impacts of the miR-18a overexpression or depression combined with irradiation on the cell growth. Flow cytometry was used to detect the cell apoptosis and cell cycle. Colony formation assay was used to evaluate the raodiosensitivities of cells. Acridine orange (AO) staining and western blot were used respectively to test the autophagy and the expressions of related proteins. Independent samples t test was used to compare the mean value between groups by using SPSS 16.0. Results: ATM mRNA was decreased significantly in CNE1 and CNE2 cells transfected with 100 or 200 nmol/L miR-18a mimics for 48 hours (CNE1: RQ=0.174±0.139 and 0.003±0.001, t=9.939 and 19 470.783;CNE2: RQ=0.024±0.008 and 0.019±0.012, t=270.230 and 137.746, respectively, all P<0.001). ATM proteins were also decreased after transfected with 100 or 200 nmol/L miR-18a mimics for 72 hours. While in the cells transfected with 100 and 200 nmol/L miR-18a inhibitor for 48 hours, the expressions of ATM mRNA were upregulated significantly (CNE1: RQ=9.419±2.495 and 2.500±1.063, t=-4.427 and -41.241; CNE2: RQ=7.210±0.171 and 115.875±15.805, t=-62.789 and -12.589, all P<0.05), and the expressions of ATM proteins increased after transfected for 72 hours. The growth of cells with miR-18a overexpression plus 4 Gy irradiation were obviously inhibited compared to that of cells with the 4Gy irradiation alone; while the growth of miR-18a-inhibited cells increased compared to that of cells with 4 Gy irradiation alone (all P<0.05). CNE1 transfected with 100 nmol/L miR-18a mimics plus 4 Gy irradiation showed the higher apoptosis rate than the cells with 4 Gy irradiation alone ((22.9±2.1)% vs. (16.3±1.0)%, t=-4.870, P<0.01). Compared to the cells with 4 Gy irradiation alone, miR-18a-overexpressed cells plus 4 Gy irradiation decreased their percentages in G1 phases ((20.2±3.0)% vs. (29.8±4.4)%, t=3.119) and G2/M phases ((21.5±0.9)% vs. (33.4±3.1)%, t=6.410, P<0.05 for both), and increased their percentages in S phases ((56.7±4.9)% vs. (36.8±6.4)%, t=-4.246, P<0.05), and these cells possessed less colony number after exposure to different doses of irradiation, more autophagy-lysosome number, and more expressions of LC3 proteins (all P<0.05). There were no significant differences in the expressions of p62 expressions between different groups of cells. Conclusion: Overexpression of miR-18a can enhance the radiosensitivities of NPC cells by targeting ATM to abrogate G1/S, G2/M arrest and to induce autophagy and apoptosis.

Downregulation of MCL-1 by Diallyl Disulfide Induces G/M Arrest in Human Leukemia K562 Cells and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the inducing effect of down-regulation of MCL-1 by diallyl disulfide(DADS) on the G/M arrest of human leukemia K562 cells and its mechanisms.</p><p><b>METHODS</b>CCK-8 was used to detect the effect of DADS on proliferation of K562 cells, flow cytometry was employed to observe the effect of cycle arrest by DADS and RNAi silencing MCL-1 gene in K562 cells. The expressions of MCL-1, PCNA and CDK1 in K562 cells treated with DADS were detected by Western blot. The amphigamy of MCL-1 with PCNA and CDK1 was detected by Coimmunoprecipitation.</p><p><b>RESULTS</b>CCK-8 detection showed that the inhibition rates of K562 cells treated with 15, 30, 60, 120, 240 µmol/L DADS were 32.48%, 59.34%, 66.42%, 77.06%, 81.05% respectively (P<0.05). Flow cytometry analysis revealed that the perecentages of G/M cells were increased to 18.6% and 34.4%, 17.5% and 28.5%, respectively at 24 and 48 h after treating K562 cells with 60 and 120 µmol/L DADS (P<0.05). And the perecentage of G/M cells of silencing MCL-1 was significantly increased (P<0.05). Silencing effects of MCL-1+DADS on the cells were enhanced more significantly as compared with DADS or MCL-1 alone (P<0.05). Western blot exhibited that DADS could markedly downregulate the expression of MCL-1, PCNA and CDK1(P<0.05). Coimmunoprecipitation revealed that MCL-1 bound with PCNA and CDK1, then forming heterodimers, which were downregulated respectively more significantly than that in the control group after treating K562 cells with DADS for 8 h (P<0.05).</p><p><b>CONCLUSION</b>DADS can inhibit the K562 cell proliferation and induce them arrest G/M through downregulation of MCL-1, then decreasing the expression of PCNA and CDK1 in hunan leukemia K562 cells. Moreover, silencing MCL-1 can enhance the effect of DADS.</p>

CMPD1 inhibited human gastric cancer cell proliferation by inducing apoptosis and G2/M cell cycle arrest

BACKGROUND: Gastric cancer occupies the fourth highest morbidity rate of cancers worldwide. Clinical therapies of gastric cancer remain limited because of uncertainty of mechanisms and shortness of effective medicine. Thus, new drug candidates for gastric cancer treatment is urgently needed. RESULTS: In this study, CMPD1 as a wildly used MK2 phosphorylation inhibitor was employed to find its impact on gastric cancer cell proliferation, apoptosis and cell cycle using colony formation assay and flow cytometry analysis. Along with its anti-proliferation effect on gastric cancer cell line MKN-45 and SGC7901, CMPD1 also induced massive apoptosis and significant G2/M phase arrest in a time-dependent and dose-dependent manner in MKN-45 cells respectively. Furthermore, Western blot confirmed that the expression of anti-apoptotic proteins Bcl-2 was decreased while BAX, cytochrome c release and cleaved PARP were increased. In addition, oncogene c-Myc was downregulated in response to CMPD1 treatment. CONCLUSIONS: Our results demonstrated that CMPD1 has anti-tumor effect on human gastric cancer cell line MKN- 45 possibly via downregulating oncogene c-Myc expression and CMPD1 could be applied as a potential candidate for treating gastric malignancy. To the best of our knowledge, it is the first report of anti-tumor effect of CMPD-1 on human gastric cancer cells.

ATM Signaling Pathway Is Implicated in the SMYD3-mediated Proliferation and Migration of Gastric Cancer Cells

PURPOSE: We previously found that the histone methyltransferase suppressor of variegation, enhancer of zeste, trithorax and myeloid-nervy-deformed epidermal autoregulatory factor-1 domain-containing protein 3 (SMYD3) is a potential independent predictive factor or prognostic factor for overall survival in gastric cancer patients, but its roles seem to differ from those in other cancers. Therefore, in this study, the detailed functions of SMYD3 in cell proliferation and migration in gastric cancer were examined. MATERIALS AND METHODS: SMYD3 was overexpressed or suppressed by transfection with an expression plasmid or siRNA, and a wound healing migration assay and Transwell assay were performed to detect the migration and invasion ability of gastric cancer cells. Additionally, an MTT assay and clonogenic assay were performed to evaluate cell proliferation, and a cell cycle analysis was performed by propidium iodide staining. Furthermore, the expression of genes implicated in the ataxia telangiectasia mutated (ATM) pathway and proteins involved in cell cycle regulation were detected by polymerase chain reaction and western blot analyses. RESULTS: Compared with control cells, gastric cancer cells transfected with si-SMYD3 showed lower migration and invasion abilities (P<0.05), and the absence of SMYD3 halted cells in G2/M phase and activated the ATM pathway. Furthermore, the opposite patterns were observed when SMYD3 was elevated in normal gastric cells. CONCLUSIONS: To the best of our knowledge, this study provides the first evidence that the absence of SMYD3 could inhibit the migration, invasion, and proliferation of gastric cancer cells and halt cells in G2/M phase via the ATM-CHK2/p53-Cdc25C pathway. These findings indicated that SMYD3 plays crucial roles in the proliferation, migration, and invasion of gastric cancer cells and may be a useful therapeutic target in human gastric carcinomas.

Ophiopogonin D inhibits cell proliferation, causes cell cycle arrest at G2/M, and induces apoptosis in human breast carcinoma MCF-7 cells / 中西医结合学报

<p><b>OBJECTIVE</b>To investigate the effects of ophiopogonin D on human breast cancer MCF-7 cells.</p><p><b>METHODS</b>Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation experiments. Cell cycle was measured with cell cycle flow cytometry and a living cell assay. Apoptosis and terminal deoxynucleoitidyl transferase-mediated dUTP nick end labeling assays were performed to detect the apoptosis of MCF-7 cells induced by ophiopogonin D. Finally, Western blotting was used to explore the mechanism.</p><p><b>RESULTS</b>Exposure of cells to ophiopogonin D resulted in marked decreases in viable cells and colony formation with a dose-dependent manner. Treatment of these cells with ophiopogonin D also resulted in cell cycle arrest at the G(2)/M phase, and increased apoptosis. Mechanistically, ophiopogonin D-induced G(2)/M cell cycle arrest was associated with down-regulation of cyclin B1. Furthermore, activation of caspase-8 and caspase-9 was involved in ophiopogonin D-induced apoptosis.</p><p><b>CONCLUSION</b>The data suggested that ophiopogonin D inhibits MCF-7 cell growth via the induction of cell cycle arrest at the G(2)/M phase.</p>

Oleanolic acid induces G₂/M phase arrest and apoptosis in human hepatocellular carcinoma Bel-7402 cells / 中国中药杂志

This study was to examine the mechanism of oleanolic acid (OA) induces G2/M phase arrest and apoptosis in human hepatocellular carcinoma Bel-7402 cells. MTT and trypan blue exclusion test assay were adopted to detect the proliferate status of cells treated with OA. We assayed the cell cycle by flow cytometry using PI staining. Apoptosis was determined by Annexin V-FITC staining and PI labeling. The expressions of cycle related proteins and apoptotic related proteins were determined by Western blot analysis. OA strongly inhibited human hepatoma cells proliferation. When Bel-7402 cells were pretreated with OA for 24 h, OA induced apoptosis and G₂/M phase cell cycle arrest in a concentration-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that OA decreased the protein levels of cyclin B1, but increased the protein levels of p-Cdk1 (Tyr15) and p-Cdc25C (Ser 216). Moreover, OA modulated the phosphorylation of protein kinases Chk1 and p2l. Western blotting assay also showed significant decrease of Bcl-2 protein expression and increase of Bax protein expression, the cytosol Cyt c level, cleaved-caspase-9 and cleaved-caspase-3 activity. These data suggest that OA produces anti-tumor effect via induction of G₂/M cell cycle arrest and apoptosis.

The ethanol extract isolated from Weiqi Decoction induces G₂/M arrest and apoptosis in AGS cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To evaluate the effects of the ethanol extract isolated from Weiqi Decoction (WQD-EE) on AGS cell proliferation and apoptosis.</p><p><b>METHODS</b>By using high-performance liquid chromatography with ultraviolet detectors (HPLC-UV) assay and MTT method, the main compounds in WQD-EE and cell viability were detected. And cell cycle distributions were determined by flow cytometry with propidium iodine (PI) staining while apoptosis was detected by flow cytometry with annexin V/PI double staining. Finally, caspase-3 activities were measured by colorimetric method and protein expression was determined by Western blotting.</p><p><b>RESULTS</b>HPLC analysis showed that naringin (35.92 μg/mg), nobiletin (21.98 μg/mg), neohesperidin (17.98 μg/mg) and tangeretin (0.756 μg/mg) may be the main compounds in WQD-EE. WQD-EE not only inhibited AGS and MCF 7 cell proliferation in a dose-dependent manner, but also blocked cell cycle progression at G2/M stage as well as inducing cell apoptosis at concentrations triggering significant inhibition of proliferation and cell cycle arrest in AGS cells. While at 0.5 mg/mL, WQD-EE significantly increased caspase-3 activity by 2.75 and 7.47 times at 24 h and 48 h, respectively. Moreover, WQD-EE in one hand reduced protein expressions of p53 and cyclin B1, and in other hand enhanced protein expressions of cytochrome c and Bax. Protein levels of Bcl-2, Fas L and Fas were not significantly affected by WQD-EE.</p><p><b>CONCLUSIONS</b>WQD-EE inhibits AGS cell proliferation through G2/M arrest due to down-regulation of cyclin B1 protein expression, and promotes apoptosis by caspase-3 and mitochondria-dependent pathways, but not by p53-dependent pathway.</p>

Fucoidan induces apoptosis of HepG2 cells by down-regulating p-Stat3 / 华中科技大学学报（医学）（英德文版）

Fucoidan is one of the main bioactive components of polysaccharides. The current study was focused on the anti-tumor effects of fucoidan on human heptoma cell line HepG2 and the possible mechanisms. Fucoidan treatment resulted in cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner detected by MTT assay, flow cytometry and fluorescent microscopy. The results of flow cytometric analysis revealed that fucoidan induced G2/M arrest in the cell cycle progression. Hoechst 33258 and Annexin V/PI staining results showed that the apoptotic cell number was increased, which was associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2 and p-Stat3. In parallel, the up-regulation of p53 and the increase in reactive oxygen species were also observed, which may play important roles in the inhibition of HepG2 growth by fucoidan. In the meantime, Cyclin B1 and CDK1 were down-regulated by fucoidan treatment. Down-regulation of p-Stat3 by fucoidan resulted in apoptosis and an increase in ROS in response to fucoidan exposure. We therefore concluded that fucoidan induces apoptosis through the down-regulation of p-Stat3. These results suggest that fucoidan may be used as a novel anti-cancer agent for hepatocarcinoma.

Knockdown of Bcl-xL Enhances Growth-Inhibiting and Apoptosis-Inducing Effects of Resveratrol and Clofarabine in Malignant Mesothelioma H-2452 Cells

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.

Triptolide induces apoptosis through extrinsic and intrinsic pathways in human osteosarcoma U2OS cells.

Triptolide, a diterpene derived from Tripterygium wilfordii Hook f., a Chinese medicinal herb, has been reported to inhibit cell proliferation and induce apoptosis in various human cancer cells, but its anticancer effects on human osteosarcoma cells have not yet been elucidated. In this study, we investigated whether triptolide induces apoptosis in human osteosarcoma cells and the underlying molecular mechanisms. We firstly demonstrated that triptolide inhibited cell growth and induced apoptosis in U2OS cells. Western blot analysis showed that the levels of procaspase-8, -9, Bcl-2, Bid and mitochondrial cytochrome c were downregulated in triptolide-treated U2OS cells, whereas the levels of Fas, FasL, Bax, cytosolic cytochrome c, cleaved caspase-3 and cleaved PARP were upregulated. These results suggest that triptolide induces apoptosis in U2OS cells by activating both death receptor and mitochondrial apoptotic pathways.

Inhibitory Effect of Melanoma Differentiation Associated Gene-7/Interleukin-24 on Invasion In Vitro of Human Melanoma Cancer Cells

The acquisition of metastasis potential is a critical point for malignant tumors. Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a potential tumor suppress gene and frequently down-regulated in malignant tumors. It has been implicated that overexpression of MDA-7 led to proliferation inhibition in many types of human tumor. Invasion is an important process which is potential to promote tumor metastasis. However, the role and potential molecular mechanism of mda-7/IL-24 to inhibit the invasion of human melanoma cancer is not fully clear. In this report, we identified a solid role for mda-7/IL-24 in invasion inhibition of human melanoma cancer LiBr cells, including decreasing of adhesion and invasion in vitro, blocking cell cycle, down-regulating the expression of ICAM-1, MMP-2/9, CDK1, the phosphorylation of ERK and Akt, NF-kappaB and AP-1 transcription activity. Meanwhile, there was an increased expression of PTEN in mda-7/IL-24 over-expression LiBr cells. Our results demonstrated that mda-7/IL-24 is a potential invasion suppress gene, which inhibits the invasion of LiBr cells by the down-regulation of ICAM-1, MMP-2/9, PTEN, and CDK1 expression. The molecular pathways involved were the MAPK/ERK, PI3K-Akt, NF-kappaB, and AP-1. These findings suggest that mda-7/IL-24 may be used as a possible therapeutic strategy for human melanoma cancer.

Radiation-induced G2 phase arrest may contribute to the radioresistance of breast cancer stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate radiation-induced cell cycle changes of human breast cancer stem cells enriched by suspension culture.</p><p><b>METHODS</b>The tumorigenicity of human breast cancer stem cell line MCF-7 cultured in serum-free media was confirmed in NOD/SCID mice, and the radiosensitivity of the cells was tested by clone formation assay following radiation exposure. Flow cytometry was performed to evaluate radiation-induced cell cycle changes, and the protein expression of pCDC25C (ser216) was measured by Western blotting.</p><p><b>RESULTS</b>After the exposure to 2 Gy radiation, the survived fraction of the cells in suspension culture and those in adherent culture was 0.856 ∓ 0.061 and 0.783 ∓ 0.097, respectively, and the cells in suspension culture showed an obviously greater capacity of tumorigenicity in NOD/SCID mice. The radiation exposure resulted in an obvious increase in the proportion of G2 phase cells from (22.03 ∓ 2.12)% to (45.83 ∓ 2.25)% and significantly increased the expression of pCDC25C (ser216).</p><p><b>CONCLUSION</b>Radiation- induced G2 phase arrest may contribute to the resistance of the breast cancer stem cells to radiotherapy.</p>