Hydrangea serrata extract exerts tumor inhibitory activity against hepatocellular carcinoma HepG2 cells via inducing p27/CDK2-mediated cell cycle arrest and apoptosis

Objective:To examine the inhibitory effect of Hydrangea serrata extract against hepatocellular carcinoma HepG2 cells and its underlying mechanisms.Methods:The effects of Hydrangea serrata extract on growth inhibition of tumor cells and spheroids were assessed using MTT and 3D culture assays.Quantitative real-time PCR and Western blot analyses were employed to investigate the changes in mRNA and protein expression levels of molecules related to cell cycle and apoptosis.Results:Hydrangea serrata extract effectively inhibited the growth of both tumor cells and spheroids.The extract also significantly upregulated p27 mRNA expression and downregulated CDK2 mRNA expression,leading to cell cycle arrest.Moreover,increased BAX/Bcl-2 ratio as well as caspase-9 and-3 were observed after treatment with Hydrangea serrata extract,indicating the induction of tumor cell apoptosis.Conclusions:Hydrangea serrata extract has the potential to alleviate tumors by effectively modulating cell-cycle-related gene expressions and inducing apoptosis,thereby inhibiting tumor growth.

Combinatorial effect of diclofenac with piperine and D-limonene on inducing apoptosis and cell cycle arrest of breast cancer cells

Objective:To investigate the potential synergistic activity of diclofenac with piperine and D-limonene in inducing apoptosis and cell cycle arrest in breast cancer MCF-7 cells.Methods:Molecular docking study was conducted to evaluate the binding affinity of diclofenac with piperine and D-limonene against p53,Bax,and Bcl-2.The MTT assay was used to determine IC50,and the Chou-Talay method was used to determine the synergistic concentration of the combination treatment of diclofenac plus piperine and diclofenac plus D-limonene.Apoptosis detection,cell cycle arrest,reactive oxygen species production,and mitochondrial membrane potential were also investigated.Results:Diclofenac,piperine,and D-limonene showed potent binding affinity for p53,Bax,and Bcl-2.Diclofenac plus piperine and diclofenac plus D-limonene enhanced the formation of reactive oxygen species,which also had an effect on the mitochondrial membrane’s integrity and caused DNA fragmentation.Diclofenac plus piperine and diclofenac plus D-limonene arrested the cells in the sub-G0phase while drastically lowering the percentage of cells in the G2/M phase.Furthermore,the elevated apoptosis in the combined therapy was confirmed by annexin V/propidium iodide staining.Conclusions:The combined therapy prominently enhanced the antiproliferative and apoptotic effects on MCF-7 cells compared with treatment with diclofenac,piperine,and D-limonene alone.

Intrinsic apoptotic pathway and G2/M cell cycle arrest involved in tubeimoside I-induced EC109 cell death

Objective： Squamous esophageal carcinoma is highly prevalent in developing countries, especially in China. Tu Bei Mu （TBM）, a traditional folk medicine, has been used to treat esophageal squamous cell carcinoma （ESCC） for a long term. tubeimoside I （TBMS1） is the main component of TBM, exhibiting great anticancer potential. In this study, we investigated the mechanism of TBMS1 cytotoxic effect on EC109 cells. Methods： Comparative nuclear proteomic approach was applied in the current study and we identified several altered protein spots. Further biochemical studies were carried out to detect the mitochondrial membrane potential, cell cycle and corresponding proteins＇ expression and location. Results： Subcellular proteomic study in the nucleus from EC109 cells revealed that altered proteins were associated with mitochondrial function and cell proliferation. Further biochemical studies showed that TBMSl-induced molecular events were related to mitochondria-induced intrinsic apoptosis and P21-cyclin B 1/cdc2 complex-related G2/M cell cycle arrest. Conclusions： Considering the conventional application of TBM in esophageal cancer, TBMS1 therefore may have a great potential as a chemotherapeutic drug candidate for ESCC.

Silibinin induces hepatic stellate cell cycle arrest via enhancing p53/p27 and inhibiting Akt downstream signaling protein expression

BACKGROUND： Proliferation of hepatic stellate cells （HSCs） plays a pivotal role in the progression of liver fibrosis conse- quent to chronic liver injury. Silibinin, a flavonoid compound, has been shown to possess anti-fibrogenic effects in animal models of liver fibrosis. This was attributed to an inhibition of cell proliferation of activated HSCs. The present study was to gain insight into the molecular pathways involved in silibinin anti-fibrogenic effect. METHODS： The study was conducted on LX-2 human stellate cells treated with three concentrations of silibinin （10, 50 and 100 μmol/L） for 24 and 96 hours. At the end of the treatment cell viability and proliferation were evaluated. Protein expression of p27, p21, p53, Akt and phosphorylated-Akt was evaluated by Western blotting analysis and Ki-67 protein expression was by immunocytochemistry. Sirtuin activity was evaluated by chemiluminescence based assay. RESULTS： Silibinin inhibits LX-2 cell proliferation in doseand time-dependent manner; we showed that silibinin upregulated the protein expressions of p27 and p53. Such regulation was correlated to an inhibition of both downstream Akt and phosphorylated-Akt protein signaling and Ki-67 protein expression. Sirtuin activity also was correlated to silibinin- inhibited proliferation of LX-2 cells. CONCLUSION： The anti-proliferative effect of silibinin on LX-2 human steUate cells is via the inhibition of the expres- sions of various cell cycle targets including p27, Akt and sir- tuin signaling.

Carbon ion irradiation-induced DNA damage evokes cell cycle arrest and apoptosis via the pRb/E2F1/c-Myc signaling pathway in p53-deficient prostate cancer PC-3 cells

Carbon ion radiotherapy has the advantages of better therapeutic effect and fewer side effects compared with those of X-rays in many kinds of tumors,including prostate cancer,and thus is an attractive treatment approach for prostate cancer.However,the biological effects and underlying mechanisms of carbon ion irradiation in prostate cancer are not yet fully understood.Therefore,this study systematically compared the effects of carbon ion irradiation with those of X-ray irradiation on DNA damage response and found that carbon ion irradiation was more effective than X-ray irradiation.Carbon ion irradiation can induce a high level of DNA double-strand break damage,reflected by the number of y-H2 A histone family member X foci,as well as by the foci lasting time and size.Moreover,carbon ion irradiation exhibited strong and long-lasting inhibitory effect on cell survival capability,induced prolonged cell cycle arrest,and increased apoptosis in PC-3 cells.As an underlying mechanism,we speculated that carbon ion irradiation-induced DNA damage evokes cell cycle arrest and apoptosis via the pRb/E2 F1/c-Myc signaling pathway to enhance the radiosensitivity of p53-deficient prostate cancer PC-3 cells.Collectively,the present study suggests that carbon ion irradiation is more efficient than X-ray irradiation and may help to understand the effects of different radiation qualities on the survival potential of p53-deficient prostate cancer cells.

Marsdenia tenacissima extract induces G_0/G_1 cell cycle arrest in human esophageal carcinoma cells by inhibiting mitogen-activated protein kinase(MAPK) signaling pathway

Marsdenia tenacissima extract(MTE, trade name: Xiao-Ai-Ping injection) is an extract of a single Chinese plant medicine. It has been used for the treatment of cancer in China for decades, especially for esophageal cancer and other cancers in the digestive tract. In the present study, the potential mechanism for MTE's activity in esophageal cancer was explored. The effects of MTE on the proliferation of human esophageal cancer cells(KYSE150 and Eca-109) were investigated by the MTT assay, the Brd U(bromodeoxyuridine) incorporation immunofluorescence assay, and flow cytometric analysis. MTE inhibited cell proliferation through inducing G0/G1 cell cycle arrest in KYSE150 and Eca-109. Western blot analysis was employed to determine protein levels in the MTE treated cells. Compared with the control cells, the expression levels of the cell cycle regulatory proteins cyclin D1/D2/D3, cyclin E1, CDK2/4/6(CDK: cyclin dependent kinase), and p-Rb were decreased significantly in the cells treated with MTE at 40 mg·m L-1. In addition, MTE had an inhibitory effect on the MAPK(mitogen-activated protein kinase) signal transduction pathway, including ERK(extracellular signal-regulated kinase), JNK(c-Jun N-terminal kinase), and p38 MAPK. Moreover, MTE showed little additional effects on the regulation of cyclin D1/D3, CDK4/6, and p-Rb when the ERK pathway was already inhibited by the specific ERK inhibitor U0126. In conclusion, these data suggest that MTE inhibits human esophageal cancer cell proliferation through regulation of cell cycle regulatory proteins and the MAPK signaling pathways, which is probably mediated by the inhibition of ERK activation.

Ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic-acid Inhibits Growth of Human Lung Cancer A549 Cells by Arresting Cell Cycle and Triggering Apoptosis

Objective： To examine the apoptotic effect of ent-llα-hydroxy-15-oxo-kaur-16-en-19-oic-acid （5F）, a compound isolated from Pteris semipinnata L （PsL）, in human lung cancer A549 cells. Methods： A549 cells were treated with 5F （0-80 lag/ml） for different time periods. Cytotoxicity was examined using a Ml-I- method. Cell cycle was examined using propidium iodide staining. Apoptosis was examined using Hoechst 33258 staining, enzyme-linked immunosorbent assay （ELISA） and caspase-3 activity analysis. Expression of representative apoptosis-related proteins was evaluated by Western blot analysis. Reactive oxygen species （ROS） level was measured using standard protocols. Potential interaction of 5F with cisplatin was also examined. Results： 5F inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. 5F increased the accumulation of cells in sub-G1 phase and arrested the cells in the G2 phase. Exposure to 5F induced morphological changes and DNA fragmentation that are characteristic of apoptosis. The expression of p21 was increased. 5F exposure also increased Bax expression, release of cytochrome c and apoptosis inducing factor （AIF）, and activation of caspase-3. 5F significantly sensitized the cells to cisplatin toxicity. Interestingly, treatment with 5F did not increase ROS, but reduced ROS production induced by cisplatin. Conclusion： 5F could inhibit the proliferation of A549 cells by arresting the cells in G2 phase and by inducing mitochondrial-mediated apoptosis.

The MORC2 p.S87L mutation reduces proliferation of pluripotent stem cells derived from a patient with the spinal muscular atrophy-like phenotype by inhibiting proliferation-related signaling pathways

Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal muscular atrophy-like clinical phenotype.The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies.Epithelial cells were isolated from urine samples from a spinal muscular atrophy(SMA)-like patient[MORC2 p.S87L),a CMT2Z patient[MORC2 p.Q400R),and a healthy control and induced to generate pluripotent stem cells,which were then differentiated into motor neuron precursor cells.Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAP K/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells.Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control.G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient.MORC2 p.S87Lspecific antisense oligonucleotides(p.S87L-ASO-targeting)showed significant efficacy in improving cell prolife ration and activating the PI3K/Akt and MAP K/ERK pathways in induced pluripotent stem cells.Howeve r,p.S87L-ASO-ta rgeting did not rescue prolife ration of motor neuron precursor cells.These findings suggest that downregulation of the PI3K/Akt and MAP K/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype.p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.

Cell Cycle Arrest, Apoptosis, and Autophagy Induced by chabamide in Human Leukemia Cells

Objective To investigate the antitumor effect of chabamide in K562 （human leukemia cell line） cells. Methods The cytotoxicity was assessed by a standard colorimetric assay using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT）. The morphological changes were observed by Hoechst 33258 staining. Induction of apoptosis, loss of the mitochondrial membrane potential （A ~＇m）, and cell cycle analysis were evaluated by flow cytometry （FCM） analysis. Levels of apoptosis-related proteins, ceil cycle-related proteins, and LC3 protein were detected by Western blotting. Moreover, the autophagy induced by chabamide was also detected by MDC fluorescent staining. Results Chabamide significantly inhibited cell proliferation by cell cycle arrest in the Go/G1 phase. This phenomenon was associated with an obvious increase in p21 expression and decrease in cyclin D1 and cyclin-dependent kinase 2/4/6 protein expression. Moreover, chabamide could regulate the changes in mitochondrial membrane potential, increase the expression of apoptosis-related proteins, such as Bax and cytochrome C, and decrease the protein expression of Bcl-2, caspase-9, caspase-3, and PARP-1. JNK, ERK1/2, and p38 were also regulated by chabamide in K562 cells. Furthermore, induction of autophagy, marked by autophagic vacuole formation, was detected. Conversion of LC3-1 to LC3-11, a marker of active autophagosome formation, was also detected following chabamide treatment. Conclusion The antitumor activity of chabamide with the results of apoptosis and autophagy induction was confirmed in K562 cells.

ZMYND10 downregulates cyclins B1 and D1 to arrest cell cycle by trimethylating lysine 9 on histone 3

The BLU gene coding for zinc finger,MYND-type containing 10(ZMYND10)protein is mapped on chromosomal region 3p21.It is frequently lost in some kinds of cancers due to hypermethylation on its promoter region and identified as a tumour suppressor gene.The underlying mechanisms for BLU-mediated tumor suppression remain unclear.BLU has been reported to disturb cell cycle progression.The present study aims at examining whether ZMYND10 prevents progression of the cell cycle by targeting to repressive histone marks and downregulating the level of cyclins.Proteins structurally similar with ZMYND10 have been shown to recognize DNA sequence upstream of coding portion of the gene encoding cell cycle regulators.Enzymes,notably demethylases modifying the lysine residues are over-expressed line oncoproteins,and targeted in anti-cancer therapy.BLU was re-expressed in H1299 and HepG2 cells.The level of cyclin D1,cyclin B1 and trimethylate lysine 9 on histone 3(H3K9me3)and the binding of BLU with SIN3A(a component of the co-repressor)were detected.Cell cycle profile was measured.The evolutionary relationship between ZMYND10 and other ZMYND proteins was analysed by phylogenetic tree construction.We found that BLU expression induced G1 arrest in H1299 cells,and induced G1/G2 arrest in HepG2 cells.Cell cycle arrest was correlated with reduced activities and levels of cyclins;cyclin D1 was downregulated in H1299 cells;Both cyclin B1 and D1 were downregulated in HepG2 cells;and that BLU was associated with SIN3A.In both cell lines,the expression of H3K9me3 was induced.BLU was clustered with histone methyltransferase SMYD3 and SMYD1 on the same clade of the deduced phylogenetic tree.The results thus suggested that ZMYND10 encoded by BLU inhibited cyclins activity to prevent cell cycle progression through interaction with repressors and histone repressive marks to block the expression of genes coding for cyclins.

Arsenic Trioxide Inhibits Proliferation in K562 Cells by Changing Cell Cycle and Survivin Expression

To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As 2O 3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As 2O 3 induced cell apoptosis, K562 cells were cultured with As 2O 3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry. Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As 2O 3 (2-10 μmol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G 2/M phase increased in proportion to As 2O 3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As 2O 3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G 2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As 2O 3-induced apoptosis.

Gambogic acid induces mitochondria-dependent apoptosis by modulation of Bcl-2 and Bax in mantle cell lymphoma JeKo-1 cells

Objective： To study the mechanisms in gambogic acid （GA） -induced JeKo-1 human Mantle Cell Lymphoma cell apoptosis in vitro. Methods： The proliferation of GA-treated JeKo-1 cells was measured by CCK-8 assay and Ki-67 immunocytochemical detection. Apopt0sis, cell cycle and mitochondrial membrane potential were measured by flow cytometric analysis. Caspase-3, -8 and -9 were detected by colorimetric assay. Bcl-2 and Bax were analyzed by Western blotting. Results： GA inhibited cell growth in a time- and dose- dependent manner. GA induces apoptosis in JeKo- 1 cells but not in normal bone marrow cells, which was involved in reducing the membrane potential of mitochondria, activating caspases-3, -8 and -9 and decreasing the ratio of Bd-2 and Bax without cell cycle arresting. Conclusions： GA induced apoptosis in human MCL JeKo-1 cells by regulating Bcl-2/Bax and activating caspase-3, -8 and -9 via mitochondrial pathway without affecting cell cycle.

Mechanisms involved in the cytotoxic effects of berberine on human colon cancer HCT-8 cells

Berberine,a constituent of some traditional Chinese medicinal plants,has been reported to have cytotoxicity effects on different human cancer cell lines.There is no available information about the effects and mechanism of action of berberine on human colon cancer cell line HCT-8.In this paper,the cytotoxicity of berberine on HCT-8 cancer cells was investigated by MTT assay,fluorescence microscopy and flow cytometry analysis.Our data revealed that berberine could significantly inhibit the growth of HCT-8 cells in a dose-and time-dependent manner.Morphology of apoptotic cells was studied with acridine orange/ethidium bromide staining.The concentrations of lactate dehydrogenase and both acid and alkaline phos-phatases were significantly increased in cell supernatants after berberine treatment,suggesting cell death.Furthermore,flow cytometry analysis showed that berberine could arrest HCT-8 cells at S phase in a time-dependent manner.To further investigate the apoptotic molecular mechanism,reverse transcription-poly-merase chain reaction(RT-PCR)and western blotting methods were used.The up-regulated mRNA and/or protein expressions of Fas,FasL,TNF-α,caspase-3 and down-regulation of pro-caspase-3 suggest that the death receptor pathway may be involved in the apoptotic pathway induced by berberine.Decrease of Bcl-2 and increase of Bax in mRNA and/or protein expressions showed that the Bcl-2 family proteins were involved in berberine-induced apoptosis.We also found that berberine-induced apoptosis was associated with an up-regulated expressions of p53 and prohibitin(PHB),and decreased vimentin expression.These results suggest that berberine can suppress cell growth and reduce cell survival by arresting the cell-cycle and by inducing apoptosis of HCT-8 cells.

Anti-proliferation and apoptosis-inducing effects of sodium aescinate on retinoblastoma Y79 cells

AIM:To investigate the anti-proliferation and apoptosisinducing effects of sodium aescinate(SA)on retinoblastoma Y79 cells and its mechanism.METHODS:Y79 cells were cultured at different drug concentrations for different periods of time(24,48,and 72 h).The inhibitory effect of SA on proliferation of Y79 cells was detected by the cell counting kit-8(CCK-8)assay,and the morphology of Y79 cells in each group was observed under an inverted microscope.An IC50 of 48 h was selected for subsequent experiments.After pretreatment with SA for 24 and 48 h,cellular DNA distribution and apoptosis were detected by flow cytometry.Real-time qunatitative polymerase chain reaction(RT-qPCR)and Western blot were used to assess changes in related genes(CDK1,CyclinB1,Bax,Bcl-2,caspase-9,caspase-8,and caspase-3).RESULTS:SA inhibited proliferation and induced apoptosis of Y79 cells in a time-dependent and concentrationdependent manner.Following its intervention in the cell cycle pathway,SA can inhibit the expression of CDK1 and Cyclin B1 at the mRNA and protein levels,and block cells in the G2/M phase.In caspase-related apoptotic pathways,up-regulation of Bax and down-regulation of Bcl-2 caused caspase-9 to self-cleave and further activate caspase-3.What’s more,the caspase-8-mediated extrinsic apoptosis pathway was activated,and the activated caspase-8 was released into the cytoplasm to activate caspase-3,which as a member of the downstream apoptotic effect group,initiates a caspase-cascade reaction that induces cell apoptosis.CONCLUSION:SA inhibits the proliferation of Y79 cells by arresting the cell cycle at the G2/M phase,and induces apoptosis via the caspase-related apoptosis pathway,indicating that SA may have promising potential as a chemotherapeutic drug.

Nuclear-targeted therapy of nerve growth factor conjugated with ^(125)I for human glioma U251 cells

The present study investigated the nuclear transportation phenomenon of ^125I-nerve growth factor （NGF） and the DNA-damaging changes to U251 cells using microautoradiography and single cell electrophoresis. The results showed that ^125I-NGF inhibited the survival of p53 mutant U251 human glioma cell/tumor and enhanced the therapeutic effectiveness of vincristine in in vivo and in vitro models. In vitro experiments showed ^125I-NGF was transported into the nucleus and damaged the DNA in U251 cells. Moreover, ^125I-NGF locked the U251 cells in the G2 phase. Further investigation showed that ^125I-NGF decreased cyclin B1 protein levels in a dose dependent manner, but the level of cyclin B1 mRNA expression remained unchanged. ^125I-NGF increased phosphorylated Chkl, Chk2 and Cdc25c protein levels in U251 cells, but did not influence p53 and p21 protein expression. Moreover, ^125I-NGF and vincristine exhibited synergistic effects on reducing cyclin B1 protein levels. These results indicate that ^125I-NGF can provide anti-tumor effects by activating the ATM and ATR pathways through DNA damage.

Apoptotic and cytostatic actions of maslinic acid in colorectal cancer cells through possible IKK-β inhibition

Objective:To explore the anti-cancer activity of maslinic acid against colorectal cancer(CRC)cell lines and its possible mechanism.Methods:The inhibitory effect of maslinic acid was screened against five CRC cell lines(HT-29,HCT 116,SW480,SW48,and LS 174 T)via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Apoptosis and cell cycle analyses were carried out using annexinⅤ-FITC/propidium iodide staining and propidium iodide staining,respectively and subjected to fluorescence-activated cell sorting analysis.Protein expression studies of inhibitor ofκB kinase-β(IKK-β),checkpoint kinase 1(Chk1)and cyclin D1 were conducted using the JESS system.Results:Maslinic acid exhibited growth inhibitory effect in a doseand time-dependent manner in HT-29 and HCT 116 cell lines.A more prominent apoptosis induced by maslinic acid was observed in HCT 116 cell line.However,in HT-29 cell line,maslinic acid induced cell cycle arrest by inhibiting the G1-S transition,which was accompanied by the downregulation of cyclin D1.The expression of unphosphorylated IKK-βprotein was increased in both(HT-29 and HCT 116)cell lines after maslinic acid treatment.Conclusions:Maslinic acid inhibits the growth of HT-29 and HCT 116 cells in a different manner,induces cell cycle arrest in HT-29 cells and causes apoptosis in HCT 116 cells partially via NF-κB pathway inhibition.

Low Dose Hyper-radiosensitivity in Human Lung Cancer Cell Line A549 and Its Possible Mechanisms

The low dose hyper-radiosensitivity （HRS） in human lung cancer cell line A549 was investigated, the changes of ATM kinase, cell cycle and apoptosis of cells at different doses of radiation were observed, and the possible mechanisms were discussed. A549 cells in logarithmic growth phase were irradiated with ^60Co y-rays at doses of 0-2 Gy. Together with flow cytometry for precise cell sorting, cell survival fraction was measured by means of conventional colony-formation assay. The expression of ATM1981 Ser-P protein was examined by Western blot 1 h after radiation. Apoptosis was detected by Hoechst 33258 fluorescent staining, and Annexin V-FITC/PI staining flow cytometry 24 h after radiation. Cell cycle distribution was observed by flow cytometry 6, 12 and 24 h after radiation. The results showed that the expression of ATM1981Ser-P protein was observed at 0.2 Gy, followed by an increase at 〉0.2 Gy, and reached the peak at 0.5 Gy, with little further increase as the dose exceeded 0.5 Gy. Twenty-four h after radiation, partial cells presented the characteristic morphological changes of apoptosis, and the cell apoptosis curve was coincident with the survival curve. As compared with control group, the cell cycle almost had no changes after exposure to 0.1 and 0.2 Gy radiation （P〉0.05）. After exposure to 0.3, 0.4 and 0.5 Gy radiation, G2/M phase arrest occurred 6 and 12 h after radiation （P〈0.05）, and the ratio of G2/M phase cells was decreased 24 h after radiation （P〈0.05）. It was concluded that A549 cells displayed the phenomenon of HRS/IRR. The mode of cell death was mainly apoptosis. The activity of ATM and cell cycle change may take an important role in HRS/IRR.

α-Mangostin and apigenin induced the necrotic death of BT474 breast cancer cells with autophagy and inflammation

Objective: To find new compounds in order to overcome the mainstay of metastatic breast cancer due to the adverse side effects from, and increasing resistance to, current chemotherapeutic agents. Methods: 毩-Mangostin and apigenin were reported in comparison to doxorubicin, a chemotherapeutic drug. Ductal carcinoma(BT474) cell line and nontumorigenic epithelial tissue from mammary gland(MCF-10 A) were used. Cell viability assessment was calculated by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Cell morphology was investigated by light microscopy. By flow cytometry analysis, programmed cell death was observed using annexin observed using propidium iodide st桋 and propidium iodide staining while cell-cycle arrest wasaining. Change in transcriptional expression was evaluated by real-time quantitative reverse transcription PCR. Results: In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the result revealed and apigenin were more cytotoxic to BT474 cells. Longer exposure times to 毩-mangostin enin caused more floating cells and a lower density of adhered cells wi毩-mangostin and apigth more vacuoles present in the colonies in BT474 only. 毩-Mangostin and apigenin caused necrosis in BT474 cells in a 24 h exposure, but a small amount of early apoptotic cells could also be detected at 24, 48 and 72 h exposure, whereas doxorubicin caused early apoptosis to BT474 cells at 24 h. Transcript expression and activity analysis supported caspase-3 was involved in the death of BT474 cells treated by all compounds. Moreover, 毩-mangostin and apigenin arrested the cellcycle at the G1-phase, but at the G2/M-phase by doxorubicin. All three compounds induced a change in transcript expression levels of inflammation-associated, proto-oncogene, autophagyassociated and apoptosis-associated genes. Conclusions: ntial new sources of chemotherapeuti毩-Mangostin and apigenin are worth investigating as potec agents for breast cancer treatment.

Genistein-induced Anticancer Effects on Acute Leukemia Cells Involve the Regulation of Wnt Signaling Pathway Through H4K20mel Rather Than DNA Demethylation

Objective:To investigate the effects and mechanisms of genistein on the gene expression in the Wnt pathway in acute leukemia(AL)cells.Methods:The expression of Wnt pathway genes and cell cycle-related genes were analyzed in two AL cell lines.Pyrophosphate sequencing was performed to determine the methylation degree.Then,the enrichment of H4K20mel and H3K9ac was determined using ChIP-qPCR.Flow cytometry was used to analyze the cell cycle.Results:The IC_(50) of genistein in the two AL cell lines was lower than that for the bone marrow mesenchymal stem cell line.Genistein upregulated H4K20mel,KMT5A and Wnt suppressor genes,including Wnt5a,and downregulated the downstream target genes of Wnt,such as c-myc and β-catenin.The methylation degree and H3K9ac enrichment in the Wnt5a promoter region remained unchanged.However,the enrichment of H4K20mel in the Wnt5a promoter and coding regions increased.In addition,genistein upregulated Phospho-cdc2,Mytl,Cyclin A,Cyclin E2,p21 and Phospho-histone H3,but downregulated Phospho-weel.Cell cycle arrest was induced in the G2/M phase.Conclusion:Genistein inhibits the activation of the Wnt pathway by promoting the expression of Wnt5a through the activation of KMT5A and enrichment of H4K20mel in the Wnt5a gene promoter and coding regions,rather than demethylation.Genistein also blocks the cell cycle in the G2/M phase.Therefore,genistein is a potential anti-leukemia drug.

Effects of Arsenic Trioxide on Human Renal Cell Carcinoma Lines in Vitro

Objective: To observe the effects of arsenic trioxide (As2O3) on human renal cell carcinoma (RCC) lines in vitro and to explore its possible molecular mechanisms. Methods: The microculture tetrazoli-um (MTT) assay was used to determine the anti-proliferative effects of As2O3 on human RCC lines. Flow cy-tometry was performed to investigate the effects of As2O3 on cell cycle and cell apoptosis. The reverse tran-scription-polymerase chain reaction (RT-PCR) was conducted to detect mRNA expression of Bcl-2, Bax, p53 and c-myc. Results: As2O3 inhibited the growth of RCC lines in vitro in a concentration-dependent manner. At the concentrations of 0.5, 1.0, 2.0 and 4.0μmol/L, the inhibition rates of As2O3 on RCC-WCS cells were 27.60%, 30.09%, 41.03% and 50.77%, respectively. Compared with untreated RCC-WCS, there was significant difference at each concentration (P<0.01). As2O3 induced a G1 phase arrest in RCC-LSL cells, but a G2/M phase arrest in RCC-WCS and RCC-SHK. As2O3 induced cell apoptosis in these cell lines. The mRNA level of p53 and c-myc decreased, but no detectable changes of Bcl-2 and Bax were observed after As2O3 treatmen. Conclusion: As2O3 in therapeutic concentrations inhibited the in vitro growth of RCC lines via cell cycle arrest and apoptosis. One of its possible mechanisms was down-regulation of p53 and c-myc. Our results suggest that As2O3 is probably a new candidate agent for the treatment of human renal carcinoma.