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.

Antitumor Effect of Apcin on Endometrial Carcinoma via p21-Mediated Cell Cycle Arrest and Apoptosis

Objective Endometrial carcinoma(EC)is a prevalent gynecological malignancy characterized by increasing incidence and mortality rates.This underscores the critical need for novel therapeutic targets.One such potential target is cell division cycle 20(CDC20),which has been implicated in oncogenesis.This study investigated the effect of the CDC20 inhibitor Apcin on EC and elucidated the underlying mechanism involved.Methods The effects of Apcin on EC cell proliferation,apoptosis,and the cell cycle were evaluated using CCK8 assays and flow cytometry.RNA sequencing(RNA-seq)was subsequently conducted to explore the underlying molecular mechanism,and Western blotting and coimmunoprecipitation were subsequently performed to validate the results.Animal studies were performed to evaluate the antitumor effects in vivo.Bioinformatics analysis was also conducted to identify CDC20 as a potential therapeutic target in EC.Results Treatment with Apcin inhibited proliferation and induced apoptosis in EC cells,resulting in cell cycle arrest.Pathways associated with apoptosis and the cell cycle were activated following treatment with Apcin.Notably,Apcin treatment led to the upregulation of the cell cycle regulator p21,which was verified to interact with CDC20 and consequently decrease the expression of downstream cyclins in EC cells.In vivo experiments confirmed that Apcin treatment significantly impeded tumor growth.Higher CDC20 expression was observed in EC tissue than in nonmalignant tissue,and increased CDC20 expression in EC patients was associated with shorter overall survival and progress free interval.Conclusion CDC20 is a novel molecular target in EC,and Apcin could be developed as a candidate antitumor drug for EC treatment.

Induction of apoptosis and G2/M cell cycle arrest by oridonin in human gastric cancer BGC-823 cells

Aim To investigate in vitro apoptosis-induction effects of oridonin on gastric tumor cells BGC-823 and its effects on cell cycle, mitochondrial membrane potential and intracellular Ca^2＋ to shed light on the mode of its anticancer action. Methods The MTT method was used to investigate the inhibitory effect of oridonin on BGC-823 cells. The apoptosis-induction effect was evaluated by confocal laser microscopy and flow cytometry. The change of mitochondrial membrane potential and the increase of intracellular Ca^2＋ were assessed by fluorescence probe rhodamine123 and Fluo 3-AM, respectively, with flow cytometry. The expression of apoptosis and cell cycle related proteins was studied using western blotting. Results Oridonin inhibited BGC-823 cells growth with IC50 of 22.21 p, mol.L^-1. It induced apoptosis in a dose-dependent manner. In addition, it decreased mitochondria membrane potential, increased intracellular Ca^2＋, and activated pro-caspase 3. BGC-823 cells were arrested in G2/M cell cycle phase with lower expression of cyclin A protein. The up-regulation of p53 was observed before apoptosis and cell cycle arrest occurred. Conclusion Oridonin inhibits the proliferation of BGC-823 cells through G2/M cell cycle arrest and apoptosis induction, which is mediated by influx of Ca^2＋, up-regulation of p53, activation of caspase-3, and down-regulation of cyclin A.

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.

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.

Paris chinensis dioscin induces G2/M cell cycle arrest and apoptosis in human gastric cancer SGC-7901 cells

AIM:To investigate the anti-tumor effects of Paris chinensis dioscin(PCD)and mechanisms regarding cell cycle regulation and apoptosis in human gastric cancer SGC-7901 cells.METHODS:Cell viability was analyzed by the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.Cell apoptosis was evaluated by flow cytometry and laser scanning confocal microscope(LSCM)using Annexin-V/propidium iodide(PI)staining,and the cell cycle was evaluated using PI staining with flow cytom-etry.Intracellular calcium ions were detected under fluorescence microscope.The expression of cell cycle and apoptosis-related proteins cyclin B1,CDK1,cytochrome C and caspase-3 was measured by immunohistochemical staining.RESULTS:PCD had an anti-proliferation effect on human gastric cancer SGC-7901 cells in a dose-and time-de-pendent manner.After treatment of SGC-7901 cells with PCD,apoptosis appeared in SGC-7901 cells.Morpho-logical changes typical of apoptosis were also observed with LSCM by Annexin V/PI staining,and the cell number of the G0/G1 phase was decreased,while the number of cells in the G2/M phase was increased.Cell cycle-related proteins,such as cyclin B1 and CDK1,were all down-regulated,but caspase-3 and cytochrome C were up-regulated.Moreover,intracellular calcium accumulation occurred in PCD-treated cells.CONCLUSION:G2/M phase arrest and apoptosis induced by PCD are associated with the inhibition of CDK-activating kinase activity and the activation of Ca2+-related mitochondrion pathway in SGC-7901 cells.

15-PGDH is reduced and induces apoptosis and cell cycle arrest in gastric carcinoma

AIM: To investigate the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in human gastric cancer and it's mechanism in apoptosis and cell cycle arrest. METHODS: Expression of 15-PGDH mRNA and protein was examined by immunohistochemistry, immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting in tissue from human gastric cancer, gastric precancerous state (gastric polyps and atrophic gastritis), normal stomach, and gastric cancer cell lines. The relationship between gastric cancer, gastric precancerous state and 15-PGDH expression was determined. The association between expression of 15-PGDH and various clinicopathological parameters in gastric cancer was evaluated. Human gastric cancer cell line SGC-7901 was transfected with 15-PGDH expression plasmids. The effect of 15-PGDH on the cell cycle was examined by flow cytometry. The effect of 15-PGDH on apoptosis was examined by transmission electron microscopy, flow cytometry and transferasemediated nick end labeling (TUNEL) assay. Expression of cell cycle (p21, p27, p16 and p53) and apoptosis (Survivin, BCL-2, BCL-XL, BAK and BAX) genes was analyzed by RT-PCR. RESULTS: Expression of 15-PGDH mRNA and protein in human gastric cancer tissues was significantly lower than in normal gastric tissues (P < 0.01). Expression in human gastric cancer cell lines MKN-28 and MKN-45 was reduced, and absent in SGC-7901 cells (P < 0.05). Reduction of 15-PGDH expression was also found in precancerous tissues, such as gastric polyps and atrophic gastritis (P < 0.01). There was a significant difference in expression of 15-PGDH among various gastric cancer pathological types (P < 0.05), with or without distant metastasis (P < 0.05) and different TNM stage (P < 0.01). Flow cytometry demonstrated a significant increase in apoptotic cells in SGC-7901 cells transfected with pcDNA3/15-PGDH plasmid for 24 and 48 h (P < 0.01), and an increased fraction of sub-G1 phase after transfection (P < 0.05). TUNEL assay showed an increased Apoptotic Index in cells overexpressing 15-PGDH (P < 0.01). After transfection, expression of proapoptotic genes, such as BAK (P < 0.05), BAX and p53 (P < 0.01), was increased. Expression of antiapoptotic genes was decreased, such as Survivin, BCL-2 and BCL-XL (P < 0.01). Expression of cyclin-dependent kinase inhibitors p21 and p16 (P < 0.01) was significantly upregulated in cells overexpressing 15-PGDH. CONCLUSION: Reduction of 15-PGDH is associated with carcinogenesis and development of gastric carcinoma. 15-PGDH induces apoptosis and cell cycle arrest in SGC-7901 cells.

Mechanisms involved in ceramide-induced cell cycle arrest in human hepatocarcinoma cells

AIM： To investigate the effect of ceramide on the cell cycle in human hepatocarcinoma Bel7402 cells. Possible molecular mechanisms were explored. METHODS： [3- （4, 5）-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide （MTT） assay, plasmid transfection, reporter assay, FACS and Western blotting analyses were employed to investigate the effect and the related molecular mechanisms of C2-ceramide on the cell cycle of Bel7402 cells. RESULTS： C2-ceramide was found to inhibit the growth of Bel7402 cells by indudng cell cycle arrest. During the process, the expression of p21 protein increased, while that of cyclinD1, phospho-ERKl/2 and c-myc decreased. Furthermore, the level of CDK7 was downregulated, while the transcriptional activity of PPARy was upregulated. Addition of GW9662, which is a PPARy specific antagonist, could reserve the modulation action on CDK7. CONCLUSION： Our results support the hypothesis that cell cycle arrest induced by C2-ceramide may be mediated via accumulation of p21 and reduction of cyclinD1 and CDK7, at least partly, through PPARy activation. The ERK signaling pathway was involved in this process.

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.

Ethanol extract of Kalopanax septemlobus leaf inhibits HepG2 human hepatocellular carcinoma cell proliferation via inducing cell cycle arrest at G_1 phase

Objective:To investigate the effects of an ethanol extract of Kalopanax septemlobus(Thunb.)Koidz.leaf(EEKS) on cell proliferation in human hepatocellular carcinoma cells and its mechanisms of action.Methods:Cells were treated with EEKS and subsequently analyzed for cell proliferation and flow cytometry analysis.Expressions of cell cycle regulators were determined by reverse transcriptase polymerase chain reaction analysis and Western blotting,and activation of eyclin-associaled kinases studied using kinase assays.Results:The EEKS suppressed cell proliferation in both HepG2 and Hep3 B cells,but showed a more sensitive anli-proliferative activity in HepG2 cells.Flow cytometry analysis revealed an association between the growth inhibitory effect of EEKS and with G_1 phase cell cycle arrest in HepG2 cells,along with the dephosphorylation of retinoblastoma protein(pRB) and enhanced binding of pRB with the E2 F transcription factor family proteins.Treatment with EEKS also increased the expression of cyclin-dependent kinase(CDK) inhibitors,such as p21WAF1/CIP1 and p27KIP1.without any noticeable changes in G_1 cyclins and CDKs(except for a slight decrease in CDK4).Treatment of HepG2 cells with EEKS also increased the binding of p21 and p27 with CDK4 and CDK6.which was paralleled by a marked decrease in the cyclin D- and cyclin E-associated kinase activities.Conclusions:Overall,our findings suggest that EEKS may be an effective treatment for liver cancer through suppression of cancer cell proliferation via G_1,cell cycle arrest Further studies arc required to identify the active compounds in EEKS.

Magnetic resonance imaging tracing of transplanted bone marrow mesenchymal stem cells in a rat model of cardiac arrest-induced global brain ischemia

BACKGROUND： Numerous studies have shown that magnetic resonance imaging （MRI） can detect survival and migration of super paramagnetic iron oxide-labeled stem cells in models of focal cerebral infarction. OBJECTIVE： To observe distribution of bone marrow mesenchymal stem cells （BMSCs） in a rat model of global brain ischemia following cardiac arrest and resuscitation, and to investigate the feasibility of tracing iron oxide-labeled BMSCs using non-invasive MRI. DESIGN, TIME AND SETTING： The randomized, controlled, molecular imaging study was performed at the Linbaixin Medical Research Center, Second Affiliated Hospital, Sun Yat-sen University, and the Institute of Cardiopulmonary Cerebral Resuscitation, Sun Yat-sen University, China from October 2006 to February 2009. MATERIALS： A total of 40 clean, Sprague Dawley rats, aged 6 weeks and of either gender, were supplied by the Experimental Animal Center, Sun Yat-sen University, China, for isolation of BMSCs. Feridex （iron oxide）, Gyroscan Inetra 1.5T MRI system, and cardiopulmonary resuscitation device were used in this study. METHODS： A total of 30 healthy, male Sprague Dawiey rats, aged 6 months, were used to induce ventricular fibrillation using alternating current. After 8 minutes, the rats underwent 6-minute chest compression and mechanical ventilation, followed by electric defibrillation, to establish rat models of global brain ischemia due to cardiac arrest and resuscitation. A total of 24 successful models were randomly assigned to Feridex-labeled and non-labeled groups （n = 12 for each group）. At 2 hours after resuscitation, 5 ×10^8 Feridex-labeled BMSCs, with protamine sulfate as a carrier, and 5 ×10^6 non-labeled BMSCs were respectively transplanted into both groups of rats through the right carotid artery （cells were harvested in 1 mL phosphate buffered saline）. MAIN OUTCOME MEASURES： Feridex-labeled BMSCs were observed by Prussian blue staining and electron microscopy. Signal intensity, celluar viability, and proliferative capacity of BMSCs were measured using MRI, Trypan blue test, and M-IT assay, respectively. Distribution of transplanted cells was observed in rats utilizing MRI and Prussian blue staining prior to and 1, 3, 7, and 14 days after transplantation. RESULTS： Prussian blue staining displayed many blue granules in the Feridex-labeled BMSCs. High density of iron granules was observed in the cytoplasm under electron microscopy. According to MRI results, and compared with the non-labeled group, the signal intensity was decreased in the Feridex-labeled group （P 〈 0.05）. The decrease was most significant in the 50 pg/mL Feridex-labeled group （P 〈 0.01）. There were no significant differences in celluar viability and proliferation of BMSCs between the Feridex-labeled and non-labeled groups after 1 week （P 〉 0.05）. Low-signal lesions were detected in the rat hippocampus and temporal cortex at 3 days after transplantation. The low-signal lesions were still detectable at 14 days, and positively stained cells were observed in the hippocampus and temporal cortex using Prussian blue staining. There were no significant differences in signal intensity in the non-labeled group. CONCLUSION： BMSC transplantation traversed the blood-brain barrier and distributed into vulnerable zones in a rat model of cardiac arrest-induced global brain ischemia. MRI provided a non-invasive method to in vivo dynamically and spatially trace Feridex-labeled BMSCs after transplantation.

Dose-dependent effects of lead on cell-cycle arrest, DNA damage, and cyclin D1 expression in primary cultured rat hippocampal neurons

BACKGROUND： Previous studies have suggested that the hippocampus is one of the neurotoxic target sites for lead. However, the molecular mechanisms of action, including the effect of lead on cell-cycle arrest, remain poorly understood. OBJECTIVE： To investigate the effects of different lead concentrations on cell-cycle arrest, DNA damage, and cyclin D1 expression in primary cultured rat hippocampal neurons. DESIGN, TIME AND SETTING： A randomized, controlled, in vitro experiment was performed at the China Medical University between July 2008 and May 2009. MATERIALS： Antibodies specific to cyclin D1 and actin were synthesized and purified by Santa Cruz Biotechnology, USA. FACStar flow cytometer was purchased from Becton Dickinson, San Jose, California, USA. METHODS： Wistar rat hippocampal neurons were primary cultured for 7 days. Neurons in the control group were treated with 0.01 mol/L phosphate buffered saline. Neurons in the 0.2, 1.0, and 10 umol/L lead acetate groups were subjected to 0.2, 1.0, and 10 umol/L lead acetate. Subsequently hippocampal neurons in each group were cultured for 24 hours. MAIN OUTCOME MEASURES： The effects of lead on cell cycle were measured by flow cytometry, DNA damage was measured using the comet assay, and cyclin D1 expression was measured using Western blot analysis. RESULTS： Treatment of hippocampal neurons with 0.2 umol/L lead acetate did not significantly alter cell cycle phase distribution, i.e., sub-G1, S, G0/G1, G2/M, whereas treatment with 1.0 and 10 umol/L lead acetate significantly increased the percentage of S and sub-G1 phase cells （P 〈 0.05）. Olive tail moment in all lead-treated groups and the percentage of DNA in the tail in 1.0 umol/L and 10 umol/L lead acetate groups were significantly greater compared with the control group （P 〈 0.05）. In addition, the percentage of tail DNA was greater in the 0.2 umol/L lead acetate group compared with the control group （P 〉 0.05）. Following incubation with 0.2, 1.0, and 10 umol/L lead acetate for 24 hours, cyclin D1 expression gradually decreased with exposure to increasing lead acetate concentrations （1.0-10 umol/L）. CONCLUSION： Lead exposure to primary cultured rat hippocampal neurons resulted in dose-dependently disturbed cellular homeostasis, including DNA damage, reduced cyclin D1 expression, and stagnation of cell-cycle progression.

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.

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.

A traditional Chinese medicine Huaier triggers G1 cell cycle arrest and apoptosis through cyclins-CDKs-CKIs machinery in MOLT4 cells

Objective: The purpose of the study was to study the effect of Huaier, a traditional Chinese medicine, on the cell cycle adjustment in MOLT4 cells in vitro. Methods: We used MTT assay to test cell viability, flow cytometry to detect cell cycle and apoptosis and western blot to examine the expression of cell-cycle and apoptotic proteins in MOLT4 cells induced by Huaier. Results: Huaier could reduce the viability of MOLT4 cell by inducing G1 arrest and apoptosis. The induction of apoptosis after treatment with Huaier for 24 h was demonstrated in a dose- and time-dependent manner by flow cytometry analysis. G1 arrest induced by Huaier was modulated through the increased expression of Cdki proteins(p21cip/waf1 and p27kip1) with a simultaneous decrease in Cdk2, Cdk4, Cdk6, cyclin D1 and cyclin E expression. Huaier also induced Bax and Bcl-2 expression and activation of Caspase-3. Conclusion: It is firstly demonstrated that Huaier can inhibit proliferation of MOLT4 cells via G1 arrest and apoptosis. These results suggest that Huaier is a cell-cycle anti-cancer drug.

Non-aqueous extracts of Curcuma mangga rhizomes induced cell death in human colorectal adenocarcinoma cell line(HT29) via induction of apoptosis and cell cycle arrest at G_0/G_1 phase

Objective:To investigate the cytotoxic activity of the hexane and ethyl acetate extracts of Curcuma mangga rhizomes against human colorectal adenocarcinoma cell lines(HT29).Methods:The cytotoxic activity of the hexane and ethyl acetate extracts of Curcuma mangga rhizomes against human colorectal adenocarcinoma cell lines(HT29) was determined by using the SRB assay.Results:The ethyl acetate extract showed a higher cytotoxic effect compared to the hexane extract.Morphological changes of the HT29 cells such as cell shrinkage,membrane blebbling and formation of apoptotic bodies while changes in nuclear morphology like chromatin condensation and nuclear fragmentation were observed.Further evidence of apoptosis in HT29 cells was further supported by the externalization of phosphatidylserine which indicate early sign of apoptosis.Conclusions:The early sign of apoptosis is consistent with the cell cycle arrest at the G_0/G_1 checkpoint which suggests that the changes on the cell cycle lead to the induction of apoptosis in HT29.

Growth arrest-specific gene 2 suppresses hepatocarcinogenesis by intervention of cell cycle and p53-dependent apoptosis

BACKGROUND Growth arrest-specific gene 2(GAS2)plays a role in modulating in reversible growth arrest cell cycle,apoptosis,and cell survival.GAS2 protein is universally expressed in most normal tissues,particularly in the liver,but is depleted in some tumor tissues.However,the functional mechanisms of GAS2 in hepatocellular carcinoma(HCC)are not fully defined.AIM To investigate the function and mechanism of GAS2 in HCC.METHODS GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting.Cell proliferation was analyzed by counting,MTS,and colony formation assays.Cell cycle analysis was performed by flow cytometry.Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting.RESULTS GAS2 protein expression was lower in HCC than in normal tissues.Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53,while knockdown of GAS2 promoted the proliferation of hepatocytes(P<0.05).Furthermore,GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells,particularly the elevation of sub G1(P<0.01).Apoptosis induced by GAS2 was dependent on p53,which was increased by etoposide addition.The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated,but became diminished upon downregulation of GAS2.In the clinic specimen,GAS2 was downregulated in more than 60%of HCCs.The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues(P<0.05).CONCLUSION GAS2 plays a vital role in HCC cell proliferation and apoptosis,possibly by regulating the cell cycle and p53-dependent apoptosis pathway.

Cucurbitacin E inhibits the proliferation of hepatoma cells in vitro and in vivo through induction of G2/M phase arrest

Objective Cucurbitacins are the highly oxygenated tetracyclic triterpenes,which are predominantly found in the Cucurbitaceae family but are also present in several other families of the plant kingdom.A number of compounds of this group have been investigated for their cytotoxic,hepatoprotective,anti-inflammatory,cardiovascular and anti-diabetic activities.In China,the cucurbitacin preparation,which contains mostly cucurbitacin B and cucurbitacin E,has been clinically used for the treatment of the primary liver carcinoma.It has been previously reported that cucurbitacin E could produce cytotoxicity against a variety of cancer cells,and various mechanisms were implicated in its cytotoxic effect.The present study is to investigate the effect of cucurbitacin E on hepatoma cells in vitro and in vivo and to study their potential mechanisms of action.Methods The MTT assay was used to assess the viability of human HepG2 and BEL7402 hepatoma cells in vitro after treatment with different concentrations of cucurbitacin E.The cell cycle distribution was determined by flowcytometric analysis after propidium iodide(PI)staining.The cell cycle-related proteins were detected using western blotting analysis.Implanted mouse hepatoma H22 model was built to evaluate the growth inhibitory effect of cucurbitacin E in vivo in mice.Results Our studies found that cucurbitacin E(10-300 nM)produced anti-proliferative effect on human HepG2 and BEL7402 hepatoma cells in vitro without cytotoxicity.According to flowcytometric analysis,cucurbitacin E arrested the cell cycle at G2/M phase in both HepG2 and BEL7402 hepatoma cells after 24 h treatment.Cucurbitacin E induced the decrease in the level of CDK1 protein and the increase in the level of p21 protein,but had no effect on the levels of cyclin A,cyclin B1 and Cdc25C protein.In in vivo anti-tumor experiment,cucurbitacin E had significant inhibitory effects on the growth of mouse H22 hepatoma cells.Conclusions Cucurbitacin E inhibited the proliferation of hepatoma cells in vitro and in vivo,at least in part,through induction of cell cycle arrest at G2/M phase,which was mediated by concomitant upregulation of p21 and downregulation of CDK1.We consider that cucurbitacin E may be useful in the treatment of liver cancer.

Long non-coding RNA GAS5 promotes PC12 cells differentiation into Tuj1-positive neuron-like cells and induces cell cycle arrest

Growth arrest-specific 5 (GAS5) is an anti-oncogene that has been extensively studied in tumors. However, research on GAS5 in the context of nervous system disease is rare at present. This study aimed to investigate the role of the long non-coding RNA GAS5 in rat pheochromocytoma cells (PC12 cells). GAS5-overexpressing lentivirus was transfected into PC12 cells, and expression levels of GAS5 and C-myc were detected by real-time PCR. Ratios of cells in S phase were detected by 5-ethynyl-2′-deoxyuridine. Immunohistochemical staining was used to detect the immunoreactivity of neuron microtubule markers Tuj1, doublecortin, and microtubule-associated protein 2. Apoptosis was detected by flow cytometry, while expression of acetylcholine in cells was detected by western blot assay. We found that GAS5 can promote PC12 cells to differentiate into Tuj1-positive neuron-like cells with longer processes. In addition, cell proliferation and cell cycle were significantly suppressed by GAS5, whereas it had no effect on apoptosis of PC12 cells. Our results indicate that GAS5 could increase the expression of choline acetyltransferase and acetylcholine release. Thus, we speculate that GAS5 is beneficial to the recovery of neurons and the cholinergic nervous system.