Morphological Changes Of Apoptotic Cells Research Articles

Endoplasmic reticulum stress mediates sulforaphane-induced apoptosis of HepG2 human hepatocellular carcinoma cells.

Sulforaphane (SFN) is a naturally occurring chemopreventive agent, which effectively inhibits proliferation of HepG2 human hepatocellular carcinoma cells via mitochondria‑mediated apoptosis. Endoplasmic reticulum stress is considered the most important cause of cell apoptosis; therefore, the present study aimed to determine whether the endoplasmic reticulum pathway was involved in SFN-induced apoptosis of HepG2 cells. An MTT assay was used to detect the inhibitory effects of SFN on HepG2 cells. Fluorescence microscopy was used to observe the morphological changes in apoptotic cells, and western blot analysis was conducted to detect the expression of binding immunoglobulin protein (Bip)/glucose-regulated protein78 (GRP78), X‑box binding protein‑1 (XBP‑1) and BH3 interacting domain death agonist (Bid). Furthermore, flow cytometry was used to determine the apoptotic rate of HepG2 cells, and the protein expression of C/EBP homologous protein (CHOP)/growth arrest‑ and DNA damage‑inducible gene153 (GADD153) and caspase-12 in HepG2 cells. The results indicated that SFN significantly inhibited the proliferation of HepG2 cells; the half maximal inhibitory concentration values were 32.03±0.96, 20.90±1.96 and 13.87±0.44µmol/l, following treatment with SFN for 24, 48and72h, respectively. Following 48h of SFN treatment (10, 20and40µmol/l), the apoptotic rates of HepG2 cells were 31.8, 61.3and77.1%, respectively. Furthermore, after 48h of exposure to SFN, the cells presented typical morphological alterations of apoptosis, as detected under fluorescence microscopy. Treatment with SFN for 48h also significantly upregulated the protein expression levels of Bip/GRP78, XBP‑1, caspase‑12, CHOP/GADD153 and Bid in HepG2 cells. In conclusion, endoplasmic reticulum stress may be considered the most important mechanism underlying SFN-induced apoptosis in HepG2 cells.

Naringenin ameliorates hypoxia/reoxygenation-induced endoplasmic reticulum stress-mediated apoptosis in H9c2 myocardial cells: involvement in ATF6, IRE1α and PERK signaling activation.

Naringenin, a flavanone mainly derived from grapes and citrus fruits, has been reported to exhibit cardioprotective effects. Accumulating evidence has confirmed that endoplasmic reticulum (ER) stress-mediated apoptosis participates in the process of myocardial ischemia/reperfusion injury and inhibiting ER stress is a potential therapeutic target/strategy in preventing cardiovascular diseases. Herein, the current study was designed to investigate whether naringenin protects H9c2 myocardial cells against hypoxia/reoxygenation (H/R) injury via attenuating ER stress or ER stress-mediated apoptosis. Our results showed that naringenin treatment resulted in obvious increases in the viability of H9c2 cells and the expression of Bcl-2 (anti-apoptotic protein), and decreases in the morphological changes of apoptotic cells, the activity of caspase-3 and the expression of Bax (pro-apoptotic protein) in H/R-treated H9c2 cells, implying the protective effects of naringenin against H/R-induced injury. In addition, naringenin also significantly reversed H/R-induced ER stress as evidenced by the up-regulation of Glucose-regulated protein 78, C/EBP homologous protein and Cleaved caspase-12 proteins. Meanwhile, naringenin remarkably reversed H/R-induced the increases in the expression of cleaved activating transcription factor 6 (ATF6) and phosphorylation levels of phospho-extracellular regulated protein kinases (PERK) and inositol-requiring enzyme-1α (IRE1α) in H9c2 cells. Finally, we found that ATF6 siRNA, PERK siRNA or IRE1α siRNA abolished H/R-induced cytotoxicity and apoptosis in H9c2 cells. In conclusion, these results confirmed that ER stress-mediated apoptosis contributes to the protection effects of naringenin against H/R injury, which is potentially involved in ATF6, IRE1α and PERK signaling activation.

Study on effect and mechanism of ellagic acid on the proliferation and apoptosis of pancreatic cancer cell line PANC-1

Objective To investigate the effect of ellagic acid on the proliferation and apoptosis of pancreatic cell line PANC-1 in vitro and explore possible mechanism of ellagic acid's effect on PANC-1 cells. Methods PANC-1 cells were treated with ellagic acid at concentrations of 0, 2, 4, 6, 8 μg/ml for 24, 48 and 72 h respectively. Inverted Microscope showed cells' morphological changes. The cell proliferation was determined by methyl thiazolyl tetrazolium (MTT) assay. When the cells were treated with ellagic acid at concentrations of 0, 2, 4, 6 μg/ml for 48 h, the morphological changes of apoptotic cells were observed by cell staining, apoptotic rate and cell cycle were detected by flow cytometry, the protein expression of cyclo-oxgenase 2 (COX-2) and nuclear factor-κB (NF-κB) were measured by Western blotting. Results Ellagic acid could significantly inhibited the growth of PANC-1 cells in a concentration-dependent and time-dependent manner. The difference among these groups were statistically significant(P<0.05). After treatment with ellagic acid, apoptosis of PANC-1 cells was seen. Apoptosis rates in the different concentration groups were respectively (11.18±0.99)%, (20.70±2.32)%, (39.68±0.69)%, the apoptosis rate of control group [(0.62±0.23)%] compared with statistically significant difference among the different concentration groups (P<0.05). The percentages of G0/G1 phase cells were respectively(48.98±2.36)%, (60.80±1.40)%, (72.82±0.59)%, the percentage of control group [(42.78±0.74)%] compared with statistically significant difference among the different concentration groups(P<0.05). The protein expression of COX-2 and NF-κB measured by Western blotting were significantly down-regulated dose-dependently. Conclusion Ellagic acid can inhibit the proliferation of pancreatic cancer cell line PANC-1 and induce apoptosis. The mechanism may depend on arresting cell cycle and decreasing protein expression of COX-2 and NF-κB. Key words: Ellagic acid; Pancreatic neoplasm; Proliferation; Apoptosis; Cyclo-oxgenase 2; Nuclear factor-κB

Effects and Mechanism of Baicalin on Apoptosis of Cervical Cancer HeLa Cells I n -v itro.

The objective of this study was to observe the apoptosis-inducing effect and mechanism of baicalin on human cervical cancer HeLa cells. The inhibitory effect of baicalin on the growth of HeLa cells was measured by MTT assay, and cell proliferation and migration was analyzed by cell scratch assay. Morphological changes of apoptotic cells were viewed by the light microscope and electron microscope, and cell growth arrest was confirmed by flow cytometry. Moreover, Western blot was used for investigating the expression of apoptosis related proteins; spectrophotometry was used to examine Caspase-3 activation. Our results showed that baicalin could inhibit the proliferation of HeLa Cells via induction of apoptosis in a time and dose-dependent manner (P<0.01). Apoptotic signaling induced by baicalin was characterized by up-regulating Bax, Fas, FasL and Caspase-8 protein expression, and down-regulating of Bcl-2 protein expression. These results indicated that baicalin-induced apoptosis involved activation Caspase-3 in HeLa cells through the intracellular mitochondrial pathway and the surface death receptor pathway.

Baicalein induces apoptosis of human cervical cancer HeLa cells in vitro.

A number of studies have shown that baicalein shows high antitumor activity in vitro and in vivo. In this study, the inhibitory effect of baicalein on human cervical cancer HeLa cells was studied in vitro. HeLa cells were treated with high (100 µg/ml) and low (50 µg/ml) doses of baicalein, and cell growth inhibition rates were examined by the MTT assay. The morphological changes of apoptotic cells were observed under the light and electron microscope, while the rate of cell apoptosis was examined by flow cytometry. The expression of apoptosis-related proteins was analyzed by western blot, and caspase-3 activation was examined by a caspase-3 activity assay and spectrophotometry. The results demonstrated that baicalein inhibits the proliferation of HeLa cells and induces apoptosis in a caspase-3-dependent pathway, through downregulation of the B-cell lymphoma 2 (Bcl-2) protein and upregulation of the Bcl-2-associated X protein (Bax), Fas, Fas ligand (FasL) and caspase-8. Thus, we conclude that baicalein induces apoptosis of HeLa cells via the mitochondrial and the death receptor pathways. Cell apoptosis in HeLa cells was most likely promoted by the activation of the proteolytic enzyme caspase-3 in both pathways.

P38 MAPK and ERK1/2 pathways are involved in the pro-apoptotic effect of notoginsenoside Ft1 on human neuroblastoma SH-SY5Y cells

AimsThis study aims to investigate the effect and the mechanisms of notoginsenoside Ft1, a natural compound exclusively found in P. notoginseng, on the proliferation and apoptosis of human neuroblastoma SH-SY5Y cells. Main methodsCCK-8 assay was used to assess the cell proliferation. Flow cytometry was performed to measure the cell cycle distribution and cell apoptosis. Hoechst 33258 staining was conducted to confirm the morphological changes of apoptotic cells. Protein expression was detected by western blot analysis and caspase 3 activity was measured by colorimetric assay kit. Key findingsAmong the saponins examined, Ft1 showed the best inhibitory effect on cell proliferation of SH-SY5Y cells with IC50 of 45μM. Ft1 not only arrested the cell cycle at S, G2/M stages, but also promoted cell apoptosis, which was confirmed by Hoechst 33258 staining. Further studies demonstrated that Ft1 up-regulated the protein expressions of cleaved caspase 3, phospho-p53, p21, and cyclin B1, but down-regulated that of Bcl-2. Moreover, Ft1 enhanced the phosphorylation of ERK1/2, JNK and p38 MAPK. However, the phosphorylation of Jak2 and p85 PI3K was reduced by Ft1. Inhibitors of p38 MAPK and ERK1/2 but not JNK abrogated the up-regulated protein expressions of cleaved caspase 3, p21 and down-regulated protein expression of Bcl-2 as well as elevated caspase 3 activity induced by Ft1. SignificanceFt1 arrested the proliferation and elicited the apoptosis of SH-SY5Y cells possibly via p38 MAPK and ERK1/2 pathways, which indicates the potential therapeutic effect of it on human neuroblastoma.

Enhanced sensitivity of breast cancer MDA-MB-231 cells to chemotherapy by silencing forkhead box C2 gene with RNA interference technology

Objective To observe the effects of forkhead box C2 gene (FOXC2) short hairpin RNA on docetaxel chemotherapy sensitivity of human breast cancer MDA-MB-231 cells.Methods MDA-MB-231 cells were transfected with recombinant expression plasmid FOXC2-small interfering RNA (siRNA) by lipofectamine 2000,and the expression of FOXC2 mRNA and protein was detected at 48 h after transfection.The morphological changes of apoptotic cells were observed under fluorescent microscope at 72 h after transfection.Methyl thiazol tetrazolium (MTT) assay was used to examine the inhibitory rate and the 50％ inhibitory concentration (IC50).Flow cytometry was applied to examine apoptosis and cell cycle at 72nd h after transfection.Results As compared with negative control plasmid and FOXC2-siRNA non-transfection group,the typical apoptotic morphology of MDA-MB-231 cells was observed,and the mRNA and protein expression levels of FOXC2 were obviously reduced in FOXC2-siRNA transfection group (P ＜ 0.05).Silencing FOXC2 increased sensitivity of MDA-MB-231 cells to docetaxel (P ＜ 0.05),and IC50 was decreased from (6.08 ±1.23) mg/L to (0.65 ±0.01) mg/L.Meanwhile,the apoptosis rate of MDA-MB-231 cells transfected with pFOXC2-shRNA was (20.01 ± 0.19)％,which was significantly higher than other three groups,and the proportion of cells in S phase and G0/G1 phase was decreased and that in G2/M phase was increased (P ＜0.05).Conclusion Down-regulation of FOXC2 expression by siRNA in breast cancer MDA-MB-231 cells could effectively induce apoptosis of MDA-MB-231 cells and increase their sensitivity to docetaxel. Key words: Breast cancer; Docetaxel; Apoptosis; Sensitizing effect

Apoptosis inducing effect of cell exogenous ceramide on human bladder cancer cells and mechanism

Objective To study the apoptosis inducing effect of cell exogenous ceramide on human bladder cancer cells and the mechanism.Methods Human bladder cancer T24 cells were treated with 10,20,40 μmol/L of C2-ceramide (C2-cer),and observed by inverted microscope.The morphological changes of apoptotic cells were examined by fluorescent staining with acridine orange (AO).Flow cytometry (FCM) was used to detect apoptosis rate,and Western blotting was done to detect the change of intracellular distribution of apoptosis inducing factor (AIF).Results Under the action of C2-cer,significant morphological changes of apoptosis appeared in T24 cells.The apoptosis rate in control group and C2-cer-treated (10,20,40 μmol/L) groups was 2.95％,14.23％,29.12％ and 48.46％,respectively.The apoptosis rate in C2-cer-treated groups was significantly higher than in control group (P ＜ 0.01).AIF in control group was located only in mitochondrion.The AIF amount in mitochondria of C2-cer-treated groups was significantly less than in control group (t =25.3,P ＜0.01).At the saMe time,AIF could be detected in the nucleus of C2-cer-treated groups.Conclusion Cell exogenous ceramide may induce dose-dependent apoptosis of human bladder cancer cells.The transferring of AIF from mitochondria to cell nucleus may play a critical role during this process. Key words: Ceramide ; Bladder neoplasms ; Apoptosis inducing factor

Curcumin induces apoptosis in gallbladder carcinoma cell line GBC-SD cells

BackgroundGallbladder carcinoma is a malignant tumor with a very low 5-year survival rate because of the difficulty with its early diagnosis and the very poor prognosis of the advanced cancer state. The aims of this study were to determine whether curcumin could induce the apoptosis of a gallbladder carcinoma cell line, GBC-SD, and to clarify its related mechanism.MethodsFirst, the anti-proliferative activities of curcumin-treated and untreated GBC-SD cells were determined using the MTT and colony formation assays. Then, the early apoptosis of cells was detected by the annexin V/propidium iodide double-staining assay and Hoechst 33342 staining assay. Detection of mitochondrial membrane potential was used to validate the ability of curcumin on inducing apoptosis in GBC-SD cells. Cell cycle changes were detected by flow cytometric analysis. Finally, the expressions of the apoptosis-related proteins or genes caspase-3, PARP, Bcl-2, and Bax were analyzed by western blot and quantitative real time PCR assay. Statistical analyses were performed using the Student’s t-test for comparison of the results obtained from cells with or without curcumin treatment.ResultsThe MTT assay revealed that curcumin had induced a dose- and a time-dependent decrease in cell viability. Colony counting indicated that curcumin had induced a dose-dependent decrease in the colony formation ability in GBC-SD cells. Cells treated with curcumin were arrested at the S phase, according to the flow cytometric analysis. A significant induction of both the early and late phases of apoptosis was shown by the annexin V-FITC and PI staining. Morphological changes in apoptotic cells were also found by the Hoechst 33342 staining. After treatment with curcumin fluorescence shifted from red to green as ΔΨm decreased. Furthermore, western blot and quantitative real time PCR assays demonstrated that the curcumin induced apoptosis in GBC-SD cells by regulating the ratio of Bcl-2/Bax and activating the expression of cleaved caspase-3.ConclusionsTaken together, the results indicate that curcumin may be a potential agent for the treatment of gallbladder cancer.

Anti-metastatic Effects on B16F10 Melanoma Cells of Extracts and Two Prenylated Xanthones Isolated from Maclura amboinensis Bl. Roots

Inhibitory effects of Maclura amboinenesis Bl, one plant used traditionally for the treatment of cancers, on metastatic potential of highly metastatic B16F10 melanoma cells were investigated in vitro. Cell proliferation was assessed using the MTT colorimetric assay. Details of metastatic capabilities including invasion, migration and adhesion of B16F10 melanoma cells were examined by Boyden Chamber invasion and migration, scratch motility and cell attachment assays, respectively. The results demonstrated that n-hexane and chloroform extracts exhibited potent anti-proliferative effects (p<0.01), whereas the methanol and aqueous extracts had less pronounced effects after 24 h exposure. Bioactivity-guided chromatographic fractionation of both active n-hexane and chloroform extracts led to the isolation of two main prenylated xanthones and characterization as macluraxanthone and gerontoxanthone-I, respectively, their structures being identified by comparison with the spectral data. Interestingly, both exhibited potent effective effects. At non-toxic effective doses, n-hexane and chloroform extracts (10 and 30 μg/ml) as well as macluraxanthone and gerontoxanthone-I (3 and 10 μM) significantly inhibited B16F10 cell invasion, to a greater extent than 10 μM doxorubicin, while reducing migration of cancer cells without cellular cytotoxicity. Moreover, exposure of B16F10 melanoma cells to high concentrations of chloroform (30 μg/ml) and geratoxanthone-I (20 μM) for 24 h resulted in delayed adhesion and retarded colonization. As insights into mechanisms of action, typical morphological changes of apoptotic cells e.g. membrane blebbing, chromatin condensation, nuclear fragmentation, apoptotic bodies and loss of adhesion as well as cell cycle arrest in the G1 phase with increase of sub-G1 cell proportions, detected by Hoechst 33342 staining and flow cytometry were observed, suggesting DNA damage and subsequent apoptotic cell death. Taken together, our findings indicate for the first time that active n-hexane and chloroform extracts as well as macluraxanthone and gerontoxanthone-I isolated from Maclura amboinensis Bl. roots affect multistep of cancer metastasis processes including proliferation, adhesion, invasion and migration, possibly through induction of apoptosis of highly metastatic B16F10 melanoma cells. Based on these data, M. amboinensis Bl. represents a potential candidate novel chemopreventive and/or chemotherapeutic agent. Additionally, they also support its ethno-medicinal usage for cancer prevention and/or chemotherapy.

Experimental study of emodin inducing apoptosis of bile duct carcinoma cell QBC939

Objective To investigate the effect and mechanism of emodin inducing the apoptosis of human bile duct carcinoma cell QBC939 in vitro. Methods The inhibition of cell proliferation was detected by MTT. The morphological changes of apoptotic cells were observed under fluorescence microscopy.The apoptosis rates and intracellular reactive oxygen species levels were detected by flow cytometry.The intracellular relative activities of caspase-9 and caspase-3 were tested through colorimetric method.Results Emodin inhibited cell proliferation of human bile duct carcinoma cell QBC939 in time-dose dependent fashion.Apoptotic cells displayed bright,nuclear presented divided leaves,debris and a set of edges under fluorescence microscopy.Treated with 30 μmol/L and 50 μmol/L emodin,the apoptosis rates of 24 hours were respectively 38.9％ ± 9.07％ and 67.09％ ± 4.08％ (P ＜ 0.05).The intracellular reactive oxygen species levels after 30 minutes treatment were 1.65 ± 0.08 and 2.28 ± 0.04folds of the control group (P ＜ 0.05). Emodin could activate caspase-9 and caspase-3,leading to elevations of their activities (P ＜ 0.05).Conclusion Emodin inhibites cell proliferation of human bile duct carcinoma cell QBC939 through inducing apoptosis.The mechanism is associated with the elevated levels of reactive oxygen species and the activation of caspase-9 cells and caspase-3. Key words: Emodin; Apoptosis; Reactive oxygen species; Caspases

Nimesulide inhibited the growth of hypopharyngeal carcinoma cells via suppressing Survivin expression

BackgroundThe objective of this study was to evaluate the efficacy of Nimesulide, a selective cyclooxygenase-2 (COX-2) inhibitor, on the growth of hypopharyngeal carcinoma cells (FaDu) in vitro, and investigate its potential mechanism.MethodsAfter FaDu cells were treated with graded concentrations of Nimesulide for divergent time, sensitivity of cells to drug treatment was analyzed by MTT assay. Morphological changes of FaDu cells in the presence of Nimesulide were observed by acridine orange cytochemistry staining. Proliferating cells were detected using the 5-Bromo-2'-deoxy-uridine (BrdU) incorporation assay. Following cells were subjected to Nimesulide (500 μmol/l) for 6 h, 12 h and 24 h, the percentage of apoptosis was examined by flow cytometry. We detected COX-2 and Survivin expression change by RT-PCR and Western blot, and analyzed the correlation of them with the growth of FaDu cells. Additionally, we also analyzed Caspase-3, Bcl-2 and Bax expressions as markers to investigate the related pathway of Nimesulide-indued apoptosis.ResultsCompared with the control group, the viabilities rates were decreased by Nimesulide in time- and dose-dependent manners, typical morphological changes of apoptotic cells were observed in the Nimesulide-treatment groups, Nimesulide could suppress the proliferation of FaDu cells significantly. The percentage of apoptosis in FaDu cells were markedly increased after Nimesulide-treatment for 6 h, 12 h and 24 h. Nimesulide down-regulated the Survivin and COX-2 expressions at mRNA and protein levels in FaDu cells. Additional analyses indicated that Bcl-2 expression was significantly decreased and the expressions of Caspase-3 as well as Bax were increased at both mRNA and protein levels.ConclusionsBased on the induction of apoptosis and suppression of proliferation, Nimesulide could inhibit the growth of FaDu cells. Furthermore, the suppression of Survivin expression may play an important role in Nimesulide-induced growth inhibition. Nimesulide could act as an effective therapeutic agent for hypopharyngeal carcinoma therapy.

Molecular mechanism of Aurora kinase inhibitor PHA739358 in inhibited proliferation and induced apoptosis of breast cancer cells

To explore the molecular mechanisms of Aurora kinase inhibitor PHA739358 in inhibited proliferation and in vitro induced apoptosis of breast cancer cells. The in vitro cultured T47D cells in logarithmic growth phase were used. The effects of PHA739358 on cell proliferation were examined by MTT (methyl thiazolyl tetrazolium) assay. The variety of nuclear and spindle morphologies was examined by immunofluorescence. And G2/M arrest was determined by flow cytometry. The morphological changes of apoptotic cells were observed by fluorescent microscope. The levels of Aurora kinase relative protein expression phosphonate-AuroraA (p-AuroraA), AuroraA, phosphonate-histone H3 (p-histone H3), histone H3, cell cycle-specific protein expression CyclinB1, cell cycle regulative protein expression Cdc2, Cdc25c, phosphonate-Cdc2 (p-Cdc2), phosphonate-Cdc25c (p-Cdc25c) and apoptosis relative protein expression PARP, Bcl-2 and Bax were detected by Western blot. The apoptotic rate was examined by flow cytometer. PHA739358 obviously inhibited the proliferation of T47D cells after a 24 h or 48 h treatment in a dose-dependent and time-dependent manner. Their IC50 values were (3.44 ± 0.54) and (0.21 ± 0.67) µmol/L respectively. Flow cytometry showed that G2/M arrested in a dose-dependent manner. PHA739358 dose-dependently and time-dependently promoted the dissection of PARP (poly (ADP-ribose) polymerase); down-regulated the expressions of Bcl-2, p-AuroraA, p-histone H3, CyclinB1 and up-regulated the levels of Bax, p-Cdc25c, p-Cdc2, P21 and P53 protein. PHA739358 had no significant effects on the expressions of AuroraA and histone H3. Flow cytometry and fluorescence microscope showed that PHA739358 significantly induced apoptosis. Flow cytometry showed the rate of apoptosis significantly increased from 0.31% ± 0.03% to 40.6% ± 0.81%. The proliferation-inhibiting and apoptosis-inducing effects of PHA739358 may provide new therapeutic approaches of breast cancer.

Expression of p53 in the Effects of Artesunate on Induction of Apoptosis and Inhibition of Proliferation in Rat Primary Hepatic Stellate Cells

BackgroundActivation of hepatic stellate cells (HSCs) plays an important role in the development of cirrhosis through the increased production of collagen. p53, the “guardian of the genome”, is a transcription factor that can bind to promoter regions of hundreds of genes where it either activates or suppresses gene expression. Thereby, p53 serves as a tumor suppressor by inducing cell cycle arrest, apoptosis, senescence and DNA repair. Artesunate is a derivative of Artemisinin, Scholars had found it had more extensive pharmacological effects past 10 years. However, little is known about the expression of p53 in the effects of Artesunate on induction of apoptosis and inhibition of proliferation in rat HSCs.Methodology/Principal FindingsIsolated and cultured rat primary HSCs in the flask for 10 days to make cells activated. HSCs were divided into two groups: experimental groups and control groups, experimental groups included with various concentrations of Artesunate (125, 150, 175, 200, 225 µmol/L) for 24, 48 and 72 hours. Analysis of MTT revealed that activated HSCs treated with various concentrations of Artesunate (150–225 µmol/L) were inhibited on dose and time-effect relationships; Concentration of hydroxyproline in supernatant was detected by digestive method; Analysis of flow cytometry demonstrated that Artesunate could arrest cell cycle in G1 and induce apoptosis; The nuclear morphological changes in apoptotic cells were evaluated with DNA staining by Hoechst 33258 dye; The expression of p53 were up-regulated showed by western blotting and RT-PCR.ConclusionArtesunate could inhibit HSCs proliferation in dose-dependent and time-dependent manners in vitro through increase the expression of p53.

Eriocaulon buergerianum extract protects PC12 cells and neurons in zebrafish against 6-hydroxydopamine-induced damage

BackgroundEricaulon buergerianum (Gujingcao) is an ophthalmic, anti-inflammatory and antimicrobial Chinese medicinal herb. This study aims to investigate the neuroprotective effects of Ericaulon buergerianum ethanol extract (EBE) and to elucidate its underlying action mechanism.MethodsThe viability of dopaminergic (DA) neuron in zebrafish was examined by anti-tyrosine hydroxylase (TH) immunostaining. The locomotor activity of zebrafish was assessed with a digital video tracking system. The viability and cellular damage of the PC12 cells were determined by MTT and LDH assays respectively. The nuclear morphological changes in apoptotic cells were evaluated with DNA staining by Hoechst 33342 dye. Intracellular nitric oxide (NO) was quantified by DAF-FM diacetate staining. The expression of inducible nitric oxide synthase (iNOS) was determined by Western blot.ResultsEBE inhibited the 6-OHDA-induced decrease in total distance of movement in zebrafish. Pretreatments of EBE (25, 50, 100 and 200 μg/ml) increased the viability of 6-OHDA-damaged PC12 cells in a dose dependent manner. Protection against 6-OHDA-induced nuclear fragmentation and accumulation of apoptotic bodies was also observed in EBE pretreated cells. Anti-oxidative (inhibition of NO production and iNOS expression in PC12 cells in vitro) activities of EBE are related to its neuroprotective effects in 6-OHDA-induced DA neuron damage.ConclusionEBE exhibited significant neuroprotective activities in zebrafish, including recovery of dopaminergic neuron loss caused by 6-OHDA in a dose-dependent manner in vivo, inhibition of 6-OHDA-induced decrease of total distance in movement in zebrafish. The iNOS-NO pathway may be involved.

Nimesulide induced apoptosis and inhibited metastasis of hypopharyngeal carcinoma cells

To investigate the effects of Nimesulide, a selective Cox-2 inhibitor, on the apoptosis, invasion and migration of hypopharyngeal carcinoma cell line (FaDu). Viabilities of FaDu cells treated with various concentrations of Nimesulide were detected by MTT assay. Morphological changes were observed by acridine orange cytochemistry staining. Apoptosis was examined by flow cytometry. The ability of invasion and migration of cells was detected by Transwell chambers. The mRNA and protein expressions of Cox-2, MMP-9 and caspase-3 in response to Nimesulide were examined by RT-PCR and Western blot, respectively. MTT assay showed that the cell surviving rates significantly decreased in time- and concentration-dependent manners (P < 0.05). The typical morphological changes of apoptotic cells were observed. Percent of apoptosis after 6 h Nimesulide treatment was (32.4 ± 6.1)%. The metastatic cells were decreased by Nimesulide to about 20%. Nimesulide decreased the expressions of Cox-2 and MMP-9, whereas increased expression the of Caspase-3. Nimesulide could induce the apoptosis and inhibit metastasis of FaDu cells.

Abstract 211: New synthetic flavone exerts antitumor activity on leukemia cell lines

Abstract Background: Flavonoids are polyphenolic compounds found in dietary components of vegetable origin. They exhibit multiple biological activities, such as antiproliferative, anxiolytic, antiallergic, antiangiogenic and antioxidant actions. We have previously demonstrated that the synthetic flavone 2′-nitroflavone (2′-NF) exerts a strong antimitogenic activity in vitro in various human and murine adenocarinoma cell lines, without disturbing the proliferation of non-neoplastic cells. We also studied the in vivo efficacy of this compound in a breast cancer murine model. In order to evaluate the effect of 2′-NF in haematological diseases, we first tested its antiproliferative potency in different human and murine leukaemia and lymphoma cell lines. In addition, to elucidate the mechanism of action we explored the cell cycle progression and apoptosis induction either in a human as well as a murine cell line. Methods and results: The antiproliferative activity of 2’-NF in several haematological cell lines was initially evaluated at a 20 µM concentration by the MTS assay. IC50 values were then determined from dose-response curves. Among human cell lines, HL-60 cells (acute promyelocytic leukaemia) were the most sensitive to 2′-NF treatment, showing an IC50 of 1±0.5 µM. Among murine cell lines, similar behaviour was observed for NFS-60 (myelogenous leukaemia) and LB02 (T cell leukaemia) cells (IC50 ≈ 8 µM). When peripheral blood mononuclear cells (PBMC) from healthy donors were used as controls, it was shown that PBMC growth was not significantly affected up to a concentration of 80 µM 2’-NF, suggesting a selective action of the nitroflavone derivative on tumor cells. Different experimental assays were then performed to evaluate the in vitro apoptotic response to 2′-NF. Thus, when HL-60 cells were treated in the presence or absence of 20 µM 2′-NF, cell cycle arrest at G2/M phase was detected after 6 h, followed by an evident increment in the proportion of hypodyploid cells in a time dependent manner (3±1% control vs. 38±1% at 14 h; 3±1% control vs. 54±5% at 24 h). A typical DNA ladder fragmentation pattern was visualized by agarose gel electrophoresis at 24 h and morphological changes of apoptotic cells, such as chromatin condensation, cell shrinkage and membrane blebbing, were observed by fluorescence microscopy. Although the expression of antiapoptotic Bcl-2 and Bcl-XL proteins remained unchanged, a 2 fold increment of the pro-apoptotic protein Bax was obtained after 9 h of exposure to 2′-NF. A significant increase in caspase-3 activity was also detected after 9 h of treatment. By using various experimental approaches, we also demonstrated that 2’-NF induced an apoptotic response in murine LB02 cells. Conclusion: Our results suggest that 2’-NF is a potent and selective antitumor compound, which induces cell cycle arrest and apoptosis in human leukaemia cells. The potential therapeutic use of 2’-NF will be next explored in a leukaemia mouse model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 211.

Suppressive effects of genomic imprinted gene PEG10 on hydrogen peroxide-induced apoptosis in L02 cells

The effects of PEG10 on hydrogen peroxide (H2O2)-induced apoptosis in human normal liver cell line L02 were investigated. The PEG10 gene was transfected into L02 cells by lipofectamine, the positive clone was screened by G418 and defined as L02/PEG10, while the cell transfected with empty expression vector (pEGFP-N1) was defined as L02/vector. L02/vector and parental L02 cells served as control. RT-PCR and Western blotting were employed to detect the expression of target genes. H2O2 (50-400 mmol/L) was administered to induce the apoptosis of L02 cells. Cells viability was measured by MTT and the morphological changes of apoptotic cells were determined by fluorescence microscopy using hoechst33342 nuclei staining. DNA fragmentation was observed by agarose gel electrophoresis. PEG10 mRNA and protein levels in L02/PEG10 cells were significantly increased as compared with those in the control cells. After treatment with 400 mmol/L H2O2 for 24 h, the cellular growth inhibition rate of L02/PEG10 cells was significantly lower (58.2%) than that of L02 (92.5%) and L02/vector (88%). Distinct morphological changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were not observed in L02/PEG10. Ladder-like DNA fragmentation in a dose-dependent manner was observed in both L02 and L02/vector cell lines, but not in L02/PEG10. PEG10 over-expression significantly inhibited cytotoxicity induced by H2O2 on human normal liver cell line L02 by antagonizing H2O2-induced apoptosis.

Alteration in TWIST Expression: Possible Role in Paclitaxel-induced Apoptosis in Human Laryngeal Carcinoma Hep-2 Cell Line

To explore the relationship between alteration in the expression of TWIST, highly conserved transcription factor from the basic helix-loop-helix family, and apoptosis of Hep-2 cells induced by chemotherapeutic agent paclitaxel. Morphological changes of Hep-2 cells were observed by acridine orange cytochemistry staining. Viability of Hep-2 cells treated with various concentrations of paclitaxel was examined by cell proliferation assay. Apoptosis was examined by flow cytometry. The mRNA and protein expression of TWIST in response to paclitaxel at 24 hours, 48 hours, and 72 hours was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Typical morphological changes of apoptotic cells at 24 hours, 48 hours, or 72 hours after treatment wiyth paclitaxel (10x10(-9) mol/L) were observed. The cell survival rates significantly decreased in a concentration- and time-dependent manner (P=0.001). Paclitaxel-induced apoptosis increased with culture time (22.6+/-5.3% after 24 hours, 38.7+/-7.9% after 48 hours, and 52.4+/-14.3% after 72 hours; P=0.002). Both mRNA and protein expression of TWIST was markedly decreased at both mRNA levels and protein levels, at 24 hours, 48 hours, and 72 hours in the paclitaxel-induced apoptosis of Hep-2 cells (P<0.001). TWIST, which has a significantly decreased expression in response to paclitaxel in Hep-2 cells, may play a pivotal role in paclitaxel-induced apoptosis of Hep-2 cells.

Role of TWIST in the apoptosis of Hep-2 cells induced by paclitaxel

To explore the relationship between the expression of transcription factor TWIST and apoptosis of Hep-2 cells induced by paclitaxel. Morphological changes of Hep-2 cells were observed by reserved microscopy and acridine orange cytochemistry staining. Viability of Hep-2 cells treated with various concentrations of paclitaxel was detected by MTT assay. Apoptosis was examined by flow cytometry. The expressions of transcription factor TWIST at both mRNA and protein level in response to paclitaxel at 24 h, 48 h and 72 h were then examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. Typical morphological changes of apoptotic cells, i.e., cellular rounding-up, cytoplasmic contraction, chromatin condensation and, particularly, apoptotic body, the main morphological characteristic of apoptosis, were observed by reserved microscopy and acridine orange cytochemistry staining. The cell surviving rates significantly decreased in a concentration- and time-dependent manner as evidenced by MTT assay (P < 0.05). Percent of apoptosis after 24 h, 48 h or 72 h paclitaxel-treatment was (22.6 +/- 5.3)%, (38.7 +/- 7.9)% and (52.4 +/- 14.3)%, respectively, whereas the percent of control was (9.85 +/- 5.83)%. There existed a statistically significant difference between treatment and control (F = 12.621, P < 0.05). The expression of TWIST at both mRNA and protein levels for 24 h, 48 h or 72 h in the paclitaxel-induced apoptosis of Hep-2 cells were decreased by 16.7%, 46.8%, 76.9% (F = 10.407, P < 0.05) and 16.4%, 33.6%, 69.6% (F = 18.013, P < 0.05) respectively. TWIST, which is significantly decreased in expression in response to paclitaxel in Hep-2 cells, may play a pivotal role in paclitaxel-induced apoptosis of Hep-2 cells.