Drug-induced Apoptosis by Anti-microtubule Agent,Estramustine Phosphate on Human Malignant Glioma Cell Line,U87MG; in vitro Study

The drug effect of estramustine phosphate (EMP), an anti-microtubule agent on human glioma cells has been studied with the focus being mainly its cytotoxity or its targeting of organelles. However, the pharmacological knowledge of estramustine with respect to its cytotoxity and mechanism is limited. To acquire such knowledge, the present study investigates the ability of EMP to induce apoptosis in a human malignant glioma cell line. Transmission electron microscope (TEM) images were examined to monitor periodic changes. Agarose gel electrophoresis was also examined. Cellular DNA fragmentation ELISA was performed to investigate the DNA fragmentation rates and an MTT assay was studied to evaluate the ID₅₀. A TEM study revealed condensing and fragmentation of the chromatin. Laddering of the bands was observed in all EMP exposure groups in agarose gel electrophoresis. DNA fragmentation in all EMP groups began at 0.5h following an exposure with EMP and increased in a dose- and time-dependent manner as revealed by DNA ELISA fragmentation. ID₅₀ at 24h was 5.0µM according to the MTT assay, a value close to 4.8µM of ID₅₀ was revealed by the DNA fragmentation assay. None of the above mentioned changes was observed in the control group. These results indicated that EMP caused a drug-induced apoptosis in the human malignant glioma cell line, U87MG.     

Extract of Saccharina japonica Induces Apoptosis companied by Cell Cycle Arrest and Endoplasmic Reticulum Stress in SK-Hep1 Human Hepatocellular Carcinoma Cells

Saccharina japonica is a family member of Phaeophyceae (brown macro-alga) and extensively cultivatedin China, Japan and Korea. Here, the potential anti-cancer effect of n-hexane fraction of S. japonica wasevaluated in SK-Hep1 human hepatocellular carcinoma cells. The N-hexane fraction reduced cell viability andincreased the numbers of apoptotic cells in a both dose- and time-dependent manner. Apoptosis was activatedby both caspase-dependent and independent pathways. The caspase-dependent cell death pathway is mediatedby cell surface death receptors and activated caspase-8 amplified the apoptotic signal either through directactivation of downstream caspase-3 or pro-apoptotic proteins (Bad, Bax and Bak) subsequently leading to therelease of cytochrome c. On the other hand, caspase-independent apoptosis appeared mediated by disruptionof mitochondrial membrane potential and translocation of AIF to the nucleus where they induced chromatincondensation and/or large-scale DNA fragmentation. In addition, the n-hexane fraction induced endoplasmicreticulum (ER)-stress and cell cycle arrest. The results suggested that potential anti-cancer effects of n-hexaneextract from S. japonica on SK-Hep1 cells.     

BCL-2 Family Proteins Modulate Radiosensitivity in Human Malignant Glioma Cells

Radiotherapy is the standard treatment for glioblastoma. Here, we assessed the radiosensitivity of 12 human malignant glioma cell lines in vitro and correlated these data with irradiation-induced cell cycle changes, chemosensitivity profiles and BCL-2 family protein expression. Irradiation at 3Gy failed to cause major cell cycle perturbations. Radioresistance was associated with collateral sensitivity to the topoisomerase II inhibitors, teniposide and doxorubicin. High levels of BCL-X_L and low levels of BAX were independently linked to radioresistance. Ectopic expression of a BAX transgene induced radiosensitization in the LN-18 cell line. Thus, BCL-2 family protein expression modulates radiosensitivity in human glioma cells and targeted alterations in BCL-2 family protein expression are a promising strategy to improve the therapeutic efficacy of radiotherapy for gliomas.     

神经节苷脂诱导U251细胞内质网应激相关凋亡

目的：探讨神经节苷脂（ GM1）对人脑星形胶质瘤细胞U251内质网应激（ ERS）相关凋亡的调控。方法用含不同浓度的GM1培养基培养U251细胞,MTT法检测细胞存活率,乳酸脱氢酶（ LDH）检测细胞损伤,流式细胞术检测细胞凋亡率,Western blotting检测ERS凋亡相关蛋白CHOP及caspase-12表达变化。结果与对照组比较,GM1孵育48 h后,U251细胞存活率明显降低,且具有剂量依赖性（ P＜0.05）;U251细胞培养基中LDH量明显增高,且具有剂量依赖性（P＜0.05）;GM1诱导U251细胞凋亡率明显增高,且具有剂量依赖性（P＜0.05）;GM1诱导细胞内CHOP及caspase-12的表达上调,且有剂量依赖性（P＜0.05）。结论 GM1可诱导U251细胞ERS相关凋亡,为恶性脑胶质瘤的治疗提供新的方向。     

UHRF2 mRNA Expression is Low in Malignant Glioma but Silencing Inhibits the Growth of U251 Glioma Cells in vitro

UHRF2 is a member of the ubiquitin plant homeo domain RING finger family, which has been proven to befrequently up-regulated in colorectal cancer cells and play a role as an oncogene in breast cancer cells. However,the role of UHRF2 in glioma cells remains unclear. In this study, we performed real-time quantitative PCR on32 pathologically confirmed glioma samples (grade I, 4 cases; grade II, 11 cases; grade III, 10 cases; and gradeIV, 7 cases; according to the 2007 WHO classification system) and four glioma cell lines (A172, U251, U373, andU87). The expression of UHRF2 mRNA was significantly lower in the grade III and grade IV groups comparedwith the noncancerous brain tissue group, whereas its expression was high in A172, U251, and U373 glioma celllines. An in vitro assay was performed to investigate the functions of UHRF2. Using a lentivirus-based RNAinterference (RNAi) approach, we down-regulated UHRF2 expression in the U251 glioma cell line. This downregulationled to the inhibition of cell proliferation, an increase in cell apoptosis, and a change of cell cycledistribution, in which S stage cells decreased and G2/M stage cells increased. Our results suggest that UHRF2may be closely related to tumorigenesis and the development of gliomas.     

Quercetin Induces Cell Cycle Arrest and Apoptosis in YD10B and YD38 Oral Squamous Cell Carcinoma Cells

Objective: Oral squamous cell carcinoma (OSCC) exhibits the highest lethality among head and neck cancers. Treatment for OSCC is limited due to diverse side effects. Quercetin is a natural flavonoid compound found in many kinds of plants and foods. Quercetin has been reported to be a modulator of proliferation and survival in various types of cancers due to its cytotoxic effects. We aimed to investigating chemopreventative roles of quercetin in YD10B and YD38 OSCC cells. Methods: For our study, two different types of OSCC cells were used. YD10B cells are tongue SCC cells with the p53 mutation and YD38 cells are lower gingiva SCC cells without the p53 mutation, respectively. The anticancer effects of quercetin were examined by cell viability, cell cycle, annexin-PI staining, and western blot. Result: Our results showed that quercetin decreased cell viability and induced G1 cell cycle arrest in YD10B and YD38 OSCC cells. Moreover, quercetin remarkably decreased the expression of cell cycle upregulating proteins and increased the expression of a CDK inhibitor. Quercetin also significantly increased the number of annexin-V-positive cells in a dose-dependent manner in both types of OSCC cells. This apoptotic potential of quercetin triggered cleavage of PARP followed by activation of p38 MAPK signaling pathway. Conclusion: In conclusion, this study demonstrates that quercetin shows different anti-cancer responses in OSCC with and without p53 mutation, respectively. Despite different p53 status in OSCC cells, quercetin led to apoptotic signals in both cells. Quercetin repressed cell proliferation with G1 cell cycle arrest and apoptosis by activating the p38 signaling pathway in two OSCC cells with different p53 status. These findings might provide new strategy for OSCC therapy by quercetin.     

The Antimicrotubule Drug Estramustine but not Irradiation Induces Apoptosis in Malignant Glioma Involving AKT and Caspase Pathways

Irradiation is one of the cornerstones used in the treatment of malignant glioma. However, the effect is modest and glioma cells generally display a pronounced radio-resistance. In this study, the effect of irradiation, alone and in combination with the antimicrotubule drug estramustine (EaM), was investigated in vitro using the BT4C rat glioma cell line, and in vivo the BT4C rat intracerebral glioma model was used. Apoptosis was detected by analysing DNA laddering, in situ end labelling (ISEL) and Annexin V reactivity. In addition, phosphorylation status of MAPK, JNK, p38, and AKT, proteins involved in pro- and anti-apoptotic signalling pathways was analysed by Western blotting. Irradiation did not induce apoptosis, neither in vitro nor in vivo. EaM, however, induced apoptosis in vivo and in vitro, regardless of whether EaM was given alone, before or after irradiation. When BT4C cells were treated with the caspase-3 inhibitor Ac-DEVD-CHO prior to EaM, the number of apoptotic cells was decreased, indicating an involvement of caspase-3. The signalling pathways regulating apoptosis are complex and involve kinases such as MAPK, JNK, p38 and AKT. Irradiation did not induce any changes in the expression levels or phosphorylation status of these proteins. On the other hand, the phosphorylation level of AKT was reduced after EaM treatment, which might, in part, propose how EaM induces apoptosis in glioma cells.     

Protein Kinase C Inhibition by UCN-01 Induces Apoptosis in Human Glioma Cells in a Time-dependent Fashion

Recent studies in our laboratory have shown that UCN-01 (7-hydroxystaurosporine), which is a derivative of the non-selective protein kinase inhibitor staurosporine that exhibits relative selectivity for protein kinase C (PKC), is a potent inhibitor of glioma growth in in vitro and in vivo models. This agent exhibits both cytotoxic and cytostatic effects, depending on the time period of drug exposure. In the present study, we examined whether UCN-01-induced cytotoxicity correlated with the induction of apoptosis, and characterized further the time course of this process as a prelude to application of UCN-01 in clinical trials. We first demonstrated that the cytotoxic effects of UCN-01 were associated with the induction of morphological features of apoptosis. Secondly. we identified electrophoretic features of apoptosis semiquantitatively at a series of time points using field inversion gel electrophoresis. These studies showed a peak in the induction of high-molecular-weight DNA fragmentation after 3–6 days of drug treatment. Thirdly, we measured the percentage of cells undergoing apoptosis at various time points using a terminal transferase-catalyzed in situ end-labeling technique, which confirmed a time- and concentration-dependent increase in apoptotic cell numbers. This correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion. Cell killing peaked within 2–4 days after beginning UCN-01 treatment, but continued at a lower level in the ensuing days. Taken together, these studies demonstrated that extended periods of exposure to UCN-01 are needed for optimal manifestation of cytotoxic effects against glioma cells, a factor that must be taken into consideration in the design of future clinical trials with this agent for malignant gliomas.     

Synergistic Cytotoxicity, Apoptosis and Protein-linked DNA Breakage by Etoposide and Camptothecin in Human U87 Glioma Cells: Dependence on Tyrosine Phosphorylation

In this study, simultaneous administration of certain inhibitors of topoisomerase I and topoisomerase II produced synergistic cytotoxicity in a series of human glioma cell lines. Camptothecin (CPT) and etoposide (VP-16) produced combination indices (CI) <1.0 in all glioma cell lines tested, including those that were relatively resistant to the two topoisomerase inhibitors individually. In contrast, CPT and VP-16 produced additive cytotoxicity in HT-29 and SW-620 colon carcinoma cell lines. To explore the molecular basis for synergy in glioma cells, we focused on one glioma cell line (U87) in which even sub-cytotoxic doses of CPT potentiated the action of VP-16. Except for genistein (a topo II agent with tyrosine kinase inhibitory function), all topo II inhibitors tested (doxorubicin, ellipticine, and m-AMSA) were synergistic with CPT. While CPT and VP-16 produced cytotoxicity and protein-linked DNA breaks (PLDB) that were supra-additive in U87 glioma cells, CPT and genistein produced additive results. Pretreatment of U87 cells with the tyrosine kinase inhibitor tyrphostin-A23 or the tyrosine phosphatase activator O-phospho-L-tyrosine (OPLT) reduced combination PLDB from synergistic to additive levels, but had no effect on the formation of PLDB induced by either CPT or VP-16 alone. CPT and VP-16 also produced a synergistic accumulation of sub-G0 (apoptotic) cells which was blocked by tyrphostin-A23. No significant increase in topoisomerase protein levels could be detected in response to combination treatment. Thus, synergistic effects between topoisomerase I and topoisomerase II inhibitors in U87 glioma cells may depend upon phosphorylation of cellular proteins other than the topoisomerases themselves.     

Xanthoxyletin,a Coumarin Induces S Phase Arrest and Apoptosis in Human Gastric Adenocarcinoma SGC-7901 Cells

This study was conducted to explore the novel anticancer compounds from Chinese herbs. During the processof screening, to evaluate the potential chemopreventive effect of natural compounds, Xanthoxyletin was isolatedfrom Erythrina variegata. It has been reported that Xanthoxyletin possesses antibacterial, fungicidal, and algicidalproperties. In this study, we examined the antiproliferative effects of Xanthoxyletin against SGC-7901 cells andits ability to induce apoptosis and cell cycle arrest for the first time. We observed that its inhibitory effects oncells were associated with the DNA damage, apoptosis through mitochondrial dysfunction, and cell cycle arrestat S phase in a dose-dependent manner. Additionally, Xanthoxyletin also increased the production of reactiveoxygen species in SGC-7901 cells. These results suggest that Xanthoxyletin may be promising anticancer agentand has worth for further mechanistic and therapeutic studies against gastric cancer.     

Iso-suillin Isolated from Suillus luteus,Induces G1 Phase Arrest and Apoptosis in Human Hepatoma SMMC-7721 Cells

Iso-suillin, a natural product isolated from Suillus luteus, has been shown to inhibit the growth of somecancer cell lines. However, the molecular mechanisms of action of this compound are poorly understood. Thepurpose of this study was to investigate how iso-suillin inhibits proliferation and induces apoptosis in a humanhepatoma cell line (SMMC-7721). We demonstrated the effects of iso-suillin on cell proliferation and apoptosisin SMMC-7721 cells, with no apparent toxicity in normal human lymphocytes, using colony formation assaysand 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Western blotting was usedto examine the expression of G1 phase-regulated and apoptosis-associated protein levels in iso-suillin treatedSMMC-7721 cells. The results indicated that iso-suillin significantly decreased viability, induced G1 phase arrestand triggered apoptosis in SMMC-7721cells. Taken together, these results suggest the potential of iso-suillin asa candidate for liver cancer treatment.     

Calotroposid A: a Glycosides Terpenoids from Calotropis gigantea Induces Apoptosis of Colon Cancer WiDr Cells through Cell Cycle Arrest G2/M and Caspase 8 Expression

Objective: This study aims to isolate the active anticancer compound from ethyl acetate fraction extracted fromthe roots of Calotropis gigantea and to determine the operating mechanism of the isolates towards WiDr colon cancercells. Methods: the isolation was conducted by using bioassay guided isolation approach method. The cytotoxicpotential was determined by using MTT method. The chemical structure was identified by using UPLCMS/MS andNMR-1H spectroscopy. The cell cycle arrest and apoptosis induction were determined by flow cytometry method.The expression of caspase-8 was determined by immunocytochemistry method. Results: The results showed thatthe active compounds are obtained calotroposid A compound which is glycosides terpenoids. Calotroposide Ais capable of inhibiting the growth of WiDr colon cancer cells at IC50 17.23μg/ml. Cell apoptosis induction took placeand was indicated by cell apoptosis increase, S and G2/M accumulation and by caspase-8 expression. Conclusion:Calotroposide A induces anticancer activity against WiDr colon cancer cells by means of apoptosis induction mechanismthrough extrinsic pathway with increased expression of caspase-8.     

Silibinin Inhibits Proliferation,Induces Apoptosis and Causes Cell Cycle Arrest in Human Gastric Cancer MGC803 Cells Via STAT3 Pathway Inhibition

Background: To investigate the effect of silibinin on proliferation and apoptosis in human gastric cancer cell line MGC803 and its possible mechanisms. Materials and Methods: Human gastric cancer cell line MGC803 cells were treated with various concentration of silibinin. Cellular viability was assessed by CCK-8 assayandapoptosis and cell cycle distribution by flow cytometry. Protein expression and mRNA of STAT3, and cell cycle and apoptosis regulated genes were detected by Western blotting and real-time polymerase chain reaction, respectively. Results: Silibinin inhibits growth of MGC803 cells in a dose- and time-dependent manner. Silibinin effectively induces apoptosis of MGC803 cells and arrests MGC803 cells in the G2/M phase of the cell cycle, while decreasing the protein expression of p-STAT3, and of STAT3 downstream target genes including Mcl-1, Bcl-xL, survivin at both protein and mRNA levels. In addition, silibinin caused an increase in caspase 3 and caspase 9 protein as well as mRNA levels. Silibinin caused G2/M phage arrest accompanied by a decrease inCDK1 and Cyclin B1 at protein and mRNA levels.. Conclusions: These results suggest that silibinin inhibits the proliferation of MGC803 cells, and it induces apoptosis and causes cell cycle arrest by down-regulating CDK1, cyclinB1, survivin, Bcl-xl, Mcl-1 and activating caspase 3 and caspase 9, potentially via the STAT3 pathway.     

Naringenin (Citrus Flavonone) Induces Growth Inhibition, Cell Cycle Arrest and Apoptosis in Human Hepatocellular Carcinoma Cells

Search for new substances with antiproliferative activity and apoptosis inducing potential towards HepG2 cells is important since HCC is notoriously resistant to conventional chemotherapy. Dietary phytochemicals with significant anti-proliferative and apoptosis inducing potential are considered as agents promising for cancer therapy. Naringenin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess strong cytotoxic activity in numerous types of cancer cells. However, the detailed molecular mechanisms of its antiproliferative effects and apoptosis induction are still unclear. In this study, we investigated antiproliferative and apoptosis-inducing effect of naringenin in human hepatocellular carcinoma HepG2 cells. Naringenin was shown to inhibit the proliferation of HepG2 cells resulted partly from an accumulation of cells in the G0_/G1 and G2/M phase of the cell cycle. Naringenin induced a rapid accumulation of p53, which might account for the naringenin-induced G0_/G1 and G2/M phase arrests in Hep G2 cells. In addition, naringenin have been shown to induce apoptosis as evidenced by nuclei damage and increased proportion of apoptotic cells detected by flow cytometry analysis. Naringenin triggered the mitochondrial-mediated apoptosis pathway as shown by an increased ratio of Bax/Bcl-2, subsequent release of cytochrome C, and sequential activation of caspase-3. Our results showed that naringenin had inhibitory effect on the growth of HepG2 cell line through inhibition of cell proliferation and apoptosis induction. The elucidation of the drug targets of naringenin on inhibition of tumor cells growth should enable further development of naringenin for liver cancer therapy.     

SPARC Modulates Cell Growth,Attachment and Migration of U87 Glioma Cells on Brain Extracellular Matrix Proteins

We have identified secreted protein acidic and rich in cysteine (SPARC) as a potential glioma invasion-promoting gene. To determine whether SPARC alters the growth, attachment, or migration of gliomas, we have used U87T2 and doxycycline-regulatable SPARC-transfected clones to examine the effects of SPARC on (1) cell growth, (2) cell cycle progression, (3) cell attachment, and (4) cell migration, using growth curves, flow cytometry, attachment, and migration analyses on different brain ECMs, including collagen IV, laminin, fibronectin, vitronectin, hyaluronic acid, and tenascin. Our data indicate that SPARC delays tumor cell growth in the log phase of the growth curve. The clones secreted different levels of SPARC. The clone secreting the lowest level of SPARC was associated with a higher percentage of cells in G₂M, whereas the clones secreting the higher levels of SPARC were associated with a greater percentage of cells in G₀/G₁. In comparison to the parental U87T2 clone, the SPARC-transfected clones demonstrated increased attachment to collagen, laminin, hyaluronic acid, and tenascin, but not to vitronectin or fibronectin. SPARC-transfected clones also demonstrated altered migration on the different extracellular matrix proteins. The modulation of migration, either positive or negative, was associated with changes in the level of secreted SPARC. These data suggest that SPARC may modulate glioma proliferation and invasion by modulating both the growth and migration of glioma cells.     

miR-192在胶质瘤U251细胞中的表达及对其增殖、迁移及凋亡能力的影响

目的 探讨miR-192在胶质瘤U251细胞中的表达及对其增殖、迁移及凋亡等生物学能力的影响.方法 采用RT-PCR检测胶质瘤细胞株U251、LN18、U373和正常人脑胶质细胞株中HEB的表达水平.采用脂质体转染法将miR-192类似物(miR-192 mimics)和阴性对照(miR-negtive control...     

Glaucocalyxin A Activates FasL and Induces Apoptosis Through Activation of the JNK Pathway in Human Breast Cancer Cells

This study was conducted to analyze the molecular mechanisms responsible for anti-proliferation effects ofglaucocalyxin A in cultured MCF-7 and Hs578T breast cancer cells. The concentration that reduced cell viabilityto 50% (IC50) after 72 h treatment was derived and potential molecular mechanisms of anti-proliferationusing the IC50 were investigated as changes in cell cycle arrest and apoptosis. Gene and protein expressionchanges related to apoptosis were investigated by semi-quantitative RT-PCR and western blotting, respectively.Involvement of phosphorylated mitogen-activated protein kinases and JNK signaling in regulation of thesemolecules was characterized by western blotting. Cell viability decreased in a concentration-dependent mannerand the IC50 was determined as 1μM in MCF-7 and 4μM in HS578T cell. Subsequently, we demonstrated thatthe GLA-induced MCF-7 and Hst578T cell death was due to cell cycle arrest at the G2/M transition and wasassociated with activation of the c-jun N-terminal kinase (JNK) pathway. We conclude that GLA has the potentialto inhibit the proliferation of human breast cancer cells through the JNK pathway and suggest its applicationforthe effective therapy for patients with breast cancer.     

Kaempferol Suppresses Proliferation and Induces Cell Cycle Arrest,Apoptosis, and DNA Damage in Breast Cancer Cells

Kaempferol is a flavonoid that has been extensively investigated owing to its antitumor effects. Nevertheless, little is known about its underlying mechanisms of action. We aimed to explore the role of kaempferol in breast cancer (BC), and thus we investigated how kaempferol suppresses the growth of BC cells. The cells were treated with kaempferol, and the effects on multiple cancer-associated pathways were evaluated. The MTS assay was used to study the cell growth inhibition induced by kaempferol. The cell cycle was analyzed by flow cytometry. Western blotting was used to analyze cellular apoptosis and DNA damage. We found that the proliferation of the triple-negative BC (TNBC) MDA-MB-231 cells was suppressed effectively by kaempferol. Interestingly, the suppressive effect of kaempferol on cell proliferation was stronger in MDA-MB-231 cells than in the estrogen receptor-positive BT474 cell line. Furthermore, after the treatment with kaempferol for 48 h, the population of cells in the G1 phase was significantly reduced, from 85.48% to 51.35%, and the population of cells in the G₂ phase increased markedly from 9.27% to 37.5%, which indicated that kaempferol contributed to the induction of G₂/M arrest. Kaempferol also induced apoptosis and DNA damage in MDA-MB-231 cells. Kaempferol increased the expression levels of H2AX, cleaved caspase 9, cleaved caspase 3, and p-ATM compared to those of the control group. Collectively, these results showed that kaempferol may be a potential drug for the effective treatment of TNBC.