Shallot and licorice constituent isoliquiritigenin arrests cell cycle progression and induces apoptosis through the induction of ATM/p53 and initiation of the mitochondrial system in human cervical carcinoma HeLa cells

This study is the first to investigate the anticancer effect of isoliquiritigenin (ISL) in human cervical carcinoma HeLa cells. The results reveal that ISL inhibits HeLa cells by blocking cell cycle progression in the G2/M phase and inducing apoptosis. Blockade of cell cycle is associated with increased activation of ataxia telangiectasia‐mutated (ATM). Activation of ATM by ISL phosphorylated p53 at Serine15, resulting in increased stability of p53 by decreasing p53 and murine double minute‐2 (MDM2) interaction. In addition, ISL‐mediated G2/M phase arrest was also associated with decreases in the amounts of cyclin B, cyclin A, cdc2, and cdc25C, and increases in the phosphorylation of Chk2, cdc25C, and cdc2. The specific ATM inhibitor caffeine significantly decreased ISL‐mediated G2/M arrest by inhibiting the phosphorylation of p53 (Serine15) and Chk2. ISL induced apoptotic cell death is associated with changes in the expression of Bax and Bak, decreasing levels of Bcl‐2 and Bcl‐X_L, and subsequently triggering mitochondrial apoptotic pathway. In addition, pretreatment of cells with caspase‐9 inhibitor blocked ISL‐induced apoptosis, indicating that caspase‐9 activation is involved in ISL‐mediated HeLa cell apoptosis. These findings suggest that ISL may be a promising chemopreventive agent against human uterine cervical cancer.     

松乳菇多糖刺激免疫细胞增殖及诱导肿瘤细胞凋亡的研究

为研究松乳菇多糖(Lactarius deliciosus Gray polysaccharide,LDG-A)对特异性免疫细胞的增殖效应活性及对肿瘤细胞凋亡的影响,在LDG-A作用下检测T淋巴细胞、B淋巴细胞的增殖效应,并观察T淋巴细胞、B淋巴细胞的细胞形态,检测T淋巴细胞、B淋巴细胞在LDG-A的作用下处于细胞周期各个时期的细胞数量及B淋巴细胞分泌抗体的情况,观察LDG-A作用于肺癌细胞A549的凋亡和细胞骨架情况。结果表明:随着LDG-A质量浓度的增加,能够促进T淋巴细胞、B淋巴细胞的增殖,并呈现出一定的剂量依赖关系;同时LDG-A能够减少T淋巴细胞、B淋巴细胞在G0/G~1期的细胞数量百分比,促进细胞进入G~2/M期,具有促进B淋巴细胞分泌抗体免疫球蛋白M、免疫球蛋白D和免疫球蛋白E的作用。在12.5~50.0μg/m L范围内,LDG-A能够诱导肺癌细胞A549凋亡,并呈现出一定的剂量依赖关系;在细胞凋亡时F-肌动蛋白丝断裂,蛋白纤维网络结构被破坏。综上所述,在体外实验中,LDG-A对特异性免疫细胞具有促增殖效应活性,能够促进肿瘤细胞的凋亡。     

Characterisation of antioxidant and antiproliferative acidic polysaccharides from Chinese wolfberry fruits

Wolfberry fruit polysaccharides (WFPs) were isolated by hot-water extraction and ethanol precipitation. With HPLC analysis, WFPs were for the first time identified as acidic polysaccharides with galacturonic acid being the main component monosaccharide (24.9%), followed by galactose (21.3%), arabinose (18.5%), and glucose (15.9%), accounting for up to 80.6% of the molar percentage. WFPs exhibited a high antioxidant activity and a dose-dependent antiproliferative effect, with IC₅₀ values of 134.9, 70.1 and 138.4 μg/mL against A549, MCF-7 and LoVo cancer cells after 48 h of incubation as estimated by MTT assay, respectively. Flow cytometry analysis showed that WFPs exerted a stimulatory effect on apoptosis of MCF-7 cells, and induced the cell-cycle arrest at the G0/G1 phase, with the observation of intracellular ROS production and DNA damage. The present study demonstrated that these polysaccharides might have the potential to provide significant natural defence against human cancer.     

Anti-proliferative and anti-tumour effect of active components in donkey milk on A549 human lung cancer cells

The anti-proliferative and anti-tumour effects of donkey milk on A549 human lung cancer cells were investigated in vitro, and its effects on cell cycle progression, apoptosis and cytokine production were examined. Donkey milk active fractions reduced the viability of A549 cells in dose-dependent and time-dependent manners. Fraction-IV, a fraction of whey protein with a molecular mass >10 kDa, was the most effective fraction (P < 0.05) in inducing an accumulation of A549 cells in the G0/G1 and G2/M phases, indicating a potent cytotoxicity and causing cell death by apoptosis. The anti-proliferative activity of conditioned medium, prepared with fraction-IV-stimulated murine splenocytes, was significantly higher than that of fraction-IV alone (P < 0.05). Moreover, it could increase the secretion of Interleukin-2 (IL-2), Interferon-γ (IFN-γ), Interleukin-6 (IL-6), tumour necrosis factor α (TNF-α) and Interleukin 1β (IL-1β). The active components of donkey milk not only could directly suppress tumour proliferation in vitro, but may also indirectly kill tumours through activation of lymphocytes and macrophages. A high content of lysozyme in fraction-IV may contribute to its anti-tumour activity.     

Antioxidant and anti-proliferative activity of Rhizoma Smilacis Chinae extracts and main constituents

Rhizoma Smilacis Chinae (RSC) is a widely used herbal material in functional food and folk medicine. In this study, methanol extract (ME), water extract (WE), polysaccharide fraction and ethyl acetate fraction (EF) of RSC were prepared and the constituents were analysed by HPLC. Different antioxidant tests were employed to evaluate the antioxidant activities of RSC extracts and its main constituents, astilbin and chlorogenic acid. The results showed that RSC extracts possessed comparable antioxidant activity to butylated hydroxyanisole in a dose-dependent manner. The radical-scavenging capacity of ME and EF was even stronger than astilbin and chlorogenic acid. The EF and ME of RSC also showed stronger anti-proliferative activity on HepG2 cells than astilbin and chlorogenic acid, with IC₅₀ values of 47 and 32 μg/mL for 24 h treatment, respectively. Flow cytometric analysis revealed that RSC extracts induced cell cycle arrest at G2/M phase and a late apoptosis of the cells.     

苦荞茶多糖诱导人肺腺癌A549细胞凋亡的线粒体机制

目的：从线粒体调控机制的角度探讨苦荞茶多糖（tartary buckwheat tea polysaccharide,TBTP）对人肺腺癌A549细胞凋亡的影响。方法：应用水提醇沉法结合超声辅助技术提取的TBTP处理A549细胞,分为空白对照组和不同质量浓度TBTP处理组。采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐法和流式细胞术检测细胞增殖和凋亡水平,采用DHE和Mito SOX染色检测细胞和线粒体活性氧簇水平,采用JC-1染色检测线粒体膜电位变化,采用Western blot法检测凋亡相关蛋白Bcl-2相关X蛋白（Bcl-2 associated X protein,Bax）、Bcl-2、剪切型半胱氨酸蛋白酶（Caspase）-3、细胞色素c的水平。结果：TBTP呈剂量和时间依赖性抑制A549细胞增殖。TBTP处理A549细胞24 h可呈剂量依赖性地促进细胞凋亡。TBTP处理能够显著增加A549细胞和线粒体内活性氧水平,降低线粒体膜电位;同时,TBTP能够增加促凋亡蛋白Bax和剪切型Caspase-3的表达、抑制抗凋亡蛋白Bcl-2的表达,促进A549细胞线粒体细胞色素c外流。结论：TBTP能够促进活性氧簇生成、降低线粒体膜电位,进而抑制A549细胞增殖,促进细胞凋亡。     

Gallic acid induces G2/M phase cell cycle arrest via regulating 14‐3‐3β release from Cdc25C and Chk2 activation in human bladder transitional carcinoma cells

Scope: Cell cycle regulation is a critical issue in cancer treatment. Previously, gallic acid (GA) has been reported to possess anticancer ability. Here, we have evaluated the molecular mechanism of GA on cell cycle modulation in a human bladder transitional carcinoma cell line (TSGH‐8301 cell). Methods and results: Using flow cytometer analysis, exposure of the cells to 40 μM GA resulted in a statistically significant increase in G2/M phase cells, which was accompanied by a decrease in G0/G1 phase cells. GA‐treated cells resulted in significant growth inhibition in a dose‐dependent manner accompanied by a decrease in cyclin‐dependent kinases (Cdk1), Cyclin B1, and Cdc25C, but significant increases in p‐cdc2 (Tyr‐15) and Cip1/p21 by western blotting. Additional mechanistic studies showed that GA induces phosphorylation of Cdc25C at Ser‐216. This mechanism leads to its translocation from the nucleus to the cytoplasm resulting in an increased binding with 14‐3‐3β. When treated with GA, phosphorylated Cdc25C can be activated by ataxia telangiectasia‐mutated checkpoint kinase 2 (Chk2). This might be a DNA damage response as indicated by Ser‐139 phosphorylation of histine H2A.X. Furthermore, treatment of the cells with a Chk2 inhibitor significantly attenuated GA‐induced G2/M phase arrest. Conclusion: These results indicate that GA can induce cell cycle arrest at G2/M phase via Chk2‐mediated phosphorylation of Cdc25C in a bladder transitional carcinoma cell line.     

The anticarcinogenic potential of essential oil and aqueous infusion from caper (Capparis spinosa L.)

The present study assessed the influence of essential oil and aqueous infusion from wild-grown caper (Capparis spinosa L.) on cell growth, NF-κB activation, apoptosis and cell cycle in the human colon carcinoma cell line, HT-29. Methyl isothiocyanate (92.06%), a degradation product of glucosinolate glucocapparin, was detected as major component of essential oil from caper leaves and flower buds. Aqueous infusion of caper showed an interesting and variegate compositional pattern containing several phenolic compounds, among which a flavonol glycoside, rutin (quercetin 3-O-rutinoside, 50.7%) and 5-caffeoyl-quinic acid (chlorogenic acid, 17.5%) were detected as dominant. Caper essential oil and aqueous infusion showed time- and dose-dependent high inhibitory effect on HT-29 cell proliferation. In addition, they induced the inhibition on nuclear factor κB (NF-κB) activity in a dose-dependent manner, while they did not show any effect on apoptosis in HT-29 cells. Flow cytometric analysis indicated that treatment with caper essential oil and aqueous infusion resulted in G₂/M cell cycle arrest in a dose-dependent manner. Presented results suggest that caper contains volatile and non-volatile compounds which potentially can play an important role in colon cancer prevention.     

Preparative separation of flavonoids in Adinandranitida leaves by high-speed counter-current chromatography and their effects on human epidermal carcinoma cancer cells

Preparative separation of camellianins A and B in Adinandra nitida leaves, which is a flavonoid-rich plant source with great practical prospects, was conducted by high-speed counter-current chromatography (HSCCC) with a solvent system composed of ethyl acetate–ethanol–water (5:1:5, v/v). Apigenin (their aglycone) was prepared by hydrolysing a water extract and recrystallising. The purity and identity of these flavonoids was confirmed by HPLC-ESI/MS, and ¹H and ¹³C NMR. These flavones were used in cultures of the A431 cell line. Their inhibitory effect was evaluated by using the MTT assay. The results showed that three flavones could significantly inhibit the growth of human epidermal carcinoma cancer cells in a concentration-dependent manner. After 48 h of treatment, IC₅₀ values of camellianin A, camellianin B and apigenin against A431 tumour cells were 9.09 μM, 12.5 μM, 21.0 μM, respectively.     

Japanese apricot extract attenuates nuclear factor-κB activation and proteasomal activity in A549 lung cancer cells

The present study investigated the mechanism of Japanese apricot extract (JAE) in inhibiting lung cancer cells proliferation. JAE inhibited A549 lung cancer cell proliferation at non-cytotoxic doses and suppressed nuclear factor-κB (NF-κB) activation induced by TNF-α at a dose of 1 mg/mL (p<0.05). Proteasome activities of A549 cells were blocked by JAE in a dose-dependent manner at concentrations of 0.67 and 1 mg/mL (p<0.05). These results indicate that JAE has anti-proliferative activity against A549 cells and suppresses NF-κB activation, partially due to inhibiting proteasome activity.     

食用色素藻蓝蛋白对非小细胞肺癌A549细胞体外增殖和凋亡的影响

本研究以人类非小细胞肺癌A549细胞系为模型,采用藻蓝蛋白处理体外培养的细胞,分别从细胞存活率、细胞形态、细胞凋亡程度以及周期分布4?个方面阐述了藻蓝蛋白对A549细胞的影响。结果表明,藻蓝蛋白能够显著降低A549细胞的存活率与生长速率（P＜0.05）,引起细胞形态发生非正常改变;同时上调促凋亡基因Bax、Bad,下调抑凋亡基因Bcl-2、Bcl-xl的表达而诱导细胞凋亡;此外,藻蓝蛋白能够异常调控细胞周期G1/S转换点相关基因的表达而引起细胞发生G1期阻滞。研究结果揭示了藻蓝蛋白对A549肺癌细胞具有显著的抑制作用,为开发新型抗癌类功能性食品着色剂提供了必要的理论依据。     

Antitumor,Antioxidant, and Nitrite Scavenging Effects of Chinese Water Chestnut (Eleocharis dulcis) Peel Flavonoids

The preparation, quantification, and characterization of flavonoid compounds from Chinese water chestnut peel (CWCP) flavonoid extract and ethyl acetate fraction (EF), n‐butanol fraction, and water fraction were studied. Among these, EF showed the maximum free radical levels (IC₅₀ values of 0.36, 0.40, and 0.37 mg/mL for DPPH?, ABTS? ⁺ , and ?OH, respectively), nitrite scavenging effects (IC₅₀ = 1.89 mg/mL), and A549 cell inhibitory activities (IC₅₀ = 776.12 μg/mL) with the highest value of total flavonoid content (TFC, 421.32 mg/g). Moreover, the contents of 8 flavonoids in this fraction were quantified using high‐performance liquid chromatography, and fisetin, diosmetin, luteolin, and tectorigenin were the 4 major flavonoids with levels of 31.66, 29.91, 13.69, and 12.41 mg/g, respectively. Luteolin produced a greater inhibition of human lung cancer A549 cells (IC₅₀ = 59.60 μg/mL) than did fisetin, diosmetin, and tectorigenin. Flow cytometry revealed that the cellular mechanisms of luteolin inhibition of A549 cells were achieved via the induction of cell proliferation arrest at G₁ phase and apoptosis/necrosis. Our findings suggest that flavonoids are closely associated with antitumor, antioxidant, and nitrite scavenging effects of CWCP.     

Neferine induces reactive oxygen species mediated intrinsic pathway of apoptosis in HepG2 cells

Evidence has accumulated concerning the medicinal application of Nelumbo nucifera in the treatment of various diseases. Neferine, an alkaloid from N. nucifera was found to exert cytotoxicity on liver cancer cells HepG2 in a dose-dependent manner. We evaluated its anticancer potential by studying its effect on mitochondrial membrane potential, intracellular calcium levels [Ca²⁺]_i, cell membrane integrity, apoptotic body formation and DNA fragmentation in cultured HepG2 cells. The reactive oxygen species level has been increased upon neferine treatment with concomitant decrease in reduced glutathione. Our data further indicate reduction of ΔψM and increased [Ca²⁺]_i during apoptosis induction by neferine with increased expression of apoptotic proteins such as Bax, Bad, cleaved forms of caspase 3, caspase 9 and PARP, with the downregulation of anti-apoptotic protein Bcl2 in HepG2 cells. Moreover, the expressions of tumour suppressor proteins p53 and PTEN were upregulated along with the downregulation of P-Akt. In addition, expression levels of TNF-α, p38 and ERK1/2 MAP kinases were increased upon neferine treatment. These results imply that mitochondrial-mediated ROS generation induced by neferine leads to caspase-dependent apoptosis in HepG2 cells.     

Methyl jasmonate enhances antioxidant activity and flavonoid content in blackberries (Rubus sp.) and promotes antiproliferation of human cancer cells

The effects of preharvest methyl jasmonate (MJ) application on fruit quality, antioxidant activity and flavonoid content in blackberries (Rubus sp.) were determined. Anticancer activity against human lung A549 cells and HL-60 leukemia cells was also evaluated. Three blackberry cultivars (Chester Thornless, Hull Thornless and Triple Crown) were used in these experiments. Blackberries treated with MJ (0.01 and 0.1 mM) had higher soluble solids content, and lower titratable acids than untreated fruit as well as enhanced content of flavonoids and increased antioxidant capacity. Extracts of treated fruit showed enhanced inhibition of A549 cell and HL-60 cell proliferation and induced the apoptosis of HL-60 cells. Cultivar Hull Thornless had higher soluble solids and lower titratable acids compared to cv. Chester Thornless and Triple Crown. On the basis of fresh weight of fruit, Hull Thornless also had significantly higher anthocyanin, total phenolic content, antioxidant and antiproliferation activity than other two cultivars.     

三羟异黄酮对人乳腺癌细胞株MCF-7增殖的影响机制

研究了三羟异黄酮(geinstein，Gen)对乳腺癌细胞株MCF-7增殖的影响机制。结果发现Gen对MCF-7细胞生长有显著抑制作用，使细胞生长阻滞于G2／M期，并使cyclin B蛋白表达增加，且呈剂量．效应关系；对cyclin E蛋白表达无影响。Gen降低了bcl-2蛋白的表达，促进了bax蛋白的表达并且降低了bcl-2／bax比值。     

Effect of selected phytochemicals on cell proliferation in A549 lung cancer cells

Effects of quercetin 3-β-d-glucoside, resveratrol, and curcumin on A549 lung cancer cell proliferation and the mechanism of these phytochemicals in regulating apoptosis and cell cycle arrest were investigated. A549 cells were treated with quercetin 3-β-d-glucoside, resveratrol, or curcumin at 37°C for 96 hr and cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Proteins related to apoptosis and cell cycle in A549 cells were quantified with Western blotting assay. Quercetin 3-β-d-glucoside, resveratrol, and curcumin inhibited A549 cell proliferation in a dose-dependent manner (p<0.05). Quercetin 3-β-Dglucoside significantly decrease the expression level of CDK4 at concentrations of 5 mM and above (p<0.05). Curcumin lowered expression level of Bcl-2, CDK4, and cyclin D1 at concentrations of 100, 50, and above, and 50 mM and above, respectively (p<0.05). These results suggest that phytochemicals, which can be found in a normal diet, inhibit lung cancer cell proliferation and regulate the expression of the proteins involved in apoptosis and cell cycle.     

硒化卡拉胶联合表阿霉素对肝癌HepG-2细胞增殖及细胞周期的影响

目的观察硒化卡拉胶(KSC)和表阿霉素(EPI)联合应用对人肝癌HepG-2细胞生长及细胞周期的影响,并探讨其作用机制。方法 MTT(噻唑蓝)比色法测定KSC和EPI对HepG-2细胞增殖的影响,计算半数抑制浓度IC50值及金式指数q判断两者联合作用效果;用倒置显微镜观察细胞形态学变化;流式细胞仪检测药物对细胞周期的影响;Western blot法检测细胞周期蛋白Cyclin A和Cdc25A、细胞周期蛋白依赖性激酶Cdk2的表达水平。结果 KSC和EPI可明显抑制HepG-2细胞增殖,且有剂量依赖性。联合组的抑制效果更显著,且优于单一用药组。EPI单独用药48 h的IC50值为3.612 mg/L,与30mg/L KSC联合用药的IC50值降至0.807 mg/L,q值判断两药联合表现为相加作用。倒置显微镜观察给药后细胞形态发生变化,联合组细胞膜破裂呈坏死状。流式细胞术结果表明,两药均可导致S期周期阻滞。Western blot结果显示两药均可使Cyclin A、Cdc25A和Cdk2蛋白表达下调,联合给药组下降更明显。结论 KSC和表阿霉素可抑制肝癌HepG-2细胞增殖,引起细胞周期S期阻滞,两药联合具有相加效应,这与其调节细胞周期相关蛋白的生成关系密切。     

Diosmin induces cell apoptosis through protein phosphatase 2A activation in HA22T human hepatocellular carcinoma cells and blocks tumour growth in xenografted nude mice

This study investigated the diosmin effect on HA22T human hepatocellular carcinoma cells in vitro and in an invivo mouse xenograft model. HA22T cells were treated with different concentrations of diosmin and analysed with Western blot analysis, TUNEL, JC-1 staining and siRNA transfection assays. Additionally, the HA22T-implanted xenograft nude mice model was applied to confirm the cellular effects. Diosmin induced apoptosis, up-regulated death receptor apoptotic pathway markers as well as mitochondrial proteins. Pro-survival Bcl-2 family proteins were inhibited and the pro-apoptotic ones were increased. Protein phosphatase 2A (PP2A) siRNA or okadaic acid reversed the diosmin effects, confirming the role of PP2A in diosmin-induced HA22T apoptosis. The HA22T-implanted nude mice model revealed that diosmin inhibited tumour cell proliferation and enhanced tumour cell apoptosis. All our experimental evidence indicates that diosmin significantly promotes HA22T apoptosis and reduces tumour sizes in xenograft nude mice via PP2A in a dose-dependent manner.     

蜂胶醇提物对人肝癌细胞Hep G2增殖及凋亡的影响

为探讨蜂胶醇提物对人肝癌细胞Hep G2 增殖和凋亡的影响,采用MTT 法检测蜂胶醇提物对Hep G2 细胞增殖的抑制作用,用荧光倒置显微镜和流式细胞仪分析蜂胶醇提物对Hep G2 细胞凋亡的影响。结果表明：12.5～200μg/mL 质量浓度的蜂胶醇提物作用24、48h 后,均能不同程度地抑制Hep G2 细胞的增殖,呈明显的时间、剂量依赖关系,半数抑制质量浓度(IC50)分别是115、78μg/mL。作用8h 后,Hep G2 细胞出现不同程度的凋亡,凋亡率由6.36% 上升到21.9%,呈现出剂量依赖关系。故蜂胶醇提物可显著抑制人肝癌细胞Hep G2 的生长,诱导其凋亡。     

亚硒酸钠与白藜芦醇联合应用对肺癌细胞的协同效应分析

目的:观察亚硒酸钠与白藜芦醇单独及联合应用对肺癌细胞的影响,并通过细胞周期探讨其作用机理。方法:用四甲基偶氮唑盐法检测亚硒酸钠和白藜芦醇单独及联合应用对细胞增殖和存活的影响;用流式细胞术测定细胞的凋亡率及细胞周期。结果:亚硒酸钠和白藜芦醇对肺癌细胞A549均有拮抗作用,且随浓度的增加,拮抗效果越显著,当亚硒酸钠浓度大于等于5μmol/L时,拮抗效果极显著(P0.01);当白藜芦醇浓度大于等于65μmol/L时,拮抗效果极显著(P0.01)。亚硒酸钠对A549细胞的半致死率(lethal dose of 50%,LD_(50))为4.759μmol/L,白藜芦醇对A549细胞的LD_(50)为45.24μmol/L。然而亚硒酸钠和白藜芦醇联合用药作用于A549细胞的拮抗效果优于单独用药,且低剂量的亚硒酸钠和白藜芦醇联合就能发挥出高剂量亚硒酸钠对A549细胞的拮抗作用效果,有效地降低了亚硒酸钠对细胞的毒副作用。另外,亚硒酸钠联合白藜芦醇作用可以阻滞A549细胞G0/G1期,最终导致细胞凋亡。