白皮杉醇抗肿瘤作用研究进展

白皮杉醇是一种化学结构类似于白藜芦醇的天然小分子化合物,具有抗氧化、清除自由基、抗菌、抗炎、抗白血病、抗细胞增殖、提高免疫调节能力、抗癌、防癌等生物活性。白皮杉醇的抗氧化、抗侵袭等生物活性强于白藜芦醇,而其抗肿瘤作用机制尚未明确。近年,白皮杉醇在抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡及抑制肿瘤细胞侵袭和迁移方面的作用越来越受到广泛关注,本文通过文献查阅后对白皮杉醇抗肿瘤作用的最新研究进展进行归纳总结,旨在为白皮杉醇进一步开发成抗肿瘤药物奠定理论基础和实验依据。     

白藜芦醇与呋咱氮氧化物偶联物的合成及抗肿瘤活性研究

白藜芦醇为一天然茋类成分,具有多样生物活性。一氧化氮作为气体信使分子,高浓度时可以抑制肿瘤生长,逆转肿瘤多药耐药性,呋咱氮氧化物即为一类较稳定的一氧化氮供体。本实验合成白藜芦醇与呋咱氮氧化物偶联物,并通过MTT法测定其抗肿瘤活性。结果白藜芦醇与呋咱氮氧化物偶联物抗肿瘤活性比单纯的白藜芦醇、呋咱氮氧化物及白藜芦醇与呋咱氮氧化合物的混合物具有更好的抗癌活性,结果表明白藜芦醇与呋咱氮氧化物的偶联物表现出显著的协同效应。     

白藜芦醇通过降低galectin-3的表达抑制宫颈癌SiHa细胞的增殖并促进其凋亡

目的：探讨白藜芦醇对人宫颈癌SiHa细胞增殖和凋亡的影响及galectin-3在其中的作用。方法：以体外培养的人宫颈癌SiHa细胞为研究对象,实验分为对照组、白藜芦醇处理组及[白藜芦醇＋galectin-3（5、10、20μg/mL）]共5组,分别给予生理盐水、300μmol/L白藜芦醇及[300μmol/L白藜芦醇＋Galectin-3（5、10、20μg/mL）]处理。48小时后,分别收集各组细胞,采用MTT法检测细胞的增殖情况,Caspase-3活性试剂盒测定细胞的凋亡情况,Western Blot技术测定细胞内galectin-3的蛋白表达水平。结果：300μmol/L的白藜芦醇明显抑制SiHa细胞的增殖,并显著增加其凋亡水平,同时减少了细胞内galectin-3的蛋白表达。在白藜芦醇处理的同时,给予5、10及20μg/mL的Galectin-3,随着Galectin-3蛋白浓度的增加,SiHa细胞的增殖抑制率逐渐降低,凋亡水平也逐渐下降,但是VEGF-R3受体的磷酸化水平却逐渐升高。结论：白藜芦醇通过降低galectin-3的表达抑制宫颈癌SiHa细胞的增殖并促进其凋亡。     

白藜芦醇通过降低galectin-3 的表达抑制宫颈癌SiHa细胞的增殖并促进其凋亡

目的：探讨白藜芦醇对人宫颈癌SiHa 细胞增殖和凋亡的影响及galectin-3 在其中的作用。方法：以体外培养的人宫颈癌 SiHa 细胞为研究对象,实验分为对照组、白藜芦醇处理组及[白藜芦醇+galectin-3(5、10、20 ug/mL)]共5 组,分别给予生理盐水、 300 umol/L白藜芦醇及[300 umol/L白藜芦醇+Galectin-3 (5、10、20 ug/mL)]处理。48 小时后,分别收集各组细胞,采用MTT法检 测细胞的增殖情况,Caspase-3 活性试剂盒测定细胞的凋亡情况,Western Blot技术测定细胞内galectin-3 的蛋白表达水平。结果： 300 umol/L的白藜芦醇明显抑制SiHa 细胞的增殖,并显著增加其凋亡水平,同时减少了细胞内galectin-3 的蛋白表达。在白藜芦 醇处理的同时,给予5、10 及20 ug/mL 的Galectin-3,随着Galectin-3 蛋白浓度的增加,SiHa 细胞的增殖抑制率逐渐降低,凋亡水 平也逐渐下降,但是VEGF-R3 受体的磷酸化水平却逐渐升高。结论：白藜芦醇通过降低galectin-3 的表达抑制宫颈癌SiHa 细胞 的增殖并促进其凋亡。     

白藜芦醇抑制上皮肿瘤细胞侵袭和迁移的机制

白藜芦醇是一种多酚类的植物抗毒素,具有多种生物学活性,其中,抗肿瘤作用受到人们越来越多的关注。大量实验表明,白藜芦醇具有靶向抑制癌细胞侵袭和迁移的能力。EMT过程使上皮细胞获得间充质表型,从而促进癌细胞的侵袭和迁移。Micro RNAs可靶向EMT的转录因子以及上皮和间充质标志物来调节EMT过程。MMP-2和MMP-9是细胞外基质的两个重要成分,均参与肿瘤细胞的转移。白藜芦醇通过影响EMT过程、调控micro RNAs家族的表达以及抑制MMPs的蛋白表达和酶活性来影响上皮肿瘤细胞的侵袭和迁移。现对白藜芦醇抑制上皮肿瘤细胞侵袭、迁移的机制进行综述。     

白藜芦醇植物资源及其生产

白藜芦醇广泛存在于多种植物中,具有抗肿瘤、抗病毒等重要生物活性。综述了白藜芦醇在植物中的分布及其生产,特别是生物技术在生产中的应用,为白藜芦醇在药品和功能食物方面的进一步开发提供参考。     

白藜芦醇诱导皮肤癌细胞株A432细胞凋亡的机制研究

白藜芦醇（Resveratrol）是天然的含有芪类结构的非黄酮类多酚化合物,广泛存在于葡萄和花生等天然植物中,具有抗炎、抗氧化、调节脂代谢及抗肿瘤等生物活性。本研究通过应用不同浓度的白藜芦醇处理正常皮肤细胞HaCaT和皮肤癌细胞A431,探讨白藜芦醇诱导皮肤癌细胞A431凋亡的可能分子机制。MTT结果表明：白藜芦醇处理后,A431细胞的数量明显减少,     

白藜芦醇抑制小鼠肥胖的功效及相关机制探讨

目的:研究白藜芦醇抑制高脂引起的肥胖的作用机制。方法:将18只C57小鼠随机分为3组,分别为对照组、高脂以及高脂+白藜芦醇小鼠模型,给小鼠喂养一定剂量白藜芦醇(100 mg/kg/d),喂养12周。提取小鼠皮下脂肪细胞,分化成熟,加入白藜芦醇,采用q RT-PCR以及Western blot等方法检测HO-1以及棕色脂肪标志基因的表达。通过q RT-PCR检测小鼠脂肪组织炎症因子、UCP-1以及HO-1的表达。结果:白藜芦醇在体内可以明显抑制高脂引起的肥胖,糖耐量异常,同时促进棕色脂肪标志基因UCP-1,PGC-1以及PRDM16的表达。白藜芦醇还可抑制肥胖小鼠脂肪组织炎症因子的增加以及抗炎蛋白HO-1的表达。在体外分化的成熟的皮下脂肪细胞中,白藜芦醇同样可以促进棕色脂肪标志基因UCP-1,PGC-1以及PRDM16的表达。白藜芦醇通过促进抗炎蛋白HO-1的表达抑制高脂引起的脂肪炎症反应。结论:白藜芦醇可以通过促进白色脂肪棕色化以及抑制慢性低度炎症抑制高脂引起的肥胖、糖耐量异常以及改善胰岛素敏感性。     

白藜芦醇对胰腺癌细胞增殖和凋亡影响的研究

为了研究白藜芦醇对人胰腺癌细胞增殖和存活的影响,利用不同浓度白藜芦醇处理PANC-1和Bx PC-3细胞,通过MTS和软琼脂集落实验检测白藜芦醇对胰腺癌细胞的停泊依赖和停泊非依赖增殖的抑制。同时,采用免疫印迹的方法,检测不同浓度白藜芦醇对胰腺癌细胞增殖相关信号通路的调控,及不同时间点凋亡激活相关蛋白分子的表达。结果发现白藜芦醇能剂量依赖性抑制PANC-1和Bx PC-3细胞增殖,并且这种增殖抑制作用与表皮生长因子受体(epithelial growth factor receptor,EGFR)及下游信号通路的抑制有关。一定浓度的白藜芦醇能诱导胰腺癌细胞发生凋亡,caspase3和caspase7被剪切活化。通过检测Bcl-2(B-cell lymphoma-2,Bcl-2)促存活家族蛋白,证实白藜芦醇能有效抑制Mcl-1(myeloid cell leukemia-1,Mcl-1)的表达,并不改变Bcl-2和Bcl-XL的表达。实验结果初步表明,白藜芦醇抑制胰腺癌的增殖与存活与EGFR信号通路及促存活蛋白Mcl-1的表达下调有关。     

柳生金针菇胞外粗多糖对小鼠肝癌H22移植性肿瘤的抑制作用和免疫功能调节的研究

本研究通过对柳生金针菇胞外粗多糖对肿瘤生长的抑制和机体免疫调节的体内实验,来探索柳生金针菇胞外粗多糖的相关抗肿瘤机制。结果表明柳生金针菇胞外粗多糖高、中、低剂量组能不同程度抑制肝癌H22肿瘤的生长。高剂量组抑瘤率最高,为35.25%,中低剂量也能抑制肿瘤生长,缓解脾脏肿大和胸腺萎缩。同时免疫学实验结果表明柳生金针菇胞外粗多糖可明显增加血清中各种细胞因子的表达,通过增强细胞和体液免疫功能来起到抗肿瘤作用。     

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.     

Heme Oxygenase-1 Promotes Survival of Renal Cancer Cells through Modulation of Apoptosis- and Autophagy-regulating Molecules

The cytoprotective enzyme heme oxygenase-1 (HO-1) is often overexpressed in different types of cancers and promotes cancer progression. We have recently shown that the Ras-Raf-ERK pathway induces HO-1 to promote survival of renal cancer cells. Here, we examined the possible mechanisms underlying HO-1-mediated cell survival. Considering the growing evidence about the significance of apoptosis and autophagy in cancer, we tried to investigate how HO-1 controls these events to regulate survival of cancer cells. Rapamycin (RAPA) and sorafenib, two commonly used drugs for renal cancer treatment, were found to induce HO-1 expression in renal cancer cells Caki-1 and 786-O; and the apoptotic effect of these drugs was markedly enhanced upon HO-1 knockdown. Overexpression of HO-1 protected the cells from RAPA- and sorafenib-induced apoptosis and also averted drug-mediated inhibition of cell proliferation. HO-1 induced the expression of anti-apoptotic Bcl-xL and decreased the expression of autophagic proteins Beclin-1 and LC3B-II; while knockdown of HO-1 down-regulated Bcl-xL and markedly increased LC3B-II. Moreover, HO-1 promoted the association of Beclin-1 with Bcl-xL and Rubicon, a novel negative regulator of autophagy. Drug-induced dissociation of Beclin-1 from Rubicon and the induction of autophagy were also inhibited by HO-1. Together, our data signify that HO-1 is up-regulated in renal cancer cells as a survival strategy against chemotherapeutic drugs and promotes growth of tumor cells by inhibiting both apoptosis and autophagy. Thus, application of chemotherapeutic drugs along with HO-1 inhibitor may elevate therapeutic efficiency by reducing the cytoprotective effects of HO-1 and by simultaneous induction of both apoptosis and autophagy.     

Novel Aza-resveratrol analogs: Synthesis,characterization and anticancer activity against breast cancer cell lines

Novel Aza-resveratrol analogs were synthesized, structurally characterized and evaluated for cytotoxic activity against MDA-MB-231 and T47D breast cancer cell lines, which exhibited superior inhibitory activity than parent resveratrol compound. The binding mechanism of these compounds with estrogen receptor-α was rationalized by molecular docking studies which indicated additional hydrogen binding interactions and tight binding in the protein cavity. Induction of Beclin-1 protein expression in breast cancer cell lines after treatment with newly synthesized resveratrol analogs indicated inhibition of growth of these cell lines through autophagy. The study highlighted the advantage of introducing the imino-linkage in resveratrol motif in enhancing the anticancer potential of resveratrol suggesting that these analogs can serve as better therapeutic agents against breast cancer and can provide starting point for building more potent analogs in future.     

Caloric restriction and resveratrol promote longevity through the Sirtuin-1-dependent induction of autophagy

Caloric restriction and autophagy-inducing pharmacological agents can prolong lifespan in model organisms including mice, flies, and nematodes. In this study, we show that transgenic expression of Sirtuin-1 induces autophagy in human cells in vitro and in Caenorhabditis elegans in vivo. The knockdown or knockout of Sirtuin-1 prevented the induction of autophagy by resveratrol and by nutrient deprivation in human cells as well as by dietary restriction in C. elegans. Conversely, Sirtuin-1 was not required for the induction of autophagy by rapamycin or p53 inhibition, neither in human cells nor in C. elegans. The knockdown or pharmacological inhibition of Sirtuin-1 enhanced the vulnerability of human cells to metabolic stress, unless they were stimulated to undergo autophagy by treatment with rapamycin or p53 inhibition. Along similar lines, resveratrol and dietary restriction only prolonged the lifespan of autophagy-proficient nematodes, whereas these beneficial effects on longevity were abolished by the knockdown of the essential autophagic modulator Beclin-1. We conclude that autophagy is universally required for the lifespan-prolonging effects of caloric restriction and pharmacological Sirtuin-1 activators.     

白花蛇舌草多糖提取物对喉癌Hep-2细胞内质网自噬的影响*

目的:探讨白花蛇舌草多糖提取物(HDPE)对喉癌Hep-2细胞内质网自噬的影响。方法:实验分为对照组、HDPE 100、200、400 mg/L组和3-MA(自噬抑制剂)组,噻唑盐比色法(MTT)检测各组细胞培养24 h、48 h、72 h后增殖抑制率;原位末端转移酶标记法(TUNEL)法检测各组培养48 h细胞凋亡情况;单丹黄酰尸胺(MDC)染色观察各组培养48 h细胞自噬体及自噬溶酶体的变化;透射电镜观察培养48 h细胞内质网周围自噬囊泡的产生情况;蛋白印迹法(Western blot)检测各组培养48 h细胞Beclin-1蛋白(Beclin-1)、微管相关轻链蛋白3Ⅰ(LC3Ⅰ)、微管相关轻链蛋白3Ⅱ(LC3Ⅱ)、葡萄糖调节蛋白 78(GRP78)、活化转录因子6(ATF6)及CCAAT 增强子结合蛋白同源蛋白(CHOP)表达。结果:与对照组比较,HDPE 100、200、400 mg/L组和3-MA组细胞增殖抑制率、凋亡指数AI升高,MDC阳性细胞率量降低,内质网周围自噬囊泡减少,GRP78、ATF6及CHOP表达及LC3Ⅰ/LC3Ⅱ比值升高,Beclin-1表达降低(P＜0.05);与3-MA组比较,HDPE 400 mg/L组细胞增殖抑制率、凋亡指数AI升高,MDC阳性细胞率、GRP78、ATF6及CHOP表达及LC3Ⅰ/LC3Ⅱ比值升高,Beclin-1表达降低(P＜0.05)。结论:HDPE可能通过抑制喉癌Hep-2细胞内质网自噬,促进细胞内质网应激凋亡,进而抑制Hep-2细胞增殖能力。     

Apoptotic Cell Death Induced by Resveratrol Is Partially Mediated by the Autophagy Pathway in Human Ovarian Cancer Cells

Resveratrol (trans-3,4,5’ –trihydroxystilbene) is an active compound in food, such as red grapes, peanuts, and berries. Resveratrol exhibits an anticancer effect on various human cancer cells. However, the mechanism of resveratrol-induced anti-cancer effect at the molecular level remains to be elucidated. In this study, the mechanism underlying the anti-cancer effect of resveratrol in human ovarian cancer cells (OVCAR-3 and Caov-3) was investigated using various molecular biology techniques, such as flow cytometry, western blotting, and RNA interference, with a major focus on the potential role of autophagy in resveratrol-induced apoptotic cell death. We demonstrated that resveratrol induced reactive oxygen species (ROS) generation, which triggers autophagy and subsequent apoptotic cell death. Resveratrol induced ATG5 expression and promoted LC3 cleavage. The apoptotic cell death induced by resveratrol was attenuated by both pharmacological and genetic inhibition of autophagy. The autophagy inhibitor chloroquine, which functions at the late stage of autophagy, significantly reduced resveratrol-induced cell death and caspase 3 activity in human ovarian cancer cells. We also demonstrated that targeting ATG5 by siRNA also suppressed resveratrol-induced apoptotic cell death. Thus, we concluded that a common pathway between autophagy and apoptosis exists in resveratrol-induced cell death in OVCAR-3 human ovarian cancer cells.     

Beclin-1-mediated autophagy protects spinal cord neurons against mechanical injury-induced apoptosis

Apoptosis has been widely reported to be involved in the pathogenesis associated with spinal cord injury (SCI). Recently, autophagy has also been implicated in various neuronal damage models. However, the role of autophagy in SCI is still controversial and its interrelationship with apoptosis remains unclear. Here, we used an in vitro SCI model to observe a time-dependent induction of autophagy and apoptosis. Mechanical injury induced autophagy markers such as LC3 lipidation, LC3II/LC3I conversion, and Beclin-1expression. Injured neurons showed decreased cell viability and increased apoptosis. To elucidate the effect of autophagy on apoptosis, the mechanically-injured neurons were treated with the mTOR inhibitor rapamycin and 3-methyl adenine (3-MA), which are known to regulate autophagy positively and negatively, respectively. Rapamycin-treated neurons showed the highest level of cell viability and lowest level of apoptosis among the injured neurons and those treated with 3-MA showed the reciprocal effect. Notably, rapamycin-treated neurons exhibited slightly reduced Bax expression and significantly increasedBcl-2 expression. Furthermore, by plasmid transfection, we showed that Beclin-1-overexpressing neuronal cells responded to mechanical injury with greater LC3II/LC3I conversion and cell viability, lower levels of apoptosis, higher Bcl-2 expression, and unaltered Bax expression as compared to vector control cells. Beclin-1-knockdown neurons showed almost the opposite effects. Taken together, our results suggest that autophagy may serve as a protection against apoptosis in mechanically-injured spinal cord neurons. Targeting mTOR and/or enhancing Beclin-1 expression might be alternative therapeutic strategies for SCI.     

苦参素复合长春新碱对HCT-8/VCR细胞耐药性的影响及其机制*

目的: 探究苦参素复合长春新碱对HCT-8/VCR细胞耐药性的影响及其机制。方法: HCT-8/VCR体外培养,分为空白组、苦参素单药组,长春新碱单药组、苦参素和长春新碱联合组,每组6个复孔。CCK-8法检测各组HCT-8/VCR细胞的耐药性;MDC法检测细胞内自噬小泡的变化;ELISA法检测IL-6的含量;Western blot检测自噬相关基因与免疫因子TLR4蛋白的表达水平。结果: 苦参素复合长春新碱可降低HCT-8/VCR细胞的耐药性,逆转倍数为3.23;与空白组比较,苦参素组和联合组自噬体含量降低、IL-6含量也明显降低(P＜0.01),长春新碱组自噬体含量升高,IL-6含量也明显升高(P＜0.01);联合组与苦参素组比较,自噬体含量升高,而IL-6含量降低(P＜ 0.01);Western blot实验表明,与空白组比较,苦参素组P62的表达降低(P＜0.05),LC3-Ⅱ/LC3-Ⅰ、Beclin-1、TLR4均升高(P＜0.05),长春新碱组和联合组P62的表达升高(P＜0.05),LC3-Ⅱ/LC3-Ⅰ、Beclin-1、TLR4均降低(P＜ 0.05);联合组与长春新碱组比较,P62的表达升高(P＜0.05),LC3-Ⅱ/LC3-Ⅰ、Beclin-1、TLR4均降低(P＜0.05)。结论: 苦参素与长春新碱联合可显著降低HCT-8/VCR的耐药性,其机制与抑制自噬活动和TLR4信号活化有关。     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy-induced apoptosis. Here, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression, the number of GFP-LC3-II-labeled autophagosome positive cells and autophagic cell death (p < 0.05). Furthermore, doxorubicin at a high dose (IC(95), 1 microM) induced apoptosis but at a low dose (IC(50), 0.07 microM) induced only autophagy and Beclin-1 expression. When combined with Bcl-2 siRNA, doxorubicin (IC(50)) enhanced autophagy as indicated by the increased number cells with GFP-LC3-II-stained autophagosomes (punctuated pattern positive). These results provided the first evidence that targeted silencing of Bcl-2 induces autophagic cell death in MCF-7 breast cancer cells and that Bcl-2 siRNA may be used as a therapeutic strategy alone or in combination with chemotherapy in breast cancer cells that overexpress Bcl-2.