Bufalin抗癌机制研究进展

Bufalin是传统中药蟾酥成分中的蟾毒配基之一。实验研究表明 ,Bufalin在低浓度时能有效诱导肿瘤细胞分化 ,高浓度时诱导肿瘤细胞凋亡。它是细胞拓扑异构酶Ⅱ的抑制剂 ,主要作用于细胞周期的S期 ;其诱导细胞分化、凋亡的机制与细胞生长相关基因的表达有关 ,也有独特的信号转导途径 ;对血管生成具有抑制作用。

Bufalin的急性毒性研究

目的:测定Bufalin的近似半数致死量(LD50)并研究Bufalin的急性毒性。方法:采用近似LD50测定方法测定Bufalin的LD50,采用HE染色法检查Bufalin的心肌毒性。结果:本研究表明,Bufalin近似的LD50为2.35mg/kg,并可导致心肌炎细胞浸润、充血等。结论:Bufalin具有一定的心肌毒性。

Bufalin对MGC-803细胞生长、分化的影响

目的 :以低分化胃腺癌细胞系MGC 80 3为实验对象 ,观察了蟾酥提取物bufalin对其生长、分化的影响。方法 :应用台盼蓝染色法、 76 90 XU荧光染色法和电子显微镜等观察细胞生长、分化。结果 :0 0 1μmol/Lbufalin可显著抑制细胞生长 ,细胞粘附力下降 ,由梭形趋于圆形 ,核质比下降 ,细胞内核糖体和线粒体数目减少 ,内质网不如对照组细胞发达 ;76 90 XU荧光染色显示 ,用药 72h ,约 71%的细胞发生分化。结论 :bufalin能有效诱导胃癌MGC 80

bufalin调控信号转导通路抗肿瘤机制的研究进展

中药是祖国医学的瑰宝,在我国,中成药或多或少的应用在癌症患者治疗的不同阶段,但是有部分医生或患者对中药的作用持保留态度,限制了中药的推广和应用。华蟾素具有抗肿瘤、抗病毒、强心、增强免疫力、止痛、麻醉、诱导血管收缩等作用,在恶性肿瘤治疗中应用广泛。其中bufalin是其发挥功能的重要单体,而部分医生对其抗肿瘤机制并不熟悉,本文拟对bufalin的抗肿瘤机制作出综述,为其临床应用提供理论依据。

Bufalin促进人食管癌细胞ECA109凋亡的研究

目的探讨bufalin促进ECA109细胞凋亡过程中的作用。方法 Giemsa染色观察bufalin作用于ECA109细胞后,细胞形态学的变化。Western Blot法检测bufalin作用于ECA109细胞2、6、12、24、36、48小时后,细胞内凋亡相关蛋白cI AP-1和BAD的蛋白表达变化。用流式细胞仪通过PI染色进行细胞周期解析及凋亡判定。结果 1.cI AP-1的蛋白水平随bufalin作用时间的增加逐渐降低,而BAD的表达水平则逐渐升高,差异具有统计学意义(P<0.05)。2.流式结果显示,细胞凋亡率随bufalin浓度增加而逐渐升高,并出现明显的G2/M期阻滞,差异具有统计学意义(P<0.05)。结论 Bufalin作用于食管癌细胞ECA109,促进细胞凋亡,随着时间延长,BAD的表达水平逐渐升高,而cI AP-1的水平逐渐降低。Bufalin以时间、剂量依赖性方式诱导ECA109细胞凋亡。

Bufalin对食管癌ECA109细胞中FAK活化和上皮-间质转化的影响

目的探讨Bufalin对食管鳞状细胞癌ECA109细胞中FAK活化和上皮-间质转化(epithelial-mesenehymal transition,EMT)的影响。方法采用RT-PCR法检测不同浓度Bufalin对食管鳞状细胞癌ECA109细胞中FAK、E-cadherin、vimentin mRNA表达的影响。Western blot法检测不同浓度Bufalin对ECA109细胞中FAK活化水平及FAK、E-cadherin、vimentin蛋白表达的影响。采用Transwell小室实验检测不同浓度Bufalin对ECA109细胞迁移与侵袭能力的影响。结果 RT-PCR结果表明Bufalin抑制FAK、E-cadherin、vimentin的mRNA表达。Western blot结果表明Bufalin抑制E-cadherin、vimentin的表达和FAK的活化。Transwell实验结果表明Bufalin可以抑制ECA109细胞的迁移及侵袭。药物干预组(20、40、60、80、100 nmol/L Bufalin及PF562271组)与阳性对照组相比,Transwell迁移实验结果显示穿过基膜的细胞个数由107.00±8.19下降至78.67±3.06、61.67±3.06、42.67±3.512、24.33±2.517、10.33±3.215、9.00±2.65;Transwell侵袭实验结果显示穿过基膜的细胞个数由127.67±8.02下降至102.33±4.51、87.33±7.10、73.00±4.58、57.33±2.52、39.00±3.61、37.33±2.52,差异均有统计学意义(P<0.05)。结论 Bufalin可以抑制食管鳞状细胞癌中FAK的活化,提示Bufalin可能是通过下调FAK来抑制食管鳞状细胞癌EMT过程及食管癌的迁移及侵袭。

Anti-tumor activities and apoptosis-regulated mechanisms of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice

AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.METHODS: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups (n = 15). Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg (BF1), 1 mg/kg (BF2) and 0.5 mg/kg (BF3) for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues. RESULTS: The tumor volumes in each group of bufalin were reduced significantly (35.21 ± 12.51 vs 170.39 ± 25.29; 49.83 ± 11.46 vs 170.39 ± 25.29; 83.99 ± 24.63 vs 170.39 ± 25.29, P < 0.01, respectively), and the survival times were prolonged in group BF1-2 (31.8 ± 4.2 vs 23.4 ± 2.1 and 29.4 ± 3.4 vs 23.4 ± 2.1, P < 0.05, respectively), and necrosis was mainly in severe or moderate degree in group BF1-2. No morphologicalchanges were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA in each group was to be found and the density of bax mRNA was increased progressively with increase of dose of bufalin by RT-PCR. CONCLUSION: Bufalin has significant anti-tumor activities in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice with no marked toxicity and was able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated mainly via up-regulating the expression of apoptosis-regulated gene bax, which may be involved in its anti-tumor mechanism of bufalin.

Improved anti-tumor efficacy and pharmacokinetics of bufalin via PEGylated liposomes

OBJECTIVE To determine the characterization,anti-tumor efficacy and pharmacokinetics of bufalin-loaded PEGylated liposomes compared with bufalin entity.METHODS Bufalin-loaded PEGylated liposomes and bufalin-loaded liposomes were prepared reproducibly with homogeneous particle size by the combination of thin film evaporation method and high pressure homogenization method.The particle size and zeta potential of the liposomes were determined by dynamic light scattering technique.The direct imaging of morphology of liposomes was charactered by transmission electron microscope.The content of bufalin in liposomes was analysed by HPLC method.The entrapment efficiency and the particle size was applied to assess the stability profile,after storage at 4℃ on day 0,7,15,30 and 90.The in-vitro release behaviours of bufalin from liposomes were conducted using dialysis bag technique at 37℃.In-vitro cytotoxicity studies were carried out using MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay on several kinds of tumor cel lines including SW620,PC-3,MDA-MB-231,A549,U251,U87 and HepG2.In-vivo pharmacokinetic study of bufalin liposomes was evaluated by HPLC method.RESULTS Their mean particle sizes were 127.6 nm and 155.0 nm,mean zeta potentials were 2.24 m V and-18.5 m V,entrapment efficiencies were 76.31%and 78.40%,respectively.In-vitro release profile revealed that the release of bufalin in bufalin-loaded PEGylated liposomes was slower than that of bufalin-loaded liposomes.The cytotoxicity of blank liposomes has been found within acceptable range,whereas bufalin-loaded PEGylated liposomes showed enhanced cytotoxicity to U251 cells compared with bufalin entity.In-vivo pharmacokinetics indicated that bufalinloaded PEGylated liposomes could extend eliminate half-life time of bufalin in plasma in rats.CONCLUSION The results suggested that bufalin-loaded PEGylated liposomes improved the solubility and increased the drug concentration in plasma.

Anti-tumor effect of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice

Objective: To investigate anti-tumor effect of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice. Methods: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors in nude mice by subcutaneous injection. Then the subcutaneous tumors were implanted into the liver of nude mice, and the orthotopic transplantation tumor models of human hepatocellular carcinoma were established. Seventy-five models were randomized into 5 groups ( n = 15) . Bufalin was injected intraperitoneally into the 3 groups at dose of 1.5,1 and 0.5 mg/kg for day 15 - 24, respectively. NS group were injected equal volume saline as above and adriamycin were injected intraperitoneally into ADM group at dose of 8.0 mg/kg for day 15. Ten mice in each group were killed at day 25 and detected on morphological and ultrastructural changes in myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscope. The survival time in each group were observed. Results: The tumor volumes in each group of bufalin were reduced significantly compared with NS group (P < 0.01), the survival time were prolonged in group Bu 1 and Bu 2 compared with NS group ( P < 0.05), and tumor tissues were mainly necrosis in severe or moderate degree in Bu 1, Bu 2 groups, and mild degree or moderate degree in Bu 3 group. No morphological changes were detected in myocardium, brain, liver and kidney tissues, respectively. Apoptotic characteristics could be seen in tumor tissues of group Bu 1 and group Bu 2. Conclusion: Bufalin has significant anti-tumor effects on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice without marked toxicity. To guide cell apoptosis may be one of its anti-tumor mechanism of bufalin.

Two new compounds derived from bufalin

The biotransformation of bufalin by cell suspension cultures of Platycodon grandifiorus was investigated and two new biotransformed products were obtained, which was 3-epi-telocinobufagln and 3-epi-bufalin-3-O-β-D-glucoside.

The anti-nociceptive effect of bufalin,an active ingredient from toad venom via modulating voltage-gated sodium channels

OBJECTIVE Toad venom(Venenum Bufonis)isalways used for analgesia in China from ancient to modern times,but the effective component of it remains unclear.METHODS In the present study,we investigated the anti-nociceptive effect and the underlying mechanism ofbufalin,an active ingredient fromtoad venom by animal behavior,patch clamp and calcium imaging.RESULTS Bufalin could significantly relieve formalin-induced spontaneous flinching and licking response as well as carrageenan-induced mechanical and thermal hyperalgesia.Using the whole-cel patch-clamp recording,bufalincaused remarkable suppressive effect on the peak currents of Na+channels in dorsal root ganglion neuroblastoma ND7-23 cel line in a U-shaped dependent manner.In addition,bufalinprompted the voltage-dependent activationand caused a negative shift of the fast-state inactivation of Na+channels.However,bufalin produced insignificant effect not onlyon voltage-dependent Kv4.2,Kv4.3 and BK channels,but also on the capsaicin induced Ca2+influx.CONCLUSION The present results indicate bufalin is capable of producing remarkable anti-nociceptive effects whichis probably ascribed to its specific modulation of voltage-gated Na+channels.

Allosteric probe-modified liposome loading bufalin-fluorouracil complex for targeted colorectal cancer therapy

Objective Bufalin,the main active anti-tumor monomer of toad venom,is crucial in cancer treatment.However,intrinsic issues,such as poor solubility and systematic toxicity,have considerably mitigated its anticancer functions and caused unwanted side effects.It is essential to develop innovative targeting systems to precisely and efficiently deliver anticancer drugs to achieve satisfying therapeutic efficiency.Methods This work established a novel and more efficient system for simultaneously detecting and killing colorectal cancer cells.The proposed method designed two allosteric probes,a report probe and a recognize probe.The method exhibited high sensitivity towards cell detection via the recognizing probe identifying target cancer cells and the report probe’s signal report.Combining bufalin and fluorouracil endowed better tumor cell inhibition.Results We observed significantly enhanced fluorescence dots surrounding the HCT-116 cell membranes.No fluorescence increments in the other three cells were identified,indicating that the established liposome complex could specifically bind with target cells.In addition,the best ratio of bufalin to fluorouracil was 0.15 and 0.5,respectively.This improved the anti-tumor effects and achieved more than 60%tumor cell inhibition.Conclusion This method will provide new opportunities for intracellular biomolecule detection and targeted cancer cell therapy.

Bufalin通过细胞外信号调节激酶／P90核糖体S6激酶2通路对人食管癌细胞裸鼠移植瘤增殖及凋亡的影响

目的探讨bufalin通过细胞外信号调节激酶（ERK）／P90核糖体s6激酶2（RSK2）通路对裸鼠移植瘤增殖及凋亡的影响。方法建立裸鼠食管癌细胞移植瘤模型，并分为模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组，观察bufalin对裸鼠食管癌移植瘤的影响，显微镜下观察各组移植瘤的组织学表现。采用原位末端转移酶标记技术（TUNEL）检测各组移植瘤的凋亡指数，采用实时定量PCR法检测各组移植瘤组织中ERK、RSK2mRNA的表达，采用Westernblot和免疫组化法检测移植瘤组织中ERK、RSK2、糖原合成激酶3β（GSK313）、Bad及其磷酸化水平。结果模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的裸鼠肿瘤体积分别为（1．758±0．181）cm3、（1．680±0．150）cm3、（1．285±0．134）cm3、（0．873±0．095）cm3、（0．815±0．108）cm3和（0．530±0．104）cm3。HE染色显示，各组移植瘤组织均有不同程度的坏死，以联合用药组最为明显。TUNEL法显示，模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的凋亡指数分别为（6．0±0．6）％、（11．0±0．7）％、（19．1±0．9）％、（25．1±1．4）％、（20．0±1．2）％和（17．1±0．7）％，以bufalin高剂量组凋亡指数最高。实时定量PCR检测显示，模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的ERKmRNA的Act值分别为0．270±0．084、0．293±0．081、0．596±0．224、0．857±0．183、0．868±0．187和1．313±0．282；RSK2mRNA的Act值分别为0．340±0．062、0．337±0．071、0．642±0．226、0．915±0．170、0．923±0．176和1．413±0．269，ERK和RSK2mRNA表达均呈降低趋势。Westernblot和免疫组化检测显示，各组ERK、RSK2、Bad蛋白水平的差异均无统计学意义（均P〉0．05）；模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的P-ERK蛋白相对表达量分别为0．721±0．094、0．695±0．095、0．555±0．080、0．388±0．052、0．341±0．060和0．235±0．056，免疫反应评分中位数分别为8、8、6、4、5和3分。模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的p-RSK2蛋白相对表达量分别为0．613±0．085、0．612±0．084、0．427±0．089、0．305±0．056、0．258±0．051和0．158±0．058，免疫反应评分中位数分别为8、8、5、3、3和1分。模型组、bufalin低剂量组、bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组的GSK313蛋白相对表达量逐渐升高，p-GSK313和p-Bad蛋白相对表达量均呈降低趋势，bufalin中剂量组、bufalin高剂量组、PD98059组和联合用药组与模型组比较，差异均有统计学意义（均P〈0．05）。结论bufalin对裸鼠食管癌移植瘤有明显的抑制作用。Bufalin可通过抑制ERK、RSK2的磷酸化水平抑制移植瘤的生长，通过抑制GSK3β的失活而抑制肿瘤的增殖，通过下调p-Bad的表达而发挥促凋亡作用。

基于网络药理学探讨中药蟾毒灵在骨肉瘤中的抗肿瘤作用机制

目的:本研究运用网络药理学分析方法,了解蟾毒灵(bufalin)在骨肉瘤中的抗肿瘤作用机制,并进一步利用相关实验进行验证。方法:通过PharmMapper数据库预测得到蟾毒灵的潜在作用靶点,借助GeneCards数据库搜索骨肉瘤相关疾病靶点,对两个数据库中的靶点进行Venny分析,随后使用String数据库绘制蛋白互作(PPI)网络,利用David数据库对关键靶点进行基因本体(gene ontology,GO)富集及KEGG信号通路分析。最后运用Cytoscape 3.7.2及Origin 2020软件构建“化合物-通路-靶点-疾病”网络图。最后利用CCK-8实验和Western blot实验进行验证。结果:通过药理学分析一共获得蟾毒灵与骨肉瘤共同靶点107个,KEGG通路富集分析共得到相关信号通路34条,涉及到肿瘤信号通路有:PI3K-AKT信号通路、MAPK信号通路、p53信号通路、VEGF信号通路、ErbB信号通路等。将其进行PPI网络分析表明,MYC、CCND1、IL6、ESR1、CDKN2A、IGF1、CAT、CDKN1A、ANXA5、PTGS2为连接度最高、权重最大的靶点。CCK-8实验证实蟾毒灵具有抑制骨肉瘤细胞增殖的作用;Western blot发现增殖相关蛋白MYC、CCND1蛋白表达下降。回复实验进一步证实,蟾毒灵可能通过调控PI3K-AKT信号通路发挥抗骨肉瘤的作用。结论:基于网络药理学分析以及相关实验发现蟾毒灵可能通过调控PI3K-AKT信号转导通路抑制骨肉瘤的生长。

蟾毒灵抑制乏氧耐药细胞诱导的M2型巨噬细胞极化逆转结肠癌耐药

目的:在乏氧微环境中探讨蟾毒灵(bufalin,BU)抑制耐药结肠癌细胞诱导M2型巨噬细胞极化对普通结肠癌细胞的作用。方法:培养基中加入氯化钴(cobalt chloride,CoCl 2)模拟乏氧环境,佛波酯(phorbol 12-myristate 13-acetate,PMA)诱导人源单核细胞THP-1分化为M0巨噬细胞。分别采集乏氧条件下耐药结肠癌细胞、普通结肠癌细胞和BU作用于耐药结肠癌细胞的条件培养基(conditioned medium,CM),然后放入M0巨噬细胞中。通过流式细胞术、Realtime PCR、ELISA实验检测M2型巨噬细胞极化标志因子的表达;运用CCK-8和蛋白免疫印迹(western blot,WB)实验检测耐药结肠癌和普通结肠癌条件培养基诱导M2型巨噬细胞极化后的上清对普通结肠癌的作用。结果:在缺氧环境下,与普通结肠癌细胞相比,耐药结肠癌细胞CM促进M2型巨噬细胞标志物IL-10、TGF-β、CD11b、CD206升高(P<0.01,P<0.05)。极化巨噬细胞的上清液增加了普通结肠癌细胞中P-gp和Bcl-2的表达,同时降低了Bax的表达和对奥沙利铂的敏感性。耐药结肠癌细胞经BU处理后,M2型巨噬细胞标志物IL-10、TGF-β、CD11b、CD206表达降低(P<0.01,P<0.05)。此外,P-gp和Bcl-2的表达减少,而Bax的表达增加,导致对OXA的敏感性增加。结论:乏氧条件下,结肠癌耐药细胞CM促进M2型巨噬细胞极化,蟾蜍灵可通过调节M2型巨噬细胞极化过程逆转结肠癌耐药。

蟾毒灵抑制M2型巨噬细胞介导的结直肠癌迁移和上皮间质转化

目的探讨蟾毒灵(BU)抑制M2型巨噬细胞介导结直肠癌转移的作用。方法用佛波酯(PMA)诱导人急性白血病单核细胞(THP-1)分化为M0型巨噬细胞,48h后用含有白细胞介素-4(IL-4)和IL-13的培养基处理M0型巨噬细胞,通过酶联免疫吸附试验(ELISA)、形态学、实时荧光定量(RT-qPCR)实验观察其表面标志物和形态变化,RT-PCR和ELISA实验检测M2型巨噬细胞的表面标志物转化生长因子-β(TGF-β)、IL-10。通过ELISA实验比较M2型巨噬细胞和结直肠癌细胞HCT116上清液中IL-6分泌水平,通过Transwell实验、划痕实验、RT-qPCR和Western blot实验检测条件培养基对结直肠癌细胞HCT116的的影响。在条件培养基中加入BU后,通过Western blot、Transwell实验、划痕实验、和RT-qPCR实验观察可以HCT116中AKT/PI3K蛋白以及迁移和上皮间质转化能力的变化。结果将THP-1成功诱导成为M2型巨噬细胞。M2型巨噬细胞通过分泌IL-6激活了HCT116中AKT/PI3K蛋白磷酸化,促进了其迁移和上皮间质转化能力。BU可以抑制M2巨噬细胞介导的HCT116迁移和上皮间质转化。结论M2型巨噬细胞释放IL-6激活了结直肠癌细胞AKT/PI3K信号通路,促进了其迁移和上皮间质化。此外,BU可以抑制其促迁移和上皮间质化作用。

Bufalin抑制食管癌TE13细胞迁移机制探讨

目的蟾蜍灵(Bufalin)是中医抗癌药物蟾酥的有效成分之一,但其抗肿瘤的机制并不十分清楚。本研究通过分析不同浓度Bufalin对食管癌TE13细胞株迁移距离、MEK1/2及其活化形式P-MEK1/2和基质金属蛋白酶(matrix metalloproteinase,MMP)表达的影响,初步探讨Bufalin抑制食管癌TE13细胞迁移能力的作用机制。方法采用四甲基偶氮唑蓝[3-(4,5-dimethylthiazol-yl)-2,5-diphenyl tetrazolium bromide,MTT]法测定细胞药物毒性,细胞划痕实验分别测量不同浓度(0、10、25、50和100nmol/L)Bufalin处理组食管癌TE13细胞的迁移距离。蛋白质印迹法及免疫细胞化学法检测MEK1/2和P-MEK1/2蛋白的表达。逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测MMP-2、MMP-9mRNA的表达情况。结果 MTT法结果显示,Bufalin药物最大无毒浓度(TC0)为100nmol/L。细胞划痕实验结果显示,不同浓度Bufalin处理组在划痕36h后与对照组相比迁移的距离不同,随着Bufalin浓度的增加,细胞迁移距离下降,100nmol/L迁移距离最小(F=243.163,P<0.01),Bufalin能有效抑制食管癌细胞迁移距离。蛋白质印迹法及免疫细胞化学检测结果显示,Bufalin对MEK1/2蛋白的表达无影响,但能显著抑制其活化形式P-MEK1/2的表达,并呈剂量依赖性,随Bufalin浓度的升高(0、10、25、50和100nmol/L),P-MEK1/2相对表达量逐渐下降,分别为0.710±0.006、0.676±0.010、0.656±0.010、0.599±0.020和0.521±0.021,F=76.369,P<0.01。P-MEK1/2阳性细胞数随着Bufalin浓度的升高而减少,分别为(80.330±1.905)%、(68.407±2.219)%、(67.297±1.797)%、(52.617±2.368)%和(26.97±2.912)%,F=243.348,P<0.01。RT-PCR结果显示,随着Bufalin药物浓度的升高,MMP-2mRNA相对表达量分别为0.772±0.010、0.725±0.019、0.663±0.015、0.582±0.019和0.519±0.019,F=112.990,P<0.01;MMP-9mRNA相对表达量分别为0.783±0.013、0.740±0.010、0.703±0.006、0.601±0.017和0.531±0.010,均低于对照组,F=179.417,P<0.01。结论 Bufalin可能通过下调MEK1/2的活性,从而抑制食管癌TE13细胞的迁移能力。

基于网络药理学和细胞实验探讨蟾蜍灵治疗胃癌的作用机制

本研究基于网络药理学、分子对接和细胞实验探究蟾蜍灵治疗胃癌的作用机制。利用PubChem和PharmMapper数据库检索蟾蜍灵的药物靶点;GeneCards数据库收集胃癌的相关靶点,运用Venny软件和STRING数据库构建药物与疾病共同靶点的PPI网络图,通过DAVID数据库进行GO富集和KEGG通路分析。利用Cytoscape3.9.0软件进行可视化并筛选核心靶点,通过GIEPA数据库、Kaplan-Meier方法、AutoDock和PyMOL软件分析核心靶点分别与胃癌和蟾蜍灵之间的关系。最后通过细胞实验对网络药理学预测的靶点和信号通路进行实验验证。网络药理学筛选到蟾蜍灵的药物靶点285个,胃癌相关的疾病靶点909个,蟾蜍灵与胃癌的交集靶点91个,结合PPI网络度值排名、胃癌患者中表达水平以及患者生存预后相关性筛选2个核心靶点为HSP90AA1和SRC。KEGG富集分析蟾蜍灵治疗胃癌可能与PI3K-Akt、FoxO、Ras和MAPK信号通路有关。生存分析结果显示,HSP90AA1和SRC mRNA在胃癌组织中表达显著上调,且其高表达的患者总生存期显著低于低表达组。分子对接结果显示,蟾蜍灵与核心靶点具有较好的结合力。细胞实验结果表明,与对照组相比,蟾蜍灵处理胃癌细胞MGC-803后,细胞的增殖、迁移能力均下降,而细胞凋亡数增加。MGC-803细胞中HSP90AA1、SRC、Bcl-2、p-AKT和FoxO1蛋白表达水平显著下调,Bax蛋白的表达显著上调。综上所述,蟾蜍灵可能通过调控HSP90AA1和SRC核心蛋白及Akt/FoxO1信号通路发挥治疗胃癌的作用。

Bufalin-induced cardiotoxicity: new findings into mechanisms

Bufalin is one of the main pharmacological and toxicological components of Venenum Bufonis and many traditional Chinese medicine preparations.The cardiotoxicity clearly limits its application to patients living in countries.Hence,an investigation of its toxicological mechanism is helpful for new drug development and treatment of the related clinical adverse reactions.We investigate the cardiotoxicity of bufalin using human induced pluripotent stem cells-derived cardiomyocytes(hiPSC-CMs)(0.003–0.1μmol·L–1),human induced pluripotent stem cells-derived cardiomyocytes(hiPSC-CMs)(0.03–0.3μmol·L–1)and eight human cardiac ion channel currents(0.01–100μmol·L–1)combined with an impedance-based bioanalytical and patch clamp method.Biphasic effect of bufalin on the contractility in hiPSC-CMs,which has been shown to strengthen myocardial contractility,accelerate conduction,and increase beating rate at the earlier stage of administration,whereas weakened myocardial contractility,abolished conduction,and ceased beating rate at the later stage of administration.Bufalin decreased the action potential duration(Action potential duration at 30%,50%and 90%repolarization),cardiac action potential amplitude,and maximal depolarization rate and depolarized the resting membrane potential of hiPSC-CMs.Spontaneous beating rates of hiPSC-CMs were markedly increased at 0.03μmol·L–1,while were weakened at 0.3μmol·L–1 after application.Bufalin blocks INav1.5 in a concentration-dependent manner with half maximal inhibitory concentration of 74.5μmol·L–1.Bufalin respectively increased the late sodium current and Na+-Ca2+exchange current with a concentration for 50%of maximal effect of 2.48 and 66.06μmol·L–1 in hiPSC-CMs.Whereas,bufalin showed no significant effects on other cardiac ion channel currents.The enhancement of the late sodium current is one of the main mechanism for cardiotoxicity of bufalin.

Reversal effect of bufalin on multidrug resistance in K562/VCR vincristine-resistant leukemia cell line

OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI) of bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin(ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry(FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein(P-gp), multidrug-associated protein-1(MRP1), Bcl-x L and Bax protein were measured by immunocytochemistry.RESULTS: The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to bufalin. The RIs of bufalin at concentrations of 0.0002,0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77,respectively. Preincubation of 0.001 μmol/L bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07%(P<0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with bufalin. When exposed to 0.01μmol/L bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-x L and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.CONCLUSION: Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-x L/Bax ratio.