CN113384596A - 一种抑制pi3k-akt通路和emt作用的橙皮素、顺铂组合物 - Google Patents
一种抑制pi3k-akt通路和emt作用的橙皮素、顺铂组合物 Download PDFInfo
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Abstract
本发明属于药物制备技术领域,具体涉及一种抑制PI3K‑AKT通路和EMT作用的橙皮素、顺铂组合物。本发明所述抑制PI3K‑AKT通路和EMT作用的橙皮素、顺铂组合物,由2‑6μM顺铂及50‑150μM橙皮素组成。与现有技术相比,本发明橙皮素与顺铂联用可以通过调节PI3K‑AKT相关通路来干预EMT过程和顺铂的化疗敏感性进而抑制TNBC侵袭转移、增强顺铂的化疗效果;且橙皮素主要来源于芸香科柑橘属甜橙Citrus sinensis(L.)Osbeck,具有取材广泛、毒副作用小且价格低廉等优点。
Description
技术领域
本发明属于药物制备技术领域,具体涉及一种抑制PI3K-AKT通路和EMT作用的的橙皮素、顺铂组合物。
技术背景
三阴型乳腺癌(Triple-negative breast cancer,TNBC)是乳腺癌中较特殊的亚型,其在年轻患者中发生的频率更高,肿瘤体积通常更大,分级更高,在生物学上更具侵袭性。由于缺乏有效的内分泌治疗和抗Her2治疗的靶点,TNBC患者难以从以上两种乳腺癌全身治疗中获益,因此常规化疗被认为是TNBC仅剩的一种全身治疗手段。目前TNBC最常用的一线化疗药物包括蒽环类、紫杉醇、顺铂及5-氟尿嘧啶类,其中顺铂在TNBC的常规化疗及新辅助化疗中表现出了一定的优势,但由于肿瘤本身的高度恶性及其对药物的敏感性差,使得患者的临床治疗效果并不理想,死亡率居高不下。复发和转移是TNBC患者死亡的主要原因,上皮间质转化(Epithelial-mesenchymal transition,EMT)是TNBC细胞发生侵袭转移的首要步骤,越来越多的证据表明,EMT除影响癌细胞的侵袭转移能力外,在癌症进展的不同方面发挥着关键作用,如干细胞特性和化疗敏感性、耐药性等,可见抑制EMT过程对TNBC化疗疗效及复发转移具有重要的现实意义。但目前,与顺铂联用能有效起到降低三阴性乳腺癌转移及化疗敏感性的药物,还不多且较难得。
发明内容
为解决上述技术问题,本发明提供了一种抑制PI3K-AKT通路和EMT作用的橙皮素、顺铂组合物。
本发明所述的一种抑制PI3K-AKT通路和EMT作用的橙皮素、顺铂组合物,由2-6μM顺铂及50-150μM橙皮素组成。
本发明所述的一种抑制PI3K-AKT通路和EMT作用的橙皮素、顺铂组合物,由4μM顺铂及100μM橙皮素组成。
本发明所述的抑制PI3K-AKT通路和EMT作用的橙皮素、顺铂组合物的应用,用于制备降低三阴性乳腺癌转移及化疗敏感性药物的制备。
与现有技术相比,本发明橙皮素与顺铂联用可以通过调节PI3K-AKT相关通路来干预EMT过程和顺铂的化疗敏感性进而抑制TNBC侵袭转移、增强顺铂的化疗效果;且橙皮素主要来源于芸香科柑橘属甜橙Citrus sinensis(L.)Osbeck,具有取材广泛、毒副作用小且价格低廉等优点。
附图说明:
图1为橙皮素与顺铂单独和联合应用对MDA-MB-231细胞存活率的影响。A.顺铂对MDA-MB-231细胞存活率的影响。B.橙皮素对MDA-MB-231细胞存活率的影响。C.橙皮素和顺铂联用对MDA-MB-231细胞存活率的影响。实验重复三次,数据均以平均值±标准差(SD)表示。
图2为橙皮素与顺铂单独和联合应用对MDA-MB-231细胞迁移能力的影响。A.划痕实验检测橙皮素、顺铂单用及联合治疗对MDA-MB-231细胞运动能力的影响(放大倍数,×40;比例尺,200μm)。B.Transwell检测橙皮素、顺铂单用及联合治疗对MDA-MB-231细胞迁移能力的影响(放大倍数,×100;比例尺,100μm)。C.划痕实验中各组细胞平均运动距离。D.Transwell实验中平均迁移细胞数目。所有数据结果以平均值±标准差(SD)表示。*p<0.05、**p<0.01或***p<0.001,与对照组相比。
图3为橙皮素与顺铂单独和联合应用对细胞克隆形成的影响。A.克隆形成实验检测橙皮素、顺铂单用及联合治疗对MDA-MB-231细胞集落形成能力的影响。B.各组相对克隆形成数目。所有数据结果以平均值±标准差(SD)表示。***p<0.001,与对照组相比。
图4为橙皮素、顺铂单独使用和联合使用对MDA-MB-231细胞凋亡的影响。A.流式细胞术分析橙皮素、顺铂单独或联合治疗后MDA-MB-231细胞的凋亡情况。B.各组细胞凋亡率。所有数据均显示为三个独立实验的平均±SD。*p<0.05,***p<0.001,与对照组相比。
图5为橙皮素、顺铂单独使用和联合使用对MDA-MB-231细胞EMT标志物表达的影响。A.N-cadherin的相对表达。B.Snail的相对表达。C.E-cadherin的相对表达。D.ZO-1的相对表达。*p<0.05,**p<0.01,***p<0.001,与对照组相比。
具体实施方式
为了便于理解本发明,下文将结合说明书附图和实施例对本发明作更全面、细致地描述,但本发明的保护范围并不限于以下具体的实施例。除非另有定义,下文中所使用的所有专业术语与本领域技术人员通常理解的含义相同。本文中所使用的专业术语只是为了描述具体实施例的目的,并不是旨在限制本发明的保护范围。
实施例1
1.实验材料
橙皮素购于上海源叶生物有限公司,质量分数99%;一抗:E-cadherin、ZO-1、N-cadherin、Snail、β-actin购自CST生物技术公司。二抗购自北京中杉金桥生物技术有限公司。Annexin V-FITC细胞凋亡检测试剂盒购自上海碧云天生物技术研究所。Transwell小室购自美国Corning Life Science公司。10×SDS-PAGE电泳缓冲液(B1005-500ML),10×蛋白电转移缓冲液(B1006-500ml),超敏发光液(P1010-100ML)购于北京普利莱基因技术有限公司。SDS-PAGE电泳液(P0014B-10L),Western及IP细胞裂解液(P0013-100ML),蛋白酶抑制剂PMSF(100m M),BCA蛋白浓度测定试剂盒(增强型),SDS-PAGE凝胶配制试剂盒(P0012A),QuickblockTM封闭液(TBSTx)购于碧云天生物技术研究所。Blue Plus IV Protein Marker购自于北京全式金生物技术有限公司。
2.细胞实验
2.1细胞培养
人乳腺癌细胞(MDA-MB-231)购于北纳创联生物技术有限公司,细胞培养采用含有10%胎牛血清,1%青霉素和链霉素的DMEM高糖培养基进行细胞培养。培养在37℃,95%O2/5%CO2的培养箱中。实验所使用的细胞均在5-7代以内且细胞密度达到80-90%,所有实验至少平行重复3次。
2.2细胞增殖实验
取对数生长期的MDA-MB-231细胞,以0.25%胰蛋白酶消化,1000rpm离心5分钟,弃上清,在96孔板中接种100μL细胞悬液,每孔的细胞接种量为5×103个,每组五个复孔,贴壁后移去培养液,加入含不同药物的培养液,空白对照组不加药,继续培养24h,细胞贴壁后计为0天(之后依次计24h,48h,72h),加入CCK8(终浓度10μL/100μL)培养箱内培养3小时;酶标仪测其吸光度,测定波长为450nm。计算细胞增殖率。
计算公式:细胞增殖率=[(As-Ab)/(Ac-Ab)]×100%
As:实验孔(含有细胞的培养基、CCK8试剂和药物;顺铂组加4μM顺铂,橙皮素组加100μM橙皮素,联用组加4μM顺铂及100μM橙皮素)。
Ac:对照组(含有细胞的培养基和CCK8试剂,不含药物)。
Ab:空白孔(不含细胞和药物,只加培养基和CCK8试剂)。
2.3划痕实验
取对数生长期人乳腺癌MDA-MB-231细胞,以0.25%胰蛋白酶消化,1000rpm离心5分钟,弃上清,在6孔板中接种1mL细胞悬液,每孔的细胞接种量为6×105个,培养24h后,生长85%-90%,6孔板下垫着直尺,用20μL移液枪头垂直划痕。PBS缓冲液洗细胞轻柔吹打3次,去除划线而脱落的细胞,加入含不同药物的培养液,空白对照组不加药,放入37℃5%CO2培养箱,继续培养24h。取出6孔板,对划痕细胞进行拍照,每组实验重复3次。统计方法:用Image J软件打开细胞图片,测量划痕细胞间距离,计算划痕迁移率。
计算公式:划痕迁移率=[(初始时间划痕宽度值-相应时间点划痕宽度值)÷初始时间划痕宽度值]×100%
2.4细胞迁移实验
取对数生长期人乳腺癌MDA-MB-231细胞,弃去旧培养基,用PBS荡洗一遍,加入0.25%胰蛋白酶消化液,消化使贴壁细胞脱落,计数5×104个/ml,制成细胞悬液。在24孔板中放置transwell小室,外室每孔加入600μL含10%血清的培养基放入培养箱预热。MDA-MB-231细胞悬浮于500μL含药培养基中,缓慢加入小室内室,放入培养箱,迁移24h。之后,小心弃去内室培养基,用4%多聚甲醛室温固定10min。小心擦干内室多聚甲醛,在结晶紫溶液中染色10min。小心冲洗小室,并轻轻擦去内室细胞。在显微镜下拍照(取5个视野),计数,统计。
2.5克隆形成实验
将细胞按不同分组药物处理之后,以每孔800-1000个细胞种6孔板,设2个复孔,充分混匀,确保细胞在板中均匀分布。约3-4天左右给细胞换液,后期应根据细胞的长势适当调整。培养约10天左右。克隆对光时肉眼可见,显微镜下观察克隆,-般在单个克隆内细胞数多于50个细胞的情况下可结束观察。弃去培养基,沿侧壁加入PBS缓冲液冼3次,避免将细胞吹起来,每孔加入600μL甲醇固定细胞5min,弃去甲醇,加入PBS润洗一遍,再每孔加入1mL结晶紫染液作用10min,弃掉染液,加入双蒸水洗2-3次,然后对6孔板拍照计数。
2.6细胞凋亡实验
将处于对数生长期的MDA-MB-231细胞以1×105/mL的密度接种于6孔板,37℃、5%CO2培养过夜,细胞贴壁后分别予以不同药物处理24h,之后吸去培养基,PBS洗2遍,用不含EDTA的胰酶消化,离心后收集细胞。收集的细胞用冷PBS洗两遍。按照凋亡试剂盒操作说明加入Annexin V-FITC和PI染料,通过流式细胞术分析细胞凋亡情况。
2.7Westen blot实验
用含有蛋白酶抑制剂和磷酸酶抑制剂的RIPA裂解液对MDA-M-231细胞中总蛋白进行提取。所提蛋白浓度使用BCA蛋白浓度测定试剂盒进行测定,蛋白样本置于-80℃冰箱保存。根据实验样品中的具体蛋白含量选择合适的蛋白上样量,采用SDS聚丙烯酰胺凝胶进行浓缩及分离,电泳完成之后将蛋白电转至PVDF膜,随后用5%脱脂奶粉封闭1h,封闭后在4℃冰箱孵育一抗过夜。第二天使用预先配制好的TBST洗涤3次,每次5min,随后加入二抗,在室温下孵育1小时,然后使用用TBST洗涤3次,每次5min,最后使用曝光机进行显影。
2.9数据统计分析
结果以平均值±标准差(SD)表示。使用Graphpad Prism 6.0单因素方差分析(ANOVA)进行组间比较。同时使用Tukey’s多重比较来比较各组之间的差异性。p<0.05的值被认为具有统计学意义。(*p<0.05,**p<0.01,**p<0.001)
3.实验结果
3.1橙皮素和顺铂联合治疗协同抑制MDA-MB-231细胞增殖
橙皮素和顺铂均以剂量依赖的方式抑制MDA-MB-231细胞的增殖。利用CCK8法检测顺铂不同浓度对MDA-MB-231细胞活力的抑制作用。如图1A所示,顺铂1-32μM处理细胞24、48小时,呈时间和浓度依赖性地抑制细胞活力。24小时顺铂对MDA-MB-231细胞的作用IC50浓度为4μM。利用CCK8法检测橙皮素不同浓度对MDA-MB-231细胞活力的抑制作用。如图1B所示,橙皮素7.82-250μM处理细胞24、48小时,呈时间和浓度依赖性地抑制细胞活力。24小时橙皮素对MDA-MB-231细胞的作用IC50浓度为100μM。二者联用后,相对顺铂单用组,细胞存活力显著下降,表明橙皮素与顺铂联用对细胞增殖的抑制作用优于顺铂单用。
3.2橙皮素和顺铂联合治疗协同抑制MDA-MB-231细胞的迁移能力
为了观察橙皮素与顺铂联用对MDA-MB-231细胞运动能力的影响,我们利用细胞划痕实验,检测药物作用后的细胞运动能力。在培养皿底部制造一条划痕,加入含药无血清培养液,24h后拍照,观察培养皿底部划痕愈合情况。实验结果表明,顺铂4μM作用4h后,细胞运动能力减弱(图2A)。橙皮素与顺铂联用后,未愈合划痕面积大于顺铂单用组。本实验结果表明:橙皮素与顺铂联用能增强对细胞活动能力的抑制,效果优于顺铂单用。
采用Transwell小室检测橙皮素与顺铂联用对人乳腺癌MDA-MB-231细胞迁移力的影响。如图2B所示,采用4μM顺铂处理细胞24h后,细胞的迁移力显著下降(P<0.05)。橙皮素单用组对MDA-MB-231细胞的迁移也有一定的抑制作用。而橙皮素与顺铂联用后,对人乳腺癌MDA-MB-231细胞迁移的抑制作用较各自单用组更为明显。
3.2橙皮素和顺铂联合治疗协同抑制MDA-MB-231细胞的克隆形成
当单个细胞在体外增殖6代以上,其后代所组成的细胞群体,成为集落或克隆。每个克隆含有50以上的细胞,大小在0.3-1.0mm之间。集落形成率表示细胞的独立生存能力。贴壁后的细胞不一定每个都能增殖和形成克隆,而形成克隆的细胞必为贴壁和有增殖活力的细胞。克隆形成率反映细胞两个重要性状。通过检测橙皮素、顺铂单用及联合治疗对MDA-MB-231细胞群体依赖性和增殖能力的影响,我们进行了细胞克隆形成的检测。由图3可知,橙皮素和顺铂联合作用对细胞集落形成的抑制作用明显强于各自单独作用。
3.3橙皮素和顺铂联合治疗协同抑制MDA-MB-231细胞的凋亡
为研究橙皮素顺铂联用对MDA-MB-231细胞凋亡的影响,我们用流式细胞术进行了细胞凋亡的检测。如图4所示,橙皮素和顺铂单个药物治疗和药物联用均提高了MDA-MB-231细胞早期和晚期细胞凋亡的比率。此外,与单独治疗相比,二者联用在诱导细胞凋亡方面的作用更强。
3.4橙皮素和顺铂联用对MDA-MB-231细胞EMT的影响
为了验证橙皮素与顺铂联用对乳腺癌细胞MDA-MB-231EMT的逆转作用,选用Western blot实验观察上皮和标记蛋白表达的变化。如图5所示,与对照组相比,橙皮素和顺铂治疗都能对MDA-MB-231细胞的EMT过程起到一定的抑制作用(*p<0.05)。橙皮素与顺铂联用后,更多地逆转了EMT相关标记蛋白的表达,(***p<0.001)。结果显示,橙皮素与顺铂联用后,上皮细胞标志物E-cadherin、ZO-1表达明显增加,间充质分子N-cadherin和Snail的表达量明显降低,效果明显优于顺铂和橙皮素单用组。
Claims (3)
1.一种抑制PI3K-AKT通路和EMT作用的橙皮素、顺铂组合物,其特征在于:由2-6μM顺铂及50-150μM橙皮素组成。
2.如权利要求1所述的抑制PI3K-AKT通路和EMT作用的橙皮素、顺铂组合物,其特征在于:由4μM顺铂及100μM橙皮素组成。
3.如权利要求1所述的抑制PI3K-AKT通路和EMT作用的橙皮素、顺铂组合物的应用,用于制备降低三阴性乳腺癌转移及化疗敏感性药物的制备。
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