CN113730385A - 白花丹素在制备抗吉非替尼耐药非小细胞肺癌药物中的应用 - Google Patents
白花丹素在制备抗吉非替尼耐药非小细胞肺癌药物中的应用 Download PDFInfo
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Abstract
本发明公开了白花丹素在制备抗吉非替尼耐药非小细胞肺癌药物中的应用。本发明通过实验验证了白花丹素能够显著抑制人吉非替尼原发性耐药与获得性耐药肺腺癌细胞的活性和增殖,显著影响人吉非替尼原发性耐药与获得性耐药肺腺癌细胞的形态与结构,诱发凋亡,能够增加促凋亡相关蛋白的表达。本发明发现了白花丹素抗吉非替尼耐药肺腺癌的新用途,其有望成为吉非替尼耐药肺腺癌患者治疗的候选化合物。
Description
技术领域
本发明涉及一种中药单体的医药用途,具体涉及白花丹素在制备抗吉非替尼耐药非小细胞肺癌的药物中的应用。
背景技术
肺癌是全球癌症相关死亡的主要原因之一,每年新诊断患者为180万例,死亡患者为160万例。肺腺癌(lung adenocarcinoma,LUAD)是最常见的肺癌组织学亚型,发病率比其他亚型的肺癌病例占比最大(约40%),全球每年有超过50万例死亡。手术切除是早期肺癌患者的首选治疗方式,而中晚期肺癌患者的治疗通常采用放疗和依赖于细胞毒性药物的联合化疗,如顺铂、培美曲塞和吉西他滨;还有一部分致癌基因突变驱动的肺癌患者,他们受益于靶向分子治疗,如吉非替尼、厄洛替尼等。多项基因组测序项目表明,超过一半的LUAD患者存在表皮生长因子受体(EGFR)突变和间变性淋巴瘤激酶(ALK)融合,针对这些突变的个体化治疗已成为标准的治疗方法。EGFR酪氨酸激酶抑制剂(EGFR tyrosine kinaseinhibitors,EGFR-TKI)是一类以吉非替尼、厄洛替尼等为代表的分子靶向药物,临床上首选用于治疗有EGFR敏感突变的晚期LUAD患者。然而,大多数患者在接受EGFR-TKI治疗9-14个月后,都不可避免的出现耐药和疾病进展。因此,寻找发现可阻断EGFR-TKI耐药的化合物,开发新的抗肺腺癌EGFR-TKI耐药的药物,对于提高患者预后具有极其重要的意义。
天然产物在抗癌药物发现过程中发挥着重要作用,美国食品和药物监督管理局批准的实体药物中,天然产物及其衍生物占所有新药的三分之一。植物来源的天然化合物及中药单体具有强大的抗肿瘤特性,它们可以单独使用,也可以与化疗药物联合使用,以克服肿瘤细胞耐药,如槲皮素通过降低p-糖蛋白表达、抑制药物转运诱导柔红霉素耐药的胃癌细胞凋亡,杨梅素通过Bcl-2家族依赖的内源性和DR5依赖的外源性凋亡途径诱导顺铂耐药的卵巢癌细胞凋亡,而白藜芦醇则通过JNK依赖性上调p62表达和激活AMPK通路促进伊马替尼耐药的慢性髓性白血病细胞自噬死亡。白花丹素(Plumbagin,PLM)是一种黄色结晶植物化学物质,主要分布于白桦科(Plumbaginaceae)、木犀科(Ebenceae)、黄连科(Dioncophyllaceae Ancestrocladaceae)和蔷薇科(Droseraceae)等植物的根、叶和茎皮中。PLM(5-羟基-2-甲基-1,4-萘醌)的分子式为C11H8O3,其结构如下所示:
白花丹素属于醌类化合物。醌类化合物广泛存在于天然植物中,可能是由于醌环存在时通过电子传递形成自由基,含有醌结构的化合物具有一定的细胞毒性。临床上也有一些含醌类结构的药物如多柔比星和米托蒽醌因其抗癌作用而受到广泛关注。PLM作为一种天然来源萘醌,已被报道具有多种生物活性,如抗癌、抗炎症、抗氧化、抗糖尿病、抗细菌、抗真菌、抗肺纤维化、抗动脉粥样硬化和镇痛活性,其抗肿瘤活性也在多种癌症模型中得到验证,如通过AMPK信号通路促进结肠癌细胞凋亡,通过PI3K/Akt信号通路诱导子宫内膜癌细胞自噬,通过基质细胞衍生因子(SDF-1)/CXCR4-CXCR7轴抑制肝细胞癌血管生成。PLM此前被报道可单独用于诱导宫颈鳞癌和舌鳞癌顺铂耐药细胞凋亡,但PLM是否对吉非替尼原发性耐药与获得性耐药LUAD细胞也具有抗肿瘤活性,目前尚不清楚。
发明内容
本发明的目的在于为白花丹素提供一种新的医药用途。具体而言,本发明提供了白花丹素在制备抗吉非替尼耐药非小细胞肺癌药物中的应用。所述吉非替尼耐药包括原发性耐药和/或获得性耐药。
所述药物为包含有白花丹素及药学上可接受的辅料的药物。所述白花丹素在抗吉非替尼耐药非小细胞肺癌时,可以单独使用,也可以与其他药物配合同时使用,或者与其他药物一起制成复方制剂使用。所述药学上可接受的辅料,是指制备不同剂型时加入所需的各种常规辅料,例如稀释剂、黏合剂、崩解剂、助流剂、润滑剂、矫味剂、包合材料、吸附材料等。所述药物的剂型可以是颗粒剂、散剂、片剂、胶囊剂、丸剂、口服液、注射剂等。
本发明发现白花丹素能够显著抑制吉非替尼耐药肺腺癌细胞的活性和生长能力,通过线粒体途径诱导吉非替尼原发性耐药与获得性耐药肺腺癌细胞发生凋亡,发挥抗肿瘤活性,发现了白花丹素抗吉非替尼耐药肺腺癌的新用途,其有望成为吉非替尼耐药肺腺癌患者治疗的候选化合物。
附图说明
图1、吉非替尼给药72h后吉非替尼耐药LUAD细胞的IC50值。
图2、PLM处理48h后吉非替尼耐药LUAD细胞的IC50值。
图3、PLM和顺铂对吉非替尼耐药LUAD细胞克隆形成率的影响。
图4、PLM和顺铂对吉非替尼耐药LUAD细胞形态的影响。
图5、Hoechst染色观察PLM和顺铂对吉非替尼耐药LUAD细胞细胞核的影响。
图6、透射电镜观察PLM对LUAD细胞亚显微结构的影响。
图7、流式细胞术检测PLM和顺铂对吉非替尼耐药LUAD细胞凋亡率的影响。
图8、PLM和顺铂对吉非替尼耐药LUAD细胞中凋亡相关蛋白表达的影响。
具体实施方式
下面结合附图和具体实验对本发明进行详细说明,用于验证白花丹素抗吉非替尼耐药非小细胞肺癌的效果。
1、实验方法:
一、浓度梯度递增法建立吉非替尼耐药细胞株HCC827 GR
将HCC827细胞接种于6孔板或25T培养瓶中进行细胞培养,待细胞生长状态良好,处于对数生长期时,加入含0.001μM吉非替尼的完全培养基处理6-24h,即不定时观察给药后细胞状态,若漂浮细胞太多,则立即更换为1640完全培养基(不含有药物吉非替尼)对细胞进行培养,若细胞状态尚可,则24h后进行撤药处理。待细胞恢复良好生长状态时再加入0.001μM的吉非替尼处理24h,然后再更换为1640完全培养基对细胞进行培养。如此反复进行给药和撤药处理,使HCC827细胞间断性暴露于药物吉非替尼中,等到细胞能够在该给药浓度的完全培养基中稳定生长时,再梯度增加给药浓度,换用更大浓度的吉非替尼处理,按此方法连续培养细胞,中途不定时通过MTT实验检测IC50变化,直至耐药指数(ResistanceIndex,RI)至少大于10,认为得到吉非替尼获得性耐药株HCC827 GR,RI=耐药株IC50/亲本株IC50。
二、MTT实验
将吉非替尼原发性耐药细胞A549和H1975细胞以5000个/孔的浓度、HCC827和HCC827 GR细胞以8000个/孔的浓度接种到96孔板中,每孔为100μL细胞悬液,每个分组设置3个重复孔。待细胞贴壁后,以0、0.001、0.01、0.1、1、10、100μM的不同浓度吉非替尼添加到每组实验孔中,细胞培养箱内培养作用72h后,或以0、1.25、2.5、5、10、20、40、80μM的不同浓度PLM添加到每组实验孔中,细胞培养箱内培养作用48h后,每孔加入20μL 0.5mg/L的MTT溶液,培养箱孵育4h,注射器吸弃培养液,切勿吸到紫色结晶沉淀,然后每孔加入150μL DMSO溶液,放至摇床上快速振摇10min,用全波长酶标仪测定各孔溶液在490nm波长处的吸光度值(OD值)。通过SPSS软件计算各组细胞的IC50值。
三、平板克隆形成实验
(1)细胞计数与稀释:待培养瓶中的细胞处于对数生长期时,进行消化计数,调整细胞浓度为3×105个/mL,转移1mL该细胞悬液置于1.5mL离心管中,标记为A管,取两个新的1.5mL离心管,分别标记B和C,并向其中加入900μL 1640完全培养基。用移液枪吹打混匀A管后,吸取100μL加入至B管,吹打混匀约50次,吸取100μL转移至C管,此时C管所含细胞浓度为3×103个/mL。
(2)克隆形成:取12孔板,向C管吸取50μL细胞悬液接种至孔板中,每种细胞设置3个复孔,并向孔板中补齐培养基至1mL,十字法摇匀。24h后,待各组细胞贴壁,更换含有梯度浓度PLM或顺铂的完全培养基继续培养2-3周,每2-3天观察细胞克隆形成情况。到达实验终点时,弃去培养基,用PBS溶液清洗2次后,加入适量4%多聚甲醛固定,固定条件为室温下30min。弃去固定液,用PBS溶液清洗2次,加入0.1%结晶紫溶液染色,染色条件为室温下30min。弃去结晶紫溶液后用自来水缓慢清洗2次,再更换为纯水清洗1次,置于50℃烘箱中烤干,在显微镜(放大100×)下观察并记录含有超过50个细胞的克隆数。根据公式:克隆形成率=克隆形成数/接种细胞数,计算各组细胞克隆形成率。
四、HE染色
(1)细胞爬片:盖玻片(24mm×24mm)经酸化清洁及高压灭菌后,浸泡在无水乙醇中,使用前镊子夹取,过火加速酒精挥发,平放至6孔板中,盖好盖子放置一边备用;将对数期细胞消化计数,调整细胞浓度至4.0-4.5×105个/mL,每孔接种1mL细胞悬液,补齐培养基至2mL;将孔板内细胞摇匀,直至在显微镜下观察每孔细胞均匀分布;细胞培养在37℃、5%CO2的培养箱中,待细胞贴壁后,吉非替尼原发性耐药细胞A549细胞对照组加入含0.1%DMSO,实验组加入含5、10和20μM PLM或10μM顺铂的1640完全培养基,吉非替尼获得性耐药细胞HCC827 GR和H1975细胞对照组加入含0.1%DMSO,实验组加入含2.5、5和10μM PLM或5μM顺铂的1640完全培养基,继续培养48h。
(2)固定细胞:在48h检测点分别取出相应孔板,弃去培养基后用PBS溶液清洗细胞1次,缓慢向孔板中加入固定液预冷的无水乙醇约2mL,固定液需彻底没过细胞,室温或4℃冰上中静置30min固定;弃去固定液后,用PBS溶液清洗爬片3-5次。
(3)苏木素染色:彻底吸净孔板中PBS溶液,加入1mL苏木素染色液,室温下染色5min;将爬片取出,平放在载玻片上,以与水柱45°角方式,进行自来水缓慢冲洗1min以上,使核染色返蓝,同时达到彻底清洗苏木素。
(4)伊红染色:在爬片上滴加伊红染色液,室温染色5min,期间注意补充伊红染液,防止爬片干片;将爬片取出,平放在载玻片上,自来水冲洗1min,然后PBS溶液清洗爬片3次。
(5)封片及拍照记录:取洁净载玻片(24mm×50mm),用铅笔在磨砂出标记实验组,在载玻片上滴加适量甘油,将清洗干净的爬片细胞面朝下倒扣,接触到甘油后,使其缓缓盖上,不要气泡产生,用吸水纸上吸去多余液体,置于光学显微镜下拍照记录细胞形态变化。
五、Hoechst染色
取对数生长期的细胞消化,将各组细胞接种到24孔板中,待细胞贴壁后,吉非替尼原发性耐药细胞A549细胞对照组加入含0.1%DMSO,实验组加入含5、10和20μM PLM或10μM顺铂的1640完全培养基,吉非替尼获得性耐药细胞HCC827 GR和H1975细胞对照组加入含0.1%DMSO,实验组加入含2.5、5和10μM PLM或5μM顺铂的1640完全培养基,作用48h。弃去培养基,用1mL常温PBS溶液洗涤细胞1遍,每孔加入300μL Hoechst 33342染色液,于37℃恒温培养箱中避光孵育30min。取出孔板,用常温PBS溶液洗涤细胞2遍,加入适量4%多聚甲醛避光固定30min。用PBS溶液洗2遍,置于荧光显微镜下拍照观察。
六、透射电镜观察
将各组细胞分别接种到10mm2细胞培养皿中,待细胞贴壁生长至融合度约为85%后,往吉非替尼原发性耐药细胞A549、吉非替尼获得性耐药细胞HCC827 GR和H1975细胞中分别加入10μM、5μM和5μM PLM作用48h。收集上清液至15mL离心管,PBS溶液洗涤2次,收集清洗液,加入胰酶于37℃恒温箱中消化,用收集的培养基终止消化,收集消化液至离心管,再次用适量PBS洗涤培养皿,收集清洗液,在室温下1500-2000rpm离心15min。弃去上清,加入1ml PBS溶液轻柔吹打混匀,转移至1.5ml离心管中,再次在室温下1500-2000rpm离心15min。沿管壁缓慢加入1ml预冷的3%戊二醛,于4℃固定过夜。PBS溶液清洗3次,每次10-15min,1%锇酸固定1-2h。PBS溶液清洗3次,每次10-15min,乙醇-丙酮进行逐级脱水,其中50%乙醇1次(4℃)、70%乙醇1次(4℃)、90%乙醇1次、90%乙醇:90%丙酮混合液1次、90%丙酮1次、100%丙酮3次,均脱水10-15min。丙酮:包埋行浸透,其中丙酮:包埋剂=2:1浸透2h以上,丙酮:包埋剂=1:2浸透1-3h以上或过夜、全浸透纯包埋剂2h或过夜,纯包埋剂环氧树脂618进行包埋,后续的聚合(40℃-48℃-60℃,15h-2h-24h)、修块、切片、染色步骤均由电镜室老师操作,透射电子显微镜下观察并拍照。
七、流式细胞术测细胞凋亡
1)样品制备:取对数生长期的各组细胞消化计数,吸取1mL浓度为4×105个/L的细胞悬液接种于6孔板中,补齐培养基体积至2mL,继续培养至细胞贴壁、细胞形态舒展。向其中2孔加入10μM凋亡抑制剂预处理1h。根据细胞种类和分组设置分别给与不同浓度的白花丹素或顺铂处理48h。上机检测当天,取出6孔板。分别收集每孔上清液至相应的15mL离心管中,用PBS溶液洗涤细胞1次,转移清洗液至对应离心管。向各孔中加入适量不含EDTA的胰酶,使胰酶没过细胞表面,置于37℃恒温培养箱中消化细胞,使胰酶处于活性最佳状态。用此前收集的上清液终止对应孔内的胰酶,轻柔地反复吹打孔板底部,转移液体至至相应15mL离心管中,再用少量PBS溶液清洗孔板1次,以充分收集所有细胞。在室温下将各组细胞以500g低速离心10min。
离心完毕后,小心弃去上清,吸取1mL PBS溶液重悬细胞,并将重悬液转移到1.5mL离心管中,再次在室温下将各组细胞以500g低速离心10min。提前将10×Binding Buffer原液,按照1:9体积比,用纯水稀释成适量的1×Binding Buffer溶液。用移液枪小心吸干净上清,吸取100μL 1×Binding Buffer溶液重悬细胞,先后向其中加入5μL PE/Annexin V和5μL 7-AAD,室温下避光染色15min,7min时取出样品,涡旋混匀5秒,继续染色至结束。向各样品中添加300μL 1×Binding Buffer溶液,轻轻吹打混匀,过筛至样品管中,在4℃避光保存,待检测。
2)流式细胞仪检测:选择激发波长为488nm和650nm,检测104个细胞的分布比例。
八、WB实验
(1)SDS-PAGE电泳分离蛋白:按照说明书指示比例配制SDS-PAGE变性胶;向各泳道分别加入各变性蛋白样品20μL及蛋白marker,启动Bio-rad电泳仪,以80V电压分离浓缩胶中的样品,使各样品出浓缩胶之前处于同一水平;以100V电压分离压平的各蛋白样品,直至蛋白marker条带间隔清晰,此时代表蛋白样品已完全分离。
(2)电转:根据分离胶大小裁剪NC膜,保证其能完全覆盖所有样品;提前将电转液倒入托盘,放置冰上预冷,同时将三明治夹、裁剪好的NC膜完全浸泡于电转液中,使其充分润湿;小心转移分离胶至三明治夹,覆盖已浸泡好的NC膜,用一根干净的15mL离心管,滚动挤压排出NC膜与胶之间的气泡,夹紧三明治夹;将三明治夹插入电转槽中,加入预冷的电转液;整个电转过程需在冰水中进行,已保证电转液温度不至过高,导致转膜失败。
(3)封闭:用PBST溶液配制终浓度为5%的脱脂牛奶封闭液;将NC膜在PBST溶液中漂洗1次,去除膜上残留的电转液;使NC膜完全浸泡在封闭液中,置于摇床上,慢速摇晃1h,充分结合NC膜上未结合蛋白的空隙。
(4)一抗孵育:提前按照一抗说明书用PBST溶液配制稀释好的一抗孵育液;将NC膜放入PBST溶液中漂洗封闭液,根据目的蛋白分子量大小,在蛋白marker指示的相应区域裁剪NC膜,并在膜上marker处做出相应标记,做好记录;把含有目的蛋白的条带放入对应一抗孵育液中,平放至盛冰的冰盒中,使孵育过程处于4℃环境中,慢摇过夜(至少孵育8h以上)。
(5)二抗孵育:根据说明书按比例配制二抗稀释液,装入暗盒,放在4℃冰箱备用;从各一抗孵育液中取出条带,放入PBST溶液中快速摇晃清洗,每次5min,洗3次;依据一抗抗性,把各条带放入相应二抗中,室温孵育1h。
(6)印迹成像:本实验使用的二抗为荧光二抗,全程需避光进行;由二抗中取出条带,置于装有PBST溶液的暗盒中快速摇晃清洗,每次10min,洗3次;采用双色红外成像系统对NC膜进行扫描,得到蛋白条带图,用其自带分析软件Odyssey或Image J统计分析蛋白条带图灰度值;用内参归一化后,对各目的条带进行灰度值比较,实验平行重复三次以上。
2、实验结果:
一、MTT实验检测吉非替尼耐药LUAD细胞的IC50
如图1所示,吉非替尼给药72h后,HCC827、A549、HCC827 GR和H1975细胞的IC50值分别为0.26±0.08μM、36.24±6.14μM、60.92±9.23μM和11.51±3.48μM,HCC827 GR细胞的耐药指数RI=139.38。实验结果表明,实验成功构建了吉非替尼获得性耐药株HCC827GR。吉非替尼原发性耐药细胞A549和吉非替尼获得性耐药细胞H1975的IC50值均显著大于吉非替尼敏感株HCC827细胞(均P<0.001)。
如图2所示,PLM处理48h后,BEAS-2B、HCC827、A549、HCC827 GR和H1975细胞的IC50值分别为2.79±0.38μM、4.59±1.26μM、5.62±1.95μM、14.69±0.81μM和4.76±1.31μM。实验结果表明PLM对吉非替尼原发性耐药细胞A549、吉非替尼获得性耐药细胞HCC827 GR和H1975均具有细胞毒性。
二、平板克隆形成实验检测吉非替尼耐药LUAD细胞克隆形成率
如图3所示,随着PLM剂量的增加,肿瘤细胞的克隆形成率显著降低,PLM以剂量依赖性的方式抑制吉非替尼原发性耐药细胞A549、吉非替尼获得性耐药细胞HCC827 GR和H1975的克隆形成能力。
三、HE染色观察PLM对吉非替尼耐药LUAD细胞形态的影响
如图4所示,DMSO对照组的A549细胞呈三角形,HCC827 GR细胞呈椭圆形,H1975细胞呈类梭形,细胞形态完整,细胞质较丰富且无皱缩。而在低、中、高浓度的PLM处理或顺铂给药48h后,吉非替尼原发性耐药细胞A549、吉非替尼获得性耐药细胞HCC827 GR和H1975细胞数下降,细胞质减少,细胞均出现皱缩浓染,A549、HCC827 GR和H1975细胞呈分枝状、细胞变短变小、细胞核边集,白色箭头指示代表性凋亡细胞。
四、Hoechst染色观察PLM对吉非替尼耐药LUAD细胞凋亡的影响
如图5所示,阳性对照药顺铂分别作用于吉非替尼原发性耐药细胞A549、吉非替尼获得性耐药细胞HCC827 GR和H1975 48h后,这些细胞出现染色质固缩,细胞核致密浓染,现蓝色高亮的现象,并可观察到核破碎,现“半月形”核(图中序号①箭头所示)和凋亡小体(图中其它箭头所示)。阴性对照0.1%DMSO处理组的A549、HCC827 GR和H1975细胞的细胞核呈椭圆形或圆形,淡蓝色,颜色均匀,核内染色质分布均匀,细胞核形态完好。而给以低、中、高浓度PLM分别处理A549、HCC827 GR和H1975细胞48h后,细胞出现染色质固缩、细胞核破碎等现象,并可观察到“半月形”核和凋亡小体。结果提示:PLM可诱导吉非替尼原发性与获得性耐药LUAD细胞发生凋亡,且呈浓度依赖性。
五、透射电镜观察PLM对吉非替尼耐药LUAD细胞亚显微结构的影响
如图6所示,DMSO对照组的A549细胞(图6A)、HCC827 GR细胞(图6D)和H1975细胞(图6G)整体结构较完整,细胞膜、细胞核结构完整,核仁清晰明显,线粒体数目较多、结构较正常。分别用10μM、5μM和5μM PLM处理吉非替尼原发性耐药细胞A549、吉非替尼获得性耐药细胞HCC827 GR和H1975细胞48h后,细胞胞质均出现空泡(图中序号①箭头指示),空泡为单层膜结构、圆形,且空泡内基本不含细胞器等物质,空泡大小不一,空泡边缘可相互接触并逐渐融合(序号②箭头所示),由小空泡变为大空泡。吉非替尼原发性耐药细胞A549细胞(图6B)和H1975细胞(图6H)可见染色质破碎、浓缩且外周化(序号③箭头所示)。HCC827 GR细胞(图6F)和H1975细胞(图6I)可见线粒体剧烈肿胀、内部空泡化、线粒体脊结构发生溶解,受损严重(序号④箭头所示);HCC827 GR细胞(图6E)观察到凋亡小体(序号⑤箭头所示),主要包含细胞质,没有细胞器或核物质。
六、流式细胞术检测PLM对吉非替尼耐药LUAD细胞凋亡率的影响
如图7所示,20μM、10μM和10μM阳性对照药顺铂分别处理A549、HCC827 GR和H1975细胞48h后,细胞凋亡率增加(均P<0.001),凋亡抑制剂Z-VAD则可拮抗顺铂诱导的凋亡率增加(均P<0.001)。给予低、中、高浓度PLM分别处理A549、HCC827 GR和H1975细胞48h后,PLM处理组诱导的细胞凋亡率较DMSO对照组显著增加(均P<0.001),并且随着处理浓度的增加细胞的凋亡率上升(均P<0.001)。与单纯给PLM处理组相比,给与10mM凋亡抑制剂Z-VAD预处理组细胞凋亡率下降(均P<0.001)。结果表明,PLM可通过浓度依赖的形式诱导吉非替尼原发性耐药细胞A549、吉非替尼获得性耐药细胞HCC827 GR和H1975凋亡,从而发挥抗肿瘤活性。
七、PLM对吉非替尼耐药LUAD细胞中凋亡相关蛋白表达的影响
如图8所示,给予2.5μM、5μM、10μM PLM或10μM顺铂分别处理吉非替尼获得性耐药细胞H1975 48h,与DMSO对照组相比,PLM处理组随着处理浓度增大剪切型Casepase3和剪切型PARP表达量逐渐增加(P<0.05),Bax/Bcl-2比值逐渐增大(P<0.01),提示PLM可诱导吉非替尼耐药LUAD细胞凋亡,并且呈浓度依赖性。
由以上多个实验可以看出,白花丹素能够显著抑制人吉非替尼原发性耐药与获得性耐药肺腺癌细胞的活性和增殖,显著影响人吉非替尼原发性耐药与获得性耐药肺腺癌细胞的形态与结构,诱发凋亡,能够增加促凋亡相关蛋白的表达,说明了包含有白花丹素的药物可用于抗吉非替尼耐药非小细胞肺癌。
Claims (4)
1.白花丹素在制备抗吉非替尼耐药非小细胞肺癌的药物中的应用。
2.根据权利要求1所述的应用,其特征在于:所述吉非替尼耐药为原发性耐药和/或获得性耐药。
3.根据权利要求1或2所述的应用,其特征在于:所述药物包含有白花丹素及药学上可接受的辅料。
4.根据权利要求1或2所述的应用,其特征在于:所述药物的剂型包括颗粒剂、散剂、片剂、胶囊剂、丸剂、口服液或注射剂。
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