CN114558141A - 降低胰腺癌细胞恶性表型的促进剂、药物组合及其用途 - Google Patents
降低胰腺癌细胞恶性表型的促进剂、药物组合及其用途 Download PDFInfo
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Abstract
本发明涉及肿瘤医药研发技术领域,具体涉及降低胰腺癌细胞恶性表型的促进剂、药物组合物及其用途。本发明将四氢生物蝶呤作用在PANC‑1和BxPC‑3细胞,随着四氢生物蝶呤浓度的增加,癌细胞的活力逐渐降低,癌细胞的凋亡数目逐渐增多,凋亡率提高;四氢生物蝶呤可降低两种癌细胞的愈合面积,可促进癌细胞中E‑cadherin表达,抑制N‑cadherin的表达,可抑制胰腺癌细胞上皮间质转化,降低癌细胞的恶性表型,从而降低癌细胞迁移侵袭的能力。表明BH4生物活性激活剂可抑制胰腺癌细胞增殖活力和细胞迁移的能力,促进细胞凋亡,抑制癌细胞上皮间质转化,从而降低癌细胞的恶性表型,可以作为胰腺癌的治疗靶点。
Description
技术领域
本发明涉及肿瘤医药研发技术领域,具体涉及降低胰腺癌细胞恶性表型的促进剂、药物组合物及其用途。
背景技术
胰腺癌是一种发病隐匿、恶性程度极高的消化系统肿瘤,是癌症相关死亡的第四大因素,且患者死亡率以每年1%的速度缓慢上升,患者总体的5年生存率仅为10%,晚期患者其5年生存率不足3%,生存率如此低的原因主要包括其恶性程度高、起病隐匿、无典型症状、切除率低、复发率高。由于胰腺癌起病隐匿、早诊困难大多患者就诊时已经是晚期而失去了手术机会,药物治疗成为主要的治疗手段。目前胰腺癌治疗的一线用药是吉西他滨,然而其单独用药的缓解率不足10%,而吉西他滨与其他药物组成的二联疗法或5-氟尿嘧啶+奥沙利铂+伊立替康+亚叶酸钙(FOLFIRINOX)四联化疗药物虽然疗效稍有改善但副反应也大大增加,其有限的疗效与难以避免的毒副反应促使我们寻找新的药物靶点以期望能提高治疗效果,改善患者的预后。
肿瘤细胞的恶性表型包括肿瘤细胞活性的增强、增殖的较快、以及迁移作用至其他部位,因此对于多数肿瘤的治疗往往需要抑制肿瘤细胞的活性、降低肿瘤细胞的增殖,扼制肿瘤细胞的迁移。四氢生物蝶呤(tetrahydrobiopterin,BH4)属于芳香族氨基酸羟化酶的辅酶,是一氧化氮合的辅因子,参与NO的合成等许多生理过程,也参与如炎症、心血管疾病等病理过程。墨蝶呤(sepiapterin)为四氢生物蝶呤的前体,研究显示,墨蝶呤可通过促进NO的产生而促进乳腺癌细胞的凋亡,抑制乳腺癌细胞的增殖,抑制肿瘤的生长,墨蝶呤通过下调p70s6k依赖的VEGFR-2表达来抑制卵巢癌细胞的增殖和迁移。BH4处理黑色素瘤细胞使NO浓度升高,可降低肿瘤细胞活力、细胞增殖能力和肿瘤瘤细胞形成黑色素瘤球形体的能力。然而,BH4在胰腺癌细胞中的作用及可能机制尚未见报道。
发明内容
为了克服现有技术的缺陷,本发明的目的之一在于提供降低胰腺癌细胞恶性表型的促进剂,包括BH4生物活性激活剂,能够降低胰腺癌细胞的活性、促进胰腺癌细胞凋亡、降低胰腺癌细胞迁移侵袭的能力。
本发明的目的之二在于提供本发明降低胰腺癌细胞恶性表型的促进剂在制备预防和治疗胰腺癌药物方面的应用。
同时,本发明还在于提供一种药物组合物,包括BH4生物活性激活剂,对胰腺癌具有一定的预防和治疗作用。
为了实现上述目的,本发明采用的技术方案如下:
降低胰腺癌细胞恶性表型的促进剂,包括BH4生物活性激活剂。
BH4生物活性激动剂在现有技术中很容易获得,具体的作为优选的,所述BH4生物活性激动剂选自墨蝶呤、沙丙蝶呤、1'、2'-二乙酰-5、6、7、8-四氢生物蝶呤、6-甲基-5、6、7、8-四氢生物蝶呤或其类似物。
本发明在细胞培养基中添加四氢生物蝶呤分别培养胰腺癌细胞PANC-1和BxPC-3,检测四氢生物蝶呤对胰腺癌细胞的作用,CCK-8结果显示随着四氢生物蝶呤浓度的增加,癌细胞的活力逐渐降低;Hoechst法结果显示,随着四氢生物蝶呤浓度的增加,癌细胞的凋亡数目逐渐增多、凋亡率提高;划痕实验结果显示,四氢生物蝶呤可降低两种癌细胞的愈合面积;Western blot结果显示,四氢生物蝶呤可促进癌细胞中E-cadherin表达,抑制N-cadherin的表达,表明四氢生物蝶呤可抑制胰腺癌细胞上皮间质转化,降低癌细胞的恶性表型,从而降低癌细胞迁移侵袭的能力。
基于本发明的上述验证结果表明BH4生物活性激活剂能够作为药物的主要活性成分制备成药物组合物,产生促进胰腺癌细胞凋亡、降低胰腺癌细胞活性、抑制胰腺癌细胞增殖、扼制胰腺癌细胞迁移的作用,甚至预防和治疗胰腺癌。
应当可以理解的是,上述药物组合物还可以包括药学上可接受的载体或辅料。作为可选的,药学上可接受的载体或辅料选自壳聚糖、胆固醇、脂质体、环糊精、微球和微囊。
附图说明
图1为PANC-1细胞对照组和实验组细胞划痕实验结果示意图;
图2为BxPC-3细胞对照组和实验组细胞划痕实验结果示意图;
图3为PANC-1细胞对照组和实验组E-cadherin与N-cadherin蛋白表达量检测结果示意图;
图4为BxPC-3细胞对照组和实验组E-cadherin与N-cadherin蛋白表达量检测结果示意图。
具体实施方式
下面结合具体实施例对本发明做进一步的详细说明。除特殊说明的之外,各实施例及试验例中所用的设备和试剂均可从商业途径得到。
下述实施例设计实验验证BH4生物活性激活剂对胰腺癌细胞增殖、凋亡、迁移及生物学特性的影响,提示BH4生物活性激活剂能够降低胰腺癌细胞恶性表征,能够作为胰腺癌治疗靶点。
下述实施例使用的实验材料包括:
1、实验细胞胰腺癌PANC-1细胞株,BxPC-3细胞株购自于中国典型培养物保藏中心细胞库。
2、主要试剂:
四氢生物蝶呤(tetrahydrobiopterin)购自于上海芮晖化工科技有限公司;
RPMI-1640培养基,DMEM高糖培养基,胎牛血清(Fetal Bovine Serum,FBS)购自于美国Hyclone公司;
CCK-8细胞增殖及细胞毒性检测试剂盒,细胞凋亡-Hoechst染色试剂盒购自于上海碧云天生物技术有限公司;
RIPA裂解液购自于Thermo Scientific公司;
E-cadherin抗体,N-cadherin抗体,GAPDH抗体购自于武汉三鹰生物科技有限公司。
实施例1实验分组及模型构建
1.1细胞培养:
PANC-1细胞,使用DMEM高糖培养基,37℃,5%CO2恒温培养箱培养,每2-3d传代1次,培养至对数生长期的细胞用于实验;
BxPC-3细胞,使用RPMI-1640培养基(含10%FBS,100U/L青霉素及100μg/mL链霉素),37℃,5%CO2恒温培养箱培养,每2-3d传代1次,培养至对数生长期的细胞用于实验;
1.2实验分组及细胞培养方法:
1.2.1细胞活力、细胞凋亡影响实验
对数生长期的PANC-1与BxPC-3细胞分别接种于96孔中培养12h,每种细胞均分为对照组和实验组,其中对照组和实验组各组分别接种10个孔,每组重复5次,对照组正常培养,实验组分别加入终浓度为2μM、4μM、8μM、12μM的四氢生物蝶呤培养24h;
1.2.2细胞迁移能力实验
对数生长期的PANC-1与BxPC-3细胞分别接种于96孔中培养12h,每种细胞均分为对照组和实验组,其中对照组和实验组各组分别接种10个孔,每组重复5次,对照组正常培养;实验组用1ml的无菌蓝色吸头在孔的中间划痕,尽量保证每一个孔中划痕的宽度一致;弃去培养液,每孔加入1ml无血清培养液以洗去悬浮的细胞,再分别加入终浓度为2μM、4μM、8μM、12μM的四氢生物蝶呤培养24h;
1.2.3对细胞迁移相关蛋白表达量的影响实验
对数生长期的PANC-1与BxPC-3细胞分别接种于96孔中培养12h,每种细胞均分为对照组和实验组,其中对照组和实验组各组分别接种10个孔,每组重复5次,对照组正常培养,实验组加入终浓度为12μM的四氢生物蝶呤培养24h。
实施例2各对照组和实验组细胞的各项性能检测
2.1利用CCK-8实验检测BH4对PANC-1细胞、BxPC-3细胞活力的影响
按实施例1所述的实验分组和细胞培养方法进行细胞培养后,在对照组和实验组每孔中加入100μLCCK-8,37℃培养2h后,酶标仪450nm处测OD值,结果如下表1所示:
表1四氢生物蝶呤对PANC-1细胞、BxPC-3细胞活力的影响
组别 | PANC-1细胞(OD值) | BxPC-3细胞(OD值) |
对照组 | 0.974±0.023 | 0.913±0.010 |
2μMBH4 | 0.848±0.037 | 0.892±0.013 |
4μMBH4 | 0.811±0.025 | 0.853±0.022 |
8μMBH4 | 0.623±0.018* | 0.536±0.024* |
12μMBH4 | 0.512±0.021** | 0.507±0.026* |
*P<0.05,**P<0.01,与对照组相比较
由上述表1所示的结果可知,随着四氢生物蝶呤浓度的增加,细胞的活力逐渐降低,与对照组相比,在PANC-1细胞中,8μM和12μM的四氢生物蝶呤处理的PANC-1细胞,细胞活力下降明显,具有统计学意义(P<0.05,P<0.05);在BxPC-3细胞中,8μM和12μM的四氢生物蝶呤处理的BxPC-3细胞,细胞活力下降明显,具有统计学意义(P<0.01,P<0.05),表明四氢生物蝶呤可降低胰腺癌细胞的活力。
2.2检测四氢生物蝶呤对PANC-1细胞、BxPC-3细胞凋亡的影响
按实施例1所述的实验分组和细胞培养方法进行细胞培养后,按照使用说明书对对照组和试验组细胞进行Hoechst染色,封片,显微镜下选择视野,观察染色结果并拍照,每组设置5个副孔,计算凋亡指数,凋亡指数=凋亡细胞数/细胞总数×100%,结果如下表2所示:
表2四氢生物蝶呤对PANC-1细胞、BxPC-3细胞凋亡的影响
组别 | PANC-1细胞凋亡率(%) | BxPC-3细胞凋亡率(%) |
对照组 | 2.01% | 2.22% |
2μMBH4 | 3.01% | 3.31% |
4μMBH4 | 7.67% | 8.42% |
8μMBH4 | 19.69%* | 25.37%* |
12μMBH4 | 26.78%* | 33.69%** |
*P<0.05,**P<0.01,与对照组相比较
由上述表2所示的结果可知,随着四氢生物蝶呤浓度的增加,细胞的凋亡率逐渐增加,与对照组相比,在PANC-1细胞中,8μM和12μM的四氢生物蝶呤处理的PANC-1细胞,细胞凋亡率显著升高(P<0.05,P<0.05);在BxPC-3细胞中,8μM和12μM的四氢生物蝶呤处理的BxPC-3细胞,细胞凋亡率显著升高(P<0.05,P<0.01),表明四氢生物蝶呤可促进胰腺癌细胞的凋亡。
2.3检测四氢生物蝶呤对PANC-1细胞、BxPC-3细胞迁移能力的影响
按照上述实施例1所述的实验分组和细胞培养方法进行细胞培养和处理后,观察每组细胞愈合面积,如图1和图2所示,并利用Image J软件测量愈合面积,结果如下3所示:
表3四氢生物蝶呤对PANC-1细胞、BxPC-3细胞迁移能力的影响
组别 | PANC-1细胞愈合面积(%) | BxPC-3细胞愈合面积(%) |
对照组 | 50.75% | 55.46% |
2μMBH4 | 50.37% | 53.52% |
4μMBH4 | 47.64% | 48.68% |
8μMBH4 | 38.61%* | 42.39%* |
12μMBH4 | 25.93%** | 40.12%* |
*P<0.05,与对照组相比较
由图1、图2和表3所示的结果可知,在PANC-1细胞中,与对照组细胞的愈合面积(55.46%)相比较,8μM与12μM的四氢生物蝶呤处理的PANC-1细胞愈合面积下降明显(P<0.05,P<0.01);在BxPC-3细胞中,与对照组细胞的愈合面积(50.75%)相比较,8μM与12μM的四氢生物蝶呤处理的BxPC-3细胞愈合面积下降明显(P<0.05,P<0.05),表明四氢生物蝶呤可降低胰腺癌细胞的迁移能力。
2.4检测四氢生物蝶呤对PANC-1细胞、BxPC-3细胞上皮间质转化相关蛋白的影响
上皮细胞间质转化(EMT)是上皮细胞来源的恶性肿瘤细胞获得迁移和侵袭能力的重要生物学过程,在肿瘤的发生和侵袭转移中其关键作用。E-cadherin为上皮组织的标记物,N-cadherin为间叶组织的标记物,E-cadherin表达下调,N-cadherin表达上调与肿瘤的发生与转移密切相关,本实施例通过检测对照组和实验组细胞E-cadherin和N-cadherin的表达量,验证四氢生物蝶呤对PANC-1细胞、BxPC-3细胞恶性表型的影响;
按照上述实施例1所述的实验分组和细胞培养方法进行细胞培养后,弃去培养液,每皿加入500μLRIPA裂解液裂解细胞,提取总蛋白。常规SDS-PAGE电泳、转膜,5%脱脂奶粉室温封闭1h,E-Cadherin抗体(1:500),N-cadherin抗体(1:500)及GAPDH抗体(1:10000)4℃孵育过夜,荧光二抗室温孵育2h后,Odyssey曝光,Image J软件分析灰度值,结果如图3、图4和表4所示:
表4四氢生物蝶呤对PANC-1细胞、BxPC-3细胞中E-cadherin与N-cadherin蛋白表达的影响
*P<0.05,与对照组相比较
与对照组相比较,12μM的四氢生物蝶呤处理的PANC-1和BxPC-3细胞中E-cadherin蛋白的相对表达量均升高(P<0.05,P<0.05),而N-cadherin蛋白的相对表达量均降低(P<0.05,P<0.05)。提示四氢生物蝶呤可抑制胰腺癌细胞上皮间质转化,降低胰腺癌细胞的恶性表型。
本发明上述实施例检测了四氢生物蝶呤对PANC-1和BxPC-3细胞活性、凋亡性能和迁移能力的作用设计了试验验证,结果显示,随着四氢生物蝶呤浓度的增加,癌细胞的活力逐渐降低癌细胞的凋亡数目逐渐增多,凋亡率提高;四氢生物蝶呤可降低两种癌细胞的愈合面积;四氢生物蝶呤可促进癌细胞中E-cadherin表达,抑制N-cadherin的表达,提示四氢生物蝶呤可抑制胰腺癌细胞上皮间质转化,降低癌细胞的恶性表型,从而降低癌细胞迁移侵袭的能力。
综上所述,本发明验证了BH4生物活性激活剂对胰腺癌细胞的影响,BH4生物活性激活剂可抑制胰腺癌细胞增殖活力和细胞迁移的能力,促进细胞凋亡,抑制癌细胞上皮间质转化,从而降低癌细胞的恶性表型,可以作为胰腺癌的治疗靶点。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (8)
1.降低胰腺癌细胞恶性表型的促进剂,其特征在于,包括BH4生物活性激活剂。
2.如权利要求1所述的降低胰腺癌细胞恶性表型的促进剂,其特征在于,所述BH4生物活性激动剂选自墨蝶呤、沙丙蝶呤、1'、2'-二乙酰-5、6、7、8-四氢生物蝶呤、6-甲基-5、6、7、8-四氢生物蝶呤或其类似物。
3.一种如权利要求1或2所述的降低胰腺癌细胞恶性表型的促进剂在制备预防和治疗胰腺癌的药物方面的用途。
4.如权利要求3所述的用途,其特征在于,所述降低胰腺癌细胞恶性表型为促进胰腺癌细胞凋亡、降低胰腺癌细胞活性、抑制胰腺癌细胞增殖、扼制胰腺癌细胞迁移。
5.一种预防和治疗胰腺癌的药物组合物,其特征在于,包括BH4生物活性激动剂。
6.如权利要求5所述的预防和治疗胰腺癌的药物组合物,其特征在于,所述BH4生物活性激动剂选自墨蝶呤、沙丙蝶呤、1'、2'-二乙酰-5、6、7、8-四氢生物蝶呤、6-甲基-5、6、7、8-四氢生物蝶呤或其类似物。
7.如权利要求5或6所述的预防和治疗胰腺癌的药物组合物,其特征在于,还包括药学上可接受的载体或辅料。
8.如权利要求7所述的预防和治疗胰腺癌的药物组合物,其特征在于,所述药学上可接受的载体或辅料选自壳聚糖、胆固醇、脂质体、环糊精、微球和微囊。
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