CN1157567A - 口服可溶性经调节柑桔果胶抑制癌症转移的方法 - Google Patents
口服可溶性经调节柑桔果胶抑制癌症转移的方法 Download PDFInfo
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Abstract
一种治疗哺乳动物癌症的方法。本发明涉及癌症患者口服可抑制原发肿瘤转移的pH调节柑桔果胶。
Description
发明领域
本发明一般地涉及用于治疗前列腺癌的方法。
发明背景
预计随着人口老龄化多种癌症的发病率会增加。例如,美国男性中前列腺癌是最常见确诊的癌症,且也是男性癌症死亡的第二大病因。据认为1994年将有20万例新近被确诊的前列腺癌患者,且将有3万8千例患者死于前列腺癌,这些数字随着人口老龄化会继续上升。大约50%确诊患有前列腺癌的患者患有已经或将自前列腺转移的疾病。前列腺癌向骨骼系统转移,并且病人典型地死于不可阻挡的骨转移性疾病。到目前为止,还无有效的治疗方法,而且对患转移性疾病的患者仅有极少的治疗方法。
肿瘤细胞的转移过程需要细胞与原发肿瘤分离,侵入基底膜,自肿瘤细胞栓子穿过血流,与靶器官的血管内皮细胞相互作用,渗出且增殖形成如同E.C.Kohn,抗癌治疗,13,2553(1993);和L.A.Kiotta,P.S.Steeg,W.G.Stettler-Stevenson,细胞64,327(1991)所描述的继发肿瘤集落。
一般认为转移的级联过程之多个阶段包含由如糖结合蛋白的细胞表面成分介导的细胞间相互作用,所说的糖结合蛋白包括如A.Raz,R.Lotan,癌症转移回顾,6,433(1987);和H.J.Gabius,生物化学生物物理学报1071,1(1991)所述的半乳糖苷结合的凝集素(galectins)。在将B16黑素瘤与uv-2236纤维肉瘤细胞静注入同基因小鼠尾静脉之前,以抗半乳糖苷结合的凝集素单克隆抗体于体外对其进行处理可以显著抑制肿瘤肺部集落的发展,如L.Meromsky,R.Lotan,A.Raz,癌症研究46,5270(1991)所述。以半乳糖苷结合的凝集素-3cDNA对低转移性、低半乳糖苷结合的凝集素-3表达性的uv-2237-cll5纤维肉瘤细胞转染使受转染细胞的转移性表型增加,如A.Raz,D.Zhu,V.Hogan,J.Shah,T.Raz,R.Karkash,G.Pazerini,P.Carmi,国际癌症杂志46,871(1990)所述。另外,人乳头状甲状腺癌之半乳糖苷结合的凝集素-3-表达水平与人结肠直肠和胃癌肿瘤分期之间已建立了相关关系,如L.Chiariotti,M.T.Berlinjieri,P.DeRosa,C.Battaglia,N.Berger,C.B.Bruni,A.Fusco,Omeogene 7,2507(1992);L.Irimura,Y.Matsushite,R.C.Sutton,D.Carralero,D.W.Ohanesian,K.R.Cleary,D.M.Ota,国际癌症杂志51,387(1991);R.Lotan,H.Ito,W.Yasui,H.Yokozak,D.Lotan,E.Tahara,国际癌症杂志56,474(1994);及M.M.Lotz,C.W.Andrews,C.A.Korzelius,E.C.Lee,G.D.Steele,A.Clarke,A.M.Mercurio,PNAS,USA90,3466(1993)所述。
已显示如甲基-α-D乳糖苷(lactoside)和乳-N-四糖的单糖可抑制B16黑素瘤细胞的转移,而D-半乳糖和阿拉伯半乳糖抑制L-1肉瘤细胞的肝转移,如J.Beauth等癌症研究临床肿瘤学杂志113,51(1987)。
已知将B16-F1鼠黑素瘤细胞与柑桔果胶或经调节的柑桔果胶一同静脉注射至同基因小鼠中可分别引起肺部集落的显著增加或减少,如D.Platt及A.Raz,全国癌症研究所杂志84:438-42(1992)所述。在本文所公开的发现之前,还未存在一种利用口服的非细胞毒制剂抑制癌症转移的有效治疗方法。因此,存在着对基于口服一种非细胞毒制剂的治疗方法的需求。
发明概述
一方面,本发明提供了一种通过口服经调节的果胶,优选水溶性pH调节的柑桔果胶,治疗哺乳动物癌症的方法,如本文所述为抑制转移。
另一方面,本发明提供了一种适于治疗哺乳动物癌症的组合物,它包括经调节的果胶,优选pH调节的柑桔果胶,与用于口服的药物学可接受的、能消化的载体之混和物。
另一方面,本发明的方法和组合物用于治疗人及其它哺乳动物前列腺癌,以抑制原发肿癌的转移。
因此,本发明的优选实施方案提供了一种新的治疗方法,其中口服一种富含半乳糖苷残基的非细胞毒天然复合糖即pH调节的柑桔果胶(MCP),这种复合糖起有效的自发性前列腺癌转移之抑制剂的作用。
按照本发明进行治疗时,在接种106Dunning大鼠前列腺腺癌MLL细胞后第14天时切除原发肿瘤后观察到16只生长有肿瘤的大鼠中有7只经尸检无肿瘤存在(淋巴结或肺中无可见性转移灶),而对照组16/16的大鼠有转移灶。与对照组相比口服1%(w/v)MCP显著减低了其余动物的肿瘤肺部病灶的数量(对照,9±4;1%MCP,1±1),对原发肿瘤的生长无作用。于体外MCP以时间-剂量依赖方式抑制MLL细胞粘附至大鼠内皮细胞及其于半固体培养基中的集落形成。MCP作用的可能机制似涉及肿瘤细胞表面糖结合蛋白。
因此,本发明提供了一种口服MCP治疗癌症的方法,MCP为一种具有独特作用机制可成功抑制肿瘤细胞离散的非毒性药物。另外,本发明提供了一种包含了MCP与口服药剂载体的治疗哺乳动物癌症的组合物。
图1A为一显示患有肺转移大鼠数目在0.1%MCP和1.0%MCP组与对照组相比显著降低的图。
图1B为一显示以1.0%MCP治疗动物的肺中所存在的转移灶与对照组相比显著减少的图。
图1C为对照大鼠肺的显微照片。
图1D为1.0%MCP大鼠肺的显微照片。
图2为MLL细胞中大鼠半乳糖苷结合的凝集素-3表达的细胞表面染色和Western印迹分析(插图)的标图。
图3A为说明MLL细胞在MCP不存在或存在不同浓度的情况下于4℃经90分钟附着情况的图。
图3B为说明MLL细胞在存在或不存在(----)0.03%w/vMCP时附着于RAEC融合单层的时间过程图。
图3C为不存在MCP时荧光MLL细胞粘附于RAEC细胞的显微照片。
图3D为存在0.1%w/vMCP时荧光MLL细胞粘附于RAEC细胞的显微照相片。
图4A为一显示MCP于0.5%琼脂糖中对MLL集落形成之影响的图。
图4B为无MCP生长时MLL细胞位相显微照片。
图4C为含0.1%(w/v)MCP生长时MLL细胞位相显微照片。
图5为人原发前列腺腺癌组织的显微照片,它显示了半乳糖苷结合的凝集素-3的存在。
图6为说明在不同浓度CP(O)或MCP(O)存在时CP和MCP对B16F1与层粘连蛋白粘附的影响。垂直小柱表示平均值±由平均值七分布计算得到的标准差。
图7A为铺于层粘连蛋白上之B16F1细胞的位相显微片。细胞仅在DMEM中培养。
图7B为铺于层粘连蛋白上并在含0.5%CP的DMEM中培养的B16F1细胞之位相显微照片。
图7C为铺于层粘连蛋白上并在含0.5%MCP的DMEM中培养的B16F1细胞之位相显微照片。
图8为一说明在仅有20μg/ml无唾液酸胎球蛋白(asialofetuin)(A)或加有0.5%CP(B)或0.5%MCP(C)时CP及MCP对无唾液酸胎球蛋白诱导的同型凝集之作用。垂直小柱表示平均值±平均值t分布计算的标准差。
图9A为仅有20μg/ml无唾液酸胎球蛋白时B16F1细胞同型凝集的位相显微照片。
图9B为有0.5%CP和无唾液酸胎球蛋白时B16F2细胞同型凝集的位相显微照片。
图9C为有0.5%MCP和无唾液酸胎球蛋白时B16F1细胞同型凝集的位相显微照片。
图10为说明半乳糖苷结合的凝集素-3与MCP涂布孔之结合的图。
图11为说明CP及MCP对B16F1细胞在0.5%琼脂糖(CPOMCPO)中形成集落之能力影响的图。
优选实施方案之详细描述
如本文所用,术语“治疗性”治疗指给已被诊断患有癌症的个体口服预定量的经调节的柑桔果胶,此方法可有效地增加用药者之生存。
如本文所用,术语“癌症”指任何新生物疾病,包括如细胞性疾病,例如肾细胞癌、Kaposi化肉瘤、慢性白血病、乳腺癌、肉瘤、卵巢癌、直肠癌、喉癌、黑素癌、结肠癌、膀胱癌、肥大细胞瘤、肺癌、乳腺腺癌、咽鳞状细胞癌及胃肠道或胃癌。优选地在本发明中进行治疗的癌症为人前列腺癌,最优选人前列腺腺癌。
本文所用的缩略语为:CP,天然柑桔果胶;MCP,pH调节的CP;EHS,Englebreth-Holm Swarm;DMEM,Dulbecco′s改良Eagle化最低限度必需培养基;CMF-PBS,无Ca2+和Mg2+的磷酸缓冲盐液,pH7.2;BSa,牛血清白蛋白。
如在文章“柑桔果胶对B16-F1黑素细胞肺部集落化之调节”(全国癌症研究所杂志,84卷第六期,1992年3月18日)中所描述,以前已对柑桔果胶(CP)(一种富含半乳糖残基的复合多糖)及其pH调节的衍生物(MCP)对B16黑素瘤实验性的转移之作用进行了分析,该文全文引入本文作为参考。已发现将MCP与B16-F1细胞一同静注可显著抑制肿瘤细胞在受注射小鼠肺部形成集落。在下文中将更全面描述的CP之pH调节将产生与未调节CP相似但更小的非支链糖链的糖组合物。MCP在体外和体内均显示出无毒性。
本发明所用的已调节的果胶是通过对柑桔果胶部分解聚,优选pH调节,制备的。
正如本领域专业人员将理解的,未经调节的果胶分子量范围在约20,000-400,000之间。正是所有植物组织细胞壁上存在的多糖类物质起着细胞间粘接物的作用。果胶最丰富来源之一是柠檬或橙子果皮,它含有约30%的这种多糖。这种多糖以插入(1-2)-L-鼠李糖残基的α-(1-4)连接之D-聚半乳糖醛酸序列的部分甲基酯天然存在。中性糖D-半乳糖、L-阿糖、D-木糖和L-岩藻糖在果胶分子上形成侧链。D.A.Rees,A.W.Wight等人已进行了结构研究(J.Chem Soc.B,1971,1366)。以溶液形式和凝胶形式的二级和三级结构由D-A.Rees,E.J.Welsh,Angew.Chem.Int.Ed 16,214(1977)进行了描述。一篇综述及文献目录由Towle,Christensen完成(Industrial Gums,R.L.Whistler,Ed.(AcademicPress,New York,2nd,ed.,1973)P.429-461)。一本值得注意的关于果胶的书由Z.I.Kertesz完成,The Pectic Substance(Interscience,New York,1951)。
果胶是以粗或细粉、淡黄白色、实际上无气味且
起来发粘的形式存在。它在20份水中几乎完全溶解,形成一含带负电荷、水合程度非常高的颗粒的粘稠溶液。它对石蕊呈酸性并且不溶于酒精或已稀释的酒精,而且也不溶于其它的有机溶剂中。如果首先以酒精、甘油或糖浆将其润湿,或如果首先将其与3份或以上的蔗糖混和,它可更容易地溶于水。它在轻度酸性条件下保持稳定;更强的酸性或碱性条件引起解聚。
在制备本发明所用之pH调节柑桔果胶中用作起始材料的一种优选果胶可从位于圣路易斯(密苏里)的Sigma Chemical Co.处购得。该物质分子量为70-100kd,重量的约85%为半乳糖醛酸基团且重量的约9.5%为甲氧基因,并且含有少于10%的水分。它可以粉状的形式得到。柑桔果胶也可从ICN Biomedicals处以Pectin102587 RT的号码购得。
制备0.5%,更优选1.0%w/v的柑桔果胶水溶液(除非特别指明,本文所有溶液浓度均以w/v表示),并且在UV下照射48小时灭菌。为使果胶部分解聚,以NaOH(3N)增加pH至10.030分钟调节果胶溶液,然后以HCl(3N)使pH降至3.0,这是根据Alberscheim等人在文章“AMethod for Analysis of Sugars in Plant Cell Wall Polysaccharides by GasLiquid Chromatography”,Carbohydrate Research,5:340-346,1967中描述的方法进行的,该文全文引入本文作为参考。约10-24小时后,溶液的pH平衡至约6.3。然后以乙醇(70%)冲洗溶液并以丙酮(100%)干燥。这就产生了平均分子量约10kd的果胶片段,这一测定结果依照Christensen在“Methods of Grading Pectin in Relation如MolecularWeight(intrinsic viscosity of pectin)”,Food Research 19:163-165(1954)一文中的方法,即以20mM的六偏磷酸钠(pH4.5)、0.2%EDTA和(0.9%)NaCl于25℃在Ubbelohde No.1粘度计中进行粘度测定而测得,该文全文引入本文作为参考。本文所用的术语“经调节的果胶”和“MCP”应指已解聚的果胶。更优选的是,本发明所用的经调节的果胶分子量约为1-15kd,最优选为约10kd,而且优选按照上文的方法制备,并且优选为水溶性,已干燥的MCP片段可以以不含Ca2+和Mg2+的磷酸缓冲盐液(pH7.2)(CMF-PBS)再水化至0.5%(w/v)的终贮存液。
如所陈述的,本发明中MCP是经口服用的,因此本发明提供了一种含有MCP和一种可消化的药用载体的组合物。适宜的可消化药用载体包括将MCP以干燥形式包入其中的明胶胶囊,或将MCP与羟丙基纤维素、羟丙基甲基纤维素、硬脂酸镁、微晶纤维素、丙二醇、硬脂酸锌及二氧化钛等混合其中的片剂。该组合物可用纯化水、香味剂及作为可消化载体的蔗糖制成液体,从而制备出在服药者服用时具有令人愉快味道的组合物。
准确的剂量及用量方案依服用者的年龄、体重、病史等多种可变因素而不同。依据治疗癌症有效的MCP成分(即不考虑可消化载体)之重量的优选剂量及用量方案为每天每公斤体重为约10至约1000mg。将MCP以相同的间隔口服,即每24小时约10-1000mg/kg和/或每6小时2.5-250mg/kg。同样的剂量和用量方案优选用于治疗哺乳动物前列腺癌,包括人前列腺癌,以减少或抑制转移。据信同样的剂量和用量方案在作为口服预防组合物服用时,将有效地预防高危哺乳动物服药者的癌症发生。
实施例
下面的实施例进一步描述了本发明的各个方面,而不是以任何方式限制本发明。
前列腺癌的Dunning(R3327)大鼠前列腺腺癌膜型是由Dunning从一雄性大鼠上发现的自发腺腺癌发展而来,这一过程由W.F.Dunning在NatlCancer Inst Mono 12,351(1963)中进行了描述。如J.T.Isaacs,W.D.W.Heston,R.M.Weissman,D.S.Coffey,Cancer Res 38,4353(1978)所述,由原发肿瘤已发展了几种具有不同分化以转移特性的亚系。MAT-LyLu(MLL)亚系为一生长快速、分化差的腺癌细胞系,如J.T.Isaacs,W.B.Isaacs W.F.J.Feitz,J.Scheres,The Prostate 9,261(1986);和K.J.Pienta,B.C.Murphy,W.B.Isaacs,J.T.Isaacs,D.S.Coffey,The Prostate 20,233(1992)所述,将1×106MLL细胞注射入大鼠大腿中约25天内会由于继发于过强的原发肿瘤的影响而导致动物死亡。在肿瘤细胞接种约12天后,原发的MLL肿瘤开始转移,在此时间之前进行肢体截肢以去除原发肿瘤可使动物痊愈。如果截肢在第12天之后进行,如K.J.Pienta,B.C.Murphy,W.B.Isaacs,J.T.Isaacs,D.S.Coffey,The Prostate 20,233(1992)所述,大多数动物在40天内死于肺及淋巴结转移。
本发明中,将可溶性MCP溶于水中长期口服可影响MLL肿瘤产生自发转移的能力。
为更全面地说明本发明以及参见图1A,将大鼠于第0天时于其后腿注射入1×106MLL细胞。第4天当原发肿瘤大小约为1cm3时,持续地将0.01%、0.1%或1%(w/v)MCP加入大鼠饮水中(N=每组8只,实验进行两次)。于第14天将大鼠麻醉,截去后肢去除原发肿瘤。加到水中的任何浓度的MCP均不影响原发肿瘤的生长(平均肿瘤重量:对照,4.2±0.26gm,0.01%,4.7±0.7gm;0.1%,4.3±0.37gm,1.0%,5.0±0.25gm)。然后进一步追踪至第30天,此时将所有组的动物杀死并作尸检。在此期间,动物从其饮水中持续地摄入MCP。对照及治疗的动物体重均适度增加且MCP治疗组动物并未观察到毒性作用。切下肺脏,水中漂洗并于Bouin′s液中固定过夜。与对照组(15/16大鼠有转移)相比,0.1%(P<0.03)MCP组(7/14大鼠有转移)患有肺转移的大鼠数目显著降低(图1A)(p<0.001)。各组大鼠每天饮用30±4ml水。在解剖显微镜下计数测定MLL肿瘤集落的数目。1.0%MCP治疗组动物肺中平均转移瘤的数目显著少于对照组(对照组9±4;治疗组1±1)(p<0.05)(图1B)(Mann-Whitney Test)。MCP的作用显示出不依赖于其在饮水中的浓度。图1C和1D也显示了患肿瘤动物肺的情况(C-对照组,D-1.0%MCP组)且突出了MCP降低已发生的表面MLL肺病灶数目的作用。1%MCP也显著有阳性淋巴结肿瘤的动物数目(对照组55%,MCP治疗组13%,p<0.01)。受治疗的动物未遭受MCP治疗的明显毒性的作用。动物体重的增加率与对照组相同。对照与治疗组每天饮水量为30±4ml/只。毛发状况,总体行为及大便颜色均无改变。
由于以前已显示MCP可以干扰细胞间通过细胞表面糖结合的半乳糖苷结合凝集素-3分子介导的相互作用,因此研究了是否MLL细胞表达半乳糖苷结合凝集素-3的问题。MLL细胞如同许多其它癌细胞一样可在其细胞表面表达半乳糖苷结合凝集素-3,正如图2所示的以定量荧光流式细胞分析所测定的及图2(印迹插图)所示的以提取的全部细胞与单一特异性免抗半乳糖苷凝集素-3肽抗体进行免疫印迹所测定的。
肿瘤-内皮细胞粘附被认为是转移过程中一个关键的步骤,因此研究了MCP对MLL-内皮细胞相互作用的影响,图3A显示了Cr标记的MLL细胞在MCP存在或不存在时与融合的单层大鼠主动脉内皮细胞(RAEC)粘附的情况。发现MCP是一较强的MLL细胞与内皮细胞粘附的抑制剂,图3A和3B。
MLL和RAEC细胞生长于补充有10%胎牛血清的RPMI1640培养基中。RAEC在组织培养孔中长至融合。于37℃在2ml加有0.5%牛血清白蛋白的无血清培养基中将2.4×106MLL细胞与5μCiNa51CrO4温育30分钟。在较充分的洗涤后,以每孔105向一式四份的RAEC单层上加入MLL细胞。如图3A所示,评估了不同浓度MCP存在或不存在时于4℃经90分钟MLL细胞的附着情况。以冷磷酸缓冲盐液将细胞洗三次以去除未结合细胞。然后将细胞用0.1NNaOH于37℃溶解30分钟,用β-计数计测定放射活性。各点代表四个孔的平均值,实验进行同样的两次。小柱代表标准误,如图3B所见,测定了0.03%(w/v)MCP不存在(----)或存在时MLL细胞附着于RAEC融合的单层细胞的时间过程。存在0.03%MCP抑制了MLL细胞对RAEC的附着。为观察荧光MLL细胞对RAEC的粘附,将105MLL细胞于0.1%FITC中温育30分钟。然后充分冲洗,将细胞加至RAEC单层上。显示了不存在(图3C)或存在0.1%(w/v)MCP(图3D)时MLL细胞的结合情况(以放大160倍显示)。很明显MLL细胞可很快与RAEC单层粘附,而在MCP存在时只观察到有较小程度的细胞附着。图3C和3D以图像显示了MCP对粘附过程的作用。将MLL细胞与FITC一起悬浮进行荧光标记,暴露于不含(图3C)或含0.1%MCP(图3D)的0.5%牛血清白蛋白中的融合的单层大鼠内皮细胞60分钟。培养物冲洗以去除未粘附细胞,然后照相。在未处理的培养物中,荧光MLL细胞几乎一致地与内皮细胞单层粘附(图3C),而存在0.1%MCP时显微镜视野中几乎检测不出荧光细胞与RAEC单层相连(图3D)。
细胞在半固体培养基中生长的能力即锚链不依赖性可用作细胞转化的一个标准,而且药物或抗体对此过程的抑制可用于测定其效力。细胞在半固体培养基中的生长需要其以类似于体内转移步骤中许多步骤的过程移动、侵入及建立新的肿瘤灶。以前已提出肿瘤细胞通过细胞表面半乳糖苷结合凝集素-3与糖蛋白糖残基的相互作用的能力,与其与琼脂糖(D-半乳糖与L-脱水-半乳糖的聚合物)半乳糖残基相互作用的能力和提供在这种半固体培养基中进行细胞增殖所需的最低支持相关。结果已被证实抗半乳糖苷结合凝集素-3单克隆抗体能抑制肿瘤细胞在琼脂糖中的生长。而且,以小鼠半乳糖苷结合凝集素-3 cDNA转染正常小鼠成纤维细胞可导致获得锚链非依赖性生长。
为测定MCP对MLL在0.5%琼脂糖中的集落形成的作用,以溶于无钙镁(CMF)-PBS的0.02%EDTA将MLL细胞自培养的单层上解离下来并以4×103细胞/ml悬于含不同浓度MCP或不含MCP的完全RPMI中。于37℃将细胞温育30分钟,然后以1∶1(vol/vol)与预先加热至45℃的溶于RPMI之1%琼脂糖(1∶4,vol/vol)溶液混和。取2ml该混和物置于预制于6cm直径皿中的1%琼脂糖层之上。于37℃温育细胞8天,然后固定,计数且照相。图4A显示了由设盲的观察者用倒置相显微镜测定的所形成集落数。0.1%MCP的存在与对照相比显著抑制了出现的MLL集落数(p<0.01,Mann-Whitney)。小柱子代表三次同样的实验的平均数与S.E.。于不含(图4B)或含0.1%(w/v)MCP(图4C)中生长之MLL细胞的位相显微照片为放大160倍。如图4A所示,MCP以剂量依赖方式抑制MLL细胞在琼脂糖中的集落形成。MCP即抑制MLL集落的数量也抑制其大小(图4B和4C)。MCP的抑制作用显示为细胞抑制而不是细胞毒性,因为它对体外培养的单层MLL细胞的生长速度无影响(数列未列出)。MCP对其它的肿瘤细胞于软质琼脂糖中形成集落的能力有相似作用,这些细胞包括B16-F黑素瘤、UV-2237纤维肉瘤;HT-1080人纤维肉瘤以及A375人黑色瘤,还不知道是否MCP阻断MLL细胞与琼脂糖的半乳糖残基的结合,或与含糖生长因子与细胞表面半乳糖苷结合凝集素-3的结合进行竞争。与此相似,还未知道是否MCP抑制体内肿瘤细胞肺集落形成与体内抑制集落形成相似,尽管这种相关似乎存在(图1和图4)。
这里所展现的结果提供了预防自发性前列腺癌转移的一种新的无毒性及口服的方法。在预实验中,我们发现半乳糖苷结合凝集素-3存在于人前列腺癌病理组织标本及人前列腺腺癌细胞系PC-3中。为进行免疫组化,将5μm甲醛固定的石蜡包埋之原发前列腺腺癌切片脱石蜡、再水化并于1mM硬脂酸钠缓冲液中用微波处理(中-高)10分钟。冲洗后,将切片在正常山羊血清中阻断30分钟,然后与第一抗体大鼠抗-半乳糖苷结合凝集素-3-TIB-166单克隆抗体温育。在DPBS中洗切片30分钟,然后与生物素化的抗-大鼠IgG温育,冲洗,并与抗生物素蛋白-生物素化的辣根过氧化酶温育,再与过氧化酶底物3′-3′-二氨基联苯胺温育。将切片以3%甲基绿复染并置于明胶-甘油上。图5显示的切片来自一患侵入性前列腺癌的病人。如图2图例所述,将PC-3细胞提取物进行免疫印迹及分析以了解人半乳糖苷结合凝集素-3的存在。用TIB-166抗半乳糖苷结合凝集素-3单克隆抗体进行的免疫组化检测人前列腺标本中半乳糖苷结合凝集素-3的表达。半乳糖苷结合凝集素-3主要在以较少基质(stromal)染色且易变的正常上皮染色的前列腺癌细胞中表达(图5)。以此抗体的半乳糖苷结合凝集素-3染色与增强的核,胞浆及细胞表面染色相关连。进一步的研究将测定半乳糖苷结合凝集素在正常及癌性前列腺组织中的作用以及MCP抑制裸鼠的人前列腺癌转移的能力。MCP分子显示出口服后可吸收入血且与对于继发肿瘤细胞集落成功的形成之必需的肿瘤细胞表面半乳糖苷结合凝集素天然配体进行竞争。由于对果胶分子特征知之甚少,目前正在进行确定MCP活性部分的特征以及其血浆水平的工作。似乎MCP的作用发生在转移的早期阶段,可能抑制了肿瘤细胞栓子的形成以及抑制了癌细胞与靶器官内皮细胞相互作用,而不是在后期的过程如转移细胞的生长,因为MCP对MLL原发肿瘤生长或血管生成无作用。
由于天然柑桔果胶(CP)和pH调节的柑桔果胶(MCP)分别是高度支链化和非支链化复合多糖,富含半乳糖残基,能够与半乳糖苷结合凝集素-3的糖结合功能区结合,我们研究了CP和MCP对糖识别介导的细胞-细胞及细胞-基质间相互作用的影响。MCP而不是CP可抑制B16-F1黑素瘤细胞粘附至层粘连蛋白及无唾液酸胎球蛋白导致的同型凝集。MCP及CP均可抑制B16-F1细胞在半固体培养基即琼脂糖中的锚链-非依赖性生长。这些结果表明由细胞表面半乳糖苷结合凝集素-3介导的糖识别可能参与细胞-细胞外基质间的相互作用且在锚链-非依赖性生长以及体内肿瘤细胞的栓塞中起作用。
更具体地说,内源性的脊椎动物半乳糖苷结合凝集素已在广泛的组织和细胞中得到鉴定并确认了特征。基于其大小,凝集素被分成两个充裕的类别,分子量大小为~14kDa和~30kDa近期已分别被称为半乳糖苷结合凝集素-1和半乳糖苷结合凝集素-3。半乳糖苷结合凝集素-3代表了很宽范围的分子,即鼠34kDa(ML-34)和人31kDa(hL-31)肿瘤相关半乳糖苷结合凝集素、35kDa或纤维细胞糖结合蛋白(CBP35),IgE-结合蛋白(εBP)、32kDa巨噬细胞非一整合蛋白粘连蛋白结合蛋白(Mac-2)以及大鼠、小鼠和人形式的29kDa半乳糖苷结合凝集素(L-29)。分子克隆研究已揭示多肽是一致的。半乳糖苷结合凝集素-3包括两个结构功能区,一个含有胶源样序列的氨基末端功能区以及包含了半乳糖苷结合位点的球形羧基末端功能区。目前还不知道是否所有上文所提及的半乳糖苷结合凝集素共有同样的天然配体。尽管已认为半乳糖苷结合凝集素-3为需要还原条件保证其糖结合活性的S型凝集素,近期的研究已获得了相反的证据。几方面的分析显示凝集素通过识别和结合互补的糖结合物(glycoconjugate)参与细胞-细胞与细胞-基质间的相互作用,且从而在不同的正常及病理过程中起关键作用。
半乳糖苷结合凝集素-3由已激活的巨噬细胞和致癌性转化及转移的细胞高度表达。该多肽增高的表达与锚链-非依赖性生长。同型凝集及肿瘤细胞肺集落形成的能力提高有关联,这表明半乳糖苷结合凝集素促进了肿瘤细胞在循环中的栓塞并增强了转移。我们以前已报道静脉注射CP增加了B16-F1鼠黑素瘤细胞肺部集落形成,而MCP减少了肺部集落形成。尽管CP增加的肺部集落形成最大可能是由于其促进同型凝集的能力,MCP通过何种机制预防肺部集落形成仍未很好地建立。
层粘连蛋白(基底膜主要的非胶原成分)为一载有聚-N-乙酰乳糖胺序列的N-连接糖蛋白,并且参与细胞粘附、迁移、生长、分化、侵润及转移。以高亲和性与含聚-N-乙酰乳糖胺序列的寡糖结合之半乳糖苷结合凝集素也以特异的糖依赖方式与层粘连蛋白的糖侧链结合。
为了进一步研究半乳糖苷结合凝集素-3的功能特性,我们利用了CP和MCP,并且检测了其是否将影响B16-F1鼠黑素瘤细胞半乳糖苷结合凝集素相关的特性。我们发现:(a)MCP,而不是CP,抑制细胞粘附至层粘连蛋白;(b)MPC抑制无唾液酸胎球蛋白诱导的同型凝集,而CP则增强凝集;和(c)CP及MCP均抑制半固体培养基中锚链-非依赖性生长。
CP及EHS层粘连蛋白购自Sigma,St.Louis,Missouri。MCP由CP通过根据Albersheim等上文描述之方法的pH修饰加以制备。无唾液酸胎球蛋白以于80℃将胎球蛋白在0.05MH2SO4中轻度酸水解(Spiro法;Grand Island Biological Co.,Grand Island,New York)60分钟。用于它处描述的经无唾液酸胎球蛋白亲和柱的一步纯化法,自细菌细胞提取重组半乳糖苷结合凝集素-3。使用前将以乳糖洗脱的重组半乳糖苷结合凝集素-3充分地对CMF-PBS透析。从R.Lotan博士(University ofTexas,M.D.Anderson)处获得抗半乳糖苷结合凝集素-3单克隆抗体。辣根过氧化物酶(HRP)-结合的免抗-鼠IgG+IgM和2,2′-连氮基-二(3-乙基苯基噻唑啉磺酸)(ABTS)底物试剂盒购自Zymed,South SanFrancisco,California。在补充有10%加热失活的胎牛血清、非必需氨基酸、2MM谷氨酰胺及抗生素的Dulbecco′s改良Eagles′最低限度培养基(DMEM)中培养B16-F1鼠黑素瘤细胞。将细胞在7%CO2和93%空气的潮湿环境下维持在37℃。
细胞与层粘连蛋白的粘附-将96孔板的组织培养孔以溶于无Ca2+和Mg2+磷酸缓冲盐液(pH7.2,CMF-PBS)的EHS层粘连蛋白(2μg/孔)于4℃预涂布过夜,于室温下以溶于CMF-PBS中的1%牛血清白蛋白(BSA)阻断残余蛋白结合位点2小时。以溶于CMF-PBS的0.02%EDTA收获细胞并悬于无血清DMEM中。将5×104细胞在含或不含不同浓度CP或MCP之DMEM中加至各孔中。于37℃温育2小时15分钟后,以CMF-PBS洗去非吸附细胞。用甲醇将吸附细胞固定并照相。按照Olier等人的方法测定吸附细胞的相对数目。简而言之,将细胞以甲基蓝染色,然后加入HCI-乙醇以释出染料。以平板阅读器测量光密度(650nm)。
无唾液酸胎球蛋白诱导的同型凝集-以溶于CMF-PBS的0.02%EDTA解离细胞并以1×106细胞/ml悬于含或不含20μg/ml无唾液酸胎球蛋白和0.5%CP或0.5%MCP的CMF-PBS中。将各份含0.5ml细胞悬液的溶液置于硅化玻璃管中于37℃以80rpm搅动60分钟。然后通过以溶于CMF-PBS的1%甲醛固定细胞终止凝集。样品用于计算单个细胞数目,且根据下列等式计算所产生的凝集:(1-Nt/Nc)×100,其中Nt和Nc分别代表存在受试成分及在对照缓冲液(CMF-PBS)中单个细胞的数目。
半乳糖苷结合凝集素-3与MCP的结合-将96孔板以含0.5%MCP和1%BSA的CMF-PBS涂布且干燥过夜。将重组半乳糖苷结合凝集素-3在含0.5%BSA的CMF-PBS中系列稀释,加入存在或不存在50mM乳糖的0.05%吐温-20(溶液A),温育120分钟,此后将孔中水分排干并以含0.1%BSA和0.05%吐温-20的CMF-PBS(溶液B)冲洗。加入溶于溶液A的大鼠抗-半乳糖苷结合凝集素-3并温育60分钟,然后以溶液B冲洗并与溶于溶液A的HRP结合的兔抗-大鼠IgG-IgM温育30分钟。冲洗后经加入ABTS确定结合于各孔中的结合酶之相对量。于405nm处测水解程度。
半固体培养基的集落形成-以溶于CMF-PBS的0.02%EDTA解离细胞并以1×103细胞/ml悬于含或不含不同浓度CP或MCP的完全DMEM中。于37℃将细胞温育30分钟并再与预先加温于45℃的溶于蒸馏水-完全DMEM(124,vol/vol)之1%琼脂糖溶液混和1∶1(vol/vol)。取2ml混和物置于6cm直径皿1%琼脂糖预铺层之上。于37℃将细胞温育14天,在以加入溶于CMF-PBS的2.6%戊二醛固定后,用倒置相显微镜测定形成集落的数目。
以前已显示层粘连蛋白可作为可溶性半乳糖苷结合凝集素-3的配体,且B16-F1细胞在其细胞表面表达半乳糖苷结合凝集素-3分子。这些结果与CP与MCP对静注B16-F1细胞的肺部集落形成的影响一起促使我们开始检测其对B16-F1细胞粘附层粘连蛋白的作用,以便评价细胞表面半乳糖苷结合凝集素-3在此过程中的可能作用。如图6和7A-C所示,MCP以剂量依赖方式显著抑制细胞粘附至层粘连蛋白,而CP对细胞结合或扩散至层粘连蛋白上均无明显作用。半乳糖苷结合凝集素-3乳糖的单糖抑制剂以高至100mM的浓度并不抑制细胞粘附至层粘连蛋白(数据未显示)。用可溶性重组半乳糖苷结合凝集素-3的竞争性结合测定不能阻断细胞粘附至层粘连蛋白且抗-Mac-2单克隆抗体也在此方面无作用(数据未显示),这提示MCP的抑制作用不能仅归于其破坏半乳糖苷结合凝集素-3与层粘连蛋白上N-乙酰乳糖胺基侧链间的相互作用,因为细胞可利用整合蛋白结合至层粘连蛋白蛋白核心。进一步地讲,抗Mac-2单克隆抗体不针对半乳糖苷结合凝集素-3的糖结合功能区,而是针对其N-末端,因此,MCP阻断粘附的确切机制与CP和乳糖相比仍不清楚。MCP的抑制作用不是由于细胞毒性,因为MCP(0.5%)不影响细胞的活力或体内的生长。
现已在肿瘤细胞体外进行同型凝集的倾向性及其体内转移潜能之间建立了良好的相关性。B16黑素瘤细胞团静脉注射后较单个的细胞可产生更多的肺部集落。而且,已显示抗半乳糖苷结合凝集素-3抗体抑制无唾液酸胎球蛋白-诱导的同型凝集(14),这提示细胞表面半乳糖苷结合凝集素多肽导致了在其与糖蛋白侧链相互作用后的同型凝集的形成。如图8和9A-C所示,MCP显著减少了同型凝集的形成,而CP则起增强作用。最可能的是非支链MCP产生类似于特异性糖抑制剂即乳糖的作用,从而遮盖了细胞表面半乳糖苷结合凝集素-3分子与无唾液酸胎球蛋白的半乳糖苷残基的相互作用,导致同型凝集的减少。相反,可以想象假设分支的糖多聚物的结构特征使CP可作为邻接细胞之间交联的桥梁,导致增强的同型凝集的形成。将其一起加以考虑,可以提示MCP通过破坏对肿瘤细胞形成转移病变起关键作用的细胞-细胞和细胞-间质相互作用来预防转移。
前文所述的MCP抑制B16-F1细胞与层粘连蛋白的粘附及同型凝集可能由于其与细胞表面半乳糖苷结合凝集素的相互作用,因为以前已显示CP以乳糖依赖方式结合B16-F1细胞表面。为说明半乳糖苷-3与MCP的结合,我们采用了一种酶联免疫吸附测定法,结果我们发现重组半乳糖苷结合凝集素-3以剂量依赖方式与固定化MCP结合且该结合被乳糖完全阻断(图9)。这些结果使我们可将MCP的同型凝集抑制作用归因于其与细胞表面半乳糖苷结合凝集素-3分子的结合。另一方面,我们不知道MCP(而不是CP)如何减弱B16-F1细胞对层粘连蛋白的粘附。由于CP(为链的复合多糖聚合物)的pH调节,同一糖组合物的非支链糖链的产生,有可能MCP较CP更倾于结合至细胞表面半乳糖苷结合凝集素-3分子。将抗-整合蛋白抗体抑制鼠B16黑素瘤细胞附着至层粘连蛋白底物的事实一并考虑,我们猜测MCP从空间上抑制层粘连蛋白受体的整合蛋白族对层粘连蛋白的识别,或者细胞表面半乳糖苷结合凝集素与层粘连蛋白上的聚-N-乙酰乳糖胺序列的相互作用可能与整合蛋白协调一致对细胞粘附至层粘连蛋白起作用。MCP与和半乳糖苷结合凝集素-3具有同样糖特异性的半乳糖苷结合凝集素-1的相互作用可能影响其削弱B16-F1细胞对层粘连蛋白的粘附和同型凝集的可能性最有可能被排除在外,因为半乳糖苷结合凝集素-1为分泌蛋白质。
将细胞于半固体培养基中生长的能力即:锚链独立性“用作细胞转化的标准,因为这一特性只有已转化的和致癌的细胞通常会显示。以前已提示肿瘤细胞通过细胞表面半乳糖苷结合凝集素-3与糖蛋白糖残基的相互作用的能力与其和琼脂糖(一种D-半乳糖和L-脱水半乳糖的聚合物)半乳糖残基的相互作用有关,且与在此半固体培养基中集落形成的效能有关。也已显示抗-半乳糖苷结合凝集素-3单克隆抗体抑制肿瘤细胞在琼脂糖中的生长,并且半乳糖苷结合凝集素-3的表达与已转化表型的抑制呈反相关。以小鼠半乳糖苷结合凝集素-3cDNA对正常小鼠成纤维细胞的转染导致获得锚链非依赖性生长特性。为进一步证实细胞表面半乳糖苷结合凝集素对细胞在半固体培养基中生长起关键作用的可能性,我们检测了CP和MCP对B16-F1黑素瘤细胞的锚链非依赖性生长的作用。如图11所示,CP和MCP以剂量依赖方式抑制了B16-F1细胞集落在半固体基质中的生长。与此类似,乳糖也以剂量依赖方式抑制锚链-非依赖性生长(数据未列出)。CP和MCP的这种剂量依赖抑制作用并不局限于对B16-F1黑素瘤细胞。UV-2237-10-3鼠纤维肉瘤细胞、HT1080人纤维肉瘤细胞及A375C1.49人黑素瘤细胞在软质琼脂糖中的生长也相同程度的被抑制。可能可溶性的CP和MCP与琼脂糖半乳糖残基竞争与半乳糖苷结合凝集素-3的结合,这以去除了细胞的细胞增殖所需基质最低限度的支持的方式产生明显的生长抑制。CP和MCP以及抗-半乳糖苷结合凝集素-3抗体可能作为-仍未识别的糖共聚物生长因子的桔抗剂起作用,该生长因子与半乳糖苷结合凝集素-3相互作用,或者它们在空间上阻碍已知生长因子靠近膜受体,这些观点仍可能有争议。然而,B16-F1细胞体外的锚链依赖性生长以及对同基因小鼠的致癌性并不为MCP(0.5%)所抑制的事实可能使我们排除前述的可能性。由于细胞在半固体培养基中生长的能力被用作细胞转化的标准,所以细胞表面半乳糖苷结合凝集素-3的获得可能是转化后过程中的一个早期步骤。
Claims (15)
1.一种治疗性治疗哺乳动物癌症的方法,它包括给患有癌症的哺乳动物口服有效量的经调节的果胶。
2.权利要求1所述的方法,其中所说的经调节的果胶为pH调节的柑桔果胶。
3.权利要求2所述的方法,其中所说的pH调节的柑桔果胶其表观平均分子量约为1-15kd。
4.权利要求1所述的方法,其中所说的癌症为前列腺癌。
5.权利要求1所述的方法,其中所说的癌症为人前列腺癌。
6.权利要求1所述的方法,其中所说的经调节的果胶是以与一种药物学可接受的可消化之载体混合的形式存在。
7.权利要求1所述的方法,其中所说癌症的治疗为抑制转移。
8.一种用于治疗性治疗哺乳动物癌症的组合物,它包括一种经调节的果胶与一种药物学可接受之可消化的口服载体的混合物。
9.权利要求8所述的组合物,其中所说的经调节的果胶为pH调节的柑桔果胶。
10.权利要求9所述的组合物,其中所说的pH调节的柑桔果胶其表观分子量为约1-15Kd。
11.权利要求3所述的方法,其中所说的pH调节的柑桔果胶之表观平均分子量为约10Kd。
12.权利要求10所述的组合物,其中所说的pH调节的柑桔果胶之表观分子量为约10Kd。
13.权利要求1所述的方法,其中所说的经调节的果胶为水溶性。
14.权利要求8所述的组合物,其中所说的经调节的果胶为水溶性。
15.一种预防癌症的方法,它包括给哺乳动物口服经调节的果胶。
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- 1995-06-14 ES ES95925253T patent/ES2227555T3/es not_active Expired - Lifetime
- 1995-06-14 BR BR9508245A patent/BR9508245A/pt not_active IP Right Cessation
- 1995-06-14 CA CA002194586A patent/CA2194586A1/en not_active Abandoned
- 1995-06-14 DK DK95925253T patent/DK0768885T3/da active
- 1995-06-14 DE DE69533550T patent/DE69533550T2/de not_active Expired - Fee Related
- 1995-06-14 CN CN95194958A patent/CN1157567A/zh active Pending
- 1995-06-14 EP EP95925253A patent/EP0768885B1/en not_active Expired - Lifetime
- 1995-06-14 PT PT95925253T patent/PT768885E/pt unknown
- 1995-06-14 AT AT95925253T patent/ATE276753T1/de not_active IP Right Cessation
- 1995-06-14 AU AU29442/95A patent/AU695677B2/en not_active Ceased
- 1995-06-14 MX MX9700118A patent/MX9700118A/es not_active IP Right Cessation
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1996
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-
1997
- 1997-01-02 US US08/735,432 patent/US5895784A/en not_active Expired - Lifetime
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2008
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100394923C (zh) * | 2006-05-26 | 2008-06-18 | 范晓青 | 低分子柑桔果胶用于调节血糖血脂和改善脂肪肝中的应用 |
CN101269087B (zh) * | 2007-11-02 | 2011-11-09 | 中国人民解放军第四军医大学 | 果胶-5-氟尿嘧啶结肠癌双靶向前体药物及制备方法 |
CN102085214A (zh) * | 2011-01-27 | 2011-06-08 | 范晓青 | 低分子柑橘果胶联和临床常用化疗药用于控制癌症和癌症转移扩散 |
Also Published As
Publication number | Publication date |
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DK0768885T3 (da) | 2004-12-06 |
BR9508245A (pt) | 1997-09-30 |
MX9700118A (es) | 1997-06-28 |
EP0768885A4 (en) | 1998-04-29 |
NO970039D0 (no) | 1997-01-06 |
CA2194586A1 (en) | 1996-01-25 |
ES2227555T3 (es) | 2005-04-01 |
JP2008111002A (ja) | 2008-05-15 |
FI965269A0 (fi) | 1996-12-31 |
NO970039L (no) | 1997-02-17 |
DE69533550T2 (de) | 2005-09-22 |
WO1996001640A1 (en) | 1996-01-25 |
PT768885E (pt) | 2005-01-31 |
AU695677B2 (en) | 1998-08-20 |
ATE276753T1 (de) | 2004-10-15 |
US5834442A (en) | 1998-11-10 |
EP0768885B1 (en) | 2004-09-22 |
EP0768885A1 (en) | 1997-04-23 |
AU2944295A (en) | 1996-02-09 |
NO311493B1 (no) | 2001-12-03 |
DE69533550D1 (de) | 2004-10-28 |
FI965269A (fi) | 1997-03-07 |
US5895784A (en) | 1999-04-20 |
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