【作者】 王旭芳；

【导师】 徐铜文； 张劲松；

【作者基本信息】 中国科学技术大学 ， 应用化学， 2008， 博士

【摘要】 哺乳动物硫氧还蛋白还原酶(thioredoxin reductase,TrxR)是含硒酶,其主要功能是催化NADPH还原硫氧还蛋白。它在促进与肿瘤发生发展相关的多重细胞活动中有很重要的作用,这些活动包括细胞增殖、凋亡和细胞信号传导等。作为含硒酶,硒代半胱胺酸残基位于TrxR分子羧基端的倒数第二位,暴露于酶的表面,具有很高的活性且在催化作用中很容易被接近。而且,硒代半胱胺酸残基的pKa值约为5-2,因而在生理pH时处于非质子化的硒醇盐状态。这两个特点使得TrxR易被烷化剂或者亲电化合物修饰,导致失活。这种氧化还原酶在肿瘤中高表达,近几年大量研究表明它是抗癌剂治疗癌症的有效分子靶。体外实验发现临床上经常使用的烷化类抗癌剂能有效抑制TrxR活力。环磷酰胺是临床上治疗恶性肿瘤最常用的烷化剂代表药物之一。它在体外无活性,在体内经过肝细胞色素P450酶代谢活化才能发挥作用,因此我们在动物体内研究环磷酰胺对肿瘤TrxR活力的影响,发现环磷酰胺是TrxR有效的抑制剂。而且,我们还观察到TrxR活力在被迅速抑制后,又逐渐恢复到正常水平,这可能与肿瘤细胞耐药性相关。我们的结果提示在使用环磷酰胺治疗时,使用药理或者遗传学方法控制TrxR活力恢复可能是克服肿瘤细胞耐药性的一个合理治疗策略。我们还使用腹水肝癌动物模型研究环磷酰胺抑制癌细胞TrxR对癌细胞增殖的影响,发现TrxR对肿瘤生长很重要,而且环磷酰胺对肿瘤细胞TrxR活力的抑制与其抗癌效果密切相关。这些结果表明环磷酰胺对癌细胞TrxR活力的抑制可以认为是其抗癌效果的一个重要机制。心力衰竭是使用高剂量的环磷酰胺经常出现的威胁生命的并发症,但是其发病机理还不是很清楚。我们发现环磷酰胺引发的心脏损伤可能是它对细胞质硫氧还蛋白还原酶(cytoplasmic thioredoxin reductase,TrxR1)和非蛋白游离巯基的共同抑制引起的。TrxR1曾经被报道在大多数组织但是不包括心脏发挥很重要的作用。我们的研究发现心脏TrxR1有其独特的两个方面。第一,肿瘤、膀胱和肾中的TrxR1超过一半活力被抑制,将会产生相应的毒性后果。但是,当心脏中TrxR1活力被强烈抑制了68%,没有看到明显的心脏损伤。第二,肿瘤组织、腹水癌细胞、膀胱和肾脏中TrxR1活力在被环磷酰胺或者异环磷酰胺抑制后能快速恢复到正常水平,但是心脏TrxR1活力恢复很慢甚至不恢复。心脏中TrxR1活力失活在环磷酰胺引起的心脏损伤中仍然发挥作用,因为失活TrxR1能获得细胞毒素的功能,在大量非蛋白游离巯基被消耗的时候,这种细胞毒素的功能会产生很明显的效应,引发心脏损伤。当高剂量的CTX或者低剂量的CTX与谷胱甘肽合成的抑制剂一起使用的时候,能引起心脏TrxR1和非蛋白游离巯基的共同显著抑制,导致心脏损伤的发生。异环磷酰胺是磷酰胺类衍生物,具有广谱的抗肿瘤活性。它需要在体内经过肝细胞色素P450酶代谢活化才能发挥作用。我们已经发现环磷酰胺能有效抑制肿瘤TrxR活力。作为环磷酰胺的异构体,异环磷酰胺与哺乳动物TrxR之间的相互作用还没有被研究。因此,我们在小鼠上研究异环磷酰胺对TrxR活力影响。我们发现异环磷酰胺能抑制肿瘤TrxR活力,导致肿瘤细胞增殖速度显著减慢。而且,异环磷酰胺优先抑制体内TrxR活力,而不是其它抗氧化参数。经过异环磷酰胺作用的癌细胞,其TrxR活力被显著抑制,当把这些细胞移植到小鼠体内时,发现它们不再具有致肿瘤的属性。异环磷酰胺还能抑制肾脏、膀胱和脑组织TrxR活力。这些结果说明异环磷酰胺对肿瘤TrxR活力的抑制与其抗癌效果高度相关,并且异环磷酰胺的全身毒性可能与其抑制多器官TrxR活力有关。异环磷酰胺对TrxR活力抑制可以看做其抗癌效应的一个很重要的机制。上面的结果说明TrxR酶的靶向保护可能是一个减少或者缓解化疗药对正常组织毒性的有效方法,这样可以提高化疗药的剂量,获得好的治疗效果。阿米福汀是美国FDA批准的用于减轻顺铂多次治疗引起的肾毒性的细胞保护剂。体外实验证明顺铂能抑制TrxR,但是药理剂量的顺铂在体内能否抑制TrxR活力至今还没有报道。我们发现药理剂量的顺铂能抑制小鼠腹水肝癌细胞TrxR的活力,压制肝癌细胞增殖,并且能抑制肾脏TrxR活力,产生肾脏毒性。阿米福汀能保护肾脏TrxR活力不受顺铂的抑制,但是不干扰顺铂对肿瘤TrxR活力的抑制。阿米福汀能显著提高顺铂对腹水肝癌模型的治愈率,可能是由于它对TrxR的选择性保护。更多还原

【Abstract】 Mammalian thioredoxin reductase(TrxR)is a selenoenzyme,its major function is catalysis NADPH-dependent reduction of the redox-active disulfide in thioredoxin (Trx).It plays an important role in multiple cellular events related to carcinogenesis including cell proliferation,apoptosis,and cell signaling.As a selenoprotein,TrxR contains a selenocysteine on its flexible C-terminal ann which is very reactive and well-accessible during catalysis,and the pKa value of selenocysteine is 5.2,making selenocysteine in TrxR molecule present in the form of -Se~- at physiological pH。Therefore,TrxR has the propensity to be conjugated with alkylating or electrophilic agents.This oxidoreductase is over-expressed in many tumor cells;recent reports indicate it is a potential molecular target of anticancer agents.It is reported that prototype compounds representative for the major classes of clinically used anticancer alkylating agents could effectively inhibit TrxR in vitro. Cyclophosphamide(CTX)is in the nitrogen mustard group of alkylating antineoplastic chemotherapeutic agents.It has no activity against cancer cells in vitro until it undergoes metabolic activation catalyzed by liver cytochrome P450 enzymes. Therefore we investigated the influence of CTX on tumor TrxR activity in vivo and we found that CTX was a potent inhibitor of TrxR.Furthermore,we found the phenomena that TrxR recovery back to normal level in CTX-treated tumors after its inactivation,which was not seen in vitro.That may be related to the resistance of tumor cells.Our results suggest utilizing pharmacologic or genetic methods to control the recovery of TrxR activity in the treatment of CTX may be a rational therapeutic strategy to overcome the CTX-resistance.We also used ascites H22 cells to investigate cancer cells response after TrxR was inhibited by CTX in vivo。The observations provide direct evidence that TrxR is essential to tumor growth,and inhibition of TrxR activity in malignant cells by CTX is tightly connected with its anticancer effect.The TrxR inhibition could thereby be considered as a crucial mechanism contributing to anticancer effect seen upon clinical use of CTX.Heart failure is a life-threatening complication of high-dose CTX chemotherapy, but little is known about its mechanism.The present study demonstrates CTX-evoked heart failure involves pronounced co-suppression of cytoplasmic TrxR(TrxR1) activity and non-protein free thiol(NPFT)level.It has been reported that TrxR1 is essential in most tissues except heart.The present study also found cardiac TrxR1 was unique in two aspects.Firstly,our previous studies showed that half inactivation of TrxR1 due to CTX or ifosfamide treatment is associated with a toxicological consequence.In contrast,in the present study,when cardiac TrxR1 activity was suppressed by 68%,no cardiac toxicity was observed.Secondly,our previous studies showed that TrxR1 activity in tumor tissue,ascitic cancer cells,bladder and kidney rapidly recovered back to normal level after being inactivated by CTX or ifosfamide, while in the present study,TrxR1 activity in heart recovered less efficiently.TrxR1 inactivation plays a role in CTX-evoked heart failure,because inactivated TrxR1 gains cytotoxic function,which would emerge under certain circumstance,such as NPFT depletion.Indeed,the present study revealed heart failure broke out when TrxR1 activity and NPFT level were pronouncedly co-suppressed by two ways,one is the high-dose CTX itself,and the other is the moderate-dose CTX plus buthionine sulfoximine,an inhibitor of glutathione synthesis.Ifosfamide is an oxazaphosphorine alkylating agent with a broad spectrum of antineoplastic activity.It is a prodrug that has first to be transformed by hepatic cytochrome P-450 enzymes.We have found that CTX potently inhibited the activity of tumor TrxR in vivo.As a structural analog of CTX,ifosfamide has not yet been studied for possible interaction with mammalian TrxR.Therefore we investigated the effect of ifosfamide on TrxR activity in mice.We demonstrated ifosfamide inhibited TrxR activity in vivo,and led to a subsequently dramatic deceleration in tumor progression.In addition,ifosfamide preferentially inhibited the activity of TrxR in vivo over other antioxidant parameters.Interestingly,the ifosfamide-treated cancer cells with decreased TrxR activity lost total tumorigenicity after being inoculated into mice.Ifosfamide could also inhibit TrxR activity in kidney,bladder and bladder. These observations provide direct evidence that inhibition of TrxR activity in malignant cells by ifosfamide is highly associated with its anticancer effect and the mechanism of ifosfamide systemic toxicity may be related to multi-organ inhibition of TrxR activity.The TrxR activity inhibition could be considered as a pivotal mechanism contributing to anticancer effect of ifosfamide.In view of the results mentioned above,the TrxR enzyme target protection may be an effective means to reduce or alleviate the tissue toxicity of anticancer agents, and then higher dose anticancer agents can be used to harvest better therapeutic effect. Amifostine is a pharmacological antioxidant used as a broad-spectrum cytoprotective agent in cancer chemotherapy and radiotherapy.FDA approval was granted in 1995 for a substantial attenuation of cumulative nephrotoxicity in patients receiving repeated doses of cisplatin(CDDP).It has been demonstrated via ex vivo experiments that CDDP inactivates TrxR,but whether CDDP at pharmacological doses inhibits TrxR activity in vivo has not been reported hitherto.Present study in mice revealed CDDP at pharmacological doses inhibited TrxR activity in both ascitic hepatoma 22 (H22)cells and kidney,leading to suppression of ascitic H22 cells growth along with nephrotoxicity.Furthermore,amifostine deterred CDDP-generated inhibition of renal TrxR activity but did not interfere with TrxR inactivation by CDDP in ascitic H22 cells.The large enhancement of CDDP cure rate in H22 ascites model by using amifostine is,at least in part,ascribed to its selective modulation on TrxR.更多还原