抗肿瘤药物的代谢产物N-乙酰基-S-（对氯苯甲酰基）半胱氨酸抑制恶性黑色素瘤生长的作用和机理研究

N-Acetyl-S-(p-chlorophenylcarbamoyl)cysteine (NACC) is one of the major metabolites in rats of anticancer drug-sulofenur developed by Elli Lilly research laboratories. Sulofenur has exhibited decent and broad anticancer activity against human solid tumors in clinical trials. However, due to its high protein binding, a high dosage of sulofenur was required for clinical responses. As a consequence, its side effect-anemia was observed in clinical trials.. Our earlier study showed NACC had much lower protein binding compared to sulofenur and it had anticancer activity against, especially, human melanoma, also UV-induced skin cancer in hairless mouse model. It induces apoptosis in melanoma cells but has no effect on cell cycle. It is able to inhibit thioredoxin and glutaredoxin activity. It inhibits metastasis of melanoma cells in vitro. It is known that sulofenur is able to elevate cytosolic Ca2+ concentration and decrease mitochondrial membrane potential by which apoptosis could be induced. It is very possible that the mechanism of action of NACC will be elevation of cytosolic Ca2+ concentration and then induction of apoptosis in cancer cells through mitochondrial pathway. . In this study, the anticancer activity of NACC against melanoma will be further evaluated in vitro and in vivo by using cultured human melanoma cells and melanoma xenograft mouse model. By using flow cytometry, ELISA, Western blot, RT-PCR, fluorescent microscopy and spectrometery methods etc, the effects of NACC on induction of apoptosis in melanoma cells and its mechanism of action will be investigated to confirm that whether NACC induces apoptosis is through elevation of cytosolic Ca2+ concentration, decreasing mitochondrial membrane potential or inhibition of thioredoxin, glutaredoxin and thioredoxin reductase. And the effects of NACC on melanoma invasion and metastasis and its mechanism of action will be studied in vitro and in vivo by using cell and mouse models.

NACC是抗肿瘤药物磺氯苯脲的主要代谢产物，其蛋白结合较磺氯苯脲大幅降低。前期研究表明：NACC对体外培养人恶性黑色素瘤细胞、小鼠皮肤癌模型均有显著的抑制作用，而对正常猴肾细胞无明显毒性。NACC对细胞周期无影响，但能诱导细胞凋亡；能抑制谷氧还蛋白和硫氧还蛋白的活性和黑色素瘤细胞的转移。.本项目拟在前期研究的基础上，采用细胞培养和裸鼠肿瘤移植实验，进一步评价NACC对黑色素瘤细胞的抑制作用。采用Annexin-V/PI双染法考察NACC对黑色素瘤细胞凋亡的影响；采用荧光光度法、酶动力学分析、流式细胞术、ELISA、Western blot、RT-PCR等方法考察NACC对肿瘤细胞胞浆钙离子浓度、线粒体跨膜电位，肿瘤细胞谷氧还蛋白、硫氧还蛋白和硫氧还蛋白还原酶活性，促凋亡蛋白表达的影响；利用细胞和裸鼠模型考察NACC对黑色素瘤细胞侵袭和转移的影响；以明确NACC抑制黑色素瘤细胞生长的机理。

恶性黑色素瘤恶性程度高、进展快、对治疗不敏感。目前，仍然缺少有效的化学药物。凋亡通路失活导致其对治疗不敏感、产生耐药性。NACC能使UACC-62细胞中caspase3和9活性升高，但不影响caspase8活性，表明NACC可能通过线粒体通路诱导凋亡。NACC能上调Bax，下调Mcl-1但是对Bad和Bcl-2无影响，表明NACC提升了Bax/Bcl-2比值，从而导致细胞色素c的释放。NACC使UACC-62细胞线粒体跨膜电势显著降低。黑色素瘤细胞短时间暴露于NACC产生大量ROS，说明诱导ROS产生是NACC破坏线粒体跨膜电势的主要机制之一。NACC诱导的细胞凋亡不依赖于PTP开合。恶性黑色素瘤的高转移性导致了极差的疗效和低生存率，因此降低黑色素瘤细胞的迁移能力是治疗过程中一个关键因素。NACC能显著抑制黑色素瘤细胞迁移，呈药物浓度和时间依赖关系，IC50值为4.93 ± 2.64 uM。凝血酶通过其蛋白酶激活受体（PAR-1）调节黑色素瘤细胞对血小板和内皮细胞的黏附能力，促进肿瘤细胞血管生成，生长和转移。实验表明UACC-62细胞迁移不依赖于凝血酶调节, NACC对其迁移的抑制不受影响。TrxR系统对肿瘤细胞转移和侵袭能力及恶性化程度关系密切，所以推测NACC对迁移的抑制和TrxR活性降低有一定关系。.TrxR在恶性黑色素瘤等许多肿瘤中过表达，和肿瘤恶性程度、细胞凋亡、转移和耐药性等密切相关。NACC对TrxR具有很好的抑制能力并呈时间和浓度依赖关系，是TrxR1的竞争性不可逆抑制剂。NACC对TrxR1的抑制是通过作用在其酶活位点上，并且需要酶活中心Cys497和Sec498残基处于还原态。质谱分析得出NACC通过氨基甲酰化Sec498残基上的硒代硫醇，而非Cys497上的硫醇，使TrxR1失活。有别于其它TrxR1抑制剂，NACC只作用在Sec498残基的硒代硫醇上，显示了它独特的抑制机制。.通过本项目的研究，我们明确了NACC对黑色素瘤细胞生长的抑制作用；明确了NACC抑制TrxR1的酶动力学性质及其作用机制；明确了NACC诱导UACC-62细胞凋亡的具体作用机制；明确了NACC对黑色素瘤细胞迁移的抑制作用。本项目的研究成果为抗黑色素瘤药物NACC的后续开发和相关新药的研究提供了前期实验基础，为TrxR过表达型黑色素瘤及其它肿瘤的治疗提供了重要信息。