Kruczynski A, Barret J M, Etiévant C, Colpaert F, Fahy J, Hill B T
Division of Experimental Cancer Research I, Centre de Recherche Pierre Fabre, Castres, France.
Biochem Pharmacol. 1998 Mar 1;55(5):635-48. doi: 10.1016/s0006-2952(97)00505-4.
This study aimed to define the mechanism of action of vinflunine, a novel Vinca alkaloid synthesised from vinorelbine using superacidic chemistry and characterised by superior in vivo activity to vinorelbine in preclinical tumour models. In vitro vinflunine cytotoxicity proved dependent on concentration and exposure duration, with IC50 values (72-hr exposures) generally ranging from 60-300 nM. Vinflunine induced G2 + M arrest, associated with mitotic accumulation and a concentration-dependent reduction of the microtubular network of interphase cells, accompanied by paracrystal formation. These effects, while comparable to those of vincristine, vinblastine or vinorelbine, were achieved with 3- to 17-fold higher vinflunine concentrations. However, vinflunine and the other Vincas all inhibited microtubule assembly at micromolar concentrations. Vinflunine, like vinblastine, vincristine and vinorelbine, appeared to interact at the Vinca binding domain, as judged by proteolytic cleavage patterns, and induced tubulin structural changes favouring an inhibition of GTP hydrolysis. However, vinflunine did not prevent [3H]vincristine binding to unassembled tubulin at concentrations < or = 100 microM, and only weakly inhibited binding of [3H]vinblastine or [3H]vinorelbine. Indeed, specific binding of [3H]vinflunine to tubulin was undetectable by centrifugal gel filtration. Thus, the comparative capacities of these Vincas to bind to or to interfere with their binding to tubulin could be classified as: vincristine > vinblastine > vinorelbine > vinflunine. By monitoring alkylation of sulfhydryl groups, differential effects on tubulin conformation were identified with vinflunine and vinorelbine acting similarly, yet distinctively from vinblastine and vincristine. Overall, vinflunine appears to function as a definite inhibitor of tubulin assembly, while exhibiting quantitatively different tubulin binding properties to the classic Vinca alkaloids.
本研究旨在明确长春氟宁的作用机制。长春氟宁是一种新型长春花生物碱，由长春瑞滨经超强酸化学合成，在临床前肿瘤模型中其体内活性优于长春瑞滨。体外实验表明，长春氟宁的细胞毒性取决于浓度和暴露时间，其IC50值（72小时暴露）通常在60 - 300 nM范围内。长春氟宁诱导G2 + M期阻滞，伴有有丝分裂积累以及间期细胞微管网络浓度依赖性减少，并伴有副晶形成。这些效应虽与长春新碱、长春碱或长春瑞滨的效应相似，但长春氟宁的浓度要高出3至17倍。然而，长春氟宁和其他长春花生物碱在微摩尔浓度下均抑制微管组装。从蛋白水解裂解模式判断，长春氟宁与长春碱、长春新碱和长春瑞滨一样，似乎在长春花生物碱结合域相互作用，并诱导微管蛋白结构变化，有利于抑制GTP水解。然而，在浓度≤100 μM时，长春氟宁并不阻止[3H]长春新碱与未组装微管蛋白的结合，且仅微弱抑制[3H]长春碱或[3H]长春瑞滨的结合。实际上，通过离心凝胶过滤无法检测到[3H]长春氟宁与微管蛋白的特异性结合。因此，这些长春花生物碱与微管蛋白结合或干扰其与微管蛋白结合的相对能力可分类为：长春新碱＞长春碱＞长春瑞滨＞长春氟宁。通过监测巯基的烷基化，发现长春氟宁和长春瑞滨对微管蛋白构象的影响相似，但与长春碱和长春新碱不同。总体而言，长春氟宁似乎是微管蛋白组装的明确抑制剂，同时与经典长春花生物碱相比，其微管蛋白结合特性在数量上有所不同。
