In the search of small molecules that can target MDM2/p53 pathway in testicular germ cell tumors (TGCTs), we identified sempervirine (2,3,4,13-tetrahydro-1H-benz[g]indolo[2,3-a]quinolizin-6-ium), an alkaloid of Gelsemium sempervirens, that has been previously proposed as an inhibitor of MDM2 that targets p53-wildtype (wt) tumor cells. We found that sempervirine not only affects cell growth of p53-wt cancer cells, but it is also active in p53-mutated and p53-null cells by triggering p53-dependent and independent pathways without affecting non-transformed cells. To understand which mechanism/s could be activated both in p53-wt and -null cells, we found that sempervirine induced nucleolar remodeling and nucleolar stress by reducing protein stability of RPA194, the catalytic subunit of RNA polymerase I, that led to rRNA synthesis inhibition and to MDM2 block. As shown for other cancer cell models, MDM2 inhibition by nucleolar stress downregulated E2F1 protein levels both in p53-wt and p53-null TGCT cells with the concomitant upregulation of unphosphorylated pRb. Finally, we show that sempervirine is able to enter the nucleus and accumulates within the nucleolus where it binds rRNA without causing DNA damage. Our results identify semperivirine as a novel rRNA synthesis inhibitor and indicate this drug as a non-genotoxic anticancer small molecule.

在睾丸生殖细胞肿瘤（TGCTs）中可以靶向MDM2 / p53途径的小分子的搜索中，我们确定了sempervirine（2,3,4,13-tetrahydro-1 H -benz [ g ] indolo [2,3- a [quinolizin-6-ium），一种短发葛根的生物碱，先前已被提议作为靶向p53野生型（wt）肿瘤细胞的MDM2抑制剂。我们发现，sempervirine不仅影响p53-wt癌细胞的细胞生长，而且在p53突变和p53-null上也有活性通过触发p53依赖和独立的途径而不影响未转化的细胞来激活细胞。为了了解在p53-wt和-null细胞中哪些机制可以被激活，我们发现sempervirine通过降低RPA194（RNA聚合酶I的催化亚基）的蛋白质稳定性来诱导核仁重塑和核仁应激，从而导致rRNA合成抑制并转到MDM2块。如其他癌细胞模型所示，核仁应激对MDM2的抑制下调了p53-wt和p53-null中E2F1蛋白的水平TGCT细胞伴有未磷酸化的pRb的上调。最后，我们证明了sempervirine能够进入细胞核并在核仁中积累，并与rRNA结合而不会引起DNA损伤。我们的结果确定了semperivirine是一种新型的rRNA合成抑制剂，并表明该药物为非遗传毒性的抗癌小分子。