Gross Annika, Alborzinia Hamed, Piantavigna Stefania, Martin Lisandra L, Wölfl Stefan, Metzler-Nolte Nils
Department of Chemistry and Biochemistry, University of Bochum, Universitätsstrasse 150, D-44801 Bochum, Germany.
Metallomics. 2015 Feb;7(2):371-84. doi: 10.1039/c4mt00255e.
Compounds which are able to destabilize the lysosomal membrane have been proposed as interesting candidates for targeted anticancer drugs due to the pronounced lysosomal changes in cancer cells. For this purpose, metallocene derivatives of a cell penetrating polyarginine peptide M–(Arg)9(Phe)2Lys–NH2 (where M = ferrocene carboxylate or ruthenocene carboxylate) were designed and their biological activities were investigated in detail. The ferrocenoyl- and ruthenocenoyl polyarginine bioconjugates were synthesized via Fmoc solid-phase peptide synthesis (SPPS) protocols on a microwave-assisted synthesizer. After HPLC purification >98% purity was observed for all conjugates. Their interaction with supported biomimetic membranes was investigated on a quartz crystal microbalance (QCM) and revealed a very strong binding of the metallocene peptides and their metal-free congeners to an artificial eukaryotic membrane model (DMPC–cholesterol). To demonstrate their antiproliferative utility as cytotoxic compounds for a targeted anticancer drug, cell viability (by the crystal violet assay), apoptosis (flow cytometry, Ann V/PI staining), induction of reactive oxygen species (ROS, by flow cytometry with dihydroethidium staining), and changes in cancer cell metabolism, e.g. respiration and glycolysis, were studied. Our results reveal only a weak toxicity for the metal-free polyarginine peptide, which could be significantly enhanced (to ca. 50 μM against HeLa cells in the best case) by coupling ferrocene or ruthenocene carboxylates to the N-terminus of the peptide. The investigation of the cellular uptake and intracellular localization by fluorescence microscopy revealed an enhanced vesicular disruption by the metallocene bioconjugate compared to the metal-free derivative which could be triggered by light and chemicals. Further studies of apoptosis, respiration, glycolysis and ROS formation reveal the superior characteristics of the metallocene compounds. While most cells remain viable even at 300 μM of the metal free bioconjugate 1, most cells are dead or in late stages of apoptosis at 200 μM of the ruthenocene derivative 3, and at 100 μM of the most active ferrocene derivative 2, however, all show very little sign of necrosis. Also, the metal free compound 1 does not induce ROS formation but both metallocene–polyarginine bioconjugates are clearly associated with enhanced intracellular ROS levels, with levels for the redox-active ferrocene derivative being two times higher than for the structurally very similar but redox-silent ruthenocene derivative. We propose that such metallocene–polyarginine peptides induce lysosomal membrane permeabilization and thereby could be developed towards targeted anticancer drugs.
由于癌细胞中明显的溶酶体变化，能够破坏溶酶体膜稳定性的化合物被认为是靶向抗癌药物的有趣候选物。为此，设计了一种细胞穿透性聚精氨酸肽M–(Arg)9(Phe)2Lys–NH2（其中M = 二茂铁羧酸盐或钌茂羧酸盐）的金属茂衍生物，并详细研究了它们的生物活性。通过在微波辅助合成仪上采用Fmoc固相肽合成（SPPS）方案合成了二茂铁酰基和钌茂酰基聚精氨酸生物共轭物。经过高效液相色谱纯化后，所有共轭物的纯度均超过98%。在石英晶体微天平（QCM）上研究了它们与支持的仿生膜的相互作用，结果表明金属茂肽及其无金属同类物与人工真核膜模型（二肉豆蔻酰磷脂酰胆碱–胆固醇）有非常强的结合。为了证明它们作为靶向抗癌药物的细胞毒性化合物的抗增殖效用，研究了细胞活力（通过结晶紫测定法）、细胞凋亡（流式细胞术，Annexin V/PI染色）、活性氧（ROS，通过二氢乙锭染色的流式细胞术）的诱导以及癌细胞代谢的变化，例如呼吸作用和糖酵解。我们的结果表明，无金属的聚精氨酸肽只有微弱的毒性，通过将二茂铁或钌茂羧酸盐偶联到肽的N端，其毒性可以显著增强（在最佳情况下对HeLa细胞约为50 μM）。通过荧光显微镜对细胞摄取和细胞内定位的研究表明，与无金属衍生物相比，金属茂生物共轭物能增强囊泡破坏，这种破坏可由光和化学物质触发。对细胞凋亡、呼吸作用、糖酵解和ROS形成的进一步研究揭示了金属茂化合物的优越特性。虽然即使在300 μM的无金属生物共轭物1存在下，大多数细胞仍能存活，但在200 μM的钌茂衍生物3存在下，大多数细胞死亡或处于细胞凋亡后期，而在100 μM的最具活性的二茂铁衍生物2存在下，所有细胞几乎没有坏死迹象。此外，无金属化合物1不会诱导ROS形成，但两种金属茂–聚精氨酸生物共轭物都明显与细胞内ROS水平升高有关，氧化还原活性二茂铁衍生物的水平比结构非常相似但氧化还原沉默的钌茂衍生物高两倍。我们认为，这种金属茂–聚精氨酸肽可诱导溶酶体膜通透性增加，因此有望开发成为靶向抗癌药物。
