Zinc Protoporphyrin Binding to Telomerase Complexes and Inhibition of Telomerase Activity

Abstract

\n Background: Zinc protoporphyrin (ZnPP) is a naturally occurring metalloprotoporphyrin (MPP) that is currently under development as a chemotherapeutic agent although its mechanism is unclear. Similar to natural and synthetic porphyrins, MPPs are thought to bind DNA and stabilize secondary structures such as guanine quadruplexes (G-4) and thus potentially impact telomerase activity and DNA synthesis which are important targets for chemotherapy. Interactions of MPPs with telomerase have not been previously reported. Methods: We wished to evaluate the effects of common MPPs, i.e., ZnPP, tin protoporphyrin (SnPP), and iron protoporphyrin (FePP), on cellular proliferation, apoptosis, and telomerase activity in hepatoma cells. The cytotoxicities of porphyrins were determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Native agarose gel electrophoresis was used to identify ZnPP binding of telomerase complexes. Inhibition of telomerase activity by ZnPP was assessed by conventional telomeric repeat amplification protocol (TRAP) and direct telomerase activity assays. Colocalization of ZnPP with telomerase was analyzed with immunofluorescence staining and confocal microscopic analysis. Results: ZnPP was the most effective MPP for decreasing DNA synthesis and cellular proliferation, while promoting apoptosis in cultured hepatocytes. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and in vitro assays, with IC50 and EC50 values of ca 2.5 and 6 µM respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (> 600kD) that contained telomerase and dyskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Taken together, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase enzymatic activity. Conclusions. ZnPP can actively interfere with telomerase activity in neoplastic cells, thus eliciting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for a number of neoplasms.