Delivery of Chemically Glycosylated Cytochrome c Immobilized in Mesoporous Silica Nanoparticles Induces Apoptosis in HeLa Cancer Cells

Abstract

Cytochrome c (Cyt c) is a small mitochondrial heme protein involved in the intrinsic apoptotic pathway. Once Cyt c is released into the cytosol, the caspase mediated apoptosis cascade is activated resulting in programmed cell death. Herein, we explore the covalent immobilization of Cyt c into mesoporous silica nanoparticles (MSN) to generate a smart delivery system for intracellular drug delivery to cancer cells aiming at affording subsequent cell death. Cyt c was modified with sulfosuccinimidyl-6-[3′-(2-pyridyldithio)-propionamido] hexanoate (SPDP) and incorporated into SH-functionalized MSN by thiol-disulfide interchange. Unfortunately, delivery of Cyt c from the MSN was not efficient in inducing apoptosis in human cervical cancer HeLa cells. We tested whether chemical Cyt c glycosylation could be useful in overcoming the efficacy problems by potentially improving Cyt c thermodynamic stability and reducing proteolytic degradation. Cyt c lysine residues were modified with lactose at a lactose-to-protein molar ratio of 3.7±0.9 using mono-(lactosylamido)-mono-(succinimidyl) suberate linker chemistry. Circular dichroism (CD) spectra demonstrated that part of the activity loss of Cyt c was due to conformational changes upon its modification with the SPDP linker. These conformational changes were prevented in the glycoconjugate. In agreement with the unfolding of Cyt c by the linker, a proteolytic assay demonstrated that the Cyt c-SPDP conjugate was more susceptible to proteolysis than Cyt c. Attachment of the four lactose molecules reversed this increased susceptibility and protected Cyt c from proteolytic degradation. Furthermore, a cell-free caspase-3 assay revealed 47% and 87% of relative caspase activation by Cyt c-SPDP and the Cyt c-lactose bioconjugate, respectively, when compared to Cyt c. This again demonstrates the efficiency of the glycosylation to improve maintaining Cyt c structure and thus function. To test for cytotoxicity, HeLa cells were incubated with Cyt c loaded MSN at different Cyt c concentrations (12.5, 25.0, and 37.5 μg/mL) for 24 to 72 h and cellular metabolic activity determined by a cell proliferation assay. While MSN-SPDP-Cyt c did not induced cell death, the Cyt c-lactose bioconjugate induced significant cell death after 72 h, reducing HeLa cell viability to 67% and 45% at the 25 μg/mL and 37.5 μg/mL concentrations, respectively. Confocal microscopy confirmed that the MSN immobilized Cyt c-lactose bioconjugate was internalized by HeLa cells and that the bioconjugate was capable of endosomal escape. The results clearly demonstrate that chemical glycosylation stabilized Cyt c upon formulation of a smart drug delivery system and upon delivery into cancer cells and highlight the general potential of chemical protein glycosylation to improve the stability of protein drugs.

Chemical glycosylation of cytochrome c improves physical and chemical protein stability

“…The Cotton effect, named after its discoverer Aimé Cotton (1869–1951), is the characteristic change in circular dichroism in the vicinity of an absorption band of a substance characterized by a positive and negative CD signal and a zero crossing at the absorption maximum. Structural changes in the heme-binding pocket lead to the disappearance of the cotton effect as reported by others and us for Cyt c [46,61]. This is in agreement with the activity data in Table 1, which show that all bioconjugates had a similar pseudo enzyme activity than Cyt c. We can surmise that for all bioconjugates the structure was native-like with the exception of a somewhat more lose tertiary structure packing in case of the dextran conjugates.…”

Curcumin Induces Apoptosis in SGC-7901 Gastric Adenocarcinoma Cells via Regulation of Mitochondrial Signaling Pathways

“…Two pathways are identified to be involved in apoptosis induction including death receptor-mediated extrinsic and mitochondria-mediated intrinsic pathways (Tomek et al, 2012). The intrinsic apoptotic pathway is characterized by permeability of the mitochondria and release of cytochrome c from mitochondria into the cytoplasm; and the extrinsic apoptotic pathway is activated by DOI:http://dx.doi.org/10.7314/APJCP.2014.15.9.3987 Curcumin Induces Apoptosis in SGC-7901 Gastric Adenocarcinoma Cells via Mitochondrial Signaling death receptors on the plasma membrane such as tumor necrosis factor receptor 1 (TNFR1) and Fas/CD95 (Liu et al, 2013;Méndez et al, 2014;Sharoar et al, 2014;Tyagi et al, 2014). Both pathways ultimately lead to the activation of the executioner Caspases-3 via diverse proapoptotic signals and finally cell death (Ma et al, 2014).…”

Purification and characterization of a cytochrome c with novel caspase-3 activation activity from the pathogenic fungus Rhizopus arrhizus

“…If this turns out to be the case, designing of fungi specific apoptosis inducing drugs may be possible. Although results of targeting human cancer cells by actively inducing apoptosis using mammalian cyt c delivery have been promising [28, 29], replicating them in the fungal cells could be very challenging due to the presence of their cell wall. Our results show the ability of R. arrhizus cyt c to activate caspase-3.…”

“…HeLa cells were grown to 90 % confluency, harvested, and disrupted as described [29]. Prior to disruption, approximately a total of 2 × 10 7 cells were suspended in 2 mL of lysis buffer consisting of 20 mM HEPES at pH 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM Na-EDTA, 1 mM Na-EGTA, 1 mM DTT, 250 mM sucrose, and 0.1 %  v / v protease inhibitor cocktail (2 mM AEBSF, 0.3 μM aprotinin, 130 μM bestatin, 14 mM E-64, 1 mM leupeptin, 1 mM EDTA).…”