COL-3-Induced Molecular and Ultrastructural Alterations in K562 Cells.

Mona Fares,Kjell Hultenby,Sandra Oerther,Danica Gubrianska,Manuchehr Abedi-Valugerdi,Moustapha Hassan,Ying Zhao

doi:10.3390/jpm12010042

Abstract

Tetracycline-3 (4-dedimethylamino sancycline, COL-3) is a non-antibiotic tetracycline derivative. COL-3 exerts potent anti-metalloproteinase activity and its antitumor effects have been reported both in vitro and in vivo. In this study, we investigated the mechanisms of COL-3-induced cytotoxicity in a chronic myeloid leukemia cell line, K562, characterized by the BCR–ABL fusion protein. COL-3 induced K562 cell death in a concentration-dependent manner with an IC50 of 10.8 µg/mL and exhibited features of both apoptosis and necrosis. However, flow cytometry analysis revealed that necrotic cells dominated over the early and late apoptotic cells upon treatment with COL-3. Transmission electron microscopy analysis in combination with Western blotting (WB) analysis revealed early mitochondrial swelling accompanied by the early release of cytochrome c and truncated apoptosis inducing factor (tAIF). In addition, ultrastructural changes were detected in the endoplasmic reticulum (ER). COL-3 affected the levels of glucose-regulated protein-94 (GRP94) and resulted in m-calpain activation. DNA double strand breaks as a signature for DNA damage was also confirmed using an antibody against γH2AX. WB analyses did not demonstrate caspase activation, while Bcl-xL protein remained unaffected. In conclusion, COL-3-induced cell death involves DNA damage as well as mitochondrial and ER perturbation with features of paraptosis and programmed necrosis.

Highlights

A balance between cell proliferation and cell death is mandatory for normal development and tissue homeostasis and disturbing this balance may contribute to the development of malignancy [1]
Our findings concerning COL-3-induced cell-death can be added to the reported mechanism of action and pharmacological activities of COL-3 as a potent inhibitor of several matrix metalloproteinases (MMPs), including MMP-1, MMP-2, MMP-8, MMP-9 and MMP-13 [34]
Based on this latter ability of COL-3, clinical trials have been performed to evaluate the treatment efficacy of the compound in patients with HIV-related Kaposi’s sarcoma, where MMPs play a pivotal role in the pathogenesis of the disease [35,36]

Summary

Introduction

A balance between cell proliferation and cell death is mandatory for normal development and tissue homeostasis and disturbing this balance may contribute to the development of malignancy [1]. Cell death may be divided into apoptosis and necrosis based on the morphological and biochemical features of dying cells [2]. Apoptosis is a highly conserved programmed cell death controlled by caspases, a family of cysteine proteases [3]. Depending on the initiating factor(s), apoptosis is mediated by extrinsic or intrinsic pathways [2]. While the extrinsic pathways are initiated by extracellular stressors through trans-membrane receptors, the intrinsic pathways are associated with intracellular stressors that cause DNA damage, oxidative stress or perturbation of intracellular organelles [2,4]. Necrosis is considered to be an uncontrolled cell death characterized by a loss of membrane integrity and dilatation of cellular organelles or as a programmed event that is mediated by receptor-interacting protein kinases (RIP)-1 and RIP-3 [5]

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Conclusion