Lysosomal-Mitochondrial Axis in Zoledronic Acid-induced Apoptosis in Human Follicular Lymphoma Cells

Laura M Mitrofan,Ferran B Castells,Jukka Pelkonen,Jukka Mönkkönen

doi:10.1074/jbc.m109.038935

Abstract

Bisphosphonates (BPs) are potent inhibitors of osteoclast function, widely used to treat excessive bone resorption associated with bone metastases, that also have anti-tumor activity. Zoledronic acid (ZOL) represents a potential chemotherapeutic agent for the treatment of cancer. ZOL is the most potent nitrogen-containing BPs, and it inhibits cell growth and induces apoptosis in a variety of cancer cells. Recently we demonstrated that accumulation of isopentenyl pyrophosphate and the consequent formation of a new type of ATP analog (ApppI) after mevalonate pathway inhibition by nitrogen-containing BPs strongly correlates with ZOL-induced cell death in cancer cells in vitro. In this study we show that ZOL-induced apoptosis in HF28RA human follicular lymphoma cells occurs exclusively via the mitochondrial pathway, involves lysosomes, and is dependent on mevalonate pathway inhibition. To define the exact signaling pathway connecting them, we used modified HF28RA cell lines overexpressing either BclXL or dominant-negative caspase-9. In both mutant cells, mitochondrial and lysosomal membrane permeabilization (MMP and LMP) were totally prevented, indicating signaling between lysosomes and mitochondria and, additionally, an amplification loop for MMP and/or LMP regulated by caspase-9 in association with farnesyl pyrophosphate synthetase inhibition. Additionally, the lysosomal pathway in ZOL-induced apoptosis plays an additional/amplification role of the intrinsic pathway independently of caspase-3 activation. Moreover, we show a potential regulation by Bcl-XL and caspase-9 on cell cycle regulators of S-phase. Our findings provide a molecular basis for new strategies concomitantly targeting cell death pathways from multiple sites.

Highlights

Indicates that bisphosphonates, especially zoledronic acid (ZOL),3 have anti-tumor activity in vitro and in vivo [5]
The optimal concentration of ZOL to study apoptosis in this cell line was found to be 50 ␮M, and this concentration was used in all subsequent experiments
Typical apoptotic morphology, including chromatin condensation, nuclear fragmentation, and cell shrinkage was observed only in the HF28RA GFP cell line using Hoechst staining, whereas again, overexpression of Bcl-XL and DN caspase-9 abolished these apoptotic features (Fig. 4B). These results suggest that the mitochondrialdependent pathway represents the major step required for complete activation of caspase-3 and apoptosis induced by ZOL in lymphoma cells

Summary

Introduction

Indicates that bisphosphonates, especially zoledronic acid (ZOL), have anti-tumor activity in vitro and in vivo [5]. Nitrogen-containing bisphosphonates, including ZOL, act by inhibiting farnesyl pyrophosphate (FPP) synthase, one of the key enzymes of the intracellular mevalonate pathway [9] This leads to a block in the production of the isoprenoid lipids, FPP, and geranylgeranyl pyrophosphate (Fig. 1) followed by loss of prenylated small signaling proteins (Rho, Ras) [10] and, apoptosis [11, 12]. This is the widely accepted molecular mechanism for nitrogen-containing bisphosphonates to inhibit osteoclast activity and bone resorption [13]. Despite the emerging data on ZOL-induced programmed cell death, its molecular mediators still remain under debate

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