The BH3 mimetic compound BH3I-1 impairs mitochondrial dynamics and promotes stress response in addition to its pro-apoptotic key function

Highlights

• BH3I-1 is a typical Bcl-2 antagonist and stimulates apoptosis.

• BH3I-1 additionally promotes mitochondrial fragmentation and HO-1 expression.

• The mitochondrial fission protein Drp-1 is linked to basal and inducible HO-1 levels.

• BH3I-1 effects on apoptosis are late events compared to its early effects on mitochondrial morphology and stress response.

Abstract

BH3 mimetics, such as BH3I-1, act as Bcl-2 antagonists, promote apoptosis and are used in basic research studies on apoptotic signaling and are currently tested as experimental anti-tumor agents. The present study addresses time- and dose-dependent responses of BH3I-1 on apoptosis, cellular stress defense mediated by heme oxygenase-1 (HO-1), and mitochondrial morphology. As expected, treatment of normal human dermal fibroblasts with BH3I-1 induced apoptosis as determined by typical markers including cytochrome c release, loss of procaspase-3, and PARP cleavage. Induction of the cellular stress response marker HO-1 precedes apoptosis induction whereas fragmentation of the mitochondrial network was triggered even more rapidly. No difference in apoptosis induction was found upon depletion of HO-1 by siRNA compared to controls suggesting that apoptosis induction by BH3I-1 is not affected by HO-1. To evaluate the functional interplay between mitochondrial fragmentation and HO-1 induction, murine embryonic fibroblasts lacking the fission factor Drp1 were used. In Drp1 knock out cells, HO-1 levels were low compared to wild type cells, both in untreated controls as well as after BH3I-1 exposure, demonstrating that Drp1 is at least in part required for determining basal and inducible HO-1 levels. Considering the sequence of events, it was shown here that BH3I-1 dependent apoptosis is a rather late event, while effects on mitochondrial morphology and cellular stress response (HO-1 induction) are observed rapidly after exposure of cells to the compound. We propose that BH3I-1 is a valuable tool for studying cellular stress responses as well as mitochondrial dynamics in future studies. Since BH3 mimetics are promising experimental anticancer drugs, our data further imply that additional biological effects such as upregulation of detoxifying systems or changes in mitochondrial dynamics could interfere, in combination therapy, with selective drug toxicity and thus need to be taken into account for drug development.

Introduction

Apoptosis plays a key role in cellular homeostasis and tumorigenesis. BH3 mimetics trigger apoptosis and are currently developed as anti-cancer drugs (Delbridge and Strasser, 2015). They mechanistically act via interfering with the equilibrium between pro- and anti-apoptotic proteins of the Bcl-2 family resulting in the initiation of apoptosis (Lessene et al., 2008; Baell and Huang, 2002). This occurs via permeabilization of the mitochondrial outer membrane (pore formation), cytochrome c release (Kuwana et al., 2002; Liu et al., 1996), and activation of initiator (e.g. caspase-9) and effector caspases (e.g. caspase-3) and endonucleases. Caspase-9 is also involved in mitochondrial remodeling during apoptosis (Cepero et al., 2005). BH3 mimetics trigger specifically the intrinsic pathway of apoptosis, providing a well-defined starting point of apoptotic signaling. BH3I-1 (Fig. 1) is an experimental anti-tumor agent (Roa et al., 2005) which acts as a Bcl-xL antagonist and induces apoptosis in a dose- and time-dependent manner. It was shown to inhibit binding of a Bak BH3 peptide to the anti-apoptotic protein Bcl-xL via targeting its BH3-binding pocket in a cell free assay and additionally disrupting the heterodimerization of Bcl-xL-CFP and Bax-YFP fusion proteins in HEK293 cells as determined by fluorescence resonance energy transfer (Degterev et al., 2001). Thus, BH3I-1 and other BH3 mimetics likely provide suitable tools for basic research and represent interesting molecules for experimental tumor therapy. Some are already used in clinical trials as anti-tumor agents (Kipps et al., 2015), however, compounds having a high specificity regarding their mode of action and at the same time having only minor side effects are required.

Along with apoptosis, the mitochondrial network shows a change in morphology and reorganizes from long filamentous tubules into small punctate spheres. In healthy cells, mitochondria form a highly dynamic network characterized by continuous cycles of fission and fusion. The shape of the mitochondrial network is determined by a balance between fission and fusion events, which are essential to maintain mitochondrial integrity and proper function including an established membrane potential (Bereiter-Hahn and Voth, 1994). The main factors involved in mitochondrial fission and fusion belong to the dynamin protein family of GTPases. Mitochondrial fusion depends on mitofusins (MFN1 and MFN2) and OPA1, while fission depends on the activity of Drp1 (Chan, 2012; Archer, 2013). Mitochondrial dysfunction was shown to result in fragmentation of the mitochondrial network in part by proteolytic inactivation of the fusion factor OPA1 (Duvezin-Caubet et al., 2006; Ishihara et al., 2006). There are opposing views whether mitochondrial fission is mandatory for mitochondria-dependent activation of apoptosis (Martinou and Youle, 2006), yet, deletion of the fission factor Drp1 was clearly shown to inhibit apoptosis in several studies. Apoptosis is part of the cellular stress response towards damage or exposure to noxious compounds (Ugarte-Uribe and Garcia-Saez, 2014). Another key component of the cellular defensive strategy against stressors comprises the activation of the Nrf2-Keap1 system which initiates the rapid expression of defense enzymes such as the NAD(P)H quinone dehydrogenase 1 (NQO1), members of the glutathione S-transferase (GST) protein family, and heme oxygenase-1 (HO-1) (Alam et al., 1999; Itoh et al., 1997; Thimmulappa et al., 2002). HO-1 levels are frequently analyzed in numerous studies as a reliable marker of cellular stress as HO-1 is well known to be strongly induced after electrophilic challenges or oxidative insult (Raninga et al., 2016; Foresti et al., 2005; Maydt et al., 2013).

The present study addresses temporal stress-response effects of the pro-apoptotic BH3-mimetic compound BH3I-1 related to changes in mitochondrial morphology, the expression of the cellular defense enzyme HO-1 and possible interconnections.