Our previous studies have established that HerGa binds target HER2+ cells in vitro
specifically through HER ligation, as confirmed through competitive inhibition by free ligand, and undergoes receptor-mediated endocytosis in response to receptor binding 12
. These same studies also showed that HerPBK10 enabled corrole toxicity at low (submicromolar) doses in contrast to the free corrole (not attached to HerPBK10), which required daily doses as high as 35 uM for 3 days to induce appreciable cytotoxicity. These previous studies suggest that the relatively low dose required for HerGa therapeutic efficacy is due to the known internalization and membrane-penetration attributes of HerPBK10 that also enable gene delivery elsewhere 23
, while the inability of the free corrole to penetrate the cell membrane prevents effective toxic impact on target cells, even if it can undergo endocytosis through attachment to or co-uptake with serum proteins 12
. In support of that hypothesis, we show here that HerGa undergoes internalization, as seen by the accumulation of intracellular corrole fluorescence over time, whereas free, untargeted corrole (S2Ga) at equivalent concentration (1 uM) showed no detectable accumulation microscopically. Correspondingly, HerGa elicited cytotoxicity whereas S2Ga did not, altogether indicating that HerPBK10 is needed for corrole internalization and mediating corrole toxicity, especially at low and pharmacologically relevant doses.
One unexpected discovery while conducting these studies has been the direct relationship between HerGa fluorescence lifetime and pH. This unique property has been useful here for reporting the intracellular environmental conditions during uptake and could possibly be exploited in future studies for diagnostic monitoring of the microenvironment in vivo. Together with two-photon imaging and intracellular depth analysis, the fluorescence lifetime changes of HerGa during cell uptake indicate that HerG accumulates into membrane-juxtaposed vesicles of slightly acidic pH, followed by transit away from the plasma membrane and deeper within the cell that corresponds to escape from endosomal vesicles. Cytosolic exposure would enable HerGa to transit along cytoskeletal pathways connecting the plasma membrane to organelles, including the mitochondria. In agreement, the taxol-mediated reduction of intracellular HerGa indicates that dynamic microtubules are required for initial transit into the cell and throughout the cytoplasm. The prevention of ΔΨ (m) disruption and O2− elevation by taxol also indicates that the cytoskeletal network is necessary for transport of HerGa to the proximity of oxygen sources, such as the mitochondria. The prevention of cytoskeletal collapse by Tiron indicates that HerGa-mediated actin and tubulin disruption lies downstream of O2− elevation. These findings altogether suggest that the cytoskeletal network is necessary to provide the initial means of HerGa transport in the cell to oxygen-generating sources (such as the mitochondria) where HerGa-mediated O2− elevation takes place, and as a result, ultimately becomes a target of oxidative damage.
Another unexpected finding was that a pH as low as 5.0 does not induce corrole release from the HerGa complex, thus suggesting that the environment encountered through receptor-mediated endocytosis and endocytic maturation does not affect complex integrity. Either the corrole stays bound to the carrier throughout cell entry or is released through a pH-independent process. Whereas a microscopic analysis of corrole release from HerGa in cells could verify these findings, the processing required for immunofluorescence assay has produced fixation artefacts showing false nuclear accumulation of corrole fluorescence (not shown), thus confounding appropriate evaluation.
Here we find that HerGa, but not its individual constituents, induce ΔΨ (m) collapse that is mediated through the elevation of superoxide. Moreover, we find through the use of endosomolytic-deficient mutants that direct exposure of HerGa to the cytosol is necessary for these events to occur. Taken altogether, these studies have identified the following stepwise pathway mediating the cytotoxic mechanism of HerGa: HerPBK10 is required to mediate uptake (Fig. S1, step a
) and early endosomal release (Fig. S1, step b
), thus enabling HerGa to avoid low pH compartments such as the late endosome and lysosome after cell entry. Cytosolic entry is necessary to facilitate downstream O2−
elevation (Fig. S1, step c
), which in turn mediates both cytoskeletal and ΔΨ (m) disruption (Fig. S1, steps d and e
). As superoxide generation would cause oxidative damage to cellular processes and structures, and ΔΨ (m) loss would disrupt cellular metabolic activity, these specific phenomena explain how HerGa mediates death to target cells.
How HerGa directly interacts with the mitochondrion to elicit these cell-death mediators remains to be elucidated. Consistent with observations in our previous studies 3, 12
, corrole fluorescence is observable throughout the cytoplasm after HerGa uptake and is not necessarily specifically targeted to mitochondria. Given the structural similarity of corroles to porphyrins, HerGa may indirectly affect mitochondrial ROS levels by activating the mitochondrial benzodiazapine receptor, for which several porphyrins are endogenous ligands 29
. On the other hand, HerGa could impact extra-mitochondrial sources of superoxide generation, including NADPH oxidase on the plasma membrane and cytoplasmic enzymes such as xanthine oxidase and nitric oxide synthase 30
, which could lead to mitochondrial damage as well as other downstream apoptotic-like events. Our cell-free studies show that the corrole itself cannot directly catalyze ROS generation, hence interactions with host factors are required.
Whereas ROS can activate the intrinsic apoptotic pathway 31
, our initial investigations on the contribution of apoptosis have yielded atypical findings. Mitochondrial permeabilization typically releases cytochrome c, which activates the caspase cascade resulting in cell death. Here, moderate but significant elevation in DNA fragmentation, an indicator of apoptotic cell death, was observed in HerGa but not control –treated cells (Fig. S2, A
). However, other ‘classical’ markers of apoptosis were lacking, including phosphatidylserine (PS) externalization (Fig. S2, A
), and elevation of activated caspase 9 and 3 (Fig. S2, B
). Moreover, cytochrome c release was not detected unless the cells were exposed to HerGa at a 10-fold higher dose over the therapeutic range (Fig. S2, C
). Alternative cell death pathways may account for these anomalies. For example, caspase-independent fragmentation of DNA in the absence of PS exposure has been observed in HER2+ T-47D tumor cells undergoing autophagy 32
. Elsewhere, selective release of Smac/DIABLO and Omi/HtrA2, but not cytochrome c, from mitochondria can result from S100A8/A9-induced death in tumor cells 33
. Mitochondrial release of Smac/DIABLO has been observed in cells undergoing anchorage-dependent cell death, or anoikis 34
, and can function independently of cytochrome c 35
. Importantly, Schafer et al 36
demonstrated that antioxidants inhibit anoikis in breast cancer cells, suggesting that elevated ROS are required in detachment-mediated cell death. These examples indicate that further studies are warranted for determining the contribution of non-classical cell death pathways to HerGa-mediated toxicity, and are currently ongoing.
Given our recent successful demonstration that targeted corroles eliminate tumor growth 3
, the investigations presented here shed light on the mechanism of corrole-mediated cell death. These studies will direct our future efforts in engineering modifications into the corrole and carrier protein that may enable even greater potency and specificity for tumor cells, thus yielding a therapeutic with optimized efficacy and safety.