Data from the present study demonstrate that the glycolytic inhibitor, 3-BrPA selectively interacts with serum proteins. Further, it is also evident that such an interaction between 3-BrPA and serum proteins could explain the lack of toxicities when the drug is given systemically at least at the 1.75 mM dose.
Systemic administration of IA therapeutic dose of 3-BrPA shows no organ toxicity
Tissue-autoradiography of rats subjected to systemic administration of 14C-3-BrPA showed strong radioactive 14C-signal in the tissue sections of organs such as heart, liver, kidney and lung, but not the brain (Figure A). Evidently, 14C-3-BrPA did not cross the blood–brain barrier, which in turn indicates that the neuronal cells could be protected from any toxicity. Remarkably, histopathological analysis of tissue sections that were positive for 14C-signal showed normal tissue architecture indicating no signs of toxicity or pathology as evident from H&E staining (Figure B). Further, TUNEL staining of the respective tissues from 3-BrPA treated rats showed no positive-staining (Figure C), confirming the absence of any apoptosis. Since the tissues were not perfused it is likely that the 14C signal observed in tissue sections were from the serum which contained 14C-3-BrPA. This is further supported by the gel-electrophoretic autoradiogram where serum proteins showed 14C signal. Thus, the 14C-signal observed in tissue-autoradiography emanates from 3-BrPA that is non-toxic or non-reactive as it was neutralized or quenched by the interaction with serum proteins.
Figure 1 Distribution of 14C-3-BrPA during systemic delivery. (A) Autoradiogram and corresponding H & E staining of tissue sections showing radioactive signal in organs such as liver, lung, kidney and heart but not brain. (B) H & E staining showing (more ...)
Selective binding of 14C-3-BrPA with serum proteins
Autoradiogram of rat serum samples resolved on SDS-PAGE gel demonstrated that systemic administration of 14C-3-BrPA resulted in the selective incorporation of 14C in rat serum proteins (Figure A, B). Interestingly, the pellet (containing erythrocytes and other particulates) did not show any 14C incorporation even after 120 minutes of 3-BrPA administration. Based on the 14C signal, the 3-BrPA binding has been found to be with the serum peptides of molecular range ~50-60 kDa. Similarly, the autoradiogram of 14C-3-BrPA treated rat serum sample resolved on 2D-gel electrophoresis showed significant incorporation of 14C selectively in two peptide spots, with strong and weak signals (Figure A, B). The autoradiogram signal of the peptide spots from the 2D-gel also localized to the molecular range between ~50-60 kDa, as observed on the one-dimensional SDS-PAGE autoradiogram. Mass Spectrometry identification of the peptide spots corresponding to the strong and weak signals were found to be peptides of alpha-1 antitrypsin (α1-AT) and an albuminoid-family, respectively (Figure C, D).
Figure 2 Selective binding of 14C-3-BrPA with rat serum proteins. (A) Coomassie stained SDS-PAGE gel showing the protein profile of serum and pellet of 14C-3-BrPA dosed rat. (B) A corresponding autoradiogram showing time-dependent increase in autoradiogram signal (more ...)
Figure 3 14C-3-BrPA primarily binds with two peptides in rat serum. (A) A Coomassie stained 2D-gel showing serum protein spots after 120 minutes of 3-BrPA treatment and (B) a corresponding autoradiogram showing a strong and also a weak signal at ~50-60 kDa. (more ...)
In another experiment, the UV–Vis spectral analysis (180 nm to 800 nm) of mouse serum samples incubated with 3-BrPA ex vivo
, demonstrated a dose-dependent increase in the absorption maxima of certain serum components at 412, 538 and 572 nm, but not at ~200-204 nm (the absorption maxima of native 3-BrPA) (Additional file 1
: Figure S2). The spectral data refer to the total serum components that may include proteins, non-proteinaceous components, small molecules such as glutathione, cysteine, NAD/NADH etc. The objective of the spectral analysis was to demonstrate that 3-BrPA treatment-dependent changes were prominent for the serum components, which was depicted by the pronounced changes at wavelengths such as 412, 538 and 572 nm. The wavelength spectra between 200–204 nm showed the peak absorbance of aqueous 3-BrPA solution that was used as the control. In the serum samples, we did not see any peak at 200–204 nm, which implied that there was no free-3-BrPA present in the serum. Further investigations showed that free-3-BrPA in vivo
was not detectable by HPLC/mass spectrophotometer even after dose escalation (not shown). The absence of free-3-BrPA in vivo
as early as 2–3 minutes after systemic administration also provided proof for the immediate reactivity or neutralization of 3-BrPA in serum.
Taken together, the data obtained from proteomic and spectral analyses validate the interaction of 3-BrPA with serum proteins. Although the interaction of anticancer agents such as metallo-drugs with albumin has already been demonstrated [17
], binding of such agents to α1-AT has not yet been reported, especially with any anti-glycolytic agents. This report is the first to indicate a possible interaction between an anticancer (alkylating) agent (3-BrPA) and α1-AT. α1-AT has been known to be an inhibitor of neutrophil elastase, and this inhibition is required to prevent the enzymatic-degradation of elastin (in lungs). Hence, further studies are required to characterize the impact of 3-BrPA-binding on the inhibitory function of α1-AT.
Given the promising pre-clinical results on the therapeutic efficacy and mechanism(s) of action of 3-BrPA, the potential exists for translation into the clinic. As a result, it is imperative to understand the possible toxic side effect of 3-BrPA, especially if systemic administration is being contemplated. Our previous report showed that in the rabbit Vx-2 tumor model a dose that was effective given IA did not cause any significant systemic toxicity [19
]. As our findings demonstrate the interaction of 3-BrPA with serum proteins, it is likely that the particular interacting 3-BrPA molecule will no longer be available for further alkylation or toxicity. Further, owing to the irreversible alkylating property of 3-BrPA, it is unlikely that the 3-BrPA might be released from these proteins at later stages to contribute any toxicity.
Thus, this report provides an explanation for the apparent lack of systemic toxicity, which could prove extremely useful when considering the optimization of systemic therapy with 3-BrPA.