The micronutrients, vitamin E, selenium and lycopene are known to provide cells with an incredibly efficient free-radical scavenging system, therefore substantially reducing cancer-promoting DNA damage. While the literature evidence and current understanding of each supplement strongly supports this view, we report an unprecedented finding here that the combination of these three supplements together, but not individually, is also antiangiogenic by virtue of its ability to induce the expression of a megakaryocyte-specific, endogenous inhibitor of angiogenesis, PF-4. Furthermore, E/S/L has the ability to promote platelet binding to an activated endothelium and therefore concentrate this inhibitor at susceptible sites; i.e., tumor-adjacent vasculature.
The initial goal of this study was to conduct a full serum proteomic analysis to identify and characterize functional biomarkers of antioxidant supplementation likely to have an impact on the course of prostate cancer in a spontaneous PCa mouse model. We have previously reported an extended survival benefit in the Lady transgenic mouse supplemented with an E/S/L cocktail and therefore wanted to further investigate the molecular mechanisms in vivo governed by their synergistic properties that could ultimately be capable of maintaining the extrinsically mutated prostate epithelium in check [2, 13]. Individually, these antioxidants imparted no benefits compared to those observed in the combination group; i.e., reduced tumor burden and extended survival . Serum profiling was therefore restricted to compare Lady transgenic mice receiving a standard diet with those receiving E/S/L-supplementation, as the latter group alone clearly exhibited the benefits. It is important to note that while we have successfully identified PF-4 as a biomarker of dietary response by differential expression, validation and isolation, univariate data analysis of the complete proteome validated an additional 9, differentially expressed peptides (p < 0.01) between these two groups of mice (Table 1). These peptides ranged in molecular weights of 2200-7400 Da. Their identifications, however, have been hampered largely by inadequate resolving power of gel electrophoresis. It is conceivable, therefore, that the expression patterns of other proteins, in addition to PF-4, may be playing as intricate a role in controlling the progression of early-stage prostate cancer.
Having identified PF-4 as a dietary biomarker of E/S/L-supplementation implicates a very intricate biologic pathway that alters the hemostatic balance in vivo to subsequently suppress the activated endothelium in the cancerous prostate. This response likely lies initially at the level of the megakaryocyte since it is the sole source for PF-4 that is currently known in vivo [16–18]. In fact, our findings here indicate that the prostate gland stained negatively for PF-4 and that the intense staining pattern was restricted to the intravascular regions of the gland, but only in mice receiving the E/S/L cocktail. In light of a recent report suggesting that upregulation of PF-4 may play a crucial role in the early stages of several cancers , we propose here that upregulation of this peptide in our study was crucial in preventing angiogenesis at the tumor site in early prostate cancer. More importantly, this response could have only been initially mediated via a biologic modulation of the megakaryocyte located at a distant site from the tumor itself (bone marrow and spleen). Platelets, being the endpoint of megakaryocyte differentiation, now carrying an increased load of PF-4, would later bind to the activated endothelium of the cancerous prostate where it would subsequently suppress the angiogenic drive induced by the developing tumor following its angiogenic switch. The existence of such a switch has been long proposed and recently proven in tumor biology [20, 21]. Overall, our findings of elevated PF-4 and improved shuttling of the protein suggest that in vivo, such properties can preclude the angiogenic drive of most tumors, particularly following initiation mutations governing the angiogenic switch. In doing so, the tumor can be kept in check by the host during its earliest stages and prevented from further localized invasion and metastatic spread, albeit in the presence of E/S/L. This is the preclinical interpretation that we propose in light of our findings herein and the current literature pertaining to tumor angiogenesis.
We have considered an additional mechanism to our observed benefit of E/S/L-supplementation in the Lady mouse. Platelet activation and binding, which are intrinsic functions of platelets and occur irrespective of dietary supplementation, should have occurred in both groups of animals, which indeed was the case. However, mice receiving the cocktail exhibited a greater degree of platelet binding capacity (↑α2β-integrin) to the activated endothelium of the cancerous prostate. Although any mechanism suggested at this stage would be purely speculative we have considered that differences in platelet numbers alone between the two groups (reduced in tumor-bearing animals versus elevated in treated animals) may have perhaps contributed to such apparent differences. However, for E/S/L-supplementation to promote platelet production it would necessitate having to increase the number of its megakaryocyte precursors in the hematopoietic compartments of Lady mice. Analysis of spleens obtained from both groups indicated no such differences in the current study (data not shown). Furthermore, there has been no indication in our studies that changes in platelet numbers results from progressive disease or to dietary supplementation (unpublished results).