An angiogenic proteome study of black raspberry extract on neuroendocrine cancers

• Black raspberry extract is a potential phytomedicinal agent for the treatment of rare cancers, such as neuroendocrine neoplasms. However, the mode of action for black raspberry extract is unknown. • Hypothesis- Black raspberry extract reduces tumors ability to form new blood vessels (angiogenesis) by changing the expression of key proteins. Understanding which proteins are affected will allow for the development of personalized phytomedicinal screening for patients for response potential. • This study took tumors from patients and cultured them in black raspberry extract for 14days and measured new blood vessel formation. After which the levels of 55 proteins was quantified. By comparing untreated vs black raspberry treated tumors six proteins were identified as important. CXCL16, THSP2, PDGF, PF4, VASH1, and PTX3. • These proteins were confirmed in plasma from neuroendocrine neoplasm patients. Black raspberry extract (BRE) is known to contain antioxidants that protect cells from free radical damage and restrict growth of blood vessels (angiogenesis), and also are able to prevent cancer in preclinical models. However, to date we don't fully understand the underlying biochemical mechanisms driving these observations and thus are unable to accurately advocate BRE use in the control of cancers, such as rare neuroendocrine neoplasms (NEN). The purpose of this study was to address this deficiency by quantifying the impact of BRE on ex-vivo angiogenesis in NEN tumors, and BRE induced changes in angiogenic protein expression, also the angiogenic proteins in plasma from NEN patients. NEN tumors were cultured and exposed to either 0.5 mg/ml BRE, control media, or 1 mM gallic acid (GA) (positive control). Measurement of cell growth from tumors (angiogenic sprouting) was quantified microscopically after 14 d. Angiogenic proteome changes were determined using a commercial 55 protein array by comparing differences in released (media) proteins of controls vs BRE treated tumors, and control vs GA treated tumors. Plasma from NEN patients and healthy controls (living renal donors) were quantified for angiogenesis linked proteins, identified by protein array analysis, using commercially available assays. In untreated tumors the average angiogenesis initiation rate and growth score was (78 ± 1)% and (6.24 ± 0.13) arbitrary units (a.u), respectively, both of which were reduced by BRE to (61 ± 2)% ( P < 0.01) and (3.97 ± 0.13) a.u ( P < 0.01), and by the positive control (gallic acid) to (5 ± 0.63)% and (0.66 ± 0.06) a.u, respectively. There was a very weak correlation between patient overall survival and NEN tumor ex-vivo angiogenesis sensitivity to BRE ( r = 0.3). Mechanistically, six proteins, thrombospondin-2 (THBS2), chemokine-(CXC)-ligand-16 (CXCL16), platelet derived growth factor (PDGF), platelet factor-4 (PF4), vasohibin (VASH1), and pentaxin-3 (PTX3) were identified as potentially important to the impact of BRE on NEN angiogenesis. Circulating plasma concentrations for each of the 6 identified proteins from NEN patients ranged significantly. Four of the six proteins, CXCL16 ((2.9 ± 0.3) pg/ml vs (2.2 ± 0.16) pg/ml, P = 0.03), PF4 ((7.8 ± 1.3) ng/ml vs (2.9 ± 1.2) ng/ml, P = 0.004), VASH1 ((15 ± 3) pg/ml vs (0.4 ± 0.15) pg/ml, P = 0.02), PTX3 ((914 ± 232) pg/ml vs (51 ± 19) pg/ml, P < 0.01), were statistically different in plasma from NEN patients when compared to healthy controls. In contrast, the other two proteins, THBS2 ((8.2 ± 2.2) pg/ml vs (11.1 ± 0.8) pg/ml, P = 0.11), PDGF ((1.313 ± 0.248) pg/ml vs (1.383 ± 0.107) pg/ml, P = 0.25), were not significantly different in NEN patients when compared to controls. The phytomedicine compound BRE has the potential to impact cancer growth by inhibiting the formation of tumor sustaining blood vessels. However, the potential is patient/tumor specific. This variation is likely due to differences in the expression within the angiogenesis proteome. Consequently, additional investigation is required to fully comprehend the phytomedicinal links between BRE and cancer treatment. Future studies will focus on quantifying THBS2, CXCL16, PDGF, PF4, VASH1, and PTX3 levels in NEN tumors and correlate with BRE response. With this information we would be better placed to provide personalized tumor specific phytomedicinal advice to patients.