2.1. Breast
Increasing rates of obesity and poor nutrition are major contributors to breast cancer occurrence in women [14]. Several studies have shown that blueberry extract and pterostilbene inhibit breast cancer in vitro and in vivo. Pterostilbene treatment of breast cancer cells has additionally been shown to alter cellular oxidative activity that may play an important role in pterostilbene-mediated cell death.
Wu and colleagues conducted experiments to examine the effects of blueberry consumption on outcomes of early development of mammary epithelial cells in pregnant and lactating rats [15]. The authors hypothesized that blueberry consumption in pregnant rats would lead to transcriptional modification and mammary gland differentiation during the developmental period. Administering blueberry powder to pregnant and lactating rats and assessing the postnatal effects of blueberry exposure on mammary architecture and differentiation were conducted as experiments. The results of the study demonstrated that rats fed the blueberry diet exhibited higher mammary branching, increased nuclear immunoreactivity of tumor suppressor phosphatase and tensin homolog deleted in chromosome 10 (PTEN), and decreased mitotic rates. Additional experiments were performed to evaluate the effect of blueberry exposure upon in vitro expression of PTEN and antioxidant activity in nontumorigenic breast cells, and results revealed that cells treated with blueberry serum showed higher PTEN but similar antioxidant capacity to control cells. The findings show that breast cells are early targets of blueberry-derived mediators, which possess the ability to alter developmental mammogenesis.
Several studies have found that blueberry exposure inhibited breast cancer in vitro and in vivo [16–18]. The results of experiments conducted by Boivin and colleagues found that blueberry juice from high-bush, low-bush, and velvetleaf blueberries exerted an antiproliferative effect against the breast cancer cell line MDA-MB-231 [17]. Likewise, Adams and colleagues found that high-bush blueberry extract decreased proliferation of triple-receptor negative breast cancer cell lines, HCC38, HCC1937, and MDA-MB-231 but did not affect proliferation of the nontumorigenic cell line, MCF-10A [18]. In the same study, treatment with blueberry extract significantly decreased human-growth-factor (HGF-) induced activation of the PI3 K/AkT/NK-κB pathway, which is implicated in breast carcinogenesis. Blueberry treatment also inhibited the metastatic potential of breast cancer cells in vitro by inhibiting HGF-induced cell migration and matrix metalloproteinase-(MMP-) 2 and MMP-9 activity.
In experiments utilizing a breast cancer xenograft model, treatment with blueberry extract produced smaller tumors with decreased expression of Ki-67, a marker of cell proliferation, and increased expression of caspase-3, an apoptosis marker. Blueberry fed mice also exhibited decreased activation of AkT and NK-κB signaling when compared to controls [18]. The cumulative findings suggest that blueberries contain a specific chemical component capable of modifying carcinogenic pathways in breast cancer cells.
Similarly, pterostilbene has been shown to exert anticancer effects in breast cancer through alteration of multiple cancer pathways both in vitro and in vivo. The carcinogenic pathways inhibited by pterostilbene treatment are similar to pathways altered by blueberry juice making it plausible that pterostilbene is the compound responsible for the anticarcinogenic effects of blueberry treatment in breast cancer. Moreover, pterostilbene’s anticancer effects have been associated with its antioxidant-inducing capacity. Alosi and colleagues found that pterostilbene induced apoptosis and inhibited proliferation of MDA-MB-231 and MCF-7 breast cancer cells in a concentration and time-dependent manner [19]. In the same study, the antioxidant activity of pterostilbene was measured using hydroethidine (HE) to detect production. The authors hypothesized that pterostilbene when applied to cancer cells would increase production which would facilitate mitochondrial membrane depolarization triggering intrinsic mitochondriallyderived apoptosis. The results of the study show a concentration-dependent increase in production, increased mitochondrial depolarization, and associated caspase release indicating apoptosis. Mannal et al. reported that treatment with pterostilbene increased caspase release and production in breast cancer cells, consistent with the findings of Alosi [20].
Studies performed by Chakraborty and colleagues found that pterostilbene reduced cell proliferation and induced apoptosis through the induction of caspase-3, Bax, and p53 in breast cancer cells [21]. It was also demonstrated by Chakraborty et al. that pterostilbene treatment increased GPx antioxidant activity and the production of H2O2 and singlet oxygen indicating a mechanism of ROS-induced apoptosis [21, 22]. To evaluate the hypothesis of ROS-induced apoptosis, pterostilbene-treated breast cancer cells were treated with catalase, H2O2 scavenger, and cell survival ratios were compared to pterostilbene-treated controls. The results of the study show that catalase treatment inhibited pterostilbene-induced cell death in MCF-7 cells confirming that H2O2 is involved in pterostilbene cytotoxicity [22]. The findings of Alosi, Mannal, and Chakraborty imply that pterostilbene alters cellular oxidation to facilitate mechanisms of apoptosis in breast cancer.
The relationship between oxidation and apoptosis was further examined by Moon and colleagues who evaluated the effects of pterostilbene upon expression of Bcl-2-associated X protein (BAX), Cytochrome C, second mitochondria-derived activator of caspases (Smac/Diablo), and manganese superoxide dismutase (MnSOD), which are markers of intrinsic apoptosis [23]. The results of the study found that pterostilbene treatment produced dose-dependent increases in BAX, cytochrome C, and Smac/Diablo expression implying that pterostilbene induces intrinsic mitochondrially derived apoptosis. Pterostilbene was also shown to significantly increase MnSOD antioxidant activity in breast cancer cells, an antiproliferative mechanism that has been studied extensively [24–26]. The interconnected mechanism of increased MnSOD expression and breast cancer inhibition is not fully understood; however, it is likely that pterostilbene’s ability to increase antioxidant activity is related to its function as an apoptotic and antiproliferative agent.
Furthermore, pterostilbene treatment produced a synergistic inhibitory effect when combined with the chemotherapy drug Tamoxifen, demonstrating clinical potential in the treatment of breast cancer [20]. Further research is necessary to investigate the clinical potential of pterostilbene’s antioxidant activity in human cases of breast cancer.
2.2. Cardiovascular
Cardiovascular disease is currently the number one cause of mortality in the Unites States, and its high prevalence is attributed to multiple risk factors such as smoking, sedentary lifestyle and low intake of fruits and vegetables [27]. On a cellular level, vascular pathology results from dysfunction of the endothelium due to repeated exposure to mechanical stress and leukocyte generated ROS [28]. Increased mechanical stress of the endothelium predisposes to vascular injury and thrombogenicity, which is then exacerbated by increased levels of OS. The endogenous antioxidant activity of the vascular system is purported to exert a basal protective effect against pathogenesis by reducing OS; however, the antioxidant capacity of the endothelium may become exhausted from increased exposure to ROS creating an imbalance between OS and antioxidant activity [28]. Routine consumption of dietary antioxidants may therefore be protective against ROS-induced vascular injury in high-risk individuals [29].
Several studies have shown that blueberries, and pterostilbene alike, exhibit protective effects against cardiovascular disease possibly due to induction of antioxidant enzymes. In a study performed by Ahmet and colleagues, rats that were fed a three-month blueberry enriched diet had 22% smaller average myocardial infarction (MI) size measured 24 hours after ligation of the left descending coronary artery compared to rats fed a control diet [30]. The authors also determined that rats fed a blueberry diet had higher ejection fractions two weeks after MI compared to controls. A possible explanation for the differences in cardiac outcomes may be due to the antioxidant effects of blueberry-derived compounds leading to cardioprotection of ischemic cardiomyocytes. The theory is supported by findings that show that cardiomyocytes from blueberry fed rats exhibit a 24% increase in the ROS-induced threshold for mitochondrial permeability transition, thereby decreasing the likelihood of apoptosis in ischemic cells [30].
Further studies show that blueberry supplementation attenuated atherosclerosis by upregulating expression of the antioxidant enzymes SOD1, SOD2, GSR, and thioredoxin reductase- (TR-) 1 in ApoE deficient mouse models of atherosclerosis [31]. Blueberry supplementation was also shown to reduce H2O2-induced intracellular ROS production in human microvascular endothelial cells (HMVECs) [32].
The antioxidant activity of pterostilbene may play an important role in the cardioprotective effects observed in blueberry supplementation studies. Pterostilbene has demonstrated numerous protective benefits against atherosclerosis through regulation of vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs). In experimental studies, VSMCs treated with pterostilbene exhibited reduction in platelet-derived-growth-factor-(PDGF-) induced proliferation and Akt, a serine-threonine kinase [33]. Pterostilbene also produced downregulation of the cell-cycle mediators, cyclin-dependent kinase (CDK)-2, CDK-4, cyclin E, cyclin D1, retinoblastoma (Rb), and proliferative cell nuclear antigen (PCNA), all of which promote unchecked VSMC proliferation resulting in atherosclerosis [33]. The results may be clinically relevant because abnormal proliferation of VSMCs is a significant component of the pathogenesis of atherosclerosis and a major contributor to the development of vascular stenosis. The antiproliferative effects of pterostilbene in VSMCs may therefore confer a defense mechanism against atherosclerosis and subsequent complications of stenosis.
In separate studies conducted by Zhang et al., pterostilbene was shown to inhibit apoptosis and induce autophagy in VECs counteracting the proatherosclerosis effect of oxidized low-density lipoprotein (oxLDL) [34, 35]. The cytoprotective mechanism of autophagy involves removal of abnormal proteins that results from oxLDL accumulation [36]. Zhang and colleagues have demonstrated that pterostilbene treatment induces autophagy in oxLDL-stimulated VECs through activation of AMP-activated protein kinase (AMPK), intracellular calcium (Ca2+), and mammalian target of rapamycin (mTOR) signaling [34]. Apoptosis plays a central role in the pathogenesis of atherosclerosis through induction of plaque instability that occurs when oxLDL induces apoptosis in VECs through activation of the lectin-like oxLDL receptor −1 (LOX-1).
Activation of LOX-1 then triggers a cascade of pro-apoptosis events such as induction of p53, cytochrome C, and caspase activation. It has been postulated that increased exposure to OS facilitates apoptosis in VECs, which propagates the atherosclerosis process [37]. Treatment with pterostilbene was shown to inhibit oxLDL-induced apoptosis, suppress oxLDL-induced OS, and reduce expression of the pro-apoptosis proteins Bax and p53. Pterostilbene treatment also inhibited NF-κB activation, an instrumental mediator of OS in VECs [34]. Furthermore, pterostilbene treatment suppressed oxLDL-induced expression of MMP, caspase-3/9, and attenuation of LOX-1 signaling [34]. Previous studies have shown that OS leads to LOX-1 activation that further stimulates production of OS creating a positive feedback loop that is pathogenic to vascular cells [38].
Pterostilbene’s modulation of LOX-1, NF-κB, and the antioxidant enzymes SOD and catalase indicate strong antioxidant and antiatherosclerosis effects that may be clinically significant. Although, the application of dietary pterostilbene in the prevention of cardiovascular disease is currently undetermined, a study performed by McAnulty and colleagues provided evidence that consumption of 250 g blueberry diets for three weeks attenuates angiotensin converting enzyme (ACE) activity and reduce lipid hydroperoxidase (LH), a marker of OS in chronic smokers [39]. The risk of vascular disease is high in chronic smokers, and reduction of OS by blueberry consumption may decrease the risk and/or severity of atherosclerotic disease.
Future directions may include large-scale clinical trials to determine the impact of blueberry-derived pterostilbene in the prevention or therapy of atherosclerosis in smokers and other high-risk groups.