The modern-day approach to cancer management is multidisciplinary, consisting of surgery, radiation therapy and chemotherapy with potential side effects. Several investigations are underway to improve the efficacy of these treatment modalities or to find new ways to treat or prevent cancer. Proteomics technology plays an important role in finding and validating biomarkers for cancer. Bioactive compounds like RSV have multiple mechanisms of action. It is vital to discover novel targets/biomarkers of chemopreventive agents like RSV that has multiple mechanisms of action so that those targets could be harnessed to develop targeted therapies. The goal of the study was to identify RSV target proteins and mechanisms implicated in its anti-neoplastic activities. The use of the proteomics technology enabled us to identify an array of proteins modulated by RSV. Although our present analysis accessed a small window in the cellular proteome, it was possible to detect RSV modulation of 1231 proteins in the HT-29 cell line. To determine if the expression of many of these proteomic markers was modified at both low and high concentrations of RSV, our confirmatory analysis included western blots and enzyme kinetic assays using RSV at concentrations 50-150 μM alone or after pre incubation with IGF-1, a mitogen elevated during obesity.
We have previously reported that at concentrations > 100 μM RSV suppressed colon cancer proliferation and up-regulated apoptosis even in the presence of IGF-1, elevated during obesity, and that has shown to enrich colon cancer stem cell populations [6, 7]. Apoptotic levels correlated with pp53 levels and suppression of proliferation transpired via attenuation of IGF-1R/Akt/Wnt signaling pathways. The present study focuses on novel targets of RSV that govern the proliferation, apoptosis and growth of a cancer cell. Using the pathway analysis software the important targets from the pool of proteins differentially altered by IGF-1 or RSV treatment were selected to devise pathways that have not been studied.
Genego Metacore software provided a basis to evaluate pathways based on differentially expressed proteins. As expected major canonical pathways affected were from the actin cytoskeleton signaling, oxidative stress response pathways, glucose metabolism, apoptosis and cell cycle etc. Among the significantly expressed proteins, G6PDH and TKT from the pentose phosphate pathway family and talin and FAK from the cytoskeleton family were found to be differentially expressed in RSV or IGF-1 treatments (Figure 1, 2 and 4). This along with the data showing that the glycolytic pathways [45, 46] and the cell-ECM interaction  to be frequently deregulated in cancer cells, fueled interest in RSV, the PPP and the talin-pFAK interaction.
Cell division is an energy-demanding process and its correct progression depends on sufficient metabolic resources to support a doubling of cell mass. Though nutrient availability is a key factor for cell proliferation, nucleotide synthesis is a rate limiting step in cancer cell replication . Ribose-5-phosphate, which is a key nucleotide component, is synthesized from glycolytic intermediates in the PPP. PPP is considered important in tumor proliferation processes because of its role in supplying tumor cells with reduced NADP and carbons for intracellular anabolic processes . In particular, the two key enzymes G6PDH and TKT have been shown to play a critical role in cancer cell cycle progression in the HT-29 cell line [28, 29].
In this study, we have demonstrated, using two different experimental approaches, that the PPP which is specifically elevated during cell cycle progression in the highly proliferating advanced human adenocarcinoma cell line HT-29 [28, 29] is further elevated by IGF-1 but suppressed by the bioactive compound RSV. Thus, a specific decrease in the activity of 2 key enzymes G6PDH and TKT, may lead to suppression of PPP that provides precursors of nucleotides for cancer cell cycle progression. Resveratrol at high concentration (150 μM) showed pronounced suppression of G6PDH and TKT in the presence of IGF-1. IGF-1 has been shown to elevate cancer cell proliferation via elevation of ROS (reactive oxygen species) . At high concentrations where resveratrol has been shown to be pro-oxidant, the pronounced effect of resveratrol on pentose phosphate pathway in the presence of IGF-1 may be due to elevated ROS levels that promotes apoptosis [20, 44]. Detailed mechanistic reason for this potentiated effect is still need to be delineated, however, based on our earlier studies we propose that this might be due to elevated suppression of pAKT, Cyclin D1, nuclear β-catenin and SP1, proteins critical for cancer cell proliferation and cell cycle progression, in the presence of IGF-1 by resveratrol .
Suppression of PPP by RSV may be via inhibition of mTORC1. Recent evidence has shown clearly that the mTORC1 controls downstream metabolic pathways like the PPP via S6K1/SREBP-1/2 . Our earlier experiments with mTOR signaling revealed that RSV activated tuberous sclerosis protein 2 (TSC-2, an mTORC1 inhibitor) and suppressed two best characterized downstream targets of mTORC1 (mammalian target of rapamycin complex 1), p70S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) .
Talin-FAK interaction has been well established and is implicated in various cancers [49, 50]. Talin plays an essential role in integrin activation and acts as a link between cell and ECM to regulate cancer cell kinetics . Talin has been shown to engage in focal adhesion interactions with Akt signaling as the intracellular survival mechanism to confer anoikis (anchorage independent apoptosis) [51, 52] resistance and promote cancer cell invasion, especially in prostate cancer . IGF-1 has been consistently linked to increased cell proliferation and cell migration, elevating cell invasion and metastatic properties of cancer cells . Our earlier work with systems biology to identify biomarkers in metastatic progression of cancer featured talin as one of the differentially expressed genes in metastatic tumors in the context of cytoskeleton remodeling pathway . Even the current proteomics data had talin as one of the differentially expressed proteins in IGF-1 and RSV treatments (Figure 4). RSV elevated talin levels at low concentration (50 μM) and suppressed talin, and concurrently elevated apoptosis at high concentration (150 μM). This may be due to the action of RSV as an antioxidant at low concentrations and pro-oxidant at high concentrations [38, 44, 54]. Anti-oxidant action at lower doses could protect DNA damage via scavenging of ROS, whereas at high concentration RSV acts as pro-oxidant leading to oxidative breakage of cellular DNA in the presence of transition metal ions such as copper causing apoptosis [44, 54]. This could possibly explain differences in talin activity at low vs high concentrations of RSV (RSV at 50 μM is not sufficient to induce apoptosis ). However, RSV was effective in suppressing IGF-1 stimulated talin expression, irrespective of concentration used. "FAK carries out protein-protein interaction adaptor functions at sites of cell attachment to the extracellular matrix (ECM), thereby contributing to focal-adhesion 'scaffolding'. FAK also transmits adhesion-dependent and growth-factor-dependent signals into the cell interior [50, 55]." The synergistic signaling between growth-factor receptors like IGF-1R and FAK might be particularly relevant as both are often up-regulated in tumor cells [36, 55]. FAK has also been shown to work similar to the IGF-1R to activate common pathways, leading to increased proliferation and cell survival. At least in pancreatic cancer, it has been shown that dual inhibition of FAK and IGF-1R led to a synergistic decrease in cell proliferation and increase in cell detachment and apoptosis compared with inhibition of either pathway alone [56, 57]. We have shown previously that RSV inhibits IGF-1R in HT-29 cells . RSV suppressed FAK activation in the presence and absence of IGF-1. These results indicate that RSV suppression of cell proliferation and elevation of apoptosis involves modulation of FAK signaling, considering that the integrin-mediated FAK signaling regulates both proliferative and apoptotic signaling pathways [58, 59].