Target Identification of Grape Seed Extract in Colorectal Cancer Using Drug Affinity Responsive Target Stability (DARTS) Technique: Role of Endoplasmic Reticulum Stress Response Proteins

Abstract

Various natural agents, including grape seed extract (GSE), have shown considerable chemopreventive and anti-cancer efficacy against different cancers in pre-clinical studies; however, their specific protein targets are largely unknown and thus, their clinical usefulness is marred by limited scientific evidences about their direct cellular targets. Accordingly, herein, employing, for the first time, the recently developed drug affinity responsive target stability (DARTS) technique, we aimed to profile the potential protein targets of GSE in human colorectal cancer (CRC) cells. Unlike other methods, which can cause chemical alteration of the drug components to allow for detection, this approach relies on the fact that a drug bound protein may become less susceptible to proteolysis and hence the enriched proteins can be detected by Mass Spectroscopy methods. Our results, utilizing the DARTS technique followed by examination of the spectral output by LC/MS and the MASCOT data, revealed that GSE targets endoplasmic reticulum (ER) stress response proteins resulting in overall down regulation of proteins involved in translation and that GSE also causes oxidative protein modifications, specifically on methionine amino acids residues on its protein targets. Corroborating these findings, mechanistic studies revealed that GSE indeed caused ER stress and strongly inhibited PI3k-Akt–mTOR pathway for its biological effects in CRC cells. Furthermore, bioenergetics studies indicated that GSE also interferes with glycolysis and mitochondrial metabolism in CRC cells. Together, the present study identifying GSE molecular targets in CRC cells, combined with its efficacy in vast pre-clinical CRC models, further supports its usefulness for CRC prevention and treatment.

Design, Synthesis and Structure-Activity Relationship Studies of Novel Survivin Inhibitors with Potent Anti-Proliferative Properties

“…To investigate that whether the survivin down-regulation effect is caused by direct interaction between survivin protein and UC-112 analogs, we performed DARTS assay which is a well-established target identification method [28–31]. DARTS assay relies on the increasing of proteolysis resistance of the target protein generated by the interaction with small molecular ligand.…”

“…Drug affinity responsive target stability (DARTS) assay was performed to identify the protein targets of UC-112 analogs in A375 or M14 cell lysates following the protocols described in the literature [28–31]. Briefly, A375 or M14 lysates were prepared in non-denaturing M-PER lysis buffer (Thermo Fisher Scientific, Waltham, MA) with protease and phosphatase inhibitors.…”

Food-based natural products for cancer management: Is the whole greater than the sum of the parts?

“…GSE also inhibited aromatase activity and expression in a breast cancer xenograft animal model [31]. In colorectal cancer (CRC), the anticancer activity of GSE was shown to be mediated by induction of endoplasmic reticulum stress and inhibition of the PI3K/AKT/mTOR pathway [32]. Strikingly, use of GSE supplements was associated with 41% reduction in risk of developing low-grade prostate cancer in 35,239 members of the VITamins And Lifestyle (VITAL) cohort participants [33].…”

Discovery of Novel Tubulin Polymerization Inhibitors and Survivin Inhibitors as Potential Anticancer Agents

“…To investigate that whether the survivin down-regulation effect is caused by direct interaction between survivin protein and UC-112 analogs, we performed DARTS assay which is a well-established target identification method [242][243][244][245]. DARTS assay relies on the increasing of proteolysis resistance of the target protein generated by the interaction with small molecular ligand.…”

“…Drug affinity responsive target stability (DARTS) assay was performed to identify the protein targets of UC-112 analogs in A375 or MDA-MB-435 cell lysates following the protocols described in the literature [242][243][244][245]. Briefly, A375 or MDA-MB-435 lysates were prepared in non-denaturing M-PER lysis buffer (Thermo Fisher Scientific, Waltham, MA) with protease and phosphatase inhibitors.…”