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In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line.

ABSTRACT: To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe (Cur-P) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin in situ. iTRAQ(TM) quantitative proteomics approach was applied to distinguish the specific binding targets from the non-specific ones. In total, 197 proteins were confidently identified as curcumin binding targets from HCT116 colon cancer cell line. Gene Ontology analysis showed that the targets are broadly distributed and enriched in the nucleus, mitochondria and plasma membrane, and they are involved in various biological functions including metabolic process, regulation, response to stimulus and cellular process. Ingenuity Pathway Analysis(TM) (IPA) suggested that curcumin may exert its anticancer effects over multiple critical biological pathways including the EIF2, eIF4/p70S6K, mTOR signaling and mitochondrial dysfunction pathways. Functional validations confirmed that curcumin downregulates cellular protein synthesis, and induces autophagy, lysosomal activation and increased ROS production, thus leading to cell death.

SUBMITTER: Wang J

PROVIDER: S-EPMC4768257 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line.

Wang Jigang J Zhang Jianbin J Zhang Chong-Jing CJ Wong Yin Kwan YK Lim Teck Kwang TK Hua Zi-Chun ZC Liu Bin B Tannenbaum Steven R SR Shen Han-Ming HM Lin Qingsong Q

Scientific reports 20160226

To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe (Cur-P) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin in situ. iTRAQ(TM) quantitative proteomics approach was applied to distinguish the specific binding targets from the ...[more]

PMID: 26915414

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Project description:BackgroundAccumulating evidence suggests that the androgen receptor (AR) and its endogenous ligands influence disease progression in breast cancer (BCa). However, AR-mediated changes in BCa differ among the various BCa subtypes according to their hormone receptor profile [i.e., presence/absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2, (HER2)]. Thus, we explored the androgen-regulated transcriptomic changes in the ER+PR+HER2+ BCa cell line, BT-474, and compared them with PR-mediated changes.MethodsWe performed RNA sequencing analysis in treated BT-474 cells with dihydrotestosterone (DHT) and progesterone. Validation of the top ten differentially androgen-regulated genes and a number of other genes found in enriched signaling pathways was performed by qRT-PCR in BT-474 and other BCa cell lines. In addition, a parallel reaction monitoring targeted proteomic approach was developed to verify selected transcripts at the protein level.ResultsIn total 19,450 transcripts were detected, of which 224 were differentially regulated after DHT treatment. The increased expression of two well-known androgen-regulated genes, KLK2 (p < 0.05) and KLK3 (p < 0.001), confirmed the successful androgen stimulation in BT-474 cells. The transcription factor, ZBTB16, was the most highly upregulated gene, with ~ 1000-fold change (p < 0.001). Pathway enrichment analysis revealed downregulation of the DNA replication processes (p < 0.05) and upregulation of the androgen signaling and fatty acid metabolism pathways (p < 0.05). Changes related to progesterone treatment showed opposite effects in gene expression than DHT treatment. Similar expression profiles were observed among other BCa cell lines expressing high levels of AR (ZR75.1 and MBA-MB-453). The parallel reaction monitoring targeted proteomic analysis further confirmed that altered protein expression (KLK3, ALOX15B) in the supernatant and cell lysate of DHT-treated BT-474 cells, compared to control cells.DiscussionOur findings suggest that AR modulates the metabolism of BT-474 cells by affecting the expression of a large number of genes and proteins. Based on further pathway analysis, we suggest that androgen receptor acts as a tumor suppressor in the BT-474 cells.

Dataset Information

In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line.

Publications

In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line.

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