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

Wang, Jigang; Zhang, Jianbin; Zhang, Chong‐Jing; Wong, Yin Kwan; Lim, Teck Kwang; Zhang, Hua; Liu, Bin; Tannenbaum, Steven R.; Shen, Han‐Ming; Lin, Qingsong

doi:10.1038/srep22146

Cited by 84 publications

(61 citation statements)

References 39 publications

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“…The Curcumin-probe (Curcumin-P) was readily synthesized by mono-alkylation of curcumin by propargyl bromide and its structure was verified by H-NMR, C-NMR and high resolution mass spectrometry [40]. …”

Section: Methodsmentioning

confidence: 99%

Curcumin targets the TFEB-lysosome pathway for induction of autophagy

Wang

et al. 2016

Oncotarget

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113

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Curcumin is a hydrophobic polyphenol derived from the herb Curcumalonga and its wide spectrum of pharmacological activities has been widely studied. It has been reported that Curcumin can induce autophagy through inhibition of the Akt-mTOR pathway. However, the effect of Curcumin on lysosome remains largely elusive. In this study, we first found that Curcumin treatment enhances autophagic flux in both human colon cancer HCT116 cells and mouse embryonic fibroblasts (MEFs). Moreover, Curcumin treatment promotes lysosomal function, evidenced by the increased lysosomal acidification and enzyme activity. Second, Curcumin is capable of suppressing the mammalian target of rapamycin (mTOR). Interestingly, Curcumin fails to inhibit mTOR and to activate lysosomal function in Tsc2−/−MEFs with constitutive activation of mTOR, indicating that Curcumin-mediated lysosomal activation is achieved via suppression of mTOR. Third, Curcumin treatment activates transcription factor EB (TFEB), a key nuclear transcription factor in control of autophagy and lysosome biogenesis and function, based on the following observations: (i) Curcumin directly binds to TFEB, (ii) Curcumin promotes TFEB nuclear translocation; and (iii) Curcumin increases transcriptional activity of TFEB. Finally, inhibition of autophagy and lysosome leads to more cell death in Curcumin-treated HCT116 cells, suggesting that autophagy and lysosomal activation serves as a cell survival mechanism to protect against Curcumin-mediated cell death. Taken together, data from our study provide a novel insight into the regulatory mechanisms of Curcumin on autophagy and lysosome, which may facilitate the development of Curcumin as a potential cancer therapeutic agent.

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Section: Methodsmentioning

confidence: 99%

Curcumin targets the TFEB-lysosome pathway for induction of autophagy

Wang

et al. 2016

Oncotarget

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113

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“…Curcumin is a versatile natural molecule that is possessing multitude of confirmed therapeutic activities but it is very safe and can be tolerated in high doses up to 10g/day [19, 20]. Regardless intensive research that confirmed the medicinal benefits of curcumin, the yellow condiment continues to intrigue more researchers to explore its activities on recently validated therapeutic targets [21–23]. Curcumin ameliorates response of cancer cells towards several cytotoxic drugs.…”

Section: Introductionmentioning

confidence: 99%

Molecular Mimics of Classic P-Glycoprotein Inhibitors as Multidrug Resistance Suppressors and Their Synergistic Effect on Paclitaxel

et al. 2017

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P-glycoprotein (Pgp) is a membrane bound efflux pump spread in a variety of tumor cells and considered as a main component of multidrug resistance (MDR) to chemotherapies. In this work, three groups of compounds (imidazolone, oxazolone and vinyl dipeptide derivatives) were synthesized aiming to develop a molecular framework that effectively suppresses MDR. When tested for their influence on Pgp activity, four compounds coded Cur1-01, Cur1-12V, Curox-1 and Curox-3 significantly decreased remaining ATP concentration indicating Pgp substrate site blocking. On the other hand, Cur-3 and Cur-10 significantly increased remaining ATP concentration, which is indicative of Pgp ATPase inhibition. The cytotoxicity of synthesized compounds was examined against Pgp expressing/highly resistant colorectal cancer cell lines (LS-174T). Compounds Cur-1 and Cur-3 showed considerable cytotoxicity with IC50 values of 7.6 and 8.9 μM, respectively. Equitoxic combination (at IC50 concentrations) of PTX and Cur-3 greatly diminished resistant cell clone from 45.7% to 2.5%, albeit with some drop in potency from IC50 of 7.9 nM to IC50 of 23.8 nM. On the other hand, combination of PTX and the non-cytotoxic Cur1-12V (10 μM) significantly decreased the IC50 of PTX to 3.8 nM as well as the resistant fraction to 16.2%. The combination test was confirmed using the same protocol but on another resistant CRC cell line (HCT-116) as we obtained similar results. Both Cur-3 and Cur1-12V (10 μM) significantly increased the cellular entrapment of Pgp probe (doxorubicin) elevating its intracellular concentration from 1.9 pmole/cell to 3.0 and 2.9 pmole/cell, respectively.

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“…To determine the cutoff threshold for considering the fold changes of proteins identified from the iTRAQ study as significantly differentiated, 2 equal amounts of 6-protein mixtures (Applied Biosystems, 4352135) were trypsin-digested and labeled with the iTRAQ reagents. 29,[50][51][52] The standard deviation of all the ratios of the labeled peptides was computed as 0.15. Thus, with the use of a 1 § 2 standard deviation formula, the ratio-change cutoff thresholds were set as 1.3 for upregulated proteins and reciprocally 0.77 for downregulated proteins.…”

Section: Resultsmentioning

confidence: 99%

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

et al. 2016

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Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ TM ). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/ MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvationmediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.

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

Cited by 84 publications

References 39 publications

Curcumin targets the TFEB-lysosome pathway for induction of autophagy

Curcumin targets the TFEB-lysosome pathway for induction of autophagy

Molecular Mimics of Classic P-Glycoprotein Inhibitors as Multidrug Resistance Suppressors and Their Synergistic Effect on Paclitaxel

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

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