Abstract
Carnosic acid (CA) and carnosol (CS) are two structurally related diterpenes present in rosemary herb (Rosmarinus officinalis). Although several studies have demonstrated that both diterpenes can scavenge free radicals and interfere in cellular processes such as cell proliferation, they may not necessarily exert the same effects at the molecular level. In this work, a shotgun proteomics study based on stable isotope dimethyl labeling (DML) and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) has been performed to identify the relative changes in proteins and to gain some light on the specific molecular targets and mechanisms of action of CA and CS in HT-29 colon cancer cells. Protein profiles revealed that CA and CS induce different Nrf2-mediated response. Furthermore, examination of our data revealed that each diterpene affects protein homeostasis by different mechanisms. CA treatment induces the expression of proteins involved in the unfolded protein response in a concentration dependent manner reflecting ER stress, whereas CS directly inhibits chymotrypsin-like activity of the 20S proteasome. In conclusion, the unbiased proteomics-wide method applied in the present study has demonstrated to be a powerful tool to reveal differences on the mechanisms of action of two related bioactive compounds in the same biological model.
Highlights
Carnosic acid (CA)1 and carnosol (CS) are two structurally related orthodiphenolic compounds with abietane carbon skeleton containing hydroxyl groups at positions C-11 and C-12 that belong to the abietanes family of naturally occurring diterpenes in the popular Lamiaceae herbs, rosemary (Rosmarinus officinalis), and sage (Salvia officinalis) [1]
The recent application of comprehensive proteomics based on nano-liquid chromatography-tandem mass spectrometry combined with stable isotope dimethyl labeling (DML) has generated new insights regarding the role of autophagy and proteostasis in the cellular response to rosemary polyphenols, demonstrating the suitability of this proteomics strategy for the investigation of the mechanisms of action of dietary compounds in cancer cells [30]
A recent study in our laboratory suggested that the effects of a CA-enriched rosemary extract on cell cycle distribution are highly dependent on the extract concentration [11]
Summary
Carnosic acid (CA) and carnosol (CS) are two structurally related orthodiphenolic compounds with abietane carbon skeleton containing hydroxyl groups at positions C-11 and C-12 that belong to the abietanes family of naturally occurring diterpenes in the popular Lamiaceae herbs, rosemary (Rosmarinus officinalis), and sage (Salvia officinalis) [1]. It has been recognized that CA and CS quinones react with a critical thiol in Keap, causing it to release Nrf transcription factor that may enter the nucleus for subsequent activation of ARE (antioxidant-response element)-mediated transcription of an array of proteins that protect against oxidative stress [3, 4] This effect has been considered as the predominant cause for the observed protective activity of both diterpenes in studies on their role in central nervous system [5]. Foodomics has demonstrated to be a useful strategy to cover the identification of a wide range of molecular changes induced by rosemary compounds in in vitro cell models In this line of work, comprehensive transcriptomic and metabolomic analyses helped on identifying global changes induced by rosemary polyphenols on colon cancer and leukemia cells [27,28,29]. The objectives of this study were to: (i) identify changes in relative abundance of proteins altered by CA and CS exposures over the time; and (ii) detect differences between the protein profiles obtained in CA- and CS-treated cells in order to shed light on the specific molecular targets and mechanisms of action of each diterpene in colon cancer cells
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