Cysteine-Conjugated Metabolites of Ginger Components, Shogaols, Induce Apoptosis through Oxidative Stress-Mediated p53 Pathway in Human Colon Cancer Cells

Shogaols, the major constituents of thermally processed ginger, have been proven to be highly effective anticancer agents. Our group has identified cysteine-conjugated shogaols (M2, M2′, and M2″) as the major metabolites of [6]-, [8]-, and [10]-shogaol in human and found that M2 is a carrier of its parent molecule [6]-shogaol in cancer cells and in mice, while being less toxic to normal colon fibroblast cells. The objectives of this study are to determine whether M2′ and M2″ behave in a similar manner to M2, in both metabolism and efficacy as anticancer agents, and to further explore the biological pro-apoptotic mechanisms of the cysteine-conjugated shogaols against human colon cancer cells HCT-116 and HT-29. Our results show that [8]- and [10]-shogaol have similar metabolic profiles to [6]-shogaol and exhibit similar toxicity toward human colon cancer cells. M2′ and M2″ both show low toxicity against normal colon cells but retain potency against colon cancer cells, suggesting that they have similar activity to M2. We further demonstrate that the cysteine-conjugated shogaols can cause cancer cell death through the activation of the mitochondrial apoptotic pathway. Our results show that oxidative stress activates a p53 pathway that ultimately leads to p53 up-regulated modulator of apoptosis (PUMA) induction and down-regulation of B-cell lymphoma 2 (Bcl-2), followed by cytochrome c release, perturbation of inhibitory interactions of X-linked inhibitor of apoptosis protein (XIAP) with caspases, and finally caspase 9 and 3 activation and cleavage. A brief screen of the markers attenuated by the proapoptotic activity of M2 revealed similar results for [8]- and [10]-shogaol and their respective cysteine-conjugated metabolites M2′ and M2″. This study highlights the cysteine-conjugated metabolites of shogaols as novel dietary colon cancer preventive agents

Metabolites of ginger component [6]-shogaol remain bioactive in cancer cells and have low toxicity in normal cells: chemical synthesis and biological evaluation.

Our previous study found that [6]-shogaol, a major bioactive component in ginger, is extensively metabolized in cancer cells and in mice. It is unclear whether these metabolites retain bioactivity. The aim of the current study is to synthesize the major metabolites of [6]-shogaol and evaluate their inhibition of growth and induction of apoptosis in human cancer cells. Twelve metabolites of [6]-shogaol (M1, M2, and M4-M13) were successfully synthesized using simple and easily accessible chemical methods. Growth inhibition assays showed that most metabolites of [6]-shogaol had measurable activities against human cancer cells HCT-116 and H-1299. In particular, metabolite M2 greatly retained the biological activities of [6]-shogaol, with an IC(50) of 24.43 µM in HCT-116 human colon cancer cells and an IC(50) of 25.82 µM in H-1299 human lung cancer cells. Also exhibiting a relatively high potency was thiol-conjugate M13, with IC(50) values of 45.47 and 47.77 µM toward HCT-116 and H-1299 cells, respectively. The toxicity evaluation of the synthetic metabolites (M1, M2, and M4-M13) against human normal fibroblast colon cells CCD-18Co and human normal lung cells IMR-90 demonstrated a detoxifying metabolic biotransformation of [6]-shogaol. The most active metabolite M2 had almost no toxicity to CCD-18Co and IMR-90 normal cells with IC(50)s of 99.18 and 98.30 µM, respectively. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay indicated that apoptosis was triggered by metabolites M2, M13, and its two diastereomers M13-1 and M13-2. There was no significant difference between the apoptotic effect of [6]-shogaol and the effect of M2 and M13 after 6 hour treatment

Carbohydrate Intake Does Not Counter the Post-Exercise Decrease in Natural Killer Cell Cytotoxicity

In a study using a randomized crossover approach, cyclists (n = 20, overnight fasted) engaged in three 75 km time trials while ingesting water (WAT) or carbohydrate (0.2 g/kg every 15 min) from bananas (BAN) or a 6% sugar beverage (SUG). Blood samples were collected pre-exercise and 0 h, 1.5 h, and 21 h post-exercise and analyzed for natural killer (NK) cytotoxicity activity (NKCA) using pure NK cell populations. The two carbohydrate trials (BAN, SUG) compared to WAT were associated with higher post-exercise glucose and lower cortisol, total blood leukocyte, neutrophil, and NK cell counts (interaction effects, p < 0.001). The immediate post-exercise increase in NK cell counts was higher in WAT (78%) compared to BAN (32%) and SUG (15%) trials (p ≤ 0.017). The 1.5 h post-exercise decrease in NK cell counts did not differ after WAT (−46%), BAN (−46%), and SUG (−51%) trials. The pattern of change in post-exercise NKCA differed between trials (p < 0.001). The 1.5 h post-exercise decreases in NKCA were 23%, 29%, and 33% in the WAT, BAN, and SUG trials, respectively, but trial contrasts did not differ significantly. Carbohydrate ingestion from BAN or SUG attenuated immediate post-exercise increases in leukocyte, neutrophil, and NK cell counts, but did not counter the 1.5 h decreases in NK cell counts and NKCA

Growth inhibitory effects of [<b>6</b>]-shogaol and its metabolites (M1, M2, and M4–M13) against human colon cancer cells H-1299 and human normal lung cells IMR-90.

<p><i>Bar</i>, standard error (n = 6).</p

Synthesis of thiol-conjugates M1, M2, M4, M5, and M13.

<p>Reagents and conditions: i) L-cysteine, NaHCO3 (cat.), MeOH/H2O, rt, 24 h; ii) NaBH4, MeOH, 0°C, 2 h; iii) N-acetyl-L-cysteine, NaHCO3 (cat.), MeOH/H2O, rt, 72 h; iv) L-glutathione reduced, NaHCO3 (cat.), MeOH/H2O, rt, 3 h.</p

Key NOESY correlations in M13-1 and M13-2 and the major differences of the ¹H and ¹³C NMR data of M13-1 and M13-2.

<p>Key NOESY correlations in M13-1 and M13-2 and the major differences of the ¹H and ¹³C NMR data of M13-1 and M13-2.</p

Structures of the major gingerols and shogaols in ginger; and key HMBCcorrelations of thiol-conjugates M1, M2, and M4; and key ¹H-¹H COSY and HMBC correlations of M13.

<p>Structures of the major gingerols and shogaols in ginger; and key HMBCcorrelations of thiol-conjugates M1, M2, and M4; and key ¹H-¹H COSY and HMBC correlations of M13.</p

Growth inhibitory effects of [<b>6</b>]-shogaol, M13, M13-1, M13-2, and a physical mixture of M13-1 and M13-2 (molar/molar = 1∶2) against human colon cancer cells HCT-116.

<p><i>Bar</i>, standard error (n = 6).</p

Growth inhibitory effects of [<b>6</b>]-shogaol and its metabolites (M1, M2, and M4–M13) against human colon cancer cells HCT-116 and human normal colon cells CCD-18Co.

<p><i>Bar</i>, standard error (n = 6).</p

Synthesis of metabolites M6–M12.

<p>Reagents and conditions: i) H2, Pd/C (10% w/w), THF, rt, 18 h; ii) BBr3, DCM, −78°C -rt, 2 h; iii) NaBH4, MeOH, 0°C -rt, 2 h; iv) aq. 15% NaSCH3, THF, rt, 6 h; v) Na, MeOH, 0°C -rt, 4 h; vi) NaBH4, CeCl3.7H2O, MeOH, −78°C, 30 min.</p