Curcumin Oxidation Is Required for Inhibition of Helicobacter pylori Growth, Translocation and Phosphorylation of Cag A

Abstract

Curcumin is a potential natural remedy for preventing Helicobacter pylori-associated gastric inflammation and cancer. Here, we analyzed the effect of a phospholipid formulation of curcumin on H. pylori growth, translocation and phosphorylation of the virulence factor CagA and host protein kinase Src in vitro and in an in vivo mouse model of H. pylori infection. Growth of H. pylori was inhibited dose-dependently by curcumin in vitro. H. pylori was unable to metabolically reduce curcumin, whereas two enterobacteria, E. coli and Citrobacter rodentium, which efficiently reduced curcumin to the tetra- and hexahydro metabolites, evaded growth inhibition. Oxidative metabolism of curcumin was required for the growth inhibition of H. pylori and the translocation and phosphorylation of CagA and cSrc, since acetal- and diacetal-curcumin that do not undergo oxidative transformation were ineffective. Curcumin attenuated mRNA expression of the H. pylori virulence genes cagE and cagF in a dose-dependent manner and inhibited translocation and phosphorylation of CagA in gastric epithelial cells. H. pylori strains isolated from dietary curcumin-treated mice showed attenuated ability to induce cSrc phosphorylation and the mRNA expression of the gene encoding for IL-8, suggesting long-lasting effects of curcumin on the virulence of H. pylori. Our work provides mechanistic evidence that encourages testing of curcumin as a dietary approach to inhibit the virulence of CagA.

Keywords: CagA; Helicobacter pylori; curcumin; metabolic transformation; reduced curcumin.

Figure 1

Effects of curcumin and phospholipid encapsulated curcumin (Meriva) on growth of four prototype strains of H. pylori. (A) Four strains of H. pylori (26695, PZ5056G, PMSS1, and 7.13) (0.1 OD) were grown in the presence of vehicle (ethanol) or increasing concentrations of curcumin, and growth was determined at the indicated time points. (B) Growth curve of H. pylori strains 26695, PZ5056G, PMSS1, and 7.13 cultured in the presence of a phospholipid formulation of curcumin (Meriva, Mer) or vehicle control (Veh, ethanol). (C) Generation time of H. pylori PMSS1 and 7.13 as determined from the growth curves (**P value < 0.005 ***P value < 0.005).

Figure 2

Effect of Meriva on growth of E. coli, Citrobacter rodentium, and H. pylori, and intracellular accumulation of curcumin and its derivatives. (A–C) Bacteria were grown in Brucella broth containing 20 µM Meriva or vehicle (ethanol), and growth was monitored by optical density for indicated time. (D–F) Bacteria were harvested and the concentration of curcumin, dihydro-(D-curc,DHC)-, tetrahydro-( T-curc-THC)-, hexahydro-(H-curc,HHC)- and octahydro-(O curc,OHC)-curcumin as well as bicyclopentadione (BCP) was quantified using LC-MS. ***P ≤ 0.001.

Figure 3

Deletion of curA gene in E. coli results in growth inhibition by curcumin. (A) Growth curve of E. coli and its deletion mutant (ΔcurA) in the presence of curcumin or vehicle (**P value < 0.005 ***P value < 0.005). The deletion of curA was done by PCR splicing method in which a pair of primers flanking the region where the deletion to be made, two complementary primers comprising a region of -15 bp to +15 bp related to the junction point were used and a high fidelity polymerase was used for PCR. (B) Quantification of curcumin and its reduced metabolites dihydro-curcumin (D-curc), tetrahydro-curcumin (T-curc), hexahydro-curcumin (H-curc), and octahydro-curcumin (O-curc) in E. coli and its curA deletion mutant. (C) RT-PCR analysis of curA expression in genomic DNA isolated from H. pylori or E. coli. ^§§§P ≤ 0.001.

Figure 4

Effect of curcumin and its stable analogs on growth of H. pylori and intracellular accumulation of bicyclopentadione (BCP) in H. pylori, E. coli, and Citrobacter rodentium (A) H pylori was grown in the absence or presence of curcumin, dimethyl-, acetal-, or diacetal-curcumin, and growth was determined by measuring optical density. (B) Intracellular accumulation of curcumin and BCP in H. pylori. (C) After 24hr treatment the ratio of accumulation of curcumin and BCP in E. coli, Citrobacter rodentium (Citro), and H. pylori (Hp). ^§§P ≤ 0.01.

Figure 5

Curcumin prevents cagA phosphorylation. (A) Gastric epithelial cells were infected with H. pylori strains PZ5056G or PMSS1 in the absence or presence of Meriva (20 μM). CagA and phospho-CagA were determined by Western blotting using β-actin as loading control. Representative results from three independent experiments are shown. (B) Mouse stomach glands were isolated and plated in matrigel in the presence of growth factors. H. pylori was grown for 6 h with or without Meriva, harvested, and microinjected into the lumen of the gastroids. Infected gastroids were cultured and stained using an anti-phospho-cSrc antibody followed by Alexa Fluor 488-conjugated anti-rabbit IgG antibody (green). Nuclei were stained with DAPI (blue). (C) Western blot analysis of c-Src phosphorylation in stomach organ cultures used in panel (B). (D) Q-PCR analysis (% fold change) of the expression of the genes encoding for the Cag proteins A, F, T, Y, L, N, Z, M, D, and E in H. pylori (Shaffer et al., 2011; Frick-Cheng et al., 2016) treated with different concentrations of Meriva. *P ≤ 0.05; **P ≤ 0.01.

Figure 6

Inhibition of phosphorylation of cagA and Src requires oxidative transformation of curcumin. (A) Western blot analysis of CagA and Src phosphorylation in gastric epithelial cells upon infection with H. pylori treated with curcumin or its stable analogs acetal-curcumin (ACurc) and diacetalcurcumin (DCurc). The blot was probed with anti-phosphoCagA (top) or anti-phosphoSrc antibody (below), and β actin was used as loading control. Representative results from three independent experiments are shown. (B, C) Quantification of Western blotting results described in (A). (D) Quantification of CXCL8 mRNA level in gastric tissue after infection with H. pylori treated with curcumin and its stable analogs. ***P ≤ 0.001, ^§§§P ≤ 0.001.

Figure 7

H. pylori output strains isolated from dietary curcumin-treated mice induce attenuated host response. (A) Western blot analysis of cSrc phosphorylation in AGS cells treated with H. pylori isolated from mice fed control chow (vehicle) or chow containing 1% Meriva. H. pylori-infected mice fed with chow containing 1% Meriva for 4 weeks shows attenuation of phosphorylation of c-Src. (B) Quantification of p-Src/cSrc in the Western blot samples in (A) showing attenuation. (C) Quantification of CXCL8 mRNA when co-cultured with AGS cells showing reduced level. (D) CFU per gram of tissue showing no colonization. ***P ≤ 0.001, ^§§§P ≤ 0.001.