An In Vitro Model of Gastric Inflammation and Treatment with Cobalamin

Abstract

Pernicious anaemia (PA) is an autoimmune condition where antibodies target intrinsic factor and parietal cells, reducing the patient's ability to absorb cobalamin promoting atrophic gastritis. Treatment guidelines are based on excretion data of hydroxocobalamin from healthy individuals obtained 50 years ago. This manuscript describes the use of phorbol 12-myristate 13-acetate (PMA) to stimulate low grade inflammation in an epithelial colorectal cell line to assess the efficacy of methylcobalamin and hydroxocobalamin. Nitric oxide increased significantly in cells exposed to higher doses of PMA (100 ng/ml, 150 ng/ml, and 200 ng/ml) accompanied by a loss of the characteristic cobblestone morphology with no negative effect on cell activity or viability. A significant reduction in nitric oxide production was associated with the addition of 200 pg/ml hydroxocobalamin, alongside a return to the characteristic cobblestone morphology. This study highlights the use of PMA to promote low grade inflammation in human cell lines to model gastric inflammation associated with autoimmunity; furthermore it raises questions regarding the concentration of cobalamin administered clinically to restore cell functionality, feasibly allowing the patient to receive reduced quantity of the vitamin more regularly, providing the patient with levels which are akin to dietary intake.

Figure 1

Nitric oxide production from colorectal adenocarcinoma (Caco-2) cells after 24-hour culture with phorbol 12-myristate 13-acetate (PMA).

Figure 2

Cell metabolic activity as measured by MTT assay after 24-hour and 48-hour incubation in culture with phorbol 12-myristate 13-acetate (PMA) (^∗significantly increased (p = 0.0013)).

Figure 3

Colorectal adenocarcinoma (Caco-2) cells after 24-hour incubation with (a) cell culture medium and (b) cell culture medium spiked with 150 ng/ml phorbol 12-myristate 13-acetate (PMA). White arrows highlight nuclear changes.

Figure 4

Colorectal adenocarcinoma (Caco-2) cells after 24-hour culture with methylcobalamin (0, 200, 500, 750, 1000, and 5000 pg·ml). (a) Nitric oxide production; (b) cell activity as measured by MTT.

Figure 5

Colorectal adenocarcinoma (Caco-2) cells after 24-hour culture with 150 ng/ml phorbol 12-myristate 13-acetate (PMA) and methylcobalamin (0, 200, 500, 750, and 1000 pg/ml). (a) Cell activity as measured by MTT; (b) nitric oxide production (^∗significantly increased p = 0.0061).

Figure 6

Colorectal adenocarcinoma (Caco-2) cells after 24-hour culture with (a) 150 ng/ml phorbol 12-myristate 13-acetate (PMA) (b) and 200 pg/ml methylcobalamin (c) or 1000 pg/ml methylcobalamin (d).

Figure 7

Colorectal adenocarcinoma (Caco-2) cells after 24-hour culture with 150 ng/ml phorbol 12-myristate 13-acetate (PMA) and hydroxocobalamin (0, 200, 500, 750, and 1000 pg/ml): (a) cell activity as measured by MTT; (b) nitric oxide production.

Figure 8

Colorectal adenocarcinoma (Caco-2) cells after 24-hour culture with (a) 150 ng/ml phorbol 12-myristate 13-acetate (PMA) (b) and 150 pg/ml hydroxocobalamin (c) or 1000 pg/ml hydroxocobalamin (d).