Anticancer properties of curcumin-treated Lactobacillus plantarum against the HT-29 colorectal adenocarcinoma cells

Abstract

Probiotic bacteria with functions of importance to the health and well-being of the host exhibit various medicinal properties including anti-proliferative properties against cancer cells. There are observations demonstrating probiotic bacteria and their metabolomics can be different in various populations with different eating habits. Here, Lactobacillus plantarum was treated with curcumin (the major compound of turmeric), and its resistance to the curcumin was determined. After then the cell-free supernatants of untreated bacteria (CFS) and bacteria treated with curcumin (cur-CFS) were isolated and their anti-proliferative properties against HT-29 colon cancer cells were compared. The ability of L. plantarum treated with curcumin to combat a variety of pathogenic bacterial species and its ability to survive in acidic conditions were evidence that the probiotic properties of the bacterium were unaffected by the curcumin treatment. L. plantarum treated with curcumin and intact L. plantarum were both able to live in acidic conditions, according to the results of the resistance to low pH test. The MTT result showed that CFS and cur-CFS dose-dependently decreased the growth of HT29 cells with a half-maximal inhibitory concentration of 181.7 and 116.3 µL/mL at 48 h, respectively. Morphological alteration of DAPI-stained cells also exhibited significant fragmentation in the chromatin within the nucleus of cur-CFS-treated cells compared to CFS-treated HT29 cells. Moreover, flow cytometry analyses of apoptosis and cell cycle confirmed DAPI staining and MTT assay results and stipulated the increased occurrence of programmed cell death (apoptosis) in cur-CFS-treated cells (~ 57.65%) compared to CFS-treated cells (~ 47%). These results were more confirmed with qPCR and exhibited the upregulation of Caspase 9-3 and BAX genes, and downregulation of the BCL-2 gene in cur-CFS- and CFS-treated cells. In conclusion, turmeric spice and curcumin may affect the metabolomics of probiotics in intestinal flora which could subsequently influence their anticancer properties.

Figure 1

Adaptation of Lactobacillus plantarum to curcumin food additive.

Figure 2

The diameter of the zone of inhibition formed by metabolites derived from untreated and curcumin-treated L. plantarum bacteria against Staphylococcus aureus and Shigella dysenteriae. (A) CFS, (B) CFS + proteinase k, (C) Phosphate buffer as a negative control, (D) cur-CFS + proteinase k, (E) cur-CFS, (F) Azithromycin, (G) Ampicillin.

Figure 3

The effect of different concentrations of CFS and cur-CFS on the viability of HT-29 cells after 24 h. (****p ˂ 0.0001) (*p < 0.05).

Figure 4

Flow cytometry quantification of apoptosis and necrosis in HT-29 colon cancer cells treated with CFS and cur-CFS in comparison with unstained and positive groups.

Figure 5

Cell cycle analysis shows the population of cells inters to the Sub-G1 phase in CFS and cur-CFS-treated cells compared to the control group (p ˂ 0.0001).

Figure 6

Chromatin changes in HT-29 colon cells after 24 h of treatment with metabolites of intact L. plantarum (CFS) and curcumin-treated L. plantarum (cur-CFS).

Figure 7

Expression ratios of BAX, Bcl-2, Caspase-9, and Caspase-3 genes in HT-29 colon cancer cells treated with CFS, cur-CFS. The control group was designated as untreated cells (****p ˂ 0.0001).