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. 2020 Jul;23(7):886-893.
doi: 10.22038/ijbms.2020.43016.10115.

Anti-cancer properties of Escherichia coli Nissle 1917 against HT-29 colon cancer cells through regulation of Bax/Bcl-xL and AKT/PTEN signaling pathways

Siamak Alizadeh  1   2 , Abolghasem Esmaeili  1 , Yadollah Omidi  2   3
Affiliations

Anti-cancer properties of Escherichia coli Nissle 1917 against HT-29 colon cancer cells through regulation of Bax/Bcl-xL and AKT/PTEN signaling pathways

Siamak Alizadeh et al. Iran J Basic Med Sci. 2020 Jul.

Abstract

Objectives: Chemotherapies used to treat colon cancer might often fail due to the emergence of chemoresistance and side effects. Escherichia coli Nissle 1917 (EcN) is a beneficial probiotic, whose molecular mechanisms in the prevention of colon cancer are yet to be fully understood. The present study assessed the anti-cancer effects of EcN treatments in human colorectal cancer, HT-29 cell line, with the analysis of related mechanisms.

Materials and methods: The co-culture conditioned-media (CM) of EcN with adenocarcinoma HT-29 cells and heat-inactivated bacteria (HIB) were applied for the treatment of the HT-29 cells. To study the inhibition potential of CM and HIB on cancer cells, various cellular/molecular analyses were implemented, including DAPI-staining and DNA ladder assays, flow cytometry and Real-time quantitative PCR (qPCR), as well as Western blotting analyses.

Results: Our results indicated that EcN could elicit apoptotic impacts on the colon cancer HT-29 cells by up-regulating PTEN and Bax and down-regulating AKT1 and Bcl-xL genes.

Conclusion: Based on our findings, EcN is proposed as a useful supplemental probiotic treatment against colon cancer.

Keywords: AKT; Apoptosis; Cell signaling; Colon cancer; Escherichia coli Nissle1917.

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Figures

Figure 1
Figure 1
Morphological changes of HT-29 cells. The cells were treated with CM and observed under light microscopy (10X). (A) Untreated HT-29 cells (control), (B) HT-29 cells treated with CM (OD600: 1.0) after 24 hr, and (C) HT-29 cells treated with CM after 48 hr. The cells showed typical features of apoptosis including nuclear fragmentation (NF), membrane blebbing (MB), and cellular shrinkage (CS) in Figure 1C. CM: Conditioned-media
Figure 2.
Figure 2.
Cell viability evaluated by MTT assay in HT-29 cells treated with CM or HIB. (A) Treated cells with CM after 24 hr. (B) Treated cells with CM after 48 hr. (C) Treated cells with HIB after 24 hr. (D) Treated cells with HIB after 48 hr. Each cell viability assay was performed in triplicate. Data are presented as mean SD * and **. P<0.05 (*) and P<0.01 (**). CM: Conditioned-media; HIB: Heat-inactivated bacteria; CT: Control; OD: Optimal density
Figure 3
Figure 3
DNA fragmentation assay for detection of apoptosis. Gel electrophoresis of DNA isolated from HT-29 cells in the control and CM-treated cells. Lane 1: ladder (100bp), Lanes 2 and 3: the untreated control cells after 24 hr and 48 hr, respectively. Lanes 4 and 5: the HT- 29 cells treated with CM after 24 hr and 48 hr, respectively. Tr: Treated cells; Ct: Control; CM; Conditioned-media
Figure 4
Figure 4
DAPI staining and flow cytometry analysis of HT-29 cells treated with CM or HIB. DAPI staining was performed to analyze changes in the nucleus. (A) Untreated control cells. (B) Treated cells with CM after 48 hr. (C) Treated cells with HIB after 48 hr. FITC-labeled annexin V/PI flow - cytometry was done to analyze apoptosis. (D) Untreated control cells. (E) Treated with CM after 48 hr. (F) Treated cells with HIB after 48 hr. CM: Conditioned-media; HIB: Heat-inactivated bacteria; NC: Necrotic cells; EA: Early apoptosis; LA: Late apoptosis; LC: Living cells
Figure 5
Figure 5
The real-time PCR analysis HT-29 cells treated with CM or HIB. The cells were treated and analyzed after 48 hr. Panels A, B, C, and D represent the mRNA expression of AKT, PTEN, Bax, and Bcl-xL, respectively. Panels E and F show the Bax/Bcl-xL expression ratio in the cells treated with CM or HIB, respectively. Data expressed as fold change. CT: Control; CM: Condition-media; HIB: Heat-inactivated bacteria. Data are presented as mean SD * and **. P<0.05 (*) and P<0.01 (**)
Figure 6
Figure 6
Western blot analysis of AKT, PTEN, Bax, and GAPDH proteins in HT-29 cells treated with CM or HIB. The cells were treated and probed with the indicated antibodies after 48 hr. (A) Western blot results. (B) The expression levels normalized based on GAPDH as the control. CT: Control; CM: Condition-media; HIB: Heat-inactivated. Data are presented as mean SD * and **. P<0.05 (*) and P<0.01 (**)
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