Assessment of Cell Cycle and Induction of Apoptosis by Shiga-like Toxin Produced by Escherichia coli O157:H7 in T47D Breast Cancer Cells Using Flow Cytometry.

BACKGROUND: The low general toxicity against tumors expressing globotriaosylceramide (Gb3) and Shiga-like toxins produced by E. coli have been proposed as an anti-cancer therapy because of their specific target. This study aimed to determine the potency of the local strains of E. coli O157:H7 isolated from humans and cattle as a new breast cancer therapy by analyzing the cell cycle's inhibition and apoptosis induction. MATERIAL AND METHODS: Approximately 10 cultured T47D cells were subjected to Shiga-like toxin produced by four local isolates of E. coli O157:H7, including KL-48 (2) from humans, and SM-25 (1), SM-7 (1), DS-21 (4) from cattle. Using ATCC 43894 as a control, the treatment was observed for 24 h by two replications. In addition, a FITC-Annexin V and PI assay were used to observe apoptosis and necrosis effect, as well as to analyze the cell cycle using propidium iodide (PI) staining. RESULTS: The results showed the toxicity effect of Shiga in the human T47 D cells line. The viability of the cells is subjected to Shiga-like toxins produced by KL-48 (2), SM7 (1), ATCC 43894, SM-25 (1), and DS-21 (4) isolates decreased with 15.20, 16.36, 22.17,  22.64, and 33.86%, in contrary to control of 94.36%. These were supported by the cells entering the late apoptosis of the cell cycle through each isolate with 67.66, 62.60, 63.68, 63.90, and 54.74%, and a control of 0.01%. Also, the necrosis cell for each treatment of 12.73, 19.3, 10.84, 10.53, and 4.86% was higher than the control of 5.51%. These were confirmed by the higher percentage of the cells treated with toxins of KL-48 (2), SM7(1), ATCC 43894, SM-25 (1), and DS-21 (4), which entered G0-G1 of the cell cycle phase with 66.41, 63.37, 61.52, 55.36, and 47.28%, respectively, than control of 40.69%. Additionally, the toxicity effect was supported by an increase in the cells entering the S and the G2-M phase of the cycle for each treatment. CONCLUSION: It is concluded that the Shiga-like toxin produced by E. coli O157:H7 local isolates can be developed as a drug against breast cancer based on its effect to arrest induction of the cell cycle and inducing apoptosis.

Shiga-Like Toxin Produced by Local Isolates of Escherichia coli O157:H7 Induces Apoptosis of the T47 Breast Cancer Cell Line.

PURPOSE: It has been suggested that Shiga-like toxins produced by Escherichia coli O157:H7 could be used as novel therapeutic agents against malignant tumors. In addition, the antitumor potency of local isolates from Indonesia, which are known to be less toxic than the control isolate ATCC 43894, has not yet been tested. The study aimed to analyze local strains of E. coli O157:H7 as a proapoptosis agent on the T47 breast cancer cell line. METHODS: As many as 30 culture cells of T47D breast cancer cell line were subjected to purified extracts of Shiga-like toxin originating from 5 local isolates of E. coli O157:H7: KL-48(2), SM-25(1), SM-7(1), DS-21(4), and 1 isolate ATCC 43894 which was used as a control. Toxin production of each isolate was detected using a sandwich enzyme-linked immunosorbent assay, and the treatment of cell lines was observed for 24 hours, with 2 replications; 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide tests and acridine orange/ethidium bromide double staining assays were used for detection and analyses of apoptosis. RESULTS: The study showed 2 local strains of E. coli O157:H7 (codes KL-48(2) and SM-25(1)) had toxins positive at titer 5 and 10 µg/100 µL. These titers were lower than the control isolate ATCC 43894, but they had a necrosis effect higher (P < .05), ie, 80.3%, than control isolate, ie, 63.3%. Other local strain SM-25(1) also had a good necrosis effect. It has a nondifferent necrosis effect (P > .05) with the control isolate ATCC 43894, ie, 13.0% from 13.3%. CONCLUSION: This study concludes that the Shiga toxin produced by E. coli O157:H7 local isolate (Indonesia) has potential as a proapoptotic and/or necrotic agent for treating T47 breast cancer cell lines, as effectively as ATCC 43894 control isolates.

Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in Escherichia coli O157:H7.

BACKGROUND/PURPOSE: Shiga-like toxin (Stx) is an important factor in the pathogenesis of Escherichia coli O157:H7 infection and is responsible for some severe complications. Stx2 is usually associated with hemolytic uremic syndrome in humans. Its expression is regulated by elements located upstream of the stx2 gene, including stx2-promoter sequence, ribosome binding site, and the antiterminator q gene. The present study aimed to find the correlation between regulatory elements and the expression level of Stx2 in two local isolates of E. coli O157:H7. METHODS: Two local E. coli O157:H7 strains SM-25(1) and KL-48(2), originating from human and cattle feces, respectively, and an E. coli reference strain, ATCC 43894, were investigated. The complete stx2 gene covering the sequences of promoter, ribosome binding site, and open reading frame and q gene of each strain was analyzed. The magnitude of Stx2 production was detected with a reverse passive latex agglutination method and Stx mediated cellular damage was determined with the Vero cell assay. RESULTS: A comparison of the complete stx2 gene contained stx2-promoter, ribosome binding site, and q genes of two local strains KL-48(2) and SM25(1), and the E. coli ATCC 43894 showed that the amino acid sequences were identical. Both local isolates were Stx negative in the reverse passive latex agglutination test and nontoxic in the Vero cell assay. CONCLUSION: The expression level of Shiga-like toxin of the two local isolates of E. coli O157:H7 did not only depend on the regulatory elements of the stx2 gene.