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
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Cell Cycle-specific Measurement of γH2AX and Apoptosis After Genotoxic Stress by Flow Cytometry
Published on: September 1, 2019
I Wayan Suardana1, Ida Bagus Ngurah Swacita1, Komang Januartha Putra Pinatih2
1Department of Preventive Veterinary Medicine, Faculty of Veterinary Medicine, Udayana University. Jl. P.B. Sudirman, Denpasar-Bali. 80234. Indonesia.
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.
Insights
Shiga-like toxins from local E. coli O157:H7 strains show promise as a breast cancer therapy. These toxins induce apoptosis and arrest the cell cycle, offering a targeted anti-cancer approach.
Area of Science:
- Oncology
- Microbiology
- Biochemistry
Background:
- Shiga-like toxins from E. coli exhibit specific toxicity towards globotriaosylceramide (Gb3)-expressing tumors.
- This presents a potential targeted anti-cancer therapy approach.
Purpose of the Study:
- To evaluate the efficacy of local E. coli O157:H7 strains as a breast cancer therapy.
- To analyze the impact of Shiga-like toxins on cancer cell cycle inhibition and apoptosis induction.
Main Methods:
- Cultured T47D breast cancer cells were treated with Shiga-like toxins from human and cattle E. coli O157:H7 isolates.
- Apoptosis, necrosis, and cell cycle progression were analyzed using FITC-Annexin V and propidium iodide (PI) staining.
Main Results:
- Shiga-like toxins significantly reduced T47D cell viability.
- Treated cells showed increased late apoptosis and necrosis compared to controls.
- Cell cycle analysis revealed arrest at the G0-G1 phase and increased S and G2-M phase entry.
Conclusions:
- Shiga-like toxins from local E. coli O157:H7 isolates demonstrate potential as a breast cancer therapeutic agent.
- The toxins effectively induce apoptosis and arrest the cell cycle, supporting their anti-cancer properties.
