Attenuated Salmonella typhimurium L forms suppress tumor growth and promote apoptosis in murine ovarian tumors

Abstract

To study the effects of attenuated Salmonella typhimurium L forms on the in vivo tumorigenicity and apoptosis of murine epithelial ovarian cancer cells, as well as the related mechanisms. Attenuated Salmonella typhimurium VNP20009 was induced into bacterial L forms by using antibiotic ceftriaxone. CCK-8 cell proliferation assay showed that attenuated S. typhimurium L forms can inhibit the proliferation of murine ovarian epithelial cancer ID8 cells. Attenuated ST L forms can induce apoptosis and inhibit invasion ability of epithelial ovarian cancer cells in vitro. TUNEL assay showed that attenuated ST L forms can induce apoptosis of ID8 cells in murine ovarian tumors. Meanwhile, attenuated ST L forms inhibit tumor growth in murine ovarian tumors. The tumorigenicity-related proteins of xenograft tumors detected by immunohistochemistry and fluorescence quantitative RT-PCR assays showed that attenuated ST L forms can reduce the expression of proteins that promote tumor growth and metastasis, such as Lgals9 and MMP9. This study confirmed that attenuated ST L forms can suppress tumor growth and promote apoptosis in murine ovarian tumors. Attenuated ST L forms may serve as a novel biological agent for bacterial-mediated tumor therapy in epithelial ovarian cancer.

Keywords: ST; Salmonella typhimurium; Apoptosis; Epithelial ovarian cancer; L forms; Tumorigenicity.

Figure 1

Salmonella typhimurium and its L forms. Note: (A) Attenuated ST VNP20009 strain original bacterial colony, Observing under a ×40 magnification microscope; (B) the L-form colonies of the attenuated Salmonella typhimurium VNP20009 strain induced by antibiotic ceftriaxone were observed under a ×40 magnification microscope. The L-form colonies were fried egg-like small colonies; (C) by Gram staining and Magnification: ×1000, ST original bacteria are observed to be red gram-negative bacteria, short rod-shaped, and dispersed in arrangement; (D) ST L-form bacteria are unevenly colored red gram-negative bacteria, with long filamentous bodies that intertwine into clusters, Gram staining and Magnification: ×1000.

Figure 2

Attenuated ST L forms inhibit ID8 cell proliferation and induce apoptosis. Note: (A) CCK-8 assay result analysis. Attenuated S. typhimurium VNP20009 L-form bacteria can inhibit the growth of mouse epithelial ovarian cancer ID8 cells, *P < 0.05. (B) FCM apoptosis detection results. (C) Statistical chart analysis of apoptosis detection. Attenuated S. typhimurium VNP20009 L-form bacteria can induce mouse EOC ID8 cell apoptosis. *P < 0.05.

Figure 3

Invasiveness evaluation via transwell invasion experiment. Note: (A) Transwell invasion experiment in the blank control group of mouse EOC ID8 cells, scale bar: 50 μm; (B) Transwell invasion experiment was conducted in the experimental group of mouse EOC ID8 cells treated with attenuated S. typhimurium VNP20009 L-form bacteria, scale bar: 50 μm; (C) Statistical analysis of the transwell invasion experiment results, *P < 0.05.

Figure 4

Attenuated ST L forms suppress ID8 transplanted tumor growth. Note: (A) HE staining of tissue sections of the transplanted tumors of mouse EOC ID8 cells in the blank control group, magnification: ×400, showed active growth of cancer cells, significant nuclear heterogeneity, and large nuclear plasma ratio, scale bar: 50 μm; (B) After treatment with attenuated ST VNP20009 L-form bacteria, HE staining was performed on the tissue sections of the transplanted tumors of mouse EOC ID8 cells, scale bar: 50 μm. When magnified 400 times and observed, they were found that the growth of cancer cells was not active; (C) 60 days after transplantation of mouse epithelial ovarian cancer ID8 cell line, the actual tumor image; (D) Statistical analysis of the experimental results of two groups of tumor growth volume detection, *P < 0.05, **P < 0.01.

Figure 5

Attenuated ST L forms promote apoptosis in murine ovarian tumors. Note: (A) TUNEL apoptosis detection in the blank control group showed that apoptosis in the tumors of mouse EOC ID8 cell line was very rare, scale bar: 50 μm; (B) After treatment with attenuated S. typhimurium VNP20009 L-form bacteria, there was significant apoptosis in the tumors of mouse EOC ID8 cells, scale bar: 50 μm. TUNEL-positive apoptosis cell nuclei of DAB color rendering were brown. Attenuated S. typhimurium VNP20009 L-form bacteria can induce cancer cell apoptosis in murine ovarian tumors.

Figure 6

Expression of Lgals9 in the transplanted tumors of mouse EOC ID8 cells. Note: (A) The tissue adjacent to the transplanted tumors of mouse EOC ID8 cells was the low expression of Lgals9, IHC score (+), scale bar: 50 μm; (B) Lgals9 was highly expressed in the transplanted tumors of mouse EOC id8 cells, IHC score (++), scale scale bar: 50 μm.

Figure 7

Effect of attenuated ST VNP20009 L forms on the expression of Lgals9 in transplanted tumors of mouse EOC ID8 cells. Note: (A) High expression of Lgals9 in transplanted tumors of mouse EOC ID8 cells, IHC score (++), scale bar: 50 μm; (B) After treatment with attenuated ST VNP20009 L-form bacteria, the expression of Lgals9 in the transplanted tumors of mouse EOC ID8 cells decreased, IHC score (+), scale bar: 50 μm; (C) qRT-PCR detection showed that attenuated ST L forms inhibited the expression of Lgals9 mRNA in mouse ID8 transplanted tumors (*P < 0.05).

Figure 8

Effect of attenuated ST L forms on the expression of MMP9 in mouse EOC ID8 tumors. Note: (A) High expression of MMP9 in transplanted tumors of mouse EOC ID8 cells, IHC score (++), scale bar: 50 μm; (B) After treatment with attenuated ST VNP20009 L-form bacteria, the expression of MMP9 in the transplanted tumors of mouse EOC ID8 cells decreased, IHC score (+), scale bar: 50 μm; (C) qRT-PCR detection showed that attenuated ST L forms inhibited the expression of MMP9 mRNA in mouse ID8 transplanted tumors (*P < 0.05).

Figure 9

Effect of attenuated ST L forms on the expression of E-cadherin in mouse EOC ID8 tumors. Note: (A) High expression of E-cadherin in transplanted tumors of mouse EOC ID8 cells, immunohistochemical score (+++), scale bar: 50 μm; (B) After treatment with attenuated ST VNP20009 L-form bacteria, the expression of E-cadherin in the transplanted tumors of mouse EOC ID8 cells decreased, IHC score (++), scale bar: 50 μm; (C) qRT-PCR detection showed that attenuated ST L forms inhibited the expression of E-cadherin mRNA in mouse ID8 transplanted tumors (*P < 0.05).