A Study Of Biological Characteristics Of PA-MSHA In Human Breast Cancer Cells And Its Possible Mechanism

ObjectiveTo investigate the effects of PA-MSHA (Pseudomonas aeruginosa-mannose sensitive hemagglutinin) on inhibiting proliferation and inducing apoptosis of breast cancer cell lines and to explore its therapeutic targets and detailed mechanisms of action in human breast cancer cells both in vitro and in vivo.MethodsPart 1:MDA-MB-468 and MDA-MB-231HM cells were treated with PA-MSHA or PA (Heat-killed Pseudomonas aeruginosa) at different concentrations and different times. Changes of cell super-microstructure were observed by transmission electron microscopy. Cell cycle distribution and apoptosis induced by PA-MSHA were measured by flow cytometry (FCM) with PI staining, ANNEXIN V-FITC staining and Hoechst33258 staining under fluorescence microscopy. Western blot was used to evaluate the expression level of apoptosis-related molecules.Part 2:PA-MSHA was administered to EGFR-positive human breast cancer cell lines MDA-MB-231HM and MDA-MB-468 in vitro and to mice bearing tumor xenografts. Western Blotting and ELISA were used to detect the effect of PA-MSHA on EGFR pathway and metastasis associated proteins. si-RNA was used to downregulate EGFR expression to determine whether EGFR expression is critical for the effect of PA-MSHA on breast cancer cells in vitro. D-mannose was added to a final concentration of 100 mM in the PA-MSHA suspension, or the assay was carried out with 100 mM D-glucose in the PA-MSHA suspension to serve as a control. The mannose co-cultured test was used to detect the effect of mannose on PA-MSHA-induced cell proliferation, cell cycle arrest, apoptosis and EGFR pathway signaling.ResultsPart 1:A time-dependent and concentration-dependent cytotoxic effect of PA-MSHA was observed in both cell lines. The advent of PA-MSHA changed cell morphology, that is to say, increases in autophagosomes, reduction in the number of pseudopodium and microvilli, and vacuoles in the cytoplasm could also be observed. FCM with PI staining, ANNEXIN V-FITC and Hoechst33258 staining showed that the different concentrations of PA-MSHA could all induce the apoptosis and G0-G1 cell cycle arrest of breast cancer cells. Cleaved caspase 3,8,9 and Fas protein expression levels were strongly associated with an increase in apoptosis of the breast cancer cells. There was a direct relationship with increased concentrations of PA-MSHA but not of PA.Part 2:We found that cells stimulated with PA-MSHA exhibited a downregulation of EGFR pathway signaling. Downregulation of EGFR using EGFR specific siRNA is involved in PA-MSHA-induced signal pathway regulation, and EGFR expression is critical for the effect of PA-MSHA on breast cancer cells in vitro. PA-MSHA was notably unable to cause more cell death in cells transfected with EGFR siRNA. PA-MSHA inhibits the invasive abilities of MDA-MB-231HM and MDA-MB-468 cells both in vitro and in vivo. The addition of mannose partially inhibited the adherence of PA-MSHA to the cell surface and the PA-MSHA-stimulated cell anti-proliferative effect, cell apoptosis, cell cycle arrest, activation of apoptosis-associated caspases and even downregulation of the EGFR signaling pathway. In vivo, PA-MSHA treatment significantly suppressed mammary tumorigenesis in xenograft in mice and decreased lung metastasis in MDA-MB-231HM cell-transplanted mice. Tumor sample analyses confirmed inhibition of the EGFR pathway in the PA-MSHA-treated mice.ConclusionsCompletely different from PA, PA-MSHA may impart antiproliferative effects against breast cancer by inducing apoptosis mediated by at least a death receptor-related cell apoptosis signal pathway, and affecting the cell cycle regulation machinery. The involvement of the mannose-mediated EGFR pathway plays a critical role in the preclinical rationale for the development of PA-MSHA for the treatment of human breast cancer. It also suggests the potentially beneficial use of PA-MSHA in adjuvant therapy for breast tumors with EGFR overexpression.