Pseudomonas aeruginosa mannose-sensitive hemagglutinin inhibits proliferation and invasion via the PTEN/AKT pathway in HeLa cells

Abstract

We investigated the effects of Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) on the proliferation and invasion of human cervical cancer cell lines, as well as the molecular pathways underlying these effects. MTT cell proliferation assays revealed a time- and concentration-dependent cytotoxic effect of PA-MSHA on HeLa cells but not H8 cells. Flow cytometry with propidium iodide and annexin-V-fluorescein isothiocyanate labeling (FITC) indicated that various concentrations of PA-MSHA could induce apoptosis and G2-M cell cycle arrest in HeLa cells. PA-MSHA also impaired the migration and invasion abilities of HeLa cells in Wound healing and Transwell invasion assays. Western blot results demonstrated that PA-MSHA reduced the expression of p-AKT, p-GSK3β, BCL-2, Vimentin and β-catenin, but increased the levels of PTEN, BAD, BAX and E-cadherin in HeLa cells. Importantly, PTEN siRNA induced the activity of p-AKT, while PA-MSHA partly inhibited this induction, indicating that PA-MSHA may reduce the cell proliferation and invasion potential by activating PTEN and thus inhibiting the AKT pathway in vitro. These data suggest the potential application of PA-MSHA to the treatment of human cervical cancer.

Keywords: PA-MSHA; PTEN; cervical cancer; p-AKT; p-GSK3β.

Figure 1. Effect of PA-MSHA or PA on cell proliferation

Values are given as percentages of the values in untreated control cells. The data are presented as the averages of triplicate results from a representative experiment; bars, standard deviation (SD). (A) The dose-dependent effect of PA-MSHA on HeLa cell proliferation. (B) The time-dependent effect of PA-MSHA on HeLa cell proliferation. (C) The dose-dependent effect of PA on HeLa cell proliferation. (D) The time-dependent effect of PA on HeLa cell proliferation. (E) The dose-dependent effect of PA-MSHA on H8 cell proliferation. (F) The time-dependent effect of PA-MSHA on H8 cell proliferation.

Figure 2. Effects of PA-MSHA on cell cycle arrest and apoptosis

The distribution of HeLa cells in the three phases of the cell cycle following treatment with PA or PA-MSHA is depicted in representative plots (A) and as percentages (B). **p < 0.01 for cells treated with PA-MSHA versus PA in the G0–G1, S and G2-M phases. (C) The percentages of apoptotic cells were measured in HeLa cells treated with PA or PA-MSHA, which are shown in representative plots. (D) The percentages of apoptotic cells were measured in HeLa cells treated with PA or PA-MSHA. The data are presented as the mean ± SD (N = 3, **p < 0.01).

Figure 3. Effects of PA-MSHA on cell migration and invasion

(A) Wound healing assay. The changes in the wound edges are illustrated with broken lines. Upper panel: PA or PA-MSHA group at 0-h timepoint. Bottom panel: PA or PA-MSHA group at 24-h timepoint. (B) The wound edges were measured in HeLa cells treated with PA or PA-MSHA. Data are presented as the mean ± SD (N = 3, **p < 0.01). (C) The numbers of invasive cells per field were counted in HeLa cells treated with PA or PA-MSHA, and are shown in representative pictures. (D) The numbers of invasive cells per field were counted in HeLa cells treated with PA or PA-MSHA. Data are presented as the mean ± SD (N = 3, **p < 0.01).

Figure 4. Effects of PA-MSHA on apoptosis- and EMT-related proteins

HeLa cells were treated with PA-MSHA or PA. The cells were harvested and the total protein was extracted. (A) The effects of PA-MSHA on the protein levels of BAD, BAX and BCL-2 were assessed by Western blot. (B) The fold-changes in the relative protein levels of BAD, BAX and BCL-2 were calculated with reference to the control levels. Data are presented as the mean ± SD (N = 3, **p < 0.01). (C) The effects of PA-MSHA on the protein levels of E-cadherin, Vimentin and β-catenin were assessed by Western blot. (D) The fold-changes in the relative protein levels of E-cadherin, Vimentin and β-catenin were calculated with reference to the controls. Data are presented as the mean ± SD (N = 3, **p < 0.01).

Figure 5. PA-MSHA inhibits the activation of the AKT signaling pathway by increasing PTEN expression

HeLa cells were treated with PA-MSHA or PA. The cells were harvested and the total protein was extracted. (A) The effects of PA-MSHA on the protein levels of AKT, p-AKT, GSK3β and p-GSK3β were assessed by Western blot. (B) The fold-changes in the relative protein levels of AKT, p-AKT, GSK3β and p-GSK3β were calculated with reference to the controls. Data are presented as the mean ± SD (N = 3, **p < 0.01). (C) Cells were transfected with PTEN siRNA or empty vectors, and Western blot analysis was performed to detect PTEN protein levels. (D) The fold-changes in the relative protein levels of PTEN were calculated with reference to the siRNA controls. Data are presented as the mean ± SD (N = 3, **p < 0.01). (E) The inhibitory effect of PA-MSHA on the PTEN siRNA-induced activation of the AKT pathway. (F) The fold-changes in the relative protein levels of PTEN, AKT, p-AKT, GSK3β and p-GSK3β were calculated with reference to the controls. Data are presented as the mean ± SD (N = 3, **p < 0.01).

Figure 6. PA-MSHA inhibits proliferation and invasion through the PTEN/AKT pathway

(A) The inhibitory effect of PA-MSHA on PTEN siRNA-induced HeLa cell proliferation. (B) The numbers of invasive cells per field were measured in PTEN siRNA-treated HeLa cells treated with PA or PA-MSHA, which are depicted in representative pictures. (C) The numbers of invasive cells per field were measured in PTEN siRNA-treated HeLa cells treated with PA or PA-MSHA. Data are presented as the mean ± SD (N = 3, **p < 0.01).