LeftyA decreases Actin Polymerization and Stiffness in Human Endometrial Cancer Cells

Abstract

LeftyA, a cytokine regulating stemness and embryonic differentiation, down-regulates cell proliferation and migration. Cell proliferation and motility require actin reorganization, which is under control of ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1). The present study explored whether LeftyA modifies actin cytoskeleton, shape and stiffness of Ishikawa cells, a well differentiated endometrial carcinoma cell line. The effect of LeftyA on globular over filamentous actin ratio was determined utilizing Western blotting and flow cytometry. Rac1 and PAK1 transcript levels were measured by qRT-PCR as well as active Rac1 and PAK1 by immunoblotting. Cell stiffness (quantified by the elastic modulus), cell surface area and cell volume were studied by atomic force microscopy (AFM). As a result, 2 hours treatment with LeftyA (25 ng/ml) significantly decreased Rac1 and PAK1 transcript levels and activity, depolymerized actin, and decreased cell stiffness, surface area and volume. The effect of LeftyA on actin polymerization was mimicked by pharmacological inhibition of Rac1 and PAK1. In the presence of the Rac1 or PAK1 inhibitor LeftyA did not lead to significant further actin depolymerization. In conclusion, LeftyA leads to disruption of Rac1 and Pak1 activity with subsequent actin depolymerization, cell softening and cell shrinkage.

Figure 1. AFM analysis of stiffness and shape of Ishikawa cells with and without LeftyA treatment.

Representative optical phase contrast image (a), AFM contact height image (b) and AFM stiffness image (c) of an untreated human endometrial cancer Ishikawa cell. Representative optical phase contrast image (d), AFM contact height image (e) and AFM stiffness image (f) of an Ishikawa cell treated for 2 hours with 25 ng/ml LeftyA. Representative optical phase contrast image (g), AFM contact height image (h) and AFM stiffness image (i) of an Ishikawa cell 15 min after addition of 10 μM cytochalasin D. Mean cell stiffness 〈E〉 (j), mean cell area 〈A〉 (k) and mean cell volume 〈V〉 (l) of untreated, LeftyA and cytochalasin D treated Ishikawa cells (〈E〉_Contr = 0.73 kPa, 〈E〉_LeftyA = 0.40 kPa, 〈E〉_CytoD = 0.21 kPa, 〈A〉_Contr = 2084 μm², 〈A〉_LeftyA = 1579 μm², 〈A〉_CytoD = 1580 μm², 〈V〉_Contr = 2934 μm³, 〈V〉_LeftyA = 2403 μm³, 〈V〉_CytoD = 1974 μm³). Error bars represent SEM of geometric mean (j) and SEM of arithmetic mean (k,l). *P < 0.05; **P < 0.01; ***P < 0.001 using one-way ANOVA followed by two-tailed Tukey’s test.

Figure 2. Effect of LeftyA on actin polymerization in Ishikawa cells.

(a) Representative original Western blot of soluble G-actin over F-actin in human endometrial cancer Ishikawa cells after a 2 hour treatment without (−LeftyA) and with (+LeftyA) LeftyA (25 ng/ml). (b) Arithmetic means ± SEM (n = 6; arbitrary units) of soluble G-actin over F-actin ratio in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml). (c) Representative original histogram of DNase1 (G-actin; Left) and Phalloidin (F-actin; Right) binding in Ishikawa cells after a 2 hour treatment without (dark grey) and with (grey) LeftyA (25 ng/ml). (d) Arithmetic means ± SEM (n = 6; arbitrary units) of G-actin over F-actin ratio in Ishikawa cells after a 2 hours treatment without and with LeftyA (25 ng/ml). (e) Original confocal images of eflour660-phalloidin binding to F-actin (red) and SYTOX Green for nuclei (blue) in Ishikawa cells treated with or without LeftyA (white bar 20 μm) (f) arithmetic means  ±  SEM (n  =  6) of actin fluorescence in Ishikawa cells with and without LeftyA treatment. (g) Averaged FRAP curve (n = 21) of control cells or cells treated for 2h with LeftyA. Error bars denote SEM of arithmetic mean of normalized fluorescence intensity at each time point. (h) Mean half-time of recovery for LeftyA-treated (Green) and untreated Ishikawa cells (Control; Blue) obtained by FRAP. *P < 0.05; **P < 0.01; ***P < 0.001 using Student’s t-test.

Figure 3. Effect of LeftyA on Rac1 transcript levels and activity in Ishikawa cells.

(a) Arithmetic means ± SEM (n = 4) of Rac1 normalized to L19 transcript levels in human endometrial cancer Ishikawa cells following treatment with LeftyA (25 ng/ml) for 2 hours. Data are depicted as fold induction relative to transcript levels of untreated samples. (b) Representative original Western blots showing activated Rac1 and total Rac1 protein abundance in human endometrial cancer Ishikawa cells after 2 hours culture in the absence or presence of LeftyA (25 ng/ml). (c) Arithmetic means ± SEM (n = 4, arbitrary units) of phospho-Rac1 protein ratio normalized to GAPDH in Ishikawa cells after 2 hours culture in the absence or presence of LeftyA (25 ng/ml). *P < 0.05; **P < 0.01; ***P < 0.001 using Student’s t-test.

Figure 4. Effect of LeftyA on actin polymerization in Ishikawa cells in absence or presence of Rac1 inhibitor.

(a) Representative original Western blot of soluble G-actin over F-actin in human endometrial cancer Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the Rac1 inhibitor NSC23766 trihydrochloride (100 μM). (b) Arithmetic means ± SEM (n = 3; arbitrary units) of soluble G-actin over F-actin ratio in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the Rac1 inhibitor NSC23766 trihydrochloride (100 μM). (c) Representative original histogram of DNase1 (G-actin; Upper) and Phalloidin (F-actin; Lower) binding in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the Rac1 inhibitor NSC23766 trihydrochloride (100 μM). (d) Arithmetic means ± SEM (n = 5 arbitrary units) of G-actin over F-actin ratio in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the Rac1 inhibitor NSC23766 trihydrochloride (100 μM) *P < 0.05; **P < 0.01; ***P < 0.001 using Student’s t-test.

Figure 5. Effect of LeftyA on actin polymerization in Ishikawa cells in absence or presence of PAK1 inhibitor.

(a) Representative original Western blot of soluble G-actin over F-actin in human endometrial cancer Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the PAK1 inhibitor IPA-3 (50 μM). (b) Arithmetic means ± SEM (n = 6; arbitrary units) of soluble G-actin over F-actin ratio in Ishikawa cells after a 2 hour treatment without and with Lefty A (25 ng/ml) in the absence and presence of the PAK1 inhibitor IPA-3 (50 μM). (c) Representative original histogram of DNAse1 (G-actin; Upper) and Phalloidin (F-actin; Lower) binding in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the PAK1 inhibitor IPA-3 (50 μM). (d) Arithmetic means ± SEM (n = 5; arbitrary units) of G-actin over F-actin ratio in Ishikawa cells after a 2 hour treatment without and with LeftyA (25 ng/ml) in the absence and presence of the PAK1 inhibitor IPA-3 (50 μM). *P < 0.05; **P < 0.01; ***P < 0.001 using Student’s t-test.

Figure 6. Schematic showing how stiffness of Ishikawa cells is affected by treatment with LeftyA via Rac1 and PAK1 pathways.