Vasopressin induces apoptosis but does not enhance the antiproliferative effect of dynamin 2 or PI3K/Akt inhibition in luminal A breast cancer cells

Abstract

Breast cancer cells abnormally express vasopressin (AVP) and its receptors. The effect of AVP is largely orchestrated through its downstream signaling and by receptor-mediated endocytosis (RME), in which Dynamin 2 (Dyn2) plays an integral role in vesicle closure. In this work, luminal A breast cancer cells were treated with AVP, and then Dynasore (DYN) was employed to inhibit Dyn2 to explore the combined effect of AVP and Dyn2 inhibition on the survival of breast cancer cells. The results revealed that DYN alone demonstrated a concentration-dependent cytotoxic effect in AVP untreated cells. Apoptosis developed in 29.7 and 30.3% of cells treated with AVP or AVP+DYN, respectively, compared to 32.5% in cells treated with Wortmannin (Wort, a selective PI3K pathway inhibitor). More apoptosis was observed when cells were treated with DYN+Wort in presence or absence of exogenous AVP. Besides, 2 or 4- fold increases in the expression of Bax and Caspase-3, were observed in cells exposed to AVP in absence or presence of DYN, respectively. This was associated with higher levels of the autophagy marker (LC3II protein). Meanwhile, the activation of Akt protein, sequentially decreased in the same pattern. Cell's invasion decreased when they were exposed to AVP alone or combined with DYN or/and Wort. Conclusively, although many reports suggested the proliferative effect of AVP, the results predict the antiproliferative and antimetastatic effects of 100 nM AVP in luminal A breast cancer cells. However, the hormone did not enhance the cytotoxic effect of Dyn 2 or PI3K pathway inhibition. Summary of the Dynamin 2 independent AVP antiproliferative effects. Breast cancer cells expresses AVP as a Prohormone (A). At high dose of AVP, the hormone is liganded with AVP receptor (B) to initiate RME, where the endosomed complex (C) is degraded through the endosome-lysosome system, as a part of signal management. These events consume soluble Dyn2 in neck closure and vesicle fission (D). This makes the cells more substitutable to the direct apoptotic effect of DYN (E). Alternatively, at lower AVP doses the liganded AVP may initiate cAMP-mediated downstream signaling (F) and cellular proliferation. In parallel, Wort inhibits PIP2-PIP3 conversion (G) and the subsequent inhibition of PI3K/Akt/mTOR pathway leading to cell death.

Keywords: Arginine vasopressin; Breast cancer; Dynamin 2; Dynasore; Wortmannin.

Fig. 1

Cytotoxic effect of Dynamin inhibition. Dynasore (A) induced a concentration-dependent cytotoxic effect in breast cancer cells. MCF-7 cells were incubated for 24 h with varying concentrations of Dynasore and cells metabolic activity was determined by MTT assay. Data are expressed as means ± SD of multiple experimental replicates (n = 5) (B). C–H are representative phase contrast photomicrographs of cells treated with AVP (C), DYN after cells stimulation with AVP (D), Wort (E), DYN in combination with Wort (F) or DYN in combination with Wort and AVP (G). Apoptotic morphological abnormalities including cell shrinking, rounding and detachment (magnification of 400×)

Fig. 2

Apoptotic effect of dynamin 2 inhibition in invasive breast cancer cells. Plots (A) through (F) show Annexin V-FITC and PI-stained MCF 7 cells untreated (A), treated with AVP (B), DYN after stimulation with AVP (C), Wort (D), DYN in combination with Wort (E) or prestimulated with AVP then treated with a combination of Wort and AVP (F). In each scatter plot, the lower left quadrant, the upper left quadrant, the lower right quadrant and upper right quadrant represent the percent of viable cells, dead cells, early apoptotic cells and late apoptotic cells, respectively. Apoptosis was observed in DYN treated cells and more apoptosis developed in presence of AVP or Wort combined with DYN treatment (G). Results are presented as mean ± SD. DYN Dynasore; Wort Wortmannin, AVP arginine vasopressin; (*) refers to P < 0.001 and indicating highly significant viability or total apoptosis relative to DMSO-treated cells

Fig. 3

AVP stimulates autophagy and enhances the autophagy effect of Dynamin inhibition, promotes cytotoxic PI3K-independent autophagy in breast cancer cells. McF-7 cells were seeded with an initial cell density 4×10⁴ grown in nutrient-rich conditions and left untreated (A), transiently treated with AVP (B), DYN after cells stimulation with AVP (C), Wort (D), DYN in combination with Wort (E) or DYN in combination with Wort and AVP (F). Following treatments, the expression of the autophagy marker (LC3II protein) was determined by flow cytometry. The autophagy was significantly induced in Dynamin inhibited cells and reduced in Wort-treated cells. Dyn2 inhibition induced significant increase in LC3II even in cells in which PI3K was inhibited by Wort. Data are presented as mean ± SD (G). DYN Dynasore; Wort Wortmannin, AVP arginine vasopressin; (_***) and (**): refer to extremely significant (P < 0.001) or highly significant differences (P < 0.01) between the indicated group compared to DMSO-treated cells

Fig. 4

Dynamin inhibition induces cell cycle arrest in G0/G1 phase in breast cancer cells. MCF-7 were treated with AVP (C), DYN after cells stimulation with AVP (D), Wort (E), DYN in combination with Wort (F) or DYN in combination with Wort and AVP (G). After cell fixation, they were PI-stained and analyzed for cell cycle by flow cytometry. A–E are representative scatter plot of cell cycle analysis and (F) is bar graph of cell fractions distributed in different phases. DYN and AVP arrested cells in G0/G1 phase

Fig. 5

Expression of total Akt and phosphorylated Akt (P-Akt) proteins at (Ser473) in breast cancer cells stimulated with AVP and treated with Dynamin 2 and/or PI3/AKT inhibitors by Western blot analysis. Top panel (A) MCF-7 cells in which V2R receptor was prestimulated with AVP or PI3K pathway was inhibited by Wort in combination with the dynamins inhibitor (DYN). Cell lysate was analyzed using Akt or P-Akt monoclonal antibodies. Bottom (B) represents the mean of bands intensities of the quantification of Total Akt or P-Akt (Ser 473) as means ± SD of data from independent experiments (*) refers to P<0.001 and indicates significant changes in the corresponding cells compared to DMSO-treated cells; DYN Dynasore; Wort Wortmannin, AVP arginine vasopressin

Fig. 6

Relative expression of Bax, Caspase-3 and Pro-AVP genes in dynamin 2 inhibited breast cancer cells. MCF-7 cells were treated with DYN alone, after their prestimulation with AVP, or cotreated with Wort. Cells mRNA was isolated, reverse transcribed, and the cDNA was used as a template in qRT-PCR to determine the relative expression. Dynamin inhibition was associated with up-regulation of apoptosis-related genes and Pro-AVP expression (*) indicates significant difference between the indicated cells versus control cells; (ns) indicates non-significant

Fig. 7

AVP reduced the invasion potential of breast cancer cells assessed by transwell assay. Untreated MCF-7 cells (A), cells stimulated with AVP (B), AVP + DYN (C), DYN + Wort (E), or AVP + DYN + Wort (F), respectively. After treatments, cells were resuspended in serum-free media, added to the upper chamber (inserts), kept at 37 °C for 18 h, fixed with formaldehyde, permeabilized by methanol, washed and then stained with Giemsa stain. Images were captured under inverted microscope and analyzed by Image J. (E) represents the relative changes in the mean area, representing the migrated cells, where treatments significantly (P < 0.001) reduced cell invasiveness