Low‑intensity pulsed ultrasound promotes apoptosis and inhibits angiogenesis via p38 signaling‑mediated endoplasmic reticulum stress in human endothelial cells

Abstract

Aberrant increase in angiogenesis contributes to the progression of malignant solid tumors. An alternative anti‑angiogenesis therapy is critical for cancer, since the current anti‑angiogenesis drugs lack specificity for tumor cells. In the present study, the effects and mechanisms of low‑intensity pulsed ultrasound (LIPUS) on human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMECs) were investigated, and the therapeutic potential of this technology was assessed. HUVECs and HMECs were treated with LIPUS (0.5 MHz; 210 mW/cm2) for 1 min and cultured for 24 h. Flow cytometry and Cell Counting Kit‑8 assays demonstrated that LIPUS treatment at a dose of 210 mW/cm2 promoted apoptosis and decreased the viability in HUVECs and HMECs. Real‑time cell analysis also revealed that LIPUS did not affect the proliferation or migration of HUVECs. An endothelial cell tube formation assay indicated that LIPUS treatment inhibited the angiogenic ability of HUVECs and HMECs. Furthermore, LIPUS increased the protein levels of the apoptosis‑associated cleaved Caspase‑3 and decreased the B‑cell lymphoma‑2 levels. LIPUS increased the phosphorylation of p38 mitogen‑activated protein kinase (MAPK), and the levels of endoplasmic reticulum (ER) stress‑associated markers, including activating transcription factor‑4 (ATF‑4) and phosphorylated eukaryotic initiation factor 2α (eIF2α). The p38 inhibitor SB203580 reversed the pro‑apoptotic and anti‑angiogenic effects of LIPUS in cells. Finally, inhibition of p38 decreased the LIPUS‑induced elevation of p‑eIF2α and ATF‑4 levels. Taken together, these results suggested that LIPUS promoted apoptosis and inhibited angiogenesis in human endothelial cells via the activation of p38 MAPK‑mediated ER stress signaling.

Figures

Figure 1.

LIPUS promoted apoptosis and inhibited cell viability in HUVECs and HMECs. Flow cytometry analysis of apoptosis rate in (A) HUVECs and (B) HMECs treated with different dose of LIPUS (70–280 mW/cm2). Quantification of early apoptosis in (C) HUVECs and (D) HMECs. Viability of (E) HUVECs and (F) HMECs treated with different doses of LIPUS (70–280 mW/cm2) was assessed by CCK-8 assay. Real-time monitoring of the (G) proliferation and (H) migration of HUVECs by real-time cell analysis. All values are expressed as the mean ± standard error of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001. LIPUS, low-intensity pulsed ultrasound; HUVECs, human umbilical vein endothelial cells; HMECs, human microvascular endothelial cells; CCK-8, Cell Counting Kit-8; Ctrl, control; NS, not significant.

Figure 2.

Effects of LIPUS on HUVEC and HMEC tube formation. Following incubation for 8 h, images (magnification, 10×) of tube-like structures in the Ctrl and LIPUS-treated cells were obtained using a microscope camera, and quantification of branches was performed with ImageJ software. (A) Images of HUVEC tube formation and (B) quantified results. (C) Images of HMEC tube formation and (D) quantified results. All values are expressed as the mean ± standard error of three independent experiments (scale bars=100 µm). **P

Figure 3.

Low-intensity pulsed ultrasound regulated the expression of apoptosis-associated proteins. (A) Cleaved Caspase-3, Bcl-2, Bax and LC3B levels, measured by western blotting. (B) Quantification of the relative levels of cleaved Caspase-3, Bax and LC3-II, which were respectively normalized to β-tubulin, Bcl-2 and LC3-I levels. (C) Phosphorylation and total protein levels of p38, ERK and JNK were assessed by western blotting. (D) Quantification of p-p38, p-ERK and p-JNK protein levels, normalized to their total protein levels. (E) Protein levels of ATF-4 and p-eIF2α were assessed by western blotting. (F) Quantification of ATF-4 and p-eIF2α protein levels, normalized to β-tubulin. All values are expressed as the mean ± standard error of three independent experiments. **P

Figure 4.

Inhibition of p38 phosphorylation rescued the pro-apoptotic and anti-angiogenic effects of low-intensity pulsed ultrasound. (A) Flow cytometry analysis of apoptosis rates in HUVECs treated with the p38 inhibitor SB203580. (B) Quantification of the percentage of early apoptotic HUVECs. (C) Western blots and (D) quantified protein levels of cleaved Caspase-3, Bcl-2 and Bax in HUVECs treated with S203580. (E) Images (magnification, 10×) of tube-like structures and (F) quantified results in HUVECs. (G) Images of tube-like structures and (H) quantified results in HMECs. Quantification of branches was conducted with ImageJ software. All values are expressed as the mean ± standard error of three independent experiments (scale bars=100 µm). **P##P<0.01 and ###P<0.001, vs. ultrasound group. HUVECs, human umbilical vein endothelial cells; HMECs, human microvascular endothelial cells; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; Ctrl, control.

Figure 5.

Low-intensity pulsed ultrasound-induced apoptosis was dependent on p38 phosphorylation. (A) Western blots and (B) quantified protein levels of p-p38 and p-ERK in HUVECs treated with S203580, normalized to the corresponding total protein levels. (C) Western blots and (D) quantified protein levels of p-eIF2α and ATF-4 in HUVECs treated with S203580, normalized to β-tubulin. All values are expressed as the mean ± standard error of three independent experiments. ***P###P<0.001 vs. ultrasound group. HUVECs, human umbilical vein endothelial cells; ERK, extracellular signal-regulated kinase; ATF-4, activating transcription factor-4; eIF2α, eukaryotic initiation factor 2α; p-, phosphorylated; Ctrl, control.