Role of transforming growth factor-β1 pathway in angiogenesis induced by chronic stress in colorectal cancer

Figures & data

Figure 1. Chronic restraint stress promoted tumor growth and weakened the efficacy of bevacizumab.

Tumor-bearing mice received normal saline, bevacizumab, propranolol, or CRS for 21 d. The tumor volume curve (a) and representative tumor-bearing mice in each group (b) indicated that CRS accelerated tumor growth and attenuated the efficacy of bevacizumab. Propranolol blocks the effects of CRS. Similar results were observed for tumor weight (c). *p < .05, vs. the NS group; #p < .05, vs. the Beva group.

Figure 2. Propranolol inhibited chronic restraint stress-stimulated angiogenesis in vivo.

Frozen sections of HT-29 tumor tissues from mice that received normal saline, bevacizumab, propranolol, or CRS were stained with an anti-CD31 antibody (a) (brown; magnification, ×200). MVD was estimated by averaging the total number of immunoreactive endothelial cells from five random fields (b). Bevacizumab decreased MVD, and CRS significantly increased MVD. The efficacy of bevacizumab was attenuated by CRS, whereas the efficacy of CRS was reversed by propranolol. *p < .05, vs. the NS group; #p < .05, vs. the Beva group.

Figure 3. Chronic restraint stress and norepinephrine induced TGF-β1 secretion.

CRS significantly increased TGF-β1 levels in mouse serum, and propranolol inhibited this elevation (a). Norepinephrine stimulated TGF-β1 secretion in SW480 cell culture supernatant and was blocked by propranolol (c). However, Ly2157299 or 2-ME did not reverse the NE effect. After NE treatment for 48 h, TGF-β1 levels were not significantly increased in HT-29 cells, but exhibited an increasing trend (b). ∆, p < .05 versus the NS group; •, p < .05 versus the Beva group; *, p < .05 versus the control group; #, p < .05 versus the NE group.

Figure 4. The mRNA and protein expression of TGF-βRI in HT-29 and SW480 cells after norepinephrine treatment.

NE induces TGF-βRI mRNA expression in HT-29 cells but not in SW480 cells (a). TGF-βRI protein expressions were similar in each group, both in HT-29 and SW480 cells (b). Quantification of proteins is presented in columns as the mean ± SD and showed no statistical difference (c). *p < .05, versus the control group; #p < .05, versus the NE group.

Figure 5. Propranolol or Ly2157299 blocked norepinephrine-induced mRNA and protein expression of HIF-1α.

After 48 h of treatment with NE, propranolol, or Ly2157299, mRNA (a) and protein (b) expression of HIF-1α were detected by RT-PCR and Western blotting, respectively. Quantification of HIF-1α protein is shown in the columns as the mean± SD (c). NE increased HIF-1α expression, whereas propranolol and Ly2157299 blocked this effect. *p < .05, versus the control group; #p < .05, versus the NE group.

Figure 6. Norepinephrine stimulated HT-29 and SW480 cells migration and invasion.

HT-29 and SW480 cells were treated with NE, propranolol, and Ly2157299. Representative images of cells that migrated to the lower membrane are shown in (HT-29) and (b) (SW480). The mean numbers of migratory (c and e) and invasive cells (d and f) were calculated from five random views and are presented in columns as mean± SD. Propranolol and Ly2157299 block NE-stimulated cell migration and invasion. *p < .05, versus the control group; #p < .05, versus the NE group.

Data availability statement

The data supporting the findings of this study are available from the corresponding author, JY, upon reasonable request.

The primer sequences used in this study were obtained from the National Center for Biotechnology Information (NCBI): https://www.ncbi.nlm.nih.gov/guide/dna-rna/.