Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide

Figure 1 Parathyroid hormone-related peptide modulates the protein expression of Met receptor in HCT116 cells. Cells were treated with or without 10^-8 mol/L parathyroid hormone-related peptide (PTHrP) for different times. The protein levels of pro-Met (Met precursor) and Met [tyrosine kinase receptor (RTK) mature form] were analyzed by Western blot to investigate whether the molecular mechanisms trigged by PTHR type 1 (PTHR1) after binding of PTHrP are capable of modulating the expression of the mature form of the RTK Met in HCT116 cells. GAPDH protein levels were determined as a control of the amount of proteins present in the membrane, since this protein is not substantially modified with the treatment by the cytokine. Graph bars represent the average of the results obtained from three independent experiments. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; Met: Receptor tyrosine kinase Met; PTHrP: Parathyroid hormone-related peptide. ^aP < 0.05; ^bP < 0.01.

Figure 2 Parathyroid hormone-related peptide increases Met phosphorylation and activation through Src kinase in HCT116 cells. A: Cells were exposed to 10^-8 mol/L parathyroid hormone-related peptide (PTHrP) 10^-8 mol/L for 3 min, 5 min, and 10 min. The protein levels of Met and p-Met (phosphorylated in the residues Tyr1234 and Tyr1235) were assessed by Western blot to investigate whether PTHrP is capable of modulating Met phosphorylation and activation in HCT116 cells. Graph bars represent the average of the results obtained from three independent experiments; B: Cells were pre-treated with PP1, a selective Src inhibitor, for 30 min and then exposed to 10^-8 mol/L PTHrP for 3 min to evaluate whether Src kinase mediates the effect of PTHrP on Met activation in HCT116 cells. Controls were run by adding an equivalent volume of DMSO, the vehicle of the inhibitor. The protein levels of p-Met and Met were evaluated by Western blot. Graph bars represent the average of the results obtained from three independent experiments. In all experiments, GAPDH protein levels were determined as a control of the amount of proteins present in the membrane, since this protein is not substantially modified with the treatment by the cytokine. DMSO: Dimethylsulfoxide; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; Met: Receptor tyrosine kinase Met; p-Me: Phospho-Met (Tyr1234/1235); PTHrP: Parathyroid hormone-related peptide. ^aP < 0.05; ^bP < 0.01.

Figure 3 Parathyroid hormone-related peptide modulates Met activation through the mitogen-activated protein kinase signaling pathway in HCT116 cells. Cells were treated with PD-98059 or SB-203580, a selective mitogen-activated protein kinase (MAPK) kinase (MEK) or P38 MAPK inhibitor, respectively, for 30 min and then exposed to 10^-8 mol/L PTHrP for 1 h to evaluate whether ERK1/2 MAPK and/or p38 MAPK mediate the effect of PTHrP on Met activation in HCT116 cells. Controls were run by adding an equivalent volume of dimethylsulfoxide, the vehicle of the inhibitors. The protein levels of Met phosphorylated in the residues Tyr1234 and Tyr1235 were assessed by Western blot. These phosphorylation sites constitute activating domains of the receptor. GAPDH protein levels were determined as a control of the amount of proteins present in the membrane, since this protein is not substantially modified with the treatment by the cytokine. Graph bars represent the average of the results obtained from three independent experiments. DMSO: Dimethylsulfoxide; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; Met: Receptor tyrosine kinase Met; p-Met: Phospho-Met (Tyr1234/1235); PTHrP: Parathyroid hormone-related peptide. ^aP < 0.05.

Figure 4 Parathyroid hormone-related peptide increases mRNA levels of Met in HCT116 cells. Colon cancer cells were exposed to parathyroid hormone-related peptide (PTHrP) for 15 min, followed by real-time polymerase chain reaction analysis to detect Met mRNA levels as described in Materials and Methods to evaluate whether PTHrP modulates Met mRNA levels in HCT116 cells. Graph bars represent the average of the results obtained from three independent experiments. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; Met: Receptor tyrosine kinase Met; PTHrP: Parathyroid hormone-related peptide. ^aP < 0.05.

Figure 5 Parathyroid hormone-related peptide promotes events related to the aggressive behavior of HCT116 cells through the Met signaling pathway. HCT116 cells were pre-incubated with SU11274, a specific Met inhibitor, for 30 min and then treated with or without parathyroid hormone-related peptide (PTHrP; 10^-8M). A: Trypan blue technique showed that Met inhibition decreased the cell proliferation induced by PTHrP at 24 h; B: Images from wound healing assay show that Met inhibition reverted the wound closure promoted by PTHrP at 5 h and 24 h; C: E-cadherin protein levels analyzed by Western blot to investigate whether Met is involved in the decrease of this epithelial-mesenchymal transition (EMT) program marker induced by PTHrP in HCT116 cells. Using the Image J-NIH program, we performed the analysis of the parameters related to cell morphology. The arrows indicate the morphological changes corresponding to the transition from a polygonal structure to spindle-like structure related to EMT program progress observed when the cells were treated for 24 h with PTHrP or PTHrP plus dimethylsulfoxide (DMSO). In all experiments, a control with DMSO, the vehicle of the inhibitor, was performed. Graph bars represent the average of the results obtained from two independent experiments. DMEM: Dulbecco’s Modified Eagle Culture Medium; DMSO: Dimethylsulfoxide; EMT: Epithelial to mesenchymal transition; PTHrP: Parathyroid hormone-related peptide. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 6 Analysis of the aspect ratio (major/minor axis) of HCT116 cells, obtained using the Image J-NIH program. The table shows values of parameters associated to cell morphology: Area; perimeter; round; major axis; minor axis; and aspect radio, relation between major axis and minor axis (AR). Graph bars show the AR for each condition. The increase of the degree of cell elongation at 24 h of exposure to parathyroid hormone-related peptide was significantly reversed in the presence of the Met inhibitor. DMSO: Dimethylsulfoxide; PTHrP: Parathyroid hormone-related peptide; SU: SU11274. ^bP < 0.01.

Figure 7 Parathyroid hormone-related peptide attenuates the cytotoxicity of CPT11, oxaliplatin, and doxorubicin through the Met signaling pathway. A-C: HCT116 cells were pre-incubated with SU11274, a specific Met inhibitor, and then treated with PTHrP (10^-8 M) and/or irinotecan (CPT-11, 10 μM) (A), oxaliplatin (OXA, 10 μM) (B), or doxorubicin (DOXO, 5 μM) (C) for 24 h. The number of viable cells was quantified by Trypan blue technique. In each experiment, a control with DOXO, the vehicle of the inhibitor, was performed. Graph bars represent the average of the results obtained from two independent experiments. CPT-11: Irinotecan; DMSO: Dimethylsulfoxide; DOXO: Doxorubicin; OXA: Oxaliplatin; PTHrP: Parathyroid hormone-related peptide; SU: SU11274. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 8 Parathyroid hormone-related peptide increases the protein expression of Met and parathyroid hormone receptor type 1 in tumor xenografts. Met and parathyroid hormone-related peptide receptor type 1 (PTHR1) protein levels were evaluated by the immunohistochemistry technique in the HCT116 cell tumor-bearing nude mouse model. Tumor sections were stained with an anti-Met antibody or anti-PTHR1 antibody. Images (400 ×) are from the tumor treated with saline solution (left) or with PTHrP (right). The immunostaining was quantified with the Fiji image processing package of the Image J-NIH program. Scale bar: 50 μM. Met: Receptor tyrosine kinase Met; PTHR1: Parathyroid hormone receptor type 1; PTHrP: Parathyroid hormone-related peptide. ^aP < 0.05; ^bP < 0.01.

Figure 9 Relation of Met and parathyroid hormone receptor type 1 staining intensity with tumor histological differentiation. A: Quantification of the immunostaining by optical density analysis using the FIJI plug-in from the Image J program; B: Representative images (200 ×) obtained by hematoxylin-eosin staining and by the immunohistochemistry technique performed for Met and PTHR1 for normal colorectal tissue and colorectal cancer tissue, with histological grades G1, G2, and G3. Scale bar: 10 μM. H-E: Hematoxylin-eosin staining; Met: Receptor tyrosine kinase Met; NT: Normal tissue; PTHR1: Parathyroid hormone receptor type 1.

Figure 10  Possible mechanism based on the action of parathyroid hormone-related peptide in the regulation of Met gene expression and its activation. The binding of parathyroid hormone-related peptide (PTHrP) to PTHR type 1 (PTHR1) in HCT116 cells promotes the up-regulation of Met gene expression. PTHrP also induces the activation of Met through Src kinase and the mitogen-activated protein kinases (MAPKs) pathway (Erk 1/2 MAPK and p38 MAPK). Once activated, Met signaling leads to molecular changes in the tumor cell that promote events related to the aggressive behavior of colorectal cancer cells. CRC: Colorectal cancer; Erk 1/2: Extracellular signal-regulated kinases 1/2; Met: Receptor tyrosine kinase Met; P: Activators domains phosphorylated; PTHR1: Parathyroid hormone receptor type 1; PTHrP: Parathyroid hormone-related peptide.