A high throughput screen identifies Nefopam as targeting cell proliferation in β-catenin driven neoplastic and reactive fibroproliferative disorders

Abstract

Fibroproliferative disorders include neoplastic and reactive processes (e.g. desmoid tumor and hypertrophic scars). They are characterized by activation of β-catenin signaling, and effective pharmacologic approaches are lacking. Here we undertook a high throughput screen using human desmoid tumor cell cultures to identify agents that would inhibit cell viability in tumor cells but not normal fibroblasts. Agents were then tested in additional cell cultures for an effect on cell proliferation, apoptosis, and β-catenin protein level. Ultimately they were tested in Apc1638N mice, which develop desmoid tumors, as well as in wild type mice subjected to full thickness skin wounds. The screen identified Neofopam, as an agent that inhibited cell numbers to 42% of baseline in cell cultures from β-catenin driven fibroproliferative disorders. Nefopam decreased cell proliferation and β-catenin protein level to 50% of baseline in these same cell cultures. The half maximal effective concentration in-vitro was 0.5 uM and there was a plateau in the effect after 48 hours of treatment. Nefopam caused a 45% decline in tumor number, 33% decline in tumor volume, and a 40% decline in scar size when tested in mice. There was also a 50% decline in β-catenin level in-vivo. Nefopam targets β-catenin protein level in mesenchymal cells in-vitro and in-vivo, and may be an effective therapy for neoplastic and reactive processes driven by β-catenin mediated signaling.

Figure 1. Nefopam inhibits cell viability in cultures from aggressive fibromatosis and hypertrophic cutaneous wounds.

Data from the Sulforhodamine B assay. The mean value for the cell culture treated with carrier was arbitrarily defined as 100 for each cell type. A) Data for a dose of 10 µM of Nefopam. There was a significantly lower level of cell viability in the aggressive fibromatosis or the hypertrophic scar cultures compared to that seen in normal tissues. B) Data from different doses of Nefopam in cell cultures from aggressive fibromatosis or from hypertrophic scars, showing a dose dependent decrease in cell viability. C) Time course data for cell viability after days of treatment with 10 µM of Nefopam in cell cultures from aggressive fibromatosis or from hypertrophic scars. Data is given as means and 95% confidence intervals. Control data is generated by treatment with diphenhydramine. Treatment with carrier alone gave identical results as for diphenhydramine. An asterisk over a data point indicates a significant difference form the control cell cultures.

Figure 2. Nefopam inhibits cell proliferation in fibroblast cell cultures from hypertrophic cutaneous wounds and from aggressive fibromatosis tumors.

Percent BrdU incorporation or annexin V staining from the various cell cultures treated with 10 µM of Nefopam. Data is given as means and 95% confidence intervals. An asterisk over a data point indicates a significant difference form the control cell cultures. There was a significant difference in BrdU incorporation in the tumor and hyperplastic scar cultures treated with Nefopam compared to treatment with carrier alone. Treatment with diphenhydramine resulted in the identical findings as for treatment with the carrier.

Figure 3. Nefopam modulates β-catenin protein level.

A) Western blot analysis for β-catenin in aggressive fibromatosis and hypertrophic scar cell cultures treated with Nefopam. A representative Western blot for total β-catenin is shown at the top of the panel. The data is shown in graphical form in the lower part of the panel, as mean and 95% confidence intervals for the relative density compared to the loading control for six pairs of cultures for each condition. An asterisk over a data point indicates a significant difference form the control cell cultures. There is a decrease in β-catenin level with Nefopam treatment in cell cultures from both conditions. B) A representative Western blot for β-catenin and serine-9-phosph-GSK-3-β in normal fibroblasts treated with Wnt3a is shown at the top of the panel. Composite data is shown in graphical form in the lower part of the panel. Actin and total GSK3-β are used as loading controls. There is an increase in serine-9-phosph-GSK-3-β and β-catenin with Wnt3a treatment, which is significantly lowered, close to the baseline level, with Nefopam treatment. C) A representative Western blot for β-catenin in fibroblasts expressing a stabilized form of β-catenin lacking exon three is shown on the top of the panel. Composite data is shown in graphical form on the bottom of the panel. There is a decrease in β-catenin with Nefopam treatment.

Figure 4. Nefopam suppresses the neoplastic phenotype in murine aggressive fibromatosis tumors.

The number of tumors found in male Apc1638N mice. Data is given as the mean and 95% confidence intervals for the number of tumors formed in male mice in no treatment; 0.1% DMSO (carrier); or Nefopam treatment groups. Data is given as means and 95% confidence intervals. An asterisk over a data point indicates a significant difference form the control cell cultures. Mice treated with the carrier showed a comparable number of tumors to mice that received no treatment, while mice treated with Nefopam developed significantly fewer tumors.

Figure 5. Nefopam regulates scar size and β-catenin level in cutaneous wound repair.

A to D) Representative histologic sections through the widest margin of scars 14 days after wounding. A) A wound from a mouse treated with TGF-β. B) A wound in a mice treated with carrier only. C) A wound from a mouse treated with TGF-β and Nefopam. D) A wound from a mouse treated with Nefopam. Arrows show the widest width of the scar. The black line is 500 µm in length. E) A graphical representation of the mean and 95% confidence intervals of the widest diameter of the scars in µms. F) A representative Western blot mice showing the β-catenin protein levels in wounds from mice treated with TGF-β, Nefopam, or both, showing that Nefopam decreases β-catenin protein level in the wounds.