Overexpression of connexin 43 reduces melanoma proliferative and metastatic capacity

Abstract

Background: Alterations in connexin 43 (Cx43) expression and/or gap junction (GJ)-mediated intercellular communication are implicated in cancer pathogenesis. Herein, we have investigated the role of Cx43 in melanoma cell proliferation and apoptosis sensitivity in vitro, as well as metastatic capability and tumour growth in vivo.

Methods: Connexin 43 expression levels, GJ coupling and proliferation rates were analysed in four different human melanoma cell lines. Furthermore, tumour growth and lung metastasis of high compared with low Cx43-expressing FMS cells were evaluated in vivo using a melanoma xenograft model.

Results: Specific inhibition of Cx43 channel activity accelerated melanoma cell proliferation, whereas overexpression of Cx43 increased GJ coupling and reduced cell growth. Moreover, Cx43 overexpression in FMS cells increased basal and tumour necrosis factor-α-induced apoptosis and resulted in decreased melanoma tumour growth and lower number and size of metastatic foci in vivo.

Conclusions: Our findings reveal an important role for Cx43 in intrinsically controlling melanoma growth, death and metastasis, and emphasise the potential use of compounds that selectively enhance Cx43 expression on melanoma in the future chemotherapy and/or immunotherapy protocols.

Figure 1

Connexin 43 expression in human melanoma cells. Total Cx43 expression pattern was evaluated by immunofluorescence staining and analysed by flow cytometry (A) or confocal microscopy (B and C) in four different human melanoma cell lines (FMS, HF, DFW and DFB). (A) The histogram shows the relative expression of Cx43 in the four human melanoma cell lines. Filled grey histogram correspond to the isotype control staining. (B) Representative images of Cx43 (green) distribution. Blue: nucleus staining (Hoechst). The Cx43 accumulated in GJ plaques are indicated with white arrows. Scale bars, 5 μM. (C) Quantification of Cx43 fluorescence signal intensities. *P<0.05; **P<0.01.

Figure 2

Connexin 43-mediated cell coupling and proliferation level in human melanoma cells. (A) Melanoma cells were preloaded with calcein-AM (GJ-diffusible dye) or Dil-CM (non-GJ-diffusible dye) and cocultured for 60 min in a 1 : 1 ratio (calcein⁺Dil⁻ donor cells : calcein⁻Dil⁺ acceptor cells) in the presence of control (gap20) or inhibitory (1848) Cx43-specific mimetic peptide. The calcein transfer from the Dil⁻ to Dil⁺ cells was assessed by flow cytometry. (A, lower panel) Bar graphs show the percentage of calcein⁺Dil⁺ cells and correspond to results from three independent experiments. (B) Proliferation was determined by CFSE (carboxyfluorescein succinimidyl ester) dilution and flow cytometry in synchronised melanoma cell cultures. Percentages of proliferating cells are indicated in the representative densities plots and in the bar graphs (n=3). (C) Nonobese diabetic (NOD)/LtSz-scid IL2Rγ^null mice received a single subcutaneous injection of FMS or DFB (500 000 cells), and the growth of primary tumours was monitored for 15 days (n=3 mice per group). The growth curves are statistically different (P<0.01). *P<0.05; **P<0.01; ***P<0.001.

Figure 3

Connexin 43 overexpression increases GJ coupling and reduces melanoma cell proliferation. (A–C) Connexin 43 expression was measured in FMS cells stably transfected with Cx43 or in control cells (transfected with the control empty vector (EV)) by western blot (A), flow cytometry (B) or immunofluorescence microscopy (C). (A) β-Actin was used as a loading control. (B) Grey histogram corresponds to isotype control. (C, left) Cx43 (green)-positive GJ plaque structures are indicated with white arrows. Blue: nucleus staining (Hoechst). Scale bars, 5 μM. (C, right) Bar graphs show the quantification of the Cx43 fluorescence intensities. (D) FMS-EV or FMS-Cx43 melanoma cells were preloaded with calcein-AM or Dil-CM and cocultured for 60 min in a 1 : 1 ratio (calcein⁺Dil⁻ donor cells : calcein⁻Dil⁺ acceptor cells) in the presence of control (gap20) or inhibitory (1848) Cx43-specific mimetic peptides. Alternatively, cells were treated with a hemichannel-specific Cx43-mimetic inhibitory peptide (P5). Bar graphs show the percentage of calcein⁺Dil⁺ cells (n=3). (E) The incorporation of [³H]thymidine assays were carried out to determine the proliferative rate of two FMS-Cx43 clones (C1 and C2) or the control FMS-EV cells. Cells were cultured in the presence of control gap20 (−) or inhibitory 1848 peptides (+) (n=3). *P<0.05; ***P<0.001.

Figure 4

Connexin 43 overexpression increases basal and tumour necrosis factor-α (TNF-α)-induced apoptosis in melanoma cells. Representative dot plots (A) and graphs show the distribution of propidium iodide (PI) and annexin-V (AV) staining in FMS-EV or FMS-Cx43 cells, untreated or treated for 24 h with 200 ng ml⁻¹ of TNF-α. (B) Bar graphs show the early apoptotic (AV⁺PI⁻) or total dying (AV⁺) cells represented as the percentage of positive cells (upper panels) or as fold change relative to untreated cells (lower panels). (C) The basal level of early apoptotic (AV⁺PI⁻) or total dying (AV⁺) FMS-Cx43 cells are shown as fold change relative to FMS-EV cells (n=3). *P<0.05; **P<0.01; ***P<0.001.

Figure 5

Connexin 43 overexpression reduces melanoma tumour growth and prevents metastasis in a xenotransplantation model. (A) Nonobese diabetic NOD/LtSz-scid IL2Rγ^null (NOD/SCID) mice received a single subcutaneous injection of FMS-EV or FMS-Cx43 (500 000 cells), and the growth of primary tumours was monitored for 15 days (n=3 mice per group). (B) Nonobese diabetic/SCID mice received a single intravenous injection of FMS-EV, FMS-Cx43 (250 000 cells) or PBS (n=6 mice per group). The mice were terminated on day 18 after inoculation and the lungs were examined. The graph bar shows the number of metastasis counted in the lungs of six mice per group. Scale bar, 1 cm. (C) Immunohistochemical staining of Mart-1 in melanoma lung metastasis biopsies of NOD/SCID mice injected with FMS-EV or FMS-Cx43 cells (overview of six mice per group). Scale bar, 25 μM. (D) Immunofluorescence analysis showed in vivo Cx43 expression (green) and Cx43-based GJ formation (white arrows) on melanoma lung metastasis biopsies of NOD/SCID mice injected with FMS-EV or FMS-Cx43 cells. Blue: nucleus staining (Hoechst). Enlarged images (3 × ) marked by the white boxes are shown on the right. Scale bar, 30 μM. (E) Immunohistochemical staining of active caspase-3 (black arrows) in melanoma lung metastasis biopsies of NOD/SCID mice injected with FMS-EV or FMS-Cx43 cells (overview of six mice per group). Below and upper each photograph showed the number of active capsase-3 positive cells per 14 μM². Scale bar, 60 μM. *P<0.05; ***P<0.001.