Occurrence of PACAP and PAC1R in human melanoma

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84 PACAP 27 and PACAP 38, and for PAC1R expression a specific rabbit polyclonal anti-human PAC1R antibody were used, respectively. For the consecutive series, PACAP staining was compared in side-by-side fashion with HMB-45. All 8 cases of metastatic melanoma and 4 out of 5 cases of primary melanoma showed positive PACAP 27 expression at different levels and only one case of primary melanoma was observed with PACAP 27-negative result. The total rate of more than 30% of the melanoma area staining positive for PACAP 27 in metastatic melanoma was almost twice higher than it in primary melanoma. On the other hand, positive immunoreactivity to PACAP 38 was consistently found in primary melanoma rather than in metastatic melanoma. Although more cases of primary and metastatic melanoma are needed to be evaluated, to investigate which of the PACAP subtypes will predominantly be produced in each melanoma stage, observations in this study showed that there are differential PACAP subtype expressions upon melanoma stages and that in the late phase of melanoma PACAP 38 is reduced. The pattern of distribution and the intensity of PACAP 27 staining differ in both melanoma stages as well. A strong PACAP 27 expression in metastatic melanoma was observed in a certain subsets of melanoma cells rather than in all melanoma cells, whereas primary melanoma cells exhibited diffuse staining of PACAP 27 overall and the total rate of PACAP 27 expression is still higher than it in primary melanoma. The present result reflects various subclasses of melanoma cells in a tumor cluster, which might help to understand the progression of melanoma.

Garcia-Fernandez et al reported PACAP expression in breast or prostate cancer, in which epithelial cells and stromal cells both showed an immunohistochemical staining with PACAP antibody, while PACAP was detected only in epithelial cells in normal breast and prostate tissues (Garcia-Fernandez, 2002 and 2004). Stromal cells in metastatic melanoma area, such as immune cells also showed PACAP immunoreactivity but it should be elucidated, whether infiltrated immune cells produce PACAP themselves or bind to PACAP produced by melanoma via PACAP receptors. Different from other healthy tissues, in healthy skin containing melanocytes in epithelial layer no expression of PACAP 27 or PACAP 38 was observed (data not shown, tested by immunofluorescence method). In addition, in metastatic melanoma a co-localization of PACAP 27 or PACAP 38 with melanoma marker HMB 45 was not found, whereas PACAP distribution is better associated with HMP 45 expression in primary melanoma. Furthermore, PACAP expression was not related to age and gender of patients or anatomical location of melanoma occurrence.

PACAP expression was detected in metastatic melanoma cell lines as well. Immunoblotting analysis, using specific mouse anti-PACAP monoclonal antibody to detect PACAP protein was performed in five established melanoma cell lines, a sample of melanoma metastasis in lymph node, and melanoma primary cell culture obtained from one patient. The results provided evidence of PACAP expression corresponding to the molecular size at 23 kDa, which accounts for around 200 amino acids and is slightly higher than the expected band (18 kDa; 176 amino acids). One of the reasons for a higher molecular weight of PACAP may be a different post-translational modification of the protein in melanoma. Using an inappropriate percentage of gel can be as well responsible for poor separation of protein, but the latter reason can be excluded because a prestained protein marker spanned a range

85 even down to 15 kDa on the membrane. Additionally, denaturation of protein can be a possible cause for the different size of targeted protein. However, PACAP expression in melanoma lines was confirmed by immunohistochemical analysis. Two melanoma lines, SK-Mel 37 and NW-Mel 450 were selected for detailed studies. These lines produced both PACAP-subtypes, PACAP 27 and PACAP 38.

Similar to metastatic melanoma tissues of patients, melanoma lines showed a stronger positive immunoreactivity for PACAP 27 rather than PACAP 38, supposing that PACAP 27 might be a main product in both melanoma lines. PACAP 27 or PACAP 38 was observed in cytoplasmic area in both melanoma lines, except the localization of PACAP 38 in SK-Mel 37. Another approach using an immunofluorescence showed again that SK-Mel 37 presented PACAP 38 in or near of the nucleic area but not in the cytoplasmic area. The localization of PACAP 38 in the cytoplasma has been demonstrated by Hegg et al (2003). The synthesis of each PACAP subtype might be separately regulated in a time dependent manner.

Further, immunohistochemical staining with specific PAC1R antibody demonstrated that healthy melanocytes did not express PAC1R, which however was detected for the first time in primary and metastatic melanoma with diverse distribution. Only two out of five cases of primary melanoma responded positively to specific PAC1R antibody, whereas seven out of eight cases of metastatic melanoma showed PAC1R expression with variable staining intensities, supposing that PAC1R expression in metastatic melanoma seems to be an important mediator for biological activity.

Furthermore, PAC1R gene transcripts could be observed in melanoma lines and melanoma primary cell culture and its expression was verified by Western Blot using a monoclonal mouse anti-human PAC1R antibody yielding ~50 kDa peptide. In 1994 Fabre showed that the VIP-related peptide recognized receptor in the melanoma cell line IGR37 and six years later Moody and his colleagues reported the VPAC1R expression in melanoma cell lines (Fabre, 1994; Moody, 2000). Accordingly, also in this study metastatic melanoma cell lines including primary melanoma cell culture were shown to express VPAC1R at different mRNA expression levels but not VPAC2R. Despite lacking in information with regard to VPAC1R expression at protein level, preferential expression of PACAP receptor types on mRNA level was noticeable in melanoma cell lines, especially VPAC1R expression in NW-Mel 450 and PAC1R expression in SK-Mel 37. PACAP receptor variants result in differential signal transduction responses, coupling to cAMP or PI (McCulloch, 2000; Dickson, 2006). This different expression level of receptors in both melanoma lines leads to the speculation that each melanoma line might have a preferential expression of VPAC1R or PAC1R, which connects with different characteristic features. In addition, expression of PAC1R splice variant was examined with specific primer encoding hip or hop area and only hop area was detected in melanoma cell lines (PCR product of hip area; data not shown). Although identifying splice variants of PAC1R on melanoma cell lines was not the purpose of this study, it should be interesting to reveal, which PAC1R splice variant could be expressed in melanoma cells besides hop area because it may explain the different characters of melanoma lines.

As reported by Le and the colleagues showing an autocrine or paracrine effect of PACAP on breast

86 cancer cells expressing PAC1R, co-expression of PACAP and PAC1R in melanoma cell lines suggested the involvement of PACAP in the biological regulation of melanoma in an autocrine or paracrine manner (Le, 2002).