Investigations On The Dynamic Expressions And Significience Of Wnt/Ca²⁺ Signaling Pathway In Different Stages Of Melanoma

Cutaneous melanoma is known as its highly malignant nature. However, the specific markers to identify whether melanoma has more ingressive abilities are uncertain. Recently, it's known that the Wnt signaling pathway can participate in development and tumorigenesis. At present, Wnt signaling processes include at least three distinct pathways: the canonicalβ-catenin pathway, the non-canonical Wnt/Ca2+ pathway and the Wnt/JNK pathway. The published literature and our recent studies have shown that activation of the canonicalβ-catenin pathway might involve in the melanoma progression. However, the roles of non-canonical Wnt/Ca2+ pathway, especially on the dynamic changes and functional significance in melanoma, are not well understood. Therefore, by using RT-PCR methods, we detect the genes levels encoding Wnt5a and Wnt11 which are the key ligands activating Wnt/Ca2+ signaling pathway in normal melanocyte and six melanoma cell lines. Taken together with calcium imaging methods and pharmacological experiments, carbachol-induced intracellular Ca2+ mobilization in different melanoma cells and negative regulation of protein kinase C are also investigated. Part 1 Comparison of morphological characteristics and aggressive abilities between melanocytes and melanoma cellsIt is shown that normal melanocytes and the melanoma cells shared differences in both morphological characteristics and cell growth. The cultured epidermal melanocytes showed clear nucleoli. The confluent areas were consisted mainly of bipolar cells in a whirling pattern. WM793B cell line was established from skin primary superficial spreading melanoma (SSM) in vertical growth phase (VGP). The cells were spindle-shaped with mostly bipolar morphologies. 451Lu cell line was established from skin nodular melanoma in VGP. Most cells appeared as dendritic morphology. SK-MEL-5 cell line was derived from axillary node metastases transmitting from skin melanoma. The cells were short spindle-shaped morphologies. A2058 cell line was highly invasive and derived from lymph node metastases transmitting from skin melanoma. The cells were spindle-shaped and tended to grow in a local congregative pattern. Libr cells were spindle-shaped and tended to grow with close cell-to-cell contact. A-375 cells were derived from skin malignant melanoma and were short spindle-shaped. Scratch assays indicated that abilities of invasion and motility in melanoma cell lines were quite different. A2058 and SK-MEL-5 cell lines which derived from lymph node metastases transmitting from skin melanoma had more aggressive abilities and demonstrated them within the early 12 hr and 24 hr, respectively. However, WM793B cells which established from skin primary superficial spreading melanoma showed poor aggressive ability and nearly no cells could invade the scratch after 48 hr. The results indicated that, comparing with normal melanocytes, melanoma cells shared the typical characteristics of cancer cells, such as heteromorphisms in cell membrane and nucleolus and proliferating in various irregular patterns. In addition, melanoma cells increase the aggressive abilities with the development of tumor progress.Part 2 Dynamic change of Wnt5A and Wnt11 gene levels in different melanomas cells mimicking different stages of tumor progression and their correlations with migrationBy using RT-PCT, we detected gene levels of Wnt5A and Wnt11 in cultured normal epidermal melanocytes and six skin melanoma cell lines, including WM793B, 451Lu, SK-MEL-5, A2058, Libr and A-375, which derived from different stages of melanoma progression. We found that the characteristics of Wnt5A and Wnt11 levels were similar, both of which were dynamically changed in different cells. Comparing with the low levels in normal melanocytes, Wnt5A and Wnt11 were up-regulated with different intensities in the most investigated melanoma cell lines except for A2058. Interestingly, WM793B, 451Lu and A2058 cell lines which respectively derived from primary superficial spreading situation to nodular melanoma and then to lymph node metastases gradually decreased their Wnt5A and Wnt11 levels. The genes were even undetectable in the A2058 melanoma cells. The results indicated that the temporal activation of noncanonical Wnt/Ca2+ pathway might be crucial for different stages of melanoma progression through dynamic change of Wnt5A and Wnt11 gene expressions.Part 3 Dynamic change of carbachol-induced intracellular Ca2+ mobilization in different melanoma cells and negative regulation of protein kinase CCalcium imaging showed that 1 mM carbachol (CCh), significantly increased intracellular calcium mobilization in WM793B, 451Lu, SK-MEL-5 and A2058 cells with different intensities. However, there were no similar response in normal melanocyte. Pretreatment of cells with phorbol 12, 13-dibutyrate (PDBu, 2μM), an activator of protein kinase C (PKC), significantly suppressed CCh-induced peak reactions in WM793B, SK-MEL-5 and A2058 cells. These results indicated that muscarinic receptors mediating the carbachol-induced intracellular calcium mobilization were phenotypically or functionallly modulated by protein kinase C during melanoma progression. Wnt5A and Wnt11 levels were the lowest in A2058 melanoma cells, while the cells showed the most significant calcium mobilization. The results also indicated that the downstream molecular protein kinase C might involve in the regulation of carbachol-induced calcium mobilization.Conclusions:1. In present study, we used WM793B, 451Lu, SK-MEL-5, A2058, Libr and A-375 melanoma cells derived from skin primary superficial spreading phase to nodular melanoma and to metastases transmitting from skin. Morphological experiments and scratch assay indicated that aobove melanoma cells could mimic the dynamic change of melanoma progression.2. During the process of melanoma progression, the characteristics of Wnt5A and Wnt11 levels were similar, both of which were dynamically changed in different cells. WM793B, 451Lu and A2058 cell lines which respectively derived from primary superficial spreading situation to nodular melanoma and then to lymph node metastases gradually decreased their Wnt5A and Wnt11 levels. The result suggested that the dynamic change of Wnt5A and Wnt11 gene expressions induced different activations of noncanonical Wnt/Ca²⁺ pathway during different stages of melanoma progression. Then the different activations of noncanonical Wnt/Ca2+ pathway could modulate the ingressive ability of melanoma cells.3. Different to the less effect on normal melanocyte, the carbachol significantly increased intracellular calcium mobilization in melanoma cells, especially in A2058, with different intensities. PKC agonist could inhibit the responses indicating that muscarinic receptors mediating the carbachol-induced intracellular calcium mobilization were negatively regulated by intracellular PKC. Wnt5A and Wnt11 gene expressions were lowest in A2058 cell which derived from lymph node metastase, whereas in A2058 cell, carbachol-induced intracellular calcium mobilization was the most significant. The results indicated that PKC, the product of downstream of Wnt/Ca²⁺ pathway activation, could regulate the muscarinic receptors.