The Effect Of Tyrosine Kinase C-abl On Wnt / β-catenin Signaling Pathway In Melanoma Cells

Cutaneous melanoma is a malignant tumor with high mortality and metastatic nature. Currently the methods of its treatment are mainly surgical excision combined with chemotherapy, biological agents or other adjuvant therapy, but often can not control the development, invasion and metastasis of tumor, and side effects of chemotherapy are usually large. Therefore, to explore the targeted therapy with good efficacy and smaller side effects has become a hotspot, while the role of tyrosine kinase c-abl and the Wnt /β-catenin signaling pathway in targeted therapy is universally concerned. In recent years, studies have shown that Wnt /β-catenin signaling pathway was activated in melanoma, which is an important pathway in intracellular signal transduction system, contacting with the extracellular irritating signaling and intracellular gene transcription regulation, mediating cell growth, development, differentiation, proliferation and apoptosis. Meanwhile c-abl was also closely involved in the intracellular signal transduction, because it can specifically phosphorylate some tyrosine residues of the substrate proteins. Howerer, there are some questions whether there is the abnormal expression of c-abl in melanoma cells, whether it is involved in the Wnt /β-catenin signal transduction pathway, and then affect the biological function of melanoma, that has not been clearly established. In this experiment, normal melanocytes and six cell lines from the melanoma in different growth stages were selected as the study object, we detect the expression of c-abl using RT-PCR and Western-blot method; observe biological changes of melanoma cells after suppressing c-abl with imatinib, further study the effect of c-abl on the Wnt /β-catenin signaling pathway and its mechanism.Part 1 The expression of tyrosine kinase c-abl in malignant melanoma cells and its significanceThe expression of c-abl was detected by RT-PCR and Western-blot in the normal epidermal melanocytes, the cell line WM973B from skin primary superficial spreading melanoma in vertical growth phase and five cell lines from the metastasis of melanomas, such as A2058(from lymph nodes), WM-266-4(from tumor skin metastasis), Hs294T(from lymph nodes), SK-MEL-5(from lymph nodes), SK-MEL-1(from thoracic duct lymph), the results showed as follow:In seven cell lines, the c-abl mRNA in normal skin melanocytes was lower expressed and were higher expressed in all melanoma cells, there is no significant differences among melanoma cells. The tendency of c-abl protein expression was mostly consistent with mRNA expression, but in the cell line from melanoma in vertical growth phase, c-abl protein expression was lower than that of metastatic melanoma cells, showing a dynamic changing characteristics, c-abl protein expression gradually increased from the epidermis normal melanocytes, cell line come from melanoma in vertical growth phase to metastatic melanoma cells, which suggest that c-abl may be involved in the occurrence and development of melanoma.Part 2 The biological effects of tyrosine kinase c-abl on melanoma cellsWe chose the cell line Hs294T from the metastasis of melanoma, by the methods such as MTT, colony formation assay, flow cytometry, Matrigel invasion assay, and observed the biological behavior changes of the melanoma cells after applying Imatinib mesylate as c-abl inhibitor, the results were shown as below:1. MTT test: the absorbance values is proportional to the number of living cells, imatinib mesylate inhibited the growth of Hs294T cells in a dose-dependent manner, in which after treating cells with four concentration gradient (0, 4, 8 and 10μmol/L), the absorbance values were respectively 566.6±10.4, 517±14.8, 458.9±13.7, 420±12.2, and the difference was significant (P<0.05). The absorbance values were respectively 420±12.2, 407.4±12.5, 390.1±11.6, 389.6±9.3, after treating cells with four concentration gradient (10, 16, 20 and 24μmol/L), no significant difference (P>0.05). In the 24 ~ 72h, Imatinib mesylate inhibited the growth of Hs294T cells in a time-dependent manner.2. Further verifying MTT results, the results of Colony formation assay showed that 10μmol/L imatinib mesylate Hs294T inhibited the colony formation of melanoma cells, and the proportional of colony formation in control group and treatment group were repectively 89.5±2.3%, 53.5±3.4%, there was a significant difference (P<0.05); In additional, the size of cell clones in control group were clearly larger than that of treatment group.3. After treatment with 10μmol/L imatinib mesylate for 48h and c-abl activity was inhibited in Hs294T cells, with FITC-AnnexinV-PI staining we observed the proportion of apoptotic cells in treatment group had a marked increase compared with the control group.4. By Matrigel invasion assay, the number of invasion cells was 73±11/mer vision in control group. After treatment cells with imatinib mesylate (5μmol/L for 24h), the number of invasion cell was 38±7/mer vision and was clearly decreased compared with the control group, the difference was statistically significant (P 0.05).So these results suggest that inhibition of c-abl activity can inhibit proliferation of melanoma cells, induce tumor cell apoptosis and decrease cell invasion.Part 3 The effect of trosine kinase c-abl on Wnt /β-catenin signaling pathway in melanoma cellsThe second section shows c-abl inhibitors can affect the biological behavior of melanoma. In order to explore its mechanism, by using the method such as laser confocal, fluorescence quantitative PCR and Western-blot, we analysised the relationship between c-abl and Wnt signaling pathway in melanoma cells Hs294T, the results showed as follows:1. Observed by confocal microscopy we found that c-abl was mainly localized in the cytoplasm and nucleus in melanoma cells, and has a higher proportion of overlap withβ-catenin protein subcellular location, which indicated that they may exist co-localization and interaction. After inhibition of c-abl activity, The subcellular localization ofβ-catenin protein has a change from the cytoplasm, nucleus to the cell membrane, but the Western-blot results showed thatβ-catenin protein expression was no significant change. We suggest that c-abl has a function promotingβ-catenin protein into the nucleus.2. After inhibition of c-abl, the results obtained from Fluorescence quantitative PCR, Western-blot experiments confirmed that the expression of LEF1 mRNA, C-myc mRNA and cyclinD1 protein were significantly reduced, which they were respectively Wnt pathway transcripts molecules and target genes. Thereby we suggest that c-abl may be able to upregulate Wnt pathway.In summary, c-abl may play an important role in the occurrence and development of melanoma, its high expression may be associated with the growth, proliferation, apoptosis, invasion of melanoma. In vitro, imatinib mesylate (c -abl inhibitor ) can downregulate Wnt /β-catenin signaling pathway, inhibit the growth of melanoma cells and induce tumor cell apoptosis, suggesting that those drugs as c-abl inhibitior will likely play an important role as as therapeutic options of melanoma, and c-abl is expected to become a potential marker for melanoma targeted therapy.