Mechanisms Of The EGFR Signaling Pathway Mediates Resistance To Paclitaxel, Invasion And Metastasis In Human Melanoma A375 Cells

IntroductionHuman malanoma is a highly invasive and metastase tumor. The major cause of death from melanoma is metastases that are resistant to conventional therapies. The invasion, metastasis and resistance to conventional chemotherapy are very important factors that affect the prognosis of melanoma patients. To study the mechanism of occurrence, development and resistance to chemotherapy in human melanoma is the focus viewpoint in oncology. At present several lines of evidence have clearly indicated that the EGFR is anomalously expressed or activated in multiple human neoplasm and has been, in general, associated with a worse prognosis.The EGFR (epidermal growth factor receptor) is a 170 - kDa single poly-peptide chain composed of 1186 amino acid residues and remains activation of TPK (tyrosine protein kinase). The general molecular structure of the EGFR family includes an amino - terminal extracellar ligand binding domain, a trans-membrane anchoring domain, and an enzymatically active carboxy terminal in-tracellular domain. The EGFR family consists of 4 members of transmembrane glycoproteic receptors; EGFR/ErbB1; HER2/ErbB2; HER3/ErbB3, and HER4/ErbB4. Binding of activating ligands to the extracellular domain of the receptor results in that receptor dimerization is initiated, leading to the formation of either homodimers, which display a relatively weak biologicactivity, or het-erodimers, which have higher proliferative potency. EGFR not only has activation of TPK, but also can be phosphorylated and activated as reactors of TPK.Signal transduction and controlling is essential for highdegree animals'de-velopment and advancement. If disordered, the cell division will become uncontrolled and carcinogenesis will happen. EGFR signaling pathway is an important pathway. At present, known EGFR -regulated downstream signaling pathways include the phosphatidylinositol -3 kinase ( PI3K )/AKT, Ras/Raf/MEK/ MAPKs, mitogen - activated protein kinase ( p44/p42 ) , and protein kinase C. EGFR has been found to not only increase resistance to apoptosis but also increase cellular adhesion, invasion and metastasis. It has been demonstrated that DNA damage can activate the EGFR pathway to contribute to resistance to chemotherapy. In order to study relation and mechanisms between the EGFR signaling pathway and paclitaxel - resistance, human melanoma A375 cells were treated with or without AG1478, or PD98059, or LY290042 and paclitaxel. To study the effect and mechanism of EGFR pathway on melanoma invasion and metastasis, A375 cells were treated with EGF or AG1478.Materials and MethodsHigh EGFR expression A375 cells of human melanoma were found and cultured by the diluted clone assay and cell culture technique.light and electron microscopy were used to detect the morphological changes of the apoptotic cells.Flow cytometry analysis (FCM) was used to determine the cell cycle progression ( PI ) and the apoptosis ratios ( AnnexinV/PI ) of A375 cells.The adhesion assay to ECM was measured by MTT method.In vitro invasion assay was measured by Boyden chamber method.Expression levels of P - EGFR, P - ERK, P - AKT, Bcl -2, Bax and Caspase - 3 protein were determined by Western - blot technique.Expression levels of MMP - 2, MMP - 9, TIMP - 1 and TIMP - 2 mRNA were determined by RT - PCR technique.Results1. Paclitaxel inhibited the growth of A375 cells in both a time - dependentand dose - dependent manner.0. 001μmol/Lto 10μmol/L paclitaxel inhibited the growth of A375 cells ( P <0.01) and induced apoptosis (P<0. 05) in a dose - and time -dependent manner. IC50 of paclitaxel to A375 cells is 9. 5μmol/L, 3. 7μmol/L, 0. 06μmol/L and 0.00μmol/L at 24 hours, 48 hours, 72 hours and 96 hours respectively (P <0. 01). The cell growth inhibition rate of 0. 001μmol/L, 0. 01μmol/L, 0.1μmol/L and 1μmol/L paclitaxel at 24 hours is 0. 07% , 10. 80%, 24.4% and 32.5% respectively (P <0.05).2. Different dose paclitaxel induced apoptosis of A375 cells by different ways.Paclitaxel (0. 001 ~ 1μmol/L) could induce apoptosis of A375 cells at 24h. The cells treated with paclitaxel initially showed nomal cell cycle at 0. 001μmol/L, G0/G1 arrest at 0.01μmol/L and G2/M arrest for 0.1 ~ 1μmol/L, which were followed by apoptosis. Paclitaxel reduced the levels of Bcl - 2 proteins , increased the levels of Bax proteins and activated Caspase - 3.3. PI3K/AKT and ERK/MAPK signaling pathways mediate resistance to paclitaxel via EGFR in A375 cells.Paclitaxel (0. 01 ~0.1μmol/L) treatment upregulated the levels of P -EGFR, P - ERK and P - AKT expression at 24h. When AG1478 was administered with paclitaxel, their expressions were downregulated. It was demonstrated that paclitaxel could activate the both EGFR - ERK/MAPK and EGFR - PDK/ AKT signaling pathways, which contributed to the resistancce to paclitaxel in human melanoma.4. The EGFR inhibitor potentiates paclitaxel response synergistically in growth inhibition and apoptosis induced.The combined treatment of paclitaxel and AG1478 markedly enhanced the antiproliferative effect and apoptotic cell death induced by single - agent treatment ( P < 0.01). Though AG1478 did not induce A375 apoptosis, it resulted in 2.7 - fold, 2.8 - fold and 1.6- fold increase in early - stage apoptosis for 0. 001 ~0. 1μmol/L paclitaxel respectively ( P <0.05). AG1478 also increased 6.8- fold ( P < 0. 01) and 0 - fold in Later - period apoptosis for 0.01 - 0. 1 μmol/L paclitaxel. The EGFR inhibitor can potentiate paclitaxel response syn-ergistically, not additively.5. EGFR signaling pathway influences A375 cell growthExogenous EGF 10ng/ml stimulation upregulated P - EGFR, P - ERK and P — AKT protein expression at 24h, but had no further effect on the growth and proliferation of A375 cells until 72h(P < 0.05 ). AG1478 could abrogate EGF effect and downregular P - EGFR, P - ERK and P - AKT protein expression-Treatment of A375 cells with AG1478 or PD98059 or SB203580 or LY290042 resulted in a marked growth inhibition in time — dependent manner, which the growth inhibition rates are 24.9% , 23.0% , 17. 8% and 20.9% respectively. In summer, EGFR regulates growth of A375 cells by at least two downstream signaling pathways of MAPK and PI3K/AKT.6. EGFR signaling pathway mediates adhesion of A375 cells Exogenous EGF could enhance cell adhesion to ECM, and cell adhesionwas increased by 2. 3 - fold at 24h ( P < 0. 01 ). AG1478, PD98059 and LY290042 could abrogate EGF effect, but SB203580 could not do it. It has been demonstrated that EGFR mediates adhesion of human melanoma to ECM by ERK/MAPK and PDK/AKT downstream signaling pathways.7. EGFR signaling pathway influences in vitro invasion of A375 cells Exogenous EGF could enhance invasion significantly, and invasive cellswere increased by 26. 37% (P <0. 05). AG1478, PD98059 and LY290042 could abrogate EGF effect, and cells through Matrigel were reduced by 29. 90% , 25.10% and 33.20% respectively( P < 0. 01), but SB203580 had no inhibition effect on EGF. It has been demonstrated that EGFR mediates cell movement and invasion of human melanoma by ERK/MAPK and PDK/AKT downstream signaling pathways.8. EGFR changes the levels of mRNA of MMP - 2, MMP - 9, TIMP - 1 andTIMP-2EGF increased the levels of mRNA of MMP -2 and MMP -9 and reducedthe levels of mRNA of TIMP -1 and TIMP -2. AG1478 could reduce the levelsof mRNA of MMP -2 and MMP -9 and increased the levels of mRNA of TIMP-1, but TIMP -2 was not changed, so that MMP -2/TIMP -2 ratio and MMP-9/TIMP -1 ratio were significantly reduced( P < 0.001). EGFR signal ad-justs invasion and metastasis of human melanoma by mediating matrix metallo-proteinases and their inhibitors.DiscussionPaclitaxel is a unique antimicrotubule agent that acts by disrupting the normal microtubular network of the cell by inhibiting microtubular depolymerization and thereby forming unusually stable microtubules. This microtubular stabilization leads to a mitotic block in the late G2/M - phase of the cell cycle and is generally believed to be the mechanism by which paclitaxel exerts its cytotoxic effects. Our study found that high dose paclitaxel (>0.1μmol/L) induced ap-optosis with G2/M arrest and low dose paclitaxel ( <0.1μmol/L) with a decrease of cells in Go/G, phase and without G2/M arrest in A375 cells. These findings implied that high and low dose paclitaxel exert cytotoxic to human melanoma by different ways.Paclitaxel has a distinctive mechanism that is more effective in a variety of cancers resisted to conventional chemotherapy. It has been also a problem for paclitaxel - resistance and its side effect. The EGFR pathway has been demonstrated to contribute to resistance to radiation and chemotherapy in many tumor types. It has been demonstrated that DNA damage can activate EGFR signaling pathway. Many downstream pathways have been contributive to accelerate cell proliferation, inhibit apoptosis and increase invasion and metastasis. Our findings suggest that paclitaxel not only induces melanoma apoptosis, but also actives EGFR and its downstram ERK/MAPK and PI3K/AKT pathways, which increase anti - apoptosis. This study demonstrates that EGFR inhibitor or ERK/ MAPK inhibitor or PI3K/AKT inhibitor can potentiate paclitaxel anti - proliferation and apoptosis - induced synergistically.It is a complicated process for invasion and metastasis. Proliferation, adhesion and ECM hydrolyzed play a critical role in cell metastasis. It has been demonstrated that EGFR activation involves in human melanoma growth. It rises gradually along with melanoma progress. It is a essential condition for, cell metastasis that increases cell proliferation and changes cell adhesion. Our findings...