An Experimental Study On Galvanotaxis And Related Mechanism Of Lung Cancer A549 Cells

BackgroundLung cancer is the leading cause of cancer death in United States, accounting for about 31% of estimated cancer deaths in men and 26% in women. That percentage in women is increasing each year. Although it has made great progress in treatment of lung cancer clinically, its five-year survival rate is only 16%. Approximately 429,000 individuals in China died from lung cancer in 2005, and the mortality of lung cancer is still continuously growing. Metastasis is the top of death causes of the patients with the disease.A series of sequential steps usually occur during cancer metastasis in which tumor cells migrate from the primary tumor firstly, penetrate blood vessels and then colonize distant sites. Cell motility is an important step in progression of metastasis and cancer metastasis is dependent on increased cell motility. Chemotaxis of cancer cells plays a very important role through whole process of the metastasis. It has been studied for decades, remaining the galvanotaxis driven by exogenous or endogenous direct-current electric fields (dcEFs) of cancer cells as a fresh item to further study. Most organs and embryos are surrounded by one layer or multiple layers of epithelial cells that produce transepithelial potentials (TEPs) of a few millivolts to tens of millivolts. The invasion of lung cancer cells into bronchi would deform the epithelium, thus the TEPs of bronchi epithelium will drive currents out of regions and the invasive cancer cells would be exposed to these endogenous electric fields.A variety of motile cell types have been found to exhibit galvanotaxis in applied dcEFs, yet not all cells exhibit this behavior. Among the human motile vertebrate cell types exhibiting galvanotactic ability, human cornea epithelial cells, retina pigment epithelial cells and human keratinocytes migrate to the cathode of dcEFs. However, human granulocytes human vascular endothelial cells migrate to the anode of dcEFs. There is limited background knowledge about galvanotaxis of cancer cells. Two rat prostate cancer cell lines were recently studied, interestingly, the highly metastatic cell line responded strongly to applied dcEFs by migrating towards the cathode. In contrast, the weakly metastatic cell line did not respond to the similar dcEFs strengths. The galvanotactic mechanism of cancer cells is still largely unknown.By studying the normal cells, it has been found that epidermal growth factor receptors (EGFRs) are critical for directed migration of cultured corneal epithelial cells in 1.5 V/cm physiological dcEFs. EGFRs activation and polarization are also required for the directed migration of keratinocytes in dcEFs. In keratinocytes and neutrophils, dcEFs directed cell migration through PI(3)Kγand PTEN signaling which is crucial for regulating the EGFR downstreams. EGFRs mediate chemotaxis and migration in many cancer cell types. Recently it is demonstrated that EGFRs signalling is essential for the galvanotactic responses of breast cancer cells; however, their role in galvanotaxis of lung cancer cells is unknown. Cytoskeleton play important roles in intracellular motion, fixation of cell organelle, maintenance of cell morphology, cell migration, signal transduction and cell division. Integrinβ1 is a vital receptor which can induce the reassignment of cytoskeleton, and bi-direct the signal transduction between inside and outside of cell. Integrinβ1 extensively affects a series of cell biological behaviors, such as survival, growth, differentiation, invasion and metastasis. Integrin and cytoskeleton are tight connected with bi-directional signal transductions between them. This also can affect a series of cell biological behaviors. FAK is a kind of non-receptor protein tyrosine kinase. It is a fundamental signal transduction molecule of Integrin. It connects with the Integrin and assembles other molecules to the conjunction site forming signal complex. The signal complexes then transduce the extracellular signal to the cytoskeleton. This mediates the cell adhesion and migration. Nowadays, Integrinβ1 and FAK are the hot issue on the studying of chemotaxis, leaving their effects on the galvanotaxis fresh item to further study.PTEN (phosphatase and tensin homologue deleted on chromosome ten) is the first anti-oncogene being a phosphatase. There are both syngenic catalytic center sequences of protein-tyrosine-phosphatase and Ser/Thr phosphatase in PTEN. PTEN also has phosphatase effect; therefore it is a double specific phosphatase. PTEN protein can depress cell growth and development, induce apoptosis, and inhibit cell migration, cell stretching and focal adhesion through PI3K-AKT, Ras-MAPK-ERK and Integrin-FAK signal pathways. Although PTEN has just been discovered for years, effects of PTEN on galvanotaxis of cells is emphasized due to the characteristic and function of PTEN.AimsTo establish an experimental model of human lung adenocacinoma A549 cells exposed to dcEFs, and investigate the effects of dcEFs on the apoptosis, proliferation, shape and migration of A549 cells, as well as the alteration of EGF-EGFR, cytoskeleton, Integrin, PTEN and their signal transductions and their regulating effects on galvanotaxis.Methods1) An experimental model of human lung adenocacinoma A549 cells exposed to dcEFs was established as the reference description to investigate the effects of dcEFs on apoptosis, proliferation, shape and migration of apoptosis, proliferation, shape and migration. (1). The A549 cells were divided into 3 groups according to whether exposed to dcEFs and exposed time. Group A unexposing to dcEFs was used as control. Group B was exposed to 1V/cm dcEFs for 12h. Group C was exposed to 1V/cm dcEFs for 24h. The number and rate of apoptosis were measured by fluorescent staining with Hochest33258 and flow cytometry. Cell cycle was detected by flow cytometry too. Western blot method was performed to determine the quantity of pERK and pAKT protein, the cell cycle related proteins including cyclin D1, cyclin E, P21 and P27 as well. (2). The A549 cells were cultured in DMEM+10%FBS. Cells in experimental groups were exposed to the direct current electric field at 2V/cm, 4V/cm, 6V/cm and 8V/cm. Cells unexposed to the electric fields were used as control. Images were taken per 5 min within 3 h and the correlated parameters were measured.2) To observe the effects of EGF and EGFR on galvanotaxis of the A549 cells. (1). The A549 cells were divided into 4 groups according to whether applied EGF or exposed direct-current electric fields. Group A was used as control. Group C and D were exposed to dcEFs at 6V/cm. Group B and D were applied 20μg/L EGF respectively. The movement images were taken per 15 min within 2 h by micro-videotape system. Total length of cell displacement and average directional cosineγwere measured. Western blot method was used to determine the expression of p-Akt and p-stat 3 protein. (2). The A549 cells were divided into 6 groups according to whether exposing to dcEFs and different EGFR inhibiters. Group A was used as control (0V/cm), Group B was exposed into dcEFs (3V/cm), Group C was given Cetuximab only (0V/cm), Group D was given Cetuximab with dcEFs (3V/cm), Group E was given AG1478 (0V/cm) only, Group F was given AG1478 with dcEFs (3V/cm). Total length of cell displacement, The average rate of cell displacement and average directional cosineγwere measured. The distribution of EGFRs and F-actin were measured by fluorescent staining. Western blot method was performed to determine the quantity of Integrinβ1 downstream signal pathway proteins.3) The effects of dcEFs on cytoskeleton and Integrin of A549 cells. According to whether exposed to 5V/cm dcEFs,the exposed time and whether applied cytochalasin B, the A549 cells were divided into 6 groups. Group A was used as control (0V/cm), Group B was exposed to dcEFs (5V/cm) for 1h, Group C was exposed to dcEFs (5V/cm) for 2h, Group D was exposed to dcEFs (5V/cm) for 2h and then cultured for 2h off the dcEFs, Group E was given cytochalasin B (0V/cm), Group F was given cytochalasin B and dcEFs (5V/cm) for 2h. Total length of cell displacement, The average rate of cell displacement and average directional cosineγwere measured. The distribution of Integrinβ1 andβ-tubulin were measured by indirect immunofluorescent staining. The distribution of F-actin was measured by direct fluorescent staining. Western blot was performed to determine the quantity of EGFRs downstream signal pathway proteins.4) The effect of PTEN on the galvanotaxis of A549 cells. With liposome mediated method, eukaryotic expression vectors with PTEN or without PTEN were transfected into A549 cells. The transfected cells were selected with 600 μg/ml of G418 and positive clones were picked and expanded. The A549 cells were divided into 3 groups. Group A without transfection was used as control. Group B was transfected with empty vector pEGFP-C1. Group C was transfected with pEGFP-C1-PTEN vector. The movement images were taken per 15min within 2h by micro-videotape system. Total length of cell displacement and average directional cosineγwere measured. Western blot was used to detect the expression of pFAK, pERK and pAKT in the cells.Results1) The dcEFs-exposure model with deferent volume of votage for the observation of lung cancer A549 cells was successfully established and the model was stable and reliable. (1). Fluorescent staining showed that the apoptosis rate of group A, B and C respectively were 0.40%, 0.43% and 0.52% (P>0.05). Flow cytometry showed that the apoptosis rate of group A, B and C respectively were 0.61%, 0.82% and 0.68% (P>0.05). Compared with group A, the proliferation index of cells were increased by 9.72% in group B and by 20.08% in group C. Western blot test illustrated that phosphorylated ERK and Akt were promoted. The expression of cyclin D1 and cyclin E were promoted too, otherwise the expression of P21 and P27 were inhibited. (2). A549 cells unexposed to the electric field showed slightly random movements during 3h. On the contrary, most of the cells in experimental groups aligned and elongated perpendicularly to the electric fields lines and began to migrate to the cathode. The speed of migration increased while voltage increased. The Average directional cosineγof cell migration was getting bigger with the time lapse(P<0.05).2) (1). Cells in group A and B unexposed to the electric fields showed slightly random movements during 2h. Cells in group C and D exposed to the electric fields migrate toward the cathode direction. The average directional cosineγof the cells in group D was larger than that in other groups(0.83±0.03,P<0.05). The total length of cell displacement in group D was longer than that in other groups also(63±16.9μm,P<0.05). The expression of p-Akt and p-stat 3 protein was increased with the treatment of EGF and electric fields. (2). Cells in group A, C and E unexposed to the electric fields showed slightly random movements during 2 hours. Cells in group E, D and F exposed to the electric fields migrate to the cathode direction. Both Cetuximab (4 nM) and AG1478 (8μM) could significantly produced blocking effect on the directed migration of A549 cells in the dcEFs. Fluorescent staining showed that EGFR and F-actin polarized to the cathode of the dcEFs. Western blot showed that striking enhancement of pERK and pAKT in A549 cells was induced in the dcEFs. Both Cetuximab (4 nM) and AG1478 (8μM) could significantly reduce the enhancement of pERK and pAKT in dcEFs-treated A549 cells, but neither was inhibited completely.3) The A549 cells exposed to the electric fields migrate to the cathode contiguously and stopped migrating after detach the dcEFs. The A549 cells recovered to be wafer. Fluorescent staining showed that Integrinβ1, F-actin andβ-tubulin polarized to the cathode of the dcEFs. Cytochalasin B treated A549 cells lost their sterical structure, and presented to be more flat. Cytochalasin B treated A549 cells also lost their tendency to migrate directly to the cathode. Integrinβ1 and F-actin presented to be depositive contiguously. No stronger fluorescence intensity and polarization of fluorescence were discovered.4) PTEN expressing vector was successfully transfected into A549 cells with liposome and the transfection efficiency was quite high. A549 cells with PTEN transfection took on no changes in shape during the exposed time in dcEFs. A549 cells without PTEN transfection groups A and B migrated directly to the cathode of dcEFs, the ATLD are 26±5.9μm and 28±5.1μm separately, theΣNcosγ/N were 0.78±0.15 and 0.76±0.11 separately. While A549 cells with PTEN transfection group C only migrated slightly to the cathode of dcEFs, the ATLD andΣNcosγ/N were 5±1.9μm and 0.21±0.18. Western blot analysis revealed that the expression of pFAK, pERK and pAKT in A549 cells were obviously increased in group A and B after exposing in dcEFs for 2h. Nevertheless, the expression of pFAK, pERK and pAKT in A549 cells were not increased in group C after the same treatment.Conclusions1) After exposing to 1 V/cm dcEFs, the apoptosis rate of A549 cells was not changed ultimately. However the flow cytometry revealed that cell cycle underwent great changes with enhancement of cellular proliferation, this might due to the promotion of ERK and Akt signal conduction pathway, which affect the cell cycle related proteins of the A549 cells. Electric field was able to induce cathodal directed migration of the A549 cells. And this effect was correlated to the voltage other than the duration of the cells exposure in the field.2) EGF could enhance the dcEFs directed migration of A549 cells. EGFRs played an important role in the galvanotactic mechanism of lung cancer cells by being polarized to the cathodal side and activated. Signal transduction pathways Akt, ERK and stat might be involved in the process.3) The directed migration of A549 cells in dcEFs relied on the maintenance of the electric fields. Cytoskeleton and Integrinβ1 were polarized in the dcEFs. Integrin-FAK-ERK signal pathway mught play a very important role in galvanotaxis.4) PTEN could inhibit the Galvantaxis of A549 cells obviously. The up-regulation of Phosphorylated FAK, ERK and AKT in A549 cells by dcEFs could be inhibited by PTEN.