| Invasion and metastasis are common phenomena in cancer cells.Metastasis is generally defined as the spread of malignant cells from the primary tumor through the circulation to establish secondary growth in a distant organ.In most cases,metastasis,not the primary tumor per se,is the main cause of mortality in cancer patients.The incidence of lung cancer is one of the highest worldwide among all cancers.Because of the difficulty of early diagnosis,high transfer and high recurrence rate,lung cancer leads to high fatality.The A549 cell line,which is derived from the tissues of patients with non-small cell lung cancer,has similar biological characteristics as cancer cells within the patient’s body.The H460 cell line,which is derived from the tissues of patients with lung adenocarcinoma,has a similar morphology and growing environment as A549 cell line.Thus,A549 and H460 cells are considered as good research models with which to study the pathogenesis of lung cancer cells.Accumulating evidence indicates that intracellular Ca2+release and cytosol Ca2+ homeostasis are crucial regulators of cell migration.However,if Ca2+participates in the migration of A549 and H460 cells,how does Ca2+release regulate A549 and H460 cell migration and what is the relationship between Ca2+release and the migration of A549 and H460 cellsERP44,endoplasmic reticulum protein 44,which has been reported to regulate calcium release inside of the endoplasmic reticulum(ER),in cell migration.However,the role and mechanism of ERP44 in regulating cell migration are unknown.In the present study,we have investigated the role of ERP44 overexpression in the regulating of A549 and H460 cell migration.We demonstrate that ERP44 overexpression inhibits A549 and H460 cell migration by regulating intracellular Ca2+release from ER Ca2+stores and the inhibitory effect of ERP44 overexpression on A549 and H460 cell migration is mainly dependent of IP3R2.Observation of Ca2+signaling in A549 and H460 cellsTo determine whether A549 and H460 cancer cell migration is regulated by intracellular Ca2+signaling,we first detected Ca2+release events using confocal microscopy.According to the results of experiments,spontaneous calcium transients were detected only in A549 cells.However,no calcium signaling was found in the leading area of both cell lines.To determine the relationship between Ca2+signaling and cell migration of A549 and H460,we constructed a calcium indicator protein GCaMPJ that was stably expressed in the A540 and H460 cell lines.A long-term(120 min)recording of A549/GCaMPJ and H460/GCaMPJ cell migration and the scanning images of Ca2+ signaling were recorded using the API live cell imaging system.Spontaneous calcium transients were observed in A549 cells,but there was no spontaneous calcium transient in H460 cells.Roles of Ca2+ signaling in A549 and H460 cell migrationIt has been previously reported that cell adhesion plays an important role in cell migration.Thus,we first investigated whether extracellular Ca2+ buffered with EGTA affects A549 and H460 cell adhesion.According to time-lapse recordings of both cell lines,results revealed that cell adherence was altered under different conditions Moreover,5 mM EGTA in the extracellular solution significantly inhibited A549 and H460 cell adhesion compared to the controls,and the inhibitory effects of EGTA on A549 and H460 cell adhesion was abolished by EGTA washout.In the next series of experiments,we examined the effects of intracellular Ca2+ release on A549 and H460 cell migration.First,we investigated the effects of the IP3Rs(Inositol 1,4,5-trisphosphate receptors)inhibitor 2-APB on Ca2+release induced by ATP in A549 and H460 cells.Compared to the controls,the peak of Ca2+transients was remarkably reduced in response to the application of 30 μM 2-APB(P<0.01).Similarly,in the presence of 2-APB(30 μM),the wound healing and transwell migration assays of both cell lines were greatly retarded,which suggest that Ca2+ release plays a role in regulating A549 and H460 cell migration and that the inhibition of Ca2+release delays A549 and H460 cell migration.The results indicated that IP3Rs are involved in regulating the migration of A549 and H460 cancer cells.2-APB inhibits A549 and H460 cell migration via altering cell polarizationIn the next series of experiments,we investigated how inhibiting the release of intracellular Ca2+ retarded A549 and H460 cell migration.Cell polarization is the basis for cell migration.Therefore,we first examined the effect of Ca2+release inhibition on cell polarization by staining cells with Phalloidin-FITC.Under the stimulation of scratch,the A549 and H460 cells displayed a trend of cell polarization.More pseudopodium protrusion and stress fibers were observed within 2 h after scratching in A549 and H460 cells.In contrast,in the presence of 2-APB(30 μM),the polarization and pseudopodium protrusion of A549 and H460 cells were significantly inhibited,suggesting that 2-APB inhibited A549 and H460 cell migration by affecting cell polarization.We exposed cells to 2-APB at different time periods in cell scratch experiments.The results indicated that cells treated with 2-APB during the first 4 h(0 h-4 h)led to a significant inhibition of wound healing in both cell lines;conversely,cells treated with 2-APB in the latter 4 h(4 h-8 h)did not affect cell wound healing,which confirmed that 2-APB inhibited A549 and H460 cell migration by inhibiting cell polarization.Next,we examined the effect of 2-APB on A549 and H460 cell random motility.During the first 1.5 h,the cells were treated with 10%FBS only;in the latter 1.5 h,cells were treated without or with 30 μM 2-APB except for 10%FBS.The centre of gravity was calculated based on the outlines of the cells,and the change in the coordination of the centre represented the tracking of cell movement.Compared to the controls,the number of cells with declining migratory rate was increased when the cells were treated with 30 μM 2-APB during the latter 1.5 h,indicating that 2-APB decreased the migration of A549 and H460.Taken together,these results indicated that IP3R inhibitor,2-APB,inhibited A549 and H460 cancer cell migration by affecting cell polarization by reducing intracellular Ca2+ release.ERP44 overexpression inhibits A549 and H460 cell migration by inhibiting intracellular Ca2+releaseBased on our observation that 2-APB inhibited the migration of A549 and H460 cells,we postulated that the inhibitory effect of 2-APB on A549 and H460 cell migration was mediated by IP3Rs.Thus,in the next series of experiments we examined the role of ERP44 overexpression in A549 and H460 cells.By using the adenovirus infection approach,ERP44 was highly expressed in ER of A549 and H460 cells.Compared to controls,ERP44 overexpression significantly inhibited ATP-induced Ca2+ release.Further studies indicated that the overexpression of ERP44 remarkably inhibited the migration in A549 and H460 cells.Long-term real-time tracking analysis of cell movement indicated that the overexpression of ERP44 inhibited A549 and H460 cell random motility,and similar to the effect of 2-APB on A549 and H460 cells,the cells exhibited a "fried egg"morphology indicating that the polarization and pseudopodium protrusion of A549 and H460 cells were inhibited by overexpression of ERP44.The physical center of gravity in ERP44 overexpressed A549 and H460 cells was nearly maintained at its original location during the 1.5 h tracking timeAs we noted above,2-APB inhibited Ca2+release and resulted in an inhibitory effect on A549 and H460 cell migration by affecting the cell cytoskeleton.Therefore,we examined whether ERP44,similar to 2-APB,also inhibited cell migration by affecting the cell cytoskeleton.In the control,A549 and H460 cells stained with Phalloidin-FITC exhibited a clear structure consisting of F-actin microfilaments and polarized cells presented a network arrangement of microfilaments at the forefront of the cells.In addition,stress fibers were observed throughout of cells.However,the microfilaments were not clearly observed or only some circular microfilaments were observed around the edge of the cells in ERP44 overexpressed A549 and H460 cells,suggesting that ERP44,similar to 2-APB,inhibited A549 and H460 cell migration by affecting the cell cytoskeletonER stress mediates the inhibition of ERP44 overexpression on cell migrationIt was previously reported that ER stress results in an up-regulation of ERP44 expression.However,no study has indicated that the overexpression of ERP44 can induce ER stress.Therefore,we examined whether the inhibitory effect of ERP44 overexpression on A549 and H460 cell migration is related to ER stress.First,we examined two ER stress related markers,GRP78 and CHOP.The real-time PCR data of A549 and H460 cells demonstrated that the overexpression of ERP44 resulted in a significant up-regulation of both markers,suggesting that ER stress occurred due to the ERP44 overexpression in ER.Next,western blot assay revealed that gradient expression of ERP44 resulted in GRP78 gradient up-regulation in both cell lines;although gradient treated A549 and H460 cells with DTT(0-2 mM,for 12 h)resulted in a gradient up-regulation of GRP78,the ERP44 expression level was not altered,which indicated the molecule mechanism of ERP44 overexpression induced ER stress is different from that of DTT mediated ER stress.The specificity of ERP44 on A549 and H460 cellsIt has been reported that ERP44 inhibits intracellular Ca2+release by binding to IP3R1.We confirmed that all three types of IP3R were expressed in A549 and H460 cells.However,the subtype of IP3Rs that mediates the inhibitory effect of ERP44 on A549 and H460 cell migration remains unknown.To clarify this,we performed RNA interference studies.We synthesized siRNAs for IP3R1,IP3R2 and IP3R3 according to a previously reported method and the real-time PCR results indicated the interference efficiency of single IP3Rs siRNA to be>50%after transfection for 72 h.Wound healing and transwell studies demonstrated that all types of IP3Rs exhibited a inhibition of cell migration on A549 and H460 cell lines compared to the controls(P<0.001 vs.control).However,among these receptors,IP3R2 displayed a remarkable inhibitory effect on A549 cell migration(P<0.001 vs.IP3R1 and IP3R3),in addition,IP3R2 also displayed a clearly inhibitory effect on H460 cell migration(P<0.001vs.IP3R1 and IP3R3).To further confirm,we carried out wound healing and transwell studies with combined IP3Rs siRNA of>30%interference efficiency.Wound healing and transwell assays in both cell lines with treatment involved IP3R2 siRNA was markedly inhibited while in A549 and H460 cell lines with IP3R1 and IP3R3 siRNA was mildly inhibited.These results suggested that IP3R2 plays a predominant role in mediating the inhibitory effect of ERP44 on A549 and H460 cell migration.Moreover,we performed wound healing and transwell experiments in ERP44 stably transfected SH-SY5Y cells,which mainly express IP3R1,indicated that the overexpression of ERP44 did not significantly inhibit cell migration,confirming that ERP44 inhibiiton of cell migration is independent of IP3R1.DiscussionTo date,there are two approaches to studying the relationship between calcium and cell migration.The first approach is to record the calcium signaling in real time to reveal the mechanism of cell migration.Another indirect approach to studying the role of calcium in cell migration is to modulate the intracellular calcium using RNA interference or treatment with inhibitors of calcium channels.In the present study,we have combined the two methods described above to examine the effect of intracellular calium activities on A549 and H460 cell migration.We observed spontaneous Ca2+signaling in A549 cells,which confirmed there are spontaneous calcium activities in non-excitable cells.Moreover,we found that intracellular Ca2+release via IP3Rs played an important role in regulation of A549 and H460 cell migration by treatment with the inhibitor 2-APB,overexpression of inhibitory protein ERP44 or RNA interference of IP3Rs.In the presence of 2-APB,the migration of A549 and H460 cells was significantly inhibited and the inhibition of 2-APB on A549 and H460 cell migration was mediated by a reduction in the intracellular Ca2+.Moreover,the inhibitory effect of 2-APB on cancer cell migration is achieved by the inhibition of cell polarization and pseudopodium protrusion.It has been reported that ERP44 inhibits intracellular Ca2+release by binding to IP3R1 inside of ER.Based on this effect of ERP44 on Ca2+,we speculate that ERP44 is involved in cancer cell migration.Indeed,the overexpression of ERP44 inhibited intracellular Ca2+release followed by significantly retarded A549 and H460 cell migration.As with 2-APB,the inhibitory effect of ERP44 on A549 and H460 cell migration was achieved by altering the polarization and pseudopodium protrusion of both cell lines.Similarly,IP3Rs knockdown experiments resulted in the inhibition of A549 and H460 cell migration,and IP3R2 displayed an unexpected significance in inhibiting A549 and H460 cell migration,which indicates that IP3R2 plays the most important role in regulating A549 and H460 cell migration.This finding was confirmed by the observation that the stable overexpression of ERP44 did not affect SH-SY5Y cell migration.On the basis of the findings above,we conclude that intracellular Ca2+release via IP3Rs plays an important role in the regulation of A549 and H460 cell migration and the Ca2+release of A549 and H460 cells is regulated by ERP44 mainly via IP3R2. |
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