Voltage Gated Sodium Channel Nav1.5 Functionally Expressed In Human Ovarian Caner

Ovarian cancer is one of the three most common cancers among gynecologicalmalignancies in the world.Because of no significant symptom in the early stage, theoverwhelming majority of patients are found at the advanced stage and its five-year survivalrate is also the lowest in gynecological malignancies.It is necessary to find effective therapymethods to increase its survival rate.Ion channels are functionally expressed in amolst every tissue and cell as the mainsignaling molecules, and they also participate in determining many cellular basic behaviors.With the development of patch clamp technique and molecular biology, many researcheshave revealed that ion channels were associated with different aspects of carcinogenicprocess by involveing in cell proliferation, multidrug resistance, migration and invasion.The functional expression of potassium, calcium and chloride channels have been widelyinvestigated over the past few decades, but little is known about sodium channel intumorigenesis.Recently study have reported that there were a window of voltage at cancercellular normal membrane potential, where there is a little and continuous entry of sodiumbecause of the partial opening or closing of voltage-gated sodium channel (VGSC), and theentry sodium might be responsible for the disrupting intracellulare ion homeostasis andsome signaling pathways.Therefore, it is important to investigat the roles of VGSC incarcinogenic process.VGSC are manly expressed in excitable cells and play importantroles in the initial and propagate of action potentials, which are composed of principalαsubunit and auxiliaryβsubunit.As we have known, based on the differences of phylogeny,αsubunit consists of Nav1 family and Nax.Recently studies have reported that theincreased functional expressions of Nav1 family (Nav1.1-1.9) were involved in thedevelopment and progression of cancer.Nav1 family consists of 4 homologous domains(D1-D4) and every homologous domain having 6 transmembrane segments (S1-S6).Alternative splicing is the important and ubiquitous process in regulating multiplicitygene expression of eucaryote.It is important for cell differentiation, so aberrations of alternative splicing are important for tumorigenesis.VGSC genes also code diversity andplasticity functional splice variants protein thought alternative splicing.Among thesesplice variants, the D1:S3 5' genomic splice have been found potentiation of cancermetastasis.In addition the splice variants of D1:S3 5' were also expressed in neonatal orfetal rat, while it rapidly disappeared with the development of organism.Therefore, VGSCmight be embryonic gene, silent in mature cells and re-expressed in cancer cells.At present, the majority sodium channel blockers on sodium channel subtypes are notspecific, which not only inhibit the abnormal subtypes but the normal subtypes.Due to thedifferent subtypes of VGSC participating in the development of different cancers, it isnecessary to design new antibodies targeting extracellular peptide of VGSC.Part One Functionally Expressed of Voltage-gated SodiumChannel Nav1 Family in Human Ovarian CanerObjective: Voltage-gated sodium channel (VGSC) subtypes play important roles in thebionomics of many tumors.This study was to explore the effect of VGSC on metastasisbehaviors of human ovarian cancer cell lines.Methods: RT-PCR and SBFI confacol assayswere respectively used to detect the functional expression of VGSC Nav1 family in ovariancancer cells (Caov-3, SKOV3, Anglne) and normal ovary tissues; MTT and Transwellassays were respectively used to detect the effect of specific VGSC inhibitor tetrodotoxin(TTX) on cell proliferation, migration and invasion of ovarian cancer in vitro.Results: ThemRNA expression levels of Nav1.1, Nav1.3, Nav1.4, Nav1.5 and Nav1.7 wereup-regulation in ovarian cancer cell lines; the distribution of intracellular sodium wasmainly in cell membrane.30μM TTX could decrease the migration and invasion ofstrongly metastatic ovarian cancer cell Caov-3 and SKOV3 to 40%-60%; while TTX hadno obvious effects on weakly metastatic cell Anglne.Conclusion: The functionalup-regulation of Nav1 family in human ovarian cancer might participate in the matastasis of ovarian cancer and be a new therapeutic target.Part Two Functional Expressions of Voltage-gated SodiumChannel SCN5A/Nav1.5 in Human Ovarian CanerObjective: The different subtypes of voltage-gated sodium channel (VGSC) are known tobe correlated with the initiation and progression of many malignant cancers.This study wasto investigate the functional expressions and roles of SCN5A/Nav1.5 on human ovariancancer.Methods: Real-time PCR, Western Blot, Immunocytochemistry andImmunohistochemistry assays were respectively used to detect the expression ofSCN5A/Nav1.5 in mRNA and protein levels in epithelial ovarian tumor specimens andovarian cancer cells lines Caov-3.Results: SCN5A/Nav1.5 was over-expressed in ovariancancer specimens and ovarian cancer cells lines Caov-3.Compared with normal ovaryspecimens, their relative mRNA expression were increased by 13.79±2.78 fold and14.57±1.49 fold, P<0.05; while the Nav1.5 mRNA in benign ovarian tumor specimens haveno obvious up-regulation than that in normal ovary (1.02±0.26 vs 1.18±0.40).Their proteinlevels differences among Caov-3 cells and ovarian tumor specimens were similar to themRNA expression.Conclusion: SCN5A/Nav1.5 plays important roles in the progression ofovarian cancer, which might be a therapeutic target in anti-ovarian cancer research.Part Three Functional Expression of Voltage-gated SodiumChannel Nav1.5 Splice Variants in Ovarian CancerObjective: Aberrations of alternative splicing play important roles in tumorigenesis.Thisstudy was to explore the functional expression of VGSC Nav1.5 splice variants in ovarian cancer cell Caov-3.Methods: RT-PCR, DAN sequencing and Western blot assays wererespectively used to detect the alternative splicing of VGSC Nav1.5 in ovarian cancer cells.Results: DNA sequencing of RT-PCR products revealed that Nav1.5c, the D1:S3 5' spliceform of Nav1.5 was the mainly splice variants existed in Caov-3 cells.Western blot assayrevealed that the Nav1.5 splice variants were up-regulation in Caov-3 cells but absent innormal ovary tissues.Conclusions: D1:S3 5' Nav1.5 might play important roles in themetastatic process of human ovarian cancer and could serve as a therapeutic target incancer research.Part Four E3-targeted anti-Nav1.5 antibody inhibits themigration and invasion of ovarian cancer cells in vitroObjective: The purpose of this study was to investigate the effects of the third extracellulartargeted anti-Nav1.5 antibody (Nav1.5-E3 Ab) on in vitro metastatic cascades of ovariancancer cells Caov-3.Methods: Using confocal microscopy detect the specific and sensitiveof Nav1.5-E3-Ab in Nav1.5 expressed in Caov-3 cells; using CCK-8 kit and Transwellassays analyzed the effects of Nav1.5-E3-Ab on the migration and invasion of ovariancancer cell Caov-3 in vitro.Results: Nav1.5-E3-Ab could specific recognize the Nav1.5existed in Caov-3 cells and Nav1.5-E3 Ab (16μg/ml) reduced the migration and invasion ofCaov-3 cells to (64.14±8.39) % and (52.80±7.99) % with the same extent as tetrodotoxin(TTX), a specific blocker of VGSC.Conclusion: Nav1.5-E3-Ab could significantlysuppress the metastasis behaviours of ovarian cancer cell Caov-3 and it might be a newpromise for the treatment of ovarian tumors.