Inhibitory Effect Of The Na⁺/Ca²⁺ Exchanger （NCX） Blockers On The Growth Of Glioblastoma Cells:A Pharmacological Study

Objection:Glioblastoma is the most common primary malignant tumor of the central nervous system and currently lacks effective treatment.Intracellular calcium(Ca²⁺)is involved in the regulation of various physiological activities of cells such as cell proliferation,differentiation and death.The plasma membrane calcium transporter ATPase（PMCA）and the sodium-calcium exchanger（NCX）on the cell membrane are responsible for the extracellular transport of Ca²⁺,and NCX is the main protein machinery for buffering intracellular Ca²⁺overload.Therefore,inhibition of NCX function may cause intracellular Ca²⁺overload and cell death.This study aims to explore the potential value of NCX as a target for the treatment of glioma by identifying the inhibitory effects of NCX blockers on glioma cells.Methods:1)Select the 6 NCX blockers and observe their effects on the proliferation of human glioma cell lines（GBMs）and astrocytes（HA）;2)Using Ca²⁺imaging technology to observe the effect of NCX blocker on intracellular free Ca²⁺concentration;3)Using flow cytometry and western blot analysis experiments to explore the molecular mechanism of NCX inhibitors killing GBMs;4)Using western blot experiments identified three isoforms of NCX（NCX1-3）expressed in GBMs and HA;5)Human glioma cells were implanted in the brain of nude mice,and the inhibitory effect of NCX blockers on intratumoral tumors was observed by small animal in vivo imaging technique.Results:1)When the drug selectively blocks the reverse mode of NCX,the growth of GBMs is not affected.When the forward mode of NCX is blocked,the growth of GBMs is significantly inhibited;2)Bepridil and CB-DMB do not inhibit the viability of HA cells,and the functional expression of NCX1 in HA cells is significantly higher than that of GBMs;3)NCX inhibitors blocks intracellular Ca²⁺efflux and causes intracellular Ca²⁺overload,induces Ca²⁺-mediated apoptosis and cell cycle arrest in GBMs,and BAPTA-AM can significantly reverse the cell death signal;4)Ca²⁺-activated MAPK signaling pathway is involved in cell cycle arrest and apoptosis induced by NCX inhibotors;5)Bepridil can pass the blood-brain barrier and significantly inhibit the growth of glioma in nude mouse brain.Conclusion:1)Blocking NCX forward mode can inhibit the growth of glioma,while blocking NCX reverse mode has no effect on glioma;2)NCX inhibotors do not inhibit HA cell activity,which may be due to the different functional expression of NCX between HA and GBMs;3)NCX has potential to become a new target for the treatment of glioma,and bepridil and CB-DMB are expected to be lead drugs.