| Background: T-type Ca2+ channels are often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation and death. T-type Ca2+ channel antagonists significantly inhibited cell proliferation and induced cell apoptosis of tumors with high expression T-type Ca2+ channels. There is increasing evidence suggest that cancer metastasis is controlled by voltage-gated sodium channel(VGSC) activity. Accordingly, the specific VGSC blocker TTX(tetrodotoxin) suppresses a variety of metastatic cell behaviors in vitro. Interestingly, parallels have been drawn between lymphocyte trafficking and cancer metastasis. A variety of ion channels has been discovered in lymphocytes, including voltage-gated potassium channels(KV1.3), calcium-activated potassium channels(KCa), calcium release-activated calcium channels(CRAC), and swelling-activated chloride channels(Clswell). However, there is no report about the expression and functional roles of T-type Ca2+ channels and VGSCs in normal lymphocytes and acute lymphocytic leukemia(ALL) cells. The aim of the study is to claim the expression and functions of T-type Ca2+ channels and VGSCs in ALL cell lines and human peripheral blood mononuclear cells(PBMCs).Methods: RT-PCR, Q-PCR, western blotting, immunofluorescence and whole-cell patch-clamp recording were employed to assess the expression of T-type Ca2+ channels and VGSCs in ALL cell lines and human PBMCs. Cell growth assay and si RNA were used to exploire the function of T-type Ca2+ channels in cell growth of ALL cell lines. We used cell cycle and cell apoptosis assay to detect the effect of T-type Ca2+ channel antagonists on cell cycle and cell apoptosis. The flow cytometric calcium flux assay was employed to test the effect of T-type Ca2+ channel antagonists on intercellular Ca2+ homeostasis and ER(endocytoplasmic reticulum) Ca2+ release.Mitochondrial membrane potential assay was used to assess the effect of T-type Ca2+ channel antagonists on mitochondrial membrane potential and the role of ER-mitochondrial Ca2+ flux in the depolarization of mitochondrial membrane potential. In addition, we used western blotting technique to detect the effect of T-type Ca2+ channel antagonists on ERK signaling pathway. Cell invasion assay was used to explore the role of VGSCs in ALL cell’s invasion.Results:(1) ALL cell lines expressed T-type Ca2+ channels, while human PBMCs did not express T-type Ca2+ channels.(2) T-type Ca2+ channels played an important role in cell proliferation of ALL cell lines.(3) We show that ALL cell lines exhibited reduced cell growth when treated with T-type Ca2+ channel inhibitors, mibefradil and NNC-55-0396 in a concentration-dependent manner. Mechanistically, these inhibitors played a dual role on cell viability:(i) blunting proliferation, through a halt in the progression to the G1-S phase; and(ii) promoting cell apoptosis.(4) T-type Ca2+ channel inhibitors disrupted intercellular Ca2+ homeostasis and induced ER Ca2+ release, which played an important role in cell apoptosis.(5) T-type Ca2+ channel inhibitor- NNC-55-0396 depolarized mitochondrial membrane potential of ALL cell lines, and the effect of NNC-55-0396 on depolarization of the mitochondrial membrane potential may not directly depend on ER-mitochondrial Ca2+ flux.(6) We observed a reduced phosphorylation of ERK1/2 in MOLT-4 cells in response to mibefradil and NNC-55-0396 treatment.(7) ALL cell lines and human PBMCs expressed VGSCs, and the main subunits of VGSCs were TTX-sensitive VGSCs.(8) TTX decreased the invasion of Jurkat and MOLT-4 cells ~90%.Conclusions:(1) T-type Ca2+ channels played an important role in cell proliferation of ALL cell lines.(2) These results indicate that mibefradil and NNC-55-0396 regulate proliferation and apoptosis in T-type Ca2+ channel expressing ALL cells and suggest a potential therapeutic target for leukemia.(3) The results indicate that the activity of VGSCs could represent a novel mechanism potentiating the invasive capacity of ALL cells. |
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