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T-type calcium channel antagonists, mibefradil and NNC-55-0396 inhibit cell proliferation and induce cell apoptosis in leukemia cell lines.

Huang W, Lu C, Wu Y, Ouyang S, Chen Y - J. Exp. Clin. Cancer Res. (2015)

Bottom Line: T-type Ca(2+) channels are often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation and death.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, partially dependent on the endoplasmic reticulum Ca(2+) release.In addition, we observed a reduced phosphorylation of ERK1/2 in MOLT-4 cells in response to mibefradil and NNC-55-0396 treatment.

View Article: PubMed Central - PubMed

Affiliation: Fujian Institute of Hematology, Fujian Medical University Union Hospital, Fuzhou, 350004, People's Republic of China. hwf0625@163.com.

ABSTRACT

Background: T-type Ca(2+) channels are often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation and death.

Methods: RT-PCR, Q-PCR, western blotting and whole-cell patch-clamp recording were employed to assess the expression of T-type Ca(2+) channels in leukemia cell lines. The function of T-type Ca(2+) channels in leukemia cell growth and the possible mechanism of the effect of T-type Ca(2+) channel antagonists on cell proliferation and apoptosis were examined in T-lymphoma cell lines.

Results: We show that leukemia cell lines exhibited reduced cell growth when treated with T-type Ca(2+) 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, partially dependent on the endoplasmic reticulum Ca(2+) release. In addition, we observed a reduced phosphorylation of ERK1/2 in MOLT-4 cells in response to mibefradil and NNC-55-0396 treatment.

Conclusions: These results indicate that mibefradil and NNC-55-0396 regulate proliferation and apoptosis in T-type Ca(2+) channel expressing leukemia cell lines and suggest a potential therapeutic target for leukemia.

No MeSH data available.


Related in: MedlinePlus

Effect of T-type Ca2+ channel antagonist, NNC-55-0396 on intracellular Ca2+ levels in Jurkat T cells. a Jurkat cells stained with Fluo-4 were preincubated with 2.5-10 μM NNC-55-0396 in the presence of extracellular Ca2+. For each sample, after the 10 min treatment with different concentrations of NNC-55-0396 baseline Ca2+ measurements were taken, cells were then stimulated at the 2 min mark with 10 μg/ml soluble anti-CD3 monoclonal antibody (mAb), OKT3 (R&D Systems, Minneapolis, MN, USA) to activate Ca2+ influx, and the analysis was immediately resumed. b Fluo-4 loaded Jurkat cells were treated with 2.5-10 μM NNC-55-0396 and stimulated in the absence of extracellular Ca2+. Results are representative of 3 independent experiments
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Fig6: Effect of T-type Ca2+ channel antagonist, NNC-55-0396 on intracellular Ca2+ levels in Jurkat T cells. a Jurkat cells stained with Fluo-4 were preincubated with 2.5-10 μM NNC-55-0396 in the presence of extracellular Ca2+. For each sample, after the 10 min treatment with different concentrations of NNC-55-0396 baseline Ca2+ measurements were taken, cells were then stimulated at the 2 min mark with 10 μg/ml soluble anti-CD3 monoclonal antibody (mAb), OKT3 (R&D Systems, Minneapolis, MN, USA) to activate Ca2+ influx, and the analysis was immediately resumed. b Fluo-4 loaded Jurkat cells were treated with 2.5-10 μM NNC-55-0396 and stimulated in the absence of extracellular Ca2+. Results are representative of 3 independent experiments

Mentions: Disruption of intracellular Ca2+ homeostasis is one of the primary processes in the early development of cell injury [31–33], and NNC-55-0396 had stronger cytotoxicity than mibefradil, especially for Jurkat cells. Thus, we examined the effect of NNC-55-0396 on intracellular Ca2+ level in Jurkat cells using flow cytometry. After NNC-55-0396 treatment, a dose-dependent increase in cytosolic Ca2+ concentration was seen in the absence of extracelluar Ca2+ (Fig. 6b). Moreover, low concentration NNC-55-0396 (<5 μM) decreased intracellular baseline Ca2+ levels, while high concentration NNC-55-0396 (>5 μM) diminished or abolished the inhibiting effect of intracellular baseline Ca2+ levels in the present of extracelluar Ca2+ (Fig. 6a). In addition, 10 μM NNC-55-0396 induced sustained Ca2+ overload (Fig. 6a, Green line). In fact, high concentration mibefradil and NNC-55-0396 also induced intracellular Ca2+ overload in MOLT-4 cells (Additional file 3: Figure S3).Fig. 6


T-type calcium channel antagonists, mibefradil and NNC-55-0396 inhibit cell proliferation and induce cell apoptosis in leukemia cell lines.

Huang W, Lu C, Wu Y, Ouyang S, Chen Y - J. Exp. Clin. Cancer Res. (2015)

Effect of T-type Ca2+ channel antagonist, NNC-55-0396 on intracellular Ca2+ levels in Jurkat T cells. a Jurkat cells stained with Fluo-4 were preincubated with 2.5-10 μM NNC-55-0396 in the presence of extracellular Ca2+. For each sample, after the 10 min treatment with different concentrations of NNC-55-0396 baseline Ca2+ measurements were taken, cells were then stimulated at the 2 min mark with 10 μg/ml soluble anti-CD3 monoclonal antibody (mAb), OKT3 (R&D Systems, Minneapolis, MN, USA) to activate Ca2+ influx, and the analysis was immediately resumed. b Fluo-4 loaded Jurkat cells were treated with 2.5-10 μM NNC-55-0396 and stimulated in the absence of extracellular Ca2+. Results are representative of 3 independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4443536&req=5

Fig6: Effect of T-type Ca2+ channel antagonist, NNC-55-0396 on intracellular Ca2+ levels in Jurkat T cells. a Jurkat cells stained with Fluo-4 were preincubated with 2.5-10 μM NNC-55-0396 in the presence of extracellular Ca2+. For each sample, after the 10 min treatment with different concentrations of NNC-55-0396 baseline Ca2+ measurements were taken, cells were then stimulated at the 2 min mark with 10 μg/ml soluble anti-CD3 monoclonal antibody (mAb), OKT3 (R&D Systems, Minneapolis, MN, USA) to activate Ca2+ influx, and the analysis was immediately resumed. b Fluo-4 loaded Jurkat cells were treated with 2.5-10 μM NNC-55-0396 and stimulated in the absence of extracellular Ca2+. Results are representative of 3 independent experiments
Mentions: Disruption of intracellular Ca2+ homeostasis is one of the primary processes in the early development of cell injury [31–33], and NNC-55-0396 had stronger cytotoxicity than mibefradil, especially for Jurkat cells. Thus, we examined the effect of NNC-55-0396 on intracellular Ca2+ level in Jurkat cells using flow cytometry. After NNC-55-0396 treatment, a dose-dependent increase in cytosolic Ca2+ concentration was seen in the absence of extracelluar Ca2+ (Fig. 6b). Moreover, low concentration NNC-55-0396 (<5 μM) decreased intracellular baseline Ca2+ levels, while high concentration NNC-55-0396 (>5 μM) diminished or abolished the inhibiting effect of intracellular baseline Ca2+ levels in the present of extracelluar Ca2+ (Fig. 6a). In addition, 10 μM NNC-55-0396 induced sustained Ca2+ overload (Fig. 6a, Green line). In fact, high concentration mibefradil and NNC-55-0396 also induced intracellular Ca2+ overload in MOLT-4 cells (Additional file 3: Figure S3).Fig. 6

Bottom Line: T-type Ca(2+) channels are often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation and death.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, partially dependent on the endoplasmic reticulum Ca(2+) release.In addition, we observed a reduced phosphorylation of ERK1/2 in MOLT-4 cells in response to mibefradil and NNC-55-0396 treatment.

View Article: PubMed Central - PubMed

Affiliation: Fujian Institute of Hematology, Fujian Medical University Union Hospital, Fuzhou, 350004, People's Republic of China. hwf0625@163.com.

ABSTRACT

Background: T-type Ca(2+) channels are often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation and death.

Methods: RT-PCR, Q-PCR, western blotting and whole-cell patch-clamp recording were employed to assess the expression of T-type Ca(2+) channels in leukemia cell lines. The function of T-type Ca(2+) channels in leukemia cell growth and the possible mechanism of the effect of T-type Ca(2+) channel antagonists on cell proliferation and apoptosis were examined in T-lymphoma cell lines.

Results: We show that leukemia cell lines exhibited reduced cell growth when treated with T-type Ca(2+) 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, partially dependent on the endoplasmic reticulum Ca(2+) release. In addition, we observed a reduced phosphorylation of ERK1/2 in MOLT-4 cells in response to mibefradil and NNC-55-0396 treatment.

Conclusions: These results indicate that mibefradil and NNC-55-0396 regulate proliferation and apoptosis in T-type Ca(2+) channel expressing leukemia cell lines and suggest a potential therapeutic target for leukemia.

No MeSH data available.


Related in: MedlinePlus