Ca(2+)-dependent potassium channels play important roles in regulatory volume decrease in human nasopharyngeal carcinoma cells / 生理学报

It has been shown that cell volume regulation mechanisms play important roles in various cell functions. We demonstrated previously that volume-activated chloride channels were involved in cell volume regulation. The present study aimed to clarify the roles of various types of potassium channels in regulatory volume decrease (RVD) induced by hypotonic challenges in human nasopharyngeal carcinoma cells (CNE-2Z cells). The whole-cell patch clamp technique was used to record hypotonic challenge-induced potassium currents. During current recordings, cells were held at 0 mV and stepped to +/-46 and +/-92 mV, repeatedly. The cell volume was computed from cell diameters. The changes of cell volume were monitored and analyzed by the time-lapse imaging technique. The results showed that the exposure to 160 mOsm/L hypotonic solution caused the cells to swell by (144.5+/-4.2)%, activated a potassium current (59.2 pA/pF+/-13.3 pA/pF at 92 mV), and induced RVD. Cell volume was recovered from hypotonic challenge-induced swelling by (48.9+/-4.6)% after 20 min. The potassium current (at 92 mV) and RVD were inhibited by the calcium-dependent potassium channel blocker, clotrimazole (100 mumol/L), by (98.5+/-2.8)% and (89.3+/-4.9)%, respectively. Depletion of extracellular calcium prevented the activation of the hypotonic challenge-induced potassium current and inhibited the process of RVD. The voltage-gated potassium channel blocker, 4-AP (5 mmol/L), partially inhibited the hypotonic challenge-activated potassium currents by (66.6+/-5.3)% (at 92 mV). These results suggest that the Ca(2+)-dependent potassium channel is the main component of volume-activated potassium channels and plays an important role in volume regulation of CNE-2Z cells. The voltage-gated potassium channels may also contribute in part to the formation of the volume-activated potassium current.

Effect of p21-targeted shRNA on curcumin-induced apoptosis of human hepatoma Huh7 cells / 药学学报

In the present study, shRNA plasmid of pSi-p21 targeting p21 mRNA was constructed and the effect of p21 shRNA on curcumin-induced apoptosis of human hepatoma Huh7 cells was investigated. The effect of curcumin on the expression of p21 mRNA and protein and the silence efficiency of pSi-p21 were detected with RT-PCR and Western blotting. The effect of pSi-p21 on curcumin-induced apoptosis of Huh7 cells was evaluated with DAPI staining. The results showed that curcumin significantly upregulated p21 mRNA and protein expression, which was knocked down by pSi-p21 of Huh7 cells. DAPI staining results showed that pSi-p21 significantly decreased curcumin-induced apoptosis of Huh7 cells. The data suggested that curcumin induced apoptosis of Huh7 cells via upregulation of p21 expression.

Effect of curcumin on JAK-STAT signaling pathway in hepatoma cell lines / 药学学报

The effect of curcumin on JAK-STAT signaling pathway was investigated in hepatoma cell lines Huh7 and Hep3B. Curcumin inhibited cell proliferation and induced apoptosis of both cell lines, but Huh7 cells were more sensitive to curcumin than Hep3B cells. Curcumin (50 micromol x L(-1)) significantly increased phosphorylations of p38 (T180/Y182) and STAT-1 (S727) in Huh7 and Hep3B cells, and caused relocalization of phosphorylated-STAT-1 (Y701) from cytoplasm to nucleus in Hep3B cells. In addition, curcumin (25 and 50 micromol x L(-1)) dramatically suppressed the phosphorylation level of STAT-1 (Y701) and resulted in a significant reduction of nuclear phosphorylated-STAT-1 (Y701) in Huh7 cells.

Background chloride currents in fetal human nasopharyngeal epithelial cells / 生理学报

To characterize the background current in fetal human nasopharyngeal epithelial cells and clarify its relationship with volume activated Cl(-) currents (I(Cl,vol)), whole-cell patch clamp and cell imaging techniques were employed. Under isotonic conditions, a background current [(5.9+/-2.1) pA/pF at +80 mV, n=21] was detected. The current presented a weak outward rectification and a negligible time-dependent inactivation. The current-voltage relationship showed that the reversal potential of the background current [(-0.73+/-1.7) mV, n=21] was close to the calculated equilibrium potential for Cl(-)(-0.9 mV). Application of extracellular hypertonic stimulation (440 mOsmol/L) suppressed the current by (59.6+/-7.1)% and the inhibition was reversible after returned to isotonic conditions. Bathing the cells in hypotonic solution (160 mOsmol/L) induced a volume-sensitive Cl(-) current. The Cl(-) channel blockers, tamoxifen (20 micromol/L) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (100 micromol/L), inhibited the background current by (74.0+/-5.2)% (P<0.01, n=5) and (60.9+/-8.9)% (P<0.01, n=6) at +80 mV and increased basal cell volume by (107.7+/-2.9)% (P<0.01, n=25) and (104.4+/-2.4)% (P<0.01, n=19), respectively. The data indicate that Cl(-) current is an important component of the background current in fetal human nasopharyngeal epithelial cells. The background Cl(-) current is involved in volume activated Cl(-) current and basal cell volume regulation.

Activation of chloride current and decrease of cell volume by ATP in nasopharyngeal carcinoma cells / 生理学报

Whole-cell patch clamp and cell volume measurement techniques were used to investigate the ATP-activated chloride current and the ATP effect on cell volume in nasopharyngeal carcinoma cells. Extracellular application of ATP in micromolar concentrations activated a current with the properties of modest outward rectification and negligible time-dependent inactivation in a dose-dependent manner. The current reversed at a potential [(-0.05+/-0.03) mV] close to the Cl- equilibrium potential (-0.9 mV). Substitution of Cl- with gluconate in the extracellular solution decreased the ATP-activated current and shifted the reversal potential positively. NPPB, one of the chloride channel blockers, inhibited the current by (81.03+/-9.36)%. The current was also depressed by the P2Y purinoceptor antagonist, reactive blue 2, by (67.39+/-5.06)%. ATP (50 micromol/L) decreased the cell volume under the isotonic condition. Depletion of extracellular and intracellular Cl- abolished the ATP effect on cell volume. The results suggest that extracellular ATP of micromolar scales can induce a chloride current associated with cell volume regulation by activation of chloride channel through binding to purinoceptor P2Y.

Volume-activated Cl- current in migrated nasopharyngeal carcinoma cells / 生理学报

The transwell chamber migration assay and the patch-clamp technique were used to investigate the volume-activated Cl(-) current (I(Cl.vol)) in migrated nasopharyngeal carcinoma cells (CNE-2Z). 47% hypotonic solution activated a ICl.vol in the migrated CNE-2Z cells. Compared with the control cells (non-migrated), the properties of this current and the sensitivity to Cl(-) channel blockers were changed. The current density in migrated CNE-2Z cells was higher than that in non-migrated cells. The current was almost completely inhibited by extracellular application of adenosine-5'-triphosphate (ATP, 10 mmol/L), 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB, 100 mmol/L) and tamoxifen (30 mmol/L) in all voltage steps applied. The inhibition of NPPB and tamoxifen on the current was stronger in migrated cells than that in non-migrated cells. The permeability sequence of the four anions was Br(-)>Cl(-)> I (-)>Gluconate. The sequence was different from that of the non-migrated cells (I(-)> Br(-)> Cl(-)> Gluconate). The results suggest that volume-activated chloride channels may be involved in the CNE-2Z cell migration.