Antiepileptic drug-induced multidrug resistance P-glycoprotein overexpression in astrocytes cultured from rat brains

Background Intractable epilepsy may be due to multidrug resistance induced by conventional antiepileptic drugs. The phenomenon is sometimes associated with an overexpression of multidrug resistance gene 1 (MDR 1). The purpose of this study was to determine if the overexpression of MDR 1 could be induced in astrocytes from rat brains in vitro using antiepileptic drugs.Methods Astrocyte cell cultures from postnatal Wistar rats (within 24 hours of birth) were established. Different concentrations of the antiepileptic drugs phenytoin, phenobarbital, carbamazepine, and valproic acid were added to the cultures for 10, 20, or 30 days. The expression of P-glycoprotein (Pgp), the protein product of MDR 1, was investigated with immunocytochemistry. Results Less than 5% of normal, untreated astrocytes had detectable Pgp staining at any time point. Phenytoin, phenobarbital, carbamazepine, and valproic acid induced the overexpression of Pgp in astrocytes in a dose- and time-dependent manner. Significantly higher levels of Pgp staining were detected at therapeutic concentrations of certain antiepileptic drugs (20 μg/ml phenobarbital, 40 μg/ml phenobarbital, and 20 μg/ml phenytoin) on day 30. Upregulation of Pgp was detected when using higher concentrations of phenytoin, phenobarbital, and valproic acid on day 20 and when using higher concentrations of any of the four antiepileptic drugs on day 30. Conclusions Treatment with antiepileptic drugs may contribute to the overexpression in astrocytes of MDR 1 and its protein product, Pgp. The mechanism leading to MDR must be considered in patients undergoing long-term treatment with antiepileptic drugs.

Chemosensitization of HepG2 cells by suppression of NF-κB/p65 gene transcription with specific-si RNA

AIM: To investigate small interfering RNA(si RNA)-mediated inhibition of nuclear factor-kappa B(NF-κB) activation and multidrug-resistant(MDR) phenotype formation in human Hep G2 cells. METHODS: Total RNA was extracted from human Hep G2 or LO2 cells. NF-κB/p65 m RNA was amplified by nested reverse transcription polymerase chain reaction and confirmed by sequencing. NF-κB/p65 was analyzed by immunohistochemistry. Specific-si RNA was transfected to Hep G2 cells to knock down NF-κB/p65 expression. The effects on cell proliferation, survival, and apoptosis were assessed, and the level of NF-κB/p65 or P-glycoprotein(P-gp) was quantitatively analyzed by enzyme-linked immunosorbent assay.RESULTS: Hep G2 cells express NF-κB/p65 and express relatively less phosphorylated p65(P-p65) and little P-gp. After treatment of Hep G2 cells with different doses of doxorubicin, the expression of NF-κB/p65, P-p65, and especially P-gp were dose-dependently upregulated. After Hep G2 cells were transfected with NF-κB/p65 si RNA(100 nmol/L), the expression of NF-κB/p65, P-p65, and P-gp were downregulatedsignificantly and dose-dependently. The viability of Hep G2 cells was decreased to 23% in the combination NF-κB/p65 si RNA(100 nmol/L) and doxorubicin(0.5 μmol/L) group and 47% in the doxorubicin(0.5 μmol/L) group(t = 7.043, P < 0.001). CONCLUSION: Knockdown of NF-κB/p65 with si RNA is an effective strategy for inhibiting Hep G2 cell growth by downregulating P-gp expression associated chemosensitization and apoptosis induction.