The Effects and Mechanisms of Periplaneta americana Extract Reversal of Multi-Drug Resistance in BEL-7402/5-FU Cells

Abstract

The present study reports the reversing effects of extracts from P. americana on multidrug resistance of BEL-7402/5-FU cells, as well as a preliminary investigation on their mechanism of action. A methylthiazolyl tetrazolium (MTT) method was applied to determine the multidrug resistance of BEL-7402/5-FU, while an intracellular drug accumulation assay was used to evaluate the effects of a column chromatography extract (PACC) and defatted extract (PADF) from P. americana on reversing multi-drug resistance. BEL-7402/5-FU reflected high resistance to 5-FU; PACC and PADF could promote drug accumulation in BEL-7402/5-FU cells, among which PADF was more effective than PACC. Moreover, results from the immunocytochemical method showed that PACC and PADF could downregulate the expression of drug resistance-associated proteins (P-gp, MRP, LRP); PACC and PADF had no effects on the expression of multidrug resistance-associated enzymes (GST-π), but PACC could increase the expression of multidrug resistance-associated enzymes (PKC). Results of real-time fluorescence quantitative PCR revealed that PACC and PADF were able to markedly inhibit the expression of multidrug resistance-associated genes (MDR1, LRP and MRP1); PACC presented a significant impact on the gene expression of multidrug resistance-associated enzymes, which increased the gene expression of GST-π and PKC. However, PADF had little impact on the expression of multidrug resistance-associated enzymes. These results demonstrated that PACC and PADF extracted from P. americana could effectively reverse MDR in BEL-7402/5-FU cells, whose mechanism was to inhibit the expression of P-gp, MRP, and LRP, and that PADF was more effective in the reversal of MDR than did PACC. In addition, some of extracts from P. americana altered (sometimes increasing) the expression of multidrug resistance-associated enzymes.

Keywords: P. americana; hepatocellular carcinoma; multidrug resistance; reversion.

Figure 1

Doubling time of BEL-7402 and BEL-7402/5-FU cells.

Figure 2

The inhibitory effect of chemotherapeutic drugs on BEL-7402 cells and BEL-7402/5-FU cells. (a) 5-FU; (b) ADM; (c) DDP.

Figure 3

The cytoxicity of PACC and PADF on the proliferation of BEL-7402/5-FU.

Figure 4

The effects of PACC and PADF on the accumulation of ADM in BEL-7402/5-FU cells (×400). (a) BEL-7402 cells, (b) BEL-7402/5-FU cells, and (c) BEL-7402/5-FU cells were treated with 50 μg/mL PACC; (d) BEL-7402/5-FU cells were treated with 85 μg/mL PACC; (e) BEL-7402/5-FU cells were treated with 23 μg/mL PADF; (f) BEL-7402/5-FU cells were treated with 45 μg/mL PADF.

Figure 4

The effects of PACC and PADF on the accumulation of ADM in BEL-7402/5-FU cells (×400). (a) BEL-7402 cells, (b) BEL-7402/5-FU cells, and (c) BEL-7402/5-FU cells were treated with 50 μg/mL PACC; (d) BEL-7402/5-FU cells were treated with 85 μg/mL PACC; (e) BEL-7402/5-FU cells were treated with 23 μg/mL PADF; (f) BEL-7402/5-FU cells were treated with 45 μg/mL PADF.

Figure 5

The relative expression of multidrug resistance-associated genes in BEL-7402 cells and BEL-7402/5-FU cells. Values are presented by mean ± SD (n = 6). (a) Gene expression of multidrug resistance-associated proteins; (b) gene expression of multidrug resistance-associated enzymes. * p < 0.05, ** p < 0.01 vs. BEL-7402.

Figure 6

The Effects of PACC and PADF on the expression of multidrug resistance-associated proteins (×400). (A) Expression of P-gp; (B) expression of MRP; (C) expression of LRP; (D) expression of GST-π; (E) expression of PKC. (a) blank control group, (b) BEL-7402 cells, (c) BEL-7402/5-FU cells, and (d) BEL-7402/5-FU cells were treated with 50 μg/mL PACC; (e) BEL-7402/5-FU cells were treated with 85 μg/mL PACC; (f) BEL-7402/5-FU cells were treated with 23 μg/mL PADF; (g) BEL-7402/5-FU cells were treated with 45 μg/mL PADF.

Figure 6

The Effects of PACC and PADF on the expression of multidrug resistance-associated proteins (×400). (A) Expression of P-gp; (B) expression of MRP; (C) expression of LRP; (D) expression of GST-π; (E) expression of PKC. (a) blank control group, (b) BEL-7402 cells, (c) BEL-7402/5-FU cells, and (d) BEL-7402/5-FU cells were treated with 50 μg/mL PACC; (e) BEL-7402/5-FU cells were treated with 85 μg/mL PACC; (f) BEL-7402/5-FU cells were treated with 23 μg/mL PADF; (g) BEL-7402/5-FU cells were treated with 45 μg/mL PADF.

Figure 6

The Effects of PACC and PADF on the expression of multidrug resistance-associated proteins (×400). (A) Expression of P-gp; (B) expression of MRP; (C) expression of LRP; (D) expression of GST-π; (E) expression of PKC. (a) blank control group, (b) BEL-7402 cells, (c) BEL-7402/5-FU cells, and (d) BEL-7402/5-FU cells were treated with 50 μg/mL PACC; (e) BEL-7402/5-FU cells were treated with 85 μg/mL PACC; (f) BEL-7402/5-FU cells were treated with 23 μg/mL PADF; (g) BEL-7402/5-FU cells were treated with 45 μg/mL PADF.

Figure 7

The relative expression of PACC and PADF on multidrug resistance-associated genes; Values are presented by mean ± SD (n = 6). (a) Effects of PACC and PADF from P. americana on the expression of multidrug resistance-associated genes; (b) Effects of PACC and PADF from P. americana on the gene expression of multidrug resistance-associated enzymes. * p < 0.05, ** p < 0.01 vs. BEL-7402, ^▲ p < 0.05, ^▲▲ p < 0.01 vs. PACC.

Figure 7

The relative expression of PACC and PADF on multidrug resistance-associated genes; Values are presented by mean ± SD (n = 6). (a) Effects of PACC and PADF from P. americana on the expression of multidrug resistance-associated genes; (b) Effects of PACC and PADF from P. americana on the gene expression of multidrug resistance-associated enzymes. * p < 0.05, ** p < 0.01 vs. BEL-7402, ^▲ p < 0.05, ^▲▲ p < 0.01 vs. PACC.