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10.1080/13556210500122995 Flavin Adenine Dinucleotide Disodium [PubMed] [CrossRef] [Google Scholar] 26. an ATP-dependent broad-spectrum medication efflux pump, and the drug concentration in cells is usually down-regulated by P-gp. It is involved with lots of structurally uncorrelated anti-cancer drugs, for instance, paclitaxel, docetaxel, doxorubicin, and vincristine [12], and that can lead to multidrug resistance (MDR). The relationship between cancer chemotherapy resistance and P-gp expression has been confirmed in numerous clinical studies [13C15]. In recent years, there has been a certain degree of success in the development of P-gp-mediated paclitaxel resistance reversal brokers [16C18]. Many clinical studies have provided evidence that III-tubulin overexpression results in another type of paclitaxel resistance in tumor cells [19C21]. Generally, mainly in neuronal cells, III-tubulin is usually expressed and it is rarely detectable in other tissues. However, III-tubulin has abnormally high expression in certain drug-resistant tumor cells originating from breast, lung, prostate, and stomach tissues [19, 22]. The exact mechanism of this type of resistance is not fully elucidated. To overcome the drug resistance mentioned above, our lab pursued novel MDR reversal brokers from natural products that can resume the sensitivity to chemotherapy drugs for MDR tumor cells. Among these compounds, EM-E-11-4 is a lathyrane-type diterpenoid from that could markedly reverse the sensitivity of drug-resistant cells from different tissues to paclitaxel at its concentration without cytotoxicity. Those tissues we investigated include the human lung adenocarcinoma cell line A549 and its P-gp overexpression drug-resistant counterpart A549/Tax, the same as the paclitaxel-resistant cell line Hela/III and the human cervical cell line Hela. Hela/III is usually originated from III-tubulin gene transfection. Therefore, we also explored the mechanism of action for the effectiveness of EM-E-11-4 in MDR reversal. RESULTS Reversal effect of EM-E-11-4 in Flavin Adenine Dinucleotide Disodium drug-resistance cells The cytotoxicity of EM-E-11-4 was examined by an MTT assay in A549/Tax (overexpression of P-gp, Physique 1B) and Hela/III (overexpression of III-tubulin, Physique 1C) cell lines. As shown in Table 1, EM-E-11-4 at 20-30 exerted considerable cytotoxicity. In the MDR cell lines, A549/Tax and Hela/III exhibited the same sensitivity to EM-E-11-4 as their parental cells. More than 90% of cells survived at a concentration of 10 EM-E-11-4 in all assays. According to the results from the cytotoxicity assay mentioned above, EM-E-11-4 at 2.5, 5, and Flavin Adenine Dinucleotide Disodium 10 M was chosen to evaluate the reversal activity. As shown in Table 2, EM-E-11-4 markedly decreased IC50 KEL values for paclitaxel in A549/Tax, Hela/III, and their parental cells. EM-E-11-4 strengthened the effect of paclitaxel better than verapamil in A549/Tax cells, and it had similar effects in Hela/III cells. These results indicate that EM-E-11-4 could reverse paclitaxel-resistance Flavin Adenine Dinucleotide Disodium mediated through P-gp or III-tubulin. Open in a separate window Physique 1 The expression of P-gp and III-tubulin in cells. (A) Chemical structure of EM-E-11-4. (B) P-gp levels in A549 and A549/Tax cells. (C) III-tubulin levels in Hela and Hela/III cells. (D) The effect of P-gp siRNA on P-gp expression in A549/Tax cells. (E) The effect of TUBB3 siRNA on III-tubulin expression in Hela/III cells. Cells were treated with vehicle (unfavorable control, NC) or siRNAs (P-gp siRNA, TUBB3-siRNA), and protein levels were determined by Western blot analysis. Table 1 Cytotoxic activities of EM-E-11-4 against various human tumor cell lines. CompoundIC50 (M, Mean SD)IC50 (M, Mean SD)A549A549/TaxHelaHela/IIIEM-E-11-431.5 2.340.2 2.021.1 4.625.3 5.8 Open in a separate window Data are presented as mean SD from three independent experiments. Table 2 Cytotoxic activity of paclitaxel combined with EM-E-11-4 against various human tumor cell lines. Cell linesIC50 (nM, Mean SD)/ Reverse IndexPaclitaxel+EM-E-11-4 (2.5M)+EM-E-11-4 (5M)+EM-E-11-4 (10M)+Vrp (10M)A5494.71.03.30.6 (1.4)0.590.08 (8.0)0.410.12 (11.5)4.30.9 (/)A549/Tax15598615713.4 (9.9)56.77.7 (27.5)22.9 4.7 (68.1)64.77.6 (24.1)Hela4.30.42.8 0.42 (1.5)1.40.3 (3.1)0.630.11 (6.8)/Hela/-III52.94.94.61.0 (11.5)3.60.8 (14.7)1.80.4 (29.4)/ Open in a separate window Reverse Index = IC50 (paclitaxel)/IC50 (paclitaxel+EM-E-11-4) Vrp, verapamil. Data are presented as mean SD from three impartial experiments. Through cell transfection with siRNAs targeting P-gp or III-tubulin, the expression level of P-gp or III-tubulin was suppressed (Physique 1D and ?and1E).1E). With or without EM-E-11-4 treatment, the activity of paclitaxel in the siRNA transfected cells was respectively assessed. As Table 3 shows, suppressed expression of P-gp or III-tubulin through siRNA increases the sensitivity of A549/Tax and Hela/III cells to paclitaxel. However, EM-E-11-4 did not dramatically influence the IC50 of paclitaxel in those cells. All the results confirm that EM-E-11-4 could reverse drug resistance by suppressing the functions of P-gp or III-tubulin. Flavin Adenine Dinucleotide Disodium Table 3 Cytotoxic activity of paclitaxel combined with EM-E-11-4 against A549/Tax (P-gp siRNA) and Hela/III (TUBB3 siRNA) cell lines. CompoundIC50 (nM, Mean SD)IC50 (nM, Mean SD)A549/TaxHela/IIINCP-siRNANCT-siRNAPaclitaxel14983524.43.648.74.32.40.3Paclitaxel + EM-E-11-4 10M25.43.222.52.72.10.42.30.4 Open in a separate window Data are presented as mean SD from three independent experiments. EM-E-11-4 strengthened the effect of paclitaxel-induced G2/M phase arrest.