Identification Of Compounds Targeting Pgp From Substituted Quinoxalinones And The Activity Of QA3 To Reverse MDR Of Cancer Cells

Backgrounds: Multidrug resistance (MDR) is a critical issue in cancer chemotherapy. Over-expression of Pgp is the most frequent cause of MDR. A range of agents that can reverse the MDR phenotype and restore drug sensitivity to cancer cells has been developed. However, most of these agents have proven to be intrinsic toxic or the undesired effects on the pharmacokinetics of accompanying anticancer drugs.P-glycoprotein (Pgp), a transmembrane glycoprotein, functions as an ATP-dependent drug transporter which unilaterally transports intracellular drugs out of cells to acquire drug resistance. Pgp, the product of the multidrug resistance 1 (Mdr1) gene, is one of the most important members of the ATP-binding cassette (ABC) transporter family of membrane proteins that utilises the energy of ATP hydrolysis to exclude hydrophobic compounds from cells. Structure analysis based on the Pgp sequence predicts that Pgp consists of two homologous halves, each containing six transmembrane domains (TMDs) and a cytoplasmic nucleotide-binding domain (NBD). The NBDs are responsible for the ATP hydrolysis that is associated with drug efflux. A short linker region joins NBD1 with TMD2. Four previously identified phosphorylation target sites for protein kinase C (PKC) within the linker region of Pgp are also shown.Based on the 3D structures of known Pgp and binding modes of compounds in complex with Pgp, We designed and synthesized a series of substituted quinoxalinones through efficient and simple approach. Theoretically, the compounds might selectively inhibit the activity of Pgp. Herein; we identified the compounds targeting Pgp from quinoxalinones and valued the effect of QA3, one of the substituted quinoxalinones, in reversal of MDR via modulation of Pgp function and expression.Methods: In the first part of the experiments, we aimed to identify the compounds targeting Pgp from substituted quinoxalinones. The cell growth inhibition of K5662/A02 cells was assayed using MTT method. We used MTT assay to evaluate the effects of compounds on reversal of MDR in the supernatants of K5662/A02 cell culture. The intracellular accumulation of Adr was measured using multilabel counter with excitation/emission wavelengths of 485/585 nm. Furthermore, the intracellular accumulation and efflux of Rh123 were measured using multilabel counter with excitation/emission wavelengths of 485/535 nm.In the second part of the experiments, we examined the mechanisms by which QA3 inhibited Pgp using K562/A02 cells. To examine the effects of QA3 on intracellular ATP levels, K562/A02 cells were treated with various concentrations of QA3 (2-10μg/ml) solution. ATP was determined using a luciferin/luciferase assay. We also examined Pgp- ATPase activity which was determined from the difference in inorganic phosphate released. The activity of PKC was determined according to Assay for Non-Radioactive Detection of PKC. We further examined the expression of Pgp using Western blot. The level of Mdr1 mRNA was valued by RT-PCR. The proportion of apoptosis cells was observed by analysis of annexin V/FITC plus PI staining. The levels of Pgp associated-apoptotic markers Caspase-3 and Caspase-9 were determined by Western blot.Results: Compared with K562 cell line, there were several different aspects in K562/A02 cell lines. The IC₅₀ value of Adr toward K562/A02 cells was higher than that toward K562 cells, the reversal factor was reached 233.3. The intracellular Adr and Rh123 accumulations were decreased in K562/A02 cells. However, a drastic decrease of intracellular Rhl23 levels was observed in individual time point (15, 30, 45, 60, 90 min). MTT assay showed that most of compounds weakly inhibited the growth of tumor cells. However, the compounds QA1, QA3 and QA7 increased Adr-induced cytotoxicity toward K562/A02 cells. The IC50 values of Adr toward K562/A02 were decreased in the presence of QA1, QA3 and QA7. The maximal reversal fold (RF) of QA1, QA3 and QA7 was reached 6.36, 6.48, and 6.07, respectively. The action of QA1, QA3 and QA7 was also confirmed by the increase of intracellular Adr accumulation in K562/A02 cells. An increase of intracellular Rhl23 and the decrease of efflux were observed in K562/A02 cells incubation with QA1, QA3 or QA7. What's more, a deceased activity of PKC and high levels of Pgp and Mdr1 in K562/A02 cells were abserved.Following exposure of K562/A02 to QA3, the ATP levels were depleted using luciferin-luciferase assay for ATP intracellular levels. The relative light unit was deceased 14.76%, 45.55%, 55.81%, 57.96%, 59.67% compared with that in the K562/A02 cells (100%), respectively. Furthermore, QA3 treatment stimulated Pgp-ATPase activity in membranes of K562/A02 cells. The released Pi content was increased slightly in the presence of 2, 4μg/ml of QA3, however, in the presence of 6, 8μg/ml of QA3 the released Pi contents were 28.0 and 34.28-fold compared to control (in the absence of QA3), respectively. And the released Pi contents in the presence of 8μg/ml of QA3 were equal to those of 10μg/ml of QA3. A combination of experiments examined the activity of PKC which played an important role in Pgp phosphorylation. Using the assay of non-radioactive detection of PKC, the activity of PKC was significantly decreased after the treatment of QA3. The potential ability of QA3 to modulate the expression of Pgp and Mdr1 was also evaluated using Western blot analysis and RT-PCR, respectively. The results showed that QA3 inhibited the expression of Pgp in K562/A02 cells after 72 h incubation. However, there were no effects on the expression of Mdr1. These results suggest that QA3 might reverse MDR phenotype in K562/A02 cells via direct transporter interaction and decrease of the expression of Pgp. In addition, a higher proportion of apoptosis cells was observed by analysis of annexin V/FITC plus PI staining. Western blot assay showed that the levels of Pgp associated-apoptotic markers Caspase-3 and Caspase-9 were increased. These data also supported the observation that the activity and expression of Pgp wereinhibited.Conclusion: Compounds 13 DM-3(QA1), 13DM-10(QA3) and 13DM-12(QA7) wereidentified as the inhibitors of Pgp using MTT assay in K562/A02 cells. CompoundQA3 might reverse MDR of tumor cells via modulating the activity and expression ofPgp. QA3 might be a candidate compound for reversing tumor MDR.