The Effect of Psoralen on Reversing GST-π Mediated Multidrug Resistance

Objective: Glutathione S-transferase π (GST-π) plays a very important role in resisting to tumor chemotherapy. This study investigates the role of Psoralen in reversing GST-π mediated multidrug resistance (MDR), and the possible mechanism in MCF-7/ADR cells. Methods: We measured the cell viability by CCK-8 assay to evaluate the cytotoxicity and multidrug resistance (MDR) reversal activity of Psoralen. To examine the alteration in targeted gene, RT-PCR was used to detect the expression of GST-π. Western blot was used to analyze the protein level of GST-π. Immunofluorescence method was applied to observe the activation of NF-κB. Results: The intracellular adriamycin drug concentration increased significantly after Psoralen treatment. Compared with those of the control group, Psoralen reduced the expression of GST-π at the mRNA and protein level in treatment group. The NF-κB inhibitor (SN50) can significantly inhibit the expression of GST-π in breast cancer MCF-7/ADR cells. Conclusions: Therefore, NF-κB signaling pathway may be one of the mechanisms of GST-π mediated multidrug resistance. Our results showed that Psoralen was involved in reversing GST-π mediated MDR. GST-π mediated drug resisting mechanism may be related to the NF-κB signaling pathways, and it may be the key factor of downstream in the NF-κB signaling pathways. Citation: Hua Y, Wang X, Xu C, Cheng K, Jia Z, et al. (2018) The Effect of Psoralen on Reversing GST-π Mediated Multidrug Resistance. J Blood Lymph 8: 200. doi:10.4172/2165-7831.1000200


Introduction
Breast cancer is considered to be a systemic disease, and is the most frequently diagnosed female malignancy in the world, which could cause serious damage to women's health [1]. At present, the main treatments for breast cancer include chemotherapy, surgery, radiotherapy, hormonal therapy and targeted antibodies [2]. Despite advances in breast cancer treatment, the multidrug resistance (MDR) continues to be the leading cause of cancer-related deaths [3]. It is very important to explore the molecular basis of MDR and developed clinical reagents or strategies to prevent the occurrence of resistance.
There are a number of mechanisms that induce cancer cells to become nonsensitive to cytotoxic drugs, such as mutation or overexpression of the drug's target, drug inactivation, or efflux of the drug from the cell [4]. Several evidence strongly supported that the excessive expression of GSTs is one of the primary resistance mechanisms [5]. The GST family of enzymes is divided into several classes including α, μ, π, δ, etc. [6]. The effect of GST-π involved in detoxification has been suggested as one of the mechanisms that contribute to drug resistance [7]. Batist's study found that the expression of GST-π in breast cancer drug resistant cell lines MCF-7/ADR cell was high, enzyme showed its activity as 45 folds as the original cell lines [8]. At present, studies have shown that using GST-π inhibitors to reverse the GST-π mediated MDR may be feasible for improving the efficiency of chemotherapeutic agents as well as the pharmacokinetic and pharmacodynamic profiles [9]. So far, specific reversal agents for GST-π are rare, ethacrynic acid emerged as one of the most accepted and studied agents in the world [10]. However, the diuresis, metabolic disorders and severe allergic reactions limit its clinic application [11]. So it is necessary to find suitable inhibitors to overcome GST-π mediate MDR.
Psoralen is a natural product present in several plant families [12], and it is a major active component of Psoralea corylifolia [13]. Studies have shown a broad spectrum of biological activities, including cytotoxicity, phytotoxicity, etc. [14]. Modern studies have shown that Psoralen has a significant inhibitory effect on tumor growth in a variety of animals and humans [15]. In the present study, we analyzed Psoralen showed potent cell cytotoxicity to MCF-7/ADR cells, Psoralen has identified that it plays an anti-tumor effect on MCF-7/ADR in vitro cell cytotoxicity [16]. And it enhances the sensitivity of MCF-7/ADR to adriamycin, which is consistent with previous studies conducted in other human cancer cell lines [17].
Previous studies in our laboratory have found that Psoralen can actually reverse breast cancer MCF-7/ADR cell [16,18]. The effect of Psoralen on reversing MDR was clear; however, the mechanism was not clear. We have found that Psoralen did not reduce the expression of MDR1, MRP and LRP, also we found that it can significantly reduce the expression of GST-π, an obvious effect on gene and protein level. We suspected the reversal MDR mechanism may be related to GST-π. We put forward a hypothesis that Psoralen can work as an inhibitor of GST-π. Then we carried out further study to determine the possible relevant regulatory pathways.

Reagents
Psoralen was supplied by Baoji Herbest Bio-Tech (Baoji, China). A cell culture reagent was supplied by GIBCO Life Technology (Grand Island, NY, USA). Adriamycin was supplied by KeyGen Biotech Co. Ltd. (Nanjing, China). β-actin antibody was obtained from Proteintech (Proteintech Group, CHI, USA). Antibodies specific for the NF-κB, and anti-rabbit IgG, HRP-linked antibody were purchased from Abcam

Cell lines
Cell lines were supplied by KeyGen Biological Technology Development (Nanjing, China).

Cell culture
We use the RPMI-1640 medium (KGM31800S-500) to culture MCF-7 and MCF-7/ADR cells, the medium contains 10% fetal bovine serum (heat-inactivated serum), and in order to avoid bacterial contamination 100 units/mL penicillin and 100 units/mL streptomycin were contained. Cells were cultured in a humidified atmosphere contained 5% CO 2 at 37°C incubator. MCF-7/ADR cells were cultured in the medium containing 1 µg/mL Adriamycin (Nanjing, China) to keep the multidrug resistance character. The culture medium was discarded, after 2 times PBS washed changed new medium for every 48 h.

Analysis of cell viability
The effect concentrations of Psoralen and SN50 were determined by CCK-8. We divided the experimental group into four groups (MCF-7/ADR, MCF-7/ADR+P, MCF-7/ADR+SN50, MCF-7/ ADR+P+SN50). Our preliminary experiment was to determine the concentration of Psoralen and apply it to the later experiment, the concentration of SN50 (18 μmol/L) cited from other documents [19]. The effects of Adriamycin on cell proliferation were analysised by the Cell Counting Kit 8 (CCK-8) according to the manufacturer's instructions. Exponentially growing MCF-7/ADR cells (5 × 10 3 cells/ well, 100 µL) were cultured into 96-well plates for about eight hours and cells adherent to the plates grow to about fifty percent. After 2 times PBS washed and replaced by fresh medium containing different treatments and medicines for 48 h. Then, 10 µL of CCK-8 solution was added to each well in the dark, and incubated at 37°C for an additional 3 h. Optical density (OD) was determined at a wave-length of 450 nm.

Western blot analysis
The effects of different concentrations of Psoralen on GST-π expression were studied by Western blot analysis. After the cells were treated with the indicated concentrations of Psoralen (4 µg/mL, 8 µg/ mL, 12 µg/mL and 16 µg/mL), cells were washed twice with cold PBS and then lysed using lysis buffer, we use Bradford reagent to determine the total protein concentration. Equal amounts of total protein was added to sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) then transferred to a nitrocellulose membrane (100 V, 2 h)and used 5% skim milk to block the membrane at room temperature on shaking table for 1.5 h. After the milk blocking, the membranes were then incubated with the primary antibodies (1:2000) for about 8 hours. After washed with TBST every 5 minutes for 3 times and used diluted secondary antibodies (1:2000) to incubate the membranes for about 2 h at room temperature (25°). After three washes (5 minutes every time), the membranes were analysised using a chemiluminescence kit (Millipore, Bedford, MA).

Immunofluorescence
Immunofluorescence technology was used to explore the responsible mechanisms of Psoralen on reversing GST-π mediated multidrug resistance. The activation of NF-κB was closely related with the drug resistance of tumor and we focused on NF-κB signaling. Psoralen has been demonstrated as inhibitors of NF-κB. The nucleus NF-κB expression was detected in MCF-7/ADR, MCF-7/ ADR+Psoralen cells.

Statistical analyses
Statistical Package for the Social Sciences (SPSS) software version 17.0 was used for statistical analysis. All numerical results were expressed as means ± standard error of the mean. For comparison between two groups, statistical significance was assessed using Student's t test and P<0.05 was considered to be significant. All data were calculated from three independent experiments.

Psoralen down regulates GST-π expression at both mRNA and protein levels
The GST-π expression was investigated by PCR and Western blotting. Therefore, the concentration of 4 µg/mL, 8 µg/mL, 12 µg/ mL and 16 µg/mL were chosen as the working concentration in the subsequent experiments. The GST-π mediated MDR reversal of Psoralen at different concentrations on the MCF-7/ADR cells was investigated by Western blotting both gene and protein level expression were significantly down-regulated in MCF-7/ADR+Psoralen cells.

Immunofluorescence detecting NF-κB activation was inhibited by Psoralen
The NF-κB activation was investigated by immunofluorescence and the data in Figure 2 demonstrated that Psoralen inhibited the nuclear translocation of NF-κB. The nucleus NF-κB expression was significantly down-regulated in MCF-7/ADR+Psoralen cells. These results suggested the MDR reversal effect of Psoralen possibly by inhibiting NF-κB activation.

RT-PCR was used to detect the expression of GST-π
RT-PCR analysis was performed to determine the effect of Psoralen on the gene expression of GST-π. Treatment of MCF-7/ADR cells with Psoralen or SN50 can both suppress the mNRA level of GST-π.SN50 as a specific inhibitor lead an obvious inhibitory action than Psoralen worked (Figure 3).

Western blotting was used to detect the expression of NF-κB in the nuclear
Protein concentration in the nuclear extracts was evaluated according to the Bradford protein assay. Nuclear extracts were stored at the following WB analysis. The expression of NF-κB in the nuclear between different treatment groups was very obvious (Figure 4). The expression of NF-κB in the nuclear in the group treated with Psoralen (8 µg/mL) was reduced indeed. The effect of Psoralen is very obvious, which was consistent with the result in the immunofluorescence.

Discussion
The aim of this study was to characterize the mechanism of Psoralen on reversing GST-π mediated multidrug resistance on MCF-7/ADR cells. Previous research in our laboratory found that Psoralen can reverse MDR but did not reduce the expression of MDR1, MRP and LRP, so we need to explore the reversal MDR mechanism [16,18].
In this study, we found that the expression of GST-π was inhibited after being processed for 48 hours (We found there were no difference after treated with Psoralen between 24 h and 48 h) with Psoralen. These results further suggested that the co-administration of Psoralen with anticancer drugs could enhance the intracellular concentration of chemo-therapies by inhibiting the expression of GST-π, and Psoralen can be used as an inhibitor or a reversal agent of GST-π. The effect of Psoralen on GST-π is not dose-dependent and may be influenced by other pathways; Psoralen may affect cell proliferation through the cell cycle. Further studies and discussions are needed to be done; this is also the shortcoming of our experiment.
All of the members of the highly diverse GST super family are capable of binding the tripeptide glutathione [20]. GST-π is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents [21]. High expression of GST-π and the nuclear localization of GST-π are known to correlate with the aggressiveness of tumor cells and the poor survival rate of patients [22]. Several lines of evidence indicate that the effect of GST-π in tumor cells is not on cell proliferation but the acquisition of resistance   to anticancer drugs [23]. Kamada approached the function of GST-π under the oxidative stress condition caused by the hydrogen peroxide treatment [24]. He found that GST-π prevented apoptosis by reducing DNA damage [24]. In our experiment, we found that the expression of GST-π in experimental group treated with Psoralens decreased significantly. There was evidence showing that Psoralens could significantly reduce the expression of NF-κB. The NF-κB activation was investigated by immunofluorescence and the data was consistent with previous research [16]. After using of NF-κB inhibitor SN50 in MCF-7/ADR cells, the expression of GST-π in the experimental group was lower than that in normal control group. We propose that GST-π may be the key factor in signaling pathway downstream of NF-κB. Subsequent results proved the hypothesis was established.
NF-κB signaling pathway is a kind of important transcription factor involved in stress response and immune cells activation, proliferation, differentiation, apoptosis and many genes which regulate the occurrence and progress of tumor [25]. NF-κB exists in almost all cell cytoplasm, only when it is activated from the cytoplasm to nucleus can it plays an important role [26]. In recent years the study found that the aberrant activation of NF-κB was closely related with the drug resistance of tumor [27]. According to a new study, using the NF-κB inhibitors can make the traditional antitumor treatment more effective [28]. It was shown by our previous experiments that Psoralen can reduce the NF-κB expression in the cell nucleus, strong evidence that Psoralen can reduce the transport of NF-κB from cytoplasm into nucleus.
In terms of anti-tumor, the role of traditional Chinese medicine which has less side effects aroused people's attention in recent years. This study aims to develop new anti-tumor drugs to fight the resistance of tumor cells. The experiment also has many shortcomings, such as no in vivo experiments and requires further study.

Conclusion
In conclusion, our studies provided evidence that the level of GST-π mediated MDR were significantly reduced by Psoralen in MCF-7/ADR cell by suppressing the activation of NF-κB signaling pathways. This ability of Psoralen could make Psoralen act as a chemosensitizer, which reverses MDR of cancer cells induced by chemotherapy through an efficient pathway, thus provides a new opportunity for the treatment of breast tumors.