br Quantitative real time PCR br In
3.7. Quantitative real-time PCR
In order to understand the molecular mechanism of SC in en-hancement of apoptosis ability of DOX, the expression level of im-portant MDR resistance genes were evaluated in SC treated A549 lung cancer cell line. The mRNA level of ABCB1, ABCC1, ABCC2 and Nrf2 were normalized to mRNA level of the uniformly expressed reference gene, GAPDH, within each sample. Results revealed that treatment of A549 cells with 25μM SC or SC loaded NLC-RGD cause’s considerable decrease in the expression level of ABCC1 and Nrf2 (Fig. 6). However, there was no significant alteration in the expression of ABCB1 and ABCC2 genes under influence of SC or SC loaded NLC-RGD. The RT-PCR data, also demonstrated that downregulation of ABCC1 and Nrf2 ex-pression will intensify when SC is loaded into NLC-RGD suggesting capability of NLC-RGD in the more precise delivery of SC into cells than free SC. In other word, downregulation of ABCC1 and Nrf2 in SC-loaded NLC-RGD treated cells are more than those who were treated with SC, suggesting that NLC-RGD can be considered as an appropriate delivery system for SC to overcome its therapeutic limitation and its specific delivery into cancerous cells.
Most of ABC transporter Inhibitors were found to lack significant efficacy in late-phase clinical trials . Therefore, it is clear that there is still a need to develop and test efficacious inhibitors.
Present study recognized a targeted nano-carrier system capable of
Fig. 6. Effects of SC and SC-loaded NLC-RGD on expression of multi drug re-sistance related genes (ABCB1, ABCC1, ABCC2 and Nrf2) in A459 lung cancer cells. SC down-regulates the expression of ABCC1 and Nrf2 and this down-regulation intensify when SC is loaded into NLC-RGD. The results were con-sidered as the mean ± standard deviation (n = 3), * Showing Significant Differences. (SC, Sildenafil Citrate; NLC-RGD, Arginyl-glycyl-aspartic 127604-16-4 containing nanostructured lipid carriers). Process Biochemistry 84 (2019) 172–179
delivering DOX simultaneously with SC as a MDR inhibitor. Characteristic of prepared nanoparticles (suitable Size, ZP, High EE and long physical stability) confirmed appropriateness of optimum formula. Prepared NPs were smaller than 200 nm to escape from re-ticuloendothelial system  and have reasonable ZP which not only causes repulsive forces among nanoparticles to stop it from aggregation, but also causes better permeability across cell membranes. In this study, prepared NPs contain RGD as a ligand of integrin’s for efficient delivery of drug into the cells. Regarding to elevated expression of certain pat-terns of integrins in cancerous cells and their central role in cellular adhesion and cancer development, targeting with RGD not only en-hances drug penetration into the cells through integrin related en-docytosis, but also hinders cell invasion and metastasis by blocking of integrins. In this study, highly expressed αvβ3 and αvβ6 integrin , A549 non-small cell lung cancer (NSCLC) cell line was used to compare the penetration rate of NLC and NLC-RGD. As shown in Fig. 4, accu-mulation of drug loaded NLC-RGD into cell are noticeably more than NLC which confirmed that RGD have a high binding affinity to can-cerous cells and is able to enhance endocytosis. In in-vivo systems, in addition to the active targeting, NLC-RGD may benefit passive targeting through enhanced permeability and retention (EPR) which indicates that NPs leak favorably into tumor tissue through permeable tumor vessels and then retained in the tumor tissue due to poor lymphatic drainage . Furthermore, absorbance of NPs occurs through lym-phatic system by Peyer’s patches in the small intestine [26,27] which leads to bypassing the liver and consequently reduce first pass meta-bolism . The main aim of this study was to overcome MDR by co-delivery DOX and SC. Data of MTT assay showed that A549 cells co-treatment with DOX and SC resulted in remarkably increased cyto-toxicity compared to DOX. In accordance to these data, Greish et al.  showed that combination therapy with DOX and SC causes a sta-tistically significant 4.7-fold reduction in tumor size compared to DOX. Enhanced cytotoxicity of DOX may be due to decreasing activity of ABC transporter by SC. Furthermore, Das et al.  showed that combina-tion therapy with DOX and SC increased DOX-induced apoptosis in cancer cells by upregulation of caspase-3 and caspase-9 activities, downregulation of Bcl-xL, and phosphorylation of Bad. As seen in Fig. 5, co-delivery of DOX and SC by NLC-RGD intensify apoptotic ef-fects of DOX, suggesting that Inhibition of ABC transporters would lead to sensitization of MDR cancer cells to chemotherapeutic agents. In accordance to these data, Shi et al.  showed that Sildenafil interacts with transmembrane regions of ABCB1- and ABCG2 transporter and decreases ABCB1- and ABCG2-mediated drug resistance by directly obstructing the transport function of ABCB1 and ABCG2. They also confirmed that Sildenafil itself may be a substrate of ABC transporter and may act as a competitive inhibitor of ABCB1 and ABCG2 by com-peting with other substrates. The results of this study also showed that loading SC into NPs enhances its adjuvant effects, which may benefit from increased uptake by cells and overcoming its low bioavailability. In addition to synergistic effects of SC beside DOX, several studies also revealed that SC may attenuate the DOX-induced cardiotoxicity [32,33]. Inhibition of multidrug resistance, may make low dose of an-ticancer drugs more effective, whereby could reduce the side effects of these agents . Improved targeting of SC can render DOX active at much lower concentrations in cancer treatment which in turn cause decreasing the side effects in the patient. Regarding to enhanced ex-pression of MDR related genes in NSCLC [35,36], In this study influence of SC on mRNA level of some of these genes were evaluated. Results revealed that, SC decreases the transcription of Nrf2 and ABCC1. Nrf2 is an important transcription factor which its aberrant expression is re-lated to poor outcomes in the treatment of NSCLC . in accordance to our results, Ji et al.  showed that knocking down of Nrf2 ex-pression lead to down regulation of ABCC1 in H69AR cells, which in turn increase intracellular concentrations of anticancer drugs. There-fore, in addition to direct inhibition of ABC transporter, downregulation of Nrf2 and ABCC1 are other probable mechanism, through which SC