Targeted Delivery Of MDR1-siRNA By EGF-PAMAM For Reversing Multidrug Resistance In MES-SA/DX5 Cells

BackgroundUterine sarcomas(US) are the fourth largest malignant tumor of the female reproductive system (Top three include cervical cancer, endometrial cancer and ovarian cancer). And the incidence is increasing year by year with the rapid improvement of diagnostic technology. It’s hard to cure uterine sarcomas with behaviors such as aggressive invasion, early distant metastasis and highly recurrence rate. Therefore, the key point of study for these years is the exploration of uterine sarcoma treatments.Surgery is the primary treatment for uterine sarcomas. Meanwhlie, the validity of adjuvant radiotherapy and chemotherapy can not be ignored. It has been reported that pelvic radiation therapy can significantly reduce the local recurrence rate of uterine sarcomas. Besides, retrospectively analysises pointed out that conventional chemotherapy drugs (doxorubicin, ifosfamide and cisplatin) were useful to control the disease with different reaction rates and combination therapy was able to prolong survival for patients obviously.Unfortunately, growing evidence has indicated that multidrug resistance (MDR) appears in uterine sarcomas with extensively use of chemo-therapeutic drugs. MDR is referred to the phenomenon that drug resistant cells to exhibit simultaneous resistance to a number of structurally and functionally unrelated chemo-therapeutic agents. The possiblily mechanisms for MDR are numerous, while P-glyprotein (P-gp) is one of the most essential transportants involved in MDR encoded by the MDR1 gene. P-gp constitutes the superfamily of ATP-dependent membrane transport proteins as a plasma membrane glyprotein with large molecular weight. It serves to pump anticancer drugs out of the cell, resulting in reduced intracellular drug concentrations necessary for effective therapy in malignant cells overexpressing P-gp.RNA interference (RNAi), accidentally discovered by Andrew Z. Fire and Craig Mello, has been widely used as a potent tool in specifically post-transcriptional gene silencing. Small interfering RNAs (siRNAs,21-25 bp) are the molecules that play a central role in the whole RNAi process. Only a small amount of siRNAs is needed to achieve gene silence since cascade effect in RNAi, resulted in an effective method for specific inhibition of gene expression. siRNAs are ideal gene materials for blocking expression of abnormalities or mutations proteins in tumor cells or tissues owing to the excellent features. Currently, targeting silence the expression of MDR1 gene to reversal MDR by siRNA has been demonstrated in head and neck squamous cell carcinoma, gastric cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer and other tumor therapies.However, with the dual characteristics of nucleic acids and small molecules, naked siRNA has the difficulty in penetrating the cell membrane leading to unsatisfactory transfection efficiency. Worse yet, naked siRNA is susceptible to nuclease (RNase) in vivo and vitro, which resulted in a short degradation time and poor stability. Therefore, safe and efficient delivery vehicle is indispensable to guarantee a successful entrance for siRNA. Viral and non-viral vectors are two major siRNA delivery vehicles at the present. In the light of some medicine constitute by gene materials which have been entered in clinical trials over the last decades, novel non-viral vector such as cationic liposomes and cationic polymers has become the mainstream siRNA delivery system.Polyamide-amine (PAMAM), as one of the most widely applied cationic polymers in biomedicine research and application, ethylenediamine as the core, is synthesized by divergent method through iterative Michael addition and amidation. Thanks to its unique molecular structure and surface characteristics (nanoscale dimensions, large internal cavity, monodisperse, multivalent nature, high-density surface functional groups, etc.), PAMAM has successfully attracted the attention of more and more researchers in the delivery of DNA, siRNA, oligonucleotides nucleotides and other aspects of genetic materials in recent years. Positively charged PAMAM combines with negatively charged siRNA via electrostatic attraction once they meet each other under physiological conditions. Therefore it helps protect siRNA against nuclease degradation, promotes cellular uptake by endocytosis and thus achieving the purpose of silencing expression of targeted genes. Researchers have proved that PMAMA is capable of delivering siRNA targeted heat shock protein and HIV replication into intracellular and efficiently knockdown the expression of related proteins for the treatment of prostate cancer and HIV.Receptor-mediated tumor targeting drug delivery system is based on some specifically and overexpressed receptors on tumor cell surface. Then researchers can take advantage of the unique combination of receptors and ligands due to the overexpressing of receptors in malignant cells. Undoubtedly, it’s suitable for delivering more drugs into tumor cells and increasing transport efficiency. EGFR and Her2 are receptors extensively researched during these years. Owing to its’abundant surface functional groups, PAMAM has the opportunity to use this feature to achieve targeted and efficient delivery. It has been confirmed that PAMAM has successfully delivered siRNA into tumor cells which overexpressed EGFR with the modification of EGF. Consequently, we infer that PAMAM can delivery siRNA by receptor-mediated pathway into uterine sarcomas with these encouraging results.However, whether EGF-PAMAM can effectively transport MDR1-siRNA into uterine sarcoma cells, then to knockdown the expression of MDR1 gene and its downstream molecules and then combine with chemotherapeutic drugs to induce cell apoptosis are yet to be confirmed. Therefore, we will synthesize complexes named EGF-PAMAM-MDR1-siRNA by way of self-assembly to explore whether siRNA targeted MDR1 gene could reverse MDR in uterine sarcoma resistant cells (MES-SA-DX5). Meanwhile, we also want to evaluate the effects of combination therapy (siRNA and paclitaxel) on cell proliferation, migration and apoptosis in MES-SA/DX5 cells.Purpose1.To synthesize complexes named EGF-PAMAM-MDRl-siRNA by way of self-assembly;2.To investigate the pathway of EGF-PAMAM-MDRl-siRNA penetrating in MES-SA/DX5 cells membrane, location and lysosomal escape;3.To explore the MDR1 gene silencing effect in vivo (mRNA and protein);4.To evaluate the effects of combination therapy (siRNA and paclitaxel) on cell proliferation, migration and apoptosis.Methods1.The synthesis of PAMAM-MDR1-siRNAand EGF-MDR1-siRNAAccording to our former results, the optimal charge ratio (N/P) and weight ratio of siRNA and EGF were 20:1 and 2:1. Charge ratio (N/P) was calculated based on the number of terminal amine groups on a PAMAM dendrimer and the number of phosphate groups in siRNA. PAMAM-MDR1-siRNA with charge ratio (N/P=20:1) was formed by incubating the two components together in 1 ×Opti-MEM for 20 min at 37 ℃. EGF-PAMAM-MDR1-siRNA was synthesized by adding EGF in 1 ×Opti-MEM to the preformed PAMAM-MDR1-siRNA at N/P ratio 20 and incubated for another 20 min at 37℃.2.TransfectionAccording to requirements, MES-SA/DX5 cells were seeded in appropriate plates at certain concentration and incubated in a 5% CO2 humidified atmosphere at 37℃ overnight before transfection. Cells were then treated with transfection materials for 6 h after washed by phosphate buffer solution (1×PBS) and opti-MEM respectively.Then complete culture media were replaced and the cells were cultured for an additional 18 h or 42 h. Inverted microscope were used to observe the growth state of cells after transfection 0 h,6 h,12 h,24 h,48 h.3.Cellular uptake of siRNAMES-SA/DX5 cells were transfected with Cy5-siRNA for 6 h and then incubated with complete culture media for additional 18 h. Then the cells were stained with Hoechst 33342 solutions for 10 min and the samples were examined and photographed using a laser scanning microscope LSM 710 (Zeiss, Germany).4. Silence effect investigationMES-SA/DX5 cells were transfected with MDR1-siRNA for 6 h and incubated with complete culture media for additional 42 h. Then total RNA and proteins of cells were extracted respectively. For detection of MDR1 transcript, cDNA was reversal transcribed, amplified and agarose gel electrophoresis. For assessing P-glyprotein, proteins were first concentration determined by BCA protein assay kit. Then equal amount of protein were loaded and separated on SDS polyacrylamide gels and blocked. Subsequently, membranes were incubated with antibodies and visualized by chemiluminescence via Chemi Capture software. For measuring the fluorescence intensity of fluorescein isothiocyanate (FITC)-labled P-glyprotein, all the transfected cells were collected, then incubated with FITC Mouse Anti-Human P-glycoprotein for 30 min at 4℃ in dark and analyzed by flow cytometry(FCM) with CellQuest Pro software. For evaluating the intracellular accumulation of Doxorubicin to verify the Silence effect from the other side, the transfected cells were treated with Doxorubicin for 4 h and stained with Hoechst 33342 solutions for another 10 min. Then the samples were examined and photographed using a laser scanning microscope LSM 710 (Zeiss, Germany).5.Bcl-2 and Caspase-3 detected by Western Blot(WB)For assessing protein Bcl-2 and Caspase-3, MES-SA/DX5 cells were transfected with MDR1-siRNA for 6 h and incubated with complete culture media combined with Taxol for additional 42 h. Then total proteins of cells were extracted and concentration determined by BCA protein assay kit. Then equal amount of protein were loaded and separated on SDS polyacrylamide gels and blocked. Subsequently, membranes were incubated with antibodies and visualized by chemiluminescence via Chemi Capture software.6.Cell proliferation assessed by CCK-8 KitFor detecting the influence on cell proliferation of the combined therapy, MES-SA/DX5 cells were transfected with different MDRl-siRNA concentration for 6 h and incubated with complete culture media combined with different Taxol concentration for additional 18 h or 42 h. Then cells were added with 10 μL per well CCK-8 for another 4 h befor the optical density was read using TECAN Safire2 Multimode microplate reader with the absorbance at 450 nm.7.Cell cycle and apoptosis analysis by FCMFor measuring the effects on cell cycle and apoptosis of the combined therapy, MES-SA/DX5 cells were transfected with MDR1-siRNA for 6 h and incubated with complete culture media combined with Taxol for additional 18 h or 42 h. Then cells were harvested and analyzed by FCM after staining with propidiumiodide (PI) or (and) FITC-labeled Annexin V Solution.8.Cell migration evaluated by Scratch AssayFor evaluating the influence on cell migration of the combined therapy, MES-SA/DX5 cells were seeded in a 6-well plate at a density of 5×10-5 cells per well. The plate was drew lines every 1cm beforehand in the back for positioning during photograph. Scratch firmly and evenly with a sterile pipette tip (10 μL) to produce a thin wound (about 500μm) when cells were grown to confluence and the exfoliated cells were washed away with 1 × PBS before transfection. Cells were then treated with MDRl-siRNA for 6 h and incubated with complete culture media combined with Taxol for additional 18 h. The migration of cells from the wound edge into the wound space was recorded every 6 h by time-lapse imaging using an Olympus inverted microscope and the distance of the gap was measured at three time points and analyzed with Olympus cellSens Dimension software.Results1.The growth state of cells was not affected by PAMAM-MDR1-siRNA and EGF-PAMAM-MDR1-siRNA which were synthesized by self-assembly. Meanwhile, the cellular uptake of siRNA was better delivered by PAMAM and EGF-PAMAM.2.It is feasible for EGF-PAMAM to deliver MDRl-siRNA into uterine sarcoma resistant cells (MES-SA/DX5) and significantly silence the expression of MDR1 gene on mRNA and protein levels;3.Cell proliferation and migration distance of MES-SA/DX5 cells were inhibited significantly, let alone cells were significantly arrested in S phase and the proportion of apoptosis were also notablely increased after treated by the combination of Taxol and siRNA delivered by EGF-PAMAM.ConclusionsAbove all, as a new type of non-viral vector materials, it’s feasible for PAMAM to deliver MDR1-siRNA into MES-SA/DX5 cells and significantly silence the expression of MDR1 gene on mRNA and protein levels, and the silence effect was better after the modification of EGF. Meanwhlie, the combined therapy lead to a promotion in cell apoptosis.It’s a promising targeted for reversing MDR in MES-SA/DX5 cells since MDR1 gene plays a vital role on MDR. Therefore, MDR1-siRNA delivered by EGF-PAMAM may be an effective way to reverse MDR in uterine sarcoma.