Article pubs.acs.org/molecularpharmaceutics
Cationic Lipid-Assisted Polymeric Nanoparticle Mediated GATA2 siRNA Delivery for Synthetic Lethal Therapy of KRAS Mutant NonSmall-Cell Lung Carcinoma Song Shen,† Chong-Qiong Mao,† Xian-Zhu Yang,*,† Xiao-Jiao Du,† Yang Liu,† Yan-Hua Zhu,† and Jun Wang*,†,‡,§ †
School of Life Sciences and ‡Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui 230027, P. R. China § High Magnetic Field Laboratory of CAS, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China S Supporting Information *
ABSTRACT: Synthetic lethal interaction provides a conceptual framework for the development of wiser cancer therapeutics. In this study, we exploited a therapeutic strategy based on the interaction between GATA binding protein 2 (GATA2) downregulation and the KRAS mutation status by delivering small interfering RNA targeting GATA2 (siGATA2) with cationic lipid-assisted polymeric nanoparticles for treatment of non-small-cell lung carcinoma (NSCLC) harboring oncogenic KRAS mutations. Nanoparticles carrying siGATA2 (NPsiGATA2) were effectively taken up by NSCLC cells and resulted in targeted gene suppression. NPsiGATA2 selectively inhibited cell proliferation and induced cell apoptosis in KRAS mutant NSCLC cells. However, this intervention was harmless to normal KRAS wild-type NSCLC cells and HL7702 hepatocytes, confirming the advantage of synthetic lethality-based therapy. Moreover, systemic delivery of NPsiGATA2 significantly inhibited tumor growth in the KRAS mutant A549 NSCLC xenograft murine model, suggesting the therapeutic promise of NPsiGATA2 delivery in KRAS mutant NSCLC therapy. KEYWORDS: synthetic lethal therapy, non-small-cell lung carcinoma, GATA2, siRNA delivery, cationic lipid-assisted polymeric nanoparticle
1. INTRODUCTION Conventional chemotherapies for cancer were initially discovered on the basis of their potential activity against rapidly dividing cancer cells, but the toxicity to normal tissues results in unpleasant side effects.1 It poses a great challenge to identify therapies that show greater effectiveness and fewer side effects. Cancer researchers have tried their best to exploit selective targeting agents that can block oncogenic signaling based on the deeper investigation of tumor-specific traits.2,3 As examples, STI571 (Gleevec, imatinib mesylate) and Herceptin (trastuzumad) exemplify the successful development of rationally designed, molecularly targeted therapy for the treatment of specific cancers.4,5 However, emergence of drug resistance and off-target toxicities have limited the application of targeted therapies, and so far only a few have demonstrated clinical efficacy and received regulatory approval.6,7 In recent years, explorations of synthetic lethal interactions have attracted great attention in that they provide a conceptual framework for the development of drugs that will preferentially kill cancer cells while sparing normal cells.8−12 A synthetic lethal relationship occurs when the silencing or inhibiting of one gene is only lethal in the context of specific cancer-causing © 2014 American Chemical Society
mutations; thus, it enhances the therapeutic index between the tumor and normal tissue.13−16 With the help of chemical compound libraries17 and whole-genome RNA interference (RNAi) screening platforms,18,19 researchers mapped synthetic lethal relationships in human cancer cells in the hope of discovering safer, more efficacious genotype-selective anticancer drugs and developing wiser cancer therapeutics. Stockwell and colleagues used small molecules to show that cells expressing oncogenic Ras display increased sensitivity to compounds that bind to particular voltage-dependent anion channels.20 Elledge and colleagues screened their shRNA library for genes whose inhibition constituted striking synthetic lethality with the KRAS oncogene.21 They identified a functionally diverse set of genes and demonstrated that pharmacological inhibitors targeting Special Issue: Drug Delivery and Reversal of MDR Received: Revised: Accepted: Published: 2612
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Scheme 1. A Schematic View of Non-Small-Cell Lung Cancer (NSCLC) Therapy via a Synthetic Lethal Interactiona
a A cationic lipid-assisted polymeric nanoparticle system that is capable of encapsulating siRNA and forming NPsiGATA2 is used to specifically treat NSCLC harboring mutant KRAS.
the small molecular inhibitor’s undesirable toxicity due to its nonspecific target modulation, it is of great clinical significance to find innovative avenues, such as RNAi that directly targets GATA2.38 RNAi is a naturally occurring mechanism that controls gene expression at the post-transcriptional level and results in the selective downregulation of specific proteins.39 With its powerful ability to specially suppress the expression of a crucial oncogene or cancer-related gene involved in carcinogenesis, RNAi, to a certain extent, holds great promise in the field of cancer therapy.40−42 The major limitations to application of small interfering RNA (siRNA) therapeutics include rapid intravascular degradation, off-target effects, and nonspecific biodistribution.43,44 Inspirationally, nanoscale carriers have the potential to partly circumvent these challenges that currently limit the successful translation of RNAi technology into the clinical arena, advancing the application of RNAi in cancer therapy.45−47 As a novel pharmacological approach to the treatment of human cancer, various clinical trials employing RNAi technology and nanoparticles are in progress, involving many companies.48,49 Our previous works have demonstrated that cationic lipidassisted poly(ethylene glycol)-block-polylactide (PEG-PLA) nanoparticles may be an appropriate siRNA carrier for cancer therapy in the murine models of breast and liver cancers.50 In this work, we developed a novel synthetic lethality-based method to selectively cure KRAS mutant NSCLC, mimicking the effect of GATA2 mutation with siRNA delivered by polymer nanoparticles (Scheme 1). We proved that these GATA2 siRNA-encapsulated PEG-PLA nanoparticles (NPsiGATA2) could be easily taken up by A549 (KRAS mutant) and H226 (KRAS wild-type) cells. Though NPsiGATA2 led to downregulation of GATA2 expression in both cell lines in vitro, suppression of GATA2 expression generated selective antiproliferative and proapoptotic effects in KRAS mutant A549 cells without exhibiting toxicity to wild-type H226 cells. Furthermore, NPsiGATA2 showed strikingly antitumor activity
these proteins could selectively impair the viability of KRAS mutant cells. Lung cancer is the leading cause of cancer-related mortality worldwide due to its high incidence and low survival rates.22,23 Non-small-cell lung cancer (NSCLC) accounts for about 80% of all lung cancers, and approximately half of the patients present with late-stage disease at the time of diagnosis.24 Activating KRAS mutations, which are the most frequent oncogenic mutations in human NSCLC (with a frequency of 10−30%), have been associated with resistance to cytotoxic chemotherapy, radiation therapy, and small molecular inhibitors.25−29 Although epidermal growth factor receptor (EGFR)directed therapies including gefitinib and erlotinib have become effective treatments for NSCLC harboring an EGFR mutation in clinic trials, the benefits are limited by several mechanisms of drug resistance, which include KRAS mutations.25,28,30,31 Up to now, there is still a lack of effective therapies for KRAS mutant NSCLC, although KRAS mutations were identified more than 25 years ago.32,33 Fortunately, beyond remarkably mild side effects, synthetic lethality offers an excellent therapeutic opportunity to exploit targets that have proved to be challenging to therapeutically modulate by common strategies, including NSCLC harboring KRAS mutations.34 The Downward laboratory expanded the repertoire of KRAS targets by searching for synthetic lethal genes using RNAi libraries and showed that the proliferation and survival of NSCLC cells with an activated mutant KRAS pathway depend on the transcription factor GATA2. Loss of GATA2 reduced the viability of NSCLC cells with RAS pathway mutations, whereas wild-type cells were unaffected.35 Additionally, loss of GATA2 led to dramatically reduced tumor development in a KRAS-driven NSCLC mouse model, demonstrating the therapeutic potential of targeting GATA2 using surrogate drugs or RNAi technology.36,37 Although researchers suppressed GATA2 regulated pathways with inhibitors and caused marked tumor clearance, considering 2613
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Lipofectamine 2000 carrying 50 nM FAM-siRNA was used as the positive control. After 4 h of incubation, cells were washed three times with phosphate-buffered saline (PBS, 0.01 M, pH 7.4), and then collected by trypsinization. Cells were resuspended in 200 μL of PBS and subjected to flow cytometric analyses using a Becton Dickinson FACS Calibur flow cytometer (Bedford, MA, USA). For each experiment, 10,000 cells were collected and analyzed using WinMDI 2.9 software. For microscopic observation, A549 and H226 cells (5 × 104) were seeded on coverslips in a 24-well tissue culture plate in 0.5 mL of DMEM containing 10% FBS. After incubation for 24 h at 37 °C in an atmosphere of 5% CO2, the medium was replaced with fresh DMEM medium containing NPFAM‑siRNA (200 nM). After a further 2 h of incubation, the cells were washed twice with PBS and fixed with 4% formaldehyde for 15 min at room temperature. Subsequently, the cells were counterstained with DAPI for cell nuclei and Alexa Fluor 568 phalloidin for the cytoskeleton according to the standard protocol provided by the suppliers. Coverslips were mounted on glass microscope slides with a drop of antifade mounting medium (Sigma-Aldrich Co.) to reduce fluorescence photobleaching. The cellular uptake of nanoparticles was visualized by a laser scanning confocal microscope (LSCM; LSM 700 Meta, Carl Zeiss Inc., Thornwood, NY). 2.5. In Vitro Gene Silencing with siRNA-Encapsulated Nanoparticles. A549, H226, or HL7702 cells (2 × 105) were seeded in a six-well tissue culture plate (Corning Inc., Corning, NY, USA) and allowed to grow until 70% confluence. The cells were transfected with nanoparticles encapsulating siGATA2 (NPsiGATA2) at different doses from 100 nM to 300 nM. Nanoparticles encapsulating siNC (300 nM, NPsiNC) and free siGATA2 were used as negative controls, and Lipofectamine 2000 carrying siGATA2 (Lipo/siGATA2) at the dose of 50 nM was used as the positive control. After incubation for 6 h at 37 °C, the medium was replaced with DMEM medium containing 10% FBS. After further incubation for 48 h (for mRNA isolation) or 72 h (for protein extraction) at 37 °C, the cellular levels of GATA2 mRNA and protein were assessed using quantitative reverse transcription real-time PCR (qRT-PCR) and Western blotting, respectively. For qRT-PCR analysis, total RNA from transfected cells was extracted using RNAiso Plus (Takara, Otsu, Japan) according to the supplied protocol. Total RNA (1 μg) was transcribed into cDNA using the PrimeScript first strand cDNA synthesis kit (Takara, Otsu, Japan). Thereafter, 2 μL of cDNA was subjected to qRT-PCR analysis targeting GATA2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using SYBR Premix Ex Taq (Perfect Real Time) (Takara, Dalian, China). Analysis was performed using the Applied Biosystems StepOne Real-Time PCR Systems. Relative gene expression values were determined by the ΔΔCT method using StepOne Software v2.1 (Applied Biosystems). Data are presented as the fold difference in GATA2 expression normalized to the housekeeping gene GAPDH as the endogenous reference, and relative to the untreated control cells. The primers used in the qRT-PCR for GATA2 and GAPDH were as follows: 5′GGTCCAGCTTTACTGTGGCTGTC-3′ (GATA2-forward), 5′-TGGTCACTACATCAGCACAATCCTC-3′ (GATA2-reverse); and 5′-TTCACCACCATGGAGAAGGC-3′ (GAPDH-forward), 5′-GGCATGGACTGTGGTCATGA-3′ (GAPDH-reverse). PCR parameters consisted of 30 s of Taq activation at 95 °C, followed by 40 cycles of PCR at 95 °C for 5 s, 60 °C for 30 s, and 1 cycle of 95 °C for 15 s, 60 °C for 60 s,
in an A549 xenograft murine model, but did not in an H226 xenograft murine model, indicating that NPsiGATA2 is promising for KRAS mutant NSCLC therapy by the synthetic lethal interaction between GATA2 downregulation and KRAS mutation.
2. MATERIALS AND METHODS 2.1. Materials. The diblock copolymer of poly(ethylene glycol) (molecular weight [MW] = 5,000) with poly(D,Llactide) (MW = 11,000) PEG5K-PLA11K and cationic lipid N,Nbis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonyl aminoethyl) ammonium bromide (BHEM-Chol) were synthesized as previously reported.50 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Ultrapurified water was prepared using a Milli-Q Synthesis System (Millipore, Bedford, MA). Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY) and L-glutamine were purchased from Gibco BRL (Eggenstein, Germany). The Lipofectamine 2000 transfection kit from Invitrogen (Carlsbad, USA) was used as suggested by the supplier. 4′,6-Diamidino-2phenylindole (DAPI) and Alexa Fluor 488 phalloidin were obtained from Invitrogen (Carlsbad, CA). Fluorescein-labeled siRNAs (FAM-siRNAs), siRNA targeting GATA2 mRNA (siGATA2) with the antisense strand 5′-UUmCCfAUCUUCAUGfCUCUCCdTsdT-3′, and scrambled siRNA (siNC) with the antisense strand 5′-GACUACUGGUCGUUGAACUdTdT-3′ were obtained from GenePharma Co. Ltd. (Shanghai, China). 2.2. Cell Culture. The human NSCLC cell line A549 (KRAS mutant) and H226 (KRAS wild-type) cell line were obtained from the American Type Culture Collection (ATCC). The human hepatocyte cell line HL7702 (KRAS wild-type) was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cell lines were maintained in Lglutamine containing DMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, HyClone, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO). These cells were incubated at 37 °C in an atmosphere of 5% CO2 and 95% air. 2.3. Preparation of Nanoparticles with siRNA Encapsulation (NPsiRNA). NPsiRNA were prepared by double emulsion according to a method reported previously. Briefly, an aqueous solution of siRNA (0.2 mg) in 25 μL of RNase-free water was emulsified by sonication for 30 s over an ice bath in 0.5 mL of chloroform containing 1.0 mg of BHEM-Chol and 25 mg of PEG5K-PLA11K. This primary emulsion was further emulsified in 5 mL of water by sonication (80 W for 2 min) over an ice bath to form a water-in-oil-in-water emulsion, and the organic solvents were removed using a rotary evaporator. The encapsulation efficiency of siRNA into the nanoparticles was determined by high-performance liquid chromatography (HPLC) analyses according to the literature. 2.4. Cellular Uptake of NPsiRNA and Intracellular Trafficking. Cellular uptake of siRNA was investigated by flow cytometric and confocal microscopy analyses. For flow cytometric analysis, A549 and H226 cells were seeded in a 24well tissue culture plate (Corning Inc., Corning, NY, USA) at a density of 5 × 104 cells/well 24 h prior to experiments. The original medium was replaced with fresh medium containing NPFAM‑siRNA. The FAM-siRNA concentration in the culture medium was 100 nM, 200 nM, and 300 nM, respectively. Free FAM-siRNA (300 nM) was used as the negative control, and 2614
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Figure 1. Cellular uptake of NPFAM‑siRNA by KRAS mutant A549 and KRAS wild-type H226 cells. Panels A and B show FACS (fluorescence activated cell sorter) histograms after internalization of NPFAM‑siRNA by A549 and H226 cells. Panels C and D show confocal fluorescence images of A549 and H226 cells after incubation with NPFAM‑siRNA at 200 nM siRNA concentration (blue, DAPI-stained nucleus; red, Alexa Fluor 568 phalloidin-stained F-actin; green, FAM-labeled siRNA).
added to each well to achieve a final concentration of 1 g L−1, with the exception of the wells used as a blank, to which the same volume of PBS (0.01 M, pH 7.4) was added. After incubation for an additional 2 h, 100 μL of extraction buffer (20% SDS in 50% N,N-dimethylformamide, pH 4.7, prepared at 37 °C) was added to the wells and incubated overnight at 37 °C. The absorbance was measured at 570 nm using a Bio-Rad 680 microplate reader (Bio-Red, USA). Cell viability was normalized to that of cells cultured in the culture medium with PBS treatment, which served as the indicator of 100% cell viability. For the colony formation assay, A549, H226, and HL7702 cells were treated for 48 h with the above-mentioned formulations, followed by trypsinization and resuspension in DMEM medium. One thousand cells were plated in each well of six-well plates. Cells were then incubated at 37 °C with 5% CO2 for 10 days. After removal of the medium, cells were washed twice with PBS, and stained with 0.5% crystal violet (Sangon Biotech., China) in methanol for 30 min with shaking at room temperature. Then, the cells were washed twice with distilled water and visualized. 2.7. In Vitro Apoptosis Induction Post NPsiGATA2 Transfection. A549, H226, and HL7702 cells were seeded in six-well plates at 1.0 × 105 per well. The cells were treated with the above-mentioned formulations 24 h post seeding. After 72 h of incubation, the cells were washed twice with PBS, collected by trypsinization, and resuspended in 500 μL of annexin V binding buffer. Subsequently, 2 μL of fluorescein isothiocyanate (FITC)-conjugated annexin V and 5 μL of propidium iodide (PI) were added to the cell suspensions. After
and 95 °C for 15 s. Standard curves were generated and the relative amount of target gene mRNA was normalized to GAPDH mRNA. Specificity was verified by melt curve analysis. For Western blot analysis, the transfected cells were washed twice with cold PBS and then resuspended in 50 μL of lysis buffer (pH 7.5, containing 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl2, 1 mM EDTA). Cell lysates were incubated on ice for 30 min and vortexed every 5 min. The lysates were clarified by centrifugation for 10 min at 12000g at 4 °C. Protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Total protein (60 μg) was separated on a 12% SDS− polyacrylamide gel (SDS−PAGE) at 90 V for 120 min and then transferred to Immobilon-P membrane (Millipore, Bedford, MA, USA) at 300 mA. After incubation in 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) in PBS with Tween-20 (PBST, pH 7.2) for 1.5 h, the membrane was then incubated in 1% BSA in PBST with a monoclonal antibody against GATA2 (1:5000, Santa Cruz Biotech., USA) overnight at 4 °C. The membrane was further incubated in 1% BSA with goat anti-rabbit immunoglobulin (IgG, 1:10,000, Santa Cruz Biotech., USA) for 30 min and visualized using the ImageQuant LAS 4000 mini system (GE Healthcare, London, U.K.). Expression levels of GATA2 protein were normalized against β-actin protein expression levels. 2.6. In Vitro Proliferation Assays. Cells were seeded in 96-well plates at 5,000 cells per well in 100 μL of complete DMEM supplemented with 10% FBS and incubated for 24 h. Subsequently, the cells were treated with the above-mentioned formulations. After 72 h treatment, an MTT stock solution was 2615
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Figure 2. Knockdown of GATA2 gene expression by NPsiGATA2. (A, B) NPsiGATA2-mediated gene silencing in A549 cell line (KRAS mutant) and H226 cell line (KRAS wild-type) examined by real-time PCR. (C, D) GATA2 expression post transfection in A549 and H226 cells, examined by Western blotting. The dose of siGATA2 for Lipo/siGATA2 was 50 nM, and the doses of free siGATA2 and siNC were 300 nM. ** p < 0.01 when compared with PBS (n = 3), * p < 0.05 when compared with PBS (n = 3).
Figure 3. Downregulation of GATA2 expression induces a significant reduction of cell proliferation in KRAS mutant A549 cells but not KRAS wildtype H226 cells. (A) Cell viability at 72 h after transfection with NPsiGATA2. (B) Colony formation ability of cells after transfection with different formulations. Photographs of crystal violet-stained colonies are shown. The dose of siGATA2 for Lipo/siGATA2 was 50 nM, and the doses of free siGATA2 and siNC were 300 nM. ** p < 0.01 when compared with PBS (n = 3), * p < 0.05 when compared with PBS (n = 3).
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Figure 4. Downregulation of GATA2 expression induces cell apoptosis in A549 cells harboring a KRAS mutation but not in KRAS wild-type H226 cells. (A, B) Annexin V and PI staining by flow cytometric analysis to analyze apoptosis cells after treatment with NPsiGATA2 for 72 h. Early apoptotic cells are presented in the lower right quadrant, and late apoptotic cells are presented in the upper right quadrant. (C, D) c-PARP expression changes in the two cell lines. The dose of siGATA2 for Lipo/siGATA2 was 50 nM, and the doses of free siGATA2 and siNC were 300 nM.
Figure 5. Low sensitivity of KRAS wild-type HL7702 hepatocytes to GATA2 suppression. (A) Reduction of GATA2 mRNA levels in HL7702 cells following transfection with different formulations. (B) Cell viability post transfection. (C) Colony formation ability of cells after transfection with siGATA2. (D) Apoptosis of HL7702 cells. The dose of siGATA2 for Lipo/siGATA2 was 50 nM, and the doses of free siGATA2 and siNC were 300 nM. ** p < 0.01 when compared with PBS (n = 3).
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Figure 6. Administration of NPsiGATA2 induces a reduction in KRAS mutant NSCLC growth. (A, B) Antitumor growth and body weight changes of mice following intravenous injection of various formulations in A549 xenograft tumors. Mice started to receive the different treatments when tumors reached a size of 100 mm3 (termed day 0). (C, D) Reduction of GATA2 mRNA and protein expression levels in A549 xenograft tumors after treatment with various formulations. Three samples of each group were randomly chosen for detection. ** p < 0.01 when compared with PBS (n = 5).
2.9. Detection of GATA2 Expression in Tumor Tissues Post Treatment. Tumor tissues were collected 24 h after the last injection. GATA2 expression at mRNA and protein levels in the tumors was analyzed by qRT-PCR and Western blot analyses, respectively. For GATA2 mRNA analysis, tumor tissues were lysed in RNAiso Plus (Takara, Otsu, Japan), and total RNA was isolated following the manufacturer’s protocol. The procedure of reverse transcription and real-time PCR was the same as that used for the in vitro analyses. For GATA2 protein analysis, tumor tissues were collected and lysed in 100 μL of tissue lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EGTA, 2.5 mM EDTA, 10% glycerol, 0.1% Tween 20, 1 mM dithiothreitol, 10 mM glycerol 2-phosphate, 1 mM NaF, and 0.1 mM Na3VO4) freshly supplemented with Roche’s Complete Protease Inhibitor Cocktail Tablets. The lysates were incubated on ice for a total of 30 min and vortexed every 5 min. The lysates were centrifuged for 10 min at 12000g. Proteins were then detected by Western blot analyses as described above. 2.10. Statistical Analysis. The statistical significance of treatment outcomes was assessed using Student’s t-test; p < 0.05 was considered statistically significant in all analyses (95% confidence level).
incubation for 10 min at room temperature in the dark, the samples were immediately analyzed using a flow cytometer. Expression levels of c-PARP (Cell Signaling, USA), a marker of apoptotic cells, were also examined by Western blotting using the above-mentioned method. 2.8. Human NSCLC Xenograft Tumor Model and Treatment. BALB/c-nu nude mice (6 weeks old) were purchased from Beijing HFK Bioscience Co., LTD (Beijing, China), and all animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. A xenograft tumor model was generated by subcutaneous injection of A549 cells (5 × 106) in 100 μL of Matrigel basement membrane matrix (Becton Dickinson, Bedford, MA) into the right flank of nude mice. When the tumor volume was around 100 mm3 at 12 days, the mice were used for subsequent experiments. The mice were randomly divided (five mice per group) and treated with PBS, free siGATA2, NPsiNC, or NPsiGATA2 by intravenous injection every other day at a dose of 40 μg or 20 μg of siRNA per mouse. The treated mice were examined for appearance, and physical activity. Tumor growth was monitored by measuring the perpendicular diameter of the tumor using calipers. The size of the tumor was measured using a vernier caliper across its longest (a) and shortest (b) diameters, and its volume was calculated using the formula of V = 0.5ab2.
3. RESULTS AND DISCUSSION 3.1. Cellular Uptake of NPsiRNA. In order to efficiently deliver siGATA2 to NSCLC cells in vitro and in vivo, we used cationic lipid-assisted PEG-PLA-based nanoparticles as the 2618
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with different formulations, and then cell proliferation was detected by the MTT proliferation assay. The same treatments were performed in KRAS wild-type H226 cells for comparison. As demonstrated in Figure 3A, NPsiGATA2 inhibited the growth of KRAS mutant A549 cells in a dose-dependent manner. When the dose of siGATA2 was increased from 100 nM to 300 nM, the viability of KRAS mutant A549 cells decreased from 75.6 ± 8.9% to 45.8 ± 6.9%. In contrast, even at the highest siGATA2 dose of 300 nM, KRAS wild-type H226 cells showed high viability, in spite of the significant downregulation of GATA2 gene expression by NPsiGATA2. These results demonstrated that inhibition of cell viability was via a synthetic lethal relationship between mutant KRAS and downregulation of GATA2 expression by NPsiGATA2. The reduction of cell proliferation in A549 cells was further corroborated by the colony formation assay. After 10 days of incubation, the clonogenicity of transfected A549 cells was observed by staining with 0.5% crystal violet. As shown in Figure 3B, clonogenicity was decreased in KRAS mutant A549 cells in a dose-dependent manner, which correlated well with the results of the MTT assay (Figure 3A), verifying the synthetic lethal relationship between mutant KRAS and downregulated GATA2 expression in A549 cells. However, the clonogenicity of transfected H226 cells was not significantly affected. Additionally, we analyzed the apoptosis of both cells to confirm the cell line specificity of proliferation inhibition. The KRAS mutant A549 cells and KRAS wild-type H226 cells were incubated with the above-mentioned formulations for 72 h, and then the cancer cells were stained with annexin V-FITC and PI to determine the level of cell apoptosis. As shown in Figures 4A and 4B, in both cell lines, NPsiNC and free siGATA2 at the siRNA dose of 300 nM did not induce remarkable cell apoptosis, while the delivery of siGATA2 with NPsiGATA2 induced dose-dependent cell apoptosis in KRAS mutant A549 cells. For example, NPsiGATA2 treatment led to 21.9% and 29.7% KRAS mutant A549 cell apoptosis at 200 nM and 300 nM, respectively. Treatment with Lipofectamine 2000 carrying siGATA2 at 50 nM also led to 24.51% apoptosis in A549 cells. However, this phenomenon was not observed in KRAS wild-type H226 cells; NPsiGATA2 at different siRNA dose and Lipofectamine 2000 carrying siGATA2 did not induce remarkable cell apoptosis compared with PBS treatment. The levels of PARP cleavage, a marker of apoptotic cell death, in KRAS mutant A549 and KRAS wild-type H226 treated with different formulations of NPsiGATA2 were analyzed by Western blotting. As shown in Figures 4C and 4D, with GATA2 depletion, we only observed dose-dependent accumulation of cPARP in A549 cells; the expression of c-PARP in H226 cells was not changed. Based on the above results, it can be summarized that siGATA2-encapsulated nanoparticles can efficiently downregulate GATA2 gene expression in both NSCLC cell lines (KRAS mutant A549 cells and KRAS wild-type H226), but inhibition of cell proliferation, reduction of clonogenicity, and remarkable cell apoptosis induction were only observed in KRAS mutant A549 cells not KRAS wild-type H226 cells after treatment with NPsiGATA2. These results indicate that NPsiGATA2 led to cytotoxicity in KRAS mutant cells rather than KRAS wild-type cells via a synthetic lethal interaction. Although RNAi therapy has emerged as a potent therapeutic agent for cancer therapy due to its ability to silence specific genes rapidly and efficiently,52,53 the siRNA-loaded delivery systems were unavoidably accumulated in the heart, spleen,
delivery system of siRNA using a previously reported double emulsion method.50 Similarly, siRNA was efficiently encapsulated into the formed nanoparticles with high encapsulation efficiency of siRNA (∼96.4%). To evaluate whether the prepared NPsiRNA could efficiently deliver siRNA into KRAS mutant A549 cells and KRAS wild-type H226 cells, we used fluorescent FAM-labeled siRNA to examine its internalization. After 5 h of incubation with NPFAM‑siRNA, the internalization of NPFAM‑siRNA by both NSLCL cell lines was examined by flow cytometry. As shown in Figures 1A and 1B, both cells incubated with NPFAM‑siRNA at different siRNA doses showed much stronger green fluorescence compared with PBS-treated cells. On the other hand, increasing the dose of NPFAM‑siRNA significantly improved the intracellular fluorescence intensity. Moreover, after incubation with the same formulation, A549 and H226 cells showed similar intracellular fluorescence intensity, implying that both cells internalized the nanoparticles at a similar level. Besides, it is noteworthy that incubation with free FAM-siRNA alone led to very minimal cell fluorescence. To further analyze the subcellular localization of NPFAM‑siRNA, we incubated NPFAM‑siRNA with A549 or H226 cells at a siRNA concentration of 200 nM. After 5 h of incubation, the F-actin cytoskeleton and cell nuclei were stained with Alexa Fluor 568 phalloidin and DAPI, respectively. Subsequently, the cells were observed by confocal laser scanning microscopy. As shown in Figures 1C and 1D, NPFAM‑siRNA signal was observed in the cytoplasm of both cells, which was consistent with the results of flow cytometric analysis. 3.2. Knockdown of GATA2 Gene Expression by NPsiGATA2. Simultaneous mutation of both genes that have a synthetic lethal interaction can lead to cell death.51 In this study, it was expected that downregulation of GATA2 in KRAS mutant cells with NPsiGATA2 would cause cell death. We then investigated whether NPsiGATA2 could downregulate GATA2 gene expression in A549 cells and H226 cells. Different formulations were incubated with the cells; then GATA2 expression at both mRNA and protein levels was determined. As shown in Figures 2A and 2B, the control NPsiNC carrying scrambled siRNA and free siGATA2 did not downregulate GATA2 expression in either cell. As expected, treatment of the cells with NPsiGATA2 significantly downregulated GATA2 expression, which was not dependent on the status of KRAS mutation in the cells. In addition, it was observed that the gene silencing efficiency of NPsiGATA2 was dose-dependent; increasing the dose of siGATA2 significantly improved the knockdown efficiency. Treatment with NPsiGATA2 at a siGATA2 dose of 300 nM led to 43.3 ± 1.7% and 42.0 ± 1.5% knockdown of GATA2 expression in KRAS mutant A549 cells and KRAS wild-type H226 cells, respectively. On the protein level, GATA2 expression was also downregulated by NPsiGATA2 in a dosedependent manner in both A549 cells and H226 cells (Figures 2C and 2D). Unlike the Lipofetamine/siGATA2, sustained release of siRNA from the NPsiGATA2 has been demonstrated.50 Although NPsiGATA2 (300 nM) and Lipo/siGATA2 exhibited similar knockdown efficiency at mRNA level 48 h post transfection, the NPsiGATA2 (300 nM) can more efficiently downregulate the GATA2 protein expression 72 h post transfection due to the released siGATA2 from 48 to 72 h. 3.3. Reduction of Cell Proliferation of KRAS Mutant A549 Cells via a Synthetic Lethal Interaction. To investigate whether treatment with NPsiGATA2 could significantly inhibit proliferation of KRAS mutant cells via a synthetic lethal interaction, we incubated KRAS mutant A549 cells for 72 h 2619
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lethality-based therapeutics can be used for NSCLC treatment and helps formulate a scheme for individualized therapy.
kidney, and especially liver, and may possess potential toxicity for normal cells.54,55 The advantage of cancer therapeutics based on synthetic lethality is the selective killing of specific cancer cells without harming normal cells, which can reduce the potential cytotoxic side effects to the greatest extent and provide new therapeutic opportunities. In view of this, we investigated the effect of GATA2 depletion on the survival and proliferation of human HL7702 hepatocytes (KRAS wild-type) in vitro. As shown in Figure 5, it was observed that HL7702 hepatocytes exhibited low sensitivity to GATA2 suppression, similar to H226 cells. The viability and proliferation of HL7702 hepatocytes were unscathed by transfection of a high concentration of NPsiGATA2. 3.4. Administration of NPsiGATA2 Induced a Reduction in KRAS Mutant NSCLC Growth by Decreasing GATA2 Expression in Vivo. We then investigated whether the inhibition of cell proliferation by NPsiGATA2 observed in vitro would also occur in vivo following systemic administration of NPsiGATA2. A KRAS mutant tumor xenograft model was generated in female athymic (nu/nu) mice by injection with A549 cells, and used to assess the efficacy of tumor growth inhibition of systemic administration of NPsiGATA2 once every other day at a siRNA dose of 1.0 mg/kg or 2.0 mg/kg per injection from 14 days post xenograft implantation. NPsiNC and free siGATA2 were used as negative controls. As shown in Figure 6A, intravenous injection of NPsiGATA2 significantly inhibited KRAS mutant A549 tumor growth, especially at the siGATA2 dose of 2.0 mg/kg per injection per mouse. However, treatment with NPsiNC or free siGATA2 did not show tumor growth inhibition in comparison with PBS treatment. The weights of mice were not significantly affected by the treatments (Figure 6B). Furthermore, we also examined the antitumor growth effect of NPsiGATA2 in mice with KRAS wildtype H226 xenografts. As shown in Figure S1A in the Supporting Information, treatment of tumor-bearing mice with NPsiGATA2 did not inhibit tumor growth in comparison with PBS treatment, implying that NPsiGATA2 is only effective in a KRAS mutant tumor model but ineffective in a KRAS wildtype tumor model. Similarly, the treatments did not significantly affect the weights of mice (Figure S1B in the Supporting Information). We further examined GATA2 expression at mRNA and protein levels in tumors by RTPCR and Western blot analyses following the treatment. As shown in Figures 6C and 6D and Figures S1C and S1D in the Supporting Information, mice treated with NPsiGATA2 showed enhanced downregulation of GATA2 gene expression at mRNA and protein levels in both KRAS mutant A549 xenografts and KRAS wild-type H226 xenografts, verifying that the effectiveness of NPsiGATA2 in the mutant KRAS tumor model was due to the synthetic lethal relationship between mutant KRAS and downregulated GATA2 expression.
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ASSOCIATED CONTENT
* Supporting Information S
Figure S1 depicting the experimental details of KRAS wild-type H226 xenograft tumors, including tumor growth, body weight changes of mice, and GATA2 mRNA and protein expression post treatment. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86 551 63600335. Fax: +86 551 63600402. E-mail:
[email protected] (X.-Z.Y.),
[email protected] (J.W.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Programs, 2013CB933900, 2012CB932500), the National High Technology Research and Development Program of China (863 Program 2012AA022501, 2014AA020708), the National Natural Science Foundation of China (51125012, 51390482, 51203145), SRFDP (20133402110019), and the Open Project of State Key Laboratory of Supramolecular Structure and Materials (SKLSSM201413).
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