Chitosan nanoparticle-mediated co-delivery of shAtg-5 and gefitinib synergistically promoted the efficacy of chemotherapeutics through the modulation of autophagy
© The Author(s) 2017
Received: 9 January 2017
Accepted: 20 March 2017
Published: 11 April 2017
Autophagy reportedly plays vital and complex roles in many diseases. During times of starvation or energy deficiency, autophagy will occur at higher levels to provide cells with the nutrients or energy necessary to survive in stressful conditions. Some anti-cancer drugs induce protective autophagy and reduce cell apoptosis. Autophagy can adversely affect apoptosis, and blocking autophagy will increase the sensitivity of cells to apoptosis signals.
We designed chitosan nanoparticles (NPs) to promote the co-delivery of gefitinib (an anti-cancer drug) and shRNA-expressing plasmid DNA that targets the Atg-5 gene (shAtg-5) as an autophagy inhibitor to improve anti-cancer effects and autophagy mediation.
The results showed that when compared to treatment with a single drug, chitosan NPs were able to facilitate the intracellular distribution of NPs, and they improved the transfection efficiency of gene in vitro. The co-delivery of gefitinib and shAtg-5 increased cytotoxicity, induced significant apoptosis through the prohibition of autophagy, and markedly inhibited tumor growth in vivo.
The co-delivery of gefitinib/shAtg-5 in chitosan NPs produced superior anti-cancer efficacy via the internalization effect of NPs, while blocking autophagy with shAtg-5 enhanced the synergistic antitumor efficacy of gefitinib.
KeywordsAutophagy Apoptosis shAtg-5 Gefitinib Nanoparticles
Current conventional cancer therapies primarily depended on single chemical drugs that defend against cancer; however, the curative effects of these agents seem to be unsatisfactory, and they often result in chemotherapy failure due to a decline in the patient’s response to drugs—a phenomenon known as acquired drug resistance [1–3]. Therefore, it is necessary to find a new synergistic strategy to strengthen the chemotherapeutic effect of a given agent. It is well known that autophagy plays an important role in the development of cell death. First, as it is an important means of material recycling, and given its involvement in cell homeostasis maintenance, autophagy removes damaged proteins and cell organelles in stress states, thus minimizing the extent of cell damage and maintaining cell stability . In tumor therapy in particular, autophagy has been shown to be one of factors associated with chemotherapy, radiotherapy, and biological immunotherapy tolerance. Therefore, to some extent, the increase in autophagy reduces the cytotoxic and apoptotic effects of anti-cancer drugs. Autophagy can adversely affect apoptosis, and blocking autophagy will increase cell sensitivity to apoptosis signals [5–7].
Nanoparticles (NPs) as nano-scaled carriers showed excellent value and potential for improving drug/gene delivery [8–14]. Owing to their small particle size and high charge potential, NPs depend on enhanced permeability and retention (EPR) effects to achieve massive accumulation of drugs around the tumor while attenuating toxic damage to healthy organs [15–18]. Seeing as both drug molecules and genes can be encapsulated within the core of NPs, safe and effective targeted co-therapies featuring a drug and genes could be successfully achieved to offer synergistic effects against tumor development [19–22].
To confirm whether autophagy regulation plays a synergistic role in enhancing the antitumor efficacy of a chemotherapeutic drug, in this work, we prepared chitosan (CS) NPs with the combined co-delivery of gefitinib and short hairpin (sh)RNA-expressing plasmid DNA targeting the Atg-5 gene (shAtg-5) to improve anti-cancer effects. It was found that free gefitinib and gefitinib NPs induced cell death, while the autophagy effects were also simultaneously enhanced to some extent, which indicated that autophagy might have both positive and negative effects on the induction of cell apoptosis. The introduction of shAtg-5 as an inhibitor of autophagy efficiently downregulated the expression of the autophagy-related protein Atg-5. Furthermore, the cytotoxicity and apoptosis of cells treated with the co-delivery of shAtg-5 and gefitinib loaded in CS NPs were significantly enhanced upon autophagy inhibition.
CS of medium molecular weight (deacetylation degree, 80%; molecular weight, 400,000) was purchased from Haixin Biological Product Co., Ltd. (Zhejiang, People’s Republic of China). Gefitinib was purchased from Eastbang Pharmaceutical Co., Ltd. (Guangdong, People’s Republic of China). The pGCsi-U6/Neo/GFP-Atg-5 shRNA-expressing plasmid (p)DNA (Atg-5 shRNA, shAtg-5) that targeted the Atg-5 mRNA sequence (TTTCATTCAGAAGCTGTTT), as well as the pGCsi-U6/Neo/GFP-shRNA-expressing pDNA (pEGFP), were purchased from Genechem Co., Ltd. (Shanghai, People’s Republic of China). All of the other purchased chemicals were of analytical grade and were obtained from a variety of vendors. A549 cells and PLC cells were obtained from Jinzhou Medical University (Liaoning, People’s Republic of China).
Preparation and characterization of gefitinib/shAtg-5 NPs
According to our previous study , gefitinib/shAtg-5 NPs were prepared through an electrostatic interaction between positively charged CS and negatively charged sodium tripolyphosphate (TPP). The particle size, zeta potential, and polymer dispersity index (PDI) were determined by dynamic light scattering (Zetasizer Nano ZS; Malvern Instruments, Malvern, UK), and the morphology of NPs was observed by means of a transmission electron microscope (JEM-1200EX; JEOL, Tokyo, Japan). The drug-release pattern was also investigated in vitro. The difference between the amount of the initially added drug and the drug in the supernatant was measured by absorbance detection using an ultraviolet (UV)/Vis spectrophotometer (model 1601; Shimadzu, Kyoto, Japan) to determine the encapsulation efficiency (EE) of the drug in the NPs.
In vitro transfection experiments
In order to evaluate the enhanced transfection efficiency of genes through their encapsulation in NPs, free enhanced green fluorescent protein (EGFP) as the reporter gene and EGFP-loaded NPs were incubated with cells at a density of 5 × 104 cells/mL for 48 h. After that, the cells were washed with ice-cold phosphate buffered saline (PBS), and they were observed using confocal laser scanning microscopy.
Cell viability assays
To investigate the regulation of cell apoptosis by autophagy, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was adopted to determine cell viability in both PLC cells and A549 cells at a density of 5 × 104 cells/mL. Free gefitinib, free gefitinib and shAtg-5, gefitinib NPs, and gefitinib/shAtg-5 NPs were chosen for incubation with the cells for 24 h at 37 °C under 5% CO2. The absorbance of the solution was quantified using a BioTek Syneray-2 microplate reader (BioTek Instruments, Winooski, VT, USA) to measure absorbance at 490 nm.
Uptake ability of different kinds of NPs in cells
Different kinds of NPs were internalized into cells, and NP distributions were observed using real-time confocal laser scanning microscopy (FluoView FV10i; Olympus Corporation, Tokyo, Japan). NP uptake rates were determined by calculating the fluorescence intensity ratio using a microplate reader (Synery-2; BioTek Instruments). Rhodamine B-labeled NPs were incubated with cells at a density of 5 × 104 cells/mL at 37 °C under 5% CO2, and the nuclei were stained with Hochest (blue) for 15 min at 37 °C [24, 25]. At a predetermined time, the cellular distribution of different kinds of NPs was traced with the help of real-time confocal laser scanning microscopy. Basing on our previous study , after the cells were treated with fluorescein isothiocyanate (FITC)-labeled NPs, cold PBS was used to wash cells to remove the uninternalized NPs, while quantification of intracellular NPs was detected using a microplate reader by checking the fluorescence of FITC, which is excited at 485 nm and emitted at 528 nm. The relative fluorescence ratio (RFR, %), which represents the relative uptake rates of NPs, was calculated by determining the ratio of the fluorescence intensity of internalized FITC-labeled NPs to that of the initially added FITC-labeled NPs.
Green fluorescent protein-light chain 3 (GFP-LC3B) plasmid transfection
Cells stably transfected with GFP-LC3 plasmid were seeded into 24-well cell culture plates for incubation for 24 h; the number of cells in each well reached a density of 5 × 104 cells/mL. Cells were treated with free gefitinib and gefitinib-loaded NPs for 24 h. Finally, these cells were washed and GFP-LC3 was able to emit bright green fluorescence by confocal laser scanning microscope.
Annexin V-FITC/propidium iodide (PI) staining by flow cytometry (FCM)
According to the protocol of our previous study , free gefitinib, gefitinib and shAtg-5, gefitinib NPs, and gefitinib/shAtg-5 NPs were chosen to determine the extent of cell apoptosis using the flow cytometer, FACS-Calibur (Becton–Dickinson, Franklin Lakes, NJ, USA).
Western blot assay
Western blot assay was performed to determine the levels of relative proteins when free gefitinib, gefitinib and shAtg-5, gefitinib NPs, and gefitinib/shAtg-5 NPs were incubated with A549 and PLC cells for 24 h. Briefly, the proteins were transferred to a membrane (typically nitrocellulose or PVDF), where they were stained with antibodies specific to the target protein. Western blotting was performed according to the manufacturer’s instructions (Cell Signaling Technology, Danvers, MA, USA). Protein bands were detected using a gel imaging system (iBox Scientia 600; UVP, LLC., Upland, CA, USA).
Mice and in vivo tumor studies
Six- to eight-week-old female nude mice (BALB/c nude mice) were purchased from Beijing Vital River Laboratory Animal Technology Company (Beijing, People’s Republic of China). PLC cells (1 × 107 cells/mL) were re-suspended in 100 μL PBS and injected subcutaneously into the anterior flank of 25 mice (five in each group). Two weeks later, when the tumor volume reached ~150 mm3, the mice were treated with intraperitoneal injections of PBS, free gefitinib, gefitinib and shAtg-5, gefitinib NPs and gefitinib/shAtg-5 NPs for 5 consecutive days each week for 3 weeks. Tumor diameters were measured twice per week with a caliper, and tumor volumes were calculated using the formula [(width)2 × length]/2 (mm3). The mice were sacrificed on day 20, the tumors were then isolated, and the tumor specimens were prepared as paraffin-embedded sections for histopathological analysis. All animal studies were conducted according to the regulations for animal experimentation issued by the State Committee of Science and Technology of the People’s Republic of China.
Preparation and characterization of gefitinib/shAtg-5 NPs
Gene transfection evaluation in vitro
Distribution of NPs in cells
Confocal microscope images of GFP-LC3-transfected A549 cells and PLC cells treated with free gefitinib and gefitinib-loaded NPs
Cell apoptosis and necrosis
Western blot assay
In vivo anti-tumor effects
Autophagy is a cellular catabolic process in which cytoplasmic target material is transported to the lysosomes for degradation through a wide array of resident acid hydrolases. It is well known that autophagy is critical in material recycling and cell homeostasis maintenance; this process removes damaged proteins and cell organelles in stress states, thus limiting the extent of cellular damage and maintaining cell stability. In tumor therapy, autophagy has been shown to be one of the factors responsible for chemotherapy, radiotherapy, and biological immunotherapy tolerance. Conversely, prolonged and uncontrolled autophagy is also involved in cell death .
In our work, we tried to clarify the role of autophagy induced by a free drug and drug-loaded NPs in the induction of apoptosis in cancer cells. It was found that free gefitinib and gefitinib-loaded NPs induced cell death, while the autophagy effects were also simultaneously enhanced to some extent, which indicated that autophagy might have some negative effects on the induction of cell apoptosis. The introduction of shAtg-5 as an inhibitor of autophagy efficiently downregulated the expression of the autophagy-related protein Atg-5; furthermore, and a series of experiments were conducted, which proved that autophagy inhibition occurred, as evidenced by the decreased ratio of LC3 II to LC3 I. Furthermore, the cytotoxicity and apoptosis of cells treated with the co-delivery of shAtg-5 and gefitinib loaded in CS NPs had been significantly enhanced in association with autophagy inhibition. When compared with free gefitinib, gefitinib and shAtg-5, and gefitinib NPs, the IC50 values of gefitinib/shAtg-5 NP-treated A549 and PLC cells within 24 h were lowest. The ratio of AV-positive and PI-positive cells treated with gefitinib/shAtg-5 NPs had increased to 81. 16% for A549 cells and to 80.04% for PLC cells. It is possible that when treating cells with the free drug or with drug-loaded NPs, autophagy was triggered and played a cytoprotective role in response to the toxicity exerted by the drug or drug-loaded NPs, which could provide a lot of nutrients and oxygen to improve tumor cell adaptability and to promote cell survival, thus preventing apoptosis and necrosis. The involvement of shAtg-5 offered specific and long-lasting gene silencing effects, and autophagy was significantly downregulated. More importantly, the cells’ sensitivity to gefitinib was enhanced by increasing the apoptosis of A549 cells and PLC cells, as induced by gefitinib. In addition, NPs protected shAtg-5 through encapsulation while avoiding degradation by enzymes, and they further improved the gene transfection efficiency and accomplished effective gene silencing.
All of these results showed that autophagy as a protective mechanism could be induced in tumor cells with the mediation of gefitinib as an anti-cancer drug; it ultimately reduced cytotoxicity during cell death, enhanced the adaptation of tumor cells to the environment, and it even led to chemotherapy tolerance. Co-delivery of shAtg-5 and gefitinib loaded in CS NPs triggered the apoptosis pathway and enhanced synergistic antitumor effects via autophagy blockade.
CS NPs prepared by an ion gelation method demonstrated excellent properties such as good drug entrapment, sustained release, smaller average particle size, a low PDI, and a high EE, and it accomplished the efficient co-delivery of shAtg-5 and gefitinib. shAtg-5 was involved as a powerful tool in the silencing of the Atg-5 protein and in the treatment of A549 cells and PLC cells, as shAtg-5 induced autophagy inhibition, while further increasing cell inhibition and apoptosis when treated with a combination of shAtg-5 and gefitinib. This finding suggested that when compared with treatment with gefitinib alone, the co-delivery of gefitinib and shAtg-5 encapsulated within the NPs greatly contributed to gefitinib-induced apoptosis among A549 cells and PLC cells by significantly inhibiting autophagy and inducing cellular apoptosis. Taken together, the co-delivery of shAtg-5 and gefitinib loaded in the CS NPs enhanced the synergistic antitumor effects via autophagy blockade.
enhanced green fluorescent protein
YZ and YS performed the preparation and characteristics of the NPs, and YZ helped with the biological study. CS and YS conducted the animal experiments. LZ and YS supervised the whole work and helped in the analysis of biological data. All authors read and approved the final manuscript.
This work was supported by Liao’ning Educational Committee (No. L2014339), Natural Science Foundation of Liaoning Province (Nos. 2014022039, 2015020692 and 201602337), and the English-language editing of this manuscript was provided by Journal Prep.
The authors declare that they have no competing interests.
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.
Ethical approval, consent to participate and publication
The submission reported data collected from animals, and all animal studies were conducted according to the regulations for animal experimentation issued by the State Committee of Science and Technology of the People’s Republic of China.
Liao’ning Educational Committee (No. L2014339) and Natural Science Foundation of Liaoning Province (Nos. 2014022039, 2015020692 and 201602337) supported this work in the design of the study and analysis of data.
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