- Open Access
Curcumin overcome primary gefitinib resistance in non-small-cell lung cancer cells through inducing autophagy-related cell death
- Ping Chen†1,
- Han-Peng Huang†1,
- Yi Wang2,
- Jun Jin1,
- Wei-Guo Long3,
- Kan Chen1,
- Xiao-Hui Zhao3,
- Chen-Guo Chen1 and
- Jian Li1Email author
© The Author(s). 2019
- Received: 26 January 2019
- Accepted: 15 May 2019
- Published: 13 June 2019
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are being wildly used as target therapy in non-small-cell lung cancer (NSCLC). However, NSCLC patients with wild-type EGFR and KRAS mutation are primary resistant to EGFR-TKIs such as gefitinib. Curcumin has been known as a potential therapeutic agent for several major human cancers. In this study, we investigated the effect of curcumin on the reversal of gefitinib resistance in NSCLC cells as well as their molecular bases.
H157 (wild-type EGFR and KARS mutation) and H1299 (wild-type EGFR and HRAS mutation) cells were treated with gefitinib or curcumin alone, or the two combination, and then cell viability, EGFR activity, expressions of Sp1 and Sp1-dependent proteins and receptor tyrosine kinases, markers of autophagy and apoptosis were examined by using CCK-8, colony formation, immunoblot, quantitative PCR, immunofluoscence, and flow cytometry assays. Also xenograft experiments were conduced to test the synergism of curcumin to gefitinib.
Our results showed that curcumin significantly enhanced inhibitory effect of gefitinib on primary gefitinib-resistant NSCLC cell lines H157 and H1299. Combination treatment with curcumin and gefitinib markedly downregulated EGFR activity through suppressing Sp1 and blocking interaction of Sp1 and HADC1, and markedly suppressed receptor tyrosine kinases as well as ERK/MEK and AKT/S6K pathways in the resistant NSCLC cells. Meanwhile, combination treatment of curcumin and gefitinib caused dramatic autophagy induction, autophagic cell death and autophagy-mediated apoptosis, compared to curcumin or gefitinib treatment alone, as evidenced by the findings that curcumin and gefitinib combination treatment-produced synergistic growth inhibition and apoptosis activation can be reversed by pharmacological autophagy inhibitors (Baf A1 or 3-MA) or knockdown of Beclin-1 or ATG7, also can be partially returned by pan-caspase inhibitor (Z-VAD-FMK) in H157 and H1299 cells. Xenograft experiments in vivo yielded similar results.
These data indicate that the synergism of curcumin on gefitinib was autophagy dependent. Curcumin can be used as a sensitizer to enhance the efficacy of EGFR-TKIs and overcome the EGFR-TKI resistance in NSCLC patients with wild-type EGFR and/or KRAS mutation.
- Non-small-cell lung cancer
- Epidermal growth factor receptor (EGFR)
- Tyrosine kinase inhibitor (TKI)
Epidermal growth factor receptor (EGFR), an oncogenic receptor tyrosine kinase, plays a key role in the development and progression of non-small-cell lung cancer (NSCLC) . First-generation EGFR-tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib reversibly bind to the ATP cleft within the EGFR kinase domain to block autophosphorylation of EGFR . Dramatic tumor response and favorable clinical outcomes led to their widespread use in the first-line setting for patients with advanced NSCLC harboring activating EGFR mutation (a deletion in exon 19 or the L858R mutation in exon 21) [3, 4]. However, NSCLC patients initially response to these EGFR-TKIs almost invariably develop drug resistance [4, 5], which commonly arise through the acquisition of a second-site mutation (T790 M) within EGFR, or via activation of compensatory signaling pathways that bypass receptor and restore downstream oncogenic signaling [6, 7]. In addition, some NSCLC patients are intrinsically resistant to EGFR-TKIs, even though their tumors harbor activating mutation of EGFR . Moreover, NSCLC overexpressing wild- type EGFR are often primary resistant, especially in tumor with concurrent KRAS mutation [9, 10]. However, the reasons for the innate resistance to EGFR-TKIs in wild-type EGFR and KRAS mutation NSCLC tumors still remain ill-defined. Therefore, the underlying mechanism of primary EGFR-TKI resistance need to be investigated in-depth. And developments of new EGFR-TKI sensitizers and combination strategies to surmount primary resistance to EGFR-TKIs are urgently required in patients with wild-type EGFR and KRAS mutation.
Several studies have showed that EGFR signaling regulates autophagy, a process involving sequestration and degradation of intracellular organelles and proteins in lysosomes, which function in cellular homeostasis and protection against a variety of disease including cancer . The downstream targets of EGFR — PI3K, AKT, and mammalian target of rapamycin (mTOR) — are well established negative regulators of autophagy . Furthermore, EGFR-TKIs induce autophagy in NSCLC [13–15] and other cancer cells . However, the relation between EGFR-TKI resistance and autophagy is poorly understood. EGFR-TKI-induced autophagy in NSCLC cells has been postulated to exert either cytoprotective  or cytotoxic  effects.
Curcumin, a compound found in the plant Curcuma longa, has been demonstrated to inhibit the growth of various cancers  and sensitize tumor cells to chemotherapeutic drugs and ionizing radiation [18, 19]. We once reported that curcumin reversed cisplatin resistance in cisplatin-resistant lung cancer cells by suppressing the FA/BRCA pathway, a DNA cross-link damage repair pathway which regulates cellular sensitivity to DNA cross-linking agents . Recently, several studies showed that curcumin is capable of inducing autophagy in different type of human cancer cells, including colon cancer, oral cancer, glioblastoma, uterine leiomyosarcoma and lung adenocarcinoma [21–25], suggesting that autophagy could play a certain role in the anticancer process of curcumin [26, 27]. A recent study reported that curcumin potentiated antitumor activity of gefitihib in gefitinib-resistant cells and xenograft mice model of NSCLC through inhibition of EGFR phosphorylation, and induction of EGFR degradation and apoptosis . However, the underlying mechanism by curcumin sensitizing the NSCLC cells to gefitinib was not clearly elucidated. Since both EGFR-TKIs and curcumin induce autophagy in cancer cells, we infer that curcumin can enhance inhibitory effect of gefitinib on primary EGFR-TKI resistant NSCLC cells through augmenting gefitinib-induced autophagy and inducing autophagy-related cell death. To demonstrate the inference, we tested the synergism of curcumin on gefitinib in primary resistant NSCLC cells and examined whether the synergism of curcumin in combination with gefitinib is associated with autophagy induction and autophagy-mediated cell death, further investigate the relevant molecular bases involved in reversing of gefitinib resistance by curcumin.
Three human NSCLC cell lines H157 (wild-type EGFR and KRAS mutation), H1299 (wild-type EGFR and NRAS mutation) and PC9 (EGFR exon 19 deletion) were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in 10% fetal bovine serum-supplemented RPMI 1640 medium (Sigma-Aldrich, St Louis, MO) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA) at 37 °C in a humidified incubator containing 5% CO2. All cells were passaged for 3 months or less before the renewal from frozen, early-passage stocks obtained from the indicated sources. Cells were regularly tested for mycoplasma with the use of a MycoAlert Mycoplasma Detection Kit (Lonza, Switzerland).
Cell viability assay
Cell viability was conducted by cell counting kit-8 (CCK-8, Dojndo, Japan) assays before and after treatment with indicated drugs according to the manufacturer’s instructions as previously described [20, 29]. For combination treatment of curcumin and gefitinib, CCK-8 assay data were converted to fraction of growth affected by the individual drug or the combination treated cells compared with untreated cells and analyzed using CalcuSyn Version 2.1 software (Biosoft, Gambride, UK) to determine whether the combination was synergistic. This program is based upon the Chou-Talalay equation , which calculates a combination index (CI). The combined effects of the two compounds can be summarized as follows: CI values <1, = 1 and >1 indicate synergistic, additive and antagonistic effects, respectively.
Colony formation assay
Colony formation assay was performed as described previously . Briefly, a density of 1000 cells per well seed onto 6-well culture plate and the indicated drugs or vehicle in DME containing 10% FBS was added to the culture at 3 day after seeding. The culture was continuously maintained for another 14 days and subjected to the colony formation assay. Colonies were visualized and countered under a microscope.
Cells were treated with the indicated drugs at various concentration, and proteins from whole cell lysates were prepared and determined using immunoblotting as previously described . The antibodies used to determine the proteins in this study were described in Additional file 1: Supplementary materials and methods.
Small interfering RNA (siRNA) siSp1, siSp3, siBeclin-1 and siATG7 and nonsilencing control siRNA were purchased from Qiagen (Duesseldorf, Germany). These siRNAs were transfected into cells with transfectamine 2000 Transfection Reagents in accordance with manufacturer’s instructions. After 48 h, the cells were harvested and subjected to immunoblot or RT-PCRassay, as previously described .
Real-time quantitative PCR and PT-PCR
For real-time quantitative PCR, total RNA was extracted from various cell samples using Trizol reagent (Invitrogen, Carlsbad, CA). Reverse transcription were conducted using the Applied Biosystem’s Power SYBR Green PCR Master Mix and the reactions were run on an ABI 7500 Fast Real-time PCR system, as previously described . For RT-PCR, products were resolved by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining. The specific primary sequences of the genes detected in this study are shown in Additional file 2: Table S1.
LC3-II puncta detection was performed as previously described . Briefly, cells were seeded on coverslips, and treated with the indicated drugs before being fixed with 2% paraformaldehyde in PBS for 20 min at 37 °C. Following permeabilization for 3 min with PBS-0.2% Triton X-100, the cells were rinsed three times in PBS and incubated overnight at 4 °C with anti-LC3 antibody diluted 1:100. After three times washes in PBS, the cells were incubated for 1 h at room temperature with Alexa Fluor 488-conjugated secondary antibody at a 1:400 dilution. Then cells were counterstained with Hoechst 33342 for 5 min for nuclear staining, and mounted on glass slides. Cells were visualized using a Leica Confocol Laser Scanhing Microscope (Leica, Wetzlar, Germany).
Sub-G1 population assay
Sub-G1 population was analyzed by flow cytometry as previously described . Briefly, cells were treated with the indicated drugs for 24 h, harvested and fixed using 7% ethanol. And then cells were stained with mixture of 50 μg/mL propidium iodide, 0.2% Triton X-100, and 100 μg/mL RNAase, and performed using a FACS flow cytometer equipped with Modfit LT for Mac V 2.0 software (BD Biosciences, NJ).
In vivo studies
Six-week-old female athymic nude BALBL/c mice were obtained from Charles River Laboratories (Wilmington, MA). All mice were handled in accordance with the protocols approved by the Institutional Animal Care and Use Committee of the Jiangsu University. Before starting the experiments, mice were maintained for 5 days in the temperature and humidity controlled condition. H157 and H1299 cells (6 × 106 cells per mouse) were subcutaneously grafted into the right flanks. When tumor volume reached approximately 70 mm ± 10 mm3, mice were randomly allocated into groups of 6 animals to receive either vehicle control, gefitinib (100 mg/kg, one daily) alone, curcumin (1 g/kg, once daily) alone, or gefitinib and curcumin together. Doses of gefitinib and curcumin used in the mice with tumor were according to Lee et al. and Xu et al. experience [28, 33]. Gefitinib was suspended in saline and curcumin was prepared properly in propylene glycol and administered orally by gavage. Tumor volume was measured every 5 days using calipers. The average tumor volume in each group was expressed in mm3 and calculated according to the equation (lenth × width2)/2. At study termination, tumor xenografts were harvested and weighted. Tumor samples obtained were snap-frozen in liquid nitrogen and stored in − 80 °C for immunoblot analysis.
Each experiment was performed at least three times, and all data are presented as the mean ± SD. Statistical analysis was performed by the unpaired 2-tailed Student t test or the Mann-Whitney U-test using the SPSS version 22.0 (SPSS Inc.; Chicago, IL). P-values <0.05 were considered significant (*, P<0.05; **, P<0.010; ***, P<0.001).
A complete description of the methods, including drugs and antibodies used in this study, cell proliferation assay, BrdUred incorporation assay, luciferase assay, plasmid transient transfection, caspase activity assay and immunofluorescence analysis are available in Additional file 1: Supplementary materials and methods.
Curcumin enhances the inhibitory effect of gefitinib on gefitinib-resistant NSCLC cells by suppressing EGFR activity
We further examined that the effect of gefitinib and curcumin on EGFR activity in these NSCLC cells. The cells pre-treated with gefitinib, or curcumin alone, or the two drug combination for 12 h, were stimulated with EGF (30 ng) for 1 h. Pre-treatment with gefitinib alone barely affect EGFR activity induced by EGF in H157 and H1299 cell lines upon EGF exposure. While curcumin alone moderately reduced EGF-induced EGFR phosphorylation, combined curcumin with gefitinib markedly decreased phosphorylated and endogenous EGFR levels induced by EGF in two gefitinib-resistant cell lines (Fig. 1f and Additional file 3: Figure S2). In PC9 cells, however, EGF-induced phosphorylated and total EGFR levels were clearly reduced by pre-treatment with gefitinib or curcumin alone. In addition, the proteasome inhibitor, MG132 partially restored curcumin and gefitinib combination-induced EGFR downregulation in H157 cells (Additional file 3: Figure S3a). These results indicate that primary resistance to gefitinib in the NSCLC cells with wild-type EGFR and KRAS mutation is associated with insensitive to gefitinib-induced EGFR downregulation, and gefitinib and curcumin combination-induced EGFR downregulation partially through proteasome-induced degradation of EGFR protein.
Combination of curcumin and gefitinib inhibits EGFR through suppressing Sp1 and HDAC1 binding induced EGFR transcription activity
To further determine if the effect of curcumin and gefitinib combination on EGFR was mediated by affecting EGFR transcription activity, we performed luciferase assay. As shown in Fig. 2g, Sp1 silencing and overexpression enhanced and reversed curcumin and gefitinib combination-induced decrease of EGFR promotor activity respectively. Likewise, quantitative PCR analysis showed that knockdown and overexpression of Sp1 potentiated and returned curcumin plus gefitinib-induced EGFR mRNA expression downregulation in H157 and H1299 cells respectively (Additional file 3: Figure S3d). Collectively, these data indicated that the diminished interaction between Sp1 and HDAC1 caused by curcumin contributes the two drugs combination-induced EGFR activity downregulation.
Curcumin and gefitinib synergistically inhibit the receptor tyrosin kinas signaling and ERK-AKT pathways
EGFR regulates multiple signaling pathways via activation of downstream kinase, such as the ERK/MEK and AKT/S6K pathways. Figure 3c and Additional file 3: Figure S5c show the effect of the drugs on the ERK/MEK and AKT/S6K phosphorylation levels in H157 and H1299 cells. Gefitinib alone had a minor affect on phosphorylation of ERK1/2, MEK1/2, AKT and S6K, the two drug combination further reduced phosphorylation levels of all kinases detected in this study. The results indicate that the combination treatment blocks EGFR downstream tyrosine kinases that have key roles in the pro-oncogenic signaling of NSCLC, thereby potentiating the inhibitory effects of gefitinib on gefitinib-resistant NSCLC cells.
Combined curcumin with gefitinib augments autophagy induction in NSCLC cells through suppressing Sp1/EGFR signaling
Since gefitinib and curcumin combination-induced EGFR downregulation was mediated by repressing Sp1, Sp1 is involved in induction of autophagy and apoptosis. To demonstrate this notion, we evaluated the effect of Sp1 overexpression on autophagy and apoptosis induced by the combination treatment. As expected, Sp1 ectopic overexpression in H157 cells attenuated the combination treatment-inducted accumulation of LC3-II and decrease of SQSTM1, and raise of cleaved-caspase-3 and cleaved-PARP (Additional file 3: Figure S7a), also reduced combination treatment-induced the increased of sub G1 population and abated the synergism of curcumin to gefitinib (Additional file 3: Figure S7b and c). Meanwhile, to prove if the induction of autophagy is associated with the downregulation of EGFR, the changes in EGFR localization were confirmed by IF microscopy using the anti-EGFR and anti-LC3 antibodies conjugated to fluorophore. As shown in Fig. 4e, the combination treatment clearly increased LC3 and EGFR co-localization compared to gefitinib or curcumin treatment alone, implying that the two drug combination-induced autophagy can entrap EGFR from plasma membrane into the autophagosome and that trapped proteins may be degraded by lysosome. To further investigate if the combination treatment-induced autophagy may lead to the downregulation of EGFR, we evaluated the levels of EGFR in the presence or absence of autophagy inhibitor (3-MA) and siBeclin-1 treatment. Interestingly, 3-MA and siBeclin-1 treatment restored the decreased activities and the total levels of EGFR induced by gefitinib plus curcumin in H157 cells (Fig. 4f and Additional file 3: Figure S6c), suggesting that there is a crosstalk between EGFR and autophagy. Taken together, these results indicate that gefitinib-induced low level autophagy can be obviously enhanced by combining curcumin and the two drug combination potentiated apoptotic induction, both the biological reactions were medicated through downregulation of Sp1/EGFR activity.
Curcumin overcomes gefitinib resistance in NSCLC cells through induction of autophagic cell death and autophagy-mediated apoptosis
Curcumin enhances therapeutic efficacy of gefitinib and in vivo through Sp1/EGFR downregulation-mediated induction of autophagy-related cell death
Although first- and second-generation EGFR-TKIs (including gefitinib, erlotinib and afatinib) produce marked tumor response and improved progression-free survival (PFS) in NSCLC patient harboring activating EGFR mutation [4, 43], patients with wild-type EGFR and KRAS mutation are not sensitive to these EGFR-TKIs [5, 9, 43]. The presence and emergence of drug resistance have highlighted the continued need for combination therapies. In this study, we designed a combinatorial strategy and showed that combination of gefitinib and curcumin exhibited potent inhibitory effect on growth of primary resistant NSCLC cells in vitro and in vivo through downregulating EGFR activity. This process concurrently induced autophagy and apoptosis reactions. We found that synergism of curcumin to gefitinib is more pronounced in gefitinib-resistant cells than in gefitinib-sensitive cells, and gefitinib induced lesser extent downregulation of EGFR activity in gefitinib-resistant cells compared to gefitinib-sensitive cells, suggesting that the insensitivity to gefitinib- induced downregulation of EGFR activity may be involved in gefitinib primary resistance in NSCLC cells, and curcumin reverse gefitinib resistance at least partially through suppressing EGFR activity. Some studies showed that curcumin and its analogs can induce EGFR degradation in EGFR-TKI-resistant and -sensitive lung adenocarcinoma cells and sensitize the TKI-resistant NSCLC cells to gefitinib [33, 47, 48]. Curcumin analog WZ35 was recently reported to induce apoptosis and suppress cell viability via reactive oxygen species (ROS)-mediated activation of the endoplasmic reticulum stress signaling and mitochondrial dysfunction in acquired TKI-resistant NSCLC cells . In human oral squamous cell carcinoma cells, curcumin or its analogs markedly increase gefitinib sensitivity by inducing apoptosis and autophagy . However, the detailed molecular bases for the results were not clarified in these studies. In our model, gefitinib and curcumin combination-induced EGFR downregulation is transcription factors Sp1 and Sp3 dependent. Since Sp1 has been found to regulated EGFR transcription through interacting with HDAC1 in cancer cells , we revealed for the first time that combination of gefitinib and curcumin decreased EGFR mRNA level and luciferase activity through suppressing Sp1 and blocking interaction of Sp1 and HDAC1 in the gefitinib-resistant NSCLC cells, and this was accompanied by decreased expression of other Sp1-dependent proteins survivin, VEGF and c-Met, and enhanced induction of autophagy and apoptosis.
EGFR activation trigger the phosphorylation of the inner tyrosine kinase portion of the receptor, then lead to cell proliferation and apoptosis inhibition through activating multiple intracellular signaling pathways, including MAPK/ERS1/2, PI3K/AKT, STAT and SRC/FAK pathways . Also bypass receptor tyrosine kinases, including c-Met, Her2, AXL and IGF1R, play important roles in NSCLC resistance to EGFR-TKIs . It is reported that downregulation of receptor tyrosine kinase by other agents such as HDAC inhibitors can sensitize resistant NSCLC cells to EGFR-TKIs [45, 46]. However, whether curcumin plus gefitinib-induced Sp1/EGFR downregulation affect the receptor tyrosine kinase is not clear. We results showed that curcumin either as a single agent or in combination with gefitinib simultaneously repressed the activity of the receptor tyrosine kinases including their protein and mRNA expressions. In addition, the enhanced activity of EGFR downstream molecules such as ERK1/2, MEK1/2, AKT and S6K was also obviously diminished by the combination treatment. These results suggest that curcumin could reverse NSCLC cell resistance to gefitinib through interfering with multiple bypass receptor tyrosine kinases and the cell proliferation pathways.
Autophagy is a cellular ‘self-eating’ degradation process in which proteins or whole organelles are degraded in lysosomes and recycle to meet the anabolic and bioenergetic need of the cell. In general, autophagy is considered a survival process, in the other condition, autophagy also induces cell death, namely autophagic cell death [11, 51, 52]. Autophagy plays the dual role in response to anticancer treatment, depending on the cellular context and the nature of the treatment. Both EGFR-TKIs and curcumin can induce autophagy [13–16, 21–25]. However, the exact role of autophagy activation induced by EGFR-TKIs remains an area of debate as some studies suggested that blocking autophagy improves the autitcancer activity of EGFR-TKIs to NSCLC cells [13, 14], whereas other studies proposed that induction of autophagy can lead to potentiation of inhibitory effect of EGFR-TKIs on NSCLC cells . In this study, the gefitinib-resistant cells treated with combination of gefitinib and curcumin showed more significant autophagy induction compared to single drug treated cells in parallel with elevation of cleaved-caspase-3 and cleaved-PARP. Importantly, autophagy inhibitors Baf A1 and 3-MA reverse decline of cell viability caused by combining gefitinib with curcumin. Meantime, co-treatment with 3-MA or Baf A1, or knockdown of ATG7 attenuated the two drug combination-induced increase of cleaved-caspase-3 and cleaved-PARP, and partly reduced the two drug combination-induced augmentation of sub G1 population and increase of caspase-3/7 activity in gefitinib-resistant NSCLC cells. Additionally, pan-caspase inhibitor (Z-VAD-FMK) can depress elevated cleaved-caspase-3 and cleaved-PARP induced by curcumin plus gefitinib, and partly returned decrease of cell viability produced by curcumin plus gefitinib, suggesting that the apoptosis induced by the combination treatment depend on autophagy induction. Therefore, we believe that gefitinib and curcumin combination-induced cell death mode consist of autophagic cell death and autophagy-mediated apoptosis. Shen et al. proposed the definition of autophagic cell death. First, cell death occurs without the involvement of the apoptosis machinery. Second, there is an increase of autophagy flux, and not just an increase of the autophagic markers in the dying cells. Finally, pharmacological inhibitors and genetic approaches (ATG knockdown by siRNA transfection) able to rescue or prevent cell death . In this study, combination of gefitinib and curcumin increased autophagy flux. Although autophagy inhibitors (Baf A1 and 3-MA) rescued two drug combination-induced cell death, co-treatment with 3-MA or knockdown of ATG (Beclin-1 and ATG7) partially reversed apoptotic reaction induced by the two drug combination. Moreover, the caspase inhibitor also partly abated increase of cleaved-caspase-3 and cleaved-PARP, and partially rescued cell death induced by the two dug combination. Taken together, these results support the idea that the cell death mode in this study included both autophagic cell death and autophagy-mediated apoptosis.
In addition, we demonstrate that the combination treatment-induced autophagy is mediated via downregulation of Sp1/EGFR as Sp1 ectopic overexpression retarded autophagy and apoptosis. Intriguingly, not only downregulation of EGFR mediated autophagy, but also autophagy induction caused the downregulation of EGFR activity, suggesting that there is an interplay between the autophagy and EGFR in activation process of the two signaling pathways.
Here, we have provided the first evidence that curcumin and gefitinib synergistically inhibit the growth of gefitinib-resistant NSCLC cells by downregulating Sp1/EGFR activity and the receptor tyrosine kinase pathways, thereby inducing autophagic cell death and autophagy-mediated apoptosis. Our data indicate that curcumin could be used as a sensitizer of EGFR-TKIs in the treatment of NSCLC with wild-type EGFR and KRAS mutation. Further studies are needed to demonstrate the efficacy of the combination treatment of curcumin and EGFR-TKIs in primary EGFR-TKI- resistant NSCLC through clinical trials.
The authors acknowledge Institute of Medical Science of Jiangsu University for their technical assistance and instrument supports.
This work was supported by grants from the National Natural Science Foundation of China (No. 81400066), Medical Research Program of Jiangsu Health and Family Planning Commission, China (No. H2017013).
PC, H-PH, and JL conceived and designed the study. PC, H-PH, YW, JJ, W-GL, KC and X-HZ carried out the experiments. PC and C-GC performed data collection and analysis. PC and JL supervised the research, and wrote and revised the manuscript. All authors read and approved the final manuscript.
All experimental procedures using animals were conducted in accordance with institutional guidelines and protocols approved by Institutional Animal Care and Use Committee of the Jiangsu University.
Consent for publication
The authors declare that they have no competing interests.
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