Nitidine chloride induces caspase 3/GSDME-dependent pyroptosis by inhibting PI3K/Akt pathway in lung cancer

Chemiacals and materials

NC (Fig. 1A) was purchased from Solarbio (Beijing, China), with the purity above 98%. DDP was purchased from Qilu Pharmaceutical Co., Ltd. (Shandong, China). Ferrostatin-1 (Fer-1), Z-VAD-FMK, Necrostatin-1 (Nec-1), Disulfiram (DSF), Z-DEVD-FMK and 740Y-P was purchased from MCE (America). 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM-54) was purchased from Abcam.

Fig. 1figure 1

A Structure of NC. B The cell viability of normal lung cells BEAS-2B and 11 lung cancer cells are measured by MTT assay for 48 h. C Colony formation assays were performed using H1688 and A549 cells with or without NC or DDP for 10–14 days. D The cell viability of H1688 and A549 cells treated with the indicated concentrations of NC for 24, 36 and 48 h respectively. *P < 0.05, **P < 0.01, ***P < 0.001 vs control

Cell lines and cell culture

NCI-H226, SK-MES-1, A549, NCI-HCC827, NCI-H1975, PC9 and BEAS-2B cells were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). NCI-H209, NCI-H446, NCI-H1688 cells were purchased from national collection of authenticated cell cultures (Shanghai, China). NCI-H196 and NCI-H1299 cells were purchased from American Type Culture Collection (ATCC). All the culture media were supplemented with 10% fetal bovine serum (FBS, Biological Industries, Israel), penicillin (100 U/mL) and streptomycin (100 μg/mL, Solarbio, Beijing, China). The cells were incubated at 37 °C with 5% CO2.

MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays

MTT assays were performed as previously described [12]. Briefly, cells (3 × 103 cells/mL) were plated in 96-well plates and treated with different concentrations of NC or DDP for 24, 36, and 48 h respectively. DMSO (0.1%) served as vehicle control. Cells were incubated with 20 μL of MTT (5 mg/ml) for 4 h to form purple formazan crystals. Then, purple formazan crystals were dissolved by 150 μL of DMSO. The absorbance of sample was measured at 490 nm using a microplate reader. Data were presented as means ± SD and the selection index (SI) was caculated as the below equation: SI = [IC50 (nomarl cells)/IC50 (cancer cells)] × 100 [34].

Clonogenic cell survival assays

Clonogenic survival was evaluated by colony formation assays. Briefly, cell suspensions were seeded into 12-well plates (300 cells per well). After adhering overnight, cells were treated with NC or DDP for 10–14 days. Eventually, colonies were fixed with formalin and stained with 0.25% (w/v) crystal violet and colonies with more than 50 cells were included in the quantification. The colony number = number of colonies formed/number of cells seeded.

Microscopy imaging

To examine the morphology of pyroptotic cells, H1688 or A549 cells were seeded in the 24-well plate at about 80% confluency. After treated with NC or DDP, the bright-field cell images were captured using an inverted microscope (EVOS FL Auto, Thermo Scientific, America). To examine the sub-morphology of pyroptotic cells, cells were seeded in 10 cm culture dishes for 12 h. After treated with NC or DDP, the pore-forming activity of drug-induced pyroptosis was examined by transmission electron microscopy (TEM, Hitachi-7650, Japan).

Hoechst 33,342/PI staining

Hoechst 33,342/PI staining assay was performed according to the manufacturer’s instructions (Apoptosis and Necrosis Assay Kit, Beyotime Institute of Biotechnology, China). H688 and A549 cells were cultured in the 24-well plate (Corning Incorporated, Corning, USA) and subjected to the indicated treatments. Then hoechst 33,342 (5 μL) and PI dye (5 μL) were added and then incubated for 20 min in the dark and visualized under an inverted fluorescence microscope (EVOS FL Auto, Thermo Scientific, USA).

Western blot (WB)

Cells or tissue samples were collected and lysed in RIPA buffer containing proteinase inhibitors (Sigma, USA) and phosphatase inhibitors (CWBio, China). The protein extracts were separated on SDS-PAGE gels, followed by electrotransfering onto polyvinylidene fluoride membranes (Sigma, USA). After blocking with 5% fat-free milk for 2 h at room temperature, the membranes were incubated with primary antibodies and subsequently incubated with secondary antibody (Thermo Scientific, MA, USA). Finally bands were scanned with Bio-Rad Imager and individual band intensity was quantified with software of ImageJ. Antibodies were used as follows: caspase 3 (CST, USA), cleaved caspase 3 (CST, USA), GSDMC (Abclonal, China), GSDMD (Abclonal, China), GSDME (Abclonal, China), PI3K (Bioss, China), p-PI3K (Bioss, China), Akt (Proteintech, China), p-Akt (CST, USA), Bcl-2 (Proteintech, China), Bax (Proteintech, China) and β-Actin (Abclonal, China).

Lactate dehydrogenase (LDH) release assays

Cell culture supernatants were collected and the LDH activity was detected using the LDH assay kit according to the manufacturer’s protocol (Beyotime Institute of Biotechnology, China). The supernatants (120 μ L/well) were transferred into a blank 96-well plate and 60 μ L of LDH detection reagents were added to each well. The plates were then incubated for 30 min at room temperature in the dark. The absorbance was measured at 450 nm on a spectrophotometric microplate reader (BioTek, USA).

Network pharmacology analysis

The key word “nitidine chloride” was searched in Herb (http://herb.ac.cn/) and Swiss TargetPrediction (https://pubchem.ncbi.nlm.nih.gov/) databases. After merging and deleting duplicate values, a total of 870 targets were obtained. With “lung cancer” as the key word, MalaCards (http://www.malacards.org/) and GeneCards (www.genecards.org/) potential disease target analysis platforms were utilized to search for human genes related to LC. After merging and deleting duplicate values, a total of 1160 targets were obtained. The overlapped targets between NC and LC were further analyzed by the KEGG enrichment analysis using the DAVID database (https://david.ncifcrf.gov/).

Cellular thermal shift assay (CETSA)

CETSA was conducted using cell lysates as previously described [13]. Cell lysis buffer treated with NC or DMSO at room temperature for 1 h were collected and the cell suspension was distributed into 0.2 ml PCR tubes, with 200 µl cell suspension in each tube. The PCR tubes were heated at the designated temperature (37, 42, 47, 52, 57 and 62 °C) for 3 min. They were then removed and incubated at 4 °C immediately after heating. The precipitated proteins were separated from the soluble fraction by centrifugation and equal portions of the supernatants were loaded onto SDS-PAGE gels for WB analysis.

Drug affinity responsive target stability (DARTS) assay

H1688 and A549 cells were lysed in 500 μL NP-40 containing freshly protease (Sigma, America) and phosphatase inhibitors (CWBio, China). The cell lysate was equally divided into two tubes and then incubated with NC (0.2 mg/mL) and an equal volume of DMSO for 1 h at room temperature, respectively. After incubation, the mixture was divided into 100 μL aliquot in tubes and digested with pronase (MCE, America) in various doses at room temperature for 30 min. Then the digestion was stopped by adding protease inhibitors, and the samples were used for WB analysis.

Cell transfection

A small interfering RNA (siRNA) that specifically targets human caspase was designed (RiboBio, China). Si-NC or si-caspase 3 was transfected into H1688 and A549 cells using Lipo3000™ (Thermo Scientific, MA, USA) according to the manufacturer’s protocol. The medium was replaced with complete growth medium after 24 h of transfection and the cells were treated with NC.

Animal experiment

Four-to-five-week-old BALB/c male mice were purchased and maintained in the animal experimental center of Guangxi Medical University. H1688 cells or A549 cells (approximately 1 × 107) were injected subcutaneously into the right flank of BALB/c mice. Considering that NC is poorly absorbed, we treated the mice with NC by intraperitoneal injection. Once the tumors volume reached an average of 50–80 mm3, the mice were randomized into 4 groups (n = 5), with either vehicle control (0.1% DMSO), lower dose of NC (8 mg/kg/week), or higher dose of NC (16 mg/kg/week) twice a day respectively. As a positive control, mice were injected with DDP (8 mg/kg/week). The tumor volume (length × width × width) and body weight of the mice were monitored every 2 days. At the end of treatment, mice were sacrificed and tumors were removed, weighted and analyzed via hematoxylin and eosin staining (H&E), immunohistochemistry (IHC) and WB.

Immunohistochemistry

After routine dewaxing and hydration, the tissue sections from mouse tumor tissue specimens of each group were boiled in a microwave oven for 10 min for antigen retrieval followed by washing three times with PBS (pH = 7.4). Then they were treated with 3% hydrogen peroxide for 25 min and washed three times with PBS (pH = 7.4). Subsequently, the tissue sections were blocked in blocking buffer containing 3% BSA and incubated with Ki67, p-PI3K, cleaved caspase 3 and GSDME‐N antibodies overnight at 4 °C followed by incubating with the secondary antibody and DAB. The sections were counterstained using hematoxylin for 3 min and washed under tap water. The images were captured by the inverted microscope (EVOS FL Auto, Thermo Scientific, USA).

Statistical analysis

Statistical analysis was performed with GraphPad Prism 9.0 software. In all experiments, comparisons between two groups were based on unpaired student’s t-test and multiple group comparisons were performed using one-way ANOVA followed by least significant difference (LSD) post hoc test. P < 0.05 was considered statistically significant. The values of the results were representative in terms of the mean ± standard deviation (SD).

留言 (0)

沒有登入
gif