Anticancer efficacy of hirsuteine against colorectal cancer by opposite modulation of wild-type and mutant p53

2.1 Reagents, antibodies and plasmids

Hirsuteine (purity: 98%) was obtained from Abphyto biotech (Chengdu, Sichuan, China). 3-(4,5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2Htetrazolium bromide (MTT) reagent was from Amresco (Solon, OH, USA). Propidium iodide (PI) was from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-FITC/PI Apoptosis Detection Kit was from BD Biosciences. TRIzol reagent was purchased from Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS) was from Biological Industries (Kibbutz Beit-Haemek, Israel). The primary antibodies against cyclin B1 (1:1000, #4135), cdc2 (1:1000, #9116), p-Rb (1:1000, #8516), Caspase-3 (1:1000, #9662), β-actin (1:1000, #8457), anti-rabbit HRP-conjugated secondary antibodies (1:2000, #7074) and anti-mouse HRP-conjugated secondary antibodies (1:2000, #7076) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against p53 (60283-1-lg), p21 (10355-1-AP), MDM2 (27883-1-AP), LC3 (14600-1-AP), p-p53 (Ser15) (67826-1-lg), p62 (18420-1-AP), Ki-67 (27309-1-AP), PUMA (55120-1-AP) were purchased from Proteintech (Wuhan, Hubei, China).

2.2 Cell lines

Human CRC cell lines SW620 (mutp53R273H) and HCT-8 (wtp53) were purchased from National Collection of Authenticated Cell Cultures (Shanghai, China). Cells were maintained in complete RPMI-1640 medium supplemented with 10% FBS, 10 µg/mL streptomycin and 100 U/mL penicillin. Cells were cultured in a standard condition: a humidified incubator with 5% CO2 at 37 °C. Cells were authenticated by short tandem repeat (STR) analysis.

2.3 siRNA

p53 knockdown was conducted using siRNA as our previous literature [15]. siRNA p53 was purchased from Genepharma (Suzhou, Zhejiang, China). Cells were seeded onto six-well plates (3 × 105 cells/well) and transfected with siRNA p53 or siRNA non targeting using Lipofectamine® 2000 (Invitrogen, Corp., Carlsbad, CA, USA) at 37 °C for 6 h. The culture medium was changed by fresh complete culture medium before subsequent experiments.

2.4 MTT assay

The cell viability was determined by MTT assay as we previously reported [14]. Cells were seeded (4 × 104 cells/well) onto 96-well plates with a volume of 200 µL per well, and treated with a series of concentrations of HST for 48 h or treated with 32 µM HST at different time points, or 32 µM HST and/or 3-MA for 48 h. After exposed with 5 mg/mL MTT, cells were further cultured at 37 °C for 4 h. The absorbance at a wavelength of 490 nm was measured by a microplate reader iMark (BIO-RAD, Hercules, CA, USA). Drug concentrations causing 50% inhibition (IC50 values) were calculated using GraphPad Software by interpolation.

2.5 Colony formation

The colony formation assay was performed as previously described [16]. Cells (1 × 103 cells/well) were seeded onto 6-well plates. After being treated with HST or DMSO for 48 h, the cells were further cultured for about 10 days in drug-free complete medium. Meantime, the fresh medium was changed every 3 days. The colonies were fixed with formalin and stained with 0.25% crystal violet (Sigma-Aldrich, St. Louis, MO, USA). The colonies were photographed and counted using Image J Software.

2.6 RNA-sequencing (RNA-seq) analysis

RNA-seq analysis was conducted according to our previous literature [17]. HCT-8 cells were exposed with DMSO or HST (32 μM) for 48 h. Total RNA from cells was extracted according to qRT-PCR and prepared total RNA was then sent to Novogene Co., Ltd (Beijing, China) for sequencing and analysis. The NovoMagic tools (https://magic.novogene.com) were used for bioinformatic analysis.

2.7 Flow cytometry

Flow cytometry was used to measure the cell cycle distribution and cell apoptosis as we reported before [17]. In brief, for cell cycle distribution analysis, after treated with HST or DMSO for 48 h, cells were collected, fixed, and permeabilized with 75% ice-cold ethanol at 4 °C overnight. Then the cells were collected and stained with propidium iodide (PI) (50 μg/mL PI, 100 μg/mL RNase A and 0.5% Triton X-100) at 4 °C for 30 min in darkness. For cell apoptosis, the treated cells were collected and stained with Annexin V/PI solution. The cell cycle distribution and cell apoptosis were further analyzed using BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA).

2.8 RNA isolation and quantitative RT-PCR

qRT-PCR was performed as our previous publication [14, 17]. Total RNA was isolated from the cells treated with HST or DMSO for 48 h using TRIzol (Life Technologies, Carlsbad, CA, USA). cDNA was synthesized from 1 μg of total RNA using StarScript II First-strand cDNA Synthesis Mix (Genstar, Beijing, China). According to the manufacturer’s instructions, qPCR was further conducted with aliquots of cDNA samples mixed with 2 × RealStar Green Fast Mixture (Genstar, Beijing, China) using a CFX96™ Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA). GAPDH was used as the reference gene for normalization using the 2−△△Ct method. The sequences of primers were as follows:

p53, Fw 5′-TAACAGTTCCTGCATGGGCGGC-3′,

Re 5′-AGGACAGGCACAAACACGCACA-3′;

p21, Fw 5′-TGTCCGTCAGAACCCATGC-3′,

Re 5′-AAAGTCGAAGTTCCATCGCTC-3′;

MDM2, Fw 5′-AGTAGCAGTGAATCTACAGGGA-3′,

Re 5′-CTGATCCAACCAATCACCTGAAT-3′;

GAPDH, Fw 5′-CATGAGAAGTATGACAACAGCCT-3′,

Re 5′-AGTCCTTCCACGATACCAAAGT-3′;

2.9 Western blotting

Western blotting was conducted as our previous publication [18]. Briefly, cells were lysed in RIPA buffer containing 1% phosphorylation inhibitors and 1% protease. Total protein was extracted, quantified, and then separated by SDS-PAGE. After electro-transfer, the polyvinylidene fluoride (PVDF) membranes were blocked, incubated with appropriate primary antibodies and HRP-conjugated secondary antibodies, and then visualized using an ECL detection kit (Thermo Fisher Scientifific, Inc., Carlsbad, CA, USA) by a ChemiDoc XRS + System (Bio-Rad, Hercules, CA, USA). β-actin was used as the internal reference.

2.10 Monodansylcadaverine (MDC) staining

MDC staining was conducted as our previous publication to detect autophagic vacuoles induced by HST in CRC cells [18]. HCT-8 and SW620 cells were seeded in 6-well plates, exposed with HST or DMSO for 48 h, then collected and stained with MDC staining buffer (50 μM) at 37 °C for 1 h in darkness. The stained cells were washed and resuspended in Wash Buffer. The cell suspension was added to the slides and then observed by fluorescence microscopy (BX51, Olympus, Japan).

2.11 Co-immunoprecipitation (Co-IP) assay

Co-IP assay was performed as previously described [17]. Prepared cells were lysed in lysis buffer (Beyotime, Shanghai, China) supplemented with phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor cocktail at 4 °C for 30 min and pelleted by centrifugation (12,000 rpm, 20 min, 4 °C). The lysates were immunoprecipitated with p53 antibody-pretreated-beads (Protein A/G Magnetic Beads) (MCE, Princeton, NJ, USA) at 4 °C over night. Proteins were eluted from the beads and subjected to Western blot analysis as described above.

2.12 Chromatin immunoprecipitation (ChIP) assay

The SW620 cells (3 × 105 cells/mL) were seeded in 10 cm dishes, and treated with HST (32 µM) or DMSO for 48 h. ChIP assay was conducted by the ChIP Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocols. The cells were fixed with 1% formaldehyde at room temperature for 10 min followed by glycine solution treatment at room temperature for 5 min to stop the cross-link. The cells were collected and lysed in lysis buffer containing 1% PMSF. Cell lysates were sonicated using Bioruptor UCD-200 (Diagenode, Liège, Belgium) to obtain 200–1000 bp chromatin fragments. The sheared chromatin samples were precleared with protein A/G beads for 30 min before they were incubated with protein A/G beads and anti-p53 antibody (10442-1-AP, Proteintech, Hubei, China) at 4 °C overnight. Anti-rabbit immunoglobulin G (IgG) was used as a negative control. After extensive washing, the bead-bound immunocomplexes were eluted using elution buffer. To reverse the crosslinks, samples were treated with 5 M NaCl, heated at 65 °C for 4 h, then treated with protease K and further incubated at 45 °C for 1 h. The chromatin complex was purified by PCR purification kit. Then the purified immunoprecipitation DNA was used for qPCR, and the 2−△△Ct method was used to calculate the p53 occupancy rate on the p21 promoter. The primer sequences of p53 binding site in p21 promoter were as follows: Fw 5′-GTTCCCAGCACTTCCTCTCC-3′; Re 5′-GAAGCAGGCAGCATAGGGAT-3′.

2.13 Xenograft in nude mice

Four to Five-week-old Female BALB/c nude mice were purchased from the experimental Animal Center of the Tianjin Medical University (Tianjin, China) and maintained under pathogen-free conditions. All animal procedures were performed according to the requirements of animal welfare ethics and approved by the Laboratory Animal Management and Use Committee (IACUC) of Tianjin Medical University (TMUaMEC 2022041).

Xenograft in nude mice was performed as we reported before [19]. Mice were subcutaneously inoculated with CRC cells (1 × 107 cells). After successful tumor bearing (about 5 mm in diameter), mice (n = 5) were randomly divided into the control group and HST treated group and intraperitoneally injected with saline or HST (20 mg/kg) once every other day for totally 18 days. The tumor volume was measured with calipers every other day, and calculated using the formula: (width2 × length)/2. The body weight was measured every other day. The nude mice were sacrificed at day 18 after the first injection. The tumor tissues were excised, and fixed with 4% paraformaldehyde for immunohistochemical detection.

2.14 Hematoxylin–eosin (H&E) and immunohistochemical (IHC) staining

H&E and IHC staining were performed as reported [17, 19]. In brief, fixing, embedding, and slicing of the tumor tissues were conducted according to the classical procedures. For IHC staining, after deparaffinization using xylene, the slides were exposed with 3% hydrogen peroxide in methanol to quench the endogenous peroxidases, then blocked using bovine serum albumin (BSA) followed by an incubation with Ki-67 or p53 primary antibodies at 4 °C for 12 h, and then cultured with Bond Polymer (anti-rabbit poly-HRP-IgG) at room temperature for 1 h. Subsequently, the slides were treated with diaminobenzidine (DAB) peroxidase, counterstained with hematoxylin, and visualized under a microscope (BX51, Olympus, Japan).

2.15 Statistical analysis

Experiments were conducted in triplicate. Data from three independent repeats were presented. The results were expressed as mean ± SD. Statistical analyses were determined by Student’s t test and one-way ANOVA using GraphPad software. p < 0.05 was regarded statistically significant.

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