Caspase 7

Que was purchased from Merck. Lecithin and cholesterol were purchased from Aladdin. DSPE-PEG2000 was purchased from Biomatrik. A549 cells and Beas-2B cells were derived from National Collection of Authenticated Cell Cultures. Enzyme-Linked Immunosorbent Assay (ELISA) detection kits were used from Shanghai Enzyme-linked Biotechnology Co., Ltd. The fetal bovine serum and DMEM for cell culture were purchased from Gibco. The antibodies of Bcl-2, caspase-3 and caspase-7 were purchased from Proteintech Group, Inc. Antibodies against TGF-β, ROCK, Rho and α-SMA were purchased from Abcam.

The relative protein expression levels of IL-1β, IL-6, ARPC3, ARPC4, HSPA1A, HSPA1L, Bax, Bcl-2, Caspase7 (proteintech, Wuhan, China), TNF-α, Caspase3, and Caspase8 (Bioss, Beijing, China) in the mammary gland tissues and MAC-T were measured using Western blot. Total protein was extracted from mammary gland tissue/cells using cold RIPA buffer (Solarbio, Beijing, China). Primary antibodies were incubated overnight at 4 °C, followed by incubation with corresponding secondary antibodies (goat anti-rabbit IgG Secondary antibody, SAB, Greenbelt, MD, USA; goat anti-mouse IgG Secondary antibody, SAB, Greenbelt, MD, USA) at 37 °C for 1 h. Band densitometry was quantified and analyzed using Image-J 1.52a.

Tianma Gouteng Decoction from Gastrodia elata 10 g, Uncaria 15 g (later fried), abalone shell 20 g (first fried), Gardenia 6 g, Scutellaria baicalensis Georgi 6 g, achyranthes root 12 g, Eucommia ulmoides Oliv. 12 g, Leonurus japonicus 12 g, Loranthus parasiticus 12 g, dried caulis of polygoni multiflori 12 g, and Poria cocos 12 g. All of the above ingredients were provided by the Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. TGD is concentrated into granules; each 1 g granule is equal to 10 g of raw materials and complies with the guidelines of Good Manufacturing Practices and Good Laboratory Practices formulated by Chinese government agencies. The TGD was dissolved in pure water to prepare solutions having a concentration of 200 mg/mL or 400 mg/mL for the experiment.
Saline was purchased from China Otsuka Pharmaceutical Co., Ltd. (Tianjin, China). The enzyme-linked immunosorbent assay (ELISA) kits, including prostacyclin (PGI2), thromboxane A2 (TXA2), angiotensin II (Ang II), and endothelin 1 (EDN1), were all obtained from Uscn Life Science, Inc. (Wuhan, China). The primary antibodies of GAPDH, AKT, p-AKT, caspase 8, caspase 7, caspase 3, and secondary antibodies were purchased from Proteintech Inc. (IL, USA). The antibodies of OPG, TRAIL, and DR5 were purchased from Abcam PLC (Cambridge, UK).

Cell lysates were obtained from 1×10⁶ cultured cells with RIPA protein extraction reagent. The protein concentration was determined by bicinchoninic acid assay (BCA). Equal amounts (30μg) of total protein lysate were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membrane. The membranes were then blocked in 5% non-fat milk at room temperature for 1 hour. Following this treatment, the membranes were probed with primary antibodies against human P62 (1:1000 dilution, Cell Signaling Technology, USA), P21 (1:1000, Proteintech), YBOX3 (1:1000, Cell Signaling Technology, USA), Histone 3 (1:1000, Proteintech), GAPDH (1:1000, Abcam, USA), CD63 (1:1000, Proteintech), CD9 (1:1000, Proteintech), Caspase 9 (1:1000, Proteintech), Cleave Caspase 9 (1:1000, Proteintech), Caspase 3 (1:1000, Proteintech), Cleave Caspase 3 (1:1000, Proteintech), Caspase 7 (1:1000, Proteintech), Cleave Caspase 7 (1:1000, Proteintech), Beclin1 (1:1000, Proteintech), Atg5 (1:1000, Proteintech), LCII (1:1000, Proteintech), LCIII (1:1000, Proteintech) at 4°C overnight. Following incubation in a solution of goat anti-rabbit (1:1000, Santa Cruz, USA) or anti-mouse IgG (1:5000 for both; Santa Cruz, USA) at room temperature for 1hour, the membranes were washed with TBST and then detected with enhanced chemiluminescence reagents (Thermo Fisher Scientific, Inc.). The bands were visualized using 1-step TM NBT/BCIP reagents (Thermo Fisher Scientific, Rockford, IL, USA) and detected by the Alpha Imager (Alpha Innotech, San Leandro, CA, USA).

Reagents and Antibodies. CBD was purchased from Push (Chengdu, China). The initial concentration of CBD powder dissolved in absolute DMSO was 51 mM and stored at −20 °C in the dark for later use. When using CBD for follow-up experiments, the DMSO concentration needed to be kept below 1‰. Poly-(lactide-co-glycolic acid-resomer) (PLGA, Mw: 24,000–38,000 Da, i.v. 0.25–0.35 dL/g) was obtained from Daigang (Jinan, China). Polyvinyl alcohol 1788 (PVA, Mw = 44.05) was purchased from Aladdin (Aladdin, Shanghai, China). RPMI-1640, DMEM, and DMEM/F12 medium were obtained from Gibco (Thermofisher, Shanghai, China). Fetal bovine serum (FBS) was supplied by BI (Israel, South America). Antibiotic-antimycotic solution and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from Solarbio (Shanghai, China). In situ cell death detection kit and RIPA buffer were purchased from Beyotime (Shanghai, China). Antibodies against PI3K (1500 μg/mL, 1:10000), Akt (2504 μg/mL, 1:3000), phospho-Akt (1683 μg/mL, 1:6000), mTOR (500 μg/mL, 1:10000), phospho-mTOR (1000 μg/mL, 1:1000), Bcl-2 (1000 μg/mL, 1:2000), Bax (1600 μg/mL, 1:4000), Cytochrome-c (1000 μg/mL, 1:4000), Caspase 7 (700 μg/mL, 1:1000), Erk1/2 (1000 μg/mL, 1:6000), phospho-Erk1/2 (370 μg/mL, 1:3000), MMP9 (400 μg/mL, 1:600), MMP2 (1000 μg/mL, 1:600), and β-actin (427 μg/mL, 1:5000) were purchased from Proteintech (Shanghai, China). Phospho-PI3K (690 μg/mL, 1:1000) was purchased from Abcolonal (Shanghai, China). HRP-conjugated secondary antibodies were purchased from Proteintech. HPLC-grade methanol and acetonitrile, were obtained from Macklin (Shanghai, China).
Cell Culture. Human T24, 5637, UM-UC-3, and SV-HUC-1 cell lines were supported by Sun Yat-Sen memorial hospital (Sun Yat-Sen University), and their biological characteristics are presented in Table S1. T24 cells and 5637 cells were cultured in RPMI-1640 medium. UM-UC-3 cells was cultured in DMEM medium, and SV-HUC-1 cells was cultured in DMEM/F12 medium. All media were supplemented with 10% FBS, and 1% Antibiotic-antimycotic solution. Cells were cultured at 37 °C under a humidified atmosphere of 5% CO₂.
Cell Viability Assay. The effect of CBD on the viability of T24, UM-UC-3, 5637, and SV-HUC-1 cells were evaluated quantitatively by the MTT assay. First, 100 µL of the cell suspension was seeded at a density of 2 × 10⁴ cells per well in 96-well culture plated and incubated overnight. Subsequently, the medium was changed, each sample well was added with 200 µL volume of different concentrations of CBD (0 and 1‰ DMSO, 10, 15, 20, 25, 30, 40, 50 µM) at 37 °C in CO₂ incubator for 48 h. Then, MTT reagent (20 µL of 5 mg/mL) was added into each well and incubated at 37 °C for an additional 4 h. Afterward, the supernatant was carefully removed, and the formed formazan crystals were dissolved by adding 150 µL DMSO to each well. The plates were shaken for 10 min at room temperature; absorbance at 490 nm was measured by using the automatic microplate reader (BioTek, Winooski, Vermont, USA).
Colony Formation Assay. One thousand cells were seeded in 6-well plates in complete medium. The next day the media were changed to fresh medium containing CBD, and after culturing for 48 h, the fresh medium was replaced, and changed every 3 days. After 2 weeks, the medium was discarded, and the cells were washed twice with PBS. The cells were fixed with 4% paraformaldehyde for 15 min, and then the fixing solution was discarded. The cells were then stained with 1‰ crystal violet for 10 min and rinsed with running water slowly. The plates were then observed for the formation of colonies.
Wound-healing Assay. Cell migration was assessed in bladder cancer cells using a wound healing assay. When the density reached 100% confluence, the wound was scratched with a sterile 200 μL pipette tip in the confluent monolayer at the center of culture plates, followed by three washes with PBS. Cells were incubated in medium supplemented with 0.1% (v/v) FBS in the presence or absence of CBD or vehicle. Images of the scratches were captured at 0, 6, 12 and 24 h to visually assess cell migration distance. The migratory distance was detected using Image J software (V1.8.0.112, National Institutes of Health, Bethesda, USA).
Hoechst 33258 Staining Assay. The nuclear morphological changes in CBD-treated bladder cancer cells were evaluated using the Hoechst 33258 stain (Solarbio, Shanghai, China). Briefly, equal number of cells was seeded in 6-well plates overnight. Cells were then washed twice with PBS and stained for 10 min with 10 µg/mL Hoechst 33258 at 37 °C in the dark after treating the cells with CBD or vehicle for 48 h. Then dyeing medium was removed and the wells were washed twice more with PBS, and the nuclear morphology of the cells was observed under a fluorescence microscope (Olympus, Tokyo, Japan).
Terminal Deoxynucleotidyl Transferase (TdT) dUTP Nick-End Labeling (TUNEL) Assay. TUNEL assay was used to detect apoptosis and was performed using in situ Cell Death Detection Kit (Beyotime, Shanghai, China). Bladder cancer cells were seeded in confocal dishes at 37 °C. After 24 h of culture, culture medium containing 12 µM of CBD was added and further cultured for 48 h in the incubator. Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min. Cells were then permeabilized with PBS containing 0.3% Triton X-100 at room temperature for 5 min. After washing the cells three times with PBS, TUNEL reaction mixture from the assay kit was used to co-incubate with cells for 1 h at 37 °C in a 5% CO₂ incubator. The dishes were washed with PBS and stained with 10 µg/mL Hoechst 33258 for 10 min at 37 °C. These cells were observed by confocal microscopy (Olympus, Tokyo, Japan).
Apoptosis Assay. The Annexin V-FITC/Propidium Iodide (PI) (Elabscience, Wuhan, China) double staining assay was performed according to the manufacturer’s manual. Briefly, bladder cancer cells were seeded at a density of 5 × 10⁵/well in 6-well plates overnight and then stimulated by 1‰ DMSO or 12 µM CBD as the vehicle control or experimental group. After treating for 48 h, the cells and culture medium were harvested using trypsin-EDTA and centrifuged at 500 rpm for 5 min at room temperature. The supernatant was discarded, and pellet was resuspended in PBS. Cell suspension was centrifuged under the same condition. The supernatant was discarded and resuspended by adding 100 µL of 1× binding buffer. Then, 2.5 µL of Annexin V-FITC and PI staining solution were added to untreated and treated cell suspension, mixed well, and incubated for 15 min at room temperature without light. Finally, 100 µL of 1× binding buffer was added to the suspension again. Fluorescence intensity was measured using the Beckman flow cytometer (Beckman Coulter, Inc.250 S.Kraemer Boulevard Brea, CA 92821, USA), and the apoptotic rates of CBD-treated cells were analyzed by using FlowJo software (FlowJoV10, Becton, Dickinson & Company, New York, NY, USA).
RNA Sequencing (RNA-seq) Assays. The extracting RNA from CBD-treated cells (Figure S3) were used to RNA-seq. The transcriptome libraries were constructed by Shanghai Majorbio Technology Co., Ltd (Shanghai, China). All quality inspection data were presented in Table S2. The mRNA expression data was uploaded to the DEseq2 software website (https://login.majorbio.com/login, accessed on 7 September 2021). To identify differential expression genes (DEGs) between two different samples, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) functional-enrichment analysis was performed to identify which DEGs were significantly enriched in metabolic pathways at Bonferroni-corrected p value ≤ 0.05 compared with the whole-transcriptome background. KEGG pathway analysis was conducted by Goatools (https://github.com/tanghaibao/Goatools, accessed on 7 September 2021) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do, accessed on 7 September 2021).
Quantitative Real-time PCR Analysis. Total RNAs extraction kit, the First-Strand cDNA synthesis kit, and qRT-PCR kit were purchased from Promega (Shanghai, China). All the indicated samples were normalized to actin and then relative mRNA levels were calculated by using the comparative Ct method. The qRT-PCR primers (Table S3) were synthesized by Sangon (Shanghai, China).
Western Blotting Analysis. Cells were lysed by Radio Immunoprecipitation Assay (RIPA) buffer with 1 mM PMSF to obtain the proteins for Western blotting. The BCA Protein Assay Kit (Sangon, Shanghai, China) was used to quantify the protein concentration. Subsequently, Western blotting experiments were performed in accordance with the previously reported laboratory procedures [25 (link)].
Preparation of CS-coated CBD-loaded PLGA Nanoparticles. Bernhard Brauner et al. [26 (link)] had been confirmed that PLGA nanoparticles (PLGA NPs) are more suitable than PLGA microparticles for instillative therapy. Hence, following a solvent evaporation technique, PLGA NPs loaded with CBD were prepared from an oil-in-water emulsion. Briefly, PLGA (25 mg) and CBD were dissolved in 2 mL of mixed reagent (V_acetone:V_{absolute ethanol} = 4:1). The oil phase was added dropwise to 20 mL of a 1% (w/v) PVA aqueous solution under stirring at 800 rpm in a magnetic stirrer for 5 h to allow solvent evaporation. Then, the NPs were collected by centrifuge and washed with 10 mL of ultrapure water three times in order to eliminate remnants of PVA, namely CBD-loaded PLGA NPs (CBD/PLGA NPs). Next, samples were resuspended in 10 mL ultrapure water and subsequently poured into equal volume of 0.16% (w/v) CS solution and stirred at 800 rpm in a magnetic stirrer for 2 h at room temperature. Finally, 500 µL ultrapure water was added as a cryoprotectant, and samples were frozen at −80 °C overnight and freeze-dried for 24 h using a freeze dryer, namely CS-coated CBD-loaded PLGA NPs (CS-CBD/PLGA NPs).
For the internalization and adhesion experiments, DiI (Beyotime, Shanghai, China) was used as the fluorescent agent, DiI-loaded PLGA NPs (DiI/PLGA NPs) and CS-coated DiI-loaded PLGA NPs (CS-DiI/PLGA NPs) were prepared using the aforementioned protocol.
Characterization of CS-CBD/PLGA NPs. The morphologies of NPs (Blank PLGA NPs, CBD/PLGA NPs and CS-CBD/PLGA NPs) were observed using transmission electron microscope (TEM) (JEM-1400, JEOL, Tokyo, Japan). For TEM examination, the lyophilized NPs were dissolved and placed onto formvar-coated copper grid and negatively stained with phosphotungstic acid. The size distribution, zeta potential, and PDI of NPs were measured by dynamic light scattering technique (DLS) (Malvern, Malvern city, UK). Each sample was measured in triplicate.
Determination of EE. The lyophilized NPs (CBD/PLGA NPs and CS-CBD/PLGA NPs) were dissolved in 1 mL DMSO for 6 h and then were centrifuged for 30 min at 4 °C at 14,000 rpm. The supernatant was collected and determined by high performance liquid chromatography (HPLC) (Agilent 1200 series, Agilent Technologies Inc., CA, USA ) equipped with a reverse-phase C18 (250 × 4.6 mm, 5 µm) column. The mobile phase consisting of acetonitrile (A) and water (B) was used for separation of CBD through isocratic elution at a flow rate of 1 mL/min and an injection volume of 10 µL. CBD detection and quantification wavelength was set at 220 nm. The EE was calculated using the following formula.
Determination of Fourier Transform Infrared (FTIR) Spectroscopy. FTIR spectra of samples homogeneously mixed with KBr and compressed into discs were traced in the range of 400 to 4000 cm⁻¹, using FTIR spectrophotometer (Shimadzu, Kyoto, Japan).
Stability Studies. The different formulations were stored at 4 and 25 °C for a period of 5 weeks. Samples were taken after 0, 2, and 5 weeks and the following physicochemical properties were examined, including particle size and encapsulation efficiency (EE,%).
In Vitro Assay of Drug Release Studies. Briefly, 10 mg of NPs were dispersed in 1 mL of phosphate buffered saline (PBS containing 1% (v/v) Tween 80) with different pH (i. e., 5.0 and 6.5) and then incubated at 37 °C at 100 rpm. At predetermined time intervals, microcentrifuge tubes containing CBD-NPs in PBS were centrifuged at 12,000 rpm for 10 min at 4 °C, and then collected 100 µL clear solution of supernatants to analyze by HPLC. The release medium was replaced by fresh dissolution medium at each interval; 100 µL samples were then withdrawn and replenished with fresh media.
In Vitro Assay of Mucoadhesion Studies. The adsorption–association of mucin with the particles was used as a method to assess mucoadhesive properties of the particles prepared [27 (link)]. Here, 2 mL of mucin suspension (0.5 mg/mL) and 2 mL of 2 mg/mL NPs dispersion (Blank PLGA NPs and CS-PLGA NPs) was mixed (vortexed) and incubated in a shaker at 37 °C at 100 rpm for 2 h. The mixtures were then centrifuged at 14,000 rpm for 30 min, and the supernatant was collected and used for the measurement of free mucin.
Bradford reagent is an effective means to detect the concentration of free mucin to further evaluate the amount of mucin adsorbed on the NPs. According to the Bradford standard curve, 10 µL of all samples (of known and unknown mucin concentration) are mixed with 190 µL Bradford reagent, and then used to detect the absorbance at a wavelength of 595 nm. Finally, the mucin content of the samples was calculated and determined.
In Vivo Assay of Mucoadhesion Studies. Mucoadhesion test was performed according to the previously described method by Martin et al. [28 (link)]. Mice were anaesthetized by intraperitoneal injection of phenobarbital sodium. External genitalia were cleansed and urine in their bladders were emptied. The bladders were then filled with PBS with or without CS-modified, DiI-loaded NPs (2 mg/mL DiI/PLGA NPs and CS-DiI/PLGA NPs) for 2 h. Mice were sacrificed, and bladders were washed extensively with PBS to remove non-adherent NPs. Samples were frozen, sliced, and captured with CLSM.
Cellular Uptake Analysis. T24 cells with density of 5 × 10⁵ cells/well were grown on confocal dishes overnight. A volume of 100 µL NPs including DiI/PLGA NPs and CS-DiI/PLGA NPs was incubated for 2 h and 6 h at 37 °C. Then, the excess NPs were washed by fresh PBS for three times. The cells were fixed by 4% paraformaldehyde for 15 min at room temperature, washed three times with PBS, and then incubated with 10 µM Hoechst 33258 dye for 5 min. After three washes with PBS, cells were captured by confocal laser scanning microscope (CLSM, Nikon, Shanghai, China).
Statistical Analysis. All data are presented as the mean ± standard deviation (SD) of the mean following analysis with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Statistical significance values were evaluated through one-way ANOVA test with post hoc contrasts by Student–Newman–Keuls test, or part of the data were conducted by Student’s t test, using SPSS 22.0 software for evaluation. p < 0.05 was defined as statistically significant difference.