Reagents and chemicals
Commercial dihydromyricelin (DHM, C15H12O8, analytical purity, CAS: 27200-12-0) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). DHM was dissolved into dimethyl sulfoxide (DMSO) to obtain a stock solution with a concentration of 200 mM, and stored at -20℃ for further applications. DMSO, absolute ethanol, methanol, crystal violet, paraformaldehyde (PFA) and paraffin were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). RPMI-1640 and DMEM high glucose medium, fetal bovine serum (FBS), penicillin, streptomycin, and trypsin were purchased from Thermofisher Scientific Co., Ltd. (Waltham, MA, USA). The other chemical and biological reagents were used as received.
Bladder cancer cell lines
The muscle invasive bladder cancer (MIBC) cell lines (T24 and UMUC3) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). T24 cell was maintained in RPMI-1640 complete medium supplied with 10% FBS and 100 U/mL penicillin/streptomycin solution. UMUC3 cell was maintained in DMEM high glucose complete medium containing FBS and antibiotics. T24 and UMUC3 cells were both cultured using a 37℃ humid incubator with 5% CO2.
Cell viability and proliferation assay
In this study, MTT assay was performed to determine the appropriate DHM drug concentration for further in vitro evaluations. Briefly, T24 or UMUC3 cells were digested using 0.25% trypsin solution, and then centrifuged at 1200 rpm for 5 min. DHM stock solution with a concentration of 200 mM was diluted using complete medium until the desired DHM concentration (0, 5, 10, 20, 30 µM) was achieved. DHM working solutions were used to re-suspend cell precipitation, and adjust cell concentration to 1.5 × 104 cells/mL. 200 µL cell suspension was added into each well of 96 well plates. After incubated at 37℃ for 48 h, 20 µL MTT reagent was added, followed by incubation for another 4 h. After that, all liquids in the 96 plates were thoroughly wiped out, and 150 µL DMSO was added into each well. The value of optical density (OD) at a wavelength of 490 nm was detected using a microplate reader (SpectraMax@M2, MD, USA). The relative cell viability and 50% inhibiting concentration (IC50) were calculated [23]. To further assess the proliferation ability of MIBC cells, T24 and UMUC3 cells were co-incubated with DHM working solution (0, 5, 20 µM) for successive 5 days. At regular time intervals, the treated cells were taken out of the incubator, and the value of OD490 was detected. Six independent samples were calculated for statistical comparison.
Clonogenic survival assay
T24 and UMUC3 cells were collected and re-suspended into DHM working solutions (0, 5 and 20 µM). These cells were seeded onto 6 well tissue culture plates with cell density of 800 cells/well, and then cultured for 12 days. 4% paraformaldehyde (PFA) solution was added to fix the cells. After that, 1 mL 0.1% crystal violet solution was applied to visualize the cell clone. The images of cell clones were captured using a digital camera (A7RⅢ, Sony, Japan), and the number of cell clones was counted from at least three independent sample.
Transwell chamber assay
In this work, transwell chambers (Millipore, Corning, USA) were used to investigate the migration ability of MIBC cells[24]. Briefly, DHM stock solution (200 mM) was diluted using FBS-free basic medium to 0, 5, 20 µM, respectively. The obtained solutions were suspended with T24 or UMUC3 cells with a concentration 4 × 105 cells/mL. 0.1 mL cell suspension was added into the upper layer of transwell chamber, and 0.7 mL complete medium was added into the lower layer. After incubated at 37℃ for 24 h, the un-migrated cells on the upper chamber layer were removed carefully using a cotton swab. The migrated cells were fixed with 4% PFA solution for 30 min, and then stained with 0.1% crystal violet solution for 15 min. The cell images were captured by an inverted fluorescence microscope (IX73, OLYMPUS, Japan), the IPP-6.0 software was used for quantitative analysis.
Wound healing assay
T24 and UMUC3 cells were seeded onto 6 well tissue culture plates with high cell density, and then cultured overnight until the cell confluence reached to 80%. A linear cell wound was created using 200 µL pipette tips. After washed twice by PBS solution, the culture plates were filled with DHM working solutions with a concentration of 0, 5, 20 µM, respectively. At regular time intervals, three representative images of wound site were captured and analyzed using an inverted fluorescence microscope (IX73, OLYMPUS, Japan).
Flow cytometric analysis
Cell cycle
The cell cycle analysis was carried out in accordance with our precious report [25]. T24 and UMUC3 cells were co-incubated with DHM working solutions (0, 5, 20 µM) for 48 h, and then collected into 1.5 mL centrifuge tubes. The obtained cells were washed using PBS solution for three times. Cell cycle staining kit was purchased from Multi Sciences Co., Ltd. (Hangzhou, China). 1 mL DNA staining solution and 10 µL permeabilization solution was added into each tube. After incubated in the dark place for 30 min, the cell cycle was detected using a flow cytometry (CytoFLEX, Beckman, USA). At least three independent samples were tested in each group.
Cell apoptosis
MIBC cells were treated with DHM working solutions (0, 5, 20 µM) for 48 h, and then collected into 1.5 mL tubes for apoptosis staining. The Annexin V-FITC/PI apoptosis kit was obtained from BD Biosciecnes Co., Ltd. (San Jose, USA). The DHM treated cells were re-suspended in the 1 × binding buffer, and then stained with Annexin V-FITC for 15 min and PI for another 10 min. The apoptosis rate of all samples was detected using a flow cytometry (CytoFLEX, Beckman, USA). At least three independent samples were tested in each group.
Phenotypic characterization
T24 cells were incubated with DHM working solutions (0, 5, 20 µM) for 12 h, and the supernatant was collected for phenotypic characterization. Human monocytic leukemia cell line (THP-1) was seeded in 6 well tissue culture plates with a density of 1 × 106 cells/well. After incubated with the supernatant for 24 h, the THP-1 cells were collected and treated with CD11b/c-APC, CD86-FITC, CD80-PE. The stained cells were then analyzed using a flow cytometry. The percentage of positive cells indicated the expression degree of the surface markers, and calculated from three independent samples [26].
Quantitative real-time PCR
According to previous report [27], the total RNA molecules from the treated MIBC cells were isolated using a HiPure RNA Mini Kit (Magen, China). The RNA concentration was then measured using an ultraviolet spectrophotometer (Nanodrop 2000, Thermo, USA). The reverse transcription reaction was carried out, and the obtained cDNA was used for quantitative real-time PCR (qRT-PCR). The iQTM SYBR®Green Supermix was purchased from Bio-Rad Co., Ltd. (Shanghai, China) and applied for qRT-PCR. Primer sequences used in this work are listed in Table S1.
Western blot and immunofluorescence (IF) staining
T24 and UMUC3 cells were incubated with DHM working solutions (0, 5, 20 µM) for 48 h before they were harvested for western blot analysis. The treated cells were completely lysed in 1 mL RIPA buffer containing 20 µL phosphatase and protease inhibitor mixture. The lysis supernatant was collected after centrifuged at 1.2 × 104 g for at least 15 min. The total protein concentration was evaluated using a BCA protein assay kit (Abcam, China). The protein samples were denatured and separated by 7.5–15% SDS - PAGE gels, and then transferred to a PVDF membrane (Millipore, USA). The PVDF membranes were blocked using 5% fat-free milk for 2 h and incubated with primary antibodies overnight and secondary antibodies for 2 h. The primary and secondary antibodies used in this work are listed in Table S2. The enhanced chemiluminescence kit was purchased from BD Biosciecnes Co., Ltd. (San Jose, USA). The protein bands were detected using a ChemiDoc™ MP Imaging System (Bio-Rad, USA). IF staining of Ki67 protein were performed according to the general protocols, and accomplished by Biofavor Biotech Co., Ltd. (Wuhan, China). The cell images of IF staining were captured by an inverted fluorescence microscope (IX73, OLYMPUS, Japan).
In vivo xenograft experiments
This work was carried out in accordance with the declaration of Helsinki. Six BALB/C57 nude mice, aging about 3 weeks, were purchased from WQJX Bio-Technology Co., Ltd. (Wuhan, China). These animals were quarantined in SPF experimental facility for 7 days to adapt the new environment.
200 µL T24 cells (1.5 × 107 cells/mL in PBS) were subcutaneously injected into the mice. The animals were fed for 15 days before DHM drug injection. DHM was firstly dissolved in absolute alcohol with a concentration of 10 mg/mL, and then diluted using normal saline to prepare the DHM working solution. DHM working solution was injected at a dose of 20 mg/Kg every third day for 21 days. For blank control, animals were injected with normal saline containing equivalent alcohol. Tumor size of the treated animals was measured using a vernier caliper, and then used to calculate the tumor volume (mm3) as AL× AW2× 0.5, where AL and AW refers to the length and width of the tumors, respectively. After sacrificing the animals, all tumor samples, blood samples and organs samples including heart, liver, spleen, lung, kidney and brain, were harvested for further tests [28].
The tumor samples and organs were fixed with 4% paraformaldehyde (PFA) solution for 48 h. These samples were embedded by paraffin and then sliced into 4 µm sections. Hematoxylin-eosin (HE) and Masson’s trichrome (MT) staining, immunofluorescence (IF) staining of Ki67, and TUNEL assay were performed according to the general protocols. An inverted fluorescence microscope (IX73, OLYMPUS, Japan) was used for data acquisition.
The concentration of alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), creatinine (CRE), and total bilirubin (TBIL) was detected. Commercial kits were purchased from BOSTER Biotechnology Co., Ltd. (Wuhan, China). The blood samples were centrifuged at 3000 rpm for 5 min, and then diluted using distilled water. The test was performed according to the manufacturers’ protocols. At least three samples were used for statistical analysis
Statistical analysis
The data from no less than three independent biological evaluations were expressed as mean ± standard deviation. One-way ANOVA as well as post hoc Tukey’s test was applied for statistical analysis. p < 0.05 was considered to be statistically different.