Polyphillin D
Polyphyllin D (PD) is a diosgenyl alpha-rhamnopyranosyl-(1→2)-[(alpha-l-arabinofuranosyl-(1→4)-β-d-glucopyranoside, with a molecular weight
of 855.02 Da and molecular structure of C44H70O16 [8] (Funakoshi Co. Ltd., Tokyo, Japan) (Fig. 1). For the preparation, we dissolved in dimethyl sulfoxide (100 mg/mL) and stored at -80°C for up to 2 weeks.
Cell culture
Human neuroblastoma cell lines IMR-32 and LA-N-2 were obtained from Sumitomo Dainippon Pharma (SD Pharma, Osaka, Japan). IMR-32 cells were cultured in Eagle's minimal essential medium (EMEM; SD Pharma) supplemented with 1% nonessential amino acids (SD Pharma), 2 mM glutamine, and 10% fetal bovine serum (FBS). LA-N-2 cells were cultured in a 1:1 mixture of Ham's F-12 (SD Pharma) and EMEM, supplemented with 2 mM glutamine and 10% FBS. All cells were incubated at 37°C in 5% CO2 and were passaged using 0.25% trypsin-ethylenediamine tetra acetic acid (EDTA) (Life Technologies, Carlsbad, CA, USA) at 70–80% confluency. All cell lines were mycoplasma-free.
Animals
Total 24 four-week-old male CAnN.Cg-Foxn1nu/CrlCrlj (BALB/c nude mice) mice were purchased from the Jackson Laboratory Japan INC (Kanagawa, Japan). Before the experiments, the mice were allowed to acclimatize for at least three days. They were housed in plastic cages with sterilized bedding in an air-conditioned room at 22°C ± 1°C and 51% ± 4% humidity with a 12 h light/12 h dark cycle and with ad libitum access to filtered water and total nutrient feed. Experiments were performed in accordance with the national guidelines for animal care and use. The present study was approved by the Ethics Committee of the Fujita Health University Hospital (AP21008, APU19055).
Each BALB/c nude mouse was subcutaneously injected with 3.0 × 10 × 6 IMR-32 and LA-N-2 cells (in phosphate-buffered saline) through the back using a 26-gauge needle. The tumor volume was measured after the subcutaneous tumor was visible. The tumor volume was calculated as follows: tumor volume = 4/3π × L/2 × W/2 × H/2. The experiment was started when the tumor volume reached 130 mm × 3. The experimental period was 14 days, and tumor volume was measured every 2 days. In the model, polyphyllin D (8 mg/kg) was orally administered daily [12], while in the non-polyphyllin D groups 200 µL distilled water (DW) was administered once a day. There were 6 mice each in the DW and polyphyllin D groups. After 14 days, all the mice were sacrificed and used for analysis. The discontinuation criteria was when the mouse body weight loss was 10% or more, and maximum tumor diameter exceeded 20 mm.
Construction of the tissue
The specimens were fixed in 4% paraformaldehyde at 4°C for 48 h. Subsequently, a paraffin block was prepared using Tissue-Tek VIP 5 Jr. (Medical equipment Kisiya Co., Ltd., Fukuoka, Japan The tissue specimens were sliced into 3 µm-thick sections and kept in a paraffin oven at 60°C overnight.
Hematoxylin and eosin (HE) staining
After deparaffinization with xylene, the sections were stained with hematoxylin and eosin (H&E; Muto Chemicals). Pathological changes in the tissues were observed under a light microscope.
Immunohistochemistry (IHC) staining
Paraffin-embedded neuroblastoma blocks were used for immunohistochemical analyses. The BOND staining protocol (Leica Biosystems, Germany) was used for all experiments. The samples were deparaffinized, heated in citrate buffer solution (pH 6.0) or EDTA (pH 8.0) to unmask the antigens, and were washed with PBS. A peroxidase-labelled polymer-conjugated anti-rabbit/mouse IgG antibody was used as the labeled polymer reagent. A 3,3'-diaminobenzidine/hydrogen peroxide solution was used to determine the peroxidase activity. Counterstaining was performed with hematoxylin. The primary antibodies used were anti-Ki-67 (1:100, Gene Tex, North America, USA), anti-RIPK3 (1:100, Abcam, Cambridge, UK), anti-MLKL phospho (ser358) (1:100, Arigo Bio, Taiwan, ROC), and Phospho-PDPK1 (Tyr376) Polyclonal Antibody (1:100, Thermo Fisher Scientific, Massachusetts, USA). The secondary antibody used was Simple Stain MAX PO (1:100; Nichirei Biosciences Inc., Tokyo, Japan). The number of tumor foci per tumor area was determined using Ki-67 staining.
Image analysis
The slide photography consisted of an Olympus BX-43 high-resolution digital camera and a digital camera (Olympus) with Net Cam software (Olympus America, Center Valley, PA, USA). An 11-megapixel cooled digital color camera (Olympus DP-70) (Olympus America, Center Valley, PA, USA) was used to capture images on a Dell (Round Rock, TX, USA) desktop computer directly connected to a microscope (Olympus BX-43) (Olympus America, Center Valley, PA, USA) [13].
Protein Extraction and Western Blot (WB)
Cell lysates were extracted from paraffin-embedded neuroblastoma blocks using the Formalin-Fixed Paraffin-Embedded Protein Isolation Kit (ITSI Biosciences, Pennsylvania, USA) and Phosphatase Inhibitor Cocktail IV (Biovision, Inc., Waltham, MA, USA). The bicinchoninic acid method (Dual Range BCA Protein Assay Ki, Visual Protein, Taiwan, ROC) was used to measure the extracted proteins.
A Jess™ Simple Western automated nano-immunoassay system (Protein Simple, San Jose, CA, USA, Bio-Techne Brand) was used. Horseradish peroxidase (HRP), peroxide/luminol-S, and secondary HRP antibodies were used according to the manufacturer's instructions. The primary antibodies used were anti-MLKL phospho (ser358) (1:10, Arigo Bio, Taiwan, ROC) and Phospho-PDPK1 (Tyr376) Polyclonal Antibody (1:10, Thermo Fisher Scientific, Massachusetts, USA). A Compass Simple Western software (version 5.0.1, Protein Simple) (Protein Simple, San Jose, CA, USA, Bio-Techne Brand) was used to automatically calculate the heights (chemiluminescence intensity), area, and signal/noise ratio and capture them as digital images.
Statistical analysis
All statistical analyses were performed using the JMP 12.2 (SAS Institute Inc., Cary, NC, USA) software. Clinical and histopathological data were analyzed using the Shapiro-Wilk test and t-test. Statistical significance was set at p < 0.05.