Expression of the Niemann-Pick C1-like 1 Protein in Gastric Cancer

Materials and Methods

Tissue samples. For this study, GC tissue samples were subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemical analyses. For the qRT-PCR analysis, Human Multiple Tissue cDNA Panel I and II (cat. no. 636742 and 636743; Takara-bio Co., Shiga, Japan) were used to represent normal human tissue; Human Digestive System Multiple Tissue cDNA Panel (cat. no.: 636746; Takara-bio Co.) was used to represent normal human digestive organ tissues; and TissueScan Gastroesophageal Cancer Tissue qPCR Panel I (cat. no.: HGRT101; OriGene Technologies, Rockville, MD, USA) was used to represent human GC tissue.

For the immunohistochemical analysis, 70 primary tumor samples were collected from patients who were diagnosed with GC and underwent gastrectomy procedures between 2005 and 2011 at Gifu University Hospital (Gifu, Japan). Another 25 primary tumor samples collected from patients diagnosed with GC who underwent gastrectomy procedures in 2014 at Gifu Municipal Hospital (Gifu, Japan) were also analyzed. Tumor staging was determined according to the Tumor, Node, Metastasis (TNM) classification system (10). Histologic classification was performed according to the Japanese Classification of Gastric Carcinoma (11). The surgery types included distal, cardiac, or total gastrectomy. All of these were resections performed with curative intent. After each surgery, follow-up and treatment procedures for recurrence were based on GC treatment guidelines (12). Blood tests (including tumor markers) were performed every three months, and computed tomography (CT) was performed every six months for three years postoperatively. After three years, blood tests and CT scans were performed every six months postoperatively. Patients were followed by their physician until death or the date of the last documented contact. Esophagogastroduodenoscopy procedures were performed annually. Patients who experienced recurrence during follow-up were treated via chemotherapy. This study was approved by the review board of the Gifu University Hospital (approval no.: 2021-B134).

Cell lines and cell culture. Five cell lines derived from human gastric cancer (GC) (MKN-1, MKN-7, MKN-45, MKN-45/F2R, and TMK-1) were used. Three cell lines (MKN-1, MKN-7, and TMK-1) were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). The MKN-45 cell line (a poorly differentiated GC cell line) was kindly provided by Hiroshima University, Japan. All cell lines were cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS; both obtained from Wako Pure Chemical Industries Ltd., Osaka, Japan) and sodium pyruvate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). MKN-45/F2R cell line is resistant to 5-FU and was established by continuously exposing the cells to increasing concentrations (0.1-2 μM) of 5-FU over one year, as was described in a previous study (13). The 5-FU used for this study was kindly provided by Kyowa Hakko (Tokyo, Japan). Prior to the study, the resistant cells were cultured in drug-free RPMI-1640 supplemented with 5% FBS for ≥2 weeks to eliminate the effects of 5-FU exposure for the experiments. During the study, they were routinely maintained in RPMI-1640 supplemented with 5% FBS and 2 μM 5-FU. Both cell lines cultured in this study were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

Microarray analysis. To search for candidate genes related to 5-FU resistance, cDNA microarray analysis was performed. Genes whose expression levels were consistently higher in 5-FU resistant cell lines than those in parental cells under three different conditions were selected. Cells were plated on 10-cm petri dishes at 1.0×10⁶ cells in a medium containing 10% FBS. Cells were harvested before 5-FU administration, 24 h after administration of 2 μM 5-FU, or 72 h after administration of 2 μM 5-FU. This was performed for the 5-FU resistant MKN-45/F2R cell lines and the MKN-45 parental cell lines, and a total of six patterns of cells were obtained. Total RNA was extracted with a RNeasy Mini kit (Qiagen, Valencia, CA, USA). Microarray analysis was outsourced to Genetic Biolab Co., Ltd. (Sapporo, Hokkaido, Japan).

Chemosensitivity assay. Cells were plated in 96-well plates at 1.0×10⁴ cells per 200 μl of medium containing 10% FBS. After 24 h of incubation at 37°C, the medium was replaced with fresh medium containing or not containing different concentrations of 5-FU. The number of viable cells was counted using a hemocytometer after 72 h.

qRT-PCR analysis. PCR was performed using TB Green Premix Ex Taq II (Takara-bio Co. Ltd). Real-time detection of the emission intensity of TB green bound to double-stranded DNA was performed using a Thermal Cycler Dice Real Time System (Takara-bio Co. Ltd). NPC1L1 primers (oligo: HA171345-F/R; Takara-bio Co.) and GAPDH primers (oligo: HA067812-F/R; Takara-bio Co.) were used for PCR. GAPDH-specific PCR products were amplified from the same cDNA samples to serve as an internal control.

Transfection and small interfering RNA experiments for NPC1L1. We used two independent small interfering RNA (siRNA) oligonucleotide sequences targeting NPC1L1 messenger RNA, as well as a negative control (Life Technologies Inc., Carlsbad, CA, USA). MKN-45 cells and MKN-45/F2R cells were cultured in RPMI-1640 supplemented 5% FBS without antibiotics for 24 h to 50-70% confluency prior to transfection. The cells were transfected with siRNA oligonucleotides using Lipofectamine RNAiMAX (Life Technologies Inc.) in serum-free Opti-MEM (Life Technologies Inc.) at a final siRNA concentration of 10 nM and analyzed 48 h after transfection.

Immunohistochemistry. Antigen retrieval treatment was performed in an autoclave using 0.01M citrate buffer (pH 6.0) for 10 min. Endogenous enzymes were blocked using 3% hydrogen peroxide (H₂O₂) for 10 min. Non-specific protein blocking was performed using Blocking One (cat. no.: 03953-95; Nacalai tesque, Kyoto, Japan) for 10 min. Sections were incubated with the primary rabbit polyclonal anti-NPC1L1 antibody (cat. no.: GTX30675; 1:500; Gene Tex Inc., Irvine, CA, USA) overnight at room temperature. They were then incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (heavy + light chains) goat polyclonal secondary antibody (cat. no.: 424144; HOSOFINE; Nichirei Biosciences, Tokyo, Japan) for 30 min. Visualization was performed following staining with 3,3′-diaminobenzidine, tetra-hydrochloride (DAB; Sensitive DAB Substrate Kit; cat. no.: 10-0048; Genemed Biotechnologies Inc., South San Francisco, CA, USA) for 1 min. The sections were counterstained with Mayer’s hematoxylin.

Tissue specimens were considered positive if the cancer cells at the most invasive fronts exhibited >10% staining, which was stronger than that of non-tumorous gland cells. Two pathologists (C.S. and T.T.), who were blinded to patients’ prognosis, performed the immunohistochemical staining and summarized the evaluations of the tumor samples.

Statistical methods. Statistical analysis was performed using JMP v14.2 (SAS Institute Inc., Cary, NC, USA). Associations between clinicopathological factors and NPC1L1 expression were analyzed using Fisher’s exact test. Kaplan–Meier survival curves were constructed for the NPC1L1-positive and NPC1L1-negative patients, and the survival rates were compared between these two groups. Differences between the survival curves were tested for statistical significance using the log-rank test. Inhibitory concentration 50% (IC₅₀) values were calculated using the Microsoft Excel 2010 software program (Microsoft Corporation, Redmond, WA, USA).