Animals and subgroups
Forty-eight 7-week male C57BL/6 mice were purchased from the Chinese Academy of Science Laboratory Animals Center in Shanghai, China. All mice were housed in standard cages with 12:12-h light-dark cycle, and free access to water and food. Mice were randomly assigned to the following four groups (n=12 in each group): control (normoxia) group (CTL), control plus TSA group (CTL+TSA), IH group, and IH+TSA. The body weight of mice in each group was measured each week. This protocol was approved by the ethics committee in Zhongshan Hospital, Xiamen University (approved number: 2017-015), and conducted in accordance with the Guide for the Care and Use of Laboratory Animals . All measure were taken to minimize the number of animals used and to reduce the pain of animals.
intermittent hypoxia exposure
The protocol of IH exposure was performed according to our previous study[22, 23] . Briefly, the mice in the IH and IH+TSA groups (n=24) were subjected to a self-made plexiglass chamber with one-way valves, and a programmable instrument regulated the times of the three gases (oxygen, nitrogen, compressed air) flowing to the chamber, making the oxygen concentration in the chamber fluctuating from 21% to nadir 6-8%. The cycle time of hypoxia and re-oxygenation is 120 seconds. The IH exposure was conducted from 8 AM to 4 PM daily for 5 consecutive weeks.
Cells culture, tumor induction and tumor measurement
Lewis lung carcinoma (LLC) cells (CoBioer Biosciences Co., Ltd. Shanghai, China) were cultured in accordance to the instruction of the manufacturer. Briefly, LLC cells were maintained at high glucose DMEM and supplemented with 10% fetal bovine serum (GIBCO, USA). A total of 1×106 LLC cells with 100μl PBS were injected into the right flank of each mouse after one week of IH exposure. When the tumor was palpable, the width (W) and length (L) was detected with an electric caliper every week. Tumor volume (V, mm3) was calculated as W2×L/2.
When tumor volume growing to about 200mm3 ( nearly 5-7 days after LLC injection), mice in the CTL+TSA and IH+TSA groups received TSA (purchased from Shanghai No.1 Biochemical & Pharmaceutical Company, Shanghai, China) intraperitoneally injection (10mg/kg) daily according to previous studies[24-27], while those in the CTL and IH groups intraperitoneal injected saline with equal volume.
After 5 weeks of experimentation, all mice were deeply anesthetized with pentobarbital and exsanguinated by cardiac puncture and plasma obtained. Tumors were excised, weighted, either frozen in liquid nitrogen and stored at -80℃ for further analysis, or fixed in buffered 10% formalin for histological examination.
Oxidative stress measurement
Malondialdehyde (MDA) and superoxide dismutase (SOD), two of the most commonly used indicators of oxidative stress were assayed with relevant kits according to the instructions of manufactures (Beyotime, Beijing, China). Briefly, frozen tumor tissues were homogenized in cold PBS (10% W/V). After lysis for 15min on ice, homogenates were centrifuged and then supernatants were obtained for further detection. MDA levels in the homogenates were determined spectrophotometrically by measuring the presence of thiobarbituric acid-reactive substances. SOD was detected by an assay kit which employs a thiazole salt for the detection of superoxide anions by producing a colored product. The levels of MDA and SOD were reported as the absorbance at a wavelength of 535nm and 560nm, respectively. The enzyme activity is reported as units/mg of protein.
Tumor tissues were homogenized with RIPA buffer (Beyotime, Beijing, China) containing protease and phosphatase inhibitor in a glass homogenizer. After centrifuged, the supernatants were extracted and detected the total protein concentrations with a bicinchoninic acid protein assay (Beyotime, Beijing, China). Equal amounts of boiled protein (40ug/lane) were subjected to 12% sodium dodecyl sulfate-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). After blocking with 5% (W/V) skim milk, the membrane was incubated with the following antibodies at 4℃ overnight: rabbit anti-Bax (1:1000, Cell Signaling Technology, USA), rabbit anti-Cleaved Caspase-3 (1:1000, Cell Signaling Technology, USA), rabbit anti-nuclear factor erythroid 2-related factor 2 (Nrf2) (1:2000, Abcam, USA), rabbit anti-NF-kB (1:1000, Cell Signaling Technology, USA) and mouse anti-b-actin (1:2000, Santa Cruz Biotechnology, USA). After a rinse with Tris-buffered saline+Tween-20 for 3 times, the PVDF membranes were incubated with goat anti-rabbit or mouse IgG-HRP at 37℃ for 1 hour. The membranes were developed and exposed using an enhanced chemiluminescence kit (Clarity™ Western ECL Substrate, Bio-Rad, USA). The experiment was repeated in triplicate of each mouse. The band densities on the membranes were estimated using the Image J analysis software (National Institutes of Health, Bethesda, MD, USA).
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay
Tumor tissues were fixed in buffered 10% formalin for 24 hours, dehydrated in graded alcohol series, cleaned with xylene, and then embedded in paraffin. Embedded tissues were sectioned to 5μm per slices. The sections were washed in PBS and incubated with TUNEL reaction solution at 37℃ for 1 hour using the apoptosis detection kits (Roche, China). After washing with PBS, then incubating with 4’, 6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA) for 5 min, the sections were analyzed and photographed with a microscope (SPII2-AOBS Leica) at 400× magnification. Both TUNEL-positive cells and total cells in 5 sections of each group were counted respectively. The apoptosis rate was calculated as following: TUNEL-positive cell number/total cell number× 100%.
GraphPad Prism software 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was conducted to analyze the data. All data are presented as mean ± standard deviation and compared using an analysis of variance (ANOVA) followed by Fisher's exact test. A p value of less than 0.05 indicated statistical significance.