Animals and cancer cells
Normal and athymic Balb/c mice (male) were bought from Hua-Fu-Kang Bioscience (Beijing, China). When mice arrived, they were 4 or 5 weeks old and weighed 17−23 g. After acclimation, they were randomly designated to experimental groups. Throughout experiment, they lived in a room with 12 h/12 h light/dark cycle and had free access to chow and water.
We bought from the Cell Bank of Chinese Academy of Science (Shanghai, China) five human pancreatic cancer cell lines, i.e. AsPC1 (#CC2404), BxPC3 (#CC2405), HPAF-2 (#CC2407), MiaPaCa2 (#CC2408), and Panc-1 (#CC2401), as well as C2C12 mouse myoblasts (#CC9003). Unless indicated otherwise, all cells were cultured at 37°C in normoxia (95% air and 5% CO2), using RPMI-1640 media and Dulbecco modified Eagle’s media (DMEM) supplemented with foetal bovine serum (FBS, 10%), glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 μg/ml). Culture media and supplements were bought from the distributor of Gibco Thermo Fisher Scientific in Beijing (China).
Pancreatic cancer cells’ Warburg effect and cytokine secretion in vitro
AsPC1, BxPC3, HPAF-2, MiaPaCa2, and Panc-1 cells were cultured till 90% confluence. After rinsing with phosphate buffered saline, these cells were cultured in serum-free media for 6 h in normoxia or hypoxia (1% O2, 5% CO2, 94% N2) [13]. After whole-cell proteins were extracted, glucose transporter-1 (Glut1), hexokinase-2 (HK-II), and phosphofructokinase-1 (PFK-1) were determined by Western blot. Glucose, lactate, TNF-α, IL-1β, and IFN-γ were assayed in removed media. Rongsheng Life Pharmacological (Shanghai, China) and Jiancheng Bio-engineering (Nanjing, China) produced the kits for the glucose and lactate assays. Human TNF-α, IL-1β, and IFN-γ were determined, using ELISA kits from Four-A Biotech (Beijing, China).
Transplantation of MiaPaCa2 and AsPC1 cells in athymic mice
MiaPaCa2 and AsPC1 cells were suspended in RPMI-1640 media and transplanted subcutaneously in athymic mice (3 × 106 cells/mouse), giving a group of MiaPaCa2-cell carriers (n = 10) and a group of AsPC1-cell carriers (n = 13). Intact athymic mice were used as normal controls (n = 14). In the next 8 weeks, food intake and body weight were recorded on a weekly basis. In the end of week 8, all mice were anesthetized, using 5% chloral hydrate. Blood was collected from the orbital sinus and centrifuged (1500 x g, 10 min, 4°C) to obtain plasma. After mice were killed by cervical dislocation, subcutaneous tumour and inguinal fat pads were removed and weighed. Skeletal muscle was removed from hind legs. The abdominal cavity was opened, epididymal fat pads were removed and weighed, and the liver was sampled. Plasma and tissue samples were kept at − 80°C.
Treating normal mice with the supernatants of MiaPaCa2 or AsPC1 cells
MiaPaCa2 and AsPC1 cells were cultured in different Petri dishes (diameter = 10 cm) till 90% confluence. Then, the cells were incubated in 15 ml serum-free RMPI-1640 medium for 24 h under normoxic conditions. The media that were conditioned by MiaPaCa2 and AsPC1 cells, respectively, were collected. The media were centrifuged to remove debris and then were saved for experiment.
Normal Balb/c mice were divided in three groups (6 mice per group). Then, they were subjected to subcutaneous injection (0.5 ml, twice a day) of normal control medium or the media that were conditioned by MiaPaCa2 and AsPC1 cells, respectively. After 7 days, all mice were sacrificed as in the preceding experiment.
In a follow-up experiment, normal Balb/c mice were divided in three groups. Mice in two groups (10 mice per group) were subjected to subcutaneous injection of normal control medium or the MiaPaCa2-cell conditioned medium as in the preceding experiment (0.5 ml, twice a day). The mice in the third group (n = 11) were subjected to subcutaneous injection of an increased amount of the MiaPaCa2-cell conditioned medium (1.0 ml, twice a day). After 7 days, all mice were sacrificed as described before.
Incubating skeletal-muscle cells with media conditioned by MiaPaCa2 cells
C2C12 mouse myoblasts were cultured in 6-well plates, using DMEM containing 10% FBS. When cells were 95% confluent, they were cultured for 48 h in DMEM with 2% horse serum so as to differentiate to skeletal-muscle cells. Then, the cells were incubated for 4 h in normal control medium or in the medium that was conditioned by MiaPaCa2 cells. Intracellular atrogin-1 and myosin (heavy chain) were determined by Western blotting.
Western blots
We performed Western blots to determine Glut1, HK-II, PFK-1, pyruvate carboxylase (PCB), glucose-6-phosphatase (G-6-Pase), LMF, PIF, atrogin-1, muscle ring finger-1 (MURF1) protein, myosin (heavy chain), insulin-like growth factor binding protein (IGFBP)-3, and adipose triglyceride lipase (ATGL). β-Actin and β-tubulin were used as loading controls. Santa Cruz Biotechnology (Santa Cruz, CA) produced the antibodies for HK-II (#6521), PFK-1 (#377346), LMF (#11238), G-6-Pase (#27196), PCB (#43228), and β-actin (#47778). Abcam (Cambridge, UK) produced the antibodies for Glut1 (#115730) PIF (#52138), MURF1 (#172479), myosin (#124205), and ATGL (#3370–1). ECM Biosciences (Versailles, KY), R&D Systems (Minneapolis, MN), and Proteintech (Chicago, IL) produced the antibodies for atrogin-1 (#AP2041), IGFBP3 (#MAB305), and β-tubulin (#66240–1), respectively.
Tissue samples were homogenized with a mechanical homogenizer, and whole-cell proteins were extracted using RIPA lysis buffer. When proteins were extracted from cultured cells, the lysis buffer was used in the first place. Protein samples were separated in polyacrylamide gel, transferred to polyvinylidene difluoride membrane, and incubated with a primary antibody at 4°C overnight. After rinsing, the membrane was incubated with a secondary antibody at room temperature for 1 h. Specific blotting was visualized, using an enhanced ECL detection kit.
Other assays
Plasma glucose and lactate were determined, using aforementioned kits. Plasma triglycerides were determined, using a kit produced by Jiancheng Bioengineering (Nanjing, China). To determine plasma TNF-α and IL-6, we used an ELISA kit for mouse TNF-α (#E02T0008, Bluegene Biotech, Shanghai, China) and an ELISA kit for mouse IL-6 (DKW12–2060, Dakewei Biotech, Shenzhen, China). When insulin-like growth factor-1 (IGF-1) was determined in skeletal muscle, we used an ELISA kit produced by Elabscience Biotechnology (Wuhan, China). Hepatic glycogen was determined using a kit produced by Jiancheng Bioengineering (Nanjing, China).
Statistics
Data are mean ± SEM. To evaluate difference in groups, we employed the analysis of variance followed with Bonferroni or student-Newman-Keuls post-hoc test. The computer programs of Statistical Product and Service Solutions (version 17.0) and Graph-Pad Prism (version 5.01) were used. P < 0.05 was considered statistically significant.