Animals
The healthy adult male nude mice (6 week-old weighting 20 to 25 g) were provided by Hunan SJA Laboratory Animal Co., Ltd (License number: SCXK (Xiang)2016-0002). The animals were fed in SPF animal house of Shenzhen University Health Science Center. Each mouse was housed in a single cage to prevent fighting and had access to food and water ad libitum. The mice were maintained at a constant temperature (23 ± 2°C) and humidity with a 12-h light–dark cycle and were acclimatized to the environment for at least a week before the experiments. All procedures, care, and handling of the rats were approved by the Ethics Committee of Shenzhen University Health Science Center.
Cell lines, Antibodies and Reagents
Human colon cancer cell line HCT-116 (BNCC339915) was purchased from BeNa Bio Technologies Inc (Beijing, China). Complete DMEM medium (KGM12800S-500) and phosphate buffer saline (PBS) (KGB5001) were purchased from KeyGEN BioTECH (Nanjing, China). Ketogenic feed was purchased from Medicience Biomedicine Co., Ltd. (Yangzhou, China). Trypsin-EDTA solution (T1300), eosin staining solution (G1100) and Scott blue/blue liquid (G1865) were purchased from Solarbio Life Sciences (Beijing, China). Hematoxylin Staining Solution (ZLI-9610), mouse anti-GAPDH Monoclonal antibody (TA-08), HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (ZB-2301) and HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (ZB-2305) were purchased from ZSGB-Bio Technologies Inc (Beijing, China). One-step TUNEL apoptosis detection kit (C1088) was purchased from Beyotime Bio Technologies Inc (Shanghai, China). Rabbit anti-beta catenin polyclonal antibody (51067-2-AP) and rabbit anti-Wnt1 polyclonal antibody (27935-1-AP) were purchased from Proteintech (Wuhan, China). Rabbit anti-Bcl-2 polyclonal antibody (A11025) and rabbit anti-survivin monoclonal antibody (A19663) were purchased from ABclonal (Wuhan, China). Rabbit anti-Bax monoclonal antibody (ab32503), rabbit anti-caspase 3 monoclonal antibody (ab197202) and rabbit anti-caspase 9 monoclonal antibody (ab32539) were purchased from Abcam (Cambridge, UK). DAB color development kit (CW0125), neutral resins (CW0136), Ultrapure RNA extraction kit (CW0581M) and TRIzon reagent (CW0580S) were purchased from Cwbio Inc (Beijing, China). HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (R223-01) was purchased from Vazyme Biotech Co.Ltd (Nanjing, China). The 2×SYBR Green PCR Master Mix (A4004M) was purchased from Lifeint Technologies Inc (Xiamen, China). Matrigel (354248) was purchased from Corning BD Biosciences (Mississauga, ON, Canada). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Colon cancer xenograft models
Subcutaneous tumor model of human colon cancer in nude mice was established according to the method described previously [24]. In brief, human colon cancer HCT-116 cells were cultured in DMEM medium containing 10% fetal bovine serum and growth to 90% confluence, then the cells collected by digesting with trypsin and suspended to a concentration of 5 × 106 cells/ml with a mixture of Matrigel and PBS at a ratio of 1:1. Subsequently, the above HCT-116 cell suspension was subcutaneous injected under the skin in the right flank of nude mice (6 week-old male nude mice weighing 20–25 g) with an amount of 0.1 ml/mouse. The growth of tumors was monitored weekly with rulers by measuring the size. The mice were randomly divided into two groups (Normal diet group and ketogenic diets group) once the tumor size reached 0.5cm3. The mice in normal diet group were fed with standard mouse feed daily, and the mice in KD group were given ketogenic diets every day. The body weights of nude mice were recorded every two days. After 30 days of ketogenic diet or normal diet, the mice of each group were euthanized by CO2 inhalation and the subcutaneous tumors were obtained and applied to the following experiments, such as the measurement of tumor size or histopathological examination.
Histopathological examination of tumor
The histological method was used to detect the pathologic changes of subcutaneous tumors and performed as described previously [25]. Briefly, mice from normal diet group and ketogenic diets group were euthanized by CO2 inhalation on day 30 after ketogenic diets or normal diets. The subcutaneous tumors were harvested and immediately fixed in 4% paraformaldehyde, then embedded with paraffin, and cutted into 3 μm thick slices. The sections were deparaffinized, rehydrated and stained with hematoxylin and eosin (H&E) successively. Subsequently, the sections were washed with flowing water, then dehydrated with high concentrations of ethanol and transparentized with xylene, sealed with neutral resins and the histopathological alterations observed under light microscopy.
TUNEL Assay
The apoptosis of tumor cells was determined by TUNEL assay via using a commercial One-step TUNEL apoptosis detection kit (Beyotime) according to the manufacturer’s instructions. Briefly, the paraffin embedded tumor sections were deparaffinized with xylene, then rehydrated with a series of concentrations of ethanol. The sections were removed to a wet box and the antigen retrieval was performed by dropping 50 μg/ml of Proteinase K working buffer on the sections and incubated at 37 °C for 30 min. Then the sections were washed three times with PBS and the residual PBS around the tissue sections was removed with absorbent paper. 50 μl of freshly prepared TUNEL test solution (5 μl of TdT enzyme with 45 μl of fluorescence-labeled solution) was added to each tissue section and incubated at 37°C in the dark for 1 h. After incubation, the sections were washed three times with PBS and the residual liquid around the tissue sections was removed with absorbent paper. Then the tissue sections were mounted with anti-fluorescence quenching mounting solution and observed under a fluorescence microscope. The excitation wavelength is 450-500nm and the emission wavelength is 515-565 nm, the green fluorescence can be observed.
Immunohistochemistry
Immunohistochemistry was applied to detect the expression of Wnt1/β-catenin signaling proteins of subcutaneous tumors and performed according to a method described previously [26]. Briefly, the above prepared tumor sections were deparaffinized with xylene and rehydrated with a series of concentrations of ethanol, then antigen repair was performed with 10 mM citrate buffer at pH 6.0. Subsequently, the endogenous peroxidase blocking solution was eliminated by incubating the sections with fresh prepared 3% hydrogen peroxide solution at room temperature for 10 min. Then the sections were washed three times with PBS and blocked in 5% BSA at 37 °C for 30 min to prevent nonspecific antigen-antibody binding. Subsequently, the diluted anti-Wnt1 antibody (1:300) and anti-β-catenin antibody (1:200) were added to the sections and incubated at 4 °C overnight. After washed three times with PBS, the sections were incubated with diluted peroxidase-conjugated goat anti-rabbit IgG (H+L) secondary antibody (1:100) at 37 °C for 30 min. The sections were wash three times with PBS and color development with 3,3 N-Diaminobenzidine Tertrahydrochloride (DAB) for 5-10 min. After washed with PBS, the sections were redyeing with hematoxylin for 3 min, differentiated with hydrochloric acid ethanol differentiation solution and back to blue. At last, the sections were washed, dehydrated, transparentized, mounted and observed under light microscopy.
Real-time quantitative PCR (RT-qPCR)
Real-time quantitative PCR (RT-qPCR) was applied to detect the mRNA expression of caspase 3 and caspase 9 of subcutaneous tumors, which performed as described previously [27]. In brief, the total RNA extraction and cDNA synthesis of subcutaneous tumors were performed by using the commercial kits according to the manufacturer’s instructions. The specific primers of caspase 3 and caspase 9 were designed by Oligo 7 software and listed in Table 1. The reaction volume of RT-qPCR was 25μl including 1 μl of cDNA template, 1 μl of forward primer, 1 μl of reverse primer, 12.5 μl of 2×SYBR Green PCR Master Mix and 9.5 μl of RNase Free dH2O. The RT-qPCR reaction condition as follows: predenaturation at 95°C for 10 min,denaturation at 95 °C for 10 sec, annealing at 58 °C for 30 sec and elongated at 72 °C for 30 sec, 40 cycles. GAPDH was as the control. The relative expression of caspase 3 and caspase 9 in the transcript levels were analyzed by 2-△△Ct method.
Western Blot
The total proteins of tumors in nude mice were shredded, ground to powder in the presence of liquid nitrogen, and lysed by protein lysate containing protease inhibitor cocktail on ice bath for 30 min. Then the lysate was centrifuged at 12000 rpm/min for 15 min, the collected supernatant was total proteins. The protein concentrations were determined by a commercial BCA quantitative kit according to the manufacturer’s instructions. The protein electrophoresis was performed by 12% SDS−PAGE and electrotransferred onto polyvinylidene difluoride membranes. After electrotransferred, the PVDF membrane was blocked in 3% BSA at room temperature for 1 h and incubated at 4°C overnight with diluted anti-caspase 3 antibody (1:1000), anti-caspase 9 antibody (1:1000), anti-Bax antibody (1:1000), anti-Bcl-2 antibody (1:1000), anti-survivin antibody (1:1000), anti-Wnt1 antibody (1:1000), anti-β-catenin antibody (1:5000) and anti-GAPDH antibody (1:2000), respectively. After washed three times with TBST, the membrane was incubated with HRP-conjugated goat anti-rabbit secondary antibodies (1:5000) at room temperature for 2 h. The protein bands were visualized with the SuperSignal WestPico chemiluminescence substrate.
Statistical analysis
All experimental values were presented as the Mean ± SD. Each experiment was repeated at least 3 times independently. Statistical analysis of data was performed using Graphpad Prism 7.0 software. One-way ANOVA was used to analyse the differences between more than two groups, and non-paired student’s t test was used to analyse the difference between two groups. P <0.05 was considered statistically significant.