Cell culture
Cervix (HeLa), and colon (HCT116 and SW480) carcinoma cell lines, and mouse Kras-V12 carcinogenic NIH-3T3 (NIH-3T3Kras) (a gift from Dr. Capella, IDIBELL, Spain) were cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillin, 10 g/ml streptomycin, and 2 mM L-glutamine (all from Biological Industries, Israel) and incubated in a humidified atmosphere of 5% CO2 at 37 °C.
For experiments, unless stated otherwise, cells were grown in DMEM media with different glucose concentrations: 25 mM glucose (high glucose), 1 mM glucose (glucose exhaustion), and 0 mM glucose (glucose deprivation). Media were supplemented with 10% FBS, 100 units/ml penicillin, 10 g/ml streptomycin, and 2 mM or 4 mM (glucose deprivation) L-glutamine.
RNA extraction and quantitative RT-PCR
Total RNA was extracted with the TRIsureTM RNA isolation system (Bioline, Memphis, TN, USA) and transcribed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). QRT-PCR was performed using the TaqMan gene expression assay and 7900HT Real-Time RT-PCR system (Applied Biosystems, Carlsbad, CA, USA). Data were analyzed by the ∆∆Ct method to normalize with TBP and GUSB expression.
Western blot
Cells were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors and centrifuged at 15,000 g for 15 min at 4 °C. Protein concentration was determined using the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA), and equal amounts of protein (20–30 μg) were subjected to 8–12% SDS-PAGE and transferred to an Immobilon membrane (Millipore, Bedford, CA, USA). Blots were treated with primary antibodies, followed by the corresponding secondary antibody with horseradish peroxidase activity. Blots were developed using Pierce ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA) in a Fujifilm LAS 3000 Intelligent Dark Box IV imaging system (Tokyo, Japan).
The following primary antibodies were used: anti-PEPCK-M (ab70359, Abcam, Cambridge, UK), anti-PEPCK-C (generous gift of Dr. Daryl K. Granner, Vanderbilt University, Nashville, TN, USA), anti-SOD2 (ab13534, Abcam), anti-p53 (ab26, Abcam), anti-p21 (sc397, Santa Cruz Biotechnology, Dallas, TX, USA), anti-gamma tubulin (T6557, Sigma-Aldrich, St. Louis, MO, USA), and anti-PKC-ζ (9372S, Cell Signaling Technology, Danver, MA, USA).
Transduction
Protocols were performed as recommended by the manufacturer. For PCK2 knockdown, HeLa cells were infected with GIPZ Lentiviral TurboGFP shRNAs (Dharmaco, Lafayette, CO, USA; clone IDs: V3LMM_427490 and V3LHS_328126) and denominated sh1-PCK2 and sh2-PCK2, respectively. GIPZ non-targeting lentiviral TurboGFP shRNA (Dharmacon; clone ID RHS-4348) was used to produce negative control cells denominated shCtrl. After transduction, cells were selected with 1 μg/ml puromycin for 1 week. For overexpression of PCK2, HeLa cells were infected with a PCK2 Human ORFeome lentiviral particles (GeneCopoeia, Rockville, MD, USA; clone ID: LP-OL06695-LX304-0200-S) and denominated L-PCK2. Cells were selected with 2 μg/ml blasticidin for 1 week.
For PKC-ζ knockdown, HeLa and SW480 cells were infected with GIPZ Lentiviral TurboGFP shRNAs (Dharmacon; clone IDs: V3LHS_635000, V3LHS_372773, V3LHS_641464) and denominated shPKCζ #37, shPKCζ #63, and shPKCζ #64, respectively. The negative control, shPKCζ #Ctrl, was produced by infecting HeLa cells with GIPZ non-targeting lentiviral TurboGFP shRNA (Dharmacon; clone ID: RHS-4348). After transduction, cells were selected with 1 μg/ml puromycin for 1 week.
Establishment of PCK2 knockout SW480 cell line with CRISPR/Cas 9 system
To generate a pool of SW480 cells lacking PCK2 (PCK2 CRISPR/Cas9 KO), guide RNAs (gRNA) designed to target PCK2 were synthesized, and annealed and cloned into the pSpCas9(BB)-2A-puro vector (Adgene, Watertown, MA, USA) as described previously [11], using an online gRNA design tool (CHOPCHOP; https://chopchop.cbu.uib.no). After 24 h post-transfection, puromycin was added for 24 h at 2 μg/ml for selection and subsequently single cells were selected in 96-well plates. The selected cells were tested for gene deletion by endonuclease assay and checked for protein knockdown by Western blot.
PEPCK-M enzymatic activity
Cell extracts from confluent 150 cm2 tissue culture dishes were washed twice in PBS, trypsinized, and centrifuged at 150 g for 3 min at 4 °C. Cells were resuspended in 200 μl of ice-cold homogenization buffer (100 mM HEPES-NaOH pH 7.2, 0.1% triton™ X-100, 2.5 mM DTT) and lysed by performing 2 freeze/thaw cycles. Homogenates were cleared by centrifugation at 100000 g for 1 h at 4 °C. PEPCK-M activity was measured in the direction of phosphoenolpyruvate formation. Briefly, the reaction consisted of 100 mM of HEPES-NaOH (pH 7.2), 3 mM malic acid, 3 mM NAD, 2 mM MgCl2, 0.2 mM MnCl2, 37 mM DTT, 6 U/ml MDH, and the reaction was started by the addition of 0.2 mM of GTP. The amount of produced NADH is proportional to PEPCK activity. Reads were measured at 340 nm at 37 °C in a total volume of 1 ml using a DU® 800 spectrophotometer (Beckman Coulter, Brea, CA, USA).
Immunohistochemistry
Tissue microarray panel (BCN962, Biomax, Rockville, MD, USA) containing multiple organ carcinoma and adjacent normal tissue was deparaffinized and rehydrated according to standard procedures. Antigen retrieval was performed by heating the slide in 10 mM sodium citrate buffer (pH 6) in a pressure cooker. The highest pressure was maintained for 3 min, and samples were let to cool down for 20 min. Endogenous peroxidase activity was inactivated by incubating samples in 6% H2O2 for 15 min.
Samples were blocked with 20% goat serum in PBS and then incubated ON with primary antibody against PEPCK-M (ab70359, Abcam) and peroxidase-based secondary anti-goat antibody. Antigen-antibody complexes were detected with a DAB peroxidase substrate kit (Dako Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol. Samples were counterstained with hematoxylin, dehydrated, and mounted with DPX. Fluorescent preparations were visualized, and images were captured with Nikon Eclipse 800 light microscope (Nikon, Tokyo, Japan).
MTT assay
To assess cell viability, 0.5 mg/ml of MTT (M2128, Sigma-Aldrich, St. Louis, MO, USA) diluted in DMEM without phenol red was added to each well, and plates were incubated at 37 °C, 5% CO2 for 2 h. The formazan product was dissolved in isopropanol, and the absorbance of samples was measured using a microplate reader at a wavelength of 570 nm with background subtraction at 650 nm.
Soft agar colony formation assay
Anchorage-independent growth was determined by plating 5000 cells in 1 ml of 0.35% agarose in 6 well plates. 0.7% agarose was mixed in a 1:1 ratio with DMEM media, supplemented with 10% FCS, 2 mM glutamine, and 1 mM or 25 mM glucose, respectively. A layer containing cells was overlaid on 0.5% agar in mQ water. Cells were fed with corresponding DMEM media and refed every 3–4 days. After 2 weeks, colonies were stained with MTT and counted.
MitoSOX staining
MitoSOX™ Red mitochondrial superoxide indicator (M36008, ThermoFisher Scientific, Waltham, MA, USA) was used to measure the production of superoxide in mitochondria. Cells were treated with 5 μM MitoSOX™ Red in HBSS/Ca/Mg for 15 min at 37 °C and 5% CO2 protected from light. Cells were washed with HBSS/Ca/Mg. Subsequently, cells were trypsinized, resuspended in corresponding DMEM media without phenol red, and analyzed by flow cytometry with Gallios™ flow cytometer (Beckman Coulter).
Metabolomics
Cells were plated in 6-well plates at 0.25 × 106 cells/well and grown overnight in a growth medium. Next day, cells were washed with PBS and pre-treated with medium lacking glucose for 3 h and glucose-deprived medium supplemented with dialyzed FCS (dFCS) and 2 mM uniformly labeled [U-13C]glutamine was added for 4 h. In the case of high-glucose media (25 mM glucose), cells were pretreated with fresh complete DMEM media for 3 h and subsequently treated with DMEM media supplemented with dFCS and 2 mM [U-13C]glutamine for 4 h. At the end of cultivation, cells were washed twice with cold PBS, snap-frozen with liquid nitrogen, and harvested using an aqueous solution of MeOH (MeOH (80%)/H2O (20%)). The concentration of metabolites was analyzed by using mass spectrometer API 3200 triple quadrupole LC-MS/MS. Enrichment studies were analyzed by using GC/MS spectrometry (GC/MS Agilent 5975C). Proline enrichment and concentration of TCA cycle intermediates were analyzed in cells harvested in 1.8 ml of cold methanol/chloroform (2:1, v:v) and analyzed by using GC/MS spectrometry.
Glucose assay
Glucose concentration in media was determined by colorimetry assay using a glucose oxidase and peroxidase method as recommended by manufacturer PGO (P7119, Sigma-Aldrich, Darmstadt, Germany). Absorbance was measured at 450 nm after 30 min of incubation at 37 °C.
Xenograft models
5 × 106 of SW480 WT or SW480 PCK2 CRISPR/Cas9 KO was injected in both flanks of female 5–6-week-old BALB/c nude mice (at least n = 3 per group). Similarly, 1 × 106 of HeLa shCtrl, sh1-PCK2, or sh2-PCK2 was injected in two mammary fat pads of female 5–6-week-old BALB/c nude mice (at least n = 8 per group). Tumor volume was measured by calipering in two dimensions and calculated as [(short length × 2) × long length)]/2. After 15 days (SW480) or 23 days (HeLa), mice were euthanized by cervical dislocation, and the tumors collected for further analysis. All the animal studies were approved by the local committee for animal care (IDIBELL, DAAM 5766).
Statistical analysis
Results are expressed as mean ± SEM. Statistical analysis was performed by one-way or two-way Anova (Sidak post hoc test) or unpaired two-tailed Student’s t test, using GraphPad Prism® software. Significance levels are one symbol = p < 0.05, two symbols = p < 0.01, and three symbols = p < 0.001.