Loss of Caspase-3 sensitizes colon cancer cells to genotoxic stress via RIP1-dependent necrosis

Caspase-3 is the best known executioner caspase in apoptosis. We generated caspase-3 knockout (C3KO) and knockdown human colorectal cancer cells, and found that they are unexpectedly sensitized to DNA-damaging agents including 5-fluorouracil (5-FU), etoposide, and camptothecin. C3KO xenograft tumors also displayed enhanced therapeutic response and cell death to 5-FU. C3KO cells showed intact apoptosis and activation of caspase-7 and -9, impaired processing of caspase-8, and induction of necrosis in response to DNA-damaging agents. This form of necrosis is associated with HMGB1 release and ROS production, and suppressed by genetic or pharmacological inhibition of RIP1, MLKL1, or caspase-8, but not inhibitors of pan-caspases or RIP3. 5-FU treatment led to the formation of a z-VAD-resistant pro-caspase-8/RIP1/FADD complex, which was strongly stabilized by caspase-3 KO. These data demonstrate a key role of caspase-3 in caspase-8 processing and suppression of DNA damage-induced necrosis, and provide a potentially novel way to chemosensitize cancer cells.


Reverse transcriptase polymerase chain reaction (RT-PCR)
Total RNA was isolated from cells using the RNAgents Total RNA Isolation System (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. First strand cDNA was synthesized using Superscript Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. PCR was performed on a Thermo Scientific Hybaid MultiBlock MBS 0.2S Thermal Cycler using gene specific primers. Primers used are listed in Table S1. PCR products were analyzed by agarose gel electrophoresis.

Immunoblotting
Cells were harvested in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton-X-100, 0.1% SDS, 1% Deoxycholate, 1 mM EDTA) supplemented with protease inhibitor cocktail (Roche Applied Sciences, San Francisco, CA). Cells were rotated for one hour at 4ºC and then centrifuged at 13,000 RPM for 30 minutes. Protein concentration was determined by the Bradford assay and 40 µg per well was loaded in 10% bis-tris gels (Life Technologies). Gels ran for 40 minutes at 180v in MOPS buffer (Life Technologies). Protein was then transferred to PVDF membranes using a TransBlot SD semi-dry transfer cell (Biorad, Hercules, CA). Membranes were blocked for nonspecific binding with 5% nonfat milk in TBS-T for one hour at room temperature and then incubated in primary antibody overnight at 4ºC. Primary antibodies were used at a 1:1000 dilution and include HMGB1 (ab18256), pMLKL (Ser358) (ab187091) (Abcam, Cambridge, England); Caspase-3 (AAP103E) (Stressgen, San Diego, CA). Following primary antibody hybridization, membranes were washed with TBS-T and incubated in appropriate HRP-conjugated secondary for 1 hour at room temperature. Secondary antibodies used include goat-anti-rabbit (31462), goatanti-mouse (31432) and mouse-anti-goat (31400) (Pierce, Rockford, IL). Presence of antibody binding was detected using Western Lighting -Plus ECL (Perkin Elmer, Waltham, MA) according to manufactures specifications. Membranes were then exposed on blue X-ray film (Phenix Research Products, Candler, NC) 3 .
Briefly, 400 pmols of siRNA duplexes were transfected into cells in 12-well plates for 4 hours, followed by incubation in medium containing 5% FBS for 20 hours, and then drug treatment in complete media containing 10% FBS.

Cell viability
Cells were plated at 20-30% confluence 24 hours prior to treatment. Unless noted otherwise, cells were treated for 48 hours. Cell proliferation was measured using Cell-Titer 96 Aqueous One Solution Cell Proliferation Assay (MTS assay), and ATP levels were measured using Cell-Titer-GLO Luminescent Viability Assay (Promega, Madison, WI) according to manufacturer's recommendations. In addition, attached and live cells were washed 3 times with HBSS after various treatments, stained with crystal violet dye (Sigma) for 10 minutes, followed by 3 more rinses with HBSS. All experiments were repeated for at least 3 times.

Mitochondrial outer membrane potential
Cells were plated at 20-30% confluence 24 hours prior to treatment. Following treatment, cells were assayed for mitochondrial outer membrane potential by MitoTracker Red CMXRos according to manufacturer's recommendations as previously described 5 . All experiments were repeated for at least 3 times.

Propidium iodide and Annexin V staining
Cells were plated at 20-30% confluence 24 hours prior to treatment. Following 48 hours treatment, cells were trypsinized and spun down at 3,000 RPM (400 g). Cells were then washed in cold PBS and reconstituted in 100µl of Annexin-binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl 2 , adjusted to pH=7.4) containing 2 µl of Annexin V-Alexa Fluor 488 and 2 µl 50 µg/ml propidium iodide (Invitrogen). Cells were then allowed to incubate in the dark at room temperature for 15 minutes before being diluted with 300 µl Annexin-binding buffer. FL1 and FL2 emission was measured on a C6 flow cytometer (Accuri Cytometers, San Jose, CA). At minimum, 30,000 events per sample were measured, all samples were done in triplicate and all experiments were performed at minimum three times.

Detection of reactive oxygen species
Cells were plated at 20-30% confluence 24 hours before treatment and then treated for 24 hours.
Following treatment, cell media was removed and replaced with fresh media containing 2 µM mitoSox reagents (Life Technologies). Cells were then incubated for 20 minutes at 37ºC/5% CO 2 .
Following incubation cells were harvested washed once in cold phosphate-buffered saline (PBS) and then reconstituted in cold 1% BSA PBS. Following excitation at 518 nm, 580 nm emission was measured on a C6 flow cytometer (Accuri Cytometers). At minimum, 30,000 events per sample were measured, all samples were done in triplicate and all experiments were performed at minimum three times.

Transmission electron microscopy
Cells grown on tissue culture plasticware were fixed in 2.5% gluaraldehyde in 100 mM PBS (8 gm/l NaCl, 0.

Immunoprecipitation
The protocol was modified from 7 . In brief, following harvesting of cells, 5 mg of protein reconstituted in 1 ml of RIPA in each experiment and used with Invitrogen Protein A and G Dynabead® immunoprecipitation system according to manufacturer's instructions. Antibodies used include Caspase-3 (sc-7148), Caspase-8 (sc-6136) (Santa Cruz) and RIP1 (610458) (BD Biosciences). Resulting precipitates were processed in the same manner as other immunoblots as described above 7 .

Xenograft studies
All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Female 5-6 week-old Nu/Nu mice (Charles River, Wilmington, MA) were housed in a sterile environment with micro isolator cages and allowed access to water and chow ad libitum. Mice were injected subcutaneously in both flanks with 4×10 6 WT or caspase-3-KO HCT116 cells. After implantation, tumors were allowed to grow 7 days and reach approximately 50mm 3 before treatment was initiated. Mice were randomized into two groups (n = 9 per group) receiving either vehicle (PBS) or 5-FU (50 mg/kg/d) every other day for 14 days. Detailed methods on tumor measurements, harvests and histological analysis are as described [8][9] .