Ascorbyl Stearate Promotes Apoptosis Through Intrinsic Mitochondrial Pathway in HeLa Cancer Cells

Materials and Methods

Chemicals. ASC-S was purchased from Tokyo Chemical Industry (Tokyo, Japan). Cell culture grade plastic-ware and chemicals such as enhanced chemiluminescence (ECL) kit were purchased from Himedia (Bangalore, India) and Life Technologies, (Bangalore, India). Cell culture grade dimethyl sulphoxide (DMSO), real-time poly merase chain reaction (PCR) primers and other analytical reagents were obtained from Sigma Chemicals (Bangalore, India). Halt protease inhibitor cocktail, bicinchoninic acid (BCA) kit for protein assay, JC-1, and verso cDNA synthesis kit were procured from Thermo Fisher Scientific (Bangalore, India). Rabbit antibodies to procaspase-9, procaspase-3, procaspase-8, BH3 interacting-domain death agonist (BID), FAS ligand (FASL), cytochrome c, nuclear factor-kappa B (NF-ĸB), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IĸBα) and β-actin, as well as horseradish peroxidase (HRP)-conjugated anti-rabbit, IgG antibody were purchased from Cell Signaling Technology (Bangalore, India) and Abcam (Kolkata, India) respectively. Polyvinylidene fluoride (PVDF) membrane was purchased from Pall Corporation (Bangalore, India). RNA purification kit and SYBRGreenreal-time PCR master mix was procured from Fermentas Life Sciences (Bangalore, India) and Biorad Laboratories (Bangalore, India)respectively. Inhibitors of caspase-8 and 9 were purchased from Calbiochem (San Diego, CA, USA).

Cell culture. HeLa cells were procured from the cell repository at National Centre for Cell Science (Pune, India) and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS) at 37°C in 5% CO₂. Cells were plated at least 48 h before drug treatment.

Preparation of ASC-S. One mM stock of ASC-S was prepared by dissolving ASC-S in DMSO and making up in DMEM by adjusting the pH to 7 with 0.1 mM sodium hydroxide in sterilized Milli Q water. Concentrations of 50-200 μM ASC-S were used for treatment of HeLa cells for 48 h at 37°C in a CO₂ incubator.

Measurement of mitochondrial membrane potential (MMP). MMP was determined using the mitochondrial-specific fluorescent probe JC-1 by a spectroflourimetric method (9). JC-1 (5 μM) was incubated with ASC-S-treated HeLa cells for 0.5 h at 37°C, prior to analysis. The change in fluorescence was measured using a spectrofluorimeter, JC-1 has dual emission, depending on the state of the MMP. A decrease in MMP resulted in a decrease in red fluorescence with a simultaneous rise in green fluorescence as the dye shifts from an aggregate to monomeric state. The ratio of red to green fluorescence was used as an index for calculation of MMP. Hyperpolarization of the mitochondria as shown by shift in the ratio of red to green fluorescence

Immunoblot analysis of proteins from ASC-S-treated HeLa cells. ASC-S-treated HeLa cells were lysed in radio-immunoprecipitation assay buffer (RIPA) buffer: 50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 2 mM EDTA with 1X protease inhibitor cocktail. Equal amounts of protein (25 μg) as quantified by BCA kit were electrophoresed on 12% SDS-p and wet transferred onto methanol pre-activated PVDF membrane. Membranes were blocked with 5% non-fat milk powder and then incubated overnight at 4°C with rabbit antibodies to procaspase-9, procaspase-3, procaspase-8, cytochrome c (whole-cell lysate) and cytosolic fraction as mentioned by Dimauro et al. (10), BID, NFĸB, IĸBα, FAS antibodies or rabbit anti-β-actin antibody and finally incubated with HRP-conjugated anti-rabbit, IgG antibody followed by exposure to ECL detection reagents and image capturing in Syngene G:Box unit (Bangalore, India). Densitometric analysis was performed using inbuilt Syngene tools electrophoresis analysis software.

FAS-ligand enzyme-linked immunosorbent assay (ELISA). ELISA was performed as described in (11) with slight modification for the detection of FAS ligand levels in the ASC-S-treated HeLa cell homogenate, which was prepared by lysing cells in RIPA buffer. Rabbit anti-FAS at 1:1,000 dilution (v/v) and HRP-conjugated anti-rabbit, IgG antibody at 1:5,000 dilution were used as primary and secondary antibody respectively followed by 3,3’,5,5’-tetramethylbenzidine/H₂O₂ as substrate for activity detection. The absorbance was measured at 450 nm with a reference wavelength of 570nm in a microplate ELISA reader. The concentration of FAS ligand expressed in ASC-S-treated HeLa was quantified with the standard FAS ligand plot.

Electrophoretic mobility-shift assay (EMSA). HeLa cells were exposed to 25-200 μM ASC-S and were harvested after 4 h for nuclear pellets prepared as described elsewhere (3). EMSA was performed by incubating 10 μg of nuclear proteins with 16 fmol of ³²P-end-labeled, 45-mer double-stranded NFĸB oligonucleotides from the human immunodeficiency virus long terminal repeat (5’-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3’; boldtype indicates NFĸB-binding sites), AP-1 (5’-CGCTTGA TGACTCAGCCGGAA-3’; boldtype indicates AP1-binding site) or NF-AT (5’-CGC CCA AAG AGG AAA ATT TGT TTC ATA-3’; boldtype indicates NFAT-binding site) in the presence of 0.5 μg of poly(2’-deoxyinosinic–2’-deoxycytidylic acid) in binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1% NP-40, 5% glycerol, and 50 mM NaCl) for 30 min at 37°C. The DNA–protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels using buffer containing 50 mM Tris, 200 mM glycine, and 1 mM EDTA, pH 8.5.The dried gel was exposed to a phosphor image plate and the radioactive bands were visualized using a phosphor image plate scanner (Amersham Biosciences, Pittsburgh, PA, USA). The images were processed by using Adobe Photoshop software (San Jose, CA, USA).

Effect of caspase inhibitors on ASC-S-induced apoptosis. HeLa cells were pre-incubated with inhibitors of caspase-8 (25 μM) or caspase-9 (40 μM) for 30 min as described in (12) and then treated with ASC-S (50, or 100 μM) at 37°C for 48 h. The cells were then harvested and incubated in buffer [PBS + 0.1% Triton-X + 0.1% sodium citrate, pH 7.4, supplemented with 50 μg/ml propidium iodide (PI) overnight at 4°C. Cell death was estimated using a flow cytometer (Partec, Görlitz, Germany).

Quantitative real-time polymerase chain reaction (RT-PCR) analysis. The expression of caspase-8, caspase-9, apoptotic protease activating factor 1 (APAF), cytochrome c, BID, NFKB, FAS, Fos proto-oncogene; AP1 transcription factor subunit (FOS) and Jun proto-oncogene, AP1 transcription factor subunit (JUN) in ASC-S-treated cells were analysed by RT-PCR. Total RNA was purified from ASC-S-treated HeLa cells using RNA purification kit. One microgram of RNA was employed for cDNA synthesis using verso cDNA synthesis kit; reaction mixtures of 20 μl were prepared with SYBR green master mix using Biorad CFX real-time PCR with validated primers as given in Table I.

The amplification parameters were as follows: initial denaturation at 95°C for 5 min prior to 39 thermal cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 30 s, with final extension of 65°C for 5 min. Standard PCR reactions were performed with 39 cycles. The PCR products were analysed and quantities calculated by inbuilt Biorad software for fold expression levels, normalized to endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Statistical analysis. Statistical analysis was performed by one-way analysis of variance (ANOVA) and Dunnett test by comparing control with ASC-S-treated cells at different doses and time intervals using Graphpad statistical software (Graph Pad Instat; La Jolla, CA, USA).