MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent was purchased from Invitrogen Life Technologies (San Diego, CA, USA). RIPA Lysis Buffer and Pierce BCA Protein Assay Kit were obtained from Thermo Fisher Scientific (HK). Twenty kDa linear PEG conjugated with maleimide functional group was purchased from NOF America Corporation (White Plains, NY).
Production of BCA-M-PEG20 and ADI
The production protocols were the same as those in our previous studies [6, 27].
Cell Proliferation Assay
Cytotoxicity of BCA-M-PEG20 against MKN-45 and BGC-823 cells was determined by MTT assay as described previously [6, 7]. Then, the results of the optical density at a wavelength of 570 nm were detected by Varioskan LUX Multimode Microplate Reader.
Western Blot Analysis
MKN-45 cancer cells with or without BCA-M-PEG20 treatments at different concentrations and times were harvested drug the log-phase. Cells and a small piece of mouse liver (as ASS and OTC positive control) were lysed with RIPA lysis buffer on ice for 15 min. Then, the extracted cellular proteins were obtained by centrifugation at 15,000 rpm for 5 min. PierceTM BCA Protein Assay was used to determine the concentration of the extracted cellular proteins. An equal amount of total protein per lane was loaded and separated by electrophoresis separation, then transferred to Immobilon-P polyvinylidene fluoride membranes. The membranes were blocked with 5% blotting-grade blocker with Tris-buffered saline (TBST, 0.1% Tween-20, 100 mM Tris-HCl, pH 7.5, 0.9% NaCl) at room temperature for 1 h. Then the membrane was incubated with a specialized primary antibody at 4 oC overnight. After incubation, membranes were washed by TBST and incubated with secondary antibodies at room temperature for 1 h. Excess secondary antibodies were removed by washing with TBST. The detection of specified protein was performed by Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA). For the primary antibodies, rabbit anti-ASS, rabbit anti-OTC, rabbit anti-PARP, rabbit anti-beta-actin (1:1000, Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-LC3 (1:1000, Abcam, HK) were utilized. HRP-conjugated goat anti-rabbit secondary antibody (1:10000, Cell Signaling Technology, Danvers, MA, USA) was applied to determine all primary antibodies. ImageJ software (National Institutes of Health) was adopted to determine the protein signal intensities.
To detect the apoptotic cells, Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA) was applied. A density of approximately 0.4 x 106 MKN-45 cells was seeded into a T25 flask and incubated overnight prior to the BCA-M-PEG20 treatments. Cells treated with or without BCA-M-PEG20 at various concentrations and time points were harvested and washed by PBS twice, followed by resuspension in 1X assay buffer and incubation with Annexin V-FITC and/or PI for about 15 min in the dark. The stained cancer cells were then subjected to BD Accuri C6 Flow Cytometer within 1 h.
Cell cycle analysis
MKN-45 cells were seeded, treated, and harvested as in apoptosis assay, followed by fixation in 60% ethanol at 4 oC overnight. Then, the fixed cells were washed by PBS twice, filtered via a 60 µm nylon mesh and incubated with PI/RNase staining buffer at room temperature for at least 1 h. Finally, fluorescence-activated cell sorting analyses were applied on the stained cells and data from each sample were collected from at least 10,000 cells. ModFit LT 3.1 (Verity Software House, Topsham, ME, USA) was used to determine the cell cycle distribution analyses.
Autophagy detection by confocal microscope
To monitor the autophagy of live MKN-45 cells induced by BCA-M-PEG20, CYTO-ID® Autophagy detection kit was used (Enzo Life Sciences, Farmingdale, NY, USA). About 6000 of MKN-45 cancer cells were seeded into confocal plates per well and incubated with or without BCA-M-PEG20 (0.58 µg/mL) for different durations. A positive control, Rapamycin (500 nM), was used in this assay. Then, cancer cells were stained with cyto-ID dye and Hoechst 33342 following the instructions of the manufacturer and detected by Leica TCS SPE confocal microscope.
Mouse xenograft model of gastric cancer treated by BCA-M-PEG20
For the preparation of MKN-45 tumor xenograft, 1 x 106 MKN-45 cells were injected into the flank of BALB/c nude mice subcutaneously. When the size of tumors achieved to 1.5-2.0 cm in diameter, they were excised, cut into tumor fragments, and implanted into the nude mice. Then, the tumor volume was measured closely. Once the stable growth of tumors was maintained, the mice were divided into 3 groups randomly. Ten mice with an average tumor volume of about 400 mm3 were assigned into one group. BCA-M-PEG20 (250 U/mouse) and 5-fluorouracil (5-FU, 10 mg/kg) were administrated twice per week and once per week to nude mice bearing tumor xenograft through i.p. injection, respectively. The control group was injected with PBS as vehicle control. Tumor dimensions were determined in situ regularly throughout the treatment period using a digital caliper and the tumor volume was estimated by the equation, 0.5 x length x (width)2. The bodyweight of the tumor bearing mice was monitored every week throughout treatment. Lastly, the mice were sacrificed by cervical dislocation and the tumors were obtained for actual tumor weight measurement.