Human CRC COLO205 cells were obtained from the Chinese Academy of Sciences Institute of Cell Resource Center (Shanghai, China). The cells were cultured in RPMI-1640 (GE Healthcare Life Sciences, Little Chalfont, UK) supplemented with 10% fetal bovine serum (FBS; Procell Life Science & Technology Co., Ltd., Wuhan, China), 100 IU/ml penicillin and 100 mg/ml streptomycin at 37°C in an atmosphere containing 5% CO₂. NP with analytical standard purities was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China), and was dissolved in absolute ethyl alcohol to 50 mM.

The siRNA oligo was synthesized by Sangon Biotech Co., Ltd., (Shanghai, China). Sequences were as follows: si-PKCζ, 5′-GGAGGACCTTAAGCCAGTT-3′ and siRNA negative control (NC) 5′-AGACTGTGAATCTAGATCAAG-3′.

For transfection, COLO205 cells were cultured for 12 h in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin, and the fragment of siRNA (50 nM) was transfected using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. After 72 h of transfection, cells were washed by PBS and then lysed with 1X radioimmunoprecipitation (RIPA) buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with 0.2 mM phenylmethylsulphonyl fluoride protease inhibitor (cat. no. 36978; Thermo Fisher Scientific, Inc.) for 30 min on ice for western blot analysis.

Cell viability was detected using the CellTiter 96^® Non-Radioactive Cell Proliferation assay (MTT; cat. no. G4000; Promega Corporation, Madison, WI, USA). COLO205 cells (1×10⁴ cells/ml) were seeded in 96-well plates for 24 h prior to treatment, with normal saline and cell culture mediums serving as the control, and three wells were prepared for each of the following groups: 0; 1×10⁻⁶; 1×10⁻⁷ and 1×10⁻⁸ mol/l NP alone (17,18); 1×10⁻⁶ mol/l NP with si-PKCζ; and 1×10⁻⁶ mol/l NP with NC. Following treatment for 0, 24, 48 and 72 h, a total of 15 µl provided dye solution was added to each well, and the 96-well plate was incubated at 37°C for 4 h, subsequent to adding 100 µl Solubilization/Stop Solution from the kit. Viability was recorded at a wavelength of 570 nm on a microplate reader (Multiskan; Thermo Fisher Scientific, Inc.). The assay was repeated in triplicate.

The effect of NP and si-PKCζ on cell cycle progression was determined by flow cytometry. After 24 h of treatment, the cells were digested and fixed with 70% ethanol for 24 h at 4°C. Following fixation, cells were stained with 50 µg/ml propidium iodide (PI) solution and 100 µg/ml RNase A in PBS for 30 min in the dark on ice and then subjected to cell cycle analysis. The apoptotic rate was measured using Annexin V/PI double staining (Annexin V-FITC Apoptosis Detection kit; cat. no. C1062; Beyotime Institute of Biotechnology). A total of 300 µl binding buffer was used for cell resuspension 1×10⁶, and 5 µl Annexin V-fluorescein isothiocyanate was added to the cell suspension for 10 min in the dark at room temperature. A total of 5 µl PI was subsequently added to the cell suspension for 5 min in the dark on ice. The samples were analyzed with a FACSCalibur flow cytometer and the FACSCalibur system (both BD Biosciences, Franklin Lakes, NJ, USA).

Total protein was extracted from COLO205 cells following treatment using RIPA buffer, and the concentrations were determined by bicinchoninic acid (Thermo Fisher Scientific, Inc.). A total of 40 µg total protein was separated by SDS-PAGE (10% spacer gel and 5% separation gel). Proteins were transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA USA), and membranes were subsequently blocked with 5% skim milk powder (BD Biosciences) for 60 min at room temperature. The membranes were subsequently incubated with the following primary antibodies: Cyclin D1 (dilution, 1:800; cat. no. ab134175); cyclin E (dilution, 1:800; cat. no. ab33911), B-cell lymphoma 2 (Bcl-2) associated agonist of cell death (Bad; dilution, 1:500; cat. no. ab62465), Bcl-2 (dilution, 1:1,000; cat. no. ab32124), cyclin-dependent kinase inhibitor (p27; dilution 1:1,000; cat. no. ab32034), PKCζ (dilution 1:1,500; cat. no. ab59364), phosphorylated (p)-PKCζ (dilution 1:1,500; cat. no. ab62372) (all from Abcam, Cambridge, MA, USA), ERK1/2 (dilution 1:1,000; cat. no. 4695; Cell Signaling Technology, Inc., Danvers, MA, USA), p-ERK1/2 (dilution 1:1,000; cat. no. 4370; Cell Signaling Technology, Inc.) and GAPDH (dilution 1:1,000; cat. no. ab8245; Abcam) overnight at 4°C. The membranes were subsequently probed with goat anti-rabbit horseradish peroxidase-labeled secondary antibody (dilution 1:10,000; cat. no. ab6721; Abcam) for 1 h at room temperature. Target proteins were detected with Clarity^™ Western enhanced chemiluminescence substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's protocols. The optical density was analyzed by AlphaEaseFC software (version 5.0, ProteinSimple, San Jose, CA, USA). GAPDH was used as the internal control. All experiments were conducted in triplicate.

Each experiment was repeated in triplicate. Differences between different groups were evaluated by one-way analysis of variance, followed by Duncan's multiple range post-hoc test using GraphPad prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Difference between two groups were analyzed by a Student's t-test using Microsoft Excel 2017 (Microsoft Corporation, Redmond, WA, USA). Results are presented as means ± standard deviation and P<0.05 was considered to indicate a statistically significant difference.

The results of MTT indicated that NP (1×10⁻⁶−1×10⁻⁸ mol/l) could significantly induce the proliferation of COLO205 cells (F=48.66; P<0.01) in a time-and dose-dependent manner, compared with the control group (Fig. 1). Compared with the NC group, the proliferation of COLO205 cells demonstrated was significantly reduced by si-PKCζ transfection in a time-dependent manner (P<0.01); however, recovery of proliferation was indicated following NP treatment (Fig. 1).

Flow cytometry was used to investigate the influence of NP and PKCζ on the cell cycle. Compared with the control group, the ratio of G0/G1 phase cells was significantly reduced by NP treatment (t=9.25, 13.17 and 14.74 for 1×10⁻⁸, 1×10⁻⁷ and 1×10⁻⁶ mol/l NP, respectively; all P<0.01; Fig. 2A), and significantly increased by the suppression of PKCζ expression (t=11.29; P<0.01), however, this elevation was suppressed by NP treatment (t=33.35; P<0.01) (Fig. 2B). Furthermore, NP treatment increased the ratio of S phase cells, compared with the control group (t=22.67, 37.02 and 47.41 for 1×10⁻⁸, 1×10⁻⁷ and 1×10⁻⁶ mol/l NP, respectively; all P<0.01; Fig. 2A). Following si-PKCζ transfection, the ratio of S phase cells was significantly decreased compared with the NC group (t=1.83; P<0.05), however, it was significantly increased following NP treatment (t=51.31; P<0.01) (Fig. 2B).

To evaluate the influence of NP and PKCζ on cell apoptosis, flow cytometry was utilized to identify any changes in the apoptotic rate of cells following NP treatment or si-PKCζ transfection. The results indicated that the ratio of viable to non-viable apoptotic cells in the NP treatment group was lower than that in the control group (P<0.01; Fig. 3A). Suppression of PKCζ expression significantly increased the ratio of viable apoptotic and non-viable apoptotic cells (P<0.01 vs. NC group), however, the ratio of apoptotic cells decreased following NP treatment (Fig. 3B).

Compared to the control group, NP treatment significantly upregulated the expression of cyclin D1 and cyclin E, and downregulated the expression of p27 (all P<0.05; Fig. 4A). Following siRNA transfection, the expression of cyclin D1 and cyclin E was significantly reduced by PKCζ suppression, and the expression of p27 was increased (all P<0.01 vs. NC group). Following NP treatment in the si-PKCζ-transfected cells, the expression of cyclin D1 and E increased, whereas the expression of p27 decreased (Fig. 4B).

Additionally, the expression of apoptosis-associated proteins Bcl-2 and Bad was examined (Fig. 5A). The results of the western blot analysis indicated that NP treatment had no significant effect on the expression of Bcl-2, but significantly reduced the expression of Bad (P<0.01 for all concentrations of NP vs. control). The expression of Bad was significantly increased following si-PKCζ transfection (P<0.01 vs. NC group) and significantly reduced by subsequent NP treatment (P<0.01 vs. si-PKCζ group) (Fig. 5B).

The results of the western blot analysis indicated that the expression of PKCζ and the phosphorylation of ERK1/2 were significantly increased by NP (P<0.05 for 1×10⁻⁸; P<0.01 for 1×10⁻⁷−1×10⁻⁶; Fig. 6A). Following si-PKCζ transfection, the expression of PKCζ and the phosphorylation of ERK1/2 were significantly reduced (both P<0.01 vs. NC), however, this was significantly recovered following NP treatment (both P<0.01 vs. siPKCζ alone) (Fig. 6B). The phosphorylation of PKCζ was also influenced by NP treatment. Following NP treatment, the phosphorylation of PKCζ was significantly increased for all concentrations of NP, compared with the control (P<0.01; P<0.05; Fig. 7A). The phosphorylation level of PKCζ did not change significantly following si-PKCζ transfection, however, subsequent NP treatment significantly increased the phosphorylation (P<0.01 vs. si-PKCζ alone; Fig. 7B).