p62 manipulation affects chlorin e6-mediated photodynamic therapy efficacy in colorectal cancer cell lines

Abstract

p62 is a multifunctional protein that mediates cell signaling pathways, autophagy and tumorigenesis, and participates in important regulation processes at the intersection between autophagy and cancer. Photodynamic therapy (PDT) is a treatment that involves photosensitizing agents and light to kill cancer cells. However, whether the efficacy of PDT depends on the expression level of p62 in colorectal cancer cell lines is not known. The present study aimed to examine the role of p62 expression levels in chlorin e6-based PDT in colorectal cancer cells. To study the effect of p62 on cancer cell death, we used PDT to treat a stable cell line overexpressing p62. Cells overexpressing p62 showed a higher cell death rate than cells not expressing this protein. Overexpression of p62 may contribute to colorectal cancer cell death. These results provide preliminary evidence for use of p62 as a therapy target to treat colorectal cancer.

Keywords: apoptosis; autophagy; colorectal cancer; p62; therapeutic agents; tumor genesis.

Figure 1.

Differences in the PDT effect depending on the p62 expression level in colon cancer cell lines. (A) Relative fold-changes in p62 mRNA levels for 58 human colon colorectal cancer cell lines (Cancer Cell Line Encyclopedia datasets) were categorized according to the classification of cells. (B) Immunoblotting analysis of whole-cell lysates of various colon cancer cell lines. (C) Colon cancer cell lines were irradiated with a PDT laser (4 J/cm²) after 6 h of pretreatment with Ce6 at the indicated concentrations. The MTT assay was performed in triplicate at 24 h following irradiation. Data are presented as the mean ± SEM of three experiments. Data were analyzed with ANOVA and Tukey's post hoc test. *P<0.05, **P<0.01, ****P<0.0001. PDT, photodynamic therapy.

Figure 2.

Effect of p62 deletion on colorectal cancer cell lines in PDT. (A) p62 siRNA-transfected cells were incubated with 100 nM chlorin e6 for 6 h prior to irradiation. Cell viability was measured by an MTT assay. (B) Level of p62 in p62 shRNA-treated cells was determined by immunoblotting. (C) Cell viability was measured by an MTT assay after applying PDT to shRNA or p62 shRNA-treated cells. Data are presented as the mean ± SEM of three experiments. *P<0.05, **P<0.01 vs. respective shCTRL. si, small interfering; PDT, photodynamic therapy; sh, short hairpin; CTRL, control.

Figure 3.

Establishment of p62 overexpressed colorectal cancer cell lines. Cells overexpressing p62 in colon cancer cell lines were confirmed by immunocytochemical assay using p62 and HA antibodies. Scale bar, 50 µm. CTRL, control.

Figure 4.

Effect of p62 overexpression on colorectal cancer cells. (A) Cells overexpressing p62 in colon cancer cell lines were confirmed by immunoblotting using a p62 antibody. (B) Cells were incubated with chlorin e6 at the indicated concentrations for 6 h, before the MTT assay. Data are presented as the mean ± SEM of three experiments. *P<0.05, **P<0.01 vs. respective control cells.

Figure 5.

p62 overexpression affects apoptotic cell death. (A) SW480 and HCT116, and (B) LoVo and DLD1 cells were incubated with chlorin e6 at indicated concentrations for the indicated period, before immunoblotting using p62, caspase 3 and β-actin antibodies.

Figure 6.

Differences in effect of oxalitin and 5-FU on p62 overexpression. (A) Cell viability was measured by an MTT assay after treatment with the indicated concentrations of oxalitin for 24 h. (B) Cell viability was measured by an MTT assay after treatment with the indicated concentrations of 5-FU for 24 h. Data are presented as the mean ± SEM of three experiments. *P<0.05, **P<0.01 vs. respective control cells. 5-FU, 5-fluorouracil.

Figure 7.

Antitumor effect of p62 overexpression in photodynamic therapy. (A) SW480 and SW480-p62 cells were collected and inoculated into BALB/c nude mice by subcutaneous injection. (B) LoVo and LoVo-p62 cells collected and inoculated into BALB/c nude mice by subcutaneous injection. Ce6 was administered to the tumors at a dose of 1.25 mg/kg, and Ce6 was applied 6 h later, followed by irradiation with a 670 nm diode light (150 J/cm²). Tumor sizes were measured using a caliper, tumor volume was calculated using the formula: 0.523 × length × width² (mm³). Data are presented as the mean ± SEM. n=4. Data were analyzed with ANOVA and Tukey's post hoc test. ***P<0.001, ****P<0.0001. Ce6, chlorin e6; IR, irradiation.