Enhancement of Radiation Sensitivity by Cathepsin L Suppression in Colon Carcinoma Cells

Abstract

Cancer is one of the main causes of death globally. Radiotherapy/Radiation therapy (RT) is one of the most common and effective cancer treatments. RT utilizes high-energy radiation to damage the DNA of cancer cells, leading to their death or impairing their proliferation. However, radiation resistance remains a significant challenge in cancer treatment, limiting its efficacy. Emerging evidence suggests that cathepsin L (cath L) contributes to radiation resistance through multiple mechanisms. In this study, we investigated the role of cath L, a member of the cysteine cathepsins (caths) in radiation sensitivity, and the potential reduction in radiation resistance by using the specific cath L inhibitor (Z-FY(tBu)DMK) or by knocking out cath L with CRISPR/Cas9 in colon carcinoma cells (caco-2). Cells were treated with different doses of radiation (2, 4, 6, 8, and 10), dose rate 3 Gy/min. In addition, the study conducted protein expression analysis by western blot and immunofluorescence assay, cytotoxicity MTT, and apoptosis assays. The results demonstrated that cath L was upregulated in response to radiation treatment, compared to non-irradiated cells. In addition, inhibiting or knocking out cath L led to increased radiosensitivity in contrast to the negative control group. This may indicate a reduced ability of cancer cells to recover from radiation-induced DNA damage, resulting in enhanced cell death. These findings highlight the possibility of targeting cath L as a therapeutic strategy to enhance the effectiveness of RT. Further studies are needed to elucidate the underlying molecular mechanisms and to assess the translational implications of cath L knockout in clinical settings. Ultimately, these findings may contribute to the development of novel treatment approaches for improving outcomes of RT in cancer patients.

Keywords: cancer; cath L; colon carcinoma; inhibitor; knockout; radiation dose; radiotherapy.

Figure 1

Assessment of cath L expression under varying radiation doses. (A) Western blot was performed to investigate the regulation differences. (B) The cellular cath L levels were compared with and without radiation treatment, and statistical analysis was performed using the t-test with GraphPad Prism software. The results indicated a highly significant difference (*** p < 0.001), and the graphs represent the mean ± standard error (SE) with a sample size of N = 5. (0 Gy = Control).

Figure 2

Cath L localization in irradiated and non-irradiated (control) caco-2 cells (63× magnification). Application of anti-cath L antibody (green) shows the lysosomal and membrane-bound cath L localization, LysoTracker red for lysosomal staining and 4′,6-diamidino-2-phenylindole (DAPI) visualizes nuclear DNA (blue) in fixed cells.

Figure 3

Impact of radiation treatment on the labeling of cysteine cathepsins’ active sites using DCG04. (A) Active-site labeling with DCG04 was carried out, and protein samples underwent electrophoresis and subsequent western blotting using streptavidin-horseradish peroxidase. (B) The cellular content of procathepsin B/L, heavy chain-form cath B/L, and mature form (two-chain form) of cath B/L in irradiated caco-2 cells labeled with DCG-04 bands was compared to cellular content from control cells labeled with DCG-04 only, non-significant (n.s). The data underwent analysis using a two-way ANOVA followed by Dunnett’s post hoc analysis. Statistical calculations were performed using GraphPad Prism. The results indicated a highly significant difference (*** p < 0.001), and the graphs represent the mean ± standard error (SE) with a sample size of N = 5. (0 Gy = Control).

Figure 4

Avidin pull-down experiment of cysteine cathepsins with and without radiation treatment. (A) Conjugated proteins to avidin sepharose beads were subjected to SDS-PAGE and western blotting with antibodies specific to human cath L. (B) Ratio of cellular contents of cath L with and without radiation treatment. The data were analyzed by t-test and statistics using GraphPad Prism. *** p < 0.001. Graphs are shown as mean ± SE, N = 5. (0 Gy= Control).

Figure 5

Cytotoxicity analysis of treated cells by radiation and a cath L inhibitor, compared to untreated cells. Statistical analysis was carried out using a t-test with GraphPad Prism, and the results indicated significant differences (*** p < 0.001), non-significant (n.s). The graphs represent the mean values along with the standard error (SE), and the study had a sample size of N = 8. (0 Gy = Control).

Figure 6

Examination of cath L expression in both control and cath L knockout cells.

Figure 7

Evaluating the effect of cath L knockout on cellular toxicity in both irradiated and non-irradiated cells. Statistical analysis was performed using a t-test with GraphPad Prism, revealing a highly significant difference (*** p < 0.001), non-significant (n.s). The graphs depict the mean values along with the standard error (SE), and the study had a sample size of N = 8. (0 Gy = Control).

Figure 8

Assessing the influence of cath L inhibition on apoptosis rates in cells exposed and non-exposed to ionizing radiation via caspase 3/7 activity assay. The data underwent analysis using a two-way ANOVA followed by Dunnett’s post hoc analysis, revealing significance levels (** p < 0.01; *** p < 0.001; N = 4), non-significant (n.s), (0 Gy = Control). Statistical calculations were carried out using GraphPad Prism.

Figure 9

Z-Phe-Tyr(tBu)-diazomethylketone is an irreversible cathepsin L inhibitor, and this compound is commonly utilized as a selective inhibitor for cath L. It is a modified peptide derivative consisting of the amino acids phenylalanine (Phe) and tyrosine (Tyr), along with a diazomethylketone functional group attached to the tyrosine residue. The tBu abbreviation indicates the presence of a tert-butyl group, which enhances stability and influences the compound’s properties. Chemical Formula: C₃₁H₃₄N₄O₅.

Figure 10

E-64 is an irreversible, potent, and highly selective broad spectrum cysteine proteinase and calpain activation inhibitor. N-[N-(L-3-Trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine. Formula: C₁₅H₂₇N₅O₅.