Hemoxygenase-1 Promotes Head and Neck Cancer Cell Viability

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a remarkably heterogeneous disease with around 50% mortality, a fact that has prompted researchers to try new approaches to improve patient survival. Hemoxygenase-1 (HO-1) is the rate-limiting step for heme degradation into carbon monoxide, free iron and biliverdin. We have previously reported that HO-1 protein is upregulated in human HNSCC samples and that it is localized in the cytoplasmic and nuclear compartments; additionally, we have demonstrated that HO-1 nuclear localization is associated with malignant progression. In this work, by using pharmacological and genetic experimental approaches, we begin to elucidate the mechanisms through which HO-1 plays a role in HNSCC. We found that high HO-1 mRNA was associated with decreased patient survival in early stages of HNSCC. In vitro experiments have shown that full-length HO-1 localizes in the cytoplasm, and that, depending on its enzymatic activity, it increases cell viability and promotes cell cycle progression. Instead, HO-1 does not alter migration capacity. Furthermore, we show that C-terminal truncated HO-1 localizes into the nucleus, increases cell viability and promotes cell cycle progression. In conclusion, we herein demonstrate that HO-1 displays protumor activities in HNSCC that depend, at least in part, on the nuclear localization of HO-1.

Keywords: cancer; head and neck; hemoxygenase-1; nucleus.

Figure 1

HO-1 mRNA expression is associated with overall survival, relapse-free survival and risk factor HPV status. (A) Kaplan–Meier overall survival curves for HNSCC patients stratified as Stage 1 + 2 according to low and high HO-1 expression level (n = 217, Breslow test, p = 0.046). (B) Kaplan–Meier relapse-free survival curves for HNSCC patients stratified as Stage1 + 2 according to low and high HO-1 mRNA expression level (n = 217, Log Rank test, p = 0.003). (C) Comparison of HO-1 mRNA levels in HNSCC according to HPV status (n = 88, ANOVA test, p = 0.034).

Figure 2

Pharmacological activation of HO-1 promotes cellular viability and cell cycle progression of HN13 cells. (A) HN13 cell viability was evaluated after treatment with hemin (20–80 uM) or its vehicle for 24 h to 72 h. (B) HN13 cell viability was evaluated after treatment with 80 uM hemin or its vehicle for 24 h to 72 h. (C) HN13 cell cycle progression and (D) HO-1 protein levels in HN13 cell line were evaluated after treatment with 80 uM hemin (H80) or vehicle (Veh) for 24 h to 72 h. (E) Protein levels of cyclin D and p27 after HO-1 activation with 80 uM hemin in HN13 cell line. (F) HaCaT cell viability was evaluated after treatment with hemin in dose-response assay at 72 h (G) and time-response assay using a 80 uM concentration, comparing the effects with those exerted in HN13. (H) HO-1 protein levels in HaCaT cell line were evaluated after treatment with 80 uM hemin (H80) or vehicle (Veh) for 24 h to 72 h. The results are expressed as cell viability percentage = (hemin group × 100/vehicle group). ** p < 0.01 and *** p < 0.001 with respect to the vehicle in HN13 cells. # p < 0.05 and ### p < 0.001 with respect to the vehicle in HaCaT cells.

Figure 3

Pharmacological inhibition of HO-1 decreases cell viability of HN13 cells. (A) HN13 cell viability was evaluated after treatment with ZnPP (1, 5 and 10 uM) or its vehicle for 24, 48 and 72 h by crystal violet assay. (B) HN13 cell viability was evaluated after treatment with 10 uM ZnPP or its vehicle for 24, 48 and 72 h by manual cell count. (C) HO-1 protein expression was evaluated after treatment with 10 uM ZnPP or vehicle for 24 h to 72 h in HN13 cell line. HaCaT cell viability was evaluated after treatment with (D) ZnPP in dose-response assay at 72 h and (E) time-response assay using 10 uM concentration, comparing the effects with HN13 cells. The results are expressed as cell viability percentage = (ZnPP group x 100 / vehicle group). * p < 0.05, ** p < 0.01 and *** p < 0.001 with respect to the vehicle in HN13 cells. # p < 0.05 and ### p < 0.001 with respect to the vehicle in HaCaT cells.

Figure 4

Pharmacological modulation of HO-1 activity does not alter HN13 cell migration. (A) HN13 cell migration was evaluated after treatment with 80 uM hemin or its vehicle for 8 h. (B) Representative images of an acellular area in 80 uM hemin-treated and vehicle-treated conditions at 0 and 8 h from (A) are shown. (C) HN13 cell migration was evaluated after treatment with 10 uM ZnPP or its vehicle for 8 h. (D) Representative images of an acellular area in 10 uM ZnPP treated and control conditions at 0 and 8 h from (C) are shown.

Figure 5

HO-1 subcellular localization in HN13 cells following pharmacological modulation. Representative images of HO-1 expression in HN13 cells after treatment with (A) 80 uM hemin or its vehicle for 24 h. (B) Semi-quantification of nuclear HO-1-expressing and cytoplasmic HO-1-expressing cells after hemin treatment. (C) Representative images of HO-1 localization in HN13 cells after treatment with 10 uM ZnPP or its vehicle for 24 h. DAPI was used to stain nucleus. (D) Semi-quantification of nuclear HO-1-expressing and cytoplasmic HO-1-expressing cells after ZnPP treatment. *** p < 0.001 respect to vehicle.

Figure 6

Subcellular localization and loss of enzymatic activity of HO-1 affects HN13 cell viability. HN13 cells were transfected with FLAG vectors bearing FL-HO-1, FL-HO-1 (H25A) or t-HO-1 constructs. (A) Representative images of direct immunofluorescence for FLAG showing differential subcellular location of HO-1 expression among stable transfectants HN13 cells. (B) Cell viability of the stably transfected HN13 cells was evaluated at 24 through 96 h. (C) Cell number of the stably transfected HN13 cells was counted at 72 h. (D) Cell cycle progression of the stably transfected HN13 cells was evaluated at 96 h. The results are expressed as cell viability percentage = (selected time group × 100/time 0 h group), being selected time 24 h to 96 h. * p < 0.05, ** p < 0.01 and *** p < 0.001 with respect to the control. # p < 0.05, ## p < 0.01 and ### p < 0.001 respect to FL-HO-1 (H25A).

Figure 7

Expression and pharmacological modulation of HO-1 in human mixed primary cell culture of squamous cell carcinoma. (A) Representative images of hematoxylin eosin staining (i) and HO-1 staining (ii) by immunohistochemistry a tumor biopsy of human SCC. (B) HO-1 expression of sample shown in (A) by immunoblot. (C) Cell viability of the human mixed primary cell culture after treatment with 80 uM hemin or 10 uM SnPP or their respective vehicles at 24 to 72 h. (D) HO-1 protein expression evaluated after treatment with 80 uM hemin or 10 uM SnPP or their respective vehicles at 48 h. The results are expressed as cell viability percentage = (selected time group × 100/time 0 h group), with a selected time from 24 h to 96 h. ** p < 0.01 and *** p < 0.001 with respect to the vehicle.