Figure 6. AZ, SFN, and AZ+SFN upend hypoxia microenvironment in H720 and H727 xenografts using Hypoxyprobe™ (pimonidazole hydrochloride) immunohistochemical analysis and western blot of HIF-1α and CAIX in xenograft lysates.

Using previous BC H727 (typical) (A, B) and H720 (atypical) (C, D) xenografts developed in NOD/SCID mice, the mice were treated with AZ (20 mg/kg), SFN (40 mg/kg), and a combination of AZ (20 mg/kg) plus SFN (40 mg/kg), daily for two weeks [3]. (A–D) Immunofluorescence labeling for pimonidazole (PIM) in tumor sections. Slides were mounted with VectaShield medium with DAPI. (E, H) Western blot analyses of freshly frozen tumors for the expression of HIF-1α and CAIX. Each lane represents a single tumor lysate. (F, G and I, J) The bars in the graphs represent the mean percentage of HIF-1α and CAIX band density ± SEM (error bars) for untreated (CTRL), and treated cohorts for 3–5 replicate measurements. These results confirm that the AZ+SFN combination markedly normalizes the hypoxic state in the xenografts coincident with loss of HIF-1α and CAIX expressions. The data represent one out of three independent experiments showing similar results. ^* p ≤ 0.05, ^** p ≤ 0.01, ^*** p ≤ 0.001, n = 3. Abbreviations: AZ: acetazolamide; SFN: sulforaphane; BC: bronchial carcinoma; IF: immunofluorescence; HIF-1α: hypoxia-inducible factor 1-alpha; CAIX: Carbonic Anhydrase 9 (CAIX).