Expression of Cathepsins B, D, and G in Isocitrate Dehydrogenase-Wildtype Glioblastoma

Figures

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  Figure 1. A schema demonstrating the proposed role of cathepsins B, D, and G, as bypass loops for the renin-angiotensin system (RAS). In the RAS, (pro)renin activation is caused by its binding to the (pro)renin receptor. Renin coverts angiotensinogen (AGN) to angiotensin I (ATI) that is acted upon by angiotensin converting enzyme (ACE) to produce angiotensin II (ATII). Aminopeptidase A converts ATII to angiotensin III (ATIII). ATII and ATIII act on angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2). Cathepsins B, D, and G and chymase are also renin-activating enzymes. Cathepsins B and D also catalyze the conversion of pro(renin) into active renin; cathepsin D converts AGN to ATI; chymase converts ATI to ATII; while cathepsin G promotes generation of ATII from ATI and directly from AGN. Adapted with permission from Integrative Cancer Science and Therapeutics (32).

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  Figure 2. Representative hematoxylin and eosin stained slide of isocitrate dehydogenase-wildtype glioblastoma sample (A). 3,3-Diaminobezidine immunohistochemical-stained slides of representative glioblastoma multiforme sample showing ubiquitous cytoplasmic staining of cathepsin B [(B), brown] and cathepsin D [(C), brown]. Cathepsin G [(D), brown] was localized to cells within the microvessels but not the tumor cells. Nuclei were counter-stained with hematoxylin [(A–D), blue]. Original magnification: 400×.

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  Figure 3. Representative immunofluorescent immunohistochemical-stained sections of isocitrate dehydogenase-wildtype glioblastoma sample demonstrating the coexpression of cathepsin B [(A,B), red] with the OCT4+ [(A), green] and SALL4+ [(B), green] cancer stem cell (CSC) subpopulations. Cathepsin D [(C,D), red] was similarly expressed on the OCT4+ [(C), green] and SALL4+ [(D), green] CSC subpopulation. Cathepsin G [(E,F), red] was not expressed by the OCT4+ [(E), green] and SALL4+ [(F), green] CSC subpopulations. Cathepsin G [(G), red] was not expressed by the tryptase+ [(G), green] cells. Cell nuclei [(A–G), blue] are displayed by 4′6′-diamidino-2-phenylindole staining. Scale bars: 20 µm.

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  Figure 4. Expression of cathepsins B, D, and G messenger RNA transcripts in isocitrate dehydogenase-wildtype glioblastoma samples from six patients. Their expression was normalized over PGK1 housekeeping gene and presented as relative units.

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  Figure 5. Representative isocitrate dehydogenase-wildtype glioblastoma samples subjected to colorimetric in situ hybridization showing an abundance of cells expressing messenger RNA transcripts of cathepsin B [(A), red] and cathepsin D [(B), red] and minimal expression of cathepsin G [(C), red]. Nuclei were counter-stained with hematoxylin [(A–C), blue]. Original magnification: 1000×.

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  Figure 6. Mean percentages of expression of cathepsins B, D, and G from cell counting of DAB IHC-stained slides of all IDHWGB samples. The level of expression of cathepsin B is significantly higher than that of cathepsin D and cathepsin G. The level of expression of cathepsin D is significantly higher than that of cathepsin G.