Expression and Localization of Cathepsins B, D, and G in Dupuytren’s Disease

Tan, Kirin; Brasch, Helen D.; Schaijik, Bede van; Armstrong, J. R.; Marsh, Reginald; Davis, Paul F.; Tan, Swee T.; Itinteang, Tinte

doi:10.1097/gox.0000000000001686

Cited by 16 publications

(13 citation statements)

References 42 publications

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“…Western blot (WB) analysis was performed on five snap-frozen WHO grade I MG samples from the original cohort of 10 patients included for DAB IHC staining. Total protein was extracted and precipitated from the tissue samples, as previously reported (35), followed by gel electrophoresis on a 4–12% 1D PAGE (cat# NW04120BOX, Thermo Fisher Scientific) with transfer to a PVDF membrane (cat# IB24001, Invitrogen, Carlsbad, CA, USA) using an iBlot 2 (cat# IB21001, Thermo Fisher Scientific). Detection of the proteins was performed on the iBind Flex (cat# SLF2000, Thermo Fisher Scientific) using the primary antibodies for cathepsin B (1:250; cat# SC-6490-R, Santa Cruz), cathepsin D (1:250; cat# SC-6486, Santa Cruz), cathepsin G (1:250; cat# ab197354, Abcam, Cambridge, UK), and α-tubulin (1:1000; cat# 62204, Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Expression of Cathepsins B, D, and G in WHO Grade I Meningioma

et al. 2019

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Aim: We have recently demonstrated the presence of putative tumor stem cells (TSCs) in World Health Organization (WHO) grade I meningioma (MG) localized to the microvessels, which expresses components of the renin-angiotensin system (RAS). The RAS is known to be dysregulated and promotes tumorigenesis in many cancer types, including glioblastoma. Cathepsins B, D, and G are isoenzymes that catalyze the production of angiotensin peptides, hence providing bypass loops for the RAS. This study investigated the expression of cathepsins B, D, and G in WHO grade I MG in relation to the putative TSC population we have previously demonstrated. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining with antibodies for cathepsins B, D, and G was performed on WHO grade I MG tissue samples from 10 patients. Three of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining to investigate co-expression of each of these cathepsins using combinations of smooth muscle actin (SMA) and embryonic stem cell marker OCT4. NanoString mRNA expression ( n = 6) and Western blotting (WB; n = 5) analyses, and enzyme activity assays (EAAs; n = 3), were performed on snap-frozen WHO grade I MG tissue samples to confirm transcriptional activation, protein expression, and functional activity of these proteins, respectively. Results: DAB IHC staining demonstrated expression of cathepsins B, D, and G in all 10 MG samples. NanoString mRNA expression and WB analyses showed transcriptional activation and protein expression of all three cathepsins, although cathepsin G was expressed at low levels. EAAs demonstrated that cathepsin B and cathepsin D were functionally active. IF IHC staining illustrated localization of cathepsin B and cathepsin D to the endothelium and SMA + pericyte layer of the microvessels, while cathepsin G was localized to cells scattered within the interstitium, away from the microvessels. Conclusion: Cathepsin B and cathepsin D, and to a lesser extent cathepsin G, are expressed in WHO grade I MG. Cathepsin B and cathepsin D are enzymatically active and are localized to the putative TSC population on the microvessels, whereas cathepsin G was localized to cells scattered within the interstitium, These results suggest the presence of bypass loops for the RAS, within WHO grade I MG.

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Section: Methodsmentioning

confidence: 99%

Expression of Cathepsins B, D, and G in WHO Grade I Meningioma

et al. 2019

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“…Total protein was extracted from three snap-frozen LMCA tissue samples from the original cohort of 16 patients used for DAB IHC staining, resolved by SDS-PAGE, and transferred to a PVDF membrane as previously described [31] , and probed using the following antibodies: PRR (1:500, cat# ab40790, Abcam), ATIIR1 (1:500; cat# sc-1173, Santa Cruz, CA, USA), ATIIR2 (1:1000; cat# ab92445, Abcam), ACE (1:200; cat# sc-12184, Santa Cruz), and β-actin (1:500; cat# ab8229, Abcam). Secondary and tertiary antibodies were goat anti-rabbit HRP (1:2000; cat# ab6721, Abcam) for PRR and ATIIR1, rabbit anti-goat Superclonal TM biotin conjugate (1:5000; cat# A27013, Thermo Fisher) and goat anti-rabbit Superclonal TM biotin conjugate (1:5000; cat# A27035, Thermo Fisher) followed by Pierce TM Streptavidin Poly-HRP (1:5000, cat# 21140, Thermo Fisher) for ACE and ATIIR2, respectively, and chicken anti-goat Alexa Fluor 647 (1:2000; cat# A21244, Life Technologies) for β-actin.…”

Section: Western Blottingmentioning

confidence: 99%

Cancer stem cells in liver metastasis from colon adenocarcinoma express components of the renin-angiotensin system

Narayanan¹,

Wickremesekera²,

Schaijik³

et al. 2019

JCMT

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Aim: We have recently identified a cancer stem cell (CSC) subpopulations within the tumor nests (TNs) and another within the peritumoral stroma (PTS) in liver metastasis from colon adenocarcinoma (LMCA). This study investigated the expression of components of the renin-angiotensin (RAS): pro-renin receptor (PRR), angiotensin converting enzyme (ACE), and angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2) in LMCA. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on 16 LMCA samples for PRR, ACE, ATIIR1 and ATIIR2. Immunofluorescence (IF) IHC staining was performed to investigate co-expression of these components of the RAS with SOX2 or OCT4. NanoString analysis (n = 6) and Western blotting (WB, n = 3) were performed on snap-frozen LMCA samples to confirm mRNA and protein expression, respectively. Results: DAB IHC staining showed the expression of PRR, ACE, ATIIR1 and ATIIR2 within all LMCA samples. NanoString analysis and WB confirmed gene and protein expression of these components of the RAS. IF IHC staining demonstrated expression of PRR, ATIIR1 and ATIIR2 by the SOX2 + CSCs within the TNs and the OCT4 + CSCs within the PTS. ACE was expressed on the endothelium of the microvessels within the PTS. Conclusion: These finding suggests the CSCs within LMCA maybe a novel therapeutic target by manipulation of the RAS.

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“…Total protein was extracted from six snap-frozen MG samples of the original cohort of 11 patients used for DAB IHC staining, separated by SDS-PAGE, and transferred to a PVDF membrane using methods previously described (16). Detection of the proteins was performed on the iBind Flex (cat# SLF2000, Thermo Fisher Scientific) using the primary antibodies for PRR (ATP6IP2, 1:500, cat# ab40790, Abcam), ATIIR1 (1:500; cat# sc-1173, Santa Cruz, CA, USA), ATIIR2 (1:1000; cat# ab92445, Abcam), ACE (1:200; cat# sc-12184, Santa Cruz), and α-tubulin (1:1000; cat# 62204, Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

“…ATII binds to ATII receptor 1 (ATIIR1) and ATII receptor 2 (ATIIR2) (14). The expression of components of the RAS on the tumor stem cells in a number of cancer types including oral cavity squamous cell carcinoma (15) and GB (16) implies a crucial role for the RAS in the maintenance and regulation of their behavior.…”

Section: Introductionmentioning

confidence: 99%

Expression of Components of the Renin-Angiotensin System by the Putative Stem Cell Population Within WHO Grade I Meningioma

et al. 2019

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Aim: We have recently demonstrated a putative stem cell population within WHO grade I meningioma (MG) that expressed embryonic stem cell (ESC) markers OCT4, NANOG, SOX2, KLF4 and c-MYC, localized to the endothelial and pericyte layers of the microvessels. There is increasing recognition that the renin-angiotensin system (RAS) plays a critical role in stem cell biology and tumorigenesis. This study investigated the expression of components of the RAS: pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) on the putative stem cell population on the microvessels of WHO grade I MG. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on WHO grade I MG tissue samples from 11 patients for PRR, ACE, ATIIR1, and ATIIR2. Two of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining to investigate co-expression of each of these components of the RAS in using combinations of CD34 and ESC marker SOX2 or OCT4. NanoString mRNA expression analysis and Western blotting (WB), were performed on six snap-frozen MG tissue samples to confirm mRNA and protein expression of these proteins, respectively. Results: DAB IHC staining demonstrated expression of PRR, ACE, ATIIR1, and ATIIR2 within all 11 MG tissue samples. WB and NanoString mRNA analyses, confirmed protein and mRNA expression of these proteins, respectively. IF IHC staining showed PRR, ATIIR1 and ATIIR2 were localized to the OCT4 + and SOX2 + endothelium and the pericyte layer of MG while ACE was localized to the OCT4 + endothelium of the microvesels. Conclusion: The novel finding of the expression of PRR, ACE, ATIIR1, and ATIIR2 on the putative stem cell population on the microvessels of WHO grade I MG, suggests that these stem cells may be a potential therapeutic target by manipulation of the RAS.

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Expression and Localization of Cathepsins B, D, and G in Dupuytren’s Disease

Abstract: Supplemental Digital Content is available in the text.

Cited by 16 publications

References 42 publications

Expression of Cathepsins B, D, and G in WHO Grade I Meningioma

Expression of Cathepsins B, D, and G in WHO Grade I Meningioma

Cancer stem cells in liver metastasis from colon adenocarcinoma express components of the renin-angiotensin system

Expression of Components of the Renin-Angiotensin System by the Putative Stem Cell Population Within WHO Grade I Meningioma

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