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Cancer Stem Cells in Moderately Differentiated Buccal Mucosal Squamous Cell Carcinoma Express Components of the Renin – Angiotensin System

View Article: PubMed Central - PubMed

ABSTRACT

Aim: We have recently identified and characterized cancer stem cell (CSC) subpopulations within moderately differentiated buccal mucosal squamous cell carcinoma (MDBMSCC). We hypothesized that these CSCs express components of the renin–angiotensin system (RAS).

Methods: 3,3′-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on formalin-fixed paraffin-embedded MDBMSCC samples to investigate the expression of the components of the RAS: (pro)renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2). NanoString mRNA gene expression analysis and Western Blotting (WB) were performed on snap-frozen MDBMSCC samples to confirm gene expression and translation of these transcripts, respectively. Double immunofluorescent (IF) IHC staining of these components of the RAS with the embryonic stem cell markers OCT4 or SALL4 was performed to demonstrate their localization in relation to the CSC subpopulations within MDBMSCC.

Results: DAB IHC staining demonstrated expression of PRR, ACE, ATIIR1, and ATIIR2 in MDBMSCC. IF IHC staining showed that PRR was expressed by the CSC subpopulations within the tumor nests, the peri-tumoral stroma, and the endothelium of the microvessels within the peri-tumoral stroma. ATIIR1 and ATIIR2 were localized to the CSC subpopulations within the tumor nests and the peri-tumoral stroma, while ACE was localized to the endothelium of the microvessels within the peri-tumoral stroma. WB and NanoString analyses confirmed protein expression and transcription activation of PRR, ACE, and ATIIR1, but not of ATIIR2, respectively.

Conclusion: Our novel findings of the presence and localization of PRR, ACE, ATIIR1, and potentially ATIIR2 to the CSC subpopulations within MDBMSCC suggest CSC as a therapeutic target by modulation of the RAS.

No MeSH data available.


Related in: MedlinePlus

Western Blot images of 1DE separated moderately differentiated buccal mucosal squamous cell carcinoma total protein extracts probed for PRR (A), ATIIR1 (B), ACE (C), ATIIR2 (D), and detected with HRP conjugated goat anti-rabbit (A,B,D) or donkey anti-goat (C), or rabbit anti-rabbit (E) secondary antibody. β-actin (E) was used as the loading control and detected using Alexafluor® 647 rabbit anti-mouse secondary antibody.
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Figure 3: Western Blot images of 1DE separated moderately differentiated buccal mucosal squamous cell carcinoma total protein extracts probed for PRR (A), ATIIR1 (B), ACE (C), ATIIR2 (D), and detected with HRP conjugated goat anti-rabbit (A,B,D) or donkey anti-goat (C), or rabbit anti-rabbit (E) secondary antibody. β-actin (E) was used as the loading control and detected using Alexafluor® 647 rabbit anti-mouse secondary antibody.

Mentions: Western Blot analysis confirmed the presence of PRR (Figure 3A) and ATIIR1 (Figure 3B) within the extracts of all five MDBMSCC samples from the original cohort of patients used for DAB IHC staining, at their expected molecular weight of 39 and 43 kDa, respectively. ACE (Figure 3C) was detected in only one of the five MDMBSCC samples analyzed, at the expected molecular weight of 195 kDa. High image exposure was required to clearly visualize the ACE signal in the MDMBSCC sample, which implies that ACE was present at very low abundance. ATIIR2 was detected in all five MDBMSCC samples at approximately 51 kDa (Figure 3D), which is larger than the native form of the protein and may signify detection of a glycosylated isoform. A band at approximately 41 kDa was detected in one of the five total protein extracts (Figure 3D, BMSCC_3) that may correspond to the native protein (27). Detection of β-actin confirmed approximately equal total protein loading across all five samples (Figure 3E).


Cancer Stem Cells in Moderately Differentiated Buccal Mucosal Squamous Cell Carcinoma Express Components of the Renin – Angiotensin System
Western Blot images of 1DE separated moderately differentiated buccal mucosal squamous cell carcinoma total protein extracts probed for PRR (A), ATIIR1 (B), ACE (C), ATIIR2 (D), and detected with HRP conjugated goat anti-rabbit (A,B,D) or donkey anti-goat (C), or rabbit anti-rabbit (E) secondary antibody. β-actin (E) was used as the loading control and detected using Alexafluor® 647 rabbit anti-mouse secondary antibody.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037224&req=5

Figure 3: Western Blot images of 1DE separated moderately differentiated buccal mucosal squamous cell carcinoma total protein extracts probed for PRR (A), ATIIR1 (B), ACE (C), ATIIR2 (D), and detected with HRP conjugated goat anti-rabbit (A,B,D) or donkey anti-goat (C), or rabbit anti-rabbit (E) secondary antibody. β-actin (E) was used as the loading control and detected using Alexafluor® 647 rabbit anti-mouse secondary antibody.
Mentions: Western Blot analysis confirmed the presence of PRR (Figure 3A) and ATIIR1 (Figure 3B) within the extracts of all five MDBMSCC samples from the original cohort of patients used for DAB IHC staining, at their expected molecular weight of 39 and 43 kDa, respectively. ACE (Figure 3C) was detected in only one of the five MDMBSCC samples analyzed, at the expected molecular weight of 195 kDa. High image exposure was required to clearly visualize the ACE signal in the MDMBSCC sample, which implies that ACE was present at very low abundance. ATIIR2 was detected in all five MDBMSCC samples at approximately 51 kDa (Figure 3D), which is larger than the native form of the protein and may signify detection of a glycosylated isoform. A band at approximately 41 kDa was detected in one of the five total protein extracts (Figure 3D, BMSCC_3) that may correspond to the native protein (27). Detection of β-actin confirmed approximately equal total protein loading across all five samples (Figure 3E).

View Article: PubMed Central - PubMed

ABSTRACT

Aim: We have recently identified and characterized cancer stem cell (CSC) subpopulations within moderately differentiated buccal mucosal squamous cell carcinoma (MDBMSCC). We hypothesized that these CSCs express components of the renin–angiotensin system (RAS).

Methods: 3,3′-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on formalin-fixed paraffin-embedded MDBMSCC samples to investigate the expression of the components of the RAS: (pro)renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2). NanoString mRNA gene expression analysis and Western Blotting (WB) were performed on snap-frozen MDBMSCC samples to confirm gene expression and translation of these transcripts, respectively. Double immunofluorescent (IF) IHC staining of these components of the RAS with the embryonic stem cell markers OCT4 or SALL4 was performed to demonstrate their localization in relation to the CSC subpopulations within MDBMSCC.

Results: DAB IHC staining demonstrated expression of PRR, ACE, ATIIR1, and ATIIR2 in MDBMSCC. IF IHC staining showed that PRR was expressed by the CSC subpopulations within the tumor nests, the peri-tumoral stroma, and the endothelium of the microvessels within the peri-tumoral stroma. ATIIR1 and ATIIR2 were localized to the CSC subpopulations within the tumor nests and the peri-tumoral stroma, while ACE was localized to the endothelium of the microvessels within the peri-tumoral stroma. WB and NanoString analyses confirmed protein expression and transcription activation of PRR, ACE, and ATIIR1, but not of ATIIR2, respectively.

Conclusion: Our novel findings of the presence and localization of PRR, ACE, ATIIR1, and potentially ATIIR2 to the CSC subpopulations within MDBMSCC suggest CSC as a therapeutic target by modulation of the RAS.

No MeSH data available.


Related in: MedlinePlus