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Extraction of Peptidoglycan from L. paracasei subp. Paracasei X12 and Its Preliminary Mechanisms of Inducing Immunogenic Cell Death in HT-29 Cells.

Tian PJ, Li BL, Shan YJ, Zhang JN, Chen JY, Yu M, Zhang LW - Int J Mol Sci (2015)

Bottom Line: X12-PG could induce the production of apoptotic bodies observed by transmission electron microscopy (TEM).X12-PG could significantly induced the translocation of calreticulin (CRT) and the release of high mobility group box 1 protein (HMGB1), the two notable hallmarks of immunogenic cell death (ICD), with the endoplastic reticulum (ER) damaged and subsequently intracellular [Ca(2+)] elevated.Our findings implied that X12-PG could induce the ICD of HT-29 cells through targeting at the ER.

View Article: PubMed Central - PubMed

Affiliation: School of Food Science and Engineering, Harbin Institute of Technology, No. 73 Huanghe Road, Harbin 150000, China. tianpei0202@gmail.com.

ABSTRACT
L. paracasei subp. paracasei X12 was previously isolated from a Chinese traditional fermented cheese with anticancer activities and probiotic potential. Herein, the integral peptidoglycan (X12-PG) was extracted by a modified trichloroacetic acid (TCA) method. X12-PG contained the four representative amino acids Asp, Glu, Ala and Lys, and displayed the similar lysozyme sensitivity, UV-visible scanning spectrum and molecular weight as the peptidoglycan standard. X12-PG could induce the production of apoptotic bodies observed by transmission electron microscopy (TEM). X12-PG could significantly induced the translocation of calreticulin (CRT) and the release of high mobility group box 1 protein (HMGB1), the two notable hallmarks of immunogenic cell death (ICD), with the endoplastic reticulum (ER) damaged and subsequently intracellular [Ca(2+)] elevated. Our findings implied that X12-PG could induce the ICD of HT-29 cells through targeting at the ER. The present results may enlighten the prospect of probiotics in the prevention of colon cancer.

No MeSH data available.


Related in: MedlinePlus

Calreticulin (CRT) exposure detected by immunofluoroscence assay after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h, then incubated with primary antibody (Anti-Calreticulin, diluted 1:200 in blocking buffer) and detected with the FITC-labeled secondary antibody. Fluorescence intensity was visualized by immunofluorescence microscopy (×200).
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ijms-16-20033-f005: Calreticulin (CRT) exposure detected by immunofluoroscence assay after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h, then incubated with primary antibody (Anti-Calreticulin, diluted 1:200 in blocking buffer) and detected with the FITC-labeled secondary antibody. Fluorescence intensity was visualized by immunofluorescence microscopy (×200).

Mentions: CRT is a high-affinity Ca2+-binding protein. The majority of cellular CRT is located in the ER, where it participates in modulating intracellular Ca2+ homeostasis and Ca2+ signaling. After stimulation (48 h) for X12-PG, CRT was exposed on the cell surface, as determined by immunofluorescence staining (Figure 5) or flow cytometry analysis (Figure 6). Sparse but clear fluorescence (Anti-CRT-FITC) on the membrane emerged when treated with 400 μg/mL X12-PG. Fluorescence intensity was enhanced when treated with a range of increasing concentrations, indicating more and more CRT translocated to the cell membrane (Figure 5). In identical conditions, the content of CRT was quantified by flow cytometry based on mean fluorescence intensity (MFI): CRT exposure significantly (p < 0.01) increased when treated with 1600 μg/mL X12-PG (Figure 6).


Extraction of Peptidoglycan from L. paracasei subp. Paracasei X12 and Its Preliminary Mechanisms of Inducing Immunogenic Cell Death in HT-29 Cells.

Tian PJ, Li BL, Shan YJ, Zhang JN, Chen JY, Yu M, Zhang LW - Int J Mol Sci (2015)

Calreticulin (CRT) exposure detected by immunofluoroscence assay after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h, then incubated with primary antibody (Anti-Calreticulin, diluted 1:200 in blocking buffer) and detected with the FITC-labeled secondary antibody. Fluorescence intensity was visualized by immunofluorescence microscopy (×200).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581339&req=5

ijms-16-20033-f005: Calreticulin (CRT) exposure detected by immunofluoroscence assay after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h, then incubated with primary antibody (Anti-Calreticulin, diluted 1:200 in blocking buffer) and detected with the FITC-labeled secondary antibody. Fluorescence intensity was visualized by immunofluorescence microscopy (×200).
Mentions: CRT is a high-affinity Ca2+-binding protein. The majority of cellular CRT is located in the ER, where it participates in modulating intracellular Ca2+ homeostasis and Ca2+ signaling. After stimulation (48 h) for X12-PG, CRT was exposed on the cell surface, as determined by immunofluorescence staining (Figure 5) or flow cytometry analysis (Figure 6). Sparse but clear fluorescence (Anti-CRT-FITC) on the membrane emerged when treated with 400 μg/mL X12-PG. Fluorescence intensity was enhanced when treated with a range of increasing concentrations, indicating more and more CRT translocated to the cell membrane (Figure 5). In identical conditions, the content of CRT was quantified by flow cytometry based on mean fluorescence intensity (MFI): CRT exposure significantly (p < 0.01) increased when treated with 1600 μg/mL X12-PG (Figure 6).

Bottom Line: X12-PG could induce the production of apoptotic bodies observed by transmission electron microscopy (TEM).X12-PG could significantly induced the translocation of calreticulin (CRT) and the release of high mobility group box 1 protein (HMGB1), the two notable hallmarks of immunogenic cell death (ICD), with the endoplastic reticulum (ER) damaged and subsequently intracellular [Ca(2+)] elevated.Our findings implied that X12-PG could induce the ICD of HT-29 cells through targeting at the ER.

View Article: PubMed Central - PubMed

Affiliation: School of Food Science and Engineering, Harbin Institute of Technology, No. 73 Huanghe Road, Harbin 150000, China. tianpei0202@gmail.com.

ABSTRACT
L. paracasei subp. paracasei X12 was previously isolated from a Chinese traditional fermented cheese with anticancer activities and probiotic potential. Herein, the integral peptidoglycan (X12-PG) was extracted by a modified trichloroacetic acid (TCA) method. X12-PG contained the four representative amino acids Asp, Glu, Ala and Lys, and displayed the similar lysozyme sensitivity, UV-visible scanning spectrum and molecular weight as the peptidoglycan standard. X12-PG could induce the production of apoptotic bodies observed by transmission electron microscopy (TEM). X12-PG could significantly induced the translocation of calreticulin (CRT) and the release of high mobility group box 1 protein (HMGB1), the two notable hallmarks of immunogenic cell death (ICD), with the endoplastic reticulum (ER) damaged and subsequently intracellular [Ca(2+)] elevated. Our findings implied that X12-PG could induce the ICD of HT-29 cells through targeting at the ER. The present results may enlighten the prospect of probiotics in the prevention of colon cancer.

No MeSH data available.


Related in: MedlinePlus