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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

Intracellular Ca2+ concentration ([Ca2+]) after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h. Cytosolic [Ca2+] was increased in a dose-dependent manner after exposure to X12-PG. HT-29 cells were loaded with Fluo-3/AM and analyzed by flow cytometry. Fluo-3/AM was excited at the 488 nm line of an argon laser and the fluorescence intensity was measured at an emission wavelength 530 nm. [Ca2+] was expressed as mean fluorescence intensity (MFI). (Error bars are mean ± SD, *p < 0.05 versus control, **p < 0.01 versus control).
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ijms-16-20033-f007: Intracellular Ca2+ concentration ([Ca2+]) after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h. Cytosolic [Ca2+] was increased in a dose-dependent manner after exposure to X12-PG. HT-29 cells were loaded with Fluo-3/AM and analyzed by flow cytometry. Fluo-3/AM was excited at the 488 nm line of an argon laser and the fluorescence intensity was measured at an emission wavelength 530 nm. [Ca2+] was expressed as mean fluorescence intensity (MFI). (Error bars are mean ± SD, *p < 0.05 versus control, **p < 0.01 versus control).

Mentions: We next examined the correlation among cytosolic free Ca2+, CRT exposure and ER dysfunction. Normally, Ca2+ is stored in the ER where physiological activities are regulated through the uptake or release of Ca2+. Intracellular Ca2+ concentration ([Ca2+]) was measured with the fluorescent probe Fluo-3/AM by flow cytometry. The MFI of [Ca2+] in the cytoplasm were increased by 18% (437.5 ± 6.5), 35% (499.5 ± 4.5) and 85% (681.5 ± 3.5, p < 0.01) respectively, compared with the control group (369.5 ± 8.5) after exposure to 400, 800, 1600 μg/mL X12-PG for 48 h (Figure 7). Before this, we had observed X12-PG-induced structural damage of ER, as well as CRT translocation. Although the mechanisms accounting for CRT exposure remain largely elusive, based on the data summarized in this work, we assumed that ER [Ca2+] homeostasis plays a major role in the regulation of CRT exposure as it is induced by X12-PG.


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)

Intracellular Ca2+ concentration ([Ca2+]) after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h. Cytosolic [Ca2+] was increased in a dose-dependent manner after exposure to X12-PG. HT-29 cells were loaded with Fluo-3/AM and analyzed by flow cytometry. Fluo-3/AM was excited at the 488 nm line of an argon laser and the fluorescence intensity was measured at an emission wavelength 530 nm. [Ca2+] was expressed as mean fluorescence intensity (MFI). (Error bars are mean ± SD, *p < 0.05 versus control, **p < 0.01 versus control).
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-20033-f007: Intracellular Ca2+ concentration ([Ca2+]) after treatment of X12-PG. (A–D) were respectively treated with 0, 400, 800, 1600 μg/mL X12-PG for 48 h. Cytosolic [Ca2+] was increased in a dose-dependent manner after exposure to X12-PG. HT-29 cells were loaded with Fluo-3/AM and analyzed by flow cytometry. Fluo-3/AM was excited at the 488 nm line of an argon laser and the fluorescence intensity was measured at an emission wavelength 530 nm. [Ca2+] was expressed as mean fluorescence intensity (MFI). (Error bars are mean ± SD, *p < 0.05 versus control, **p < 0.01 versus control).
Mentions: We next examined the correlation among cytosolic free Ca2+, CRT exposure and ER dysfunction. Normally, Ca2+ is stored in the ER where physiological activities are regulated through the uptake or release of Ca2+. Intracellular Ca2+ concentration ([Ca2+]) was measured with the fluorescent probe Fluo-3/AM by flow cytometry. The MFI of [Ca2+] in the cytoplasm were increased by 18% (437.5 ± 6.5), 35% (499.5 ± 4.5) and 85% (681.5 ± 3.5, p < 0.01) respectively, compared with the control group (369.5 ± 8.5) after exposure to 400, 800, 1600 μg/mL X12-PG for 48 h (Figure 7). Before this, we had observed X12-PG-induced structural damage of ER, as well as CRT translocation. Although the mechanisms accounting for CRT exposure remain largely elusive, based on the data summarized in this work, we assumed that ER [Ca2+] homeostasis plays a major role in the regulation of CRT exposure as it is induced by X12-PG.

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