Background Endoplasmic reticulum （ER） stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin （PQS） is able to inhibit excessive ER stress-relat...Background Endoplasmic reticulum （ER） stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin （PQS） is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured eardiomyocytes were stimulated with thapsigargin （TG）, which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration. Methods Primary cultured cardiomyocytes from neonatal rats were exposed to TG （1 μmol/L） treatment for 24 h, following PQS pre-treatment （160 μg/mL） for 24 h or pre-treatment with small interfering RNA directed against protein kinase-like endoplasmic reticulum kinase （Si-PERK） for 6 h. The viability and apoptosis rate of cardiomyocytes were detected by cell counting kit-8 and flow cytometry respectively. ER stress-related protein expression, such as glucose-regulated protein 78 （GRP78）, calreticulin, PERK, eukaryotic translation initiation factor 2α （elF2c0, activating transcription factor 4 （ATF4）, and C/EBP homologous protein （CHOP） were assayed by western blotting. Results Both PQS pre-treatment and PERK knockdown remarkably inhibited the cardiomyocyte apoptosis induced by TG, increased cell viability, decreased phosphorylation of both PERK and eIF2α, and decreased protein levels of both ATF4 and CHOP. There was no statistically significant difference between PQS pre-treatment and PERK knockdown in the cardioprotective effect. Conclusions Our data indicate that the PERK-eIF2α-ATF4-CHOP pathway of ER stress is involved in the apoptosis induced by TG, and PQS might prevent TG-induced cardiomyocyte apoptosis through a mechanism involving the suppression of this pathway. These findings provide novel data regarding the molecular mechanisms by which PQS inhibits cardiomyocyte apoptosis.展开更多
Localized prostate cancer is curable via annihilation of the entire cancer neighborhood by surgery or local radiation.Unfortunately,once metastatic,no available therapy is curative.The vast majority will die despite a...Localized prostate cancer is curable via annihilation of the entire cancer neighborhood by surgery or local radiation.Unfortunately,once metastatic,no available therapy is curative.The vast majority will die despite aggressive systemic combinational androgenablation therapies.Thus,there is an urgent need for effective systemic therapeutics that sterilize the entire microenvironment in metastatic castration-resistant prostate cancer(mCRPC).To accomplish this goal,advantage can be taken of the unique biology of mCRPC cells.Like their normal cell of origin,mCRPCs retain expression of the prostate-specific differentiation protein,prostate-specific antigen(PSA),which they abundantly secrete into their extracellular fluid(ECF).This unique,and essentially universal,secretion of enzymatically active PSA into the ECF by mCRPCs creates an exploitable therapeutic index for activation of systemically delivered highly lipophilic toxins as“molecular grenades”covalently linked to cysteine-34 of human serum albumin(HSA)via a stable maleimide containing PSA cleavable peptide such that PSA-dependent hydrolysis(i.e.,“detonation”)releases the grenades restrictively within the ECF of mCRPC.This approach decreases dose-limiting host toxicity while enhancing plasma half-life from minutes to days(i.e.,pharmacokinetic effect)and increasing the tissue concentration of the maleimide coupled albumin delivery(MAD)in the ECF at sites of cancer due to the enhanced permeability of albumin at these sites(i.e.,enhanced permeability and retention effect).This allows the MAD-PSA detonated grenades to circulate throughout the body in a non-toxic form.Only within sites of mCRPC is there a sufficiently high level of enzymatically active PSA to efficiently“pull the pin”on the grenades releasing their lipophilic cellpenetrant toxins from HSA.Thus,if a sufficient level of“detonation”occurs,this will kill mCRPC cells,and sterilize the entire PSA-rich metastatic sites via a bystander effect.In this review,two examples of such MAD-PSA detonated molecular grenades are presenteddone based upon thapsigagin and the other on niclosamide.展开更多
Effects of thapsigargin, an inhibitor of Ca2+-ATPase in surface of endoplasmic reticulum, on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron mi...Effects of thapsigargin, an inhibitor of Ca2+-ATPase in surface of endoplasmic reticulum, on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron mi-croscopy Propidium iodide staining and flow cytome- try revealed that in the serum-free condition, thapsigar-gin increased the rate of apoptosis of BEL- 7404 cells in a dose-dependent manner. Prolongation of the period of serum-free condition enhanced the apoptosis induced by thapsigargin treatment. Morphological observation with electron microscope further demonstrated that chromatin condensation and fragmentation, apoptotic bodies existed in TG-treated cells, supporting that thapsigargin is a po-tent activator of apoptosis in the cells.展开更多
基金Acknowledgements This work was supported by International Science and Technology Cooperation Project （2010DFA31690）, National Natural Science Foundation of China （81030063 and 81170140） and China Postdoctoral Science Foundation （2014M562608）. The authors declare no conflict of interests regarding the publication of this paper.
文摘Background Endoplasmic reticulum （ER） stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin （PQS） is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured eardiomyocytes were stimulated with thapsigargin （TG）, which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration. Methods Primary cultured cardiomyocytes from neonatal rats were exposed to TG （1 μmol/L） treatment for 24 h, following PQS pre-treatment （160 μg/mL） for 24 h or pre-treatment with small interfering RNA directed against protein kinase-like endoplasmic reticulum kinase （Si-PERK） for 6 h. The viability and apoptosis rate of cardiomyocytes were detected by cell counting kit-8 and flow cytometry respectively. ER stress-related protein expression, such as glucose-regulated protein 78 （GRP78）, calreticulin, PERK, eukaryotic translation initiation factor 2α （elF2c0, activating transcription factor 4 （ATF4）, and C/EBP homologous protein （CHOP） were assayed by western blotting. Results Both PQS pre-treatment and PERK knockdown remarkably inhibited the cardiomyocyte apoptosis induced by TG, increased cell viability, decreased phosphorylation of both PERK and eIF2α, and decreased protein levels of both ATF4 and CHOP. There was no statistically significant difference between PQS pre-treatment and PERK knockdown in the cardioprotective effect. Conclusions Our data indicate that the PERK-eIF2α-ATF4-CHOP pathway of ER stress is involved in the apoptosis induced by TG, and PQS might prevent TG-induced cardiomyocyte apoptosis through a mechanism involving the suppression of this pathway. These findings provide novel data regarding the molecular mechanisms by which PQS inhibits cardiomyocyte apoptosis.
基金We would like to acknowledge the Prostate Cancer Foundation,Department of Defense Prostate Cancer Research Program(W81XWH-16-1-0410)NIH Prostate SPORE(P50 CA058236)Patrick C.Walsh Prostate Cancer Research Fund,and the Hopkins-Allegheny Health Network Cancer Research Fund。
文摘Localized prostate cancer is curable via annihilation of the entire cancer neighborhood by surgery or local radiation.Unfortunately,once metastatic,no available therapy is curative.The vast majority will die despite aggressive systemic combinational androgenablation therapies.Thus,there is an urgent need for effective systemic therapeutics that sterilize the entire microenvironment in metastatic castration-resistant prostate cancer(mCRPC).To accomplish this goal,advantage can be taken of the unique biology of mCRPC cells.Like their normal cell of origin,mCRPCs retain expression of the prostate-specific differentiation protein,prostate-specific antigen(PSA),which they abundantly secrete into their extracellular fluid(ECF).This unique,and essentially universal,secretion of enzymatically active PSA into the ECF by mCRPCs creates an exploitable therapeutic index for activation of systemically delivered highly lipophilic toxins as“molecular grenades”covalently linked to cysteine-34 of human serum albumin(HSA)via a stable maleimide containing PSA cleavable peptide such that PSA-dependent hydrolysis(i.e.,“detonation”)releases the grenades restrictively within the ECF of mCRPC.This approach decreases dose-limiting host toxicity while enhancing plasma half-life from minutes to days(i.e.,pharmacokinetic effect)and increasing the tissue concentration of the maleimide coupled albumin delivery(MAD)in the ECF at sites of cancer due to the enhanced permeability of albumin at these sites(i.e.,enhanced permeability and retention effect).This allows the MAD-PSA detonated grenades to circulate throughout the body in a non-toxic form.Only within sites of mCRPC is there a sufficiently high level of enzymatically active PSA to efficiently“pull the pin”on the grenades releasing their lipophilic cellpenetrant toxins from HSA.Thus,if a sufficient level of“detonation”occurs,this will kill mCRPC cells,and sterilize the entire PSA-rich metastatic sites via a bystander effect.In this review,two examples of such MAD-PSA detonated molecular grenades are presenteddone based upon thapsigagin and the other on niclosamide.
文摘Effects of thapsigargin, an inhibitor of Ca2+-ATPase in surface of endoplasmic reticulum, on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron mi-croscopy Propidium iodide staining and flow cytome- try revealed that in the serum-free condition, thapsigar-gin increased the rate of apoptosis of BEL- 7404 cells in a dose-dependent manner. Prolongation of the period of serum-free condition enhanced the apoptosis induced by thapsigargin treatment. Morphological observation with electron microscope further demonstrated that chromatin condensation and fragmentation, apoptotic bodies existed in TG-treated cells, supporting that thapsigargin is a po-tent activator of apoptosis in the cells.
