Limits...
Endoplasmic reticulum stress-induced resistance to doxorubicin is reversed by paeonol treatment in human hepatocellular carcinoma cells.

Fan L, Song B, Sun G, Ma T, Zhong F, Wei W - PLoS ONE (2013)

Bottom Line: Interestingly, co-pretreatment with tunicamycin and Pae significantly increased apoptosis induced by doxorubicin.Furthermore, induction of ER stress resulted in increasing expression of COX-2 concomitant with inactivation of Akt and up-regulation of the pro-apoptotic transcription factor CHOP (GADD153) in HepG2 cells.However, co-pretreatment with tunicamycin and Pae decreased the expression of COX-2 and levels of activation of Akt as well as increasing the levels of CHOP in HCC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.

ABSTRACT

Background: Endoplasmic reticulum stress (ER stress) is generally activated in solid tumors and results in tumor cell anti-apoptosis and drug resistance. Paeonol (Pae, 2-hydroxy-4-methoxyacetophenone), is a natural product extracted from the root of Paeonia Suffruticosa Andrew. Although Pae displays anti-neoplastic activity and increases the efficacy of chemotherapeutic drugs in various cell lines and in animal models, studies related to the effect of Pae on ER stress-induced resistance to chemotherapeutic agents in hepatocellular carcinoma (HCC) are poorly understood.

Methodology/principal findings: In this study, we investigated the effect of the endoplasmic reticulum (ER) stress response during resistance of human hepatocellular carcinoma cells to doxorubicin. Treatment with the ER stress-inducer tunicamycin (TM) before the addition of doxorubicin reduced the rate of apoptosis induced by doxorubicin. Interestingly, co-pretreatment with tunicamycin and Pae significantly increased apoptosis induced by doxorubicin. Furthermore, induction of ER stress resulted in increasing expression of COX-2 concomitant with inactivation of Akt and up-regulation of the pro-apoptotic transcription factor CHOP (GADD153) in HepG2 cells. These cellular changes in gene expression and Akt activation may be an important resistance mechanism against doxorubicin in hepatocellular carcinoma cells undergoing ER stress. However, co-pretreatment with tunicamycin and Pae decreased the expression of COX-2 and levels of activation of Akt as well as increasing the levels of CHOP in HCC cells.

Conclusions/significance: Our results demonstrate that Pae reverses ER stress-induced resistance to doxorubicin in human hepatocellular carcinoma cells by targeting COX-2 mediated inactivation of PI3K/AKT/CHOP.

Show MeSH

Related in: MedlinePlus

Effect of co-pretreatment with Pae and tunicamycin on apoptosis induced by doxorubicin in HepG2 cells.(A) HepG2 cells were treated with 3 µmol/L tunicamycin for 8 hr, either in the absence or the presence of 31.25 mg/L Pae and then exposed to doxorubicin (2.5 mg/L) for 24 hr. Apoptosis was analyzed as the sub-G1 fraction by fluorescence-activated cell sorting (FACS). a: Untreated HepG2 cells; b: HepG2 cells treated with doxorubicin alone; c: HepG2 cells co-pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr d:HepG2 cells co-pretreated with tunicamycin and Pae, and then exposed to doxorubicin for 24 hr. (B) and(C) Cell morphology and percentage of apoptotic cells was examined by TUNEL staining. a: Untreated HepG2 cells; b: HepG2 cells treated with doxorubicin alone; c: HepG2 cells pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr d:HepG2 cells co-pretreated with tunicamycin and Pae and then exposed to doxorubicin for 24 hr.Data are presented as mean ± SD for the three independent experiments. (**P<0.01 compared with HepG2 cells treated with doxorubicin alone, ##P<0.01 compared with HepG2 cells pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr). (D) Cleaved caspase-3 as an apoptotic marker were measured by western blot using specific anti- caspase-3 antibody. β-actin in the same HepG2 cells extract was used as an internal reference.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3643935&req=5

pone-0062627-g003: Effect of co-pretreatment with Pae and tunicamycin on apoptosis induced by doxorubicin in HepG2 cells.(A) HepG2 cells were treated with 3 µmol/L tunicamycin for 8 hr, either in the absence or the presence of 31.25 mg/L Pae and then exposed to doxorubicin (2.5 mg/L) for 24 hr. Apoptosis was analyzed as the sub-G1 fraction by fluorescence-activated cell sorting (FACS). a: Untreated HepG2 cells; b: HepG2 cells treated with doxorubicin alone; c: HepG2 cells co-pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr d:HepG2 cells co-pretreated with tunicamycin and Pae, and then exposed to doxorubicin for 24 hr. (B) and(C) Cell morphology and percentage of apoptotic cells was examined by TUNEL staining. a: Untreated HepG2 cells; b: HepG2 cells treated with doxorubicin alone; c: HepG2 cells pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr d:HepG2 cells co-pretreated with tunicamycin and Pae and then exposed to doxorubicin for 24 hr.Data are presented as mean ± SD for the three independent experiments. (**P<0.01 compared with HepG2 cells treated with doxorubicin alone, ##P<0.01 compared with HepG2 cells pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr). (D) Cleaved caspase-3 as an apoptotic marker were measured by western blot using specific anti- caspase-3 antibody. β-actin in the same HepG2 cells extract was used as an internal reference.

Mentions: To determine the effects of Pae on ER stress–induced resistance to doxorubicin, HepG2 cells were pretreated with Pae for 8 hr in the presence of tunicamycin, then subjected to doxorubicin treatment for 24 hr. Apoptosis was assessed by FACS analysis (Figure 3A), TUNEL staining (Figure 3B and 3C) and Western Blot (Figure 3D). Apoptosis induced by doxorubicin was increased by pretreatment of tunicamycin and Pae together. In the group treated with tunicamycin and Pae together the sub-G1 population increased sharply to 50.19% compared to cells pretreated with tunicamycin alone. Similar results were observed with TUNEL staining. In additon, When HepG2 cells were pretreated with tunicamycin and Pae combination treatment, significantly higher levels of cleaved caspase 3 were observed compared to HepG2 cells pretreated with tunicamycin.Together, these results support the inhibitory effect of Pae on ER stress–resistance to doxorubicin.


Endoplasmic reticulum stress-induced resistance to doxorubicin is reversed by paeonol treatment in human hepatocellular carcinoma cells.

Fan L, Song B, Sun G, Ma T, Zhong F, Wei W - PLoS ONE (2013)

Effect of co-pretreatment with Pae and tunicamycin on apoptosis induced by doxorubicin in HepG2 cells.(A) HepG2 cells were treated with 3 µmol/L tunicamycin for 8 hr, either in the absence or the presence of 31.25 mg/L Pae and then exposed to doxorubicin (2.5 mg/L) for 24 hr. Apoptosis was analyzed as the sub-G1 fraction by fluorescence-activated cell sorting (FACS). a: Untreated HepG2 cells; b: HepG2 cells treated with doxorubicin alone; c: HepG2 cells co-pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr d:HepG2 cells co-pretreated with tunicamycin and Pae, and then exposed to doxorubicin for 24 hr. (B) and(C) Cell morphology and percentage of apoptotic cells was examined by TUNEL staining. a: Untreated HepG2 cells; b: HepG2 cells treated with doxorubicin alone; c: HepG2 cells pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr d:HepG2 cells co-pretreated with tunicamycin and Pae and then exposed to doxorubicin for 24 hr.Data are presented as mean ± SD for the three independent experiments. (**P<0.01 compared with HepG2 cells treated with doxorubicin alone, ##P<0.01 compared with HepG2 cells pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr). (D) Cleaved caspase-3 as an apoptotic marker were measured by western blot using specific anti- caspase-3 antibody. β-actin in the same HepG2 cells extract was used as an internal reference.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643935&req=5

pone-0062627-g003: Effect of co-pretreatment with Pae and tunicamycin on apoptosis induced by doxorubicin in HepG2 cells.(A) HepG2 cells were treated with 3 µmol/L tunicamycin for 8 hr, either in the absence or the presence of 31.25 mg/L Pae and then exposed to doxorubicin (2.5 mg/L) for 24 hr. Apoptosis was analyzed as the sub-G1 fraction by fluorescence-activated cell sorting (FACS). a: Untreated HepG2 cells; b: HepG2 cells treated with doxorubicin alone; c: HepG2 cells co-pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr d:HepG2 cells co-pretreated with tunicamycin and Pae, and then exposed to doxorubicin for 24 hr. (B) and(C) Cell morphology and percentage of apoptotic cells was examined by TUNEL staining. a: Untreated HepG2 cells; b: HepG2 cells treated with doxorubicin alone; c: HepG2 cells pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr d:HepG2 cells co-pretreated with tunicamycin and Pae and then exposed to doxorubicin for 24 hr.Data are presented as mean ± SD for the three independent experiments. (**P<0.01 compared with HepG2 cells treated with doxorubicin alone, ##P<0.01 compared with HepG2 cells pretreated with tunicamycin, and then exposed to doxorubicin for 24 hr). (D) Cleaved caspase-3 as an apoptotic marker were measured by western blot using specific anti- caspase-3 antibody. β-actin in the same HepG2 cells extract was used as an internal reference.
Mentions: To determine the effects of Pae on ER stress–induced resistance to doxorubicin, HepG2 cells were pretreated with Pae for 8 hr in the presence of tunicamycin, then subjected to doxorubicin treatment for 24 hr. Apoptosis was assessed by FACS analysis (Figure 3A), TUNEL staining (Figure 3B and 3C) and Western Blot (Figure 3D). Apoptosis induced by doxorubicin was increased by pretreatment of tunicamycin and Pae together. In the group treated with tunicamycin and Pae together the sub-G1 population increased sharply to 50.19% compared to cells pretreated with tunicamycin alone. Similar results were observed with TUNEL staining. In additon, When HepG2 cells were pretreated with tunicamycin and Pae combination treatment, significantly higher levels of cleaved caspase 3 were observed compared to HepG2 cells pretreated with tunicamycin.Together, these results support the inhibitory effect of Pae on ER stress–resistance to doxorubicin.

Bottom Line: Interestingly, co-pretreatment with tunicamycin and Pae significantly increased apoptosis induced by doxorubicin.Furthermore, induction of ER stress resulted in increasing expression of COX-2 concomitant with inactivation of Akt and up-regulation of the pro-apoptotic transcription factor CHOP (GADD153) in HepG2 cells.However, co-pretreatment with tunicamycin and Pae decreased the expression of COX-2 and levels of activation of Akt as well as increasing the levels of CHOP in HCC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China.

ABSTRACT

Background: Endoplasmic reticulum stress (ER stress) is generally activated in solid tumors and results in tumor cell anti-apoptosis and drug resistance. Paeonol (Pae, 2-hydroxy-4-methoxyacetophenone), is a natural product extracted from the root of Paeonia Suffruticosa Andrew. Although Pae displays anti-neoplastic activity and increases the efficacy of chemotherapeutic drugs in various cell lines and in animal models, studies related to the effect of Pae on ER stress-induced resistance to chemotherapeutic agents in hepatocellular carcinoma (HCC) are poorly understood.

Methodology/principal findings: In this study, we investigated the effect of the endoplasmic reticulum (ER) stress response during resistance of human hepatocellular carcinoma cells to doxorubicin. Treatment with the ER stress-inducer tunicamycin (TM) before the addition of doxorubicin reduced the rate of apoptosis induced by doxorubicin. Interestingly, co-pretreatment with tunicamycin and Pae significantly increased apoptosis induced by doxorubicin. Furthermore, induction of ER stress resulted in increasing expression of COX-2 concomitant with inactivation of Akt and up-regulation of the pro-apoptotic transcription factor CHOP (GADD153) in HepG2 cells. These cellular changes in gene expression and Akt activation may be an important resistance mechanism against doxorubicin in hepatocellular carcinoma cells undergoing ER stress. However, co-pretreatment with tunicamycin and Pae decreased the expression of COX-2 and levels of activation of Akt as well as increasing the levels of CHOP in HCC cells.

Conclusions/significance: Our results demonstrate that Pae reverses ER stress-induced resistance to doxorubicin in human hepatocellular carcinoma cells by targeting COX-2 mediated inactivation of PI3K/AKT/CHOP.

Show MeSH
Related in: MedlinePlus