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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.

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Effect of co-treatment with tunicamycin and celecoxib on the expression of COX-2 and CHOP in HepG2 cells.HepG2 cells were treated with 3 µmol/L tunicamycin in either the absence (control) or the presence of celecoxib (50 µmol/L) for 8 hr. (A)Equal amounts of cell lysates were subjected to western blot analysis using specific anti-COX-2 and anti-CHOP antibody. β-actin in the same HepG2 cells extract was used as an internal used as an internal reference. Optical density reading values of the specific protein versus the loading control protein β-actin are represented as fold of the control values. (*P<0.05, **P<0.01, compared with untreated HepG2 cells, #P<0.05, ##P<0.01, compared with HepG2 cells treated with tunicamycin alone). (B) RNA was harvested and gene expression examined by qRT-PCR. The qRT-PCR fold-changes were normalised using the expression of a housekeeping gene (GAPDH) and compared with those obtained from untreated HepG2 cells.Data are presented as mean ± SD for the three independent experiments. (**P<0.01 compared with untreated HepG2 cells, ##P<0.01 compared with HepG2 cells pretreated with tunicamycin).
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pone-0062627-g007: Effect of co-treatment with tunicamycin and celecoxib on the expression of COX-2 and CHOP in HepG2 cells.HepG2 cells were treated with 3 µmol/L tunicamycin in either the absence (control) or the presence of celecoxib (50 µmol/L) for 8 hr. (A)Equal amounts of cell lysates were subjected to western blot analysis using specific anti-COX-2 and anti-CHOP antibody. β-actin in the same HepG2 cells extract was used as an internal used as an internal reference. Optical density reading values of the specific protein versus the loading control protein β-actin are represented as fold of the control values. (*P<0.05, **P<0.01, compared with untreated HepG2 cells, #P<0.05, ##P<0.01, compared with HepG2 cells treated with tunicamycin alone). (B) RNA was harvested and gene expression examined by qRT-PCR. The qRT-PCR fold-changes were normalised using the expression of a housekeeping gene (GAPDH) and compared with those obtained from untreated HepG2 cells.Data are presented as mean ± SD for the three independent experiments. (**P<0.01 compared with untreated HepG2 cells, ##P<0.01 compared with HepG2 cells pretreated with tunicamycin).

Mentions: To confirm that COX-2, which was activated by ER stress, indeed enhances doxorubicin–induced apoptosis, HepG2 cells were pretreated with tunicamycin in the presence of celecoxib, a selective COX-2 inhibitor, for 8 hr, followed by doxorubicin treatment for 24 hr. Inhibition of cell growth was determined by the MTT assay. Celecoxib significantly reduced cell proliferation when co-pretreated with tunicamycin (Figure 5). Apoptosis was also quantified by FACS analysis (Figure 6A) and TUNEL staining (Figure 6B and 6C) and Western-blot(Figure 6D). Co-pretreatment of both celecoxib and tunicamycin significantly increased the percentage of sub-G1 cells and the number of TUNEL-positive HCC cells as well as the levels of cleaved caspase 3. CHOP (CCAAT/enhancer-binding protein homologous protein), also called GADD153, is one of the primary effectors of ER stress–mediated cell apoptosis [20]. Western blotting and qRT-PCR analysis was also performed to detect the expression of CHOP. As shown in Figure 7A and 7B, the protein expression and the expression of CHOP mRNA was markedly increased in the presence of celecoxib and tunicamycin. These data indicate that down-regulation of COX-2 could inhibit the effect of tunicamycin on doxorubicin-induced apoptosis.


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-treatment with tunicamycin and celecoxib on the expression of COX-2 and CHOP in HepG2 cells.HepG2 cells were treated with 3 µmol/L tunicamycin in either the absence (control) or the presence of celecoxib (50 µmol/L) for 8 hr. (A)Equal amounts of cell lysates were subjected to western blot analysis using specific anti-COX-2 and anti-CHOP antibody. β-actin in the same HepG2 cells extract was used as an internal used as an internal reference. Optical density reading values of the specific protein versus the loading control protein β-actin are represented as fold of the control values. (*P<0.05, **P<0.01, compared with untreated HepG2 cells, #P<0.05, ##P<0.01, compared with HepG2 cells treated with tunicamycin alone). (B) RNA was harvested and gene expression examined by qRT-PCR. The qRT-PCR fold-changes were normalised using the expression of a housekeeping gene (GAPDH) and compared with those obtained from untreated HepG2 cells.Data are presented as mean ± SD for the three independent experiments. (**P<0.01 compared with untreated HepG2 cells, ##P<0.01 compared with HepG2 cells pretreated with tunicamycin).
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pone-0062627-g007: Effect of co-treatment with tunicamycin and celecoxib on the expression of COX-2 and CHOP in HepG2 cells.HepG2 cells were treated with 3 µmol/L tunicamycin in either the absence (control) or the presence of celecoxib (50 µmol/L) for 8 hr. (A)Equal amounts of cell lysates were subjected to western blot analysis using specific anti-COX-2 and anti-CHOP antibody. β-actin in the same HepG2 cells extract was used as an internal used as an internal reference. Optical density reading values of the specific protein versus the loading control protein β-actin are represented as fold of the control values. (*P<0.05, **P<0.01, compared with untreated HepG2 cells, #P<0.05, ##P<0.01, compared with HepG2 cells treated with tunicamycin alone). (B) RNA was harvested and gene expression examined by qRT-PCR. The qRT-PCR fold-changes were normalised using the expression of a housekeeping gene (GAPDH) and compared with those obtained from untreated HepG2 cells.Data are presented as mean ± SD for the three independent experiments. (**P<0.01 compared with untreated HepG2 cells, ##P<0.01 compared with HepG2 cells pretreated with tunicamycin).
Mentions: To confirm that COX-2, which was activated by ER stress, indeed enhances doxorubicin–induced apoptosis, HepG2 cells were pretreated with tunicamycin in the presence of celecoxib, a selective COX-2 inhibitor, for 8 hr, followed by doxorubicin treatment for 24 hr. Inhibition of cell growth was determined by the MTT assay. Celecoxib significantly reduced cell proliferation when co-pretreated with tunicamycin (Figure 5). Apoptosis was also quantified by FACS analysis (Figure 6A) and TUNEL staining (Figure 6B and 6C) and Western-blot(Figure 6D). Co-pretreatment of both celecoxib and tunicamycin significantly increased the percentage of sub-G1 cells and the number of TUNEL-positive HCC cells as well as the levels of cleaved caspase 3. CHOP (CCAAT/enhancer-binding protein homologous protein), also called GADD153, is one of the primary effectors of ER stress–mediated cell apoptosis [20]. Western blotting and qRT-PCR analysis was also performed to detect the expression of CHOP. As shown in Figure 7A and 7B, the protein expression and the expression of CHOP mRNA was markedly increased in the presence of celecoxib and tunicamycin. These data indicate that down-regulation of COX-2 could inhibit the effect of tunicamycin on doxorubicin-induced apoptosis.

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