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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 tunicamycin treatment on cell apoptosis induced by doxorubicin in HepG2 cells.(A) HepG2 cells were pretreated with 3 µmol/L tunicamycin (TM) for 8 hr and then exposed to doxorubicin (2.5 mg/L) for 24 hr. Sub-G1 analysis in HepG2 cells were determined by FACS, and the data are expressed as the mean ± SD of three independent experiments. a: Untreated HepG2 cells; b: HepG2 cells treated with tunicamycin alone; c: HepG2 cells treated with doxorubicin alone; d: HepG2 cells were pretreated with tunicamycin for 8 hr, and then exposed to doxorubicin for 24 hr. (B) and (C) Cell morphology and percentage of apoptotic cells was examined by TUNEL staining. In this representative image, the cells with brown nuclei are apoptotic cells. a: Untreated HepG2 cells; b: HepG2 cells treated with tunicamycin alone; c: HepG2 cells treated with doxorubicin alone; d: HepG2 cells were pretreated with tunicamycin for 8 hr, and then exposed to doxorubicin for 24 hr. Data are presented as mean ± SD of three independent experiments (bars represent S.D.). (**P<0.01, compared with untreated HepG2 cells; ##P<0.01, compared with doxorubicin alone) (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.
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pone-0062627-g002: Effect of tunicamycin treatment on cell apoptosis induced by doxorubicin in HepG2 cells.(A) HepG2 cells were pretreated with 3 µmol/L tunicamycin (TM) for 8 hr and then exposed to doxorubicin (2.5 mg/L) for 24 hr. Sub-G1 analysis in HepG2 cells were determined by FACS, and the data are expressed as the mean ± SD of three independent experiments. a: Untreated HepG2 cells; b: HepG2 cells treated with tunicamycin alone; c: HepG2 cells treated with doxorubicin alone; d: HepG2 cells were pretreated with tunicamycin for 8 hr, and then exposed to doxorubicin for 24 hr. (B) and (C) Cell morphology and percentage of apoptotic cells was examined by TUNEL staining. In this representative image, the cells with brown nuclei are apoptotic cells. a: Untreated HepG2 cells; b: HepG2 cells treated with tunicamycin alone; c: HepG2 cells treated with doxorubicin alone; d: HepG2 cells were pretreated with tunicamycin for 8 hr, and then exposed to doxorubicin for 24 hr. Data are presented as mean ± SD of three independent experiments (bars represent S.D.). (**P<0.01, compared with untreated HepG2 cells; ##P<0.01, compared with doxorubicin alone) (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 potential effects of ER stress on the sensitivity of hepatocellular carcinoma cells to chemotherapeutic agents, HepG2 cells were pretreated with the ER stress inducer tunicamycin (TM) for 8 hr before the addition of doxorubicin for 24 hr. Cell viability was assessed using the MTT assay (Figure 1). Pretreatment with different concentrations of TM significantly decreased doxorubicin-induced cytotoxicity in HepG2 cells between 0.63 to 10 mg/L of doxorubicin. Sub-G1 analysis was conducted by fluorescence-activated cell sorting analysis (FACS) and morphological changes, indicative of apoptosis, were also assessed by TUNEL staining. Consistent with the results of the MTT assay, treatment of HepG2 cells with doxorubicin (2.5 mg/L) resulted in a marked increase in the sub-G1 cell population (33.09%), which was significantly reduced (18.84%) in the presence of tunicamycin (Figure 2A). Similar results were observed through TUNEL staining (Figure 2B and2 C). Apoptosis was also measured through western blot analysis of cleaved caspase 3. As shown in Figure 2D, There was significantly lower levels of cleaved caspase 3 in HepG2 cells pretreated with tunicamycin compared to HepG2 cells treated with doxorubicin alone.


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 tunicamycin treatment on cell apoptosis induced by doxorubicin in HepG2 cells.(A) HepG2 cells were pretreated with 3 µmol/L tunicamycin (TM) for 8 hr and then exposed to doxorubicin (2.5 mg/L) for 24 hr. Sub-G1 analysis in HepG2 cells were determined by FACS, and the data are expressed as the mean ± SD of three independent experiments. a: Untreated HepG2 cells; b: HepG2 cells treated with tunicamycin alone; c: HepG2 cells treated with doxorubicin alone; d: HepG2 cells were pretreated with tunicamycin for 8 hr, and then exposed to doxorubicin for 24 hr. (B) and (C) Cell morphology and percentage of apoptotic cells was examined by TUNEL staining. In this representative image, the cells with brown nuclei are apoptotic cells. a: Untreated HepG2 cells; b: HepG2 cells treated with tunicamycin alone; c: HepG2 cells treated with doxorubicin alone; d: HepG2 cells were pretreated with tunicamycin for 8 hr, and then exposed to doxorubicin for 24 hr. Data are presented as mean ± SD of three independent experiments (bars represent S.D.). (**P<0.01, compared with untreated HepG2 cells; ##P<0.01, compared with doxorubicin alone) (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.
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pone-0062627-g002: Effect of tunicamycin treatment on cell apoptosis induced by doxorubicin in HepG2 cells.(A) HepG2 cells were pretreated with 3 µmol/L tunicamycin (TM) for 8 hr and then exposed to doxorubicin (2.5 mg/L) for 24 hr. Sub-G1 analysis in HepG2 cells were determined by FACS, and the data are expressed as the mean ± SD of three independent experiments. a: Untreated HepG2 cells; b: HepG2 cells treated with tunicamycin alone; c: HepG2 cells treated with doxorubicin alone; d: HepG2 cells were pretreated with tunicamycin for 8 hr, and then exposed to doxorubicin for 24 hr. (B) and (C) Cell morphology and percentage of apoptotic cells was examined by TUNEL staining. In this representative image, the cells with brown nuclei are apoptotic cells. a: Untreated HepG2 cells; b: HepG2 cells treated with tunicamycin alone; c: HepG2 cells treated with doxorubicin alone; d: HepG2 cells were pretreated with tunicamycin for 8 hr, and then exposed to doxorubicin for 24 hr. Data are presented as mean ± SD of three independent experiments (bars represent S.D.). (**P<0.01, compared with untreated HepG2 cells; ##P<0.01, compared with doxorubicin alone) (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 potential effects of ER stress on the sensitivity of hepatocellular carcinoma cells to chemotherapeutic agents, HepG2 cells were pretreated with the ER stress inducer tunicamycin (TM) for 8 hr before the addition of doxorubicin for 24 hr. Cell viability was assessed using the MTT assay (Figure 1). Pretreatment with different concentrations of TM significantly decreased doxorubicin-induced cytotoxicity in HepG2 cells between 0.63 to 10 mg/L of doxorubicin. Sub-G1 analysis was conducted by fluorescence-activated cell sorting analysis (FACS) and morphological changes, indicative of apoptosis, were also assessed by TUNEL staining. Consistent with the results of the MTT assay, treatment of HepG2 cells with doxorubicin (2.5 mg/L) resulted in a marked increase in the sub-G1 cell population (33.09%), which was significantly reduced (18.84%) in the presence of tunicamycin (Figure 2A). Similar results were observed through TUNEL staining (Figure 2B and2 C). Apoptosis was also measured through western blot analysis of cleaved caspase 3. As shown in Figure 2D, There was significantly lower levels of cleaved caspase 3 in HepG2 cells pretreated with tunicamycin compared to HepG2 cells treated with doxorubicin alone.

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