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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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Related in: MedlinePlus

Effect of tunicamycin on cell viability induced by doxorubicin in HepG2 cells.HepG2 cells were pretreated with tunicamycin (0,1.5,3 and 6 µ mol/L) for 8 hr then exposed to different concentrations of doxorubicin (0, 0.63, 1.25, 2.5, 5 and 10 mg/L) for 24 hr. Cell viability of HepG2 cells was determined by the MTT assay. Data are expressed as the mean ± SD of three independent experiments (bars represent S.D.). (*P<0.05, **P<0.01, compared with HepG2 cells treated with doxorubicin alone).
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pone-0062627-g001: Effect of tunicamycin on cell viability induced by doxorubicin in HepG2 cells.HepG2 cells were pretreated with tunicamycin (0,1.5,3 and 6 µ mol/L) for 8 hr then exposed to different concentrations of doxorubicin (0, 0.63, 1.25, 2.5, 5 and 10 mg/L) for 24 hr. Cell viability of HepG2 cells was determined by the MTT assay. Data are expressed as the mean ± SD of three independent experiments (bars represent S.D.). (*P<0.05, **P<0.01, compared with HepG2 cells treated with doxorubicin alone).

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 on cell viability induced by doxorubicin in HepG2 cells.HepG2 cells were pretreated with tunicamycin (0,1.5,3 and 6 µ mol/L) for 8 hr then exposed to different concentrations of doxorubicin (0, 0.63, 1.25, 2.5, 5 and 10 mg/L) for 24 hr. Cell viability of HepG2 cells was determined by the MTT assay. Data are expressed as the mean ± SD of three independent experiments (bars represent S.D.). (*P<0.05, **P<0.01, compared with HepG2 cells treated with doxorubicin alone).
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Related In: Results  -  Collection

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

pone-0062627-g001: Effect of tunicamycin on cell viability induced by doxorubicin in HepG2 cells.HepG2 cells were pretreated with tunicamycin (0,1.5,3 and 6 µ mol/L) for 8 hr then exposed to different concentrations of doxorubicin (0, 0.63, 1.25, 2.5, 5 and 10 mg/L) for 24 hr. Cell viability of HepG2 cells was determined by the MTT assay. Data are expressed as the mean ± SD of three independent experiments (bars represent S.D.). (*P<0.05, **P<0.01, compared with HepG2 cells treated with doxorubicin alone).
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