| Background Hepatocellular carcinoma (HCC) is one of the most common solidmalignancies characterized by a high prevalence of drug resistance. The development ofdrug resistance to chemotherapeutic agents in HCC cells is the major cause ofchemotherapy failure in HCC patients. The molecular mechanisms that underliechemotherapeutic resistance are poorly understood.A number of cellular stress conditions, such as nutrient deprivation, hypoxia, andalterations in glycosylation status, lead to the accumulation of unfolded and/or misfoldedproteins in the lumen of the endoplasmic reticulum that is termed ER stress. The ERresponds to stress conditions by activating a range of signaling pathways that couples theER protein folding load with the ER protein folding capacity and is termed the unfoldedprotein response (UPR). The activation of the UPR is believed to alleviate ER stress andpromote cell survival via the activation of survival or proliferative pathways. Hypoxia andanoxia are pathophysiologic characteristics of most solid tumors.Evidence is emerging thathypoxia and anoxia play an important role in drug resistance of solid tumors, but how theseprocesses contribute to chemoresistance in HCC cells remains to be explored. Bothhypoxia and anoxia are known to cause ER stress and initiate the UPR. Therefore, ERstress from tumor hypoxia may lead to drug resistance.Paeonol (Pae), a major active component extracted from the herb Pycnostelmapaniculatum (Bunge) K Schum and the root cortex of Paeonia suffruticosa Andrews,possesses extensive pharmacological activities such as sedation, hypnosis, antipyresis,analgesic, anti-oxidation, anti-inflammation, and immunoregulation. Moreover, Pae displays oncostatic actions and increases the efficacy of chemotherapeutic drugs.However,studies related to the effect of Pae on ER stress–induced resistance tochemotherapeutic agents in HCC are limited. In this study, we examined how the inductionof ER stress results in increased resistance of hepatocellular carcinoma cells to apoptosisinduced by chemotherapeutic drugs. Furthermore, we provide evidence that Pae reversesthe effects of ER stress–induced resistance to doxorubicin by a COX-2mediatedinactivation of the PI3K/AKT/CHOP pathway.Methods:(1) To examine effect of tunicamycin on cell viability and apoptosis induced by doxorubicin in HepG2cells.Growth inhibition rate of each group was detected using MTT assay.The apoptosis rate was deteceted by flow cytometry and the morphological change of apoptosis was observed by TUNEL.(2) To examined how the induction of ER stress results in increased resistance of hepatocellular carcinoma cells to apoptosis induced by chemotherapeutic drugs，The expression of COX-2p-AKT,CHOP were assayed by Western-blot analysis.(3) To examine effect of co-pretreatment with tunicamycin and Paeonol on cell apoptosis induced by doxorubicin in HepG2cells. The apoptosis rate was deteceted by flow cytometry and the morphological change of apoptosis was observed by TUNEL. To clarify the reversing mechanism of Paeonol on ER stress induced resistance to doxorubicin-induced apoptosis in hepatocellular carcinoma cells. The expression of COX-2p-AKT,CHOP were assayed by Western-blot analysis.Results:1. Induction of ER stress protects hepatocellular carcinoma cells against apoptosis induced by doxorubicin (1) HepG2cells were pretreated with tunicamycin (3μmol/L) for8hr then exposed to different concentrations of doxorubicin(0,0.63,1.25,2.5,5,10mg/L) for24hr. Cell viability of HepG2cells was determined by the MTT assay. Pretreatment with TM significantly decreased doxorubicin-induced cytotoxicity in HepG2cells between0.31to5mg/L of doxorubicin.(2) HepG2cells were pretreated with3μmol/L tunicamycin for8hr and then exposed to doxorubicin (2.5mg/L) for24hr. Sub-G1analysis were determined by FACS and cell morphology and percentage of apoptotic cells was examined by TUNEL staining.Treatment of HepG2cells with doxorubicin resulted in a dramatic increase in the sub-G1cell population, which was significantly reduced in the presence of tunicamycin. Similar results were observed through TUNEL staining.2. The mechanism of ER stress induced resistance to doxorubicin-induced apoptosis in hepatocellular carcinoma cells (1) HepG2cells were treated with3μmol/L tunicamycin (TM) for0(control),4and8hr. Equal protein amounts of cell lysates were subjected to western blot assay using specific anti-COX-2and anti-GRP78antibody. Administration of tunicamycin to HepG2cells induced an early increase in GRP78expression in both cells, indicative of ER stress. Simultaneously, the expression of COX-2in both HepG2cells rapidly increased after treatment with tunicamycin (2) HepG2cells were pretreated with3μmol/L tunicamycin for8hr, either in the absence or the presence of Celecoxib (50μmol/L) and then exposed to doxorubicin (2.5mg/L) for24hr. Cell viability of HepG2cells was determined by the MTT assay. Apoptosis was analyzed as the sub-G1fraction by fluorescence-activated cell sorting (FACS) and Cell morphology and percentage of apoptotic cells was examined by TUNEL staining, apoptosis induced by doxorubicin was increased by co-pretreatment of tunicamycin and Celecoxib. In this group the sub-G1percentage and the number of TUNEL-positive HCC cells indicative of apoptosis were increased compared to cells pretreated with tunicamycin alone.(3) HepG2cells were treated with3μmol/L tunicamycin in either the absence (control) or the presence Celecoxib (50μmol/L) for8hr. Equal amounts of cell lysates were subjected to western blot analysis using specific anti-p-AKT and anti-CHOP antibody. HepG2cells treated with the COX-2inhibitor, Celecoxib and PI3K inhibitor,LY294002, in the presence of TM decreased the expression of p-AKT protein and increased the expression of CHOP protein. 3. Endoplasmic reticulum stress–induced resistance to doxorubicin is reversed by Paeonol treatment in human hepatocellular carcinoma cells (1) HepG2cells were treated with3μmol/L tunicamycin for8hr,either in the absence or\the presence of （31.25mg/L）Paeonol and then exposed to doxorubicin (2.5mg/L) for24hr.Apoptosis was assessed by FCM analysis and TUNEL staining.Apoptosis induced by doxorubicin was increased by co-pretreatment of tunicamycin and melatonin. In this group the sub-G1percentage and the number of TUNEL-positive HCC cells indicative of apoptosis were increased compared to cells pretreated with tunicamycin alone.(2) HepG2cells were treated with3μmol/L tunicamycin in either the absence (control) or the presence of Paeonol (31.25mg/L) for8hr. Equal amounts of cell lysates were subjected to western blot analysis using specific anti-COX-2and anti-phospho (p)-Akt antibody. HepG2cells treated with Paeonol, in the presence of TM decreased the expression of COX-2and (p)-Akt. Equal amounts of cell lysates were also subjected to western blot analysis using specific anti-CHOP antibody. HepG2cells treated with the Paeonol, in the presence of TM decreased the expression of p-AKT protein and increased the expression of CHOP protein.Conclusions:1. Endoplasmic reticulum stress induced resistance to doxorubicin by increasing CHOP via COX-2/PI3K/AKT pathway in human hepatocellular carcinoma cells.2. Paeonol reverses endoplasmic reticulum stress–induced resistance to doxorubicin by increasing CHOP viaCOX-2/PI3K/AKT pathway in human hepatocellular carcinoma cells. |
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