Anacardic Acid Induces Cell Apoptosis Associated With Induction Of ATF4-dependent Endoplasmic Reticulum Stress

Anacardic acid (6-pentadecylsalicylic acid, AA), a natural compound isolated from thetraditional medicine Amphipterygium adstringens, has been reported to possess antitumoractivities. However, its molecular targets have not been thoroughly studied. Here, we reportedthat AA was a potent inducer of endoplasmic reticulum (ER) stress, leading to apoptosis inhepatoma HepG2and melanoma U266cells. Induction of ER by AA was supported by adose-and time-dependent increase in expression of the ER signaling downstream molecules,such as GRP78/BiP, phosphorylated eIF2a, ATF4and CHOP in both HepG2and U266celllines. Blockage of ATF4expression by siRNA partially inhibited, while knockdown of CHOPexpression by siRNA slightly increased AA-induced cell death in these cells. In addition, AAsuppressed HepG2xenogratf tumor growth which is also associated with increased ER stressin vivo. These results suggest that AA induces tumor cell apoptosis associated withATF4-dependent ER stress.Material and methods1. MTS assayThe effects of compounds on cell viability were determined by the MTS assay (Cell Titer96? AQueous One Solution Cell Proliferation assay, Promega Corporation, Madison, WI,USA) as described previously’^’. Exponentially growing cells were harvested and seeded at 2500cells/well in a96-well plate. Atfer24h incubation, compounds or DMSO as theuntreated control were added, followed by continuous incubation for the indicated times.20|_il of MTS were added to each well and the incubation was continued for an additional3hours. The absorbance was measured with a microplate reader (Sunrise, Tecan) at490nm.Cell viability was calculated as follows:(absorbance of experimental well-absorbance ofblank)/(absorbance of untreated control well-absorbance of blank) x100%. We also foundthat the MTS assay demonstrated that AA inhibited the growth of t the HepG2and U266cellsin both a dose-and time-dependent manner, However, AA did not significantly inhibit thegrowiti of16HBE cells.2.Clonogenic assayThis assay was performed as we previously described [！]. HepG2and u266cells exposedto either vehicle or AA for12h were suspended in30%agarose supplemented with20%FCSand50%RPMI-1640medium then cultured in24-well plates in an atmosphere of5%COifor7days, then stained with0.3%crystal violet solution. The colonies>60jim were countedunder the light microscope. The experiments were done in triplicate. Additionally, weinvestigated the effect of AA on the suppression of long-term colony formation.3.Cell death detection assay via Flow cytometryThis was performed using Aimexin V-FITC and propidium iodide (PI) double stainingfollowed by flow cytometry as previously described [〗].Briefly, cultured HepG2、U266and16HBE cells were harvested and washed with cold PBS and resuspended with the bindingbuffer, followed by Aimexin V-FITC incubation for15min and PI staining for another15min at4°C in dark. The stained cells were analyzed with flow cytometry within30min. Theresults showed that treatment of the HepG2and U266cells with AA dramatically increasedthe population of both Aimexin V/PI-stained cells, which was not seen in the treated16HBEcells.4.Morphological characterization of cell deathTo monitor temporal changes in the incidence of cell death in the live culture condition,Aimexin V-FITC and propidium idodide (PI) were added to the cell culture medium and at the desired sequential time points, the HepG2and U266cells in the culture dish were imagedwith an inverted fluorescence microscope equipped with a digital camera (Axio Obsever Zl,Zeiss). We found that AA significantly increased Aimexin V/PI-positive cells and inducedtypical apoptotic morphological changes.5.RNA InterferenceTo knockdown CHOP or ATF-4expression in cells, siRNA targeting human CHOP orATF-4were synthesized and purchased from Santa Cruz Biotechnology Inc.(Santa Cruz, CA,USA). siRNA with non-specific sequences were used as control scrambled siRNA. DifferentsiRNAs were transfected separately into cells by using Lipofecatmine2000(Invitrogen)reagent and medium was replaced6h atfer transfection. These data indicate that CHOP is notthe predominant UPR branch regulating cell death in HepG2and U266cells treated by AA,but that induction of ATF4-dependent ER stress at least partially contributed to AA-inducedcell death.6.Establishment and treatment of HepG2xenogratfsMale Balb/c nude mice aged5weeks were purchased from Guangdong Animal Centerand housed in accordance with protocols approved by the Guangdong Animal Center. Balb/cnude mice were s.c. inoculated in the letf armpit of each mouse with HepG2cells (1x10^cells/mouse). When the tumor size reaches50-75^mm, mice were randomly divided intotwo groups (6mice per group). Nude mice bearing HepG2tumor were i.p. injected withvehicle and AA (2mg/kg, once/day), respectively, for14days. Tumors were measured bycaliper and tumor volume was calculated using standard formula: Width^x Length/2. Bodyweight, tumor weight, tumor volume were detected and summarized. The results indicate thatAA induces tumor growth inhibition and ER stress induction in vivo.7. Western blot analysisWestern blotting was performed as previously described In brief, an equal amount oftotal protein extracts from cultured cells were fractionated by12%SDS-PAGE andelectrically transferred onto polyvinylidene difluoride (PVDF) membranes. Mouse or rabbitprimary antibodies and horseradish peroxidase (HRP)-conjugated appropriate secondary antibodies were used to detect the designated proteins. The bound secondary antibodies on thePVDF membrane were reacted with ECL detection reagents (Amersham Bioscience) andexposed to X-ray films (Kodak, Japan), in our current study, AA not only significantlyactivated expression of GRP78and CHOP, similar to previtr[4ous repo], but also activatedexpression of phospho-eIF2a and ATF4in both HepG2and U266cellsConclusionsThe antitumor activity of AA both in vitro and in vivo in HepG2and U266cells is atleast partially associated with the induction of ATF4-dependent ER stress pathway.