Brefeldin A inhibits colorectal cancer growth by triggering Bip/Akt-regulated autophagy
Li Zhou, Wei Gao, Kui Wang, Zhao Huang, Lu Zhang, Zhe Zhang, Jing Zhou, Edouard C. Nice, and Canhua Huang
ABSTRACT:
Colorectal cancer (CRC) is one of the most prevalent neoplastic diseases worldwide, and effective treatmentremainsachallenge.Here,wefoundthatthemacrolideantibioticbrefeldinA(BFA)exhibitsconsiderable antitumor activity both in vitro and in vivo. Induction of completeautophagic flux is characterized asa key event in BFA-induced CRC suppression. Mechanistically, BFA provokes endoplasmic reticulum stress–mediated binding immunoglobulin protein (Bip) expression, leading to increased Bip/Akt interaction and resultant decreased Akt phosphorylation,therebyactivatingautophagy.AutophagyinhibitionorBipsuppressionrelievesBFA-inducedcell death, suggesting a key role for Bip-regulated autophagy in the antitumor properties of BFA. Moreover, BFA acts synergistically with paclitaxel or 5-fluorouracil in CRC suppression. Collectively, our study provides an important molecularbasisforBFA-inducedautophagyandsuggeststhattheantibioticBFAcouldberepositionedasapotential anticancer drug for CRC treatment.—Zhou, L., Gao, W., Wang, K., Huang, Z., Zhang, L., Zhang, Z., Zhou, J., Nice, E. C., Huang, C. Brefeldin A inhibits colorectal cancer growth by triggering Bip/Akt-regulated autophagy.
KEY WORDS: cancer therapy • ER stress • autophagic flux
Introduction
Colorectal cancer (CRC) is the third most commonly diagnosedcancerinmenandthesecondinwomenworldwide (1). With the development in the clinical treatment of CRC over the past decades, surgical resection plus chemotherapy or radiation exerts favorable effects on CRC patients (2). However,thefirst-linechemotherapeuticdrugs[including5fluorouracil(5-FU)andoxaliplatin,etc.]exhibitcompromised treatment outcomes due to side effects or drug resistance, and the overall prognosis for CRC patients remains poor (3). Assuch,thereisanurgentneedtodevelopnoveltherapeutic agents for efficient treatment of CRC patients.
Autophagy is a biologic process by which damaged organelles and macromolecules are degraded and recycled for cell survival and proliferation under physiologic conditions (4). Previous studies have found that autophagy suppresses malignant transformation in the early stages of tumor development (5). However, in the later stages, autophagy may facilitate tumor progression through promotingcellsurvival understressedconditions like nutrient deficiency (6). In addition, autophagy can be activated in response to chemotherapy to relieve the stress withdiverseclinicaloutcomes(7).Forinstance,autophagy induced by 5-FU, a commonly used chemotherapeutic agent for CRC, exhibits a protective role and causes drug resistance in CRC cells (8, 9). In contrast, a very recent study has found that a new autophagy enhancer, IR58, exerts significant anticancer effects in CRC cells via inducing autophagic cell death (10). Therefore, the role of autophagy in determining CRC cell fate is complex. Elucidating the biologic function of autophagy during CRC chemotherapy is scientifically worthwhile for providing novel therapeutic strategies.
Brefeldin A (BFA), a lactone antibiotic produced by Eupenicillium brefeldianum, was initially isolated with hopes to become an antiviral drug but is now widely used to study protein transport (11, 12). BFA inhibits protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus indirectly by preventing the binding of cytosolic coat-protein complex COP-I onto the Golgi membrane (13). It has been reported that BFA exhibits potent anticancer activity in various cancers, including melanoma, prostate, and breast cancer (14–16). However, the detailed mechanism underpinning the anticancer effect of BFA remains to be further defined. In addition, to date, limited data exist regarding the role of BFA in CRC suppression.
In the present study, we demonstrate that BFA induces CRCcelldeathbytriggeringautophagyinvitroandinvivo. The up-regulation of binding immunoglobulin protein (Bip) and its interaction with Akt are characterized as key events in BFA-induced autophagy. Notably, BFA synergistically suppresses CRC cell growth with paclitaxel and 5-FU. These findings provide a novel link between BFA andautophagy,whichisofparticularclinicalrelevancefor a potential therapeutic strategy against CRC.
MATERIALS AND METHODS
Cell culture and reagents
Human CRC cell lines HCT116, HT29, DLD-1, SW620, SW480, and NCM460 were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with10%fetalbovineserum(ThermoFisherScientific),100U/ml penicillin(MilliporeSigma,Burlington,MA,USA),and100U/ml streptomycin (MilliporeSigma) in a humidified incubatorat 37°C under 5% CO2 atmosphere.
Reagents used in this study—BFA (HY-16592), 3-methyladenine (3-MA) (HY-19312), bafilomycin A1 (Baf A1; HY100558), 5-FU (HY-90006), and paclitaxel (HY-B0015)—were purchased from MedChem Express (Monmouth Junction, NJ, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (M2128), DMSO (D2650), Crystal Violet (C0775), and chloroquine (CQ) diphosphate salt (C6628) were purchased from MilliporeSigma. BFA, Baf A1, 5-FU, and paclitaxel were dissolved in DMSO. 3-MA, MTT, Crystal Violet, and CQ diphosphate salt were dissolved in PBS.
The following antibodies were used in this study: cleaved caspase-3 (9664S; Cell Signaling Technology, Danvers, MA, USA), ATG5 (12994S; Cell Signaling Technology), ATG7 (8558S; Cell Signaling Technology), ATG14L (96752; Cell Signaling Technology), Beclin1 (3738; Cell Signaling Technology), Akt (4685; Cell Signaling Technology), phosphorylated (p-)Akt (Ser473) (4060; Cell Signaling Technology), mTOR (2972; Cell Signaling Technology), p-mTOR (Ser2448) (2971; Cell Signaling Technology), p70S6K (9202; Cell Signaling Technology), pp70S6K (Ser371) (9208; Cell Signaling Technology), 4EBP1 (9452; Cell Signaling Technology), p-4EBP1 (Ser65) (9451; Cell Signaling Technology), Bcl-2 (15071; Cell Signaling Technology), PARP (ab74290; Abcam, Cambridge, MA, USA), cleaved PARP (ab32064; Abcam), Ki67 (ab66155; Abcam), p62 (sc-48402; Santa Cruz Biotechnology, Dallas, TX, USA), b-actin (sc-1616; Santa Cruz Biotechnology), Bip (sc-376768; Santa Cruz Biotechnology), CHOP (sc-56107; Santa Cruz Biotechnology), eIF2a (sc-133132; Santa Cruz Biotechnology), p-eIF2a (sc-133227; Santa Cruz Biotechnology),PERK(sc-377400;SantaCruzBiotechnology),IRE1a (sc-390960; Santa Cruz Biotechnology), LC3 (NB100-2220; Novus, Saint Charles, MO, USA), horseradish peroxidase– conjugated anti-rabbit secondary antibody (sc-2004; Santa Cruz Biotechnology), horseradish peroxidase–conjugated anti-mouse secondary antibody (sc-2005; Santa Cruz Biotechnology). For immunofluorescence, goat anti-mouse Alexa Fluor 488, goat anti-rabbit Alexa Fluor 488, goat anti-mouse Alexa Fluor 594, andgoatanti-rabbitAlexaFluor594wereobtainedfromThermo Fisher Scientific.
Detection of cell growth
The MTT assay was used to detect short-term effects of BFA on tumor cell growth, and the detailed procedure was previously described by Wang etal. (17). Briefly, cells were seeded in 96-well plates (5 3 103 cells/well) and treated for 24 h with the indicated concentrations of BFA. The absorbance was measured at 490-nm test wavelength and 570-nm reference wavelength with ELISA multiwell spectrophotometer.
The long-term effects of BFA on tumor cell proliferation were analyzed with a colony formation assay as previously described by Dou et al. (18). Cells were seeded in 24-well plates (300 cells/ well) and treated with the indicated concentration of BFA. The medium was changed every 3 d. After 2 wk, the colonies were stained with Giemsa for 30 min and washed 3 times. The visible colonies were photographed by Molecular Imager Gel Do XR + System (Bio-Rad, Hercules, CA, USA) and counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
5-Ethynyl-20-deoxyuridine labeling assay
The 5-ethynyl-20-deoxyuridine (EdU) labeling assay was performed in 96-well plates (5 3 103 cells/well) using the EdU Cell Proliferation Assay Kit (Ribobio, Guangzhou, China). After 24 h of BFA treatment, 10 mM EdU was added to each well, and the cells were incubated for another 12 h at 37°C. Cells were then fixed with 4% paraformaldehyde in PBS and stained with reaction cocktail. DAPI was then added for nuclear staining followed by imaging with a fluorescence microscope. DAPIpositive cells and EdU-positive cells were counted in at least 3 random fields. The proportion of EdU-positive cells vs. DAPIpositive cells in the vehicle group was used as control, and percentagewasthencalculatedbycomparingthetreatedgroupwith the vehicle group.
Lactate dehydrogenase release assay
A lactate dehydrogenase (LDH) test kit (Beyotime Biotechnology, Nanjing, China) was used to evaluate the cytotoxicity of BFA. Cells were seeded in 96-well plates (5 3 103 cells/well) and incubated to about 70–80% confluency. Then, the cells were treated with various concentrations of BFA. Cells cultured in mediumwithoutBFAwereusedasnegativecontrol.After24hof incubation, the 96-well plates were centrifuged at 500 g for 3 min. The LDH maximum leakage control (positive control) was prepared by adding 10 ml of lysis solution to the control cells 60 min prior to centrifugation. After centrifugation, 80 ml of supernatant fromeachwellwastransferredtoanew96-wellplatefortheLDH assay according to the manufacturer’s instructions.
TUNEL assays and flow cytometry
Inbrief,cellswereplatedonglasscoverslipsin24-wellplates(53 103 cells/well), treated with indicated concentrations of BFA for 24 h, and fixed in 4% paraformaldehyde. TUNEL staining was performed using the DeadEnd Fluorometric TUNEL system according to the manufacturer’s instructions (G3250; Promega, Madison, WI, USA). The apoptotic and nonapoptotic signals were photographed usinga fluorescent microscope, and then the percentage of cells with DNA nick-end labeling was evaluated.
The ratio of apoptotic cells was measured with an annexin V–FITC/propidium iodide (PI) Detection Kit (KGA108; KeyGen Biotech, Nanjing, China) according to the manufacturer’s protocol. Briefly, the cells were harvested and washed twice with PBS and resuspended in 500 ml binding buffer. After adding 5 ml annexinV–FITCand2mlPIintothecellsuspension,respectively, at least 20,000 live cells were analyzed on a FACSCalibur flow cytometer(BDBiosciences,SanJose,CA,USA).Cisplatin(75mM) was used as positive control. Data were analyzed by using FlowJo software (FlowJo, Ashland, OR, USA).
Immunoblotting and immunoprecipitation
Cells were washed with ice-cold PBS and then lysed with RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF). For immunoprecipitations, cells were lysed with IP lysis buffer (20 mM Tris,137mMNaCl,10%glycerol,1%NP-40and2mMEDTA,pH = 8). The whole-cell lysates were subjected to immunoprecipitation overnight at 4°C with 1 mg of the indicated antibodies, and the immunoprecipitated protein was pulled down with Protein A agarosebeads (40 ml; GE Healthcare, Chicago,IL, USA)for 4 h. The samples were analyzed by immunoblotting with the indicated antibodies.
Immunofluorescence
Cells were plated on glass coverslips in 24-well plates (5 3 103 cells/well),treatedwithindicatedconcentrationsofBFAfor24h, and fixed in 4% paraformaldehyde. After being washed with PBS, cells were permeabilized with 0.4% Triton X-100 followed by blocking with 5% fetal bovine serum. The indicated first antibodies and Alexa Flour secondary antibodies were used to incubate with cells. Images were viewed with a confocal laser scanning microscopy (Carl Zeiss, Oberkochen, Germany).
RNA interference
ATG5, Bip, and scramble small interfering RNA (siRNA) were synthesizedbyGenePharma(Shanghai,China).Thesequencesof siRNA were as follows: human ATG5 siRNA, 59-GCAACUCUGGAUGGGAUUGTT-39; human Bip siRNA, 59-GGAGCGCAUUGAUACUAGATT-39. The siRNA was transfected with Lipofectamine 3000 reagent (Thermo Fisher Scientific) for 48 h according to the manufacturer’s protocol.
Immunohistochemistry
Tumor xenografts were formalin-fixed, paraffin-embedded, and sectioned in pre-adherent slides. Slides were subjected to the indicated first antibodies overnight. The sections were incubated with secondary antibody and then developed with 3,3’diaminobenzidine chromogen according to the protocol. All samples were visualized, and images were captured using a DM2500 fluorescence microscope (Danaher, Wetzlar, Germany). Quantitative scoring analyses were performed by multiplying the percentage of staining-positive cells (A: 0, ,5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, .75%) by the intensity (B: 0, negative; 1, weakly positive; 2,positive; 3, stronglypositive). Thefinal scorefor each slide was calculated as A 3 B.
Tumor xenograft model
All animal experiments were approved by the Institutional Animal Care and Treatment Committee of Sichuan University. Sixwk-old female nude mice (BALB/c, nonfertile, and 18–20 g each) were purchased from HFK Bioscience (Beijing, China). The mice were housed under standard conditions. For the subcutaneous xenograft model, HCT116 cells (1 3 107 cells/mouse) were suspendedinPBSandsubcutaneouslyimplantedintoflanksofmice. Whenthetumorvolumereached;50mm3,micewererandomly grouped and intraperitoneally injected with 0.1 ml vehicle (10% ricinus oil, 5% DMSO, 10% ethanol, 75% physiologic saline) or BFA (15 mg/kg/d). The tumor volumes were measured every day and evaluated according to the following formula: tumor volume (mm3) = (length 3 width2)/2. The mice wereeuthanized after 4 wk and tumors were harvested.
Statistical analysis
All statistical analysis and graphics were performed using GraphPad 6 software (GraphPad, La Jolla, CA, USA). A 1-way ANOVA or Student’s t test was used to analyze statistical differences. All data are presented as the mean 6SD from at least 3 individual experiments. A value of P , 0.05 was considered as statistically significant.
RESULTS
BFA inhibits the growth of CRC cells in vitro
To validate whether BFA exhibits an antitumor effect against CRC, we first determined the cell growth in responsetoBFAtreatmentindifferenthumanCRCcelllines. As shown in Fig. 1A, B, BFA treatment for 24 h markedly decreased the growth of various CRC cell lines (HCT116, HT29, DLD-1, SW620, and SW480), whereas the IC50 value in NCM460 (a normal human colonic epithelial cell line) cells was much higher than those in CRC cells. Consistently, the proliferation of CRC cells was significantly inhibited under BFA treatment, as evidenced by reduced colony formation and EdU incorporation (Fig. 1C, D). We then performed LDH release assay and found that BFA treatment exhibited marked cytotoxicity in HCT116 and HT29 cells (Fig. 1E). Previous studies suggested that BFA exerts antitumor activity by inducing apoptosis in breast and prostate cancer cells (16, 19). To evaluate whether apoptosis is involved in BFA-induced cytotoxicity in CRC cells, we performed an in situ TUNEL assay and observed obvious apoptotic CRC cells in response to BFA treatment (Fig. 1F). The proapoptotic effect of BFA was further evidenced by Annexin V/PI staining measured with flow cytometry(Fig.1G).Inaddition,BFA treatmentresultedin increased levels of cleaved caspase-3 and cleaved PARP, 2 special and sensitive markers of apoptosis, in CRC cells (Fig. 1H). Collectively, these results demonstrate that BFA exhibits a considerable antitumor effect in CRC cells in vitro.
BFA induces autophagic flux in CRC cells
Increasing evidence has highlighted the potential application of pharmacologically modulating autophagy for cancer treatment (5, 7). We thus investigated whether autophagy is regulated by BFA in CRC cells. To ascertain this hypothesis, we first examined the protein levels of autophagy-related genes in BFA-treated CRC cells. As expected, BFA treatment promoted the turnover of LC3-I to lipidated LC3-II in a dose- and time-dependent manner in various CRC cells (Fig. 2A and Supplemental Fig. S1A, B). However, no apparent difference in the turnover of LC3-I to lipidated LC3-II was detected between BFAtreated cells and controls in NCM460 cells (Supplemental Fig. S1B). In agreement, the autophagic phenotype was further supported by the accumulation of autophagic vesicles (LC3 puncta) in BFA-treated cells compared with control cells (Fig. 2B and Supplemental Fig. S1C–E). Also, weexamined theexpression levels of ATG5 and Beclin 1,2 autophagy-related proteins, to clarify whether BFA promoted the formation of autophagic vesicles. As shown in Fig. 2A, BFA enhanced the expression of ATG5 and Beclin 1 in a dose-dependent manner. In addition, it has been reported that the enhanced interaction of Beclin 1 with ATG14L and the diminished interaction of Beclin 1 with Bcl-2 are key events in the initiation process of autophagy (20). We found that BFA treatment increased the interaction of Beclin 1 with ATG14L and decreased the binding of Beclin 1 with Bcl-2 (Fig. 2C). Moreover, either treatment with 3-MA (an inhibitor of class III PI3K) or siRNA-mediated ATG5 silencing prominently restored elevation of LC3-II levels in BFA-treated cells (Fig. 2D and Supplemental Fig. S1F, G). Taken together, these data suggest that BFA promotes the initiation process of autophagy in CRC cells.
To determine whether BFA induces complete autophagic flux, we examined the protein levels of p62, a wellknown autophagic substrate. We observed decreased p62 levels in BFA-treated cells along with the increased LC3-II levels, implying the induction of complete autophagic flux. In addition, combinatorial treatment of BFA with autolysosome inhibitors (CQ or Baf A1) resulted in enhanced LC3-II turnover and accumulation of endogenous LC3 puncta (Fig. 2E and Supplemental Fig. S2A, B). To further confirm the fusion of autophagosome with lysosome in BFA-treated CRC cells, we examined the colocalization of LC3 with LAMP1 (lysosome marker). BFA treatment induced obvious colocalization of LC3 with LAMP1, suggesting the fusion of autophagosome with lysosome (Fig. 2F and Supplemental Fig. S2C, D). Together, these findings revealthat BFA induces complete autophagic flux in CRC cells.
Autophagy induction contributes to the antitumor activity of BFA in CRC cells
To evaluate whether autophagy was involved in the antiCRC effect of BFA, CRC cells were treated with BFA combined with CQ or 3-MA. As shown in Fig. 3A, B, combinational use of CQ or 3-MA with BFA restored BFA-induced growth inhibition.Asimilar increaseincell proliferation was also observed in BFA-treated CRC cells in combination with CQ as evidenced by colony formation analysis and EdU labeling (Fig. 3C–G). In addition, LDH release assay also revealed that CQ counteracted BFA-induced cytotoxicity (Fig. 3H, I). Consistently, similarresultswereobtainedbyinhibitionofautophagyusing siRNA-targeted ATG5 knockdown (Fig. 3J, K). Moreover, we also evaluated whether BFA-induced autophagy had a correlation with BFA-induced apoptosis. As shown in Supplemental Fig. S3A, B, inhibiting autophagy by CQ had no obvious effect on BFA-induced apoptosis. Collectively, these results suggest that autophagy is involved in BFA-induced CRC suppression.
BFA induces autophagic cell death through ER stress–mediated Bip up-regulation in CRC cells
Moon et al. (21) previously reported that BFA could stimulate ER stress, leading to the up-regulation of Bip expression. We thus investigated whether BFA-induced autophagy was attributed to ER stress stimulation and Bip up-regulation. During the process of ER stress, misfolded proteins bind and sequester the ER chaperone Bip (also called GRP78 or HSPA5). Bip sequestration then activates unfolded protein response (UPR) sensors (PERK, IRE1a, and ATF6a), leading to the increased transcription of Bip required for the UPR process (22). Thus, we examined the proteinlevelsofBipandERstress–relatedproteinsinBFAtreated cells. As expected, BFA treatment promoted the expression of several ER stress–related proteins (Bip, CHOP, PERK, IRE1a, eIF2a) in a dose-dependent manner in CRC cells (Supplemental Fig. S4A). This was further confirmed by the increased dissociation of Bip with 2 parallel UPR sensors (PERK and IRE1a) (Supplemental Fig. S4B). To investigate the role of ER stress in BFAinduced autophagy, CRC cells were treated with BFA combined with 4-phenylbutyric acid (4-PBA, a chemical chaperone for the correct folding of proteins) or siBip, respectively. As shown in Fig. 4A, B, knockdown of Bip by siBip or mitigation of ER stress by 4-PBA could markedly restore BFA-induced LC3-II accumulation. This was further supported by reduced LC3 puncta accumulation under Bip knockdown (Fig. 4C and Supplemental Fig. S4C). We then intended to evaluate whether BFA-induced ER stress stimulation and Bip up-regulation and were involvedintheanti-CRCeffectofBFA.Wefoundthat4-PBA or siBip significantly rescued BFA-induced growth inhibition in CRC cells (Fig. 4D–G and Supplemental Fig. S4D, E). Collectively, these results demonstrate that ER stress–mediated Bip up-regulation is required for BFAinduced autophagy and growth inhibition in CRC cells.
BFA activates autophagy via promoting Bip/Akt interaction and consequent Akt dephosphorylation
As Akt/mTOR signaling acts as a key negative modulator of autophagy (23), we aimed to test whether BFA could inhibit Akt/mTOR signaling to induce autophagy. As shown in Fig. 5A, BFA treatment significantly inhibited Akt/mTOR pathway as evidenced by decreased phosphorylation levels of Akt, mTOR, 4E-BP1, and p70S6K. To strengthen these results, we then transfected a constitutively active form of Akt to rescue BFA-induced Akt/ mTOR inhibition. As expected, Akt reactivation markedly reduced BFA-induced LC3-II turnover and LC3 puncta accumulation (Fig. 5B, C, and Supplemental Fig. S4F), suggesting an important role for the Akt/mTOR pathway in BFA-induced autophagy. Gao et al. (24) have reported that knockdown of Bip would regulate the activation of Akt, suggesting an intrinsic link between Bip and Akt. Consistently, we found that siRNA-mediated Bip knockdown partially restored BFA-induced inhibition of Akt phosphorylationinCRCcells(Fig.5D).Notably,Bipcould interact with Akt, and this interaction could be further increased by BFA treatment (Fig. 5E), revealing that Bip could interact withAkt,leading toAkt inactivation. Taken together, these findings suggest that BFA induces autophagy by promoting Bip/Akt interaction, which inhibits Akt phosphorylation in CRC cells. BFA exhibits antitumor effect against CRC in vivo the indicated time points. F) Hematoxylin and eosin staining of the heart, liver, lung, spleen, kidney, and intestine in mice treated with vehicle or BFA (15 mg/kg per day). Scale bars, 100 mm. G) Tumor tissues from HCT116 xenografts treated with vehicle or BFA were evaluated by immunohistochemistry analysis for the expression levels of Bip, LC3, and p-Akt. Scale bars, 50 mm. H) The expression levels of Bip, LC3, p-Akt, total Akt, ATG5, Beclin 1, CHOP, and PPARg in tumor xenografts were determined by immunoblotting. All data are means 6 SD. *P , 0.05, **P , 0.01, ***P , 0.001.
To further explore the biologic effects of BFA in vivo, a mouse xenograftmodel was generated by subcutaneously inoculating the human CRC HCT116 cell line into nude mice. As shown in Fig. 6A, xenografts treated with BFA grew at a slower rate than the vehicle-treated group. Moreover, tumor size and tumor weight were markedly reduced in BFA-treated mice compared with those of the control group (Fig. 6B, C). In addition, immunohistochemistry analysis of Ki67 was performed to confirm the change in the proliferation status of tumor cells. As shown in Fig. 6D, xenografts treated with BFA displayed weaker Ki67 intensity than those treated with placebo. These results suggest that BFA significantly inhibits the growth of CRC cells in vivo. Moreover, we found that BFA treatment had no significant effect on the body weight of mice (Fig. 6E) or the pathologic features (Fig. 6F) of major organs as well as intestine, suggesting that BFA has no obvious toxic or adverse effectin mice. We also examined the expression levels of Bip, Akt, and LC3 in tumor tissues from xenografts. Consistent with the in vitro results, BFA-treated xenografts exhibited increased Bip expression, decreased phosphorylation levels of Akt, and stronger LC3 staining intensity (Fig. 6G). In addition, the protein levels of autophagy markers (ATG5 and Beclin 1) and apoptosis-relevant proteins (CHOP and PPARg) were both up-regulated in the BFA-treated group (Fig. 6H). Collectively, these data indicatethatBFAinducesCRCsuppressionthroughBip/Aktregulated autophagy in vivo.
5-FU and paclitaxel, 2 chemotherapeutic drugs commonly employed in clinical CRC treatment, have been reported to display acquired drug resistance partially due to the activation of autophagy (25, 26). To this end, we examinedtheantitumoreffectsofBFAadditionontheresponseof CRC cells to 5-FU or paclitaxel treatment. As shown in Supplemental Fig. S5A, B, combinational treatment of BFA with 5-FU or paclitaxel markedly decreased the cell growth comparedwithmonotherapy.Moreover,theproliferationof CRC cells was significantly inhibited in response to combinationaltreatmentofBFAwith5-FUorpaclitaxelcompared with monotherapy (Supplemental Fig. S5C, D). Taken together, these results demonstrate that BFA can effectively sensitize CRC cells to the treatment of 5-FU or paclitaxel.
DISCUSSION
BFA is a fungal metabolite that hasbeen proved to display profound inhibitory effects on cellular secretory pathway and widely used for the research of protein secretion and transport (27). Earlier studies demonstrated that BFA treatment effectively suppressed cancer stem cell–like propertiesinhumanCRCColo205cells(28).However,the mechanisms underlying the growth-inhibitory effects of BFA in CRC are still elusive. In this study, our findings demonstrated that BFA stimulated ER stress and upregulated Bip expression, thereby promoting Bip/Akt interaction and inhibiting Akt phosphorylation, resulting in autophagyactivationandsubsequentgrowthinhibitionin CRC cells (Supplemental Fig. S6).
It has been previously shown that ER stress may either induce or suppress autophagy in a context-dependent manner. During physiologic ER stress, the PERK-eIF2a pathway and IRE1a-JNK pathway could be activated to promote autophagic process (29, 30). In contrast, aberrant ER stress could result in impaired autophagic flux in some pathologic conditions (31). Meanwhile, autophagy inducedbyERstressmayalsoexhibit oppositeeffectson cell fate determination, resulting in either cell survival or cell death (32). In the present study, we initially demonstrated the BFA-induced autophagy was attributed to ER stress stimulation and Bip up-regulation. Interestingly, mitigation of ER stress restored BFA-induced autophagy and rescued BFA-induced growth inhibition in CRC cells, suggesting that BFA stimulated ER stress and thereby promoted autophagy and subsequent cell death. In line with our data, ER stress induced by Shiga toxins caused autophagy induction and exhibited cytotoxic effects in intestinal epithelial cells (33). However, autophagy induced by BRAF inhibitor–stimulated ER stress could remove misfolded proteins and damaged organelles, leading to BRAF inhibitor resistance in melanoma (34). Thus, these previous studies, together with our findings, indicate that autophagy induced by ER stress may promote cell survival or cell death in different stress conditions, which appears to be context dependent.
The Akt/mTOR signaling pathway is a major pathway negatively regulating autophagy and is also involved in tumor proliferation (35). In this study, our findings revealed that Akt and mTOR signaling were inhibited by BFA as evidenced by decreased phosphorylation of Akt and mTOR. Furthermore, we found that inhibition of Akt/mTOR signaling in BFA-treated cells was due to ER stress stimulation and Bip up-regulation, whose expressioncorrelateswith thephosphorylationstatusofAkt(36). Our findings demonstrated that BFA could obviously increase the expression of Bip and inhibit the phosphorylation of Akt/mTOR, suggesting that the Bip/Akt/mTOR axisisinvolvedinBFA-inducedautophagy.Moreover,we showed that BFA inhibited the phosphorylation of Akt by promoting the interaction of Akt and Bip, which is consistent with a previous finding in human choriocarcinoma model (37). Overall, these results suggest that targeting the Bip/Akt/mTOR signaling pathway may deserve exploration as a potential therapeutic strategy for CRC treatment.
In summary, our current findings demonstrate that the lactone antibiotic BFA inhibits CRC growth by induction of Bip/Akt-mediated autophagic cell death. These findings provide new insights into the mechanisms of BFAinduced CRC suppression and support the rational utility of BFA for therapeutic treatment of CRC.
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