Induction of autophagy by oleifolioside A in HCT-116 human colorectal cancer cells
Applied Biological Chemistry volume 66, Article number: 32 (2023)
In current study, we addressed the anti-cancer effect of oleifolioside A and its mechanism on the regulation of cell death in HCT-116 human colorectal cancer cells. Oleifolioside A inhibited HCT-116 cell proliferation and caused apoptosis associated with sequential activation of caspases 8 and 3, followed by PARP cleavage. Moreover, anti-LC3-positive granules and the increased LC3-II level were observed in HCT-116 cells treated with oleifolioside A, which is the specific characteristics of autophagy. Treatment of autophagy inhibiors, 3-MA and Wort, markedly accelerated the cell death by oleifolioside A and, furthermore, knockdown of Beclin-1 and Atg7 using shRNA increased oleifolioside A-induced apoptosis, suggesting a cytoprotective function of autophagy against oleifolioside A-triggered apoptosis. Treatment of HCT-116 cells with oleifolioside A time-dependently activated extracellular signal-regulated kinase (ERK). Oleifolioside A-induced autophagy was dramatically inhibited by pretreatment with an ERK inhibitor, U0126, which resulted in a marked reduction in cell viability. These findings indicate that oleifolioside A induce autophagy through ERK activation in HCT-116 cells and that autophagy suppression enhances apoptosis induced by oleifolioside A.
Dendropanax morbifera Leveille (D. morbifera) has long been used in South Korea as a traditional medical material to treat various diseases such as infectious diseases, migraine headache, dysmenorrhea, and skin diseases . Many studies have proved that D. morbifera extracts possess various pharmacological activities, such as anti-inflammatory [2,3,4] and anti-cancer effects  as well as protective effects on kidney function  and hippocampus [7,8,9]. Although some compounds isolated from D. morbifera extracts exhibited anti-diabetic , anti-obesity , anti-thrombotic , anti-inflammatory , and anti-complementary activities , systematic studies of the pharmacological efficacy of the active ingredients of D. morbifera are still not sufficient.
It has been previously reported that oleifolioside A, a bioactive compound isolated from D. morbifera, triggered caspase-independent apoptosis through nuclear translocation of AIF and EndoG in human cervical carcinoma HeLa cells . Although this study has investigated apoptosis-inducing effects of oleifolioside A, its function in autophagy has not been addressed.
The currrent study aimed to explore whether oleifolioside A was able to generate autophagy in HCT-116 human colorectal cancer cells. In addition, mechanisms underlying interactions between autophagy and apoptosis induced by oleifolioside A was investigated.
Oleifolioside A hinders cell proliferation and triggers apoptosis in HCT-116 cells
First, we investigated the inhibitory effect of cell viability by oleifolioside A in HCT-116 cells. Cell viability was assessed by MTT assay after different concentrations of oleifolioside A were treated for 12 h or 24 h. As shown in Fig. 1A, cell viability was diminished in a time- and concentration-dependent way after treatment with oleifolioside A.
To evaluate whether the decrease in cell viability was due to apoptosis, after treatment with different concentrations of oleifolioside A for 24 h, cells were stained with Annexin V-FITC and PI. Flow cytometry analysis of Annexin V/PI-stained cells were conducted. As shown in Fig. 1B, C, oleifolioside A significantly increased apoptotic cell populations in a concentration-dependent way, suggesting induction of apoptosis by oleifolioside A in HCT-116 cells. To further confirm oleifolioside A-induced apoptosis, we examined the activation of caspase-3, a core protease in the apoptotic process and the cleavage of PARP, an endogenous caspase-3 substrate, by Western blot analysis. As shown in Fig. 1D, oleifolioside A treatment elevated the cleaved forms of caspase-3 and PARP in a concentration-dependent way. It is well documented that after synthesis as procaspase-3, caspase-3 activation is occurred by caspase-8 and caspase-9 mediating extrinsic and intrinsic apoptosis pathways, respectively [19, 20]. To investigate which of these two caspases triggers the activation of caspase-3 in oleifolioside A-induced apoptosis, therefore, caspase-8 and -9 activations in HCT-116 cells treated with oleifolioside A were monitored by immunoblotting. Oleifolioside A treatment elevated the cleaved forms (18 and 43 kDa) of caspase-8 in a dose-dependent way, but did not those of caspase-9 (Fig. 1D). To further validate caspase-dependent apoptosis by oleifolioside A, cell viability after treatment with caspase inhibitors was monitored. As shown in Fig. 1E, compared with oleifolioside A treatment alone, a marked increase of the cell viability was observed in cells pretreated with the Z-VAD-FMK. In addition, pretreatment with z-IETD-FMK also notably increased the cell viability, whereas z-LEHD-FMK did not reveal a noticeable increment compared with the oleifolioside A alone-treated group. Taken together, these results indicate that oleifolioside A generates apoptosis through caspase-8 activation followed by subsequent caspase-3 activation in HCT-116 cells.
Oleifolioside A causes autophagy in HCT-116 cells
Several natural product compounds have been reported to trigger both apoptosis and autophagy in diverse human cancer cells, including colon cancer cells [21, 22]. To address whether oleifolioside A triggers autophagy in HCT-116 cells, we analyzed the change in a key autophagy marker protein, LC3-II, in HCT-116 cells treated with oleifolioside A by immunoblotting. As shown in Fig. 2A, B, the conversion of LC3-I to LC3-II, which is a core step in autophagy induction , was markedly increased in a time-dependent way after oleifolioside A treatment. Caspase-8, caspase-3 and PARP cleavages were simultaneously elevated in a time-dependent manner. Moreover, by LC3 immunofluorescence through confocal microscope anti-LC3-positive granules were detected in oleifolioside A-treated cells compared to control, untreated cells (Fig. 2C). These results indicate that oleifolioside A causes autophagy as well as apoptosis in HCT-116 cells. Induction of autophagy by oleifolioside A was also observed in A549 human lung cancer cells and MCF-7 human breast cancer cells (Additional file 1: Figure S1).
Inhibition of autophagy decreases cell viability in HCT-116 cells
Many studies have recently reported that autophagy inhibitor, such as Wort and 3-MA, could strengthen apoptosis in human cancer cells treated with natural product compounds [23,24,25,26,27,28]. We treated 3-MA and Wort to repress the autophagy produced by oleifolioside A treatment and measured cell viability. As shown in Fig. 3A, B, cell viability was dose-dependently decreased when oleifolioside A-induced autophagy was inhibited by 3-MA and Wort (Additional file 2: Figure S2). Next, to verify whether autophagy can control apoptosis, Atg7 and Beclin-1, crucial regulators of autophagy, were knocked down using shRNA and then the changes in LC3, PARP and caspase-3 were checked by immunostaining. As shown in Fig. 3C, knockdowns of Atg7 and Beclin-1 gene remarkably repressed Atg7 and Beclin-1 protein expression. Simultaneously, caspase-3 and PARP cleavages were significantly enhanced compared with oleifolioside A-treated shControl cells. Collectively, these results indicate that autophagy inhibiton by 3-MA and Wort or by Atg7 and Beclin-1 knockdowns promotes oleifolioside A-induced apoptosis and cell death in HCT-116 cells.
Oleifolioside A generates autophagy through activation of ERK pathway
Induction of autophagy by ERK/JNK/p38 MAPK signaling pathway has been recently reported [17, 29,30,31,32,33]. To explore the pathway which contributes to autophagy induction by oleifolioside A in HCT-116 cells, we investigated MAPK signaling pathway by immunoblotting. As shown in Fig. 4A, the active phosphorylated forms of ERK and JNK were time-dependently elevated. To investigate the effects of ERK and JNK MAPK signal pathways on cell death, we measured cell viability after oleifolioside A treatment in HCT-116 cells pre-treated with MAPK inhibitors. As shown in Fig. 4B, cell viability was significantly decreased by U0126, but not by SB203580 and SP600125. In addition, the protein level of LC3-II increased by oleifolioside A treatment was also notably decreased after the combined treatment with U0126 and oleifolioside A (Fig. 4C, D). Taken together, these results indicate that oleifolioside A-triggered autophagy is mediated by ERK signaling pathway, and autophagy inhibition by U0126 augments cell death induced by oleifolioside A in HCT-116 cells.
Although a previous study reported that oleifolioside A, a new component extracted from Dendropanax morbifera Leveille, triggers caspase-independent apoptosis in human cervical carcinoma HeLa cells , its role in relation to autophagy has yet to be identified. Thus, in this study, we addressed the role of oleifolioside A on autophagy in HCT-116 human colorectal cancer cells. Here we have demonstrated for the first time that oleifolioside A can trigger autophagy and apoptosis in HCT-116 cells. Unlike a previous report  showing the caspase-independent apoptosis by oleifolioside A in HeLa cells, our current results clearly indicated that oleifolioside A triggered caspase-dependent apoptosis, as proven by the increase of caspase-8, -3 and PARP cleavages, as well as the rescue from cell death by caspase inhibitors, z-VAD and z-IETD. These findings suggest that oleifolioside A-induced apoptosis is possibly cell type-dependent.
The level of LC3-II expression is the most extensively used as a typical marker of the autophagosome formation, because LC3-I to LC3-II conversion is a hallmark showing the extent of autophagosome [34, 35]. In addition to apoptosis induction by oleifolioside A in HCT-116 cells, in current study, we demonstrated that oleifolioside A triggers autophagy in HCT-116 cells, as evidenced by LC3 immunostaining and immunoblot analysis showing the marked elevation of LC3-II expression levels. Numerous studies have shown that the suppression of autophagy by its specific inhibitor could enhance apoptosis and consequently result in the increase of cell death in human colon cancer cells treated with natural compounds [23,24,25,26,27,28]. Consistent with these observations, our present results showed that autophagy inhibition by its specific inhibitor, 3-MA or wortmannin, led to the increase in cell death in HCT-116 cells, as characterized by the significant reduction in cell viability. Moreover, knockdowns of Atg7 and Beclin-1 genes markedly enhanced oleifolioside A-triggered apoptosis in HCT-116 cells, as proven by increases of caspase-3 and PARP cleavages. These results suggest that autophagy might have cytoprotective role against apoptotic cell death in oleifolioside A-treated HCT-116 cells.
It had been previously reported that ERK, one of MAPK family members, plays a key role in regulation of autophagy [17, 36,37,38]. For instance, Sivaprasad and Basu reported that TNF induced autophagy in MCF-7 cells via the ERK signaling pathway and inhibition of ERK signaling enhanced TNF-triggered cell death . Wen et al. showed that orexin A, a class of peptides, triggered autophagy through the ERK1/2 activation in HCT-116 cells and ERK inhibition reduced the LC3 II level increased by orexin A treatment . In addition, we have also previously reported that dendropanoxide (DP), another natural compound from Dendropanax morbifera Leveille, induces autophagy through the activation of ERK1/2 in MG-63 human osteosarcoma cells and autophagy inhibition elevates DP-triggered apoptosis . In accordance with these findings, in the present study, we revealed that oleifolioside A activated ERK1/2 and ERK1/2 inhibition by U0126 notably decreased the LC3-II level enhanced by oleifolioside A, which leads to augment the cell death by oleifolioside A.
Material and methods
The ERK1/2 inhibitor U0126, was obtained from Promega (Madison, WI, USA). Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), Z-VAD-FMK (pan-caspase inhibitor), SB203580, SP600125 and wortmannin (Wort) were purchased from Calbiochem (Darmstadt, Germany). Antibodies for PARP-1/2, p-ERK and ERK were obtained from Santa Cruz Biotechnology (CA, USA); antibodies for Atg7, Beclin-1, caspase-3, caspase-8, caspase-9, LC3, p-p38, p38, p-JNK and JNK were purchased from Cell Signaling Technology (Dancers, Mass, USA); antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and polyvinylidene difluoride membrane (PVDF) were obtained from Millipore (Milford, MA, USA); fluorescein isothiocyanate (FITC)-conjugated and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Vector Laboratories (CA, USA) and Enzo Life Science (Farmingdale, NY, USA), respectively. 3-Methyladenine (3-MA) and 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich. (St. Louis, MO, USA). Annexin V-FITC apoptosis detection kit and BCA protein assay kit were purchased from BD Biosciences (San Jose, CA, USA) and Thermo (Rockford, IL, USA), respectively. Oleifolioside A isolated from D. morbifera was prepared as described previously , dissolved in dimethyl sulfoxide (DMSO) as a stock solution at 30 mM concentration, and stored in aliquots at − 20 °C.
Human colon cancer cell line HCT-116 was obtained from American Type Culture Collection (Rockville, MD, USA). Cells were cultured in plastic dishes containing Dulbecco’s modified Eagle’s medium (DMEM; WelGENE Co., Korea) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 μg/ml streptomycin and 100 U/ml penicillin at 37 °C under 5% CO2.
The cytotoxic effect of oleifolioside A on HCT-116 cells was measured by MTT assay as previously described . Briefly, when the cells reached about 70% confluence, they were treated with various concentrations of oleifolioside A for 12 and 24 h. Cell viability was determined by measuring the optical density (OD) at 590 nm and quantified as a percentage compared to the control.
Westernblot analysis was conducted as described previously . Antibodies for caspase-3, caspase-8, caspase-9, LC3, PARP-1/2, p-ERK, ERK p-p38, p38, p-JNK, JNK and GAPDH were utilized.
Immunofluorescence staining analysis was performed with anti-LC3 antibody and FITC-conjugated secondary antibody according to a procedure as previously described . Fluorescent signals in cells were observed with LSM 700 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
Annexin V and PI staining
After treatment with the indicated concentrations of oleifolioside A for 24 h, the cells were harvested, washed with cold PBS, and then stained with Annexin V-FITC and propidium iodide (PI) in accordance with the manufacturers’ instructions and finally analyzed with Beckman-Coulter Cytomics FC500 flow cytometer (Beckman-Coulter, Miami, FL, USA).
Plasmids pLKO.1 lentiviral plasmid containing shRNA against Beclin-1 (TRCN0000033552) and Atg7 (TRCN0000007587) were purchased from Sigma-Aldrich (Mission shRNA). Lentivirus productions and lentiviral infections were carried out according to the supplier's protocol. After puromycin selection, cells were treated with oleifolioside A and the silencing efficacy of shRNAs was proved using by immunoblot analysis, as described previously .
All experiments data are expressed as means ± SEM of at least three replicates in each group. Comparisons between the two groups were determined by Student’s t-test and *P < 0.05 was considered statistically significant.
Availability of data and materials
The datasets used in this study are available from the corresponding authors upon request.
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We thank Dr. Hyung-In Moon (SKEDERM Cosmetic R&D Center, Seoul, Republic of Korea) for providing oleifolioside A.
This work was supported by the Dong-A University research fund.
The authors declares that they have no competing interests.
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Induction of authophagy by oleifolioside A in A549 human lung cancer cells and MCF-7 human breast cancer cells. A Cells were treated with different concentration of oleifolioside Afor 24 h and then analyzed by immunoblotting with an antibody against LC3. GAPDH was used as loading control of immunoblotting analysis. B Cells were treated with oleifolioside Afor 24 h and then, after fixation, cells were immunostained with anti-LC3 antibodies. Nuclei were stained with DAPI. Images were taken under confocal microscope.
Effects of oleifolioside A on MAPK signalling pathway in HCT-116 cells. Cells were pre-treated with or without 10 mM Methyladenineor 10 μM Wortmanninfor 1 h and then treated with 50 μM oleifolioside A for 24 h. Immunoblot analysis was performed with antibody against LC3. Bar graphs show densitometry analysis of LC-3II/GAPDH ratio. Data were expressed as the mean ± SEM of three independent experiments. **p<0.01 compared with control; # p<0.05 compared with oleifolioside A-treated group.
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An, SY., An, HK., Kim, KS. et al. Induction of autophagy by oleifolioside A in HCT-116 human colorectal cancer cells. Appl Biol Chem 66, 32 (2023). https://doi.org/10.1186/s13765-023-00791-5
- Oleifolioside A
- HCT116 cells