Abstract
Background/Aim: MicroRNAs (miRNAs) play regulatory roles in pancreatic ductal adenocarcinoma (PDAC). However, it is still required to identify the function of miRNA-301-3p in pancreatic cancer cells. Materials and Methods: Effects of luteolin on cell growth, TRAIL cytotoxicity, and miR-301-3p levels were evaluated. The role of miRNA-301-3p in regulating cell proliferation, target gene expression, and TRAIL cytotoxicity were studied. Results: The levels of miR-301-3p were down-regulated in PANC-1 cells exposed to luteolin, which inhibits the growth of PANC-1 cells and sensitizes cells to TRAIL. The knockdown of miR-301-3p attenuates cell proliferation and enhances TRAIL cytotoxicity. In addition, caspase-8 was directly targeted by miR-301-3p. Conclusion: Our findings unveil a critical biological function of miR-301-3p in regulating cell proliferation and elevating an antiproliferative effect of TRAIL on cancer cells. Our observation of miR-301-3p/caspase-8 relationship can also serve to clarify the role of miR-301-3p in other cancer types and related diseases.
- miRNA-301-3p
- pancreatic cancer
- caspase-8
- TRAIL
- luteolin
- combination effect
Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and a highly fatal malignancy with lymph node or distant metastases upon diagnosis. PDAC is predicted to be the second principal cause of cancer-related deaths by the year 2030 (1). Despite much progress on pancreatic cancer research over the past decades, surgery is the only attempt towards a curative outcome (2). Insufficient clinical efficacy of chemotherapy and radiation therapy has been reported and attributed to drug resistance in PDAC (3-6). Several signaling pathways, including nuclear factor kappa light chain enhancer of activated B cells (NF-ĸB) signaling, are activated, leading to cell growth and metastasis in PDAC. Mutations in tumor suppressor genes are also detected in PDAC (5, 7-9). A better understanding of the molecular characteristics of PDAC can lead to development of novel treatment strategies to combat PDAC.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily. TRAIL has been known to inhibit cell growth by inducing cell-cycle arrest (10, 11). In addition, anticancer effects of TRAIL can occur through death receptors 4 and 5, which generally regulate the activation of caspases, a family of cysteine proteases (12-14). Upon TRAIL binding, activation of caspase-8 cleaves executioner caspases, such as caspase-3 and -7, by which effective caspases contribute to chromatin condensation and nuclear fragmentation with the formation of apoptotic bodies (12-15). Indeed, TRAIL-induced apoptosis is impeded by the low expression of death receptors and caspases (16-18). Moreover, TRAIL resistance is associated with FADD-like ICE inhibitory proteins (FLIP) and inhibitor of apoptosis (IAP) family, such as cIAP, XIAP, and survivin, which inhibit the activation of caspases (19). Accumulating evidence has shown that combination of TRAIL with cancer therapeutic agents is effective to subdue TRAIL resistance. For instance, treatment of kaempferol can induce death receptor expression or suppress survivin levels, thereby enhancing the cytotoxicity of TRAIL (18, 20).
MicroRNAs (miRNAs) are expressed in a tissue-specific manner and dysregulated in most cancers (21-24). miRNAs function as oncogenes or tumor suppressors based on their expression, target genes, and type of cancer. For example, over-expression of miRNA-363-3p suppresses the proliferation of breast cancer cells by targeting potassium voltage-gated channel subfamily H member 2 (KCNH2) (25). It was also demonstrated that miRNA-196b targets Homeobox A9 (HOXA9), thereby positively regulating the invasion of epithelial ovarian cancer cells (26). In addition, we previously demonstrated that miRNAs are differentially expressed in PDAC and that several miRNAs, including miRNA-17-5p, miRNA-132-3p/-212-3p, and miRNA-337-3p can directly regulate executioner caspases, thereby modulating TRAIL cytotoxicity in pancreatic cancer cells (27).
In the present study, we investigated whether miRNA-301-3p levels are negatively regulated by luteolin treatment and if knockdown of miRNA-301-3p enhances TRAIL cytotoxicity. We further identified that miRNA-301-3p directly targets caspase-8 in pancreatic cancer cells, indicating the feasibility of miRNA-301-3p modulation as a miRNA-based therapy for PDAC.
Materials and Methods
Cell culture and transfection. A human pancreatic cancer cell line, PANC-1, was obtained from the Korea Cell Line Bank (Seoul, Republic of Korea). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified 5% (v/v) CO2 atmosphere at 37°C. For antisense oligonucleotide (ASO) control (ASO-control), ASO-miRNA-301a-3p, ASO-miRNA-301b-3p, miRNA mimic control (miRNA-control), miRNA-301a-3p mimic, and miRNA-301b-3p mimic transfection (GE Healthcare, Chicago, IL, USA), cells were exposed to indicated concentrations using siRNA transfection reagent (RNAiMAX; Invitrogen, Carlsbad, CA, USA) and Opti-MEM medium (Invitrogen) as previously described (28).
Cytotoxicity measurements. Cells were transfected with antisense oligonucleotides (ASO-control or a mixture of ASO-miRNA-301a-3p and ASO-miRNA-301b-3p) or exposed to luteolin at 0.1, 5, 10, 25, 50, 100, and 200 μM for 48 or 96 h. For the combination treatments, luteolin or a mixture of ASO-miRNA-301a-3p and ASO-miRNA-301b-3p was treated for 48 h prior to the treatment of TRAIL at indicated concentrations. Following treatments, the cells were fixed with 10% trichloroacetic acid and stained with 0.4% sulforhodamine B (SRB) for 30 min. Protein-bound dye was extracted with 10 mM Tris base solution, and the optical absorbance of each well was measured at 565 nm. For bromodeoxyuridine (BrdU) staining to detect proliferation, BrdU incorporation assay was performed as previously described (29). Cells were treated with 10 μM BrdU for 1 h and then the media were replaced and incubated for an additional hour before fixing in methanol at −20°C for 10 min. Antigen retrieval was performed at 70°C in formamide retrieval solution for 30 min. After blocking in phosphate-buffered saline containing 0.01% bovine serum albumin and 0.01% Tween-20, slides were incubated for 30 min with BrdU monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). After washing, slides were incubated with Alexa 555-linked secondary antimouse IgG (Invitrogen).
Real-time quantitative polymerase chain reaction (PCR) for mRNA and mature miRNA. Total RNA was isolated by miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. To detect mRNA levels, cDNA was prepared using SuperScript III reverse transcription kit (Invitrogen). Real-time qPCR was performed on an AriaMx real-time PCR system (Agilent Technologies, Santa Clara, CA, USA) using the Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Primer sequences used in this study were designed by GENOTECH (Daejeon, Republic of Korea). Primer sequences were as follows: primary transcripts of miRNA-301a (pri-miRNA-301a), forward 5’-TTC AAC CGA TGC AAG ATG CT-3’ and reverse 5’-GTC AGA GCA TTC GTT AGC AG-3’; primary transcripts of miRNA-301b (pri-miRNA-301b), forward 5’-CTG ACG AGG TTG CAC TAC-3’ and reverse 5’-TCC CAG ATG CTT TGA CAA TAT C-3’; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5’-GAA GGT GAA GGT CGG AGT C-3’ and reverse 5’-GAA GAT GGT GAT GGG ATT TC-3’. For the detection of mature miRNAs, one hundred nanograms of total RNA was primed using gene-specific looped primers with a 20 μL reaction volume, and the cDNA was quantified with TaqMan miRNA Assays (Applied Biosystems, Foster City, CA, USA) as previously described (21).
Cell-cycle analysis. To determine cell-cycle distribution, cells were collected after trypsinization into a single cell suspension, fixed with cold ethanol at 4°C for 1 h before being stored at −20°C until further analysis. On analysis, fixed cells were washed and resuspended in 1 ml phosphate-buffered saline (PBS) containing 50 kg/ml RNase A and 50 kg/ml ethidium bromide. After incubating for 20 min at 37°C, cells were analyzed for DNA content by flow cytometry (FACS Calibur; Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). For each sample, 10,000 events were acquired, and cell cycle distributions were determined using a cell cycle analysis software (Modfit; Verity, Topsham, ME, USA).
Luciferase reporter constructs and assay. The three prime untranslated regions (3’UTR) of caspase-8, a miRNA-301-3p predicted target, were PCR amplified using human genomic DNA as a template and a LongAmp™ TaqDNA Polymerase (New England BioLabs, Ipswich, MA, USA). The PCR amplicons were ligated into the psiCHECK-2 vector (Promega, Madison, WI, USA). The mutant reporter constructs harboring mutation of the first four nucleotides of the seed-match sequence were constructed using the QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA, USA). The interaction of the target genes with miR-301-3p was screened using the psiCHECK-2 vector harboring a wild or mutant 3’UTR. Constructs were co-transfected with either miRNA-control, miRNA-301a-3p, or miRNA-301b-3p mimic into cells. Twenty-four hours after transfection, cell lysates were used to measure both firefly and renilla luciferase activities using the Dual-Luciferase® Reporter Assay System (Promega) following the manufacturer's instructions. Primer sequences used in this study were designed by GENOTECH. Primer sequences were as follows: caspase-8, forward 5’-ATG CCT CGA GGA GAC AGA ATC TCG CTC TGT C-3’ and reverse 5’-ATT AGC GGC CGC CAA GTT TGG GGA GAG CTT AT-3’.
Protein extraction and immunoblotting. Proteins were extracted with RIPA cell lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA), and phosphatase/protease inhibitor cocktail (Thermo Fisher Scientific). Tris-HCl gradient gels (4-20%) (Bio-Rad Laboratories, Hercules, CA, USA) were used to separate proteins. Blotting was performed for caspase-8 (D35G2) (Cell Signaling Technology, Danvers, MA, USA) and GAPDH (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).
Data analysis. The IC50 was defined as the drug concentration at which cell viability was equal to 50% that of the control without drug, and calculated using Eq. 1 and Eq. 2: Eq. 1 Eq. 2 where D is the drug concentration, m is the Hill-type coefficient, R is the residual unaffected fraction (the resistance fraction), and Kd is the concentration of drug that produces a 50% reduction of the drug's maximum effect (100–R) (30). The effect of combination of drugs at fixed concentrations was analyzed by comparing experimental data to the reference additivity values calculated using Bliss independence model. Values of the ratio of the experimental survival rate to the reference value between 0.8 and 1.2 was defined as additive, ≤0.8 as synergistic, and ≥1.2 as antagonistic (30, 31).
Statistical analysis. An unpaired t-test and analysis of variance were used to determine statistical significance. The data are shown as means±SD. The differences were considered significant for p-values of less than 0.05. All experiments were replicated at least three times.
Results
Luteolin induces cytotoxicity and has a synergistic effect with TRAIL in PANC-1 cells. Previous reports demonstrated that luteolin negatively regulates the proliferative status of several types of cancer cells, including gastric and breast cancer cells (32, 33). To explore the possibility of antiproliferative effects of luteolin in PANC-1 cells, we treated several concentrations of luteolin for 48 and 96 h. A significant reduction in overall cell growth was noted for PANC-1 cells (Table I and Figure 1A). This reduction of cell growth was also evidenced by a significant decrease in the number of PANC-1 in the S-phase of cell cycle (Figure 1B). In addition, we used fluorescence-activated cell sorting (FACS) analysis to investigate the cell cycle kinetics of PANC-1 cells treated with luteolin. It was found that 50.2% of the vehicle-treated cells were in G0/G1, 28.9% were in S, and 20.9% were in G2/M. In contrast, 52.4% and 50.7% of luteolin-treated cells were in G0/G1, 18.8% and 21.5% were in S, and 28.9% and 27.8% were in G2/M, at 25 μM and 50 μM, respectively, along with an increase in the percentage of sub-G1 phase (Figure 1C and D). Previous studies demonstrated that luteolin can sensitize renal cell carcinoma and liver cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (34, 35). Therefore, we next sought to investigate whether the antiproliferative effects of TRAIL are improved by luteolin in PANC-1 cells. Pretreatment of luteolin caused strong synergistic effects with 5, 25, and 50 ng/ml of TRAIL (Figure 1E).
Luteolin down-regulates miRNA-301-3p levels in PANC-1 cells. Mature miRNA-301-3p consists of miRNA-301a-3p and miRNA-301b-3p derived from the miRNA-301a and miRNA-301b precursors, respectively, and both miRNA-301a-3p and miRNA-301b-3p share identical seed sequences. We previously reported that the levels of miRNA-301-3p were significantly up-regulated in pancreatic cancer cells compared to normal pancreatic duct cells (21), indicating that miRNA-301-3p can serve as an oncogene in pancreatic cancer cells. This is in accordance with previous reports that miRNA-301-3p levels are higher in pancreatic ductal adenocarcinoma cells than normal pancreatic ductal cells (36). Therefore, we next sought to investigate whether luteolin treatment can affect the levels of miRNA-301-3p. Treatment with luteolin resulted in a decrease in the levels of primary transcripts of miRNA-301a and miRNA-301b (Figure 2A and B). We consistently observed a decrease in mature miRNA-301-3p levels (miRNA-301a-3p and miRNA-301b-3p) in luteolin exposed cells (Figure 2C and D).
Knockdown of miRNA-301-3p inhibits the growth of PANC-1 cells and potentiates TRAIL cytotoxicity. The proliferative status of PANC-1 cells was observed following the inhibition of mature miRNA-301-3p with antisense oligonucleotides (ASO). Representative dose-response curves showed that nanomolar (nM) concentrations of ASO-miRNA-301-3p significantly decreased the proliferation of PANC-1 cells in a concentration-dependent manner (Figure 3A). Antiproliferative activity parameters are summarized in Table II. Based on the IC50 value of ASO-miRNA-301-3p at 96 h, transfection of either ASO-control or ASO-miRNA-301-3p at 50 nM resulted in a reduction of both mature miRNA-301a-3p and miRNA-301b-3p (Figure 3B). Moreover, pretreatment of ASO-miRNA-301-3p at 50 nM induced potential synergistic effects with 5, 25, and 50 ng/ml of TRAIL (Figure 3C), indicating that miRNA-301-3p has a potentiality of regulating a gene involved in the regulation of TRAIL sensitivity.
Caspase-8 is directly targeted by miRNA-301-3p in PANC-1 cells. Concerning TRAIL-induced cell death, caspase-8 has been reported as one of the critical factors that determine TRAIL cytotoxicity (37). Also, among several candidate mRNAs, we found that miRNA-301-3p has miRNA: mRNA-predicted interactions for the 3’ untranslated region (UTR) of caspase-8 mRNA (Figure 4A). To experimentally validate if the 3’UTR of caspase-8 mRNA indeed contains matching sites for the interaction with miRNA-301-3p, we carried out a luciferase assay. Luciferase activity in PANC-1 cells, transfected with vectors harboring wild type 3’UTR of caspase-8 (WT), showed a significant reduction with miRNA-301a-3p and miRNA-301b-3p when compared to the miRNA-control (Figure 4B). In contrast, no significant change was observed with a vector containing the 3’UTR of caspase-8 mRNA where four core binding sites for miRNA-301-3p were mutated (Figure 4B). A significant down-regulation or up-regulation of caspase-8 protein was noted in PANC-1 cells transfected with miRNA-301-3p or ASO-miRNA-301-3p, respectively, compared to corresponding controls, indicating that caspase-8 is a bona fide target of miRNA-301-3p (Figure 4C).
Discussion
Herein we demonstrated evidence that miRNA-301-3p is down-regulated by luteolin treatment and that knockdown of miRNA-301-3p shows in vitro antiproliferative effects on PANC-1 cells, indicating the role of miRNA-301-3p as an oncogenic non-coding RNA. Moreover, miRNA-301-3p directly targets caspase-8, a critical regulator of apoptosis, and knockdown of miRNA-301-3p sensitizes PANC-1 cells to TRAIL.
TRAIL has been considered a potent anticancer agent owing to its ability to selectively destroy cancer cells. However, resistance to TRAIL is the major hindrance to achieve encouraging outcomes (38). Inhibitions of cell survival pathways, such as phosphoinositide 3-kinase (PI3K)/AKT, protein kinase C (PKC), and inhibitor of nuclear factor-ĸB (IĸB) kinase (IKK) were suggested as strategies to overcome TRAIL resistance (39). In addition, it has been demonstrated that the up-regulation and down-regulation of caspase-8 render cancer cells sensitive and resistant to TRAIL, respectively (40, 41). Indeed, epigenetic modifications can potentiate TRAIL cytotoxicity by up-regulating death receptor expression, inducing cell-cycle arrest, and elevating caspase-8 levels (38, 40).
Several genes have been identified as miRNA-301-3p targets. In pancreatic cancer cells, miRNA-301-3p directly targets phosphatase and tensin homolog (PTEN), thereby activating PI3K/AKT signaling (36). In this study, it was also noted that inhibition of miRNA-301-3p can potentiate gemcitabine cytotoxicity. Another validated target of miRNA-301-3p is NF-ĸB-repressing factor (NKRF). Over-expression of miRNA-301-3p, therefore, activates NF-ĸB signaling by repressing NKRF in pancreatic cancer cells, contributing to the growth of cancer (42). To our knowledge, our study is the first to implicate miRNA-301-3p as a direct regulator of caspase-8 expression. Taken together, these findings indicate that knockdown of miRNA-301-3p can play critical roles in modulating multiple regulatory factors of TRAIL sensitivity in cancer cells.
It has been reported that luteolin can potentiate TRAIL cytotoxicity in cancer cells. For example, luteolin treatment sensitizes liver and cervical cancer cells to TRAIL by elevating death receptor 5 levels (35, 43). Another study showed that luteolin inactivates AKT signaling, hence enhancing TRAIL-induced apoptosis in renal cell carcinoma cells (34). Our findings further explain the mechanism underlying the luteolin-mediated sensitization of cancer cells to TRAIL since luteolin down-regulates miRNA-301-3p, which target caspase-8. Although further studies are required to precisely identify how luteolin modulates the levels of miRNA-301-3p, NF-ĸB and specificity protein 1 (SP1), transcriptional activators of miRNA-301a and miRNA-301b (42, 44, 45), can serve as regulators of miRNA-301-3p expression in luteolin-treated cells since NF-ĸB and SP1 are inactivated by luteolin (46, 47).
Collectively, our findings provide new insight into how miRNA-301-3p can regulate TRAIL sensitivity in pancreatic cancer cells. We also recently reported that several miRNAs, including miRNA-337-3p, are dysregulated in pancreatic cancer and they directly regulate the expression of executioner caspases-3 and caspase-7 in PANC-1 cells (27). Therefore, it is possible to reinforce the idea that the modulation of caspase regulatory miRNAs, including miRNA-301-3p, can provide a promising opportunity for developing novel strategies to manage pancreatic cancer.
Acknowledgements
This study was supported by Hallym University Research Fund 2018 (H20180239).
Footnotes
* These Authors contributed equally to this study.
Authors' Contributions
SM, SWS, and HAS designed and performed the majority of experiments. JSL assisted in the experiments. CKK and HJK contributed to the statistical analysis and data interpretation. JKP designed the experiments and wrote the manuscript. All authors approved the final manuscript.
Conflicts of Interest
The Authors declare that they have no competing interests.
- Received December 2, 2019.
- Revision received December 12, 2019.
- Accepted December 19, 2019.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved