AU-1 from Agavaceae plants causes transient increase in p21/Cip1 expression in renal adenocarcinoma ACHN cells in an miR-34-dependent manner
Here, we show that AU-1, spirostanol saponin isolated from Agavaceae plants, causes a transient increase in cyclin-dependent kinase inhibitor (CDKI) p21/Cip1 through the upregulation of miRNAs, miR-34 and miR-21. AU-1 stimulated p21/Cip1 expression without exerting cytotoxicity against different types of carcinoma cell lines. In renal adenocarcinoma ACHN cells, AU-1 transiently elevated the expression level of p21/Cip1 protein without marked increases in p21/Cip1 mRNA levels. Rapid and transient increases in miR-34 and miR-21, both of which are known to upregulate p21/Cip1, were observed in AU-1-treated cells. Inhibitor for miR-34 and for miR-21 significantly blocked the AU-1-caused increase in p21/Cip1, indicating that elevation of p21/Cip1 protein by AU-1 is dependent on these microRNAs. We further clarified that NAD-dependent deacetylase SIRT1, a direct target of miR-34, is decreased by the treatment with AU-1. Furthermore, we found that SIRT1-knockdown increases p21/Cip1 protein levels in an miR-21-dependent manner. On the other hand, ectopic expression of p21/Cip1 resulted in the lowered expression of miR-34 and miR-21, suggesting that reciprocal regulation exists between p21/Cip1 and these miRNAs. We propose that the following feedback network composed of miR-34/SIRT1/miR-21/p21 is triggered by the treatment with AU-1: in cells treated with AU-1, transient elevation of miR-34 leads to the downregulation of SIRT1, thereby miR-21 is freed from SIRT1-dependent suppression. Then, elevated miR-21 upregulates p21/Cip1 protein, followed by the suppression of miR-34 expression.
KeywordsAgave utahensis p21/Cip1 miR-34 miR-21 SIRT1
Materials and methods
Etoposide was purchased from Wako Pure Chemicals Industries (Osaka, Japan). (25R)-5β-spirostan-3β-yl O-β-D-glucopyranosyl-(1 → 4)-β-D-galactopyranoside (AU-1) was isolated from Agave utahensis Engelm . siRNA against human SIRT1 was from Santa Cruz Biotechnology (USA). miRCURY LNA Power Inhibitor for miR-34 (4100032-100) and that for miR-21 (4100688-100) were from EXIQON (Denmark). The human renal adenocarcinoma cell line ACHN and human hepatocellular carcinoma cell line HepG2 were obtained from the American Type Culture Collection (ATCC).
ACHN, HepG2, and the human glioma cell line U-251 were maintained in Dulbecco’s modified eagle medium (DMEM) containing 10 % fetal calf serum (FCS), 50 units/mL penicillin G sodium salt, and 50 μg/mL streptomycin sulfate, and cultured in a humidified atmosphere of 8.5 % CO2 at 37 °C.
Construction of the p21/Cip1 expression vector
pcDNA-p21 expression vector was prepared as follows: cDNA was obtained using Ready-To-Go You-Prime First-Strand Beads (GE Healthcare Biosciences, Japan) using total RNA extracted from ACHN cells. Polymerase chain reaction (PCR) was then performed using the cDNA as template (forward primer: CGGGATCCGAAGTCAGTTCCTTGTGGA; reverse primer: GGAATTCAAGGCAGAAGATGTAGAGC), and the amplified product corresponding to the open reading frame of p21/Cip1 (GenBank no. NM000389) was subcloned into the BamHI/EcoRI site of pcDNA3.1(+) vector.
Quantification of mRNA
Quantification of mRNA was performed via real-time PCR. Briefly, 5 μg of total RNA was reverse-transcribed using Ready-To-Go You-Prime First-Strand Beads (GE Healthcare Biosciences). The resultant cDNA was then subjected to real-time PCR analysis using a TaqMan Gene Expression Assay kit (Applied Biosystems, Tokyo, Japan). mRNA levels were determined via TaqMan assay mixtures as follows: p21/Cip1 (Hs01121172) and β-actin (4310881E). Amplification and quantification were performed using the ABI PRISM 7000 Real-Time PCR System (Applied Biosystems). p21/Cip1 mRNA levels were normalized to those of β-actin mRNA as an internal control. Data were analyzed using Student’s t-test.
Quantification of miRNA
Quantification of miRNA was performed via real-time PCR. Briefly, total RNA enriched with microRNA was extracted from ACHN cells using a mirVana miRNA Isolation Kit (Life Technologies, Tokyo, Japan). Reverse transcription was performed using a TaqMan MicroRNA Reverse Transcription kit (Life Technologies) with 10 ng of total RNA. The resultant cDNA was then subjected to real-time PCR analysis using a TaqMan Gene Expression Assay kit (Life Technologies). miRNA levels were then determined via TaqMan assay mixtures as follows: miR-21 (000397), miR-34 (000426), miR-149 (000472), and miR-192 (000491). Amplification and quantification were performed using an ABI PRISM 7000 Real-Time PCR System (Applied Biosystems). Data were analyzed using Student’s t-test.
Cells were washed with PBS, and cell extracts were prepared using SDS sample buffer without loading dye. After normalization of protein content via the protein assay, the dye was added to samples, followed by SDS-PAGE and immunoblotting analyses. For the detection of p21/Cip1 and β-actin, the membranes were incubated with the primary antibody (Santa Cruz Biotechnology) for 2 h. Immunocomplexes on the PVDF membranes were visualized with enhanced chemiluminescence Western blotting detection reagents (GE Healthcare Biosciences).
The cytotoxic effects of AU-1 on the growth of ACHN and HepG2 cells were examined as follows: ACHN cells seeded on 60-mm dishes at a density of 2.0 × 105 cells/dish or HepG2 cells seeded on 60-mm dishes at a density of 1.0 × 105 cells/dish were incubated in 8.5 % CO2/air for 24 h at 37 °C, followed by AU-1 treatment at various concentrations (up to 50 μM) for 24 h. Negative control cells were treated with vehicle dimethyl sulfoxide (DMSO). Positive control cells were treated with etoposide for 24 h. The cell number per dish was determined using a hemocytometer after the cells were washed.
Data are presented as the mean values ± standard error of the mean (SEM) of three experiments performed in triplicate and analyzed using Student’s t-test.
Results and discussion
AU-1 causes transient increase in CDKI p21/Cip1 without exhibiting cytotoxicity towards renal adenocarcinoma cells
Cytotoxic activities of AU-1 against ACHN and HepG2 cells
ACHN IC50 (μM)a
HepG2 IC50 (μM)a
35.8 ± 1.40
40.0 ± 3.56
0.21 ± 0.05
0.32 ± 0.09
Upregulation of miR-34 and miR-21 are involved in p21/Cip1 increase by AU-1
miR-34 upregulates p21/Cip1 protein expression via the mechanism that leads to SIRT1 suppression followed by miR21 induction
Overexpression of p21/Cip1 decreases miR-21 and miR-34 levels
We previously reported that naturally occurring compounds, cardenolide glycosides, caused sustained increase in the levels of p21/Cip1 protein in hepatocellular carcinoma HepG2 and renal adenocarcinoma ACHN cells, resulting in the significant impairment of their growth . In contrast to the observation in the present study using AU-1, cardenolide glycosides-induced elevation of p21/Cip1 protein in ACHN cells accompanied the upregulation of p21/Cip1 mRNA, and, indeed, p53 expression was elevated in cardenolide glycosides-treated ACHN cells. Sustained increase in p21/Cip1 and resultant significant growth inhibition was also provided by the treatment of hepatocellular carcinoma cells with siRNA targeting FXR, a bile acid-activated nuclear receptor .
A CDKI, p21/Cip1, plays a key role in cell cycle regulation and its dysregulation may lead to tumor progression. In the present study, we have highlighted a unique signaling circuit composed of p21/Cip1, two different types of miRNAs, miR-34 and miR-21, and an NAD-dependent deacetylase, SIRT1. Among the members of this circuit, both p21/Cip1 and miR-34 are direct p53 targets and have been described as tumor-suppressive molecules . On the other hand, miR-21, which has been characterized as oncogenic miRNA , is rather growth-promotive. SIRT1, which plays a critical role in metabolic health by deacetylating many target proteins, was originally considered to be a potential tumor promoter since it negatively regulates the tumor suppressor p53 . Indeed, we have observed that SIRT1-knockdown caused the elevation of p53 protein levels in ACHN cells (data not shown). In the case of ACHN cells treated with AU-1, miR-34 is linked to p21/Cip1 through the p53-independent pathway, in which SIRT1 and miR-21 are involved, although p53 expression is slightly elevated by AU-1 (data not shown). Elucidation of the mechanism of how miR-34 is downregulated by p21/Cip1 is critical to establish AU-1 as the anti-cancer compound against solid types of tumor, since AU-1-triggered increases in miR-34 and p21/Cip1 will be sustained by blocking this negative feedback loop. In order to understand the mechanism of how certain types of tumor cells acquire resistance to tumor-suppressive compounds, further insight should be addressed to the signaling circuit demonstrated in this study.
We thank Ken Ando, Toshiyuki Oshima, and Harutaka Ichikawa for their helpful advice and discussions. This work was supported, in part, by a grant from the Japan Private School Promotion Foundation.
Compliance with ethical standards
Conflict of interest
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