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Piceatannol Induced Apoptosis Through Up-Regulation of Microrna

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Piceatannol Induced Apoptosis Through Up-Regulation of Microrna Du et al. Biol Res (2017) 50:36 DOI 10.1186/s40659-017-0141-8 Biological Research RESEARCH ARTICLE Open Access Piceatannol induced apoptosis through up‑regulation of microRNA‑181a in melanoma cells Maotao Du1*, Zhong Zhang2 and Tao Gao2 Abstract Background: Melanoma took top position among the lethal cancers and, despite there have been some great attempts made to increase the natural life of patients with metastatic disease, long-lasting and complete remis- sions are few. Piceatannol, owns the similar function as resveratrol, has been defned as an anti-cancer agent playing important role in inhibition of proliferation, migration and metastasis in various cancer. Thus, we aim to investigate the anti-cancer efect and mechanisms of piceatannol in melanoma cells. Methods: Melanoma cell lines WM266-4 and A2058 were treated either with or without piceatannol. Cell viability and cell apoptosis were assessed by using MTT and Annexin V/PI assay, respectively. Cells were transfected with spe- cifc miRNA using Lipfectamine 2000. miRNA bingding ability to 3’-UTR region within specifc gene was assed by frefy luciferase analysis. Gene and protein expression was eveluated by qRT-PCR and western blot analysis, respectively. Results: Our study showed that piceatannol inhibited WM266-4 and A2058 cells growth and induced apoptosis. Totally, 16 diferentially expressed miRNAs were screened out including 8 up-regulated and 8 down-regulated miR- NAs. Expression level of miR-181a is signifcantly higher in piceatannol-treated cells than normal control and is lower in melanoma cancer tissues than its adjacent normal tissues. Bcl-2 is a target gene of miR-181a. Moreover, silencing of miR-181a reverses the decrease of cell viability induced by piceatannol in WM266-4 and A2058 cells. Taken together, present study uncovered the ability of piceatannol to repress melanoma cell growth and clarifed the contribution of miR-181a in the anticancer role of piceatannol. Conclusion: The present study proposes that piceatannol can be taken into account to be a hopeful anticancer agent for melanoma. Keywords: Piceatannol, Apoptosis, microRNA-181-a, Melanoma cells Background show up with unresectable cancer at later stages which Melanoma is aggressive skin cancer and its incidence is usually treated with surgical operation, commonly continues to increase globally [1, 2]. It is the ffth and together with adjuvant chemotherapy [5]. By reason of sixth most common cancer in males and females, respec- its complex etiology addition to drug/chemo resistance tively [3], and is one of the most common cancers among and high metastatic potential, poor prognosis often pre- adolescents and young adults [4]. It is known that surgical sent in patients who are diagnosed with advanced mela- resection is a promising and curative intervention when noma, remaining therapeutically unsatisfactory for many melanoma is diagnosed at early stage, but many patients years [6]. For cancer chemo prevention or treatment, the discovery and development of novel medicament with *Correspondence: [email protected] low toxicity, high efciency and excellent potential is an 1 Department of Dermatology, The Second Afliated Hospital important part of cancer therapies. Tus, it is needed to of Chongqing Medical University, 76 Linjiang Road, Yuzhong District, fnd efective bio-compound by studying its molecular Chongqing 400010, China Full list of author information is available at the end of the article mechanism at the basis of melanoma pathogenesis in © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Du et al. Biol Res (2017) 50:36 Page 2 of 10 order to provide potential targets for its diagnosis and stored at − 20 °C in the dark. MTT based Cell Count- treatment. ing Kit-8 (CCK-8) (Beyotime, Haimen, China), 10% fetal Piceatannol is a strong antioxidant and owns anti- bovine serum (Gibco, UK), antibodies of Bcl-2, Bax, cas- cancer and chemo preventive abilities. Despite the pase-3 and β-actin were provided from Abcam (Cam- pharmacological actions of piceatannol, especially its bridge, MA). antioxidant, antitumor, and anti-infammatory activities, Tissue specimens were obtained from patients who including bioavailability and toxicity in humans are well are underwent melanoma surgical resection in the Sec- documented, depending on the cell type piceatannol may ond Afliated Hospital of Chongqing Medical Univer- either stimulate [7, 8] or inhibit [7] apoptosis. Although it sity and adjacent tissues, which is classifed as control was reported that the evaluation of anti-melanoma efect group, were acquired from resected neighboring area of of piceatannol on human melanoma cells SK-Mel-28 [9], melanoma. We surgically procured tumor samples from the results limited with supporting metabolic data and patients with primary cutaneous melanoma. Upon sur- therapeutic efects and underlying mechanism of picea- gical removal of the primary melanoma, a single surgi- tannol on human melanoma remains unknown. Tereby, cal oncologist (A.I.R.) used a scalpel to macrodissect these limitations are attempted to be replenished in our and procured a portion of the remaining primary tumor. study. We’ve collected 10 paired tissues, namely 10 normal tis- It is the fact that microRNAs (miRs) are a class of small sues and 10 tumor tissues, in which the level of miR- non-coding RNA that can specifcally binding to the 181a was measured. No patients underwent cutaneous 3′-untranslational region of their target mRNAs and in radiotherapy or chemotherapy before. Tis study was this way cause mRNA degradation or inhibition of pro- approved by ethics review board of Chongqing Medical tein translation [10, 11]. Altered miRNA expression has University and all the patients provided written informed been widely reported in cancer, it has been reported that consent. high levels of oncogenic miRNAs downregulate tumor suppressor genes, and conversely, decreased tumor Cell line, culture and piceatannol treatment suppressor miRNAs responsible for elevated oncogene Melanoma cells WM266-4 and A2058 were provided expression [12]. By means of regulating the expression from Wuhan cell bank of Procell. Cells were cultured in of their target genes, miRNAs afect diverse of biologi- DMEM contains 10% fetal bovine serum, 100 μg/ml of cal processes, such as cell proliferation, survival, difer- penicillin and streptomycin, and incubated in a humidi- entiation, cell cycle progression and apoptosis [13]. In fed condition of 95% air and 5% CO2 at 37 °C. After 24 addition to their prognostic or diagnostic utility as bio- and 48 h of culturing, the cells treated with piceatannol markers, miRNAs have appeared as novel biological tool were collected for following measurement. for cancer treatment [14–16]. It is complex to escape from apoptosis for cancer cells. Highly expressed levels MTT cell viability assay of anti-apoptotic protein, B cell lymphoma 2 (Bcl-2) is The 96-hole culture plates was used to seed WM266-4 known to be one of the major causal factors in tumo- and A2058 cells with 1 × 10 [4] cells/well, and the plate rigenesis. In the apoptotic process of eukaryotic cells, was placed in incubation box at 37 °C with 5% CO2. Bcl-2 plays an crucial role contributing survival in inhi- The cells were treated with different concentrations of bition of cell death [17]. Highly expressed level of Bcl-2 piceatannol and after 24-h culture, MTT assay (Gibco) has been reported in various cancers including mela- was performed to determine the cell apoptosis. The noma [18], and has been broadly linked to its anti-apop- culture media were placed and 20 μl MTT assay (5 mg/ totic activity in melanoma cells, silencing by miRNAs ml) was added to each well prior to 4 h incubation. [19]. 150 μl DMSO were added to dissolve the formazan In present study, we attempt to investigate the anti- crystals. After 10 min oscillation, the optical density cancer mechanisms of piceatannol in the highly aggres- (OD) values were measured with a microplate reader sive human melanoma cell line, WM266-4 and A2058, (Beckmann Coulters) at 490 nm. Cell viability was pre- and elucidate the inhibitory efect of piceatannol on mel- sented as a percent of MTT reduction in the treated anoma cells through attenuating miRNA-mediated Bcl-2. cells versus the controls (cells incubated in serum- free DMEM without extracts). The relative MTT level Methods (%) was calculated as [A]/[B] × 100, where [A] is the Materials and samples absorbance of the test sample and [B] is the absorb- Piceatannol with purity > 99% was obtained from ance of control sample containing the untreated cells. Chengdu Must Bio-tech, and 100 mM stock solution was Decreased relative MTT level indicates decreased cell prepared by dissolving the piceatannol in ethanol and viability. Du et al. Biol Res (2017) 50:36 Page 3 of 10 Cell apoptosis detection by fow cytometry Fisher Scientifc, Inc) following the manufacturer’s pro- In brief, WM266-4 and A2058 cells were harvested and tocol. Expression of 16 miRNAs was quantifed by PCR washed with PBS after treatment. Te density of cells array assays purchased from QIAGEN. Tis array experi- was adjusted to 3 × 10 [5], then added 400 μl binding mentally verifed representative miRNAs which regulate bufer, 5 μl Annexin V (Gibco), 10 μl PI (Bio-Science, various apoptosis related genes, and their inhibition or Shanghai, China), and placed the plate at room tempera- overexpression resulted in stimulate or attenuate apop- ture for 15 min in the dark.
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