Curcumin Inhibits Proliferation, Migration, Invasion and Promotes

Li et al. BMC Cancer (2018) 18:1230 https://doi.org/10.1186/s12885-018-5130-y

RESEARCH ARTICLE Open Access Curcumin inhibits proliferation, migration, invasion and promotes apoptosis of retinoblastoma cell lines through modulation of miR-99a and JAK/STAT pathway Yaping Li1, Weixuan Sun2, Ning Han1, Ying Zou1 and Dexin Yin3*

Abstract Background: Curcumin, a primary active ingredient extracted from the Curcuma longa, has been recently identified as a potential anti-tumor agent in multiple kinds of cancers. However, the effect of curcumin on retinoblastoma (Rb) is still unclear. Therefore, we attempted to reveal the functional impacts and the underlying mechanisms of curcumin in Rb cells. Methods: Two Rb cell lines SO-Rb50 and Y79 were pre-treated with various doses of curcumin, and then cell proliferation, apoptosis, migration, and invasion were assessed, respectively. Further, regulatory effects of curcumin on miR-99a expression, as well as the activation of JAK/STAT pathway were studied. Results: Data showed that curcumin significantly inhibited the viability, colony formation capacity, migration and invasion, while induced apoptosis of SO-Rb50 and Y79 cells. Up-regulation of miR-99a was observed in curcumin-treated cells. Curcumin suppressed the phosphorylation levels of JAK1, STAT1, and STAT3, while curcumin did not inhibit the activation of JAK/STAT pathway when miR-99a was knocked down. Conclusion: Curcumin inhibited proliferation, migration, invasion, but promoted apoptosis of Rb cells. The anti-tumor activities of curcumin on Rb cells appeared to be via up-regulation of miR-99a, and thereby inhibition of JAK/STAT pathway. Keywords: Curcumin, Retinoblastoma, miR-99a, JAK/STAT pathway

Background developing nations is still unsatisfactory. The mainstay Retinoblastoma (Rb) is a common malignant intraocular of therapy is for globe salvage. tumor among children [1], accounting for approximately Curcumin, a nature yellow pigment, is the primary 4% of all pediatric malignancies [2]. Orally chemother- active ingredient extracted from the rhizome of the East apy alone failed to cure patients with intraocular Rb [3], Indian plant Curcuma longa [5]. Currently, multiple while combination therapy, such as chemotherapy com- therapeutic properties of curcumin have been recog- bined with focal therapy, has become the treatment of nized, including anti-inflammatory, anti-neoplastic [6], choice for intraocular Rb and with high cure rate [4]. anti-oxidant [7], anti-fibrotic [8], and anti-ischemic [9] Although the overall survival rates have been signifi- effects. Specifically regarding the anti-neoplastic func- cantly improved recently, the survival rate of Rb in the tion, curcumin has been reported to suppress the secretion of gastrin-mediated acid, and thereby inhibiting the * Correspondence: [email protected] progression of gastric cancer [10]. In vitro investigation 3Department of Vascular Surgery, China-Japan Union Hospital of Jilin has showed that curcumin inhibited bladder cancer cells University, No.126, Xiantai Street, Changchun, Jilin 130033, China growth and metastasis via regulating β-catenin Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access 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. Li et al. BMC Cancer (2018) 18:1230 Page 2 of 9

expression [11]. Another study demonstrated that curcu- Cell viability min enhanced the radiosensitivity of renal cancer cells, SO-Rb50 and Y79 cells (5 × 103 cells/well) were planted in suggesting the potential application of curcumin as an 96-well plated for 24 h of incubation. Thereafter, 0–50 μM adjuvant in radiotherapy of renal cancer [12]. Although of curcumin was added to treat cells for 24 h. Cell Count- the anti-tumor potentials of curcumin in multiple kinds ing Kit-8 (CCK-8, Dojindo Molecular Technologies, of cancers has been studied [13], the role of curcumin in Gaithersburg, MD) solution (10 μL) was then added, and Rb has not been well recognized. the cultures were incubated at 37 °C for another 2 h. The Recent evidence has suggested that microRNA (miRNA) optical density was detected at 450 nm using the iMark regulation is implicated in the anti-tumor properties of microplate reader (Bio-Rad, Hercules, CA). curcumin [14–16]. miRNAs are a class of short, non-coding RNAs that play significant roles in modulating Colony formation various diseases progression. It has been reported that, SO-Rb50 and Y79 cells (500 cells/well) were seeded in miR-99a was frequently dysregulated in several human 6-well plates. After adherence and curcumin treatment, cancers, including breast cancer, nasopharyngeal carcin- the culture medium was replaced and the cells were cul- oma, esophageal squamous cell carcinoma and oral car- tured in normal medium for another two weeks. The cinoma, and the tumor-suppressive effects of miR-99a in colonies were fixed with 100% methanol and stained these tumors have been revealed [17–20]. To date, no lit- with 1% crystal violet (Sigma-Aldrich). Colonies contain- erature has focused on the tumor suppressive activities of ing more than 50 cells were defined as survivors. miR-99a in Rb. Besides, a growing number of literatures have showed that, curcumin exerts its therapeutic proper- Apoptosis assay ties through regulating the expression of cancer-related FITC-annexin V/PI detection kit (Beijing Biosea Bio- miRNAs [21, 22]. Therefore, herein we aimed to explore technology,Beijing,China)wasusedinthisstudyto whether curcumin-mediated anti-tumor activity in Rb quantify apoptotic cell rate after curcumin treatment. cells via modulation of miR-99a. In brief, SO-Rb50 and Y79 cells (5×105 cells/well) were To this end, two Rb cell lines SO-Rb50 and Y79 were planted in 6-well plates at. When cells were grown to pre-treated with curcumin, and then cell growth and about 80% confluence, cells were treated with 30 μM metastasis were evaluated, respectively. Further, the curcumin for 24 h. Thereafter, cells were collected, regulatory effects of curcumin on miR-99a expression, washed twice in PBS, resuspended in 200 μLbinding and JAK/STAT pathway were studied to explore the pos- buffer, and stained by 10 μL FITC-annexin V and 5 μL sible molecular mechanisms governing this tumor sup- PI in the dark at room temperature for 30 min. Follow- pressive activity. ing the adding of 200 μL PBS, the fluorescence intensity was measured by a FACS scan (Beckman Coulter, Ful- Methods lerton, CA). Cell lines and curcumin treatment Rb cell lines, i.e., Y79 and SO-Rb50, were respectively Transwell assay obtained from the American Type Culture Collection A modified Boyden chamber with 8.0-μm pore filters (Catalogue number: ATCC® HTB-18, ATCC, Manas- (Costar-Corning, New York) was utilized in this study to sas,VA)andtheOphthalmicCenterofSunYat-sen evaluate cell migratory capacity following curcumin University (Guangzhou, China). The base medium for treatment. Cell invasion was carried out the same as mi- both Y79 and SO-Rb50 cell lines was RPMI-1640 gration assay, except that the transwell inserts were medium (Life Technologies Corporation, Cergy Pon- pre-coated with matrigel before assay. In brief, cells pre- toise, France). To make the complete growth medium, treated with 30 μM curcumin were suspended in 200 μL 15% fetal bovine serum (FBS, Life Technologies Cor- of serum-free medium. The cell suspension was added poration), 0.1% ciprofloxacin, 2 mM L-glutamine, 1 into the upper chamber of the 24-well plates, and mM sodium pyruvate, and 4.5% dextrose were added. 600 μL complete medium was added into the lower Culture conditions were 37 °C in a humidified air chamber. After incubation at 37 °C for 24 h, with 5% CO2. non-transferred cells were wiped off by cotton swabs. Curcumin with purity greater than 98% (Sigma-- Transferred cells were stained with crystal violet and Aldrich, St. Louis, MO) was dissolved in dimethyl sulf- counted directly. oxide (DMSO, Sigma-Aldrich) at a concentration of 50 mM for stocking. The stock solution of curcumin was miRNA transfection diluted by the culture medium so that the vehicle was miR-99a inhibitor with sequences of CACAAGAUC less than 0.1%. Curcumin was used at concentration of GGAUCUACGGGUU and its scrambled control (NC) 0–50 μM for 24 h. were both purchased from GenePharma (Shanghai, Li et al. BMC Cancer (2018) 18:1230 Page 3 of 9

China). Lipofectamine 3000 reagent (Invitrogen) was analyzed using the one-way analysis of variance used for transfection following the manufacturer’s proto- (ANOVA). The SPSS version 13.0 software (SPSS Inc., col. 48 h later, transfection was stopped and the effi- Chicago, IL) was used to analyze statistical significance. ciency of transfection was quantified by western blot. A P-value of < 0.05 was considered to indicate a statisti- cally significant result. qRT-PCR Cellular RNA was extracted using Trizol reagent (Life Technologies Corporation). RNA (5 μg) from each sam- Results ple was subjected to reverse transcription using the Taq- Curcumin inhibited the proliferation of SO-Rb50 and Y79 man MicroRNA Reverse Transcription Kit (Applied cells Biosystems, Foster City, CA). Taqman Universal Master To start with, SO-Rb50 and Y79 cells were subjected – μ Mix II was used in real-time PCR and each real-time with 0 50 M of curcumin, and then the viability of PCR was carried out in triplicate for a total of 20 μL cell was tested after 24 h treatment with curcumin. reaction mixtures on ABI PRISM 7500 Real-time PCR As a result, curcumin significantly reduced the viabil- System (Applied Biosystems). Relative expression of ity of SO-Rb50 and Y79 cells (P <0.05 or P < 0.01; CyclinD1 and miR-99a was normalized to GAPDH and Fig. 1a) in a dose-dependent manner. The IC50 values U6 snRNA, respectively. The primers were as follows: of curcumin in SO-Rb50 and Y79 cells were 38.4 and μ μ CyclinD1, forward, 5′-TCC TCT CCA AAA TGC CAG 34.8 M, respectively. Thus, 30 Mwasselectedfor AG-3′, and reverse, 5′-GGC GGA TTG GAA ATG use in the follow-up experiments. Results in Fig. 1b AAC TT-3′; miR-99a, forward, 5′-GTT GGA TCC TAT displayed that curcumin significantly reduced the sur- TAA TAG GGG GCC CAT GCA AGA T-3′, reverse, vival rate of Rb cells (P < 0.001). Further results 5′-GTT CTC GAG GCA CTG TGT ATA GCA TTT showed that curcumin down-regulated the expression TGT CAG-3′. of CyclinD1, while up-regulated the expression of p21 (P <0.05 or P <0.01; Fig. 1c and d). All these data Western blot provided the evidence that curcumin exerted Cellular proteins were extracted in 1% Triton X-100 and anti-proliferating functions on Rb cells. 1 mM PMSF (pH 7.4) over ice for 30 min. The extracts were centrifuged at 1200 g for 15 min at 4 °C and the Curcumin induced apoptosis in SO-Rb50 and Y79 cells supernatant were collected. Protein concentrations were Impacts of curcumin on SO-Rb50 and Y79 cells apoptosis ™ quantified using the BCA Protein Assay Kit (Pierce, were determined. Results in Fig. 2a showed that, apoptotic Appleton, WI). Protein (0.1 mg) was resolved over cell rate was increased by addition of curcumin in both SDS-PAGE and transferred to a polyvinylidene fluoride SO-Rb50 and Y79 cells (P < 0.01). Then, western blot ana- membrane. The membranes were blocked in 5% non-fat lysis was carried out to determine the regulatory role of dry milk for 1 h at room temperature, followed by prob- curcumin in apoptosis-related protein expression. Results ing with primary antibodies at 4 °C overnight: CyclinD1 in Fig. 2b and c displayed that, the ratio of Bcl-2 to Bax (1:10000, ab134175), p21 (1:50, ab107099), Bcl-2 (1:500, was significantly declined (P < 0.001), and caspase-3 and ab59348), Bax (1:1000, ab199677), caspase-3 (1:250, -9 were significantly cleaved (P <0.001) in curcumin- ab13586), caspase-9 (1:200, ab25758), MMP2 (1:1000, treated cells. Thus, these results suggested the pro-apop- ab37150), RhoA (1:5000, ab187027), ROCK1 (1:2000, totic effects of curcumin on Rb cells. ab45171), Vimentin (1:500, ab137321), JAK1 (1:300, ab125051), p-JAK1 (1:1000, ab215338), STAT1 (1:500, ab3987), p-STAT1 (1:1000, ab109461), STAT3 (1:500, Curcumin inhibited the migratory and invasive capacities ab119352), p-STAT3 (1:1000, ab30647), and β-actin of SO-Rb50 and Y79 cells (1:1000, ab8224, all from Abcam, Cambridge, MA). After Next, transwell assay was performed to ask whether incubation with the secondary antibodies, blots were vi- curcumin also could affect SO-Rb50 and Y79 cells sualized by enhanced chemiluminescence (ECL) method. migration and invasion. Data in Fig. 3a and b re- Intensity of the positive blots was tested by Image Lab™ vealed that, relative migration and invasion of Software (Bio-Rad, Hercules, CA). SO-Rb50 and Y79 cells were both reduced by curcumin (P <0.05 or P < 0.01). Results in Fig. 3c-e Statistical analysis revealed that, the protein levels of MMP2, RhoA, Data were presented as mean ± standard derivations ROCK1, and Vimentin were all down-regulated by from three independent experiments. Statistical differ- curcumin (P <0.05 or P < 0.01), which further con- ences between two groups were analyzed using the Stu- firmedtheinhibitoryroleofcurcumininRbcells dent t test, and between three or more groups were migration and invasion. Li et al. BMC Cancer (2018) 18:1230 Page 4 of 9

Fig. 1 Curcumin inhibited SO-Rb50 and Y79 cells proliferation. a Viability of cell pre-treated with 0–50 μM curcumin. b Colony formation capacity of cell pre-treated with 30 μM curcumin. c mRNA level expression of CyclinD1 in cell pre-treated with 30 μM curcumin. d Protein level expression of CyclinD1 and p21 in cell pre-treated with 30 μM curcumin. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control group

Fig. 2 Curcumin promoted apoptosis in SO-Rb50 and Y79 cells. a Apoptotic cell rate of cell pre-treated with 30 μM curcumin. b Protein expression levels of apoptosis-associated factors in cell pre-treated with 30 μM curcumin. c Quantitative protein levels. ** P < 0.01, *** P < 0.001 vs. control group Li et al. BMC Cancer (2018) 18:1230 Page 5 of 9

Fig. 3 Curcumin inhibited the migratory and invasive capacities of SO-Rb50 and Y79 cells. a Relative migration and (b) relative invasion of cell after treatment with 30 μMcurcumin.c and d Protein expression levels of metastasis-related factors in cell pre-treated with 30 μM curcumin. e Quantitative protein levels. * P < 0.05, ** P < 0.01 vs. control group

Curcumin deactivated JAK/STAT signaling pathway via implying miR-99a-silenced cells were successfully regulating miR-99a established. Previous studies suggested that curcumin rendered its Several studies have suggested that curcumin exerted anti-tumor activities via regulating several miRNAs anti-inflammatory and anti-tumor effects through inhi- [14–16]. The present work attempted to study if biting JAK/STAT signaling pathway [23–28]. Herein, we miR-99a was a downstream gene of curcumin. The explored whether miR-99a was implicated in the regula- expression levels of miR-99a in cells pre-treated with tory effect of curcumin on JAK/STAT pathway. Results or without curcumin were detected. qRT-PCR data showed that curcumin remarkably reduced the phos- showed that, miR-99a was up-regulated in cells phorylation levels of JAK1, STAT1, and STAT3 (P < 0.05, treated with curcumin as relative to control cells P < 0.01 or P < 0.001). However, curcumin did not inhibit (without curcumin treatment) (P <0.05, Fig. 4a), the phosphorylation of these proteins when miR-99a was which indicated that curcumin could efficiently knocked down (Fig. 4c and d). up-regulate miR-99a expression in Rb cells. Then, miR-99a expression in SO-Rb50 and Y79 cells were Discussion suppressed by transfection with miR-99a inhibitor, to Curcumin has been considered as a promising approach further explore whether the anti-tumor activity of for the treatment of multiple cancers, including gastric curcumin was through enhancing miR-99a expression. cancer, bladder cancer, renal cancer, etc [6, 10–13]. Re- Results in Fig. 4b showed that, miR-99a expression in cently, several researchers also focused on the potential cells transfected with miR-99a inhibitor was much role of curcumin in Rb. Sreenivasan and his colleagues lower than that in NC-transfected cells (P <0.01), have reported that curcumin suppressed the expression Li et al. BMC Cancer (2018) 18:1230 Page 6 of 9

Fig. 4 Curcumin deactivated JAK/STAT signaling pathway. a miR-99a expression in cell pre-treated with 30 μM curcumin. b miR-99a expression in cell transfected with miR-99a inhibitor and NC. U6 snRNA was used as an internal control. c Protein expression levels of main factors in JAK/STAT pathway, after treatment with curcumin with or without miR-99a inhibitor transfection. d Quantitative protein levels. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control or NC groups. ## P < 0.01, ### P < 0.001 vs. curcumin + NC group of multidrug resistance associated protein 1 (MDR1) Y79 cells, while curcumin could induce cell apoptosis to [29], thereby rendering Rb cells more sensitive to some extent. Furthermore, miR-99a was highly chemotherapy [30]. Another investigation demonstrated expressed in curcumin-treated cells. Curcumin could that curcumin induced the apoptosis of Y79 cells pos- not block the activation of JAK/STAT signaling pathway sibly through activating JNK and p38 MAPK pathways when miR-99a was knocked down. [31]. Although these authors have identified some Previous studies showed that the anti-tumor activities anti-tumor activities of curcumin on Rb, additional stud- of curcumin are mainly due to the inhibition of prolifer- ies are required to cross-check the anti-tumor properties ation, migration and invasion, as well as due to the in- of curcumin on other Rb cell types, and to decode the duction of apoptosis [11, 15, 22]. This study revealed underlying mechanisms. Herein, we demonstrated that that curcumin significantly reduced the viability and col- curcumin significantly inhibited the viability, colony for- ony formation of both SO-Rb50 and Y79 cells, and the mation capacity, migration and invasion of SO-Rb50 and IC50 value of curcumin in these two Rb cell lines is Li et al. BMC Cancer (2018) 18:1230 Page 7 of 9

about 35 μM. Besides, based on the data in the current JAK/STAT pathway in miR-99a-silenced cells, implying study we inferred that curcumin-suppressed Rb cells curcumin inhibited JAK/STAT pathway in a miR-99a-de- proliferation might be via modulation of cell cycle, as pendent mechanism. CyclinD1 was down-regulated and p21 was up-regulated In this study, the IC50 values of curcumin in two Rb by addition of curcumin. Similar finding was reported by cell lines (SO-Rb50 and Y79) were 38.4 and 34.8 μM, re- Yu et al. [31] that curcumin induced G1 phase arrest via spectively, and 30 μM curcumin exhibited excellent down-regulations of Cyclin D3, CDK2/6, and anti-tumor activities. Due to the extensive intestinal and up-regulations of p21 and p27. CyclinD1 and p21 are hepatic metabolic biotransformation, curcumin given or- two key molecules in cell-cycle progression. CyclinD1 ally resulted in low serum levels of curcumin. The peak accelerates the G1/S transition, and p21 prevents cell serum concentration after 8000 mg of curcumin was cycle progression from the G1 to the S phase [32–34]. 1.77 ± 1.87 μM[44]. Thus, it seems to be unfeasible to In addition to inhibiting Rb cells proliferation, we also achieve 30 μM in the clinical use by the systemic route. demonstrated that curcumin was able to induce apop- Other routes of delivery of curcumin for Rb, including tosis, as apoptotic cell rate was increased, anti-apoptotic intravenous, intra-arterial, periocular and intravitreal protein Bcl-2 was down-regulated, pro-apoptotic protein techniques may be possible to achieve such a high con- Bax was up-regulated, and caspase-3 and -9 were centration in the vitreous or the retina. Clinical usage of cleaved in curcumin-treated cells. curcumin should take into account the ocular toxicity, The occurrence of distant metastasis and organ infil- ocular penetration, and systemic toxicity of Rb. tration [35] may lead to poor Rb prognosis. MMP2 is Nowadays, extensive preclinical studies over the past highly expressed in Rb [36], and pharmacological inhib- several decades have demonstrated the anti-tumor activ- ition of MMP2 reduces Rb cells migration [37]. ROCK1 ities of curcumin in human cancers [10–13]. These pre- is a downstream effector of RhoA, RhoA/ROCK pathway clinical studies suggested the potential efficacy of is critical in controlling migration [38]. Vimentin is a curcumin in clinical trials. In clinical studies, curcumin well-known marker for epithelial-mesenchymal transi- has been used either alone or in combination with other tion (EMT), which promotes cancer cells invasion and agents such as quercetin and gemcitabine to prevent or metastasis [39, 40]. Herein, data showed that curcumin treat cancers [45, 46]. Phase I/II clinical studies have reduced the relative migration and invasion of SO-Rb50 been conducted using curcumin and found that curcu- and Y79 cells, and reduced the expression levels of min was helpful for overcoming chemo-resistance [47]. MMP2, RhoA, ROCK1 and Vimentin, suggesting the Our study provided in vitro evidences that curcumin has anti-migrating and anti-invasive roles of curcumin in Rb anti-Rb activities. However, significant research includ- cells. ing animal and clinical trials is warranted to better It has been reported that curcumin exerted anti-tumor understand the clinical value of curcumin in the treat- activities via modulation of various miRNAs, including ment of Rb. miR-21 [14], miR-340 [15] and miR-98 [16]. Curcumin could alter the expression of many miRNAs in Y79 cells Conclusions [41], which may contribute to its anti-tumor properties To sum up, this study illuminated that curcumin inhib- in Rb. Herein, we for the first time demonstrated that ited proliferation, migration, invasion, and induced curcumin treatment up-regulated miR-99a expression. apoptosis of Rb cells. The anti-tumor activities of curcu- In combination with the viewpoint obtained from previ- min on Rb cells appear to be via up-regulation of ous studies that miR-99a is a tumor-suppressive miRNA miR-99a, and thereby inhibition of JAK/STAT pathway. in multiple cancers [17–20], we inferred that curcumin mediated anti-tumor activity in Rb possibly via regulat- Abbreviations ing miR-99a expression. ANOVA: Analysis of variance; CCK-8Cell: Cell Counting Kit-8; DMSO: Dimethyl The JAK/STAT pathway is one of the important sig- sulfoxide; ECL: Enhanced chemiluminescence; FBS: Fetal bovine serum; miRNA: MicroRNA; Rb: Retinoblastoma naling pathways in modulating cell proliferation, differ- entiation, migration and apoptosis [42]. Recent Acknowledgements discoveries suggested that mutated JAK proteins will be Not applicable. potent target for anti-tumor therapy [43]. In this study, curcumin suppressed the phosphorylation of JAK1, Funding STAT1 and STAT3 in both SO-Rb50 and Y79 cells. This research received no specific grant from any funding agency in the These findings were consistent with previous studies public, commercial or not-for-profit sectors. that suggested the anti-tumor effect of curcumin is – Availability of data and materials through inhibiting JAK/STAT signaling pathway [26 28]. The datasets used and analyzed during the current study are available from We additionally found that curcumin could not block the corresponding author on reasonable request. Li et al. BMC Cancer (2018) 18:1230 Page 8 of 9

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