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Hindawi Journal of Oncology Volume 2021, Article ID 4451533, 11 pages https://doi.org/10.1155/2021/4451533 Research Article Carnosic Acid Induces Antiproliferation and Anti-Metastatic PropertyofEsophagealCancerCellsviaMAPKSignalingPathways 1 2 3 4 5 6 Sicong Jiang, Yinda Qiu, Zhaozhen Wang, Yulong Ji, Xiaofang Zhang, Xiaosong Yan, and Zhiqiang Zhan Division of oracic and Endocrine Surgery, University Hospitals and University of Geneva, Geneva 41211, Switzerland School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China Department of Clinical Medicine, Jiangxi Health Vocational College of China, Nanchang, Jiangxi, China Jiangxi Key Laboratory of Translational Cancer Research, Jiangxi Cancer Hospital of Nanchang University, Nanchang, Jiangxi, China Department of Pathology, Jiangxi Cancer Hospital of Nanchang University, Nanchang, Jiangxi, China Department of oracic Surgery, Jiangxi Cancer Hospital of Nanchang University, Nanchang, Jiangxi, China Department of Oncology, Jiangxi Pingxiang People’s Hospital, Pingxiang, Jiangxi, China Correspondence should be addressed to Zhiqiang Zhan; zzq198265@163.com Received 2 July 2021; Accepted 2 November 2021; Published 16 November 2021 Academic Editor: Yuan Seng Wu Copyright © 2021 Sicong Jiang et al. -is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Carnosic acid (CA) is a polyphenolic diterpene extracted from rosemary. Reports have shown that CA possesses anticancer activity. However, whether CA inhibits esophageal squamous cell carcinoma, an aggressive type of esophageal cancer, remains untested. Methods. -e effects of CA on cell survival, migration, and apoptosis were evaluated by a combination of MTT, colony formation assay, flow cytometry, and Transwell assay. -e potential signaling pathways involved were investigated via Western blot assay. Results. CA dose-dependently inhibited cell proliferation, apoptosis, migration, and colony formation. Mechanistically, CA arrested the cell cycle at G2/M phase, promoted cell apoptosis, induced DNA damage, and inhibited the MAPK signaling pathways. Conclusion. Our results suggest that CA is a potential anticancer drug for esophageal squamous cell carcinoma. Carnosic acid (CA), a polyphenolic diterpene derived 1. Introduction from the rosemary plant, possesses anti-inflammatory, an- Esophageal cancer is one of the most common malignant tiviral, antioxidative, and antitumor activities [9]. Reports tumors in the digestive system. -ere are two main path- have shown that CA inhibited proliferation and migration of ological types of esophageal cancer: esophageal adenocar- breast cancer and melanoma cells, arrested the cell cycle at cinoma (EAC) and esophageal squamous cell carcinoma the G2/M phase through suppression of cyclin A expression (ESCC) [1–4]. -e early vague and nonspecific symptoms of in leukemia and intestinal cancer, and induced apoptosis in ESCC have contributed to its high morbidity and mortality prostate cancer and tumors of the human central nervous [5]. Despite continuous improvement in treatment regi- system [10, 11]. -is antitumor effect may be mediated mens, including surgery, chemotherapy, and radiotherapy, through inhibition of the MAPK signaling pathways. -is the 5-year survival rate remains fluctuating between 15% and pathway represents a large family of serine/threonine ki- 25% [6–8]. In addition, chemotherapeutic agents often have nases that, upon the reception of a stimuli, trigger a cascade unbearable side effects. -us, there is an urgent need to of phosphorylation leading to specific cellular responses [12] develop natural and safer compounds for the treatment of and also plays a critical role in tumor progression and ESCC. metastasis by induction of proteolytic enzymes that degrade 2 Journal of Oncology the ECM (a key marker of invasion carcinoma), enhancement iodide) and 100μg/mL RNase solution in PBS for 30 min at of cell migration, initiation of several prosurvival genes, and room temperature. Cells were analyzed on a flow cytometer maintenance of tumor growth [13]. In addition, combination (BD FACSalibur, BD Biosciences). of CA with other drugs, such as curcumin, adriamycin, and carmustine, increased the antitumor effects of the latter 2.6. Cell Apoptosis Analysis. Apoptosis was analyzed using compounds [14]. In this report, we studied the potential use of the apoptosis detection kit (BD Biosciences, USA) following CA in the treatment of ESCC and explored the molecular the vendor’s instructions. KYSE-150 cells were treated with mechanisms underlying its tumor-suppressive effect. CA (10, 20, and 40μM) for 48 h and washed with PBS once and Annexin V-binding buffer once. -e cells were si- 2. Materials and Methods multaneously incubated with fluorescein-labeled Annexin V and PI for 15 min at room temperature. Cells were then 2.1. Cell Culture. Human esophageal squamous carcinoma washed once again with Annexin V-binding buffer and cell line, KYSE-150, and normal human liver cells, MIHA, resuspended with the same buffer, which were analyzed on a were obtained from the Institute of Biochemistry and Cell FACSCalibur (BD Biosciences, MD, USA). Biology, Chinese Academy of Sciences (Shanghai, China). KYSE-150 cells were cultured in RPMI-1640 (Gibco, Eggenstein, Germany) supplemented with 10% fetal bovine 2.7. Transwell Assay. Cells were treated with CA at 10, 20, serum (FBS) (Gibco, Eggenstein, Germany) and and 40μM for 48 h, which were then digested, resuspended, 1 × penicillin/streptomycin (-ermo Fisher Scientific, and diluted with serum-free medium to a concentration of Carlsbad, CA, USA). MIHA cells were grown in Dulbecco’s 1 × 10 /100μl. Subsequently, 600μl of RPMI-1640 containing modified Eagle’s medium (DMEM) (Gibco, Eggenstein, 10% FBS was added into the lower chamber and cultured in a Germany) supplemented with 10% FBS and 1 × penicillin/ 37 C incubator. Cells on the top of the membrane were re- streptomycin. All cells were cultured at 37 C in a 5% CO moved using Q-tips, and cells on the bottom of the membrane atmosphere. were fixed with 4% paraformaldehyde for 15 min at room temperature and washed with double distilled (DD) water. -e cells were stained with crystal violet for 5 min and rinsed 2.2. Cell Viability Assay. KYSE-150 (6500 cells/well) and with DD water and 30% glacial acetic acid to dissolve crystal MIHA (6000 cells/well) were seeded in 96-well plates. Cells violet. -e plate was read at 560 nm. Cell migration rate was were incubated overnight and treated with increasing calculated as follows: average OD of treated cells/average OD concentrations of CA (10, 20, and 40μM) for 48 h. -e cell of control unit) × 100%. viability was determined using 3-(4,5-di-methylthiazol-2- yl)-2,5-diphenyl-2 tetrazolium bromide (MTT) assay. In brief, cells were incubated with MTTreagent at 37 C for 4 h, 2.8.Immunofluorescence(IF)Assay. Cells on coverslips were and 100μl DMSO was added to each well to dissolve for- fixed with 4% paraformaldehyde, permeabilized in 0.5% mazan crystals. -e absorbance was read at 490 nm on a Triton X-100 (in 1 × PBS), and incubated overnight at 4 C spectrophotometer (DTX880, Beckman Coulter, CA, USA). with primary antibody against 53BP1 (CST, Danvers, MA). Cells were washed three times and incubated with DyLight 549-conjugated anti-rabbit secondary antibodies for 1 h at 2.3.ColonyFormationAssay. KYSE-150 cells were seeded at a room temperature. Cells were stained with DAPI, and im- density of 1000cells/well in 12-well plates and allowed to attach ages were acquired using Nikon Ti microscopy. overnight. Cells were treated with different concentrations of CA (10, 20, and 40μM) for 15 days at 37 C. -e cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, 2.9. Western Blot Assay. Cells were grown on 6-well plates and stained with crystal violet 10 min at room temperature. and were treated with 10, 20, and 40μM CA for 48 h. -e Colonies were counted using a stereomicroscope. cells were then washed with PBS and lysed using cell lysis buffer. -e cell lysates were quantitated using the BCA 2.4. EdU Staining Assay. KYSE-150 cells were seeded in 6- method (BioRad, CA, USA). An equal amount of cell lysates was resolved on SDS-PAGE and electroblotted onto poly- well plates the day before the experiment. Cells were treated with 10, 20, and 40μM CA for 48 h. -e EdU solution was vinylidene difluoride membranes. -e membranes were blocked using 5% nonfat milk at room temperature for 2 h added (final concentration of 10μM), and cells were con- tinued to incubate for 2 h. -en, cells were processed for and incubated with primary antibodies at 4 C overnight. -e membranes were incubated with the peroxidase-conjugated EdU staining using the EdU Proliferation kit (Beyotime, China) following the vendor’s instructions. Cells were then secondary antibodies for 1.5 h at room temperature. -e immunoreactive bands were visualized using an ECL de- observed under fluorescence microscopy (Nikon). tection kit (BioRad Laboratories, CA, USA). 2.5. Cell Cycle Analysis. KYSE-150 cells (2.5 ×10 ) were seeded in 6-well plates and incubated with 10, 20, and 40μM 2.10. Statistical Analysis. All data were analyzed by Graph- CA for 48 h. Cells were fixed in 70% ethanol for 24 h, washed Pad Prism 7.0. -e means of multiple groups were compared with cold PBS, and incubated with 50μg/mL PI (propidium by one-way analysis of variance, and the means of two groups Journal of Oncology 3 mitosis, leading to their anticancer effects. To investigate were compared with the independent samples t-test. Values are expressed as the mean± SD of three independent ex- whether CA-induced G2/M phase arrest was caused by DNA ∗ ∗∗ ∗∗∗ damage, KYSE-150 cells were treated with CA for 48 h and periments, with p< 0.05, p< 0.01, and p< 0.001. P53 binding protein 1 (53BP1), a widely used marker for DNA double-strand breaks, was evaluated by immunoflu- 3. Results orescence (IF) assay. As shown in Figure 4, CA dose-de- 3.1.EffectsofCATreatmentonCellSurvivalofKYSE-150Cells. pendently increased the number of 53BP1-positive foci, with CA is a natural benzenediol abietane diterpene found in CA at 40µM reaching ∼ eight 53BP1-positive foci per nu- rosemary, with its chemical structure as shown in cleus. When phosphorylation of the Ser-139 residue of the Figure 1(a). To evaluate the effect of CA on cell survival, histone variant H2AX (c-H2AX) occurs, a molecular marker KYSE-150 cells were treated with increasing concentrations of DNA double-strand break was evaluated by Western blot. of CA as indicated in Figure 1(b) for 48 h and cell viability CA dose-dependently increased the expression of c-H2AX. was analyzed by MTTassay. CA at<25μM had no significant -e results suggested that CA provokes a strong DNA effect on cell viability, which was decreased to 80% at 25μM, damage repair response in KYSE-150 cells. and CA at>25μM dose-dependently decreased cell viability. To evaluate the potency of CA, the half-maximal inhibitory 3.5. CA Induces Apoptosis of KYSE-150 Cells. Severe DNA concentration (IC50) was compared with that of the two damage usually leads to cell cycle arrest and apoptosis. To most commonly used chemotherapeutic drugs, 5-fluoro- test this, KYSE-150 cells were treated with CA as indicated in uracil (5-FU) and cisplatin (DDP). As shown in Table 1, the Figure 5, and flow cytometry assay was performed to analyze IC50 of CA for KYSE-150 cells was 29.87± 4.38 μM, com- the cell apoptosis. As expected, CA-induced KYSE-150 cell pared to 65.98± 1.39 of 5-FU and 79.21± 2.02 of DDP and apoptosis in a dose-dependent manner (Figure 5(a)). When IC50 for MIHA cells was >200 μM, compared to >200 of 5- apoptosis-related proteins, Bax, Bcl2, and cleaved caspase-3 FU and 16.64± 0.39 of DDP. -ese results suggest that CA at were analyzed by Western blot, CA dose-dependently de- less than 25μM had no obvious cytotoxicity and is a potent creased the expression of Bcl2 and simultaneously increased anticancer drug in KYSE-150 cells. the expression of both Bax and cleaved caspase-3, respec- tively (Figure 5(c)). -ese results indicated that CA induces cell apoptosis by regulating the expression of apoptosis- 3.2. CA Inhibits the Proliferation of KYSE-150 Cells. To related proteins. evaluate the effect of CA on colony formation capability, the colony formation assay was performed using KYSE-150 cells as described in the Materials and Methods section. As shown 3.6. CA Inhibits Metastasis and Invasion of KYSE-150 Cells in Figures 2(a) and 2(b), CA inhibited colony formation in a via Suppressed MAPK Signaling Pathway. Cell migration dose-dependent manner. To evaluate the effect of CA on cell and invasion play an important role in cancer metastasis. To proliferation, KYSE-150 cells were treated with EdU as evaluate the effects of CA on cell migration and invasion, described in the Materials and Methods section. As shown in KYSE-150cells were treated with CA at 10, 20, and 40μM for Figures 2(c) and 2(d), CA dose-dependently inhibited cell 24 h and Transwell assay was performed. As shown in proliferation. Figures 6(a) and 6(b), CA dose-dependently decreased cell migration. Recent studies have shown that inappropriate ac- tivation of the epithelial-to-mesenchymal transition (EMT) is 3.3. CA Arrests Cell Cycle at the G2/M Phase. To further associated with increased tumor invasion and metastasis explore the molecular mechanisms of CA-induced inhibi- [15, 16]. To explore whether CA inhibits EMT, KYSE-150 cells tion of cell proliferation, KYSE-150 cells were treated with were treated with CA and Western blot assay was used to 10, 20, and 40μM of CA for 48 h and the cell cycle was determine the expression of the key proteins involved in the analyzed by flow cytometry. As shown in Figures 3(a) and EMT process. However, as shown in Figure 6(c), CA did not 3(b), CA arrested KYSE-150 cells at the G2/M phase in a inhibit tumor cell metastasis and invasion via an EMTpathway. dose-dependent manner. It is worth noting that CA treat- To investigate the potential singling pathways involved in CA- ment increased the percentage of cells at the G2/M phase and induced inhibition of cell proliferation and migration, com- decreased the percentage of cells at the G0/G1 phase relative ponents of the signaling pathways were analyzed by Western to controls. Cells were then treated with 5, 10, and 20μM of blot. As shown in Figure 6(d), CA dose-dependently decreased CA, and G2/M phase-related proteins were evaluated by the expression of p-ERK, p-JNK, and p-38 but did not affect Western blot. CA dose-dependently decreased the expres- that of total JNK, p-38, and ERK. -ese results suggest that CA sion of cyclin B1, MDM2, and CDC2 (Figure 3(c)). Taken inhibits cell metastasis and invasion through suppression of together, CA-induced inhibition of cell proliferation occurs ERK, JNK, and p-38 signaling pathways. most likely through suppression of G2/M phase-related proteins, leading to cell cycle arrest at the G2/M phase. 4. Discussion 3.4. CA Provokes Strong DNA Damage Response. DNA ESCC is one of the most aggressive cancers and ranked the damage induced by anticancer drugs can block cells at the sixth leading cause of cancer death in the world [17]. Although G2/M phase, which prevents cells from entering into progress has been made in clarifying the molecular 4 Journal of Oncology OH 120 HO *** HO *** H *** *** Carnosic acid Concentrations (µΜ) (a) (b) Figure 1: Evaluation of cytotoxicity and potency of CA. (a) -e chemical structure of carnosic acid. (b) KYSE-150 viability was assessed ∗∗∗ using the MTT assay 48 h after treatment. Data were the means± SD of three independent experiments ( p< 0.001 vs control group). Table 1: IC50 (μM) values were determined via the MTT assay. mechanisms seem to be cancer type-dependent. CA has been reported to inhibit cell proliferation through cell cycle arrest IC50 (μM) Cell lines/compounds at the G0/G1 phase in melanoma cancer [25], at the G2 phase MIHA KYSE-150 in human glioma [26], and at the G1 phase in estrogen Carnosic acid >200 29.87± 4.38 receptor (ER)-negative human breast cancer cells [27]. -is 5-Fluorouracil >200 65.98± 1.39 anticancer activity was mediated by the capabilities of CA to DDP 16.64± 0.39 79.21± 2.02 activate p21-mediated signaling pathway [25] and induced IC50 (μM) values were drug concentrations necessary for 50% inhibition of apoptosis and production of reactive oxygen species (ROS) cell viability. Data are presented as “mean± SD” from at least three in- [28], inhibited the EMT [29], enhanced the anticancer effects dependent experiments in triplicates. -e drug treatment period was 48 h. of other compounds [26], and sensitized TRAIL-mediated apoptosis [30]. Here, we showed that CA dose-dependently mechanisms of its pathogenesis, ESCC still carries high inhibited the malignant phenotypes, including cell prolif- mortality rates, early metastasis, and poor prognosis [18, 19]. eration, migration, and colony formation, in an ESCC cell CA is a functional ingredient of rosemary that has been used as line, KYSE-150 (Figure 2). In addition, CA dose-depen- dently decreased the expression of G2/M phase-related an antioxidant food for many years [20, 21]. Recently, research has shown that CA exhibits antitumor activity in colon cancer, proteins, MDM2, cyclin B1, and CDC2 (Figure 3). Anti- cancer drug-induced DNA damage can activate p53-de- breast cancer, and skin tumors through inhibition of cell proliferation, invasion, and metastasis and induction of apo- pendent pathways, blocking the cell cycle at the G2/M phase ptosis and ROS production [22]. In the present study, we and inhibiting cell proliferation [31–33]. Consistently, we demonstrated that CA suppressed the malignant phenotypes of demonstrated that CA could induce severe DNA damage, ESCC cells. Mechanistically, the tumor-suppressive effect of showing a dose-dependent increase in P53BP1-positive foci CA was mediated mainly by cell cycle arrest at the G2/M phase, per nucleus after CA treatment (Figure 4), which was promotion of severe DNA damage and apoptosis, and acti- corroborated by an increased expression of c-H2AX, a vation of MAPK multiple signaling pathways. molecular marker of DNA double-strand break. It is well- As a naturally occurring anticancer compound, CA has known that cells with irreversible DNA damage and cell cycle arrest will undergo apoptosis [34, 35]. In line with this, been studied for its cytotoxic effect in normal cells and its anticancer activity in different tumors. CA was demon- we observed that CA dose-dependently increased KYSE-50 cell apoptosis compared with a nontreated control group by strated to decrease cell viability in a dose-dependent manner, with an IC50 of 25.6 to 96µM in breast cancer MCF7 cells flow cytometry analysis (Figures 5(a) and 5(b)). -ese data and 19.6µM and 22.9 µM in prostate cancer cell lines LNCaP showed a dose-dependent decrease in the expression of and 22Rv1 cells, respectively [23, 24], suggesting it is a very apoptosis-inhibiting factor, Bcl2, and a simultaneous in- potent anticancer compound. In agreement with the pre- crease in the expression of apoptosis-promoting factors, Bax vious studies, we found that CA at less than 25µM did not and cleaved caspase-3, by Western blot (Figure 5(c)). MAPK have obvious cytotoxicity, and concentrations larger than signaling pathways play an important role in the initiation of 25µM dose-dependently decreased cell viability of an ESCC tumor cell invasion and metastasis [36]. Indeed, CA dose- cell line, KYSE-150 (Figure 1(b)). dependently inhibited cell migration (Figures 6(a) and 6(b)), which was most likely mediated by the suppression of ERK, CA has been shown to inhibit the malignant phenotypes of cancer cells in different cancers, and its molecular p-38, and JNK signaling pathways (Figure 6(d)). Cell viability (%) DMSO 1.5625 3.125 6.25 12.5 200 Journal of Oncology 5 control 10 μM 20 μM 40 μM (a) 1.5 1.0 ** 0.5 *** *** 0.0 Control 10 μM 20 μM 40 μM (b) EDU Hoechst Merge control 10 μM 20 μM 40 μM (c) Figure 2: Continued. Colon Formation 6 Journal of Oncology ** 30 *** *** Control 10 μM 20 μM 40 μM (d) Figure 2: CA inhibits proliferation of KYSE-150 cells. (a) Cells were treated with CA and grown for 15 days. CA dose-dependently decreased colony formation. (b) Quantification of colony formation in (a). (c) Representative images showing EdU-positive cells after CA treatment for 48 h. (d) Quantification of (c). -e statistical significance was calculated using the unpaired student’s two-tailed t-test with the ∗∗ ∗∗∗ p values ( p< 0.01 and p< 0.001 vs control group). Control 10 μM 20 μM 40 μM 400 300 0 0 0 0 FL2-A FL2-A FL2-A FL2-A (a) Control 10 μM 20 μM 40 μM Dip G1 (%) Dip S (%) Dip G2 (%) (b) Figure 3: Continued. Count 1K Count Edu-positive cells (%) The relative content of cells (%) 1K Count 1K Count 1K Journal of Oncology 7 KYSE-150 Control 10 μM 20 μM 40 μM MDM2 Cyclin B1 CDC2 GAPDH (c) Figure 3: CA arrests KYSE-150 cells at the G2/M phase. KYSE-150 cells were treated with different concentrations of CA for 48 h. (a) -e cell cycle was measured using flow cytometry. (b) Quantification of flow cytometry data in (a). (c) -e expressions of MDM2, cyclin B1, and ∗ ∗∗ CDC2 were measured using Western blot. Values were mean± SD of at least three independent experiments ( p< 0.05 and p< 0.01 vs control group). 53BP1 DAPI Merge 10 ** Control ** 10 μM 4 20 μM control 10 μM 20 μM 40 μM 40 μM (a) (b) KYSE-150 Control 10 μM 20 μM 40 μM Γ-H2AX GAPDH (c) Figure 4: CA provokes intense DNA damage response in KYSE-150 cells. (a) Representative images showing CA treatment provokes intense DNA damage response by immunofluorescence. (b) Quantification of (a) showing that CA dose-dependently increased the DNA damage (more than 200 cells were examined in each group). (c) Western blot showing that CA dose-dependently increased the expression of ∗ ∗∗ c-H2AX. Values were presented as the mean± SD of at least three independent experiments ( p< 0.05 and p< 0.01 vs control group). average 53bp1 foci per cell 8 Journal of Oncology Control 10 μM 20 μM 40 μM 4 4 4 4 10 10 10 10 3 3 3 3 10 10 10 10 2 2 2 2 PI 10 10 10 10 1 1 1 1 10 10 10 10 0 0 0 0 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Annexin V (a) 50 KYSE-150 Control 10 μM 20 μM 40 μM ** Bcl2 30 BAX ** Cleaved caspase-3 GAPDH Control 1.0 mM 2.0 mM 4.0 mM (b) (c) Figure 5: CA promotes apoptosis of KYSE-150 cells. (a) -e apoptosis of KYSE-150 was analyzed by flow cytometry. (b) Quantification of flow cytometry data in (a). (c) -e expression of Bcl2 was inhibited, while that of Bax and cleaved caspase-3 was activated in a dose- dependent manner by Western blot. GAPDH was used as loading control. Values are average± SD of three independent experiments ∗ ∗∗ ( p< 0.05 and p< 0.01 vs control group). Control 10 μM 20 μM 40 μM (a) Figure 6: Continued. Percentage of Apoptotic cells (%) Journal of Oncology 9 *** *** *** Control 10 μM 20 μM 40 μM (b) KYSE-150 Control 10 μM 20 μM 40 μM E-cadherin N-cadherin GAPDH (c) KYSE-150 Control 10 μM 20 μM 40 μM p-JNK JNK p-p38 p38 p-ERK ERK GAPDH (d) Figure 6: CA inhibits the migration and invasion of KYSE-150 cells via suppressing the MAPK signaling pathway. (a) CA dose-dependently inhibits cell migration as evaluated by Transwell assay. (b) Quantification of cell migration in the Transwell assay in (a). (c) CA dose- dependently decreases the expression E-cadherin and simultaneously increases the expression of N-cadherin, respectively, by Western blot. (d) CA dose-dependently decreases the levels of p-JNK, p-ERK, and p-38 but does not affect those of total JNK, p-38, and ERK by Western blot. promotion of apoptosis, inhibition of cell migration, and a 5. 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Journal of Oncology – Hindawi Publishing Corporation
Published: Nov 16, 2021
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