2‑D08 mediates notable anticancer effects through multiple cellular pathways in uterine leiomyosarcoma cells

Joung,Hosouk; Joung,Hosouk; Joung,Hosouk; Liu,Hyunju; Liu,Hyunju; Liu,Hyunju

doi:10.3892/or.2024.8756

2‑D08 mediates notable anticancer effects through multiple cellular pathways in uterine leiomyosarcoma cells

Authors:
- Hosouk Joung
- Hyunju Liu
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Affiliations: Research Institute of Medical Sciences, Chonnam National University Medical School, Hwasun, Jeonnam 58128, Republic of Korea, Department of Obstetrics and Gynecology, Chosun University College of Medicine, Gwangju 61452, Republic of Korea
Published online on: June 14, 2024 https://doi.org/10.3892/or.2024.8756
Article Number: 97
Copyright: © Joung et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

2',3',4'‑trihydroxyflavone (2‑D08), a SUMO E2 inhibitor, has several biological functions, including anticancer activity, but its effects on uterine leiomyosarcoma (Ut‑LMS) are unknown. The anticancer activity of 2‑D08 was explored in an in vitro model using SK‑LMS‑1 and SK‑UT‑1B cells (human Ut‑LMS cells). Treatment with 2‑D08 inhibited cell viability in a dose‑ and time‑dependent manner and significantly inhibited the colony‑forming ability of Ut‑LMS cells. In SK‑UT‑1B cells treated with 2‑D08, flow cytometric analysis revealed a slight increase in apoptotic rates, while cell cycle progression remained unaffected. Western blotting revealed elevated levels of RIP1, indicating induction of necrosis, but LC3B levels remained unchanged, suggesting no effect on autophagy. A lactate dehydrogenase (LDH) assay confirmed increased LDH release, further supporting the induction of apoptosis and necrosis by 2‑D08 in SK‑UT‑1B cells. 2‑D08‑induced production of reactive oxygen species and apoptosis progression were observed in SK‑LMS‑1 cells. Using Ki67 staining and bromodeoxyuridine assays, it was found that 2‑D08 suppressed proliferation in SK‑LMS‑1 cells, while treatment for 48 h led to cell‑cycle arrest. 2‑D08 upregulated p21 protein expression in SK‑LMS‑1 cells and promoted apoptosis through caspase‑3. Evaluation of α‑SM‑actin, calponin 1 and TAGLN expression indicated that 2‑D08 did not directly initiate smooth muscle phenotypic switching in SK‑LMS‑1 cells. Transcriptome analysis on 2‑D08‑treated SK‑LMS‑1 cells identified significant differences in gene expression and suggested that 2‑D08 modulates cell‑cycle‑ and apoptosis‑related pathways. The analysis identified several differentially expressed genes and significant enrichment for biological processes related to DNA replication and molecular functions associated with the apoptotic process. It was concluded that 2‑D08 exerts antitumor effects in Ut‑LMS cells by modulating multiple signaling pathways and that 2‑D08 may be a promising candidate for the treatment of human Ut‑LMS. The present study expanded and developed knowledge regarding Ut‑LMS management and indicated that 2‑D08 represents a notable finding in the exploration of fresh treatment options for such cancerous tumors.

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1	Echt G, Jepson J, Steel J, Langholz B, Luxton G, Hernandez W, Astrahan M and Petrovich Z: Treatment of uterine sarcomas. Cancer. 66:35–39. 1990. View Article : Google Scholar : PubMed/NCBI
2	Kim WY, Chang SJ, Chang KH, Yoon JH, Kim JH, Kim BG, Bae DS and Ryu HS: Uterine leiomyosarcoma: 14-year two-center experience of 31 cases. Cancer Res Treat. 41:24–28. 2009. View Article : Google Scholar : PubMed/NCBI
3	Tirumani SH, Deaver P, Shinagare AB, Tirumani H, Hornick JL, George S and Ramaiya NH: Metastatic pattern of uterine leiomyosarcoma: Retrospective analysis of the predictors and outcome in 113 patients. J Gynecol Oncol. 25:306–312. 2014. View Article : Google Scholar : PubMed/NCBI
4	Chern JY, Boyd LR and Blank SV: Uterine sarcomas: The latest approaches for these rare but potentially deadly tumors. Oncology (Williston Park). 31:229–236. 2017.PubMed/NCBI
5	Murakami M, Ichimura T, Kasai M, Matsuda M, Kawamura N, Fukuda T and Sumi T: Examination of the use of needle biopsy to perform laparoscopic surgery safely on uterine smooth muscle tumors. Oncol Lett. 15:8647–8651. 2018.PubMed/NCBI
6	Hernando E, Charytonowicz E, Dudas ME, Menendez S, Matushansky I, Mills J, Socci ND, Behrendt N, Ma L, Maki RG, et al: The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas. Nat Med. 13:748–753. 2007. View Article : Google Scholar : PubMed/NCBI
7	Babichev Y, Kabaroff L, Datti A, Uehling D, Isaac M, Al-Awar R, Prakesch M, Sun RX, Boutros PC, Venier R, et al: PI3K/AKT/mTOR inhibition in combination with doxorubicin is an effective therapy for leiomyosarcoma. J Transl Med. 14:672016. View Article : Google Scholar : PubMed/NCBI
8	Gaumann AK, Drexler HC, Lang SA, Stoeltzing O, Diermeier-Daucher S, Buchdunger E, Wood J, Bold G and Breier G: The inhibition of tyrosine kinase receptor signalling in leiomyosarcoma cells using the small molecule kinase inhibitor PTK787/ZK222584 (Vatalanib^®). Int J Oncol. 45:2267–2277. 2014. View Article : Google Scholar : PubMed/NCBI
9	Hayashi T, Horiuchi A, Sano K, Hiraoka N, Kanai Y, Shiozawa T, Tonegawa S and Konishi I: Molecular approach to uterine leiomyosarcoma: LMP2-deficient mice as an animal model of spontaneous uterine leiomyosarcoma. Sarcoma. 2011:4764982011. View Article : Google Scholar : PubMed/NCBI
10	Cancer Genome Atlas Research Network. Electronic address, . simpleelizabeth.demicco@sinaihealthsystem.ca and Cancer Genome Atlas Research Network: Comprehensive and integrated genomic characterization of adult soft tissue sarcomas. Cell. 171:950–965.e28. 2017. View Article : Google Scholar : PubMed/NCBI
11	Mäkinen N, Aavikko M, Heikkinen T, Taipale M, Taipale J, Koivisto-Korander R, Bützow R and Vahteristo P: Exome sequencing of uterine leiomyosarcomas identifies frequent mutations in TP53, ATRX, and MED12. PLoS Genet. 12:e10058502016. View Article : Google Scholar : PubMed/NCBI
12	Tsuyoshi H and Yoshida Y: Molecular biomarkers for uterine leiomyosarcoma and endometrial stromal sarcoma. Cancer Sci. 109:1743–1752. 2018. View Article : Google Scholar : PubMed/NCBI
13	Hanahan D and Weinberg RA: Hallmarks of cancer: The next generation. Cell. 144:646–674. 2011. View Article : Google Scholar : PubMed/NCBI
14	DeCensi A, Guerrieri-Gonzaga A, Gandini S, Serrano D, Cazzaniga M, Mora S, Johansson H, Lien EA, Pruneri G, Viale G and Bonanni B: Prognostic significance of Ki-67 labeling index after short-term presurgical tamoxifen in women with ER-positive breast cancer. Ann Oncol. 22:582–587. 2011. View Article : Google Scholar : PubMed/NCBI
15	Davey MG, Hynes SO, Kerin MJ, Miller N and Lowery AJ: Ki-67 as a prognostic biomarker in invasive breast cancer. Cancers (Basel). 13:44552021. View Article : Google Scholar : PubMed/NCBI
16	Tollefson MK, Karnes RJ, Kwon ED, Lohse CM, Rangel LJ, Mynderse LA, Cheville JC and Sebo TJ: Prostate cancer Ki-67 (MIB-1) expression, perineural invasion, and gleason score as biopsy-based predictors of prostate cancer mortality: The mayo model. Mayo Clin Proc. 89:308–318. 2014. View Article : Google Scholar : PubMed/NCBI
17	Maia R, Santos GAD, Reis S, Viana NI, Pimenta R, Guimarães VR, Recuero S, Romão P, Leite KRM, Srougi M and Passerotti CC: Can we use Ki67 expression to predict prostate cancer aggressiveness? Rev Col Bras Cir. 49:e20223200. 2022.(In English, Portuguese). View Article : Google Scholar : PubMed/NCBI
18	Zhang F, Zhang F, Liu Z, Wu K, Zhu Y and Lu Y: Prognostic role of Ki-67 in adrenocortical carcinoma after primary resection: A retrospective mono-institutional study. Adv Ther. 36:2756–2768. 2019. View Article : Google Scholar : PubMed/NCBI
19	Akhan SE, Yavuz E, Tecer A, Iyibozkurt CA, Topuz S, Tuzlali S, Bengisu E and Berkman S: The expression of Ki-67, p53, estrogen and progesterone receptors affecting survival in uterine leiomyosarcomas. A clinicopathologic study. Gynecol Oncol. 99:36–42. 2005. View Article : Google Scholar : PubMed/NCBI
20	Travaglino A, Raffone A, Catena U, De Luca M, Toscano P, Del Prete E, Vecchione ML, Lionetti R, Zullo F and Insabato L: Ki67 as a prognostic marker in uterine leiomyosarcoma: A quantitative systematic review. Eur J Obstet Gynecol Reprod Biol. 266:119–124. 2021. View Article : Google Scholar : PubMed/NCBI
21	Kim YS, Keyser SG and Schneekloth JS Jr: Synthesis of 2′,3′,4′-trihydroxyflavone (2-D08), an inhibitor of protein sumoylation. Bioorg Med Chem Lett. 24:1094–1097. 2014. View Article : Google Scholar : PubMed/NCBI
22	Marsh DT, Das S, Ridell J and Smid SD: Structure-activity relationships for flavone interactions with amyloid beta reveal a novel anti-aggregatory and neuroprotective effect of 2′,3′,4′-trihydroxyflavone (2-D08). Bioorg Med Chem. 25:3827–3834. 2017. View Article : Google Scholar : PubMed/NCBI
23	Choi BH, Philips MR, Chen Y, Lu L and Dai W: K-Ras Lys-42 is crucial for its signaling, cell migration, and invasion. J Biol Chem. 293:17574–17581. 2018. View Article : Google Scholar : PubMed/NCBI
24	Zhou P, Chen X, Li M, Tan J, Zhang Y, Yuan W, Zhou J and Wang G: 2-D08 as a SUMOylation inhibitor induced ROS accumulation mediates apoptosis of acute myeloid leukemia cells possibly through the deSUMOylation of NOX2. Biochem Biophys Res Commun. 513:1063–1069. 2019. View Article : Google Scholar : PubMed/NCBI
25	Liu H, Lee SM and Joung H: 2-D08 treatment regulates C2C12 myoblast proliferation and differentiation via the Erk1/2 and proteasome signaling pathways. J Muscle Res Cell Motil. 42:193–202. 2021. View Article : Google Scholar : PubMed/NCBI
26	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
27	Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M and Gingeras TR: STAR: Ultrafast universal RNA-seq aligner. Bioinformatics. 29:15–21. 2013. View Article : Google Scholar : PubMed/NCBI
28	Li B and Dewey CN: RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 12:3232011. View Article : Google Scholar : PubMed/NCBI
29	Love MI, Huber W and Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15:5502014. View Article : Google Scholar : PubMed/NCBI
30	Robinson MD, McCarthy DJ and Smyth GK: edgeR: A bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 26:139–140. 2010. View Article : Google Scholar : PubMed/NCBI
31	Guo C, Sun L, Chen X and Zhang D: Oxidative stress, mitochondrial damage and neurodegenerative diseases. Neural Regen Res. 8:2003–2014. 2013.PubMed/NCBI
32	Nishikawa M, Hashida M and Takakura Y: Catalase delivery for inhibiting ROS-mediated tissue injury and tumor metastasis. Adv Drug Deliv Rev. 61:319–326. 2009. View Article : Google Scholar : PubMed/NCBI
33	Zhitkovich A: N-Acetylcysteine: Antioxidant, aldehyde scavenger, and more. Chem Res Toxicol. 32:1318–1319. 2019. View Article : Google Scholar : PubMed/NCBI
34	Beamish JA, He P, Kottke-Marchant K and Marchant RE: Molecular regulation of contractile smooth muscle cell phenotype: Implications for vascular tissue engineering. Tissue Eng Part B Rev. 16:467–491. 2010. View Article : Google Scholar : PubMed/NCBI
35	Xie C, Ritchie RP, Huang H, Zhang J and Chen YE: Smooth muscle cell differentiation in vitro: Models and underlying molecular mechanisms. Arterioscler Thromb Vasc Biol. 31:1485–1494. 2011. View Article : Google Scholar : PubMed/NCBI
36	Amada S, Nakano H and Tsuneyoshi M: Leiomyosarcoma versus bizarre and cellular leiomyomas of the uterus: A comparative study based on the MIB-1 and proliferating cell nuclear antigen indices, p53 expression, DNA flow cytometry, and muscle specific actins. Int J Gynecol Pathol. 14:134–142. 1995. View Article : Google Scholar : PubMed/NCBI
37	Sprogøe-Jakobsen S and Hølund B: Immunohistochemistry (Ki-67 and p53) as a tool in determining malignancy in smooth muscle neoplasms (exemplified by a myxoid leiomyosarcoma of the uterus). APMIS. 104:705–708. 1996. View Article : Google Scholar : PubMed/NCBI
38	Horiuchi A, Nikaido T, Ito K, Zhai Y, Orii A, Taniguchi S, Toki T and Fujii S: Reduced expression of calponin h1 in leiomyosarcoma of the uterus. Lab Invest. 78:839–846. 1998.PubMed/NCBI
39	Owens GK, Kumar MS and Wamhoff BR: Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 84:767–801. 2004. View Article : Google Scholar : PubMed/NCBI
40	Kimura Y, Morita T, Hayashi K, Miki T and Sobue K: Myocardin functions as an effective inducer of growth arrest and differentiation in human uterine leiomyosarcoma cells. Cancer Res. 70:501–511. 2010. View Article : Google Scholar : PubMed/NCBI
41	Wang B, Wang Y, Zhang J, Hu C, Jiang J, Li Y and Peng Z: ROS-induced lipid peroxidation modulates cell death outcome: Mechanisms behind apoptosis, autophagy, and ferroptosis. Arch Toxicol. 97:1439–1451. 2023. View Article : Google Scholar : PubMed/NCBI
42	Nakamura H and Takada K: Reactive oxygen species in cancer: Current findings and future directions. Cancer Sci. 112:3945–3952. 2021. View Article : Google Scholar : PubMed/NCBI
43	Philipp-Staheli J, Kim KH, Liggitt D, Gurley KE, Longton G and Kemp CJ: Distinct roles for p53, p27Kip1, and p21Cip1 during tumor development. Oncogene. 23:905–913. 2004. View Article : Google Scholar : PubMed/NCBI
44	O'Neill CJ, McBride HA, Connolly LE and McCluggage WG: Uterine leiomyosarcomas are characterized by high p16, p53 and MIB1 expression in comparison with usual leiomyomas, leiomyoma variants and smooth muscle tumours of uncertain malignant potential. Histopathology. 50:851–858. 2007. View Article : Google Scholar : PubMed/NCBI
45	Schaefer IM, Hornick JL, Sholl LM, Quade BJ, Nucci MR and Parra-Herran C: Abnormal p53 and p16 staining patterns distinguish uterine leiomyosarcoma from inflammatory myofibroblastic tumour. Histopathology. 70:1138–1146. 2017. View Article : Google Scholar : PubMed/NCBI
46	Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW and Vogelstein B: Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science. 282:1497–1501. 1998. View Article : Google Scholar : PubMed/NCBI
47	Le Guen L, Marchal S, Faure S and de Santa Barbara P: Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration. Cell Mol Life Sci. 72:3883–3896. 2015. View Article : Google Scholar : PubMed/NCBI
48	Scirocco A, Matarrese P, Carabotti M, Ascione B, Malorni W and Severi C: Cellular and molecular mechanisms of phenotypic switch in gastrointestinal smooth muscle. J Cell Physiol. 231:295–302. 2016. View Article : Google Scholar : PubMed/NCBI
49	Chakraborty R, Chatterjee P, Dave JM, Ostriker AC, Greif DM, Rzucidlo EM and Martin KA: Targeting smooth muscle cell phenotypic switching in vascular disease. JVS Vasc Sci. 2:79–94. 2021. View Article : Google Scholar : PubMed/NCBI
50	Kim HR, Cho KS, Kim E, Lee OH, Yoon H, Lee S, Moon S, Park M, Hong K, Na Y, et al: Rapid expression of RASD1 is regulated by estrogen receptor-dependent intracellular signaling pathway in the mouse uterus. Mol Cell Endocrinol. 446:32–39. 2017. View Article : Google Scholar : PubMed/NCBI
51	Hong K and Choi Y: Role of estrogen and RAS signaling in repeated implantation failure. BMB Rep. 51:225–229. 2018. View Article : Google Scholar : PubMed/NCBI
52	Vaidyanathan G, Cismowski MJ, Wang G, Vincent TS, Brown KD and Lanier SM: The Ras-related protein AGS1/RASD1 suppresses cell growth. Oncogene. 23:5858–5863. 2004. View Article : Google Scholar : PubMed/NCBI
53	Park DJ, Nakamura H, Chumakov AM, Said JW, Miller CW, Chen DL and Koeffler HP: Transactivational and DNA binding abilities of endogenous p53 in p53 mutant cell lines. Oncogene. 9:1899–1906. 1994.PubMed/NCBI
54	Smardová J, Pavlová S, Svitáková M, Grochová D and Ravcuková B: Analysis of p53 status in human cell lines using a functional assay in yeast: detection of new non-sense p53 mutation in codon 124. Oncol Rep. 14:901–907. 2005.PubMed/NCBI
55	Mills J, Matos T, Charytonowicz E, Hricik T, Castillo-Martin M, Remotti F, Lee FY and Matushansky I: Characterization and comparison of the properties of sarcoma cell lines in vitro and in vivo. Hum Cell. 22:85–93. 2009. View Article : Google Scholar : PubMed/NCBI