Cytotoxic, anti-proliferative, and apoptotic evaluation of Ramalina sinensis (Lichenized fungus, Ramalinaceae) on oral squamous cell carcinoma cell line; in-vitro study

Cytotoxic, anti-proliferative, and apoptotic evaluation of Ramalina sinensis (Lichenized fungus, Ramalinaceae) on oral squamous cell carcinoma cell line; in-vitro study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cytotoxic, anti-proliferative, and apoptotic evaluation of Ramalina sinensis (Lichenized fungus, Ramalinaceae) on oral squamous cell carcinoma cell line; in-vitro study Hanieh Karimi, Maryam Koopaie, Mohammad Sohrabi, Hooman Norouzi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2246187/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background: In response to the poor prognosis of oral squamous cell carcinoma (OSCC), the most frequent cancer in the oral cavity, efforts have been made to create more effective treatment methods based on natural products. Lichens are a source of active pharmacologic compounds, which have lately been discovered to be helpful in cancer treatment. Current research seeks to evaluate the impact of Ramalina sinensis ( R. sinensis ) lichen on the cell viability and apoptosis of OSCC cell lines, considering lichens’ anti-inflammatory and anticancer capabilities. Methods: R. sinensis was selected for investigation of its effects on human oral squamous cell carcinoma cell line (KB cell line, NCBI Code: C152) and acetone and effect of methanol extract of R. sinensis on OSCC cell line was investigated. The chemical composition and m etabolic profiling of R. sinensis were investigated. Viability was assessed by MTT assay analysis, and apoptotic cells were measured using flow cytometry analysis. Scratch assay was used to assess cell migration. Results: The chemical composition of R. sinensis was investigated using LC-ESI-MS/MS, and 33 unique compounds in acetone and methanol extract of R. sinensis were detected. The proliferation of KB cells was gradually but significantly inhibited by 6.25, 12.5, 25, 50, 100, and 200 μg/mL in a time-dependent manner. There was a statistically significant difference between acetone and methanol extract in the inhibition of cell proliferation. Flow cytometry results indicate an increase in apoptosis of OSCC cells by acetone extract. R. sinensis significantly inhibited the migration and invasion of OSCC cells in a concentration-dependent manner. Conclusion: The results of the present study showed the positive effects of R. sinensis extract on the apoptosis of OSCC cells and the anticancer effects of R. sinensis extracts. Oral squamous cell carcinoma (OSCC) cell viability apoptosis Ramalina sinensis cell migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background The most frequent kind of cancer in the oral cavity, oral squamous cell carcinoma (OSCC), and accounts for approximately 90% of all oral malignancies (1). OSCC is a multifactorial disease in which both extrinsic and intrinsic factors, such as smoking and alcohol intake, as well as iron deficiency anemia and genetics, have a role in disease incidence and progression (2). Although surgery, radiotherapy, chemotherapy, or a mixture of them are known as the common treatment approaches of OSCC, these modalities have some cosmetic and functional side effects (3). Despite advancements in treatment modalities, the survival rate of oral cancer remains low, and attempts are underway to provide novel and effective treatment modalities considering the dismal prognosis of oral cancer (4). Lichens as a source of active pharmacologic compounds, have recently been introduced for treatment purposes (5, 6). Bioactive metabolites, such as the phenolic chemicals despides, depsidones, and dibenzofurans, are produced by lichens, which are symbiotic organisms composed of a fungus and an algae/cyanobacteria (7). As lichen-derived chemicals, over 1000 metabolites have been found, and these compounds exhibit a broad spectrum of biological actions, including antioxidant, cytotoxic, anti-inflammatory, and anti-proliferative properties (7). Lichen metabolites are classified as either primary or secondary. Proteins, lipids, and carbohydrates are primary metabolites that have a role in lichen structure and metabolism. Secondary metabolites are complex compounds having therapeutic functions (8). Several species have been identified as having traditional medicinal uses (9). Their traditional usage dates back to Ancient Greece when one of its species was used to treat inflammation, jaundice, and impetigo. Ramalina calicaris, Ramalina conduplicans, Ramalina farinacea, Ramalina inflata, Ramalina menziesii, Ramalina roesleri, and Ramalina sinensis are some of the Ramalina species that have been used in traditional medicine (10, 11). Furthermore, there is evidence for the interaction of various lichen substances with several molecular mechanisms that are important in cell death. These interactions include cell cycle arrest, apoptosis, necrosis, and inhibition of angiogenesis (12). Although there are several reports on the anticancer activity of lichen substances or organic extracts against different human cell lines, this is the first report considering the anticancer properties of Ramalina species lichens on OSCC cells. The aim of the present study is to assess the effect of Ramalina lichen on the cell viability and apoptosis of OSCC cell lines. Methods Chemicals and reagents Thermo Fisher Scientific (Massachusetts, USA) provided the acetonitrile, and DiKMA Technologies provided the formic acid (Beijing, China). Water was produced via a Milli-Q® integral water purification system (Merck, Germany). Lichen material Ramalina specimens were collected in the Hyrcanian forest in Mazandaran Provinces (Iran) in May 2018, and Ramalina Sinensis ( R. sinensis ) was selected for this study. Lichen materials were collected in accordance with the relevant guidelines and regulations of the Plant Varieties Protection, Environmental Protection Organization of Iran. The Museum of Iranian Lichens in the Iranian Research Organization for Science and Technology (Tehran, Iran) provided the principal keys to identifying species (Figure 1). The lichen was identified by Dr. Mohammad Sohrabi, lichenologist from the Iranian Research Organization for Science and Technology. The voucher specimen was deposited in the Museum of Iranian Lichens, Iranian Research Organization for Science and Technology (IROST), Iran (https://irost.org/museum/). The cleaned lichen thallus was grounded to a fine powder using a mortar and pestle under a small amount of liquid nitrogen. Cell lines and culture The human oral epidermal carcinoma cell line (KB cell line, NCBI Code: C152) were obtained from the Pasteur Institute of Iran (Tehran, Iran) (Supplementary File). Cancer cell lines were cultured in DMEM with 10% FBS and 1% PS at 37°C, 5.0% CO 2 and 95.0% humidity. Cells were cultured in a humidified atmosphere of 95.0% air, and 5.0% CO 2 at 37°C. Cell viability MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine viability, which is based on the reduction of MTT by mitochondrial dehydrogenase in intact cells into an insoluble purple formazan product. In 96-well plates, cells (1*10 4 /well) were seeded, and after 24 hours, the cells were treated with R. sinensis extract at various doses (200, 100, 50, 25, 12.5, and 6.25 μg/ml) and then R. sinensis -containing medium were carefully removed after the treatment. Cells were washed twice with PBS before each well received 100 mL media containing 0.5 mg/ml MTT in PBS, then the plate was incubated at 37°C for 4 hours. The medium was then completely removed, 200 mL of Tris-DMSO solution was added to each well, and the plate was vibrated for 30 minutes. Using an ELISA plate reader, the absorbance, as proportional to cell viability, was then measured in at least three independent measurements using a microplate reader (Bio-Rad Corp, California, USA) at 570 nm in each well and Gen 5 Version 2.07.17 software (BioTek, Winooski, USA) for data analysis. The average of measurements was reported for each well. Flow cytometry Flow cytometry analysis was performed on apoptotic cells using a Beckman Coulter (EPICS XL). The ramalina-treated and untreated cells were collected, twice-washed in PBS, fixed in 70.0% ethanol at 4°C for at least 12 hours, centrifuged, and then incubated for 30 minutes at 37°C in the dark with 0.1% Triton X-100, 200 mg/ml RNase A, and 50 mg/ml propidium iodide (PI) in PBS. More than 3*10 4 /well cells were counted in each sample, and cells with lower DNA content than those in the G0/G1 phase were labeled as apoptotic. Scratch assay After 24 hour of cultivation as confluent monolayers in the complete medium, cells were wounded by removing cells across the well with a standard 200 μL pipette tip [13]. To eliminate the non-adherent cells, the wounded monolayers were washed twice. When the wound was made, and 24 hours later, it was observed using an inverted phase contrast microscope (Leica, Wetzlar, Germany). This 24 hour was selected since it is less than the doubling time in control. Four separate fields from each sample were evaluated for quantitative measurement of the distance between the borderlines, while four different equidistant spots in each picture were measured to get a better approximation of the true breadth of the wounded region. The migration rate was estimated as the ratio of the mean distance between both borderlines generated by scratching to the distance that remained cell-free after re-growing and is represented as a percentage of the control. In quadruplicates, two separate sets of tests were carried out. Metabolic profiling of R. sinensis The chemical composition of R. sinensis was investigated according to Norouzi et al. briefly (13); lichenochemicals were separated by Waters Alliance e2695 separation module (Milford, MA, USA) and Atlantis T3 C18 column (2.1mm × 100 mm, 3 μm; Milford, MA, USA) and Column temperature set to 30℃. Samples were dissolved in methanol, and after filtration (PTFE membrane filters, 0.45 μm, Simplepure, China), 10 μL of each sample was injected into the separation module. Lichenochemicals were eluted within 25 min as follows: elution began by 95% (water + 0.1% formic acid, v/v) and gradually decreased to 5% within 20 min. The Elution process went on for another 5 min by 95% B (acetonitrile). The flow rate of eluents was adjusted to be 0.25 ml min -1 . A Quattro micro API mass spectrometer (Milford, MA, USA) was used for tandem mass analysis. The MS/MS parameters applied were as follows: source temperature and desolvation temperature were set to be 120 ℃ and 300 ℃, respectively; capillary voltage, cone voltage, and collision energy were regulated at 3.5 kV, 30 V, and 30 eV, respectively; for both nebulizing and drying the gas, N 2 was used. MassLynx 4.1 and MZmine 2.53 were used for data acquiring and analysis. Initial annotation of detected compounds was carried out based on the lichen spectral database (LDB) provided in GNPS public spectral libraries (14). Chemical structures were sketched using ChemDraw Ultra 12.0. Statistical analysis SPSS software (version 22; SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8.2.1 (GraphPad Software, San Diego, CA) were used for statistical analysis. All results are given as as mean ± standard deviation (SD), with p ≤ 0.05 considered statistically significant. Results MTT assay results To assess the impact of R. sinensis on the viability of OSCC cells, we conducted an MTT experiment in KB cells with various R. sinensis concentrations (6.25, 12.5, 25, 50,100, and 200 μg/mL) and various incubation durations (48 and 72 hour) [Additional file 1]. R. sinensis reduced the proliferation of KB cells in a time-dependent manner and the higher concentration (100 μg/mL) having a more considerable inhibitory impact than the lower 50 μg/mL concentration ((Figure 2 (cell viability after 48 hour) and Figure 3 (cell viability after 72 hour)). The cell viability of OSCC cells treated with 100 μg/mL of acetone and methanol extract was 17.65% (SD = ±7.64%) and 2.60% (SD = ±1.77%), respectively. There was a statistically significant difference between acetone and methanol extract in the cell viability of OSCC cells treated with 100 μg/mL concentration (p = 0.029). The cell viability of OSCC cells at 200 μg/ml concentration was 1.48% (SD = ±0.92), and 1.89% (SD = ±1.09), for acetone and methanol extract, respectively, indicating a statistically significant difference. The inhibitory concentration at 50% inhibition (IC50) was the parameter used to compare the cytotoxic activity, and a lower IC50 means better cytotoxic activity. The IC50 against KB cells was 50 μg/mL. Flow cytometry Results The percent of OSCC cells R. sinensis treated 24 hour (50 μg/mL of acetone extract of R. sinensi ) in the early and late apoptosis phases were 16.8% and 23.1%, respectively (Figure 4). The percent of OSCC cells of control in the early apoptosis phase was 3.43% and 8.07% in the late apoptosis phase (Figure 5). Furthermore, the percent of R. sinensis treated (50 μg/mL of methanol extract) OSCC cells (24 hour treatment) in the early and late apoptosis phase was 30.1% and 40.1%, respectively, which indicates an increase in apoptotic cells by methanol extract (Figure 6). Scratch assay Non-cytotoxic doses of R. sinensis extract (50 μg/mL) were used to treat OSCC cells to assess the effect of R. sinensis on cell migration. R. sinensis extract significantly inhibited cell migration in OSCC cells (Figure 7) [Additional file 2]. In particular, the migration of cancer cells was inhibited by acetone and methanol extract of R. sinensis. These results indicate that R. sinensis suppresses the wound-healing process in human OSCC. There was no significant difference between acetone and methanol extract in cell migration inhibition. Therefore, R. sinensis may prevent cancer metastasis in OSCC cells (Figure 8). Metabolic profiling using LC-ESI-MS/MS The chemical composition of R. sinensis was investigated using LC-ESI-MS/MS in negative ion mode (ESI - ). We could detect 33 compounds in two extracts of R. sinensis that were mainly of chromones, depsides, dibenzofurans, depsidones, terpenoids, pulvinic acid derivatives, fatty acids, polyols, monocyclic aromatic compounds, along with small numbers of unknown compounds (Table 1) . In this study, 2 chromones were detected at m/z 235 and 361 that were tentatively identified as 6-hydroxymethyl eugenitin and lepraric acid, respectively. Depsides included ramalinaic acid ( m/z 359), subsekikaic acid ( m/z 389), 2-O-Methyldivaricatic acid ( m/z 401), stenosporic acid ( m/z 415), perlatolic acid ( m/z 443), 2'-O-Methylperlatolic acid ( m/z 457), and squamatic acid ( m/z 802). Four molecular ions were tentatively identified as dibenzofurans, including isousnic acid ( m/z 342), usnic acid ( m/z 343), placodiolic acid ( m/z 375), and pseudoplacodiolic acid ( m/z 774). Usnic acid also indicated a 2M-2H+Na adduct at m/z 710. Besides this, five depsidones were also identified as virensic acid, norstictic acid, conprotocetraric acid, methylstictic acid, and gangaleoidin at m/z 356, 371, 376, 399, and 412, respectively. Ceruchinol ((-)-ent-Kauran-16 α -ol), a diterpene, was the only terpenoid identified at m/z 289. We could also identify two fatty acids, including nephrosterinic acid ( m/z 295) and arachidonic acid ( m/z 304). Atranol and olivetolic acid are two monocyclic aromatic compounds that were tentatively identified at m/z 151 and 223, respectively. Meso-erythritol was the only polyol we could identify at m/z 121. Finally, a molecular ion at m/z 350 proved to be a pulvinic acid derivative, further identified as leprapinic acid. Moreover, this compound also showed an adduct (2M-2H+Na) at m/z 725. Discussion The results of the present study showed that R. sinensis extract had a significant effect on the apoptosis of OSCC cells. MTT Assay results showed that R. sinensis reduced the survival rate of SCC cells, and scratch test results showed that it reduced the ability of cell migration in OSCC cells. These results indicate the role of R. sinensis ’ potential in apoptosis induction. R. sinensis stops the cell cycle in the gap2/mitosis (G2/m) phase. This substance regulates the expression of cydin-dependent kinase1 ( CDK1 ), cydin B1 , and cydin-dependent kinase inhibitor1 ( CDKN1A ) genes (15). In previous studies, the antioxidant properties of R. sinensis have been shown. Nazari et al. stated that R. sinensis and its secondary metabolites have antioxidant properties and the ability to remove toxic free radicals (16). In addition, the anti-proliferative effects of R. sinensis on different cancer cell lines have been shown in previous studies. Backorova et al. have shown that secondary lichen metabolites, such as usnic acid and atranurine, induce apoptosis in HT-29 cell line by activating caspase-3 (17). Lee et al. stated that R. sinensis induces apoptosis in MCF-7 and MDA-MB-231 breast cancer cells and inhibits the growth of cells in both cell groups (18). Song-Suk-Suh et al. showed that R. sinensis induces apoptosis and suppresses cell growth and proliferation. It also prevents the invasion of colorectal cancer cells, and this effect is dose-dependent (19). Nguyen et al. demonstrated the effect of the anti-cancer properties of Cucullata Flavocetraria on various cancer cell lines (20). Aoussar et al. conducted a study to evaluate the chemical composition and antimicrobial activity of the Ramalina Farnesia . Their study confirmed the ability of R. sinensis extract on the inhibition of free radicals (21). Yang et al. showed that the two compounds, including osnic acid and potassium osnate, play a role in controlling the growth of cancer cells in in-vitro studies against various cancer cell lines. Osnic acid displayed anticancer activity in colorectal cancer cells with limited in-vivo effects. Osnic acid has been reported to have cytotoxic activity against HCT116, human colon cancer cells (22). Other compounds derived from R. sinensis extract are protolichesterinic acid, which eliminate cancer cells by blocking the expression of heat shock protein 70 (HSP-70) in the prostate cancer cell line and activating caspase 3 in the HeLa cell line (23 ). In the present study, compounds such as methystictic acid and nephrosterinic acid were present in all methanolic and acetonic extracts. A compound such as atranol was available in both acetone and methanolic extracts. Considering that methanolic and acetonic extracts had similar effects on apoptosis and necrosis of OSCC cells, it can be concluded that these main compounds have anti-cancer effects of methystictic acid and nephrosterinic acid. However, further studies are necessary to investigate the cytotoxic effect of each of these extracts alone. In addition, the acetone extract of R. sinensis was more effective in inhibiting cell proliferation than the methanol extract. These results are the same as those of Nguyen et al., in which acetone extract had more cytotoxic properties than ethanolic extract, attributed to the active ingredients of acetone extract of R. sinensis . Most of the anti-cancer effects of osnic acid on human colorectal cancer cell lines were at a concentration of 12.5-100 μM, and cell viability was assessed using the MTT assay (24 ). Their results showed that the number of invasive cells in the groups treated with osnic acid was less than other cells in the control group. Osnate was undetectable in tumor tissues in mice receiving osnic acid, while 0.166 ± 1.5117 nmol/g was observed in the tissues of mice treated with potassium osnate. The composition of potassium osnate has stronger anti-cancer effects than osnic acid, which can be due to its better availability for cells. They also showed that the compound tamidoline has anti-cancer properties against colorectal cancer cells (25 ). Many lichen-derived acids have acceptable anti-cancer properties, so some of these compounds, such as lacanuric acid and valproic acid, are much more effective than conventional drugs in reducing thyroidoxin levels (20 ). Conclusion Based on our knowledge, this study is the first study investigating lichen ( R. sinensis ) on the OSCC cell line. The results of the present study showed the positive effects of R. sinensis extract on the apoptosis of OSCC cells. This study was an introduction to the anticancer effects of R. sinensis extract and is expected to be the subject of further studies with a focus on investigating the pathogenesis of lichen on the apoptosis of OSCC cells and future research is anticipated to concentrate on the anticancer properties of R. sinensis extract. Declarations Ethics approval and consent to participate All procedures performed in this studies were in accordance with relevant guidelines and regulations in the Declaration of Helsinki. This study was approved by the Tehran University of Medical Sciences Ethical Committee (ethical code: IR.TUMS.DENTISTRY.REC.1399.076). All methods were performed in accordance with the relevant guidelines and regulations. Online certification https://ethics.research.ac.ir/ProposalCertificateEn.php?id=147916&Print=true&NoPrintHeader=true&NoPrintFooter=true&NoPrintPageBorder=true&LetterPrint=true Permissions to collect Ramalina sinensis Lichen materials were collected in accordance with the relevant guidelines and regulations of the Plant Varieties Protection, Environmental Protection Organization of Iran (Tehran, Iran), and relevant permissions were taken from the plant Varieties Protection, Environmental Protection Organization of Iran to collect Ramalina sinensis. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information files]. Competing interests The authors declare that they have no competing interests Funding No funding. Authors' contributions MK, HK, and MS conceived the study idea and led data collection. MK, MS, and HK created the study protocol and wrote the original draft. MK, HK, and HN contributed to data analysis / interpretation and preparation of the manuscript. MK, MS, and HN led the writing- review & editing. All authors interpreted the results, read, and approved the final manuscript. Acknowledgements Not applicable. References Vucicevic Boras V, Fucic A, Virag M, Gabric D, Blivajs I, Tomasovic-Loncaric C, et al. Significance of stroma in biology of oral squamous cell carcinoma. Tumori Journal. 2018;104(1):9-14. Sasahira T, Kirita T. Hallmarks of cancer-related newly prognostic factors of oral squamous cell carcinoma. International journal of molecular sciences. 2018;19(8):2413. Nandini D, Rao RS, Hosmani J, Khan S, Patil S, Awan KH. Novel therapies in the management of oral cancer: an update. Disease-a-Month. 2020;66(12):101036. D'Cruz AK, Vaish R, Dhar H. Oral cancers: current status. Oral oncology. 2018;87:64-9. Popovici V, Bucur L, Vochita G, Gherghel D, Mihai CT, Rambu D, et al. In vitro anticancer activity and oxidative stress biomarkers status determined by Usnea barbata (L.) FH Wigg. dry extracts. Antioxidants. 2021;10(7):1141. Tripathi AH, Negi N, Gahtori R, Kumari A, Joshi P, Tewari LM, et al. A Review of Anti-Cancer and Related Properties of Lichen-Extracts and Metabolites. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents). 2022;22(1):115-42. Dandapat M, Paul S. Secondary metabolites from lichen Usnea longissima and its pharmacological relevance. Pharmacognosy Research. 2019;11(2). Suh S-S, Kim TK, Kim JE, Hong J-M, Nguyen TTT, Han SJ, et al. Anticancer activity of ramalin, a secondary metabolite from the antarctic lichen Ramalina terebrata, against colorectal cancer cells. Molecules. 2017;22(8):1361. Zhao Y, Wang M, Xu B. A comprehensive review on secondary metabolites and health-promoting effects of edible lichen. Journal of Functional Foods. 2021;80:104283. Crawford SD. Lichens used in traditional medicine. Lichen secondary metabolites: Springer; 2019. p. 31-97. Oh S-O, Wang XY, Wang LS, Liu PG, Hur J-S. A note on the lichen genus Ramalina (Ramalinaceae, Ascomycota) in the Hengduan Mountains in China. Mycobiology. 2014;42(3):229-40. Ristic S, Rankovic B, Stamenkovic S. Biopharmaceutical potential of two Ramalina lichens and their metabolites. Current Pharmaceutical Biotechnology. 2016;17(7):651-8. Norouzi H, Azizi A, Gholami M, Sohrabi M, Boustie J. Chemotype variations among lichen ecotypes of Umbilicaria aprina as revealed by LC-ESI-MS/MS: a survey of antioxidant phenolics. Environmental Science and Pollution Research. 2020;27(32):40296-308. Olivier-Jimenez D, Chollet-Krugler M, Rondeau D, Beniddir MA, Ferron S, Delhaye T, et al. A database of high-resolution MS/MS spectra for lichen metabolites. Scientific data. 2019;6(1):1-11. Shrestha G, St Clair LL. Lichens: a promising source of antibiotic and anticancer drugs. Phytochemistry reviews. 2013;12(1):229-44. Nazari J, Payamnoor V, Kavosi M, Asadi J. Extraction of anti-cancer triterpenoids (betulinic acid and betulin) from the birch bark-inhabiting lichen (Ramalina sinensis). Iranian Journal of Medicinal and Aromatic Plants Research. 2018;34(4):604-16. Bačkorová M, Jendželovský R, Kello M, Bačkor M, Mikeš J, Fedoročko P. Lichen secondary metabolites are responsible for induction of apoptosis in HT-29 and A2780 human cancer cell lines. Toxicology in vitro. 2012;26(3):462-8. Lee E, Lee CG, Yim JH, Lee HK, Pyo S. Ramalin‐Mediated Apoptosis Is Enhanced by Autophagy Inhibition in Human Breast Cancer Cells. Phytotherapy Research. 2016;30(3):426-38. Song X, Li Y, Zhang H, Yang Q. The anticancer effect of Huaier. Oncology reports. 2015;34(1):12-21. Nguyen TT, Yoon S, Yang Y, Lee H-B, Oh S, Jeong M-H, et al. Lichen secondary metabolites in Flavocetraria cucullata exhibit anti-cancer effects on human cancer cells through the induction of apoptosis and suppression of tumorigenic potentials. PloS one. 2014;9(10):e111575. Aoussar N, Laasri FE, Bourhia M, Manoljovic N, Mhand RA, Rhallabi N, et al. Phytochemical analysis, cytotoxic, antioxidant, and antibacterial activities of lichens. Evidence-Based Complementary and Alternative Medicine. 2020;2020. Yang P-M, Liu Y-L, Lin Y-C, Shun C-T, Wu M-S, Chen C-C. Inhibition of autophagy enhances anticancer effects of atorvastatin in digestive malignancies. Cancer research. 2010;70(19):7699-709. Xu M, Heidmarsson S, Olafsdottir ES, Buonfiglio R, Kogej T, Omarsdottir S. Secondary metabolites from cetrarioid lichens: Chemotaxonomy, biological activities and pharmaceutical potential. Phytomedicine. 2016;23(5):441-59. Eyvani H, Moghaddaskho F, Kabuli M, Zekri A, Momeny M, Tavakkoly-Bazzaz J, et al. Arsenic trioxide induces cell cycle arrest and alters DNA methylation patterns of cell cycle regulatory genes in colorectal cancer cells. Life Sciences. 2016;167:67-77. Yang Y, Bhosle SR, Yu YH, Park S-Y, Zhou R, Taş İ, et al. Tumidulin, a lichen secondary metabolite, decreases the stemness potential of colorectal cancer cells. Molecules. 2018;23(11):2968. Tables Table 1 is available in the Supplementary Files section. Additional Files Additional Files 1 & 2 are not available with this version Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.docx Table1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 26 Dec, 2022 Reviews received at journal 21 Dec, 2022 Reviews received at journal 13 Dec, 2022 Reviewers agreed at journal 01 Dec, 2022 Reviewers invited by journal 23 Nov, 2022 Editor assigned by journal 23 Nov, 2022 Editor invited by journal 22 Nov, 2022 Submission checks completed at journal 22 Nov, 2022 First submitted to journal 07 Nov, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2246187","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":154252853,"identity":"1f75e961-84e8-4ced-81aa-e7898b63a796","order_by":0,"name":"Hanieh Karimi","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hanieh","middleName":"","lastName":"Karimi","suffix":""},{"id":154252854,"identity":"f0f6bd8f-57ce-40f2-ad5d-42f8eea021d1","order_by":1,"name":"Maryam Koopaie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYJCCA0AIBIwNDB+AFBs7KVoYZ4C0MBNpDxgw84BJAop1288+PPDjjI2cOfvhxsc2v7bJ8zEzMH74mINbi9mZdIODPTfSjC17EpuNc/tuG7YxMzBLztyGR8uBNIYDPB8OJ244kNgmndtzmxGohY2ZF5+W888YDv4BaTn/sP23Zc9te8JabqQxHOa5AdRyIxGo+MftRCK0PGM4LHMG6JcZD5slextuJ7cxMzbj98v5NOaPb44BQ4w//eGHH39u285vbz744SMeLXBgACIY28BkAxHqYVoY/hCneBSMglEwCkYWAAAasFo1aPVnMAAAAABJRU5ErkJggg==","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Koopaie","suffix":""},{"id":154252858,"identity":"0a405c61-39d9-4625-a637-b242e09a64a7","order_by":2,"name":"Mohammad Sohrabi","email":"","orcid":"","institution":"Iranian Research Organization for Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Sohrabi","suffix":""},{"id":154252860,"identity":"e6ca1413-41b8-4812-b39b-490dd01d054c","order_by":3,"name":"Hooman Norouzi","email":"","orcid":"","institution":"Iranian Research Organization for Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hooman","middleName":"","lastName":"Norouzi","suffix":""}],"badges":[],"createdAt":"2022-11-07 09:29:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2246187/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2246187/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29618190,"identity":"ee9c113a-b401-4ceb-9d60-becf6eff8497","added_by":"auto","created_at":"2022-11-28 21:34:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":324294,"visible":true,"origin":"","legend":"\u003cp\u003eThallus of \u003cem\u003eR. sinensis \u003c/em\u003e(scale bar represents 10 mm).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2246187/v1/2691e625cb7618a6553e92d4.jpg"},{"id":29618653,"identity":"cc87ee3b-52c0-4cf1-b58a-bfa68b41535d","added_by":"auto","created_at":"2022-11-28 21:42:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1720097,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of cell viability (ratio of the treated group to control) after 48 hour treatment.\u003c/p\u003e\n\u003cp\u003e*: P-value (p) \u0026gt;0.05, ***: P-value (p) \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2246187/v1/dd4e10caf0fbbf123a697a6d.jpg"},{"id":29618978,"identity":"e5fcd116-f259-403b-a016-a02cc432fb45","added_by":"auto","created_at":"2022-11-28 21:58:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3372164,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of cell viability (ratio of the treated group to control) after 72 hour treatment.\u003c/p\u003e\n\u003cp\u003e*: P-value (p) \u0026gt;0.05, ***: P-value (p) \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2246187/v1/d1b0d15d7b9bcdc85f336ede.jpg"},{"id":29618191,"identity":"a064fa84-872c-46f2-b409-7071261206ac","added_by":"auto","created_at":"2022-11-28 21:34:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":807799,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry analysis after 24 hour treated with 50 μg/mL of acetone extract of \u003cem\u003eR. sinensis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eQ1: Necrotic cells, Q2: Late Apoptotic cells, Q3: Early Apoptotic cells, Q4: Viable cells.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2246187/v1/905f554ee5bbf90ac3c97e75.jpg"},{"id":29618656,"identity":"3b95ad9c-2e2f-41bd-b474-23de9f9044f4","added_by":"auto","created_at":"2022-11-28 21:42:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":589431,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry analysis after 24 hour in the control group.\u003c/p\u003e\n\u003cp\u003eQ1: Necrotic cells, Q2: Late Apoptotic cells, Q3: Early Apoptotic cells, Q4: Viable cells.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2246187/v1/60e6e97f10927600fa790a48.jpg"},{"id":29618199,"identity":"c99f3cf3-58c7-4a9f-8404-a06301368a54","added_by":"auto","created_at":"2022-11-28 21:34:37","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":793422,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry analysis after 24 hour in the treated with 50 μg/mL of methanol extract of \u003cem\u003eR. sinensis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eQ1: Necrotic cells, Q2: Late Apoptotic cells, Q3: Early Apoptotic cells, Q4: Viable cells.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2246187/v1/4d1353408803f129e76bc324.jpg"},{"id":29618196,"identity":"de7ef462-41cc-4529-8ebe-f4e8fac587cd","added_by":"auto","created_at":"2022-11-28 21:34:37","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":124153,"visible":true,"origin":"","legend":"\u003cp\u003eScratch assay of OSCC cells in control, acetone extract, and methanol extract of \u003cem\u003eR. sinensis\u003c/em\u003e at 0, 24, and 48 hour of treatment.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2246187/v1/62718f2b0954c570feabff35.jpg"},{"id":29618756,"identity":"d5b9bf6b-d234-4a14-94c0-27d69a7871e2","added_by":"auto","created_at":"2022-11-28 21:50:37","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":85313,"visible":true,"origin":"","legend":"\u003cp\u003eScratch assay of OSCC cells in control, acetone extract, and methanol extract of \u003cem\u003eR. sinensis\u003c/em\u003e at 0, 24, and 48 hour of treatment.\u003c/p\u003e\n\u003cp\u003e*: P-value (p) \u0026gt;0.05, ***: P-value (p) \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2246187/v1/ee62c9a2498817c9f6d2dfea.jpg"},{"id":29618979,"identity":"41b2276d-b183-42a6-948e-abbce386df34","added_by":"auto","created_at":"2022-11-28 21:58:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1343461,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2246187/v1/88681e87-e754-4292-8fe4-49dcb213ae3c.pdf"},{"id":29618192,"identity":"51b028f4-c098-4c1b-a5a6-5188827e7d9e","added_by":"auto","created_at":"2022-11-28 21:34:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":41483,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-2246187/v1/c58c8eb21999ecb730fdf5dd.docx"},{"id":29618197,"identity":"ee0f5eb2-9d67-42c2-8668-5efe83661b7d","added_by":"auto","created_at":"2022-11-28 21:34:37","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":598761,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-2246187/v1/2fb3ec5e25838055f872cdcd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cytotoxic, anti-proliferative, and apoptotic evaluation of Ramalina sinensis (Lichenized fungus, Ramalinaceae) on oral squamous cell carcinoma cell line; in-vitro study","fulltext":[{"header":"Background","content":"\u003cp\u003eThe most frequent kind of cancer in the oral cavity, oral squamous cell carcinoma (OSCC), and accounts for approximately 90% of all oral malignancies (1). OSCC is a multifactorial disease in which both extrinsic and intrinsic factors, such as smoking and alcohol intake, as well as iron deficiency anemia and genetics, have a role in disease incidence and progression (2). Although surgery, radiotherapy, chemotherapy, or a mixture of them are known as the common treatment approaches of OSCC, these modalities have some cosmetic and functional side effects (3). Despite advancements in treatment modalities, the survival rate of oral cancer remains low, and attempts are underway to provide novel and effective treatment modalities considering the dismal prognosis of oral cancer (4).\u003c/p\u003e\n\u003cp\u003eLichens as a source of active pharmacologic compounds, have recently been introduced for treatment purposes (5, 6). Bioactive metabolites, such as the phenolic chemicals despides, depsidones, and dibenzofurans, are produced by lichens, which are symbiotic organisms composed of a fungus and an algae/cyanobacteria (7). As lichen-derived chemicals, over 1000 metabolites have been found, and these compounds exhibit a broad spectrum of biological actions, including antioxidant, cytotoxic, anti-inflammatory, and anti-proliferative properties (7). Lichen metabolites are classified as either primary or secondary. Proteins, lipids, and carbohydrates are primary metabolites that have a role in lichen structure and metabolism. Secondary metabolites are complex compounds having therapeutic functions (8).\u003c/p\u003e\n\u003cp\u003eSeveral species have been identified as having traditional medicinal uses (9). Their traditional usage dates back to Ancient Greece when one of its species was used to treat inflammation, jaundice, and impetigo. \u003cem\u003eRamalina calicaris, Ramalina conduplicans, Ramalina farinacea, Ramalina inflata, Ramalina menziesii, Ramalina roesleri,\u003c/em\u003e and \u003cem\u003eRamalina sinensis\u003c/em\u003e are some of the Ramalina species that have been used in traditional medicine (10, 11). Furthermore, there is evidence for the interaction of various lichen substances with several molecular mechanisms that are important in cell death. These interactions include cell cycle arrest, apoptosis, necrosis, and inhibition of angiogenesis (12). Although there are several reports on the anticancer activity of lichen substances or organic extracts against different human cell lines, this is the first report considering the anticancer properties of Ramalina species lichens on OSCC cells. The aim of the present study is to assess the effect of Ramalina lichen on the cell viability and apoptosis of OSCC cell lines.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eChemicals and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThermo Fisher Scientific (Massachusetts, USA) provided the acetonitrile, and DiKMA Technologies provided the formic acid (Beijing, China). Water was produced via a Milli-Q\u0026reg; integral water purification system (Merck, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLichen material\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRamalina specimens were collected in the Hyrcanian forest in Mazandaran Provinces (Iran) in May 2018, and \u003cem\u003eRamalina Sinensis\u003c/em\u003e (\u003cem\u003eR. sinensis\u003c/em\u003e) was selected for this study. Lichen materials were collected in accordance with the relevant guidelines and regulations of the Plant Varieties Protection, Environmental Protection Organization of Iran. The Museum of Iranian Lichens in the Iranian Research Organization for Science and Technology (Tehran, Iran) provided the principal keys to identifying species (Figure 1). The lichen was identified by Dr. Mohammad Sohrabi, lichenologist from the Iranian Research Organization for Science and Technology. The voucher specimen was deposited in the Museum of Iranian Lichens, Iranian Research Organization for Science and Technology (IROST), Iran (https://irost.org/museum/). The cleaned lichen thallus was grounded to a fine powder using a mortar and pestle under a small amount of liquid nitrogen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human oral epidermal carcinoma cell line (KB cell line, NCBI Code: C152) were obtained from the Pasteur Institute of Iran (Tehran, Iran) (Supplementary File). Cancer cell lines were cultured in DMEM with 10% FBS and 1% PS at 37\u0026deg;C, 5.0% CO\u003csub\u003e2\u003c/sub\u003e and 95.0% humidity. Cells were cultured in a humidified atmosphere of 95.0% air, and 5.0% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine viability, which is based on the reduction of MTT by mitochondrial dehydrogenase in intact cells into an insoluble purple formazan product. In 96-well plates, cells (1*10\u003csup\u003e4\u003c/sup\u003e/well) were seeded, and after 24 hours, the cells were treated with \u003cem\u003eR. sinensis\u003c/em\u003e extract at various doses (200, 100, 50, 25, 12.5, and 6.25 \u0026mu;g/ml) and then \u003cem\u003eR. sinensis\u003c/em\u003e -containing medium were carefully removed after the treatment. Cells were washed twice with PBS before each well received 100 mL media containing 0.5 mg/ml MTT in PBS, then the plate was incubated at 37\u0026deg;C for 4 hours. The medium was then completely removed, 200 mL of Tris-DMSO solution was added to each well, and the plate was vibrated for 30 minutes. Using an ELISA plate reader, the absorbance, as proportional to cell viability, was then measured in at least three independent measurements using a microplate reader (Bio-Rad Corp, California, USA) at 570 nm in each well and Gen 5 Version 2.07.17 software (BioTek, Winooski, USA) for data analysis. The average of measurements was reported for each well.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry analysis was performed on apoptotic cells using a Beckman Coulter (EPICS XL). The ramalina-treated and untreated cells were collected, twice-washed in PBS, fixed in 70.0% ethanol at 4\u0026deg;C for at least 12 hours, centrifuged, and then incubated for 30 minutes at 37\u0026deg;C in the dark with 0.1% Triton X-100, 200 mg/ml RNase A, and 50 mg/ml propidium iodide (PI) in PBS. More than 3*10\u003csup\u003e4\u003c/sup\u003e/well cells were counted in each sample, and cells with lower DNA content than those in the G0/G1 phase were labeled as apoptotic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScratch assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 24 hour of cultivation as confluent monolayers in the complete medium, cells were wounded by removing cells across the well with a standard 200 \u0026mu;L pipette tip [13]. To eliminate the non-adherent cells, the wounded monolayers were washed twice. When the wound was made, and 24 hours later, it was observed using an inverted phase contrast microscope (Leica, Wetzlar, Germany). This 24 hour was selected since it is less than the doubling time in control. Four separate fields from each sample were evaluated for quantitative measurement of the distance between the borderlines, while four different equidistant spots in each picture were measured to get a better approximation of the true breadth of the wounded region. The migration rate was estimated as the ratio of the mean distance between both borderlines generated by scratching to the distance that remained cell-free after re-growing and is represented as a percentage of the control. In quadruplicates, two separate sets of tests were carried out.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolic profiling of \u003cem\u003eR. sinensis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chemical composition of \u003cem\u003eR. sinensis\u0026nbsp;\u003c/em\u003ewas\u003cem\u003e\u0026nbsp;\u003c/em\u003einvestigated according to Norouzi et al. briefly (13); lichenochemicals were separated by Waters Alliance e2695 separation module (Milford, MA, USA) and Atlantis T3 C18 column (2.1mm \u0026times; 100 mm, 3 \u0026mu;m; Milford, MA, USA) and Column temperature set to 30℃. Samples were dissolved in methanol, and after filtration (PTFE membrane filters, 0.45 \u0026mu;m, Simplepure, China), 10 \u0026mu;L of each sample was injected into the separation module. Lichenochemicals were eluted within 25 min as follows: elution began by 95% (water + 0.1% formic acid, v/v) and gradually decreased to 5% within 20 min. The Elution process went on for another 5 min by 95% B (acetonitrile). The flow rate of eluents was adjusted to be 0.25 ml min\u003csup\u003e-1\u003c/sup\u003e. A Quattro micro API mass spectrometer (Milford, MA, USA) was used for tandem mass analysis. The MS/MS parameters applied were as follows: source temperature and desolvation temperature were set to be 120 ℃ and 300 ℃, respectively; capillary voltage, cone voltage, and collision energy were regulated at 3.5 kV, 30 V, and 30 eV, respectively; for both nebulizing and drying the gas, N\u003csub\u003e2\u003c/sub\u003e was used. MassLynx 4.1 and MZmine 2.53 were used for data acquiring and analysis. Initial annotation of detected compounds was carried out based on the lichen spectral database (LDB) provided in GNPS public spectral libraries (14). Chemical structures were sketched using ChemDraw Ultra 12.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPSS software (version 22; SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8.2.1 (GraphPad Software, San Diego, CA) were used for statistical analysis. All results are given as as mean \u0026plusmn; standard deviation (SD), with p \u0026le; 0.05 considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMTT assay results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the impact of \u003cem\u003eR. sinensis\u003c/em\u003e on the viability of OSCC cells, we conducted an MTT experiment in KB cells with various \u003cem\u003eR. sinensis\u003c/em\u003e concentrations (6.25, 12.5, 25, 50,100, and 200 \u0026mu;g/mL) and various incubation durations (48 and 72 hour) [Additional file 1]. \u003cem\u003eR. sinensis\u003c/em\u003e reduced the proliferation of KB cells in a time-dependent manner and the higher concentration (100 \u0026mu;g/mL) having a more considerable inhibitory impact than the lower 50 \u0026mu;g/mL concentration ((Figure 2 (cell viability after 48 hour) and Figure 3 (cell viability after 72 hour)).\u003c/p\u003e\n\u003cp\u003eThe cell viability of OSCC cells treated with 100 \u0026mu;g/mL of acetone and methanol extract was 17.65% (SD = \u0026plusmn;7.64%) and 2.60% (SD = \u0026plusmn;1.77%), respectively. There was a statistically significant difference between acetone and methanol extract in the cell viability of OSCC cells treated with 100 \u0026mu;g/mL concentration (p = 0.029). The cell viability of OSCC cells at 200 \u0026mu;g/ml concentration was 1.48% (SD = \u0026plusmn;0.92), and 1.89% (SD = \u0026plusmn;1.09), for acetone and methanol extract, respectively, indicating a statistically significant difference. The inhibitory concentration at 50% inhibition (IC50) was the parameter used to compare the cytotoxic activity, and a lower IC50 means better cytotoxic activity. The IC50 against KB cells was 50 \u0026mu;g/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe percent of OSCC cells \u003cem\u003eR. sinensis\u003c/em\u003e treated 24 hour (50 \u0026mu;g/mL of acetone extract of \u003cem\u003eR. sinensi\u003c/em\u003e) in the early and late apoptosis phases were 16.8% and 23.1%, respectively (Figure 4).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The percent of OSCC cells of control in the early apoptosis phase was 3.43% and 8.07% in the late apoptosis phase (Figure 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, the percent of \u003cem\u003eR. sinensis\u003c/em\u003e treated (50 \u0026mu;g/mL of methanol extract) OSCC cells (24 hour treatment) in the early and late apoptosis phase was 30.1% and 40.1%, respectively, which indicates an increase in apoptotic cells by methanol extract \u0026nbsp;(Figure 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eScratch assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNon-cytotoxic doses of \u003cem\u003eR. sinensis extract\u0026nbsp;\u003c/em\u003e(50 \u0026mu;g/mL) were used to treat OSCC cells to assess the effect of \u003cem\u003eR. sinensis\u003c/em\u003e on cell migration. \u003cem\u003eR. sinensis extract\u003c/em\u003e significantly inhibited cell migration in OSCC cells (Figure 7) [Additional file 2]. In particular, the migration of cancer cells was inhibited by acetone and methanol extract of \u003cem\u003eR. sinensis.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThese results indicate that \u003cem\u003eR. sinensis\u003c/em\u003e suppresses the wound-healing process in human OSCC. There was no significant difference between acetone and methanol extract in\u0026nbsp;cell migration inhibition. Therefore, \u003cem\u003eR. sinensis\u003c/em\u003e may prevent cancer metastasis in OSCC cells (Figure 8).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e\u003cem\u003eMetabolic profiling using LC-ESI-MS/MS\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chemical composition of \u003cem\u003eR. sinensis\u003c/em\u003e was investigated using LC-ESI-MS/MS in negative ion mode (ESI\u003csup\u003e-\u003c/sup\u003e). We could detect 33 compounds in two extracts of \u003cem\u003eR. sinensis\u003c/em\u003e that were mainly of chromones, depsides, dibenzofurans, depsidones, terpenoids, pulvinic acid derivatives, fatty acids, polyols, monocyclic aromatic compounds, along with small numbers of unknown compounds (Table 1)\u003cem\u003e.\u003c/em\u003e In this study, 2 chromones were detected at \u003cem\u003em/z\u003c/em\u003e 235 and 361 that were tentatively identified as 6-hydroxymethyl eugenitin and lepraric acid, respectively. Depsides included ramalinaic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e359), subsekikaic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e389), 2-O-Methyldivaricatic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e401), stenosporic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e415), perlatolic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e443), 2\u0026apos;-O-Methylperlatolic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e457), and squamatic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e802). Four molecular ions were tentatively identified as dibenzofurans, including isousnic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e342), usnic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e343), placodiolic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e375), and pseudoplacodiolic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e774). Usnic acid also indicated a 2M-2H+Na adduct at \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e710. Besides this, five depsidones were also identified as virensic acid, norstictic acid, conprotocetraric acid, methylstictic acid, and gangaleoidin at \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e356, 371, 376, 399, and 412, respectively. Ceruchinol ((-)-ent-Kauran-16\u003cstrong\u003e\u0026alpha;\u003c/strong\u003e-ol), a diterpene, was the only terpenoid identified at \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e289. We could also identify two fatty acids, including nephrosterinic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e295) and arachidonic acid (\u003cem\u003em/z\u0026nbsp;\u003c/em\u003e304). Atranol and olivetolic acid are two monocyclic aromatic compounds that were tentatively identified at \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e151 and 223, respectively. Meso-erythritol was the only polyol we could identify at \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e121. Finally, a molecular ion at \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e350 proved to be a pulvinic acid derivative, further identified as leprapinic acid. Moreover, this compound also showed an adduct (2M-2H+Na) at \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e725.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of the present study showed that \u003cem\u003eR. sinensis\u003c/em\u003e extract had a significant effect on the apoptosis of OSCC cells. MTT Assay results showed that \u003cem\u003eR. sinensis\u003c/em\u003e reduced the survival rate of SCC cells, and scratch test results showed that it reduced the ability of cell migration in OSCC cells. These results indicate the role of \u003cem\u003eR. sinensis\u003c/em\u003e\u0026rsquo; potential in apoptosis induction.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eR. sinensis\u0026nbsp;\u003c/em\u003estops the cell cycle in the gap2/mitosis (G2/m) phase. This substance regulates the expression of \u003cem\u003ecydin-dependent kinase1\u003c/em\u003e (\u003cem\u003eCDK1\u003c/em\u003e), \u003cem\u003ecydin B1\u003c/em\u003e, and \u003cem\u003ecydin-dependent kinase inhibitor1\u003c/em\u003e (\u003cem\u003eCDKN1A\u003c/em\u003e) genes (15). In previous studies, the antioxidant properties of \u003cem\u003eR. sinensis\u003c/em\u003e have been shown. Nazari et al. stated that \u003cem\u003eR. sinensis\u003c/em\u003e and its secondary metabolites have antioxidant properties and the ability to remove toxic free radicals (16). In addition, the anti-proliferative effects of \u003cem\u003eR. sinensis\u003c/em\u003e on different cancer cell lines have been shown in previous studies. Backorova et al. have shown that secondary lichen metabolites, such as usnic acid and atranurine, induce apoptosis in HT-29 cell line by activating caspase-3 (17). Lee et al. stated that \u003cem\u003eR. sinensis\u003c/em\u003e induces apoptosis in MCF-7 and MDA-MB-231 breast cancer cells and inhibits the growth of cells in both cell groups (18).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSong-Suk-Suh et al. showed that \u003cem\u003eR. sinensis\u003c/em\u003e induces apoptosis and suppresses cell growth and proliferation. It also prevents the invasion of colorectal cancer cells, and this effect is dose-dependent (19). Nguyen et al. demonstrated the effect of the anti-cancer properties of \u003cem\u003eCucullata Flavocetraria\u003c/em\u003e on various cancer cell lines (20). Aoussar et al. conducted a study to evaluate the chemical composition and antimicrobial activity of the \u003cem\u003eRamalina Farnesia\u003c/em\u003e. Their study confirmed the ability of \u003cem\u003eR. sinensis\u003c/em\u003e extract on the inhibition of free radicals (21).\u003c/p\u003e\n\u003cp\u003eYang et al. showed that the two compounds, including osnic acid and potassium osnate, play a role in controlling the growth of cancer cells in in-vitro studies against various cancer cell lines. Osnic acid displayed anticancer activity in colorectal cancer cells with limited \u003cem\u003ein-vivo\u003c/em\u003e effects. Osnic acid has been reported to have cytotoxic activity against HCT116, human colon cancer cells (22). Other compounds derived from \u003cem\u003eR. sinensis\u003c/em\u003e extract are protolichesterinic acid, which eliminate cancer cells by blocking the expression of heat shock protein 70 (HSP-70) in the prostate cancer cell line and activating caspase 3 in the HeLa cell line\u0026nbsp;(23\u003c/a\u003e).\u003c/p\u003e\n\u003cp\u003eIn the present study, compounds such as methystictic acid and nephrosterinic acid were present in all methanolic and acetonic extracts. A compound such as atranol was available in both acetone and methanolic extracts. Considering that methanolic and acetonic extracts had similar effects on apoptosis and necrosis of OSCC cells, it can be concluded that these main compounds have anti-cancer effects of methystictic acid and nephrosterinic acid. However, further studies are necessary to investigate the cytotoxic effect of each of these extracts alone. In addition, the acetone extract of \u003cem\u003eR. sinensis\u003c/em\u003e was more effective in inhibiting cell proliferation than the methanol extract. These results are the same as those of Nguyen et al., in which acetone extract had more cytotoxic properties than ethanolic extract, attributed to the active ingredients of acetone extract of \u003cem\u003eR. sinensis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eMost of the anti-cancer effects of osnic acid on human colorectal cancer cell lines were at a concentration of 12.5-100 \u0026mu;M, and cell viability was assessed using the MTT assay\u0026nbsp;(24\u003c/a\u003e). Their results showed that the number of invasive cells in the groups treated with osnic acid was less than other cells in the control group. Osnate was undetectable in tumor tissues in mice receiving osnic acid, while 0.166 \u0026plusmn; 1.5117 nmol/g was observed in the tissues of mice treated with potassium osnate. The composition of potassium osnate has stronger anti-cancer effects than osnic acid, which can be due to its better availability for cells. They also showed that the compound tamidoline has anti-cancer properties against colorectal cancer cells\u0026nbsp;(25\u003c/a\u003e). Many lichen-derived acids have acceptable anti-cancer properties, so some of these compounds, such as lacanuric acid and valproic acid, are much more effective than conventional drugs in reducing thyroidoxin levels\u0026nbsp;(20\u003c/a\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBased on our knowledge, this study is the first study investigating lichen (\u003cem\u003eR. sinensis\u003c/em\u003e) on the OSCC cell line. The results of the present study showed the positive effects of \u003cem\u003eR. sinensis\u003c/em\u003e extract on the apoptosis of OSCC cells. This study was an introduction to the anticancer effects of \u003cem\u003eR. sinensis\u003c/em\u003e extract and is expected to be the subject of further studies with a focus on investigating the pathogenesis of lichen on the apoptosis of OSCC cells and future research is anticipated to concentrate on the anticancer properties of \u003cem\u003eR. sinensis\u003c/em\u003e extract.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in this studies were in accordance with relevant guidelines and regulations in the Declaration of Helsinki. This study was approved by the Tehran University of Medical Sciences Ethical Committee (ethical code: IR.TUMS.DENTISTRY.REC.1399.076).\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eAll methods were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOnline certification\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003ehttps://ethics.research.ac.ir/ProposalCertificateEn.php?id=147916\u0026amp;Print=true\u0026amp;NoPrintHeader=true\u0026amp;NoPrintFooter=true\u0026amp;NoPrintPageBorder=true\u0026amp;LetterPrint=true\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePermissions to collect\u0026nbsp;Ramalina sinensis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLichen materials were collected in accordance with the relevant guidelines and regulations of the Plant Varieties Protection, Environmental Protection Organization of Iran (Tehran, Iran), and relevant permissions were taken from the plant Varieties Protection, Environmental Protection Organization of Iran to collect Ramalina sinensis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files]. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNo funding.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMK, HK, and MS conceived the study idea and led data collection. MK, MS, and HK created the study protocol and wrote the original draft. MK, HK, and HN contributed to data analysis / interpretation and preparation of the manuscript. MK, MS, and HN led the writing- review \u0026amp; editing. All authors interpreted the results, read, and approved the final manuscript. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eVucicevic Boras V, Fucic A, Virag M, Gabric D, Blivajs I, Tomasovic-Loncaric C, et al. Significance of stroma in biology of oral squamous cell carcinoma. Tumori Journal. 2018;104(1):9-14.\u003c/li\u003e\n \u003cli\u003eSasahira T, Kirita T. Hallmarks of cancer-related newly prognostic factors of oral squamous cell carcinoma. International journal of molecular sciences. 2018;19(8):2413.\u003c/li\u003e\n \u003cli\u003eNandini D, Rao RS, Hosmani J, Khan S, Patil S, Awan KH. Novel therapies in the management of oral cancer: an update. Disease-a-Month. 2020;66(12):101036.\u003c/li\u003e\n \u003cli\u003eD\u0026apos;Cruz AK, Vaish R, Dhar H. Oral cancers: current status. Oral oncology. 2018;87:64-9.\u003c/li\u003e\n \u003cli\u003ePopovici V, Bucur L, Vochita G, Gherghel D, Mihai CT, Rambu D, et al. In vitro anticancer activity and oxidative stress biomarkers status determined by Usnea barbata (L.) FH Wigg. dry extracts. Antioxidants. 2021;10(7):1141.\u003c/li\u003e\n \u003cli\u003eTripathi AH, Negi N, Gahtori R, Kumari A, Joshi P, Tewari LM, et al. A Review of Anti-Cancer and Related Properties of Lichen-Extracts and Metabolites. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents). 2022;22(1):115-42.\u003c/li\u003e\n \u003cli\u003eDandapat M, Paul S. Secondary metabolites from lichen Usnea longissima and its pharmacological relevance. Pharmacognosy Research. 2019;11(2).\u003c/li\u003e\n \u003cli\u003eSuh S-S, Kim TK, Kim JE, Hong J-M, Nguyen TTT, Han SJ, et al. Anticancer activity of ramalin, a secondary metabolite from the antarctic lichen Ramalina terebrata, against colorectal cancer cells. Molecules. 2017;22(8):1361.\u003c/li\u003e\n \u003cli\u003eZhao Y, Wang M, Xu B. A comprehensive review on secondary metabolites and health-promoting effects of edible lichen. Journal of Functional Foods. 2021;80:104283.\u003c/li\u003e\n \u003cli\u003eCrawford SD. Lichens used in traditional medicine. \u0026nbsp; Lichen secondary metabolites: Springer; 2019. p. 31-97.\u003c/li\u003e\n \u003cli\u003eOh S-O, Wang XY, Wang LS, Liu PG, Hur J-S. A note on the lichen genus Ramalina (Ramalinaceae, Ascomycota) in the Hengduan Mountains in China. Mycobiology. 2014;42(3):229-40.\u003c/li\u003e\n \u003cli\u003eRistic S, Rankovic B, Stamenkovic S. Biopharmaceutical potential of two Ramalina lichens and their metabolites. Current Pharmaceutical Biotechnology. 2016;17(7):651-8.\u003c/li\u003e\n \u003cli\u003eNorouzi H, Azizi A, Gholami M, Sohrabi M, Boustie J. Chemotype variations among lichen ecotypes of Umbilicaria aprina as revealed by LC-ESI-MS/MS: a survey of antioxidant phenolics. Environmental Science and Pollution Research. 2020;27(32):40296-308.\u003c/li\u003e\n \u003cli\u003eOlivier-Jimenez D, Chollet-Krugler M, Rondeau D, Beniddir MA, Ferron S, Delhaye T, et al. A database of high-resolution MS/MS spectra for lichen metabolites. Scientific data. 2019;6(1):1-11.\u003c/li\u003e\n \u003cli\u003eShrestha G, St Clair LL. Lichens: a promising source of antibiotic and anticancer drugs. Phytochemistry reviews. 2013;12(1):229-44.\u003c/li\u003e\n \u003cli\u003eNazari J, Payamnoor V, Kavosi M, Asadi J. Extraction of anti-cancer triterpenoids (betulinic acid and betulin) from the birch bark-inhabiting lichen (Ramalina sinensis). Iranian Journal of Medicinal and Aromatic Plants Research. 2018;34(4):604-16.\u003c/li\u003e\n \u003cli\u003eBačkorov\u0026aacute; M, Jendželovsk\u0026yacute; R, Kello M, Bačkor M, Mike\u0026scaron; J, Fedoročko P. Lichen secondary metabolites are responsible for induction of apoptosis in HT-29 and A2780 human cancer cell lines. Toxicology in vitro. 2012;26(3):462-8.\u003c/li\u003e\n \u003cli\u003eLee E, Lee CG, Yim JH, Lee HK, Pyo S. Ramalin‐Mediated Apoptosis Is Enhanced by Autophagy Inhibition in Human Breast Cancer Cells. Phytotherapy Research. 2016;30(3):426-38.\u003c/li\u003e\n \u003cli\u003eSong X, Li Y, Zhang H, Yang Q. The anticancer effect of Huaier. Oncology reports. 2015;34(1):12-21.\u003c/li\u003e\n \u003cli\u003eNguyen TT, Yoon S, Yang Y, Lee H-B, Oh S, Jeong M-H, et al. Lichen secondary metabolites in Flavocetraria cucullata exhibit anti-cancer effects on human cancer cells through the induction of apoptosis and suppression of tumorigenic potentials. PloS one. 2014;9(10):e111575.\u003c/li\u003e\n \u003cli\u003eAoussar N, Laasri FE, Bourhia M, Manoljovic N, Mhand RA, Rhallabi N, et al. Phytochemical analysis, cytotoxic, antioxidant, and antibacterial activities of lichens. Evidence-Based Complementary and Alternative Medicine. 2020;2020.\u003c/li\u003e\n \u003cli\u003eYang P-M, Liu Y-L, Lin Y-C, Shun C-T, Wu M-S, Chen C-C. Inhibition of autophagy enhances anticancer effects of atorvastatin in digestive malignancies. Cancer research. 2010;70(19):7699-709.\u003c/li\u003e\n \u003cli\u003eXu M, Heidmarsson S, Olafsdottir ES, Buonfiglio R, Kogej T, Omarsdottir S. Secondary metabolites from cetrarioid lichens: Chemotaxonomy, biological activities and pharmaceutical potential. Phytomedicine. 2016;23(5):441-59.\u003c/li\u003e\n \u003cli\u003eEyvani H, Moghaddaskho F, Kabuli M, Zekri A, Momeny M, Tavakkoly-Bazzaz J, et al. Arsenic trioxide induces cell cycle arrest and alters DNA methylation patterns of cell cycle regulatory genes in colorectal cancer cells. Life Sciences. 2016;167:67-77.\u003c/li\u003e\n \u003cli\u003eYang Y, Bhosle SR, Yu YH, Park S-Y, Zhou R, Taş İ, et al. Tumidulin, a lichen secondary metabolite, decreases the stemness potential of colorectal cancer cells. Molecules. 2018;23(11):2968.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Additional Files","content":"\u003cp\u003eAdditional Files 1 \u0026 2 are not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Oral squamous cell carcinoma (OSCC), cell viability, apoptosis, Ramalina sinensis, cell migration","lastPublishedDoi":"10.21203/rs.3.rs-2246187/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2246187/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eIn response to the poor prognosis of oral squamous cell carcinoma (OSCC), the most frequent cancer in the oral cavity, efforts have been made to create more effective treatment methods based on natural products. Lichens are a source of active pharmacologic compounds, which have lately been discovered to be helpful in cancer treatment. Current research seeks to evaluate the impact of \u003cem\u003eRamalina sinensis \u003c/em\u003e(\u003cem\u003eR. sinensis\u003c/em\u003e) lichen on the cell viability and apoptosis of OSCC cell lines, considering lichens’ anti-inflammatory and anticancer capabilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e \u003cem\u003eR. sinensis\u003c/em\u003e was selected for investigation of its effects on human oral squamous cell carcinoma cell line (KB cell line, NCBI Code: C152) and acetone and effect of methanol extract of \u003cem\u003eR. sinensis\u003c/em\u003eon OSCC cell line was investigated. The chemical composition \u003cem\u003eand m\u003c/em\u003eetabolic profiling of \u003cem\u003eR. sinensis \u003c/em\u003ewere\u003cem\u003e \u003c/em\u003einvestigated. Viability was assessed by MTT assay analysis, and apoptotic cells were measured using flow cytometry analysis. Scratch assay was used to assess cell migration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The chemical composition of \u003cem\u003eR. sinensis\u003c/em\u003e was investigated using LC-ESI-MS/MS, and 33 unique compounds in acetone and methanol extract of \u003cem\u003eR. sinensis \u003c/em\u003ewere detected.\u003cem\u003e \u003c/em\u003eThe proliferation of KB cells was gradually but significantly inhibited by 6.25, 12.5, 25, 50, 100, and 200 μg/mL in a time-dependent manner. There was a statistically significant difference between acetone and methanol extract in the inhibition of cell proliferation. Flow cytometry results indicate an increase in apoptosis of OSCC cells by acetone extract. \u003cem\u003eR. sinensis\u003c/em\u003e significantly inhibited the migration and invasion of OSCC cells in a concentration-dependent manner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e The results of the present study showed the positive effects of \u003cem\u003eR. sinensis\u003c/em\u003e extract on the apoptosis of OSCC cells and the anticancer effects of \u003cem\u003eR. sinensis\u003c/em\u003eextracts.\u003c/p\u003e","manuscriptTitle":"Cytotoxic, anti-proliferative, and apoptotic evaluation of Ramalina sinensis (Lichenized fungus, Ramalinaceae) on oral squamous cell carcinoma cell line; in-vitro study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-28 21:34:32","doi":"10.21203/rs.3.rs-2246187/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-12-26T06:53:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-12-21T14:52:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-12-13T08:07:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f57d3b5a-c25c-41ff-b8a0-5447ad6c2b94","date":"2022-12-01T10:31:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-11-23T13:42:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-11-23T13:12:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-11-22T16:29:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-11-22T16:25:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Complementary Medicine and Therapies","date":"2022-11-07T09:23:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"59d2c8dd-17b4-4390-8145-733eb9514ada","owner":[],"postedDate":"November 28th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2023-08-05T06:59:12+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-28 21:34:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2246187","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2246187","identity":"rs-2246187","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}