Ginsenoside Rh2 sensitizes the anti-cancer effects of sunitinib by inducing cell cycle arrest in renal cell carcinoma

Ginsenoside Rh2 sensitizes the anti-cancer effects of sunitinib by inducing cell cycle arrest in renal cell carcinoma | 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 Article Ginsenoside Rh2 sensitizes the anti-cancer effects of sunitinib by inducing cell cycle arrest in renal cell carcinoma Hyun Ji Hwang, Seong Hwi Hong, Hong Sang Moon, Young Eun Yoon, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1480511/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Sunitinib, a VEGF blockade, is used to treat clear cell renal cell carcinoma (ccRCC). However, the anti-cancer treatment effects of sunitinib do not last long in ccRCC patients. Ginsenoside, a natural medicine extracted from ginseng, has been studied in cancer treatment and shown to have anti-tumor effects and low toxicity. We assessed cell viability and cell cycle analysis in ccRCC cell lines after treatment with ginsenoside and sunitinib. DNA damage was evaluated by measuring 8-OHdG levels and comet assay. ROS levels, reflecting cause of oxidative stress, were also measured. Ginsenoside significantly enhanced the inhibition of cell viability by sunitinib, a result that was also confirmed in the xenograft model. In cell cycle analysis, combination treatment of ginsenoside and sunitinib enhanced G2M arrest in comparison with single-treatment groups. In addition, DNA damage was increased by ginsenoside and sunitinib according to the comet assay, and the level of 8-OHdG, which reflects oxidative DNA damage, also increased. We verified that ginsenoside enhances the efficacy of sunitinib to inhibit the proliferation of ccRCC cells via induction of oxidative DNA damage. The combination therapy of sunitinib and ginsenoside is an effective new treatment strategy for RCC patients. Clear cell renal cell carcinoma DNA damage ginsenoside sunitinib ROS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Renal cell carcinoma (RCC) is the most common type of kidney cancer. Among RCCs, clear cell RCC (ccRCC) accounts for approximately 75% of RCC 1 . In ccRCC, von Hippel-Lindau (VHL) disease results from a major genetic mutation and is characterized by an E3 ubiquitin ligase that induces degradation of hypoxia inducing factor-alpha (HIF alpha) under normoxic conditions 2 . Genetic VHL inactivation of ccRCC causes constitutive HIF alpha accumulation and consequent upregulation of hypoxia-related genes 3 – 8 . Histologically, ccRCC is a hyper-vascular tumor caused by the upregulation of angiogenesis-associated genes such as vascular endothelial growth factor (VEGF), a downstream product of the hypoxia pathway 9 , 10 . VEGF binds the vascular endothelial growth factor receptor (VEGFR) which is the tyrosine kinase receptor (TKR), to induce angiogenesis 11 . Anti-angiogenic drugs are used sequentially to prolong clinical benefit in patients with recurrent disease. Among anti-angiogenic drugs inhibiting VEGF/VEGFR pathways, sunitinib, a tyrosine kinase receptor inhibitor (TKI), is the classic first-line drug for RCC 12 . However, 10–20% of advanced RCC patients are intrinsically refractory to sunitinib, and the remaining patients typically experience tumor progression after 6–15 months of therapy, meaning sunitinib fails to successfully prolong the survival of RCC patients 13 . Because RCC is difficult to completely treat with sunitinib, an alternative therapy is needed to increase the sensitivity of treatment and prevent the development of resistance to sunitinib in RCC. The use of natural products for cancer treatment has been increasing. Effective treatment with lower toxicity compared to other drugs may be achieved by using natural products. Ginsenoside, a natural material derived from ginseng, has been studied in cancer treatment due to its anti-tumor effects and low toxicity 14 . There are more than 100 types of ginsenosides extracted from ginseng, including Rg3, Rh2, Rb1, and Rb2 15 . Rg3 and Rh2 are known to represent anti-cancer effects in various cancers including lung cancer, breast cancer, and prostate cancer by inducing inhibition of proliferation and invasion and DNA damage 16 – 19 . However, not much is known about the effects of Rg3 and Rh2 on ccRCC. In this study, we investigated the effects of Rg3 and Rh2 on RCC and whether co-treatment with ginsenoside and sunitinib work in ccRCC and explored the underlying mechanisms. Results Ginsenoside Rh2 enhanced the anti-cancer effect of sunitinib in ccRCC. First, to evaluate whether ginsenoside Rg3 and Rh2 exhibit anti-cancer effects in renal cancer, we measured cell viability in three ccRCC cell lines (Caki-1, 786-O, and A498). Both ginsenoside Rg3 and Rh2 reduced ccRCC cell growth in a dose-dependent manner (Figs. S1A-B). Next, we measured cell viability to determine the most suitable concentration of sunitinib for measuring synergic effects with ginsenoside in ccRCC (Fig. S1C). We conducted combination treatment experiments using Rg3 10 µM, Rh2 10 µM, and sunitinib 10 µM, concentrations that reduced cell viability by about 50% in all three cell lines (Fig. S1A-C). Combination treatment with ginsenoside and sunitinib effectively reduced cell viability (Fig. 1 A). To assess metastatic ability, we performed invasion assays using matrigel. Compared to single treatment, invasion ability was more effectively inhibited by combination treatment (Figs. 1 B and C). Ginsenoside Rg3 and Rh2 both showed more effective anti-cancer effects when administered with sunitinib compared to single treatment. However, cell viability measured for the combination of ginsenoside and sunitinib indicates that Rh2 (Caki-1 17.8%, p < 0.001/ 786-O 23.7%, p < 0.001/ A498 11.5% p < 0.001) is superior to Rg3 (Caki-1 33.7%, p value < 0.01/ 786-O 37.8%, p < 0.05/ A498 28.3%, p < 0.001) for suppressing growth (Fig. 1 A). In addition, consistent with the cell viability results, the combination of sunitinib and Rh2 (Caki-1 23.7%, p < 0.001 / 786-O 14.6%, p < 0.001 / A498 23.0%, p < 0.001) resulted in better inhibition of metastasis than in combination with Rg3 (Caki-1 31.8%, p < 0.01 / 786-O 37.2%, p < 0.001 / A498 36.3%, p < 0.001) (Fig. 1 C). To evaluate the effectiveness of sunitinib and Rh2 combination therapy, we used the xenograft model to test effects in ccRCC cell lines. The single administrations of sunitinib and Rh2 significantly reduced tumor size and weight, but the effect of the combined administration of sunitinib and Rh2 was more remarkable (Figs. 1 D and E). These results indicate that ginsenosides, especially Rh2, effectively enhance the anti-cancer effects of sunitinib. Cell cycle arrest in ccRCC cell lines is increased by combination treatment with sunitinib and ginsenoside. To evaluate whether the combination of sunitinib and ginsenoside has a synergic effect on inhibition of ccRCC proliferation, we assessed the cell cycle using flow cytometry. Cell cycle arrest triggered by sunitinib/ginsenoside Rg3 or Rh2 was determined using PI staining. In flow cytometry analysis, each ginsenoside induced G1 arrest and sunitinib induced G2M arrest, but co-treatment by Rh2 and sunitinib further enhanced G2M phase arrest (Figs. 2 A and S2A). We conducted western blot analysis to evaluate the protein levels of the cell cycle arrest marker. Phosphorylation of P53 on serine 15 residue, which is a primary response to DNA damage, is important for P53 activation 20 , 21 and expression of P21 22,23 . The western blot data indicated that the expression levels of p-P53 and P21 increased in the single drug group compared to the control group, and increased in the combination treatment group compared to the single drug or control group (Fig. 2 B). These data showed that ginsenoside Rg3 and Rh2 significantly promoted sunitinib-induced cell cycle arrest. Combined administration of ginsenoside Rh2 and sunitinib increase DNA damage, activating ATM and ATR pathways. In order to detect DNA damage, a major cause of cell cycle arrest, we performed a comet assay to measure the length of the DNA tail, including broken DNA fragments or damaged DNA. In the co-treatment group, a remarkably long DNA tail was observed compared with Rh2 or sunitinib treatment (Fig. 3 A). Additionally, we examined the expression of γH2AX, a marker of DNA damage, 24 by immunocytochemistry (ICC). The fluorescence intensity of γH2AX (green) was significantly higher in the co-treatment group than in the single treatment group (Fig. 3 B). Next, to determine whether there was a direct link between DNA damage and cell cycle arrest due to drug treatment, we performed immunohistochemistry (IHC) using tissue from xenograft tumors. The DNA damage response (DDR) signaling pathway organized by the ATM and ATR kinases is the key regulator of the cellular process networks 25 , and phosphorylation of ATM and ATR by DDR signaling activates P53 and P21 to induce cell cycle arrest 26 . In xenograft tissues, the activities of ATM, ATR, and γH2AX, as well as P53 and P21, were significantly increased in the co-treatment group compared to Rh2 or sunitinib treatment groups (Figs. 3 C and D). These results indicate that ginsenoside Rh2 promotes sunitinib-induced DNA damage in ccRCC. Ginsenoside Rh2 increases oxidative DNA damage by increasing ROS generation by sunitinib. Oxidative damage is the main cause of damage to DNA 27 . To examine whether the combination of Rh2 and sunitinib directly induces oxidative DNA damage, we measured the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, in xenograft serum and ccRCC cell lines. Compared to the Rh2 treatment group, the sunitinib and Rh2 combination treatment groups had higher levels of 8-OHdG (Fig. 4 A). The same results were shown in ccRCC cell lines (Figs. 4 B and S3A). For serum, the sunitinib and combination groups showed no significant results but levels were remarkably increased in the cell lines. Oxidative stress that causes DNA damage reflects increases in levels of ROS 28 . ROS levels were measured with a fluorescence microscope, revealing significant increase of ROS level in the combination group (Figs. 4 C and S3B). In addition, N-acetyl-L-cysteines (NAC), a ROS inhibitor, was used to determine whether oxidative stress by Rh2 and sunitinib can be alleviated. We confirmed that cell viability decreased after combination treatment with Rh2 and sunitinib, but recovered through treatment with NAC (Figs. 4 D and S3C). The ROS level in the combination group decreased due to NAC, and as a result cell cycle arrest was also alleviated (Figs. 4 E-F and S3D-E). In addition, the protein levels of cell cycle arrest markers decreased after NAC treatment (Figs. 4 G and S3F). Taken together, our results indicate that a combination of Rh2 and sunitinib, which has been confirmed to induce cell cycle arrest by increasing ROS, is a feasible novel ccRCC treatment method. Discussion In the present study we demonstrated that ginsenoside enhances the sensitivity of sunitinib to inhibit the proliferation of renal cell carcinoma cells via induction of cell cycle arrest. Ginsenoside Rg3 and Rh2 induced G1 phase arrest, while co-treatment with sunitinib further increased G2/M arrest in ccRCC cells. These cell cycle analyses confirmed that the co-treatment of ginsenoside and sunitinib enhances cell cycle arrest. We measured DNA damage among the causes of cell cycle arrest and found that DNA damage by Rh2 and sunitinib increased by detecting changes in comet assay and γH2AX expression. Moreover, we found that the protein expressions of p-ATM and p-ATR, which are activated first when DNA damage occurs, were expressed to a greater degree in co-treatment tissues than in single-drug treatment tissues. Increases of 8-OHdG, an oxidative DNA damage marker, was found in both the serum and cell lines of the group that was treated with both drugs. The ROS-induced oxidative stress increased significantly in the combination group. To determine whether Rh2 and sunitinib reliably induce ROS, NAC was used as a ROS inhibitor. We confirmed that cell viability and cell cycle in samples treated with both drugs recovered after NAC treatment. Therefore, ginsenoside increases the oxidative DNA damage induced by sunitinib, which causes cell cycle arrest to inhibit cell growth (Fig. 5 ). DNA damage can be induced by various external stimuli, including oxidative stress, UV exposure, and chemotherapeutic drugs 29 . In addition, metabolic reactions caused by various stresses result in three types of DNA damage: DNA adducts, oxidative DNA damage, and dNTP pool alterations 30 . ROS produce oxidative DNA damage. The reaction of ROS with DNA mainly comes about due to reactions of OH with pyrimidines, purines, or sugars in DNA, and one of the most frequent oxidative DNA lesions is 8-hydroxy-2-deoxyguanosine (8-OHdG) 31 . Therefore, 8-OHdG is known to be a biomarker for oxidative damage of DNA 32 , 33 . We confirmed the level of 8-OHdG in the serum obtained mice xenograft model and in ccRCC cell lines and observed that the co-treatment group exhibited greater increases in 8-OHdG than the single treatment groups. Interestingly, H2AX is activated by DNA damage phosphorylates ATM and ATR, while inversely activating ATM and ATR phosphorylate H2AX 34 . Since both ATM (involved in double-strand breakage) and ATR (involved in single-strand breakage) 35 are increased by both drugs, ginsenoside and sunitinib comprise two ways to cause DNA breakage. To ensure that damaged DNA is not propagated to the next generation, cell cycle regulation stabilizes conditions necessary for cell growth and homeostasis 36 . Three checkpoints, G1, S, and G2M, phase control DNA replication and cell death in cancer, and are controlled by P53, a key regulator alongside CDK and P21 37,38 . In Fig. 2 , cell cycle arrest was triggered to prevent DNA damage induced by ginsenoside and sunitinib. Down-regulation of CDK and up-regulation of P21 inhibit progression through cell cycle checkpoints, resulting in cell cycle arrest 39 . These changes in the expressions of cell cycle arrest markers (Fig. 2 B) suggest that Rh2 and sunitinib induced cell cycle arrest by oxidative DNA damage in ccRCC. Co-treatment with ginsenoside and sunitinib in ccRCC caused DNA damage and cell cycle arrest, and thereby suppressed the growth of cancer. We propose that ccRCC can be effectively treated by increasing the sensitivity of sunitinib, an existing treatment for ccRCC, through co-treatment with ginsenoside, a natural product. Our findings suggests that combination therapy using sunitinib and ginsenoside Rh2 is an effective and novel treatment strategy for ccRCC patients. Methods Cell culture Caki-1 and 786-O cells (ATCC, Manassas, VA, USA) were cultivated in RPMI medium including L-glutamine (Sigma-Aldrich, #R8758, St. Louis, MS, USA) and A498 (ATCC) was cultivated in DMEM medium containing 4.5g/L glucose (Sigma-Aldrich, #D6429). Media were added to 10% fetal bovine serum (FBS) (Sigma-Aldrich, #TMS-013-BKR) and 1% antibiotic-antimycotic (GIBCO, #15240062, Waltham, MA, USA). Cells were maintained at 37℃ under 5% CO 2 . Drugs Ginsenoside Rg3 (Sigma-Aldrich #SML0184), Rh2 (Sigma-Aldrich #73658), and sunitinib malate (Sigma-Aldrich #PZ0012) were melted in DMSO (Sigma-Aldrich) at a concentration of 10 mM. N-acetyl-L-cysteine (NAC) was used as a ROS inhibitor (Sigma-Aldrich, #A9165-5G) by melting in DMSO to a concentration of 5 M and heating. Cell proliferation assay The viability of Caki-1, 786-O, and A498 cells was assessed using the EZ-CYTOX (DoGenBio, #EZ-1000, Seoul, Korea). All three cell lines were seeded in 96-well plates (1x10 4 cells/well). After incubation overnight, the cells were treated with different concentrations of ginsenoside Rg3, Rh2, and sunitinib for 24 h. Then EZ-Cytox solution was supplemented to each well. Absorbance at a wavelength of 450 nm was detected by a Microplate reader. The cell viability rates were calculated, and graphs were generated. Matrigel invasion assay Caki-1, 786-O, and A498 cells were plated in a serum-free medium for 24 h. A total of 3x10 5 cells containing fresh media were seeded into an 8 µM transparent PET membrane (FALCON, #353097, Corning, NY, USA) and placed in 24 well plates containing 20% FBS fresh media. Cells were allowed to invade for 24 h. Samples were fixed with 3.7% formaldehyde and then subjected to a permeabilization process with 100% methanol. Samples were dyed with 0.4% crystal violet assessed through a microscope. Cell cycle assay First, 5x10 5 cells were fixed in 80% ethanol for 1 h at -20℃. Then, the cells were washed twice with phosphate-buffed saline (PBS), and 500 µl PI/RNase staining buffer (BD Pharmingen, #550825, San Diego, California, USA) was added. The samples were incubated at RT for 20 min in a dark environment and analyzed by flow cytometry. Xenograft model and treatments Female BALB/c nude mice (4 weeks old) were purchased from Orient Bio (South Korea) and were housed under specific pathogen-free conditions. All animal experimental protocols were approved by the Hanyang University Institutional Animal Care and Use Committee (2020-0104A). All procedures related with the in vivo experiments and animal care were carried out in accordance with the approved guidelines. The study is compliant with the ARRIVE guideline 2.0. To establish the xenograft model, 1x10 7 A498 cells were injected subcutaneously into the side regions of nude mice. The mice were randomized into four groups (n = 5) and administered drugs when the tumor volume reached approximately 300 mm 3 . Sunitinib (10 mg/kg) was administered orally daily, and ginsenoside Rh2 (10 mg/kg) was applied by intraperitoneal injection three times a week. For the combination treatment, the two drugs were administered together. Body weights and tumor volumes were measured three times per week using calipers. After 4 weeks of treatment, the mice were sacrificed and tumor tissues were harvested and fixed in formalin for IHC staining. Immunohistochemistry (IHC) Paraffin-embedded tumor tissue specimens were sliced into 3-µm-thick sections and mounted onto slides. Then, the slides were subjected to de-paraffinization, rehydration, and antigen retrieval, and incubated with specific primary antibodies overnight at 4℃. Subsequently, the sections were incubated with secondary antibodies after washing with PBS. After staining with diaminobenzidine (DAB), the tumor sections were visualized under a microscope. In this study, the tumor sections were stained with phospho-ATM (Ser 1981) (Abcam, #ab81292, Cambridge, UK), phospho-ATR (Ser 428) (Abcam, #ab178407), P53 (Abcam #ab1101), phospho-P53 (Ser 15) (Cell signaling technology, #9284S), P21 (Abcam, #ab109520), and CDK2 (Abcam, #ab32420) to assess the expressions of proteins in ccRCC cell lines. Quantification was scored by the product of intensity and percentage of staining. Western blot analysis Caki-1, 786-O, and A498 cells were treated with sunitinib (10 µM) and ginsenoside Rh2 (10 µM) for 24h. In brief, cells were lysed in lysis buffer and protein was extracted by centrifuge. The protein was separated by SDS-PAGE and transferred from the polyacrylamide gel to the PVDF membrane. The membranes were blocked with 5% skim milk in TBS-T buffer, further incubated with specific primary antibodies (phospho-P53 (Ser 15) (Cell Signaling Technology, #9284S, Danvers, MA, USA), P53 (Abcam #ab1101), P21 (Abcam, #ab109520), and CDK2 (Abcam, #ab32420) at 4℃ overnight and followed by incubation with secondary antibodies at RT for 1 h. Protein bands were detected by a film using developer and fixer. Immunocytochemistry (ICC) Cells were fixed with 4% paraformaldehyde (Biosesang, #PC2031-100, Gyeonggido, Korea) and permeabilized using 0.1% Triton X-100 in PBS. After sufficient washing with PBS, cells were blocked for 30 min under RT using 10% normal goat serum. Cells were incubated with anti-γH2AX (phospho serine 139) antibody (Abcam, #ab81299), followed by secondary antibodies labeled with AlexaFluor488 (anti-rabbit IgG) (Invitrogen, #A32731, Waltham, MA, USA). DNA was stained with Hoechst 33342 (Thermo scientific, #62249, Waltham, MA, USA). Images were photographed utilizing a confocal microscope. Comet assay Alkaline comet assay was conducted using a comet assay kit (Biotechne, #4250-050-K, Minneapolis, MN, USA) following the manufacturer’s instructions. DNA was stained with Hoechst 33342 (Thermo Scientific, #62249) and fluorescence images were captured using a confocal microscope. Intracellular ROS production ccRCC cell lines were incubated in DMEM and RPMI in 24-well plates containing 10% FBS. ROS level was measured by fluorometric intracellular ROS kit (Sigma-Aldrich, #MAK145). 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Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1..jpg SupplementaryFigure2..jpg SupplementaryFigure3..jpg Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 07 Jun, 2022 Reviews received at journal 30 May, 2022 Reviewers agreed at journal 19 May, 2022 Reviews received at journal 19 May, 2022 Reviewers agreed at journal 18 May, 2022 Reviewers agreed at journal 25 Apr, 2022 Reviewers invited by journal 23 Apr, 2022 Editor assigned by journal 20 Apr, 2022 Editor invited by journal 30 Mar, 2022 Submission checks completed at journal 30 Mar, 2022 First submitted to journal 23 Mar, 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. 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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-1480511","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":94753644,"identity":"64b28827-4ba6-486a-bc1e-5521dbdd6f3a","order_by":0,"name":"Hyun Ji Hwang","email":"","orcid":"","institution":"Hanyang University Graduate School of Biomedical Science \u0026 Engineering","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Ji","lastName":"Hwang","suffix":""},{"id":94753645,"identity":"ed4504fb-2bec-4b97-b17e-e4c0e3ace2d0","order_by":1,"name":"Seong Hwi Hong","email":"","orcid":"","institution":"Hanyang University College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seong","middleName":"Hwi","lastName":"Hong","suffix":""},{"id":94753646,"identity":"b2391351-9135-436c-abd5-6cae4e514d10","order_by":2,"name":"Hong Sang Moon","email":"","orcid":"","institution":"Hanyang University College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hong","middleName":"Sang","lastName":"Moon","suffix":""},{"id":94753647,"identity":"87351444-6145-47cb-acba-572273d32f60","order_by":3,"name":"Young Eun Yoon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYHACxgMMDBJQdgUQMzM3ENQD1sIDZp4BaWEkSgsDRAtjG5jEr8Xg9uEDBz78srC3l0g++LhwXm00fztQy4+Kbbi1nEtLODizTyKxRyIt2XjmtuO5Mw4zNjD2nLmNW8sZHoPDvD0SCTwSOWbSvNuO5TYAtTAzthHWYg/RMudY7nyitPD8kGDsAWtpqMndQEiL5Bk2oF8agH458yzZmOfYgdyNQC0H8fmF7wzzwQcf/tTZs7cDQ4ynpi533vnDBx/8qMCtBQzA0SGQAGIeBgscwK8eBP4AMT9YXR1hxaNgFIyCUTDiAABUZ1wV68BDigAAAABJRU5ErkJggg==","orcid":"","institution":"Hanyang University College of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Young","middleName":"Eun","lastName":"Yoon","suffix":""},{"id":94753648,"identity":"ed7b193a-8f98-4312-9c5b-33c3c7757d41","order_by":4,"name":"Sung Yul Park","email":"","orcid":"","institution":"Hanyang University College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sung","middleName":"Yul","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2022-03-23 07:44:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1480511/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1480511/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19875061,"identity":"3ac3025c-100a-43cc-a8ba-6d48f99724a2","added_by":"auto","created_at":"2022-04-01 19:57:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":217303,"visible":true,"origin":"","legend":"\u003cp\u003eGinsenoside enhances the anti-proliferative effects of sunitinib in ccRCC cell lines and the xenograft model.\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(A) Cell viability was measured after 24 hours of drug co-treatment with ginsenoside and sunitinib. Caki-1, 786-O, and A498 cells were treated with Rh2 (10 μM), Rg3 (10 μM), and sunitinib (10 μM) (**p\u0026lt;0.01, ***p\u0026lt;0.001). \u003c/p\u003e\u003cp\u003e(B) The Matrigel invasion assay showed that anti-invasiveness significantly decreased in ccRCC cell lines after co-treatment with ginsenoside and sunitinib. The representative images were obtained after 24 hours of drug treatment. \u003c/p\u003e\u003cp\u003e(C) Numbers of invading cells in each group (**p\u0026lt;0.01, ***p\u0026lt;0.001).\u003c/p\u003e\u003cp\u003e(D-E) A498 cells were injected subcutaneously into balb/c nude mice (n=5). We evaluated tumor volumes and sizes in four treatment groups: DMSO, Rh2 (10 mg/kg), sunitinib (10 mg/kg), and Rh2 + sunitinib. Mice received intraperitoneal injections of ginsenoside three times per week and sunitinib was given by oral administration every day. All groups were evaluated three times per week. The tumor weights were evaluated after 4 weeks of treatment (*p\u0026lt;0.05, **p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"Figure1..jpg","url":"https://assets-eu.researchsquare.com/files/rs-1480511/v1/d5db115cacbb59c776a93340.jpg"},{"id":19875284,"identity":"8b83dbcb-7cad-4b69-a3e1-2586a324dba6","added_by":"auto","created_at":"2022-04-01 20:02:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":163910,"visible":true,"origin":"","legend":"\u003cp\u003eGinsenoside Rh2 induces cell cycle arrest by increasing P53 phosphorylation of sunitinib in ccRCC cell lines.\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\u003cp\u003e(A) Cells were treated with ginsenoside Rh2 (10 μM) and sunitinib (10 μM) and analyzed using propidium iodide staining detected by flow cytometry. Quantification of the cell cycle was performed. \u003c/p\u003e\u003cp\u003e(B) Protein levels of p-P53 (p-S15), P53, and P21 were detected by western blot. β-actin was used as a loading control.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2..jpg","url":"https://assets-eu.researchsquare.com/files/rs-1480511/v1/f00335fe4cdebb3cfb8ce5ef.jpg"},{"id":19875064,"identity":"0b606c1d-2719-4309-bf82-6ffba99947b7","added_by":"auto","created_at":"2022-04-01 19:57:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":327562,"visible":true,"origin":"","legend":"\u003cp\u003eThe combination treatment of ginsenoside and sunitinib increases DNA damage response by activating ATM and ATR in ccRCC.\u003c/p\u003e\u003cp\u003e(A) Comet assay induced by ginsenoside Rh2 and/or sunitinib. The tails indicate DNA damage. Representative images of cell nuclei stained with Hoechst blue after electrophoresis at alkaline pH. \u003c/p\u003e\u003cp\u003e(B) Immunocytochemistry (ICC) of γH2AX (green) showed DNA damage after treatment of Rh2 and sunitinib. Counterstaining with DAPI (blue) was conducted to visualize the nuclei.\u003c/p\u003e\u003cp\u003e(C-D) Immunohistochemistry staining for p-ATR, p-ATM, γH2AX, P53, and P21 proteins in A498 xenograft treated with Rh2/ sunitinib. We quantified the protein levels identified in five mice tissues per group (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure3..jpg","url":"https://assets-eu.researchsquare.com/files/rs-1480511/v1/ac34782b3edd96ec1685d250.jpg"},{"id":19875063,"identity":"a10fef08-be6c-46a7-981b-1d96f9b0a1fe","added_by":"auto","created_at":"2022-04-01 19:57:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":192432,"visible":true,"origin":"","legend":"\u003cp\u003eROS generated by ginsenoside and sunitinib increases levels of 8-OHdG, an oxidative DNA damage marker, in 786-O cells.\u003c/p\u003e\u003cp\u003e(A) Levels of 8-OHdG in serum samples of xenograft mice were measured by competitive ELISA (***p\u0026lt;0.001).\u003c/p\u003e\u003cp\u003e(B) 8-OHdG levels of 786-O cells treated with Rh2 and sunitinib (**p\u0026lt;0.01).\u003c/p\u003e\u003cp\u003e(C) Fluorescent images of ROS (red) were evaluated in live cells treated with Rh2 and sunitinib. Hoechst (blue) was used as a counter stain. \u003c/p\u003e\u003cp\u003e(D) Changes in cell viability when NAC, a ROS inhibitor, is administered with Rh2 and sunitinib. NAC (5 mM), Rh2 (10 μM), and sunitinib (10 μM) (***p\u0026lt;0.001).\u003c/p\u003e\u003cp\u003e(E) ROS induced by sunitinib and Rh2 was alleviated by treatment with NAC. Measurements were taken 24 hours after administration of the drug with a fluorescence microscope. \u003c/p\u003e\u003cp\u003e(F) Cell cycle analysis by flow cytometry of 786-O cells treated with Rh2, sunitinib, and NAC. Histogram data from the cell analysis are quantified and graphed. \u003c/p\u003e\u003cp\u003e(G) Western blot analyses to evaluate the expressions of the indicated proteins in 786-O cells that were treated with Rh2, sunitinib, and NAC. We confirmed that the protein expressions of cell cycle arrest markers were reduced by NAC.\u003c/p\u003e","description":"","filename":"Figure4..jpg","url":"https://assets-eu.researchsquare.com/files/rs-1480511/v1/1a719bc2fbe41823c2aef80c.jpg"},{"id":19875066,"identity":"d2903376-6799-43f7-b110-daf2b2c951d8","added_by":"auto","created_at":"2022-04-01 19:57:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":68135,"visible":true,"origin":"","legend":"\u003cp\u003eGinsenoside increases DNA damage due to sunitinib to induce cell death by cell cycle arrest.\u003c/p\u003e","description":"","filename":"Figure5..jpg","url":"https://assets-eu.researchsquare.com/files/rs-1480511/v1/c6265dd9e0ff6f20e3226abf.jpg"},{"id":19875286,"identity":"50259208-591e-402e-98ca-aa09a0a1a46d","added_by":"auto","created_at":"2022-04-01 20:02:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":805446,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1480511/v1/1d6441b7-e112-4081-86e0-abbb4dbe4600.pdf"},{"id":19875060,"identity":"1b102901-1590-4f2f-b186-e5e2ef165cbc","added_by":"auto","created_at":"2022-04-01 19:57:45","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":119869,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1..jpg","url":"https://assets-eu.researchsquare.com/files/rs-1480511/v1/f16c3a4f18013ac67231a8bd.jpg"},{"id":19875285,"identity":"354143dc-025e-42a8-907e-3fdb1ee91f26","added_by":"auto","created_at":"2022-04-01 20:02:45","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":179094,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2..jpg","url":"https://assets-eu.researchsquare.com/files/rs-1480511/v1/ba8455094383006002398e7b.jpg"},{"id":19875067,"identity":"e6d291c0-17e2-4a41-9b3a-8810ba6458da","added_by":"auto","created_at":"2022-04-01 19:57:45","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":280234,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3..jpg","url":"https://assets-eu.researchsquare.com/files/rs-1480511/v1/2690a7b1675efd3f992e0ca8.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ginsenoside Rh2 sensitizes the anti-cancer effects of sunitinib by inducing cell cycle arrest in renal cell carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRenal cell carcinoma (RCC) is the most common type of kidney cancer. Among RCCs, clear cell RCC (ccRCC) accounts for approximately 75% of RCC \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In ccRCC, von Hippel-Lindau (VHL) disease results from a major genetic mutation and is characterized by an E3 ubiquitin ligase that induces degradation of hypoxia inducing factor-alpha (HIF alpha) under normoxic conditions \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Genetic VHL inactivation of ccRCC causes constitutive HIF alpha accumulation and consequent upregulation of hypoxia-related genes \u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Histologically, ccRCC is a hyper-vascular tumor caused by the upregulation of angiogenesis-associated genes such as vascular endothelial growth factor (VEGF), a downstream product of the hypoxia pathway\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. VEGF binds the vascular endothelial growth factor receptor (VEGFR) which is the tyrosine kinase receptor (TKR), to induce angiogenesis \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnti-angiogenic drugs are used sequentially to prolong clinical benefit in patients with recurrent disease. Among anti-angiogenic drugs inhibiting VEGF/VEGFR pathways, sunitinib, a tyrosine kinase receptor inhibitor (TKI), is the classic first-line drug for RCC \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, 10\u0026ndash;20% of advanced RCC patients are intrinsically refractory to sunitinib, and the remaining patients typically experience tumor progression after 6\u0026ndash;15 months of therapy, meaning sunitinib fails to successfully prolong the survival of RCC patients \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Because RCC is difficult to completely treat with sunitinib, an alternative therapy is needed to increase the sensitivity of treatment and prevent the development of resistance to sunitinib in RCC.\u003c/p\u003e \u003cp\u003eThe use of natural products for cancer treatment has been increasing. Effective treatment with lower toxicity compared to other drugs may be achieved by using natural products. Ginsenoside, a natural material derived from ginseng, has been studied in cancer treatment due to its anti-tumor effects and low toxicity \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. There are more than 100 types of ginsenosides extracted from ginseng, including Rg3, Rh2, Rb1, and Rb2 \u003csup\u003e15\u003c/sup\u003e. Rg3 and Rh2 are known to represent anti-cancer effects in various cancers including lung cancer, breast cancer, and prostate cancer by inducing inhibition of proliferation and invasion and DNA damage \u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, not much is known about the effects of Rg3 and Rh2 on ccRCC. In this study, we investigated the effects of Rg3 and Rh2 on RCC and whether co-treatment with ginsenoside and sunitinib work in ccRCC and explored the underlying mechanisms.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGinsenoside Rh2 enhanced the anti-cancer effect of sunitinib in ccRCC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, to evaluate whether ginsenoside Rg3 and Rh2 exhibit anti-cancer effects in renal cancer, we measured cell viability in three ccRCC cell lines (Caki-1, 786-O, and A498). Both ginsenoside Rg3 and Rh2 reduced ccRCC cell growth in a dose-dependent manner (Figs. S1A-B). Next, we measured cell viability to determine the most suitable concentration of sunitinib for measuring synergic effects with ginsenoside in ccRCC (Fig. S1C). We conducted combination treatment experiments using Rg3 10 \u0026micro;M, Rh2 10 \u0026micro;M, and sunitinib 10 \u0026micro;M, concentrations that reduced cell viability by about 50% in all three cell lines (Fig. S1A-C). Combination treatment with ginsenoside and sunitinib effectively reduced cell viability (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). To assess metastatic ability, we performed invasion assays using matrigel. Compared to single treatment, invasion ability was more effectively inhibited by combination treatment (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB and C). Ginsenoside Rg3 and Rh2 both showed more effective anti-cancer effects when administered with sunitinib compared to single treatment. However, cell viability measured for the combination of ginsenoside and sunitinib indicates that Rh2 (Caki-1 17.8%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001/ 786-O 23.7%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001/ A498 11.5% p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) is superior to Rg3 (Caki-1 33.7%, p value\u0026thinsp;\u0026lt;\u0026thinsp;0.01/ 786-O 37.8%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05/ A498 28.3%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) for suppressing growth (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). In addition, consistent with the cell viability results, the combination of sunitinib and Rh2 (Caki-1 23.7%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 / 786-O 14.6%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 / A498 23.0%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) resulted in better inhibition of metastasis than in combination with Rg3 (Caki-1 31.8%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 / 786-O 37.2%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 / A498 36.3%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). To evaluate the effectiveness of sunitinib and Rh2 combination therapy, we used the xenograft model to test effects in ccRCC cell lines. The single administrations of sunitinib and Rh2 significantly reduced tumor size and weight, but the effect of the combined administration of sunitinib and Rh2 was more remarkable (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD and E). These results indicate that ginsenosides, especially Rh2, effectively enhance the anti-cancer effects of sunitinib.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle arrest in ccRCC cell lines is increased by combination treatment with sunitinib and ginsenoside.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate whether the combination of sunitinib and ginsenoside has a synergic effect on inhibition of ccRCC proliferation, we assessed the cell cycle using flow cytometry. Cell cycle arrest triggered by sunitinib/ginsenoside Rg3 or Rh2 was determined using PI staining. In flow cytometry analysis, each ginsenoside induced G1 arrest and sunitinib induced G2M arrest, but co-treatment by Rh2 and sunitinib further enhanced G2M phase arrest (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA and S2A). We conducted western blot analysis to evaluate the protein levels of the cell cycle arrest marker. Phosphorylation of P53 on serine 15 residue, which is a primary response to DNA damage, is important for P53 activation \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and expression of P21 \u003csup\u003e22,23\u003c/sup\u003e. The western blot data indicated that the expression levels of p-P53 and P21 increased in the single drug group compared to the control group, and increased in the combination treatment group compared to the single drug or control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). These data showed that ginsenoside Rg3 and Rh2 significantly promoted sunitinib-induced cell cycle arrest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCombined administration of ginsenoside Rh2 and sunitinib increase DNA damage, activating ATM and ATR pathways.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to detect DNA damage, a major cause of cell cycle arrest, we performed a comet assay to measure the length of the DNA tail, including broken DNA fragments or damaged DNA. In the co-treatment group, a remarkably long DNA tail was observed compared with Rh2 or sunitinib treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Additionally, we examined the expression of \u0026gamma;H2AX, a marker of DNA damage, \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e by immunocytochemistry (ICC). The fluorescence intensity of \u0026gamma;H2AX (green) was significantly higher in the co-treatment group than in the single treatment group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Next, to determine whether there was a direct link between DNA damage and cell cycle arrest due to drug treatment, we performed immunohistochemistry (IHC) using tissue from xenograft tumors. The DNA damage response (DDR) signaling pathway organized by the ATM and ATR kinases is the key regulator of the cellular process networks \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, and phosphorylation of ATM and ATR by DDR signaling activates P53 and P21 to induce cell cycle arrest \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In xenograft tissues, the activities of ATM, ATR, and \u0026gamma;H2AX, as well as P53 and P21, were significantly increased in the co-treatment group compared to Rh2 or sunitinib treatment groups (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). These results indicate that ginsenoside Rh2 promotes sunitinib-induced DNA damage in ccRCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGinsenoside Rh2 increases oxidative DNA damage by increasing ROS generation by sunitinib.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOxidative damage is the main cause of damage to DNA \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. To examine whether the combination of Rh2 and sunitinib directly induces oxidative DNA damage, we measured the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, in xenograft serum and ccRCC cell lines. Compared to the Rh2 treatment group, the sunitinib and Rh2 combination treatment groups had higher levels of 8-OHdG (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). The same results were shown in ccRCC cell lines (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and S3A). For serum, the sunitinib and combination groups showed no significant results but levels were remarkably increased in the cell lines. Oxidative stress that causes DNA damage reflects increases in levels of ROS \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. ROS levels were measured with a fluorescence microscope, revealing significant increase of ROS level in the combination group (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC and S3B). In addition, N-acetyl-L-cysteines (NAC), a ROS inhibitor, was used to determine whether oxidative stress by Rh2 and sunitinib can be alleviated. We confirmed that cell viability decreased after combination treatment with Rh2 and sunitinib, but recovered through treatment with NAC (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD and S3C). The ROS level in the combination group decreased due to NAC, and as a result cell cycle arrest was also alleviated (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE-F and S3D-E). In addition, the protein levels of cell cycle arrest markers decreased after NAC treatment (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG and S3F). Taken together, our results indicate that a combination of Rh2 and sunitinib, which has been confirmed to induce cell cycle arrest by increasing ROS, is a feasible novel ccRCC treatment method.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study we demonstrated that ginsenoside enhances the sensitivity of sunitinib to inhibit the proliferation of renal cell carcinoma cells via induction of cell cycle arrest. Ginsenoside Rg3 and Rh2 induced G1 phase arrest, while co-treatment with sunitinib further increased G2/M arrest in ccRCC cells. These cell cycle analyses confirmed that the co-treatment of ginsenoside and sunitinib enhances cell cycle arrest. We measured DNA damage among the causes of cell cycle arrest and found that DNA damage by Rh2 and sunitinib increased by detecting changes in comet assay and γH2AX expression. Moreover, we found that the protein expressions of p-ATM and p-ATR, which are activated first when DNA damage occurs, were expressed to a greater degree in co-treatment tissues than in single-drug treatment tissues. Increases of 8-OHdG, an oxidative DNA damage marker, was found in both the serum and cell lines of the group that was treated with both drugs. The ROS-induced oxidative stress increased significantly in the combination group. To determine whether Rh2 and sunitinib reliably induce ROS, NAC was used as a ROS inhibitor. We confirmed that cell viability and cell cycle in samples treated with both drugs recovered after NAC treatment. Therefore, ginsenoside increases the oxidative DNA damage induced by sunitinib, which causes cell cycle arrest to inhibit cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDNA damage can be induced by various external stimuli, including oxidative stress, UV exposure, and chemotherapeutic drugs \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In addition, metabolic reactions caused by various stresses result in three types of DNA damage: DNA adducts, oxidative DNA damage, and dNTP pool alterations \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. ROS produce oxidative DNA damage. The reaction of ROS with DNA mainly comes about due to reactions of OH with pyrimidines, purines, or sugars in DNA, and one of the most frequent oxidative DNA lesions is 8-hydroxy-2-deoxyguanosine (8-OHdG) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Therefore, 8-OHdG is known to be a biomarker for oxidative damage of DNA \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. We confirmed the level of 8-OHdG in the serum obtained mice xenograft model and in ccRCC cell lines and observed that the co-treatment group exhibited greater increases in 8-OHdG than the single treatment groups. Interestingly, H2AX is activated by DNA damage phosphorylates ATM and ATR, while inversely activating ATM and ATR phosphorylate H2AX \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Since both ATM (involved in double-strand breakage) and ATR (involved in single-strand breakage) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e are increased by both drugs, ginsenoside and sunitinib comprise two ways to cause DNA breakage.\u003c/p\u003e \u003cp\u003eTo ensure that damaged DNA is not propagated to the next generation, cell cycle regulation stabilizes conditions necessary for cell growth and homeostasis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Three checkpoints, G1, S, and G2M, phase control DNA replication and cell death in cancer, and are controlled by P53, a key regulator alongside CDK and P21 \u003csup\u003e37,38\u003c/sup\u003e. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, cell cycle arrest was triggered to prevent DNA damage induced by ginsenoside and sunitinib. Down-regulation of CDK and up-regulation of P21 inhibit progression through cell cycle checkpoints, resulting in cell cycle arrest \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These changes in the expressions of cell cycle arrest markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) suggest that Rh2 and sunitinib induced cell cycle arrest by oxidative DNA damage in ccRCC.\u003c/p\u003e \u003cp\u003eCo-treatment with ginsenoside and sunitinib in ccRCC caused DNA damage and cell cycle arrest, and thereby suppressed the growth of cancer. We propose that ccRCC can be effectively treated by increasing the sensitivity of sunitinib, an existing treatment for ccRCC, through co-treatment with ginsenoside, a natural product. Our findings suggests that combination therapy using sunitinib and ginsenoside Rh2 is an effective and novel treatment strategy for ccRCC patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eCell culture\u003c/b\u003e Caki-1 and 786-O cells (ATCC, Manassas, VA, USA) were cultivated in RPMI medium including L-glutamine (Sigma-Aldrich, #R8758, St. Louis, MS, USA) and A498 (ATCC) was cultivated in DMEM medium containing 4.5g/L glucose (Sigma-Aldrich, #D6429). Media were added to 10% fetal bovine serum (FBS) (Sigma-Aldrich, #TMS-013-BKR) and 1% antibiotic-antimycotic (GIBCO, #15240062, Waltham, MA, USA). Cells were maintained at 37℃ under 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDrugs\u003c/b\u003e Ginsenoside Rg3 (Sigma-Aldrich #SML0184), Rh2 (Sigma-Aldrich #73658), and sunitinib malate (Sigma-Aldrich #PZ0012) were melted in DMSO (Sigma-Aldrich) at a concentration of 10 mM. N-acetyl-L-cysteine (NAC) was used as a ROS inhibitor (Sigma-Aldrich, #A9165-5G) by melting in DMSO to a concentration of 5 M and heating.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell proliferation assay\u003c/b\u003e The viability of Caki-1, 786-O, and A498 cells was assessed using the EZ-CYTOX (DoGenBio, #EZ-1000, Seoul, Korea). All three cell lines were seeded in 96-well plates (1x10\u003csup\u003e4\u003c/sup\u003e cells/well). After incubation overnight, the cells were treated with different concentrations of ginsenoside Rg3, Rh2, and sunitinib for 24 h. Then EZ-Cytox solution was supplemented to each well. Absorbance at a wavelength of 450 nm was detected by a Microplate reader. The cell viability rates were calculated, and graphs were generated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMatrigel invasion assay\u003c/b\u003e Caki-1, 786-O, and A498 cells were plated in a serum-free medium for 24 h. A total of 3x10\u003csup\u003e5\u003c/sup\u003e cells containing fresh media were seeded into an 8 \u0026micro;M transparent PET membrane (FALCON, #353097, Corning, NY, USA) and placed in 24 well plates containing 20% FBS fresh media. Cells were allowed to invade for 24 h. Samples were fixed with 3.7% formaldehyde and then subjected to a permeabilization process with 100% methanol. Samples were dyed with 0.4% crystal violet assessed through a microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell cycle assay\u003c/b\u003e First, 5x10\u003csup\u003e5\u003c/sup\u003e cells were fixed in 80% ethanol for 1 h at -20℃. Then, the cells were washed twice with phosphate-buffed saline (PBS), and 500 \u0026micro;l PI/RNase staining buffer (BD Pharmingen, #550825, San Diego, California, USA) was added. The samples were incubated at RT for 20 min in a dark environment and analyzed by flow cytometry.\u003c/p\u003e \u003cp\u003e\u003cb\u003eXenograft model and treatments\u003c/b\u003e Female BALB/c nude mice (4 weeks old) were purchased from Orient Bio (South Korea) and were housed under specific pathogen-free conditions. All animal experimental protocols were approved by the Hanyang University Institutional Animal Care and Use Committee (2020-0104A). All procedures related with the in vivo experiments and animal care were carried out in accordance with the approved guidelines. The study is compliant with the ARRIVE guideline 2.0.\u003c/p\u003e \u003cp\u003eTo establish the xenograft model, 1x10\u003csup\u003e7\u003c/sup\u003e A498 cells were injected subcutaneously into the side regions of nude mice. The mice were randomized into four groups (n\u0026thinsp;=\u0026thinsp;5) and administered drugs when the tumor volume reached approximately 300 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Sunitinib (10 mg/kg) was administered orally daily, and ginsenoside Rh2 (10 mg/kg) was applied by intraperitoneal injection three times a week. For the combination treatment, the two drugs were administered together. Body weights and tumor volumes were measured three times per week using calipers. After 4 weeks of treatment, the mice were sacrificed and tumor tissues were harvested and fixed in formalin for IHC staining.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunohistochemistry (IHC)\u003c/b\u003e Paraffin-embedded tumor tissue specimens were sliced into 3-\u0026micro;m-thick sections and mounted onto slides. Then, the slides were subjected to de-paraffinization, rehydration, and antigen retrieval, and incubated with specific primary antibodies overnight at 4℃. Subsequently, the sections were incubated with secondary antibodies after washing with PBS. After staining with diaminobenzidine (DAB), the tumor sections were visualized under a microscope. In this study, the tumor sections were stained with phospho-ATM (Ser 1981) (Abcam, #ab81292, Cambridge, UK), phospho-ATR (Ser 428) (Abcam, #ab178407), P53 (Abcam #ab1101), phospho-P53 (Ser 15) (Cell signaling technology, #9284S), P21 (Abcam, #ab109520), and CDK2 (Abcam, #ab32420) to assess the expressions of proteins in ccRCC cell lines. Quantification was scored by the product of intensity and percentage of staining.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blot analysis\u003c/b\u003e Caki-1, 786-O, and A498 cells were treated with sunitinib (10 \u0026micro;M) and ginsenoside Rh2 (10 \u0026micro;M) for 24h. In brief, cells were lysed in lysis buffer and protein was extracted by centrifuge. The protein was separated by SDS-PAGE and transferred from the polyacrylamide gel to the PVDF membrane. The membranes were blocked with 5% skim milk in TBS-T buffer, further incubated with specific primary antibodies (phospho-P53 (Ser 15) (Cell Signaling Technology, #9284S, Danvers, MA, USA), P53 (Abcam #ab1101), P21 (Abcam, #ab109520), and CDK2 (Abcam, #ab32420) at 4℃ overnight and followed by incubation with secondary antibodies at RT for 1 h. Protein bands were detected by a film using developer and fixer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunocytochemistry (ICC)\u003c/b\u003e Cells were fixed with 4% paraformaldehyde (Biosesang, #PC2031-100, Gyeonggido, Korea) and permeabilized using 0.1% Triton X-100 in PBS. After sufficient washing with PBS, cells were blocked for 30 min under RT using 10% normal goat serum. Cells were incubated with anti-γH2AX (phospho serine 139) antibody (Abcam, #ab81299), followed by secondary antibodies labeled with AlexaFluor488 (anti-rabbit IgG) (Invitrogen, #A32731, Waltham, MA, USA). DNA was stained with Hoechst 33342 (Thermo scientific, #62249, Waltham, MA, USA). Images were photographed utilizing a confocal microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eComet assay\u003c/b\u003e Alkaline comet assay was conducted using a comet assay kit (Biotechne, #4250-050-K, Minneapolis, MN, USA) following the manufacturer\u0026rsquo;s instructions. DNA was stained with Hoechst 33342 (Thermo Scientific, #62249) and fluorescence images were captured using a confocal microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIntracellular ROS production\u003c/b\u003e ccRCC cell lines were incubated in DMEM and RPMI in 24-well plates containing 10% FBS. ROS level was measured by fluorometric intracellular ROS kit (Sigma-Aldrich, #MAK145). After treating with the mix solution, samples were incubated for about 1 hour and then treated with Rh2 (10 \u0026micro;M) and sunitinib (10 \u0026micro;M) for 24 hours and measured with a fluorescence microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e Statistical analysis was performed using GraphPad Prism 8. All experiments were repeated at least three times. Statistical significance was determined by t-test and one-way analysis of variance (ANOVA). A p-value of less than 0.05 was considered significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant number 2018R1D1A1B07050776).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that supports the findings of this study are available within the article [and its\u003c/p\u003e\n\u003cp\u003esupplementary material].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChoueiri, T. K. \u0026amp; Motzer, R. J. Systemic Therapy for Metastatic Renal-Cell Carcinoma. 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Cell Death Differ \u003cb\u003e25\u003c/b\u003e, 114\u0026ndash;132, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/cdd.2017.172\u003c/span\u003e\u003cspan address=\"10.1038/cdd.2017.172\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Clear cell renal cell carcinoma, DNA damage, ginsenoside, sunitinib, ROS","lastPublishedDoi":"10.21203/rs.3.rs-1480511/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1480511/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSunitinib, a VEGF blockade, is used to treat clear cell renal cell carcinoma (ccRCC). However, the anti-cancer treatment effects of sunitinib do not last long in ccRCC patients. Ginsenoside, a natural medicine extracted from ginseng, has been studied in cancer treatment and shown to have anti-tumor effects and low toxicity. We assessed cell viability and cell cycle analysis in ccRCC cell lines after treatment with ginsenoside and sunitinib. DNA damage was evaluated by measuring 8-OHdG levels and comet assay. ROS levels, reflecting cause of oxidative stress, were also measured. Ginsenoside significantly enhanced the inhibition of cell viability by sunitinib, a result that was also confirmed in the xenograft model. In cell cycle analysis, combination treatment of ginsenoside and sunitinib enhanced G2M arrest in comparison with single-treatment groups. In addition, DNA damage was increased by ginsenoside and sunitinib according to the comet assay, and the level of 8-OHdG, which reflects oxidative DNA damage, also increased. We verified that ginsenoside enhances the efficacy of sunitinib to inhibit the proliferation of ccRCC cells via induction of oxidative DNA damage. The combination therapy of sunitinib and ginsenoside is an effective new treatment strategy for RCC patients.\u003c/p\u003e","manuscriptTitle":"Ginsenoside Rh2 sensitizes the anti-cancer effects of sunitinib by inducing cell cycle arrest in renal cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-04-01 19:57:43","doi":"10.21203/rs.3.rs-1480511/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-06-07T05:34:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-05-30T17:42:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"ee8d8d36-915e-431b-a141-1bce82f040d0","date":"2022-05-19T17:08:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-05-19T16:52:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"d315cdba-0d62-4078-8245-b242227d2288","date":"2022-05-18T18:47:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22bf3dfa-a12c-47a0-a8c3-1bb059bb4605","date":"2022-04-26T02:38:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-04-23T19:47:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-04-20T14:52:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-03-30T11:35:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-03-30T11:30:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-03-23T07:40:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5bbbc30c-622f-47c8-8268-8f86609277ad","owner":[],"postedDate":"April 1st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-09-08T06:44:14+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-01 19:57:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1480511","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1480511","identity":"rs-1480511","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}