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Despite the establishment of new therapy options during the last decades, advanced renal cell carcinoma (RCC) remains an uncurable disease, due to evolving drug-resistance. New, more efficient treatment strategies are still in urgent demand. Shikonin (SHI) from Traditional Chinese Medicine has exhibited anti-tumor properties in several tumor entities. Thus, in the current investigation we evaluated the impact of SHI on progressive growth and metastatic behavior, not only in therapy-sensitive (parental) but also in therapy-resistant RCC cells. Parental and sunitinib-resistant RCC cells, Caki-1, 786-O, KTCTL-26, and A498, were treated with SHI. Tumor cell growth, proliferation, clonogenic capacity, cell cycle phase distribution, induction of cell death (apoptosis and necroptosis) and the expression and activity of regulating and signaling proteins were evaluated. Moreover, the adhesion and chemotactic activity of the RCC cells after exposure to SHI were investigated. SHI did not affect cell motility but led to significant inhibition of progressive tumor cell growth in both therapy-sensitive and therapy-resistant RCC cells. Thereby, SHI significantly inhibited the growth, proliferation and clone formation in parental and sunitinib-resistant RCC cells by G2/M phase arrest through down-regulation of cell cycle activating proteins, blocking cell division. Furthermore, SHI induced apoptosis and necroptosis by activating necrosome complex proteins. Concomitantly, SHI impaired the Akt/mTOR pathway, pivotal for cell survival and growth. Thus, SHI may hold promise as an additive option in treating patients with advanced and therapy-resistant RCC. Citation Format: Eva Juengel, Sascha D. Markowitsch, Olesya Vakhrusheva, Yasminn Akele, Jovanna Kitanovic, Patricia Schupp, Kimberly S. Slade, Anita Thomas, Igor Tsaur, Rene Mager, Thomas Efferth, Axel Haferkamp. Cell growth of sunitinib-resistant renal cell carcinoma was inhibited by Shikonin through necroptosis induction and Akt/mTOR signaling inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5384.

Although innovative therapeutic concepts have led to better treatment of advanced renal cell carcinoma (RCC), efficacy is still limited due to the tumor developing resistance to applied drugs. Artesunate (ART) has demonstrated anti-tumor effects in different tumor entities. This study was designed to investigate the impact of ART (1&ndash, 100 &micro, M) on the sunitinib-resistant RCC cell lines, Caki-1, 786-O, KTCTL26, and A-498. Therapy-sensitive (parental) and untreated cells served as controls. ART&rsquo, s impact on tumor cell growth, proliferation, clonogenic growth, apoptosis, necrosis, ferroptosis, and metabolic activity was evaluated. Cell cycle distribution, the expression of cell cycle regulating proteins, p53, and the occurrence of reactive oxygen species (ROS) were investigated. ART significantly increased cytotoxicity and inhibited proliferation and clonogenic growth in both parental and sunitinib-resistant RCC cells. In Caki-1, 786-O, and A-498 cell lines growth inhibition was associated with G0/G1 phase arrest and distinct modulation of cell cycle regulating proteins. KTCTL-26 cells were mainly affected by ART through ROS generation, ferroptosis, and decreased metabolism. p53 exclusively appeared in the KTCTL-26 cells, indicating that p53 might be predictive for ART-dependent ferroptosis. Thus, ART may hold promise for treating selected patients with advanced and even therapy-resistant RCC.

Therapy resistance remains a major challenge in treating advanced renal cell carcinoma (RCC), making more effective treatment strategies crucial. Shikonin (SHI) from traditional Chinese medicine has exhibited antitumor properties in several tumor entities. We, therefore, currently investigated SHI’s impact on progressive growth and metastatic behavior in therapy-sensitive (parental) and therapy-resistant Caki-1, 786-O, KTCTL-26, and A498 RCC cells. Tumor cell growth, proliferation, clonogenic capacity, cell cycle phase distribution, induction of cell death (apoptosis and necroptosis), and the expression and activity of regulating and signaling proteins were evaluated. Moreover, the adhesion and chemotactic activity of the RCC cells after exposure to SHI were investigated. SHI significantly inhibited the growth, proliferation, and clone formation in parental and sunitinib-resistant RCC cells by G2/M phase arrest through down-regulation of cell cycle activating proteins. Furthermore, SHI induced apoptosis and necroptosis by activating necrosome complex proteins. Concomitantly, SHI impaired the AKT/mTOR pathway. Adhesion and motility were cell line specifically affected by SHI. Thus, SHI may hold promise as an additive option in treating patients with advanced and therapy-resistant RCC.

Introduction: Prostate carcinoma (PCa) is the most common malignancy in men. Androgen-targeted therapy and chemotherapy are currently the treatment of choice for advanced stages. Due to resistance towards these therapies, prognosis remains poor and new treatment options are urgently required. Shikonin (SHI) from Traditional Chinese Medicine (TCM) might be promising, since it induces anti-tumor effects in different tumor entities. However, data on PCa are few, and data on resistant PCa are not existent. Material and Methods: Parental (=sensitive) and docetaxel-resistant PCa cell lines, PC3, DU145, LNCaP, and 22Rv1 were exposed to SHI [0.1 - 1.5 μM] for 24, 48, or 72 hours. Untreated cells served as controls. Tumor cell growth, proliferation, cell cycle, and the expression of cell cycle regulating proteins were assessed. Several cell deaths, like apoptosis, necrosis and necroptosis as well as the metabolic activity were evaluated. Results: Time- and dose-dependent exposure to SHI significantly reduced tumor cell growth and proliferation in parental and docetaxel-resistant PCa cells, compared to the untreated controls. This was accompanied by cell cycle arrest in the G2/M phase in parental PC3 and docetaxel-resistant DU145 and S phase arrest in resistant 22Rv1, associated with modulated expression of cell cycle regulating proteins. Significant apoptotic effects were observed in parental and docetaxel-resistant PC3, DU145, 22Rv1, and resistant LNCaP. Moreover, SHI induced necroptosis in all parental and resistant PCa cells, as shown by additional application to the necroptosis inhibitor necrostatin-1. In line with this, RIP-1 expression enhanced under SHI exposure. In contrast, SHI did not alter the metabolic activity of the PCa cells. Conclusion: Significant anti-tumor effects are apparent in parental but also in docetaxel-resistant PCa cells after SHI application. Thus, adding SHI to standard therapies might be a promising treatment strategy for patients with advanced prostate cancer. Further investigations are necessary to verify our findings. Funding: Friedrich-Spicker-Stiftung (2017). Acknowledgments: The main portion of the results presented here are part of the MD thesis of Kira M. Juetter at the Department of Urology and Pediatric Urology, University Medical Center Mainz, Langenbeckstraße 1, 55131 Mainz, Germany. Citation Format: Sascha D. Markowitsch, Kira M. Juetter, Patricia Schupp, Kristine Hausschulte, Olesya Vakhrusheva, Jindrich Cinatl, Martin Michaelis, Thomas Efferth, Axel Haferkamp, Eva Juengel. Shikonin impairs the growth of docetaxel-resistant prostate cancer cells by necroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1423.

Introduction: Prostate carcinoma (PCa) is the most common cancer in men. The therapeutic effect of approved compounds, such as docetaxel, is limited due to the development of therapy resistance, making new treatment options essential. Artesunate (ART), used in Traditional Chinese Medicine, has shown anti-tumor activity in several tumor types. However, little is known about the efficacy of ART on therapy-resistant PCa. Therefore, the impact of ART on docetaxel-resistant PCa cells was investigated. Material and Methods: Parental (=sensitive) and docetaxel-resistant PCa cell lines, PC3, DU145, and LNCaP, were exposed to ART [1-100 µM] for 24, 48, or 72 hours. Cells not exposed to ART served as controls. Tumor cell growth, proliferation, cell cycling, and the expression of cell cycle regulating proteins were assessed. Apoptotic, ferroptotic, and necrotic effects were also evaluated. Results: Exposure to ART significantly reduced tumor cell growth and proliferation of parental and docetaxel-resistant PCa cells in a time- and dose-dependent manner. Cell cycle arrest in the G0/G1 phase after 48h exposure to 37.5 µM ART was observed in parental LNCaP and in all three resistant cell lines. The observed changes in cell growth and cell cycle phases were accompanied by marked modulation of cell cycle regulating proteins. Significant apoptotic effects were observed in parental PC3 and both parental and resistant LNCaP, whereas ferroptosis was apparent in parental and resistant DU145 after exposure to ART. Necrotic events were not detectable in the PCa cells after ART exposure. Conclusion: ART induced anti-tumor effects, though differing according to the cell line, were apparent in not only parental but also in docetaxel-resistant PCa cells. Thus, adding ART to standard therapies might be a promising treatment strategy for patients with advanced prostate cancer. Further investigations are necessary to verify our findings. Citation Format: Vitus Braeunig, Patricia Schupp, Sascha Markowitsch, Olesya Vakhrusheva, Holger Erb, Thomas Efferth, Axel Haferkamp, Eva Juengel. Artesunate reduces tumor growth and induces different kinds of cell death in docetaxel-resistant prostate carcinoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6327.