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Epibrassinolide is a member of brassinosteroids with a polyhydroxysteroid structure similar to steroid hormones of vertebrates. It was shown that EBR decreased cell proliferation and induced apoptosis in different colon cancer cell lines without exerting a cytotoxic effect in epithelial fetal human colon cells. This finding highlighted the potential of epibrassinolide in clinical therapeutic setup. In our previous studies, we showed that epibrassinolide was able to induce apoptosis via endoplasmic reticulum stress. Recently, we also showed that endoplasmic reticulum and apoptotic stresses can be prevented via autophagic induction in non-cancerous epithelial or aggressive forms of cancer cells. Therefore, here in this study, we evaluated the anti-tumoral effect of epibrassinolide as well as the autophagy involvement in the aggressive forms of colon cancer cell lines as well as in vivo SCID mouse xenograft colon cancer model for the first time. For this purpose, SCID mouse model was used for subcutaneous injection of colon cancer cells in matrigel formulation. We found that autophagy is induced in both in vitro and in vivo models. Following tumor formation, SCID mice were treated daily with increasing concentrations of epibrassinolide for two weeks. Our findings showed that EBR inhibited the volume and diameter of the tumor in a dose-dependent manner by causing cell cycle arrest. Therefore our data suggest that epibrassinolide exerts a cytostatic effect on the agrressive form of colon cancer model in vivo, without affecting endoplasmic reticulum stress and the induction of autophagy might have role in this effect of epibrassinolide. © 2023 Elsevier Ltd 36587800

Epibrassinolide (EBR), a plant-derived polyhydroxylated derivative of 5 alpha-cholestane, structurally shows similarities to animal steroid hormones. According to the present study, EBR treatment triggered a significant stress response via activating ER stress, autophagy, and apoptosis in cancer cells. EBR could also increase Akt phosphorylation in vitro. While the activation of Akt resulted in cellular metabolic activation in normal cells to proceed with cell survival, a rapid stress response was induced in cancer cells to reduce survival. Therefore, Akt as a mediator of cellular survival and death decision pathways is a crucial target in cancer cells. In this study, we determined that EBR induces stress responses through activating Akt, which reduced the mTOR complex I (mTORC1) activation in SW480 and DLD-1 colon cancer cells. As a consequence, EBR triggered macroautophagy and led to lipidation of LC3 most efficiently in SW480 cells. The cotreatment of spermidine (Spd) with EBR increased lipidation of LC3 synergistically in both cell lines. We also found that EBR promoted polyamine catabolism in SW480 cells. The retention of polyamine biosynthesis was remarkable following EBR treatment. We suggested that EBR-mediated Akt activation might determine the downstream cellular stress responses to induce autophagy related to polyamines.

Kolon kanseri, kanserden ölümlerde akciğer kanserinden sonra yer alan yaygın bir kanser türüdür. Kolon kanserinin dünyada artan görülme sıklığına bağlı olarak yeni tedavi stratejilerine ve moleküler mekanizmaların aydınlatılmasına gerek duyulmaktadır. Epibrassinolid (EBR), bitki büyüme düzenleyicilerinden brassinosteroidlerin (BR) bir üyesi olup polihidroksisteroid yapıdadır. EBR'nin omurgalılarda bulunan steroid hormonlara da yapısal olarak benzerlik gösterdiği bilinmektedir. EBR'nin apoptotik hücre ölümünü tetiklediği farklı kanser hücre hatlarında laboratuvarımız tarafından çeşitli çalışmalarda belirlenmiştir. Ayrıca, çalışmalarımız EBR'nin normal epitel hücrelerine etki etmediğini göstererek, klinikte kullanılabilirliği açısından önemli bilgiler elde edilmiştir. EBR'nin neden olduğu apoptozun moleküler temelleri SILAC (stable-isotope labelled aminoacid in cell culture) yöntemi ile belirlenmiştir. Hücre sağkalımı, apoptoz ve endoplazmik retikulum (ER) stresi ile ilgili pek çok proteinin anlamlı bir şekilde değiştiği laboratuvarımızda gerçekleştirilen deneyler ile gösterilmiştir. Bu noktadan hareketle tez kapsamında EBR'nin potansiyel anti-tümöral etki mekanizması kolon kanseri hücrelerinde in vitro ve in vivo denemeler ile aydınlatılmak istenmiştir. Bu amaçla SW480 ve DLD-1 hücre hatları kullanılmış, SW480 hücreleri ise SCID (severe combined immunodeficiency) farelere subkutan olarak matrijel içerisinde verilerek kolon kanseri modeli oluşturulmuştur. İn vitro deneylerde EBR'nin sferoid büyümesini engelleyici etkisi tespit edilmiştir. Bunun yanı sıra, tümör oluşumunu takiben günlük EBR uygulamaları ile tümör çapı ve hacminin doz uygulamasına bağlı olarak tümör büyümesine ket vurucu etkisi tespit edilmiştir. Bunun yanısıra bu etkinin tümörü oluşturan hücrelerde apoptotik hücre ölümünün tetiklenmesinden daha ziyade hücre döngüsüne ket vurarak tetiklediği gösterilmiştir. Bu çalışma ile EBR'nin kolon kanserine karşı in vivo modelde olası anti-tümöral ve sistemik etkisi literatürde ilk defa modellenmiştir. Colon cancer is the second leading cause of cancer related deaths after lung cancer. Due to the increasing incidence of colon cancer in the world, new treatment strategies and molecular mechanisms need to be clarified. Epibrassinolide (EBR) is a member of brassinosteroids (BR) from plant growth regulators and has a polyhydroxysteroid structure. EBR is also known to be structurally similar to steroid hormones found in vertebrates. Various studies have been conducted by our laboratory in different cancer cell lines in which EBR induces apoptotic cell death. In addition, our studies showed that EBR does not affect normal epithelial cells and important information was obtained in terms of its usefulness in the clinic. The molecular basis of the EBR-induced apoptosis was determined by SILAC (stable-isotope-labeled aminoacid in cell culture) method. According to study results, EBR altered cell death and survival decision mechanism through effecting endoplasmic reticulum (ER stress). From this point of view, the scope of the thesis was to elucidate the potential anti-tumor action of EBR with in vitro and in vivo experiments using colon cancer cells. For this purpose, SW480 and DLD-1 cell lines were used, and SW480 cells were administered subcutaneously in SCID (severe combined immunodeficiency) mice and colon cancer model was generated. In vitro experiments have shown that EBR inhibits spheroid growth. In addition, daily EBR administration following tumor formation induced tumor growth and tumor volume decrease in a dose-dependent manner. In addition, this effect of EBR was due to cell cycle inhibition rather than apoptotic cell death induction in tumor-forming cells. This study, therefore, was the first of the literature showing the possible anti-tumoral and systemic effects of EBR in in vivo colon cancer model. 137

Prostate cancer is one the leading cancer type after lung cancer among male. Obesity is one of the known risk factor for developing highly aggressive and metastatic prostate cancer, which is an obstacle in cancer therapy. For these particular reasons, drugs are used in the therapy of obesity is suggested with their anti­tumoral effect, whether blocking adipogenesis and lipogenesis pathways or not. Orlistat inhibits fatty acid synthase (FASN), a large multifunctional enzyme, is responsible for de novo synthesis of long chain fatty acids. FASN activity is confirmed in the most of the xenograft models of prostate cancer cells. In this study we utilized PC3 cells, medium FASN expression profile compared to DU145 and LNCaP cells, and PNT1A prostate epithelial cells to evaluate the molecular targets of orlistat in adenosin mono phosphate kinase (AMPKa) and lipogenesis signaling axis. We demonstrated that orlistat inhibited cell proliferation and led to cell viability decrease in PC3 prostate cancer cells but not in PNT1A prostate epithelial cells. The activation of AMPKa was observed in both cell lines, which led to inhibition of protein translation through dephosphorylating mTOR at ser2448 and phosphorylating mTOR at ser2481 after treatment with Orlistat at 15 µM and 20 µM concentrations for 24h. Concomitantly, orlistat induced autophagy via phosphorylation of ULK­1 at ser555. The modulation of Acetyl­CoA carboxylase (ACC), which regulates biosynthesis and oxidation of fatty acids was observed following orlistat treatment. However, AMPK phosphorylates ACC at ser79 and inhibits its activity. These findings suggest that orlistat altered AMPK­related a number of critical pathways to exert its anti­tumor activity in prostate cancer cells. However, AMPK is a metabolic stress regulator protects epithelial normal cells against orlistat treatment. Thereby, it is crucial to have deep understanding about the role of AMPK in prostate cancer cells.