Characterisation of the Morphological, Functional and Molecular Changes in Sunitinib-Resistant Renal Cell Carcinoma Cells

Sunitinib resistance is a major clinical problem hampering the treatment of renal cell carcinoma (RCC). Studies on the comprehensive characterisation of morphological, functional and molecular changes in sunitinib-resistant RCC cells are lacking. The aim of the current study was to develop sunitinib resistance in four human RCC cell lines (786-0, Caki-1, Caki-2 and SN12K1), and to characterise the changed cell biology with sunitinib resistance. RCC cells were made resistant by continuous, chronic exposure to 10 µM of sunitinib over a period of 12 months. Cell proliferation, morphology, transmigration, and gene expression for interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), Bcl-2 and Bax were studied. There was no significant difference in growth rate or transmigration between the parental and resistant cells. Sunitinib-resistant cells were significantly hypertrophic compared with parental cells as evidenced by increases in the surface areas of the whole cells and the nuclei. IL-6 was significantly increased in all resistant cells. IL-8 was increased in sunitinib-resistant Caki-2 and SN12K1 cells and decreased in 786-0 without any significant changes in Caki-1. VEGF was increased in resistant Caki-2 and SN12K1 cells but not in 786-0 and Caki-1. The Bcl2/Bax ratio was increased in Caki-1, Caki-2 and SN12K1 cells but decreased in 786-0 cells. The increased IL-6 may contribute to sunitinib resistance either via VEGF-mediated angiogenesis or through shifting of the Bcl2/Bax balance in favour of anti-apoptosis

Exploring resistance of renal cell carcinoma to sunitinib therapy

Renal cell carcinomas (RCC) are highly metastatic and therapy-resistant. Several novel tyrosine\ua0kinase inhibitors (TKI) targeting angiogenesis have been introduced into clinical practice for RCC,\ua0the most common being sunitinib. However, use of sunitinib is limited by development of therapy\ua0resistance in RCC. Comprehensive characterisation of morphological, functional and molecular\ua0changes in sunitinib-resistant RCC cells is lacking. Understanding the mechanisms of resistance to\ua0sunitinib therapy, and developing measures to overcome resistance, may help improve patient care.The overall hypotheses of this project were: 1) that RCC models that demonstrate resistance to\ua0current RCC therapies can be utilised to investigate and define molecular pathways of resistance;\ua0and 2) that these same models can be used to test novel therapies. Specifically, the project aimed: 1) to develop and characterise RCC cell lines that are resistant to sunitinib; 2) to explore the\ua0mechanisms of resistance in the RCC cell lines with emphasis on angiogenic and apoptotic\ua0pathways; 3) to overcome sunitinib-resistance with appropriate agents using in vitro methodologies; and 4) to validate in vitro findings in a murine xenograft model of RCC.A literature review is provided in Chapter 1, and general Materials and Methods are described in\ua0Chapter 2. Chapters 3 describes development of sunitinib resistance in four human RCC cell\ua0lines: 786-0, Caki-1, Caki-2, and SN12K1 and characterise the differences in cell and molecular\ua0biology from parental RCC cell lines. The RCC cell lines were made resistant to continuous,\ua0chronic exposure to 10 μM sunitinib. Cell growth/proliferation (MTT assay), morphology and apoptosis (phase-contrast microscopy and hematoxylin-eosin staining), transmigration (migration\ua0chamber), and gene expression (qRT-PCR) for interleukin-6 and -8 (IL-6, IL-8), vascular\ua0endothelial growth factor (VEGF), Bcl2 and Bax were studied. Resistant cells were stable in 10 μM\ua0sunitinib, maintained viability in 20 μM sunitinib, and have now been in culture for over two years.\ua0Resistant cells proliferated more slowly, but were significantly hypertrophic compared with parental\ua0cells. The transmigration assay showed no difference between resistant cells and their respective\ua0parental cells. One of the most striking differences between resistant and parental RCC was that IL-6 (in all RCC) and IL-8 (in most RCC) significantly increased in resistant cells, with an associated\ua0increase in VEGF. An anti-apoptotic mechanism was also demonstrated in therapy-resistant RCC.\ua0Increased IL-6 may contribute to resistance of RCC through downstream upregulation VEGF.Thus, IL-6 increase was considered worthy of further investigation as a potential therapeutic target\ua0for therapy-resistant RCC. In Chapter 4, resistant cells were grown with and without Tocilizumab\ua0(50 μg/mL), a monoclonal antibody that blocks the IL-6 receptor (IL-6R) protein. Cell\ua0growth/proliferation, cell morphology and apoptosis, gene expression (qRT-PCR) and protein levels\ua0(ELISA) for IL-6, VEGF, Bcl2 and Bax were again studied. The in vitro results demonstrated that\ua0inhibition of IL-6R by Tocilizumab inhibited cell growth, increased apoptosis, decreased VEGF expression, and reactivated and retrieved the sunitinib sensitivity.Chapter 5 provides some preliminary data of a mouse preclinical model investigating the use of\ua0Tocilizumab in the sunitinib-resistant RCC cell lines. These were implanted sub-cutaneously in\ua0NOD-SCID immunodeficient mice (1.6 x 106\ua0cells per 100 μL of PBS). The animals were\ua0randomly divided into four groups (N=6) of sunitinib-resistant cell lines. All mice in each group\ua0were injected subcutaneously with 100 μL of cell suspension. Viability of any remaining cells was\ua0verified after injection of animals, using a Trypan blue exclusion assay and regrowth in culture.\ua0Food and water intake, body weight and tumour growth were monitored throughout the experiment.\ua0One week after RCC injection, three mice from each group were injected with Tocilizumab (100 μg\ua0in 100 μL, three times per week for five weeks, intraperitoneal/IP injection). The other mice were\ua0given 100 μL IP physiological saline. After five weeks of Tocilizumab, mice were euthanased and\ua0tumours removed. Tumour weight was measured and tumour volume estimated. All organs were\ua0checked for metastatic clones of RCC, but none were detected. Tumour tissue was collected and\ua0frozen for RNA analysis. Fixed tumour and organ tissue was prepared for morphological analyses\ua0(microscopy; ApopTag for apoptosis), and immunohistochemistry for Ki67 (proliferation), IL-6 and\ua0VEGF. Only Caki-1 and SN12K1 RCC grew. Tocilizumab did not significantly decrease tumour\ua0weight in Caki-1 tumours, but there was a significant decrease in IL-6 and VEGF mRNA.\ua0Surprisingly, in SN12K1 tumours, there was a non-significant increase in mean tumour weight, and\ua0a significant increase in VEGF mRNA, with Tocilizumab.In the concluding Chapter (Chapter 6), a summary of major findings and future directions of this\ua0project are presented. Using the in vitro model, the project has confirmed that targeting IL-6 in\ua0therapy-resistant RCC could retrieve their susceptibility to cancer therapy. The significance of the\ua0project is that improving the understanding of RCC development and therapy resistance provides\ua0the exciting potential for testing personalised adjuvant therapies for treatment of RCC

Limitations to the Therapeutic Potential of Tyrosine Kinase Inhibitors and Alternative Therapies for Kidney Cancer

Background: Renal cell carcinomas (RCCs) are the most common primary renal tumor. RCCs have a high rate of metastasis and have the highest mortality rate of all genitourinary cancers. They are often diagnosed late when metastases have developed, and these metastases are difficult to treat successfully. Since 2006, the standard first-line treatment for patients with metastatic RCC has been multitargeted tyrosine kinase inhibitors (TKIs) that include mammalian target of rapamycin (mTOR) inhibitors. RCCs are highly vascularized tumors, and their angiogenesis is controlled by tyrosine kinases that play a vital role in growth factor signaling to stimulate this process. TKI therapy was introduced for direct targeting of angiogenesis in RCC.TKIs have been moderately successful in the treatment of metastatic RCC and initially increased cancer-specific survival times. However, RCC rapidly becomes resistant to TKIs, and no current drug has produced a cure for advanced RCC.Methods: We provide an overview of RCC, explain some reasons for therapy resistance in RCC, and describe some therapies that may overcome resistance to TKIs. The key pathways that determine therapy resistance are illustrated.Results: Factors involved in the development and progression of RCC include genetic mutations, activation of hypoxia-inducible factor and related proteins, cellular metabolism, the tumor microenvironment, and growth factors and their receptors. Resistance to the therapeutic potential of TKIs can be acquired or intrinsic. Alternative therapies include other small molecule drugs and immunotherapy based on immune checkpoint blockade.Conclusion: The treatment of RCC is undergoing a paradigm shift from sole use of small molecule antiangiogenesis TKIs as first-line therapy to include newly approved agents for second-line and third-line therapy that now involve the mTOR pathway and immune checkpoint blockade drugs for patients with advanced RCC

Mikania micrantha Kunth: An Ethnopharmacological Treasure Trove of Therapeutic Potential

Mikania micrantha is utilized as a therapeutic for the treatment of various human ailments including insect bites, rashes and itches of skin, chicken pox, healing of sores and wounds, colds and fever, nausea, jaundice, rheumatism, and respiratory ailments. This study aimed at summarizing the traditional uses, phytochemical profile, and biological activities of M. micrantha based on obtainable information screened from different databases. An up-to-date search was performed on M. micrantha in PubMed, Science Direct, clinicaltrials.gov, and Google Scholar databases with specific keywords. No language restrictions were imposed. Published articles, theses, seminar/conference papers, abstracts, and books on ethnobotany, phytochemistry and pharmacological evidence were considered. Based on the inclusion criteria, this study includes 53 published records from the above-mentioned databases. The results suggest that fresh leaves and whole plant are frequently used in folk medicine. The plant contains more than 150 different phytochemicals under the following groups: essential oils, phenolics and flavonoids, terpenes, terpene lactones, glycosides, and sulfated flavonoids. It contains carbohydrates and micronutrients including vitamins and major and trace minerals. M. micrantha possesses antioxidant, anti-inflammatory, anti-microbial, anti-dermatophytic, anti-protozoal, anthelmintic, cytotoxic, anxiolytic, anti-diabetic, lipid-lowering and antidiabetic, spasmolytic, memory-enhancing, wound-healing, anti-aging, and thrombolytic activities. No clinical studies have been reported to date. M. micrantha might be one of the potential sources of phytotherapeutic compounds against diverse ailments in humans. Studies are required to confirm its safety profile in experimental animals prior to initiating clinical trials. Moreover, adequate investigation is also crucial to clarify exact mechanism of action for each biological effect.The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through large group Research Project under grant number RGP2/362/44. [RGP2/362/44]; Deanship of Scientific Research at King Khalid UniversityThe authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through large group Research Project under grant number RGP2/362/44

In vivo and in silico studies of membrane-stabilizing and clot lysis activities of Trachyspermum ammi

Trachyspermum ammi contains many important phytochemicals, including thymol, which is used to treat many diseases. This study aimed to check the membrane-stabilizing and clot-lysis capacity of the ethanol extract of T. ammi (ETA) and its main chemical component, thymol (THY). For this, we used hypotonic solution-induced erythrocyte lysing and egg-albumin protein denaturation for membrane-stabilizing capacity and human blood clot lysis for anti-atherombosis capacity. Further, we observed in silico study of THY and ntric oxide synthase (NOSs) isoform. The stabilizing effects of ETA and THY on the membrane changed depending on the concentration. At the highest concentration (160 μg/mL), they stopped the most bleeding, while the standard drug, acetylsalicylic acid, stopped less bleeding at the same concentration. In the erythrocyte lysing test, ETA showed 102.60 ± 0.01 and 75.83 ± 1.44% membrane protection. In the egg-albumin denaturation test, they showed 27.62 ± 0.02% and 56.71 ± 0.02% membrane protection, respectively. In our clot lysis experiment, ETA and THY also showed significant and concentration-dependent clot lysis capacity. The IC50 and EC50 values of THY were lower than ETA when it came to keeping the membrane stable and breaking up clots, respectively. In our in silico investigation, THY and NOSs showed significant binding interaction. Taken together, ETA and its major component, THY, exhibited potent membrane stabilizing activity and clot lysis activity in in vitro studies. Further studies are required to elucidate its other active principles and their biological effects, along with possible mechanisms

Antiemetic activity of trans-ferulic acid possibly through muscarinic receptors interaction pathway: In vivo and in silico study

Current study was conducted to assess the effectiveness of the polyphenol trans-ferulic acid (TFA) as an antiemetic agent using in vivo and in silico methods. To evaluate this, we induced emesis in 3-day-old chicks through the oral administration of copper sulfate (CuSO4·5H2O) at a dose of 50 mg/kg. To ascertain the potential antiemetic mechanism of TFA, we employed various reference drugs such as domperidone (6 mg/kg), ondansetron (5 mg/kg), and hyoscine (21 mg/kg) as positive control groups, while the vehicle acted as a negative control group. TFA was administered orally at the doses of 25, 50 and 100 mg/kg body weight. Both the TFA and reference drug provided alone or in combined groups to assess their synergistic or antagonistic activity on the chicks. Molecular docking of TFA and the selected reference drugs was conducted against 5HT3, D2, H1, NK1, and mAChRs (M1-M5) receptors for determining binding affinity to the receptors. Active binding sites and drug-receptor interactions were predicted with the aid of various computational tools. Various pharmacokinetic features and drug-likeness of all the selected ligands were determined through the SwissADME online server. The results suggest that TFA diminishes the mean number of retches and enhances latency in the chicks at lower doses. In the combined drug therapy, TFA exhibited better antiemetic effects with ondansetron and hyoscine. In silico ADME proposed that TFA retains preferable drug-likeness and better pharmacokinetic properties to be a reliable lead. Additionally, TFA revealed the elevated binding affinity against mAChRs and the ligand (TFA) expressed the highest binding affinity (−7 kcal/mol) with the M5 receptor (6OL9). In conclusion, TFA demonstrated mild antiemetic effects in chicks, possibly through the mAChRs interaction pathway