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      Antitumor Effects of L-citrulline on Hela Cervical Cancer Cell Lines

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          Abstract

          Aim:

          Cervical cancer is the deadliest gynecological malignancy. This study aims to examine the anticancer effects of L-citrulline on HeLa cell culture.

          Materials and Methods:

          HeLa cells were cultured in complete Eagle's minimum essential medium. HeLa cells were seeded in 96-well plates and incubated with L-citrulline. After incubation, MTT dye was added and incubated. Annexin-V technique was used to test the apoptosis. The activated caspases of HeLa cells by L-citrulline exposure were measured with the Caspase 3/7 technique. One-way variance analysis was conducted for statistical analysis by using GraphPad Prism 6.0 for Windows.

          Results:

          L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner in application times of 24 and 48 hours. The IC 50 dose of L-citrulline was 0.19 and 0.16 mg/mL at 24 and 48 hours, respectively. When HeLa cells were exposed to an IC 50 dose of L-citrulline for 24 hours, the percentages of the dead, early apoptotic, and late apoptotic cells were detected to be 0.75%, 23.05%, and 12.75%, respectively. The differences in the wideness of the scratch area were observed at the initial stage and after 24 hours of applying L-citrulline.

          Conclusion:

          L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner. Based on Annexin and Caspase findings, it can be concluded that L-citrulline exerted a pro-apoptotic effect on HeLa cells in only a short exposure time. L-citrulline also showed a migration inhibitory effect. The findings of this study indicate L-citrulline to be worthy of investigation for its anticancer activities in vitro and in vivo, and as a candidate for cancer therapy.

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          Most cited references33

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          In vitro cell migration and invasion assays.

          Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.
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            In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis

            Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior. In vitro assays are excellent approaches to extrapolate to in vivo situations and study live cells behavior. Here we present four in vitro protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on two-dimensional wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and three-dimensional spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.
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              Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

              Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).
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                Author and article information

                Journal
                Anticancer Agents Med Chem
                Anticancer Agents Med Chem
                ACAMC
                Anti-Cancer Agents in Medicinal Chemistry
                Bentham Science Publishers
                1871-5206
                1875-5992
                14 September 2022
                14 September 2022
                : 22
                : 18
                : 3157-3162
                Affiliations
                deptEskisehir Acibadem Hospital , Obstetrics and Gynecology Clinic , Eskisehir, , Turkey;

                deptDepartment of Biology, Faculty of Science , Eskişehir Technical University , Eskisehir, , Turkey
                Author notes
                [* ]Address correspondence to this author at the Eskisehir Acibadem Hospital, Obstetrics and Gynecology Clinic, Eskisehir, Turkey; Tel: 05324453803; E-mails: cyerenr@ 123456yaani.com ; cyeren@ 123456yaani.com
                Article
                ACAMC-22-3157
                10.2174/1871520622666220426101409
                9637975
                35473537
                9c6ac1b3-a64d-4af1-9a91-2b5701325bc1
                Copyright @ 2022

                This is an Open Access article published under CC BY 4.0 https://creativecommons.org/licenses/by/4.0/legalcode

                History
                : 31 January 2022
                : 03 March 2022
                : 07 March 2022
                Categories
                Pharmaceutical and Medicinal Chemistry,Oncology

                Oncology & Radiotherapy
                l-citrulline,hela,anticancer effects,mtt,annexin-v,migration assay
                Oncology & Radiotherapy
                l-citrulline, hela, anticancer effects, mtt, annexin-v, migration assay

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