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RESEARCH PROBLEM The effect of pinnata on the CONCLUSION Can treatment with an aqueous solution of • When in aqueous solution K. pinnata does not inhibit cell migration cell migration of astrocytomas posses antibacterial qualities. However, it does on astrocytomas? maintain its highly cytotoxic components. Author: Jorge Rosa, Escuela Secundaria de la Universidad de Puerto Rico Advisors: Magdiel Martínez, Amayla Budet and Jeffrey Marrero, Molecular Science Research Center INTRODUCTION • K. pinnata has the ability to inhibit cell migration among astrocytomas when in aqueous solution Astrocytomas, from cell line 1321N1,are METHODOLOGY 2. and small concentrations. cancerous astrocytes. These are a type of glial cell in the brain which is responsible for maintaining synaptic 1. Extraction of the components of Kalanchoe pinnata through lyophilization, APPLICABILITY contact and the signaling capacities among neurons. dilution and centrifuge 2. Realization of Antibiotic Susceptibility test utilizing the Kirby-Bauer method aer 24 hours They also represent a fundamental element in of incubaon K. pinnata’s ability to inhibit cell migration on a. The cells are seeded on a culture plate. Once this is done disks containing controlling blood flow and defining brain micro astrocytomas makes a viable way of preventing Gentamicin, Phosphorate buffered saline and the different concentrations of structure. Due to the fact that this variant is cancerous, 3. metastasis or diminishing it’s effects. a condition commonly described as an abnormal cell Kalanchoe pinnata. After being incubated for 24 hrs the inhibition zone is cycle and uncontrolled of cells, they measure in millimeters. FUTURE RESEARCH have the ability to metastasize. 3. Realization of Cytotoxicity assay in order to determine which concentrations will • Examining wound healing percentage of In the metastatic process of cancer cells the initial be used for the Wound Healing assay Kalanchoe pinnata dissolved on a non- phase is know as tumor invasion. This stage consists a. The cells are incubated for 24 hrs and their growth medium is changed for one aqueous solution mainly of the cells migrating away from the tumor and containing Kalanchoe pinnata. After another 24 hrs incubation Alamar Blue is added and a fluorescence reader determines the quantity of live and dead towards blood and lymph vessels in order to move 4. • Compare cytotoxicity and wound healing across and to different parts of the body (Clark, 2015). cells. effects of Kalanchoe pinnata on epithelial Further more, cancerous cells also spread in a more 4. Realization of Wound Healing assay cells and astrocytomas local manner by migrating among connecting or a. After the cells are seeded and incubated for 24 hrs the cell monolayer is nearby tissue. scrapped with a micropipette tip to create a 1mm wound. Following this the WHAT I LEARNED pinnatum, a crassulacean more cells are treated with Kalanchoe pinnata and incubated for 24 hrs. Once this is Thanks to this experience I learned the commonly know as Kalanchoe pinnata or life , done the wound healing percentage is determined in pixels . fundamentals of cell culture and many has shown antitumor promoting activity on the Epstein- procedures of importance when studying Barr virus due to the highly cytotoxic activities carried cells. I was also able to further familiarize out by it’s and bufadienolides. One of these myself with basic laboratory equipment and 50 is Diagremontianin, a highly cytotoxic component in RESULTS & ANALYSIS other fields of research all crassulacean (Supratam, 2001). Other FIG.3 - Wound Healing 100 40 ACKNOWLEDGEMENTS members of the Kalanchoe family, such as Kalanchoe FIG.1 –Antibiotic Assay tubiflora, have antiproliferative capabilities that make Susceptibility Test • KP-W shows the ability to 30 I am grateful to Magdeil Martínez, Amayla it a viable for cancer treatments. inhibit cell migration at Budet, Jeffrey Marrero, Keyla Soto and Myrna • KP-W (15,16) does not T24 20 Gandia for their guidance during this process IC50: 25.8 ug/ml poses antibiotic qualities and for allowing me such an impactful and HYPOTHESIS 50 10 developmental experience. % Wound Healing (Over T0) When treated with an aqueous solution from K. pinnata 0 BIBLIOGRAPHY leaves the cell migration of astrocytomas will be inhibited. T0 T24 Chen, Y. (2012). Scratch Wound Healing Asssay. N3

Cell Viability (% Over Control) Cheng, Y. B. [鄭翊翎]. (2016). Functional characterization and therapeutic role of interleukin-1 receptor-associated kinase 1 (IRAK1) in hepatocellular carcinoma. (Thesis). University of Control 0 Hong Kong, Pokfulam, Hong Kong SAR. Clark A., Matic D. (2015). Modes of cancer cell invasion and the role of the microenvironment. VARIABLES 1.0 -3 10 -2 100 -1 1000 0 1 [Drug],ug/ml Comisión Nacional para el Conocimiento y Uso de la Biodiversidad. (2009). Control Kalanchoe pinnata (Lam.) Pers. K. pinnata w.extract Facultad de Ciencias de la Universidad de Granada. SECADO POR LIOFILIZACIÓN. DEPENDENT VARIABLES- Percentage of Wound Healing Hsieh Y., Huang H., Leu Y., Peng K., Chang C., and Chang M. Anticancer Activity of Kalanchoe

tubiflora Extract Against Human Lung Cancer Cells In Vitro and In Vivo INDEPENDENT VARIABLES- K. pinnata treatment FIG.4 –Analysis of Wound Kettenmann H., Verkhratsky A. (2011). Neuroglia - Living Nerve Glue FIG.2 – Cytotoxicity Assay Healing Percentage Liang C., Park A., Guan J. (2007). In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. CONSTANTS- Initial quantity of cells per well • KP-W significantly inhibits the Majaz Q., Tatiya A.,Khurshid M.,Nazim S.,Siraj S.(2011). The miracle plant (Kalanchoe • In order to perform the pinnata): A Phitochemical and Pharmalogical Review. (1.0 x ​10↑5 ); Quantity of aqueous solution growth cell migration of astrocytomas. Singapore National Parks Board. (2013). Kalanchoe pinnata (Lam.) Pers. Wound Healing Assay the Supratman U, Fujita T, Akiyama K, Hayashi H, Murakami A, Sakai H, Koshimizu K, medium per well (500µL); Time under observation (24 K.P-W On average cells treated with Ohigashi H. (2001). Anti-tumor promoting activity of bufadienolides from Kalanchoe pinnata and K. concentration of KP-W in daigremontiana x tubiflora. hrs) 10ug/ml KP-W had a reduction in IMAGES the solution must be 10µg/ http://massspectrumbotanicals.net/wp-content/uploads/2013/04/KalPin.jpg migration of approximately https://basicmedicalkey.com/wp-content/uploads/2017/01/B9781416066279000378_fx2.jpg mL. http://www.genecopoeia.com/wp-content/uploads/Cell-Proliferation-Assay-Kits/Cell-Proliferation- 50%. Assay-Kits-32.jpg