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Multiple Effects of Hymenocrater longiflorus on human colon cancer (RKO) by using different techniques Rafal S. A. Al-Anee a, Khulood W. Al-Sammarraeb, Ghassan M. Sulaimanc, Giuliana Napolitanod , Saman A. Ahmade a b Biotechnology Research Centre, University of Al-Nahrain, Baghdad, Iraq Department of biotechnology, college of science, university of Al-Nahrain, Baghdad, Iraq c Division of Biotechnology, Department of Applied Sciences, University of Technology, Baghdad, Iraq d Department of Structural and Functional Biology, University of Naples Federico II, Naples, Italy e Department of Field Crop, College of Faculty of Agriculture Science, University of Sulaimani /Iraq. a * Author to whom correspondence should be addressed; E-mail: [email protected] Tel.: +964 (0) 7801672140. Abstract Hymenocrater longiflorus is a perennial herb. Leaves ovate- belong or lanceolate. This plant is growing above timberline or in subalpine zone between the rocks. The methanolic extract of this plant was used to study the immunoflourescence assay for γ-H2AX for RKO (colon cancer) cell line and observe Induction of DNA double-strand breaks causes phosphorylation of histone H2AX at serine 139. Then study effects on some proteins like P21,P53, P53-P, γ-H2AX and observe cleavage of PARP proteins by western blot analysis and this an indicator of DNA damage and apoptosis for RKO cell line. The results show Induction of DNA double-strand breaks causes phosphorylation of histone H2AX at serine 139 by Immunoflourescence staining for γ-H2AX on RKO cell line at 4 hrs for 80 and 100µg ml-1 H. longiflorus plant extract in compares with CPT, UV and NT cells. Also during treatment with plant extract at different times and concentrations observe increasing in proteins like P21, P53, P53-P, γ-H2AX and cleavage of PARP protein and these are signs of DNA damage which lead to apoptosis . Key word: H. longiflorus, RKO, immunoflourescence, γ-H2AX, Western blot.