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Anti-cancer activity by Thai medicinal plant associated with cell death induction and signaling pathway
Thailand (IFMSA-Thailand) - Faculty of Medicine, Srinakharinwirot University, Bangkok
Associate Professor Ramida Watanapokasin, Ph.D.
Associate Professor Ramida Watanapokasin, Ph.D.
Type of Research Project
- Basic science
What is the background of the project?
Nowadays, cancer has become one of the most important public health problems in developing countries. Treatments of cancer include chemotherapy, radiotherapy, surgery and targeted therapy which is limited by the high cost. These treatments also show side effects to normal cells in cancer patients. Therefore, finding of bioactive compounds to develop anti-cancer drug is necessary. In Thailand, we have many types of medicinal plants which normally are used in Thai cuisine for long time. Thus, we have found some active compounds that could inhibit cancer cell growth and induce cell death through signaling pathway in order to modify and develop as a therapeutic agent in the future.
What is the aim of the project?
1. To investigate the effect of bioactive compounds on cancer cell growth inhibition. 2. To investigate the mechanisms of cancer cell death induction by bioactive compound. 3. To investigate the effect of bioactive compounds on cancer cell metastasis inhibition.
What techniques and methods are used?
Molecular biology techniques; Immunofluorescence technique using Hoechst staining for nuclear morphological changes and JC-1 staining for mitochondrial membrane potential, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay for cell viability, Flow cytometry for cell cycle and apoptotic cell analysis, Western blot analysis for protein expression etc. Cancer cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained as a monolayer in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% Fetal bovine serum (FBS) (GE Healthcare, UK), 100 U/ml penicillin and 100 µg/ml streptomycin (PAA Laboratories, Pasching, Austria). The cells were cultured and subcultured 2-3 times/week. Cell proliferation and cell viability assays MTT assay was used to determine the cytotoxicity of the bioactive compound. Cells were seeded in a 96-well plate and allowed to grow for 24 h. Then, cells were treated with the compound for 24 h, whereas the control group was treated with 0.5% DMSO (Dimethyl sulfoxide). After incubation, 100 µl of 0.5 mg/ml MTT solution was added to each well and incubated, then supernatant was removed and DMSO was added to solubilize the formazan crystals. The absorbance was measured by using a microplate reader at 570 nm (Multiskan EX; Thermo Electron Corp., Vantaa, Finland), and the half maximal inhibitory concentration (IC50) value was calculated by using the GraphPad Prism 3.03 (GraphPad Software, Inc., San Diego, CA, USA). Nuclear morphological staining with Hoechst 33342. Cells were seeded for 24 h. Then, the cells were treated for 24 h, while, the control group was treated with 0.5% DMSO. After incubation, cells were stained with 5 µM Hoechst 33342 and examined under a fluorescence microscope (IX73; Olympus, Tokyo, Japan). Cell cycle analysis to examine apoptosis induction via upregulation of sub-G1 population, flow cytometry was carried out. Measurement of mitochondrial membrane potential (ΔΨm) JC-1 was used to determine the function of the ΔΨm, which specific to mitochondria that is incorporated into the mitochondrial membrane. Western blot analysis. Cells were treated with the compound for 24 h, whereas the control group was treated with 0.5% DMSO and harvested at designated time points. The pellet cells were lysed with Radioimmunoprecipitation assay (RIPA) lysis buffer supplemented complete mini protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany).
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- The tasks of the student will be performed on his/her own
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
Lecture and lab work will be taught and done in parallel by post-doctorol fellow. The student will manipulate samples and perform lab work, as described above in steps/ stages, and write lab reports by themself under supervision. In brief, the student is expected to gain knowledge, laboratory experiences and understanding in molecular techniques which are immunofluorescence technique using Hoechst staining and JC-1 staining, MTT assey, Flow cytometry and western blot analysis. The student will understand the mechanisms of apoptosis induction in cancer cells by using medicinal plant extracts in order to be able to apply in the clinical research in the future. The student will have to give a scientific report by the end of the exchange.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
The student will be taught by post-doctoral fellow, both lecture and lab work. Lecture and lab work will be done in parallel.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student’s name will be mentioned in a future publication
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system - Students in biomedical fields - Dental medicine students (IADS members)
- 1. Thuncharoen W; Chulasiri M; Nilwarangkoon S; Nakamura Y; Watanapokasin R. Apoptotic induction of skin cancer cell death by plant extracts. J Med Assoc Thai. 2013 Jan; 96 (Suppl 1): S60-4. PMID: 23724457.
- 2. Sophonnithiprasert T; Mahabusarakam W; Nakamura Y; Watanapokasin R. Goniothalamin induces mitochondria-mediated apoptosis associated with endoplasmic reticulum stress-induced activation of JNK in HeLa cells. Oncol Lett. 2017 Jan; 13(1): 119-128. doi: 10.3892/ol.2016.
- 3. Chowchaikong N; Nilwarangkoon S; Laphookhieo S; Tanunyutthawongse C; Watanapokasin R. p38 inhibitor inhibits the apoptosis of cowanin-treated human colorectal adenocarcinoma cells. Int J Oncol. 2018 Jun; 52(6): 2031-2040. doi: 10.3892/ijo.2018.4353.
- 4. Tangchirakhaphan S; Innajak S; Nilwarangkoon S; Tanjapatkul N; Mahabusrakum W; Watanapokasin R. Mechanism of apoptosis induction associated with ERK1/2 upregulation via goniothalamin in melanoma cells. Exp Ther Med. 2018 Mar; 15(3): 3052-3058. doi: 10.3892/etm.2018.5762.
- 5. Krajarng A; Chulasiri M; Watanapokasin R. Etlingera elatior Extract promotes cell death in B16 melanoma cells via down-regulation of ERK and Akt signaling pathways. BMC Complement Altern Med. 2017 Aug 22; 17(1): 415. doi: 10.1186/s12906-017-1921-y.
- In addition; the student can search “Watanapokasin” (Assoc. Prof. Ramida Watanapokasin) in Pubmed for further information.
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