Effect of Pre-Low-Dose Irradiation on the Anticancer Activity of gallic acid in leukemia K562 and K562/Dox cells: Cell viability and cellular energetic state studies
•Download as PPTX, PDF•
0 likes•16 views
Effect of Pre-Low-Dose Irradiation on the Anticancer Activity of gallic acid in leukemia K562 and K562/Dox cells: Cell viability and cellular energetic state studies
Recommended
Recommended
More Related Content
Similar to Effect of Pre-Low-Dose Irradiation on the Anticancer Activity of gallic acid in leukemia K562 and K562/Dox cells: Cell viability and cellular energetic state studies
Similar to Effect of Pre-Low-Dose Irradiation on the Anticancer Activity of gallic acid in leukemia K562 and K562/Dox cells: Cell viability and cellular energetic state studies (20)
More from Ktn Aye
Recently uploaded
Recently uploaded (20)
Effect of Pre-Low-Dose Irradiation on the Anticancer Activity of gallic acid in leukemia K562 and K562/Dox cells: Cell viability and cellular energetic state studies
- 1. Khin TheNu Aye Ph.D. scholar in Biomedical Sciences CMU Presidential Scholarship Program Department of Radiologic Technology, Faculty of Associated Medical Sciences 1 AMS Symposium, 2022
- 2. Headlines • Introduction • Objective • Materials and Methods • Results and Discussion • Conclusion • References2 AMS Symposium, 2022
- 3. • Malignant clonal disease • Hematopoietic stem cells • Myeloid and Lymphoid: Acute or Chronic • Most morbidity and mortality • 10 million deaths in 2020 3 Source: Globocan 2020 https://gco.iarc.fr/today/ Introduction : Leukemia (Multidrug Resistant Cancer)
- 4. AMS Symposium, 2022 4 chemotherapy radiotherapy Bone Marrow Transplant Immunotherapy Targeted therapy Multidrug Resistance : Drug Efflux Pirarubicin Natural Polyphenols Curcumin Resveratrol Gallic acid Treatments
- 5. https://www.mdpi.com Gallic Acid (3,4,5- Trihydroxy benzoic acid) Gallic Acid Phenolic acid antioxidant Anti-cancer Anti- inflammation Gallnuts tea leaves oak bark Chemical formula 5 AMS Symposium, 2022
- 6. 6 High-Dose ( > 0.1 Gy) Low Dose Radiation: ( dose ≤ 0.1 Gy) Radiation Hormesis Adaptive Response Bystander effect (RIBE) Hyperradiosensitivity (HRS) Deleterious effects to Cancer cells Radiotherapy AMS Symposium, 2022
- 7. Objective • To determine the effect of pre-low-dose irradiation followed by gallic acid on cell viability and cellular energetic state of leukemic K562 and K562/Dox cells 7 AMS Symposium, 2022
- 8. AMS Symposium, 2022 Materials and Methods Experimental Design 8
- 9. 1. Cell line and cell culture 9 K562 K562/Dox Leukemic cell lines K562 = myelogenous leukemic cell line: drug-sensitive type K562/DOX = doxorubicin resistant type Complete Media (RPMI 1640 + 10% FBS + Penicillin-Streptomycin 1%) AMS Symposium, 2022
- 10. Absorbed Dose kV mA sec 1 0.02 Gy 100 200 0.14 2 0.05 Gy 100 200 0.36 3 0.1 Gy 100 200 0.71 X-irradiation: Medical diagnostic X-ray machine (Shimadzu, Japan, 125kV, 500 mA) 10 2. Irradiation System
- 11. 3. Cell viability assay 11 X-radiation: 0.02 , 0.05 , 0.1Gy Gallic acid: 10 µM , 100 µM 24-hr incubation 24-hr incubation Resazurin assay AMS Symposium, 2022 Principle Luminescence Spectrofluorometer
- 12. 4. Succinate Dehydrogenous (SDH) Activity 12 X-radiation: 0.02 , 0.05 , 0.1Gy Gallic acid: 10 µM , 100 µM 1:30 hr incubation 1:30 hr incubation MTT assay AMS Symposium, 2022 Principle Spectrophotometer
- 13. 5. Mitochondrial membrane potential (∆Ψm) measurement 13 X-radiation: 0.02 , 0.05 , 0.1Gy Gallic acid: 10 µM , 100 µM 1:30 hr incubation 1:30 hr incubation AMS Symposium, 2022 ∆Ψm Luminescence Spectrofluorometer
- 14. Cells Fluorescence Emission (Ex at 360 nm) ATP Extraction By Boiling (60 °C) Extraction ATP Purification By Thin layer chromatography (TLC) 14 6. ATP Determination
- 15. AMS Symposium, 2022 15 7. Statistical Analysis Mean ± standard error of the mean (S.E.) Independent Student’s t-test P-value < 0.05
- 16. 16 Results and Discussion AMS Symposium, 2022
- 17. Cell viability assay Figure 1: Effect of pre-irradiation to low-dose X-rays on percentage cell viability • Pre-low dose irradiation and Gallic Acid treatment • ↓ cell viability • No significant effect if compared with GA alone 17 AMS Symposium, 2022 LDR and GA 10 µM LDR and GA 100 µM
- 18. Succinate Dehydrogenous (SDH) Activity Figure 2: Effect of pre-irradiation to low-dose X-rays on absorbance • ↓ SDH activity, mitochondrial activity • More significant effect of combined effect in 10 μM GA treatment with LDR 18 AMS Symposium, 2022 LDR and GA 10 µM LDR and GA 100 µM
- 19. Mitochondrial membrane potential (∆Ψm) Figure 3: Effect of pre-irradiation to low-dose X-rays on (∆Ψm) • ↓ mitochondrial membrane potential • Less energetic state, especially in K562/DOX • ↑chemotherapeutic effect of Gallic acid 19 AMS Symposium, 2022 LDR and GA 10 µM LDR and GA 100 µM
- 20. ATP Determination Figure 4: percentage of ATP change of treated and non-treated cells • ↓ % of ATP change • K562/Dox cells • ↓ mitochondrial activity • Less energetic state 20 AMS Symposium, 2022 LDR and GA 10 µM LDR and GA 100 µM
- 22. Reduction in SDH activity, ∆Ψm, ATP Significant effect in Drug-resistant type Mitochondrial impairment Enhance cellular energetic damage Potential of Gallic acid-induced energetic damage Multidrug resistant cancer treatment Gallic Acid 10 and 100 µM 22 AMS Symposium, 2022 MDR LDR Low dose radiation (0.02, 0.05, 0.1 Gy) LDR Leukemic cell lines Drug-sensitive (K562) & Drug-resistant (K562/Dox)
- 23. References • https://gco.iarc.fr/today/ • https://seer.cancer.gov/statfacts/html/leuks.html • https://www.mdpi.com 23 AMS Symposium, 2022
- 24. • Advisor: Associate Professor Dr. Montree Tungjai • Associate Professor Dr. Suchart Kothan • Dr. Singkome Tima • Ms. Benji • Seniors and friends • CMU Presidential Scholarship Program 24 AMS Symposium, 2022
- 25. 25 Shwedagon Pagoda, Yangon, Myanmar AMS Symposium, 2022
- 26. Mitochondrial membrane potential (MMP) Principle Rho B+ Rho B+ Rho B MTT MTT Formazan Rho B+ Rho B+ Rho B+ + + + - - - Accumulation of Rho B in mitochondrial is related to MMP Formazan quench FI of Rho B in mitochondrial Extracellular Intracellular Mitochondrial 26
- 27. Mitochondrial membrane potential Slope = Vi Rho B (40nM) MTT (200 µM) Cells Time FI 27
- 28. Introduction Low Dose Radiation Hypersensitivity Less damage repair (DNA & cell cycle arrest) P53 stimulation ↓ Antiapoptotic genes and NF-КB Damage propagation through cell cycle Mitotic cell death P-53 independent apoptosis ↑DNA DSB 28
- 29. LDR in cell biology Radiation Dose Response Models 29
- 30. LDR to Normal Cells LDR ≤ 0.1 Gy Rae 1 upregulation Adaptive Response 30
- 31. LDR to Immune System 31
- 32. Inhibit Thymidine kinase population about 4 or 5 hours More time for DNA repair/recover induced by initial radiation Reduced propagation of genomic instability Free radical detoxification Free radical scavengers production reduction of free radicals concentration LDR ≤ 0.1 Gy LDR to reduce genomic instability 32
- 33. Bystander Effect (RIBE) • Cellular damage in non-irradiated neighboring cells near to irradiated cells • Diffusion of soluble molecules (ROS, CO, Ca2+ etc) into the medium cause of proteolysis 33
- 34. LDR MDR cells (P53 mutation) ROS production P53 tumor suppressor genes activation P53 tumor suppressor gene expression Bax-independent pathway Fas upregulation Mitochondrial Apoptotic Pathway Germ cells LDR in Tumor Treatment LDR 34
- 35. SDH + + + + - - - - Ym ATP P-glycoprotein (Kinetic) DNA ROS IR 1.5 h post, with / without GA THP Cell Death Cell Cycle arrest Apoptosis pHi
- 36. SDH + + + + - - - - Ym ATP P-glycoprotein (Kinetic) DNA THP Vary THP, GA inhibit 10,50,100 uM GA induce Mito damage Cell Death Cell Cycle arrest Apoptosis pHi
- 37. P-glycoprotein/ P-gp Introduction Cell membrane proteins, energy-dependent mechanism 170 kDa transmembrane glycoprotein which contains 10-15 kDa of N-term glycosylation Two nucleotide-binding domains (NBD) and two transmembrane domains (TMD) 37
- 38. P-glycoprotein/ P-gp https://doi.org/10.1533/9781908818317.115 Figure: Possible mechanism for ATP-binding cassette (ABC) protein-mediated transport. Introduction 38
- 39. Introduction Overcome MDR There are two main strategies leading to increased intracellular concentration of antitumor drugs in MDR cells: (1) the development of resistance reversing agents (chemosensitizers) that can inhibit the transporter-mediated efflux of antitumor drugs. (2) the development of antitumor agents that will be retained by the MDR cell at a concentration sufficient to inhibit cell proliferation. 39
- 40. Low Dose X-irradiation (0.02, 0.05 , 0.1 Gy) Cell Viability Assay SDH Activity MMP (∆Ψm) measurement ATP determination Statistical analysis 40 Leukemia cells K562 K562/Dox Gallic Acid ( 10, 100 µM) AMS Symposium, 2022
Editor's Notes
- GA: C₆H₂(OH)₃CO₂H/ C7H6O5
- erythroleukemia type, derived from a 53-year-old female CML patient, to maintain resistance, 400 nM of Doxorubicin in 30-day cycle
- At irradiation, the cells are placed in the center of an x-ray beam at XXX cm from the x-ray tube. A medical diagnostic x-ray machine operated at 100 kV, 200 mA is used. Table shows the parameters obtained by this medical x-ray machine to generate radiation dose at 0.02, 0.05 or 0.1 Gy.
- Resazurin assay: 0.1 mg/ml (4 hr incubation)/ to determine cell viability. Resazurin (blue and non-fluorescent) is reduced by dehydrogenase enzymes in living cells to form the red fluorescent dye resorufin. The amount of resorufin can be monitored by measuring fluorescence or absorbance, proportional to the number of living cells in the sample.
- MTT assay : 200 uM concentration, 4 hr incubation, MTT reduced by mito SDH, purple formazan. For mitochondrial activity, by measuring activity of mitochondrial enzymes such as succinate dehydrogenase. In this assay, MTT is reduced to a purple formazan by NADH. This product can be quantified by light absorbance. Treated cells will be incubated with MTT solution. After 20 mins, DMSO is added and absorption intensity is recorded at 560 nm.
- Luckhoff buffer: HEPES-Na+ buffer, pH 7.25, 37’C, Rho B 40nM …2o mins later, MTT 200 uM. The principle of MMP determination by rhodamine B. Cell can be divided by extracellular, intracellular and mitochondria. Rho-B enters cell by passive diffusion, cytosolic and enters mitochondria. Accumulation of Rho B in mitochondria is related to MMP. After addition MTT, MTT enters Mito, SDH reductase to Formazan, that can Quench Rho-B fluorescence in mitochondria. The fluorescence of RhoB can be used to calculate MMP.
- For ATP determination, Cells will be centrifuged and discard supernatant. Then, vortex cell pellets after 60’C boiling water addition, cell destruct and intracellular ATP is released. Use supernatant, purify ATP from other molecules with thin layer chromatography. The ATP will be determined by fluorescence technique. Mobile phase, Butanol: Acetic acid: Water – 4:1:5 v/v
- The principle of MMP determination by rhodamine B or Rho B. Cell can be divided by extracellular, intracellular and mitochondria. Rho-B enters cell by passive diffusion, cytosolic and enters mitochondria. Accumulation of Rho B in mitochondria is related to MMP After addition MTT, MTT enters Mito, SDH reductase to Formazan, that can Quench Rho-B fluorescence in mitochondria. The fluorescence of RhoB can be used to calculate MMP.
- Cells (2x10^6) ’re incubated in 2 ml of HEPES-Na1 buffer with 40 nM rhodamine B in 1 cm quartz cuvette and vigorously stirred at 37 °C. The rhodamine B fluorescence at 582 nm (excited at 553 nm) was monitored as a function of time. After 20 min of incubation, 200 uM MTT was added to the solution, yielding a progressive decrease in rhodamine B fluorescence. The slope of rhodamine B fluorescence decrement after addition MTT is Vi, the initial rate of the decrease of rhodamine B fluorescence intensity. The MMP can be calculated by using this equation….Nernst Equ:
- Retinoic acid early inducible 1 (RAE-1) family of murine cell surface glycoproteins ; Rae 1 combines with NKG2D
- Proteolysis is important for generation of numerous biologically active molecules (growth factors and cytokines)
- LDR in purinergic signalling : depend on tumor types (cell death or cell proliferation)
- P-glycoprotein or P-gp is Cell membrane protein, functioning in energy-dependent mechanism. 170 kDa transmembrane glycoprotein which contains 10-15 kDa of N-term glycosylation. - The Membrane topology of P-glycoprotein is displayed in the slide. There are Two nucleotide-binding domains (NBD) and two transmembrane domains (TMD).
- The propose mechanism of P-gp is shown in the slide. Fig 1. Drug passively diffuses into the cell. 2. and 3. P-gp bind with drug and ATP bind with ATP binding site on P-gp 4. Then P-gp efflux drug out of the cells by using ATP hydrolysis.
- To overcome multidrug resistance. There are two main strategies to overcome MDR by increased intracellular concentration of antitumor drugs in MDR cells include; (1) the development of resistance reversing agents that can inhibit the transporter-mediated efflux of antitumor drugs. (2) the development of antitumor agents that will be retained by the MDR cell at a concentration sufficient to inhibit cell proliferation.
- Resazurin= reduction of non-fluorescent dye resazurin to fluorescent dye resorufin via mitochondrial reductase