wei lu & fiona 25 feb 2015 fyp presentation! np
TRANSCRIPT
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Cultivation and Screening of Microalgae Isolates for Anti-Tumour Bioactive Compounds
Chin Wei LuLim Wei Xin Fiona SUPERVISOR: Dr. New Jen YanCO-SUPERVISOR: Dr. Charmaine Lloyd
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Microalgae Microalgae are photoautotrophic, unicellular
algae cells
Being explored as alternative sources, over terrestrial plants, of high-value products, such as renewable biofuels and nutritional supplements (e.g. dietary anti-oxidants, vitamins)
Great diversity
Simple growth requirements
High growth efficiencies
Mass cultivation offshore
Introduction
Lugol’s iodine-stained microalgae isolated from local water bodies
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Nitrogen-deficient cultivation increased lipid content in Chlorella vulgaris microalgae from 14.5% to 24.6% of dry weight (Mujtaba et al., 2012)
Ultraviolet-A irradiation increased both lipid content and degree of unsaturation, in Nitzschia closterium (Bacillariophyceae) and Isochrysis zhangjiangensis (Chrysophyceae) microalgae (Huang and Cheung., 2011)
Introduction
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Flexibility to alter biomass composition
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Introduction
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Anti-cancer activity in
macroalgae
Reduction in tumour size of Agrobacterium tumefaciens-infected potato discs, after treatment with ethanol crude extracts from Jania rubens algae (Ibrahim et al., 2005)
Jania rubens red algae
Pepsin-digested extracts from Caulerpa microphysa algae induced tumour shrinkage in immunocompromised mice transplanted with human leukaemia cell lines (Lin et al., 2012)
Caulerpa microphysa algae
http://www.umema.it/Alghe/album/Rosse/slides/02%20Jania%20Rubens.html
http://biogeodb.stri.si.edu/pacificalgae/specie/19
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I. To determine if crude extracts from local water bodies-originated microalgae species (vs38, vs88, vs31 and KK6) have anti-tumour effects against cancerous basophils KU812 and oestrogen receptor-negative breast cancer cell line MB231
II. To compare the efficacy of these crude extracts with anti-cancer antibiotic Actinomycin-D in inhibiting KU812 and MB231 proliferation
Objectives
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Cultivation of microalgae strains• 4 morphologically-distinct
microalgae strains isolated from local water bodies
• Incubated in an enclosed room at (25±1)⁰C with illumination
• Half of the respective algal cultures were irradiated under UV-C for 4 hours (with swirling every 10 minutes) -> 24 hours recovery timevs38
KK6
vs88
vs31
Materials and Methods
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Harvesting of microalgae strains Washing -> Lyophilisation -> Solvent
extraction (Hexane & Ethanol & dH2O)-> Ultra-sonication -> Vortex with micro glass beads -> Incubation -> Evaporate to dryness
Mass of dried extracts were recorded
Re-dissolve dried extracts in DMSO to achieve a Master stock of microalgae extract(2000mg/mL)
Materials and Methods
Microtube
Glass tube
2000mg/mL6
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Anti-tumour test for microalgae crude extracts and Actinomycin-D
1. Cancer cells were seeded at a density of 2.0x105 living cells/mL in each well on a 96-well plate, in replicates of five for each working concentration
2. Cells then incubated at 37◦C, 5% CO2 for 24 hours to acclimatise/adhere to substratum
3. Cells then added with microalgae crude extract master stocks, to the following working concentrations:
o 2.0mg/mLo 1.0mg/mLo 0.5mg/mLo 0.25mg/mLo 0.0625mg/mL
Materials and Methods
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4. Treated cells then incubated at 37◦C, 5% CO2 for 48 hours
5. Cell viability at each microalgae crude extract working concentration assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay
Anti-tumour test for microalgae crude extracts and Actinomycin-D
Statistical analysis Significant differences in cell viability between
samples were tested for using Student’s t-test for non-paired samples and Mann-Whitney-U test
Materials and Methods
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Negative controlCancer cells cultured in pure culture medium only
Safe DMSO controlCancer cells cultured in 0.1% (v/v) DMSO
MTT controlCancer cells cultured in 50% (v/v) DMSO
Materials and Methods
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Analysis of results Growth inhibitory or stimulatory effects of microalgae crude
extracts assessed only at 0.25mg/mL working concentration
Growth stimulatory effect population of living cancer cells in microalgae test is significantly larger than in negative control
Growth inhibitory effect population of living cancer cells in microalgae test is significantly smaller than in negative control
Materials and Methods
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Growth inhibition efficiency =
[(cell viability in negative control – cell viability in microalgae test)/cell viability in negative control] x 100%
MB231 cell viability in stressed and non-stressed microalgae vs88 distilled water crude extract tests, and in their respective negative controls, after 48-hour exposure (representative bar chart)
Materials and Methods
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Viable breast cancer MB231 cells (after MTT assay) seen under microscope. LEFT: incubation with inhibitory microalgae crude extract. RIGHT: negative control
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Results and Discussion
Effects of test microalga on cancerous basophils KU812, BY STRAIN
Y-axis = Population of living cancer cells presentBlue – test microalgae crude extractPale blue – negative control 12
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Results and DiscussionEffects of test microalga on breast cancer cell line MB231, BY STRAIN
Dark grey – test microalgae crude extractPale grey – negative controlY-axis = Population of living cancer cells present 13
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Growth of cancerous basophils KU812 is significantly inhibited by microalgae strain vs31, but not by vs38, vs88 and KK6.
Growth of breast cancer cell line MB231 is significantly inhibited by microalgae strains vs88 and vs38, but not by vs31 and KK6.
1) Microalgae strains vs31 and vs88 are promising candidates for further exploration into their anti-tumour potentials against cancerous basophils and breast cancer respectively
Possible implicationsResults and Discussion
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Possible implications2) Modes of action of anti-tumour bioactive compounds
from vs31, vs38 and vs88 possibly specific and targeted
Since proliferation of cancerous basophils KU812 and breast cancer cells MB231 each inhibited by different microalgae strains
Interference only with oncogenic events pertaining to respective cancer cell line, not general proliferative mechanisms (e.g. disruption of DNA replication) (National Cancer Institute, 2014)
Results and Discussion
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Test microalga with growth inhibitory effects, BY EXTRACTION SOLVENT
Hexane crude extract
Ethanol crude extract
Distilled water crude extract
Results and Discussion
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Possible implications3) Anti-tumour bioactive compounds from vs38, vs88 and vs31
are likely of medium to high chemical polarity
12 out of 15 microalgae crude extracts inhibiting cancer growth were isolated using polar ethanol and distilled water extraction solvents
Extraction solvents “capture” compounds of like polarity
Results and Discussion
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37.1% 13.4%
30.4% 20.8% 34.2% 18.9%
Results and Discussion
Effects of test microalga on cancerous basophils KU812, BY GROWTH CONDITIONS
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Non-stressedStressed
Non-stressedStressed StressedNon-stressed
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Results and Discussion
Effects of test microalga on breast cancer cell line MB231, BY GROWTH CONDITIONS
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Possible implications4) Bioactive compounds inhibiting KU812 and MB231 cancer
proliferation from vs31, vs38 and vs88 likely NOT lipids in nature
Non-stressed crude extracts should theoretically comprise smaller lipid content than stressed counterparts, due to lack of ultraviolet-C irradiation
Greater proportion of non-lipids stronger growth inhibition efficiencies
Results and Discussion
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Nature of microalgae crude extracts (inhibits or stimulates cancer cell growth) on KU812 cell line (purple) and MB231 cell line (blue)
Results and Discussion
Test microalga with growth stimulatory effects, BY GROWTH CONDITIONS
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5) Lipids may play an assistive role in cancer cell proliferation
Crude extracts from stressed microalgae should theoretically comprise greater lipid content from ultraviolet-C irradiation
Greater abundance of lipids in stressed crude extracts translated to poorer cancer growth inhibition
Findings corroborate other literature reporting growth-stimulating effects of lipids on cancerous tumours (Baenke et al., 2013, Huang and Cheung., 2011)
Signalling molecules for cancer cell proliferation, migration, angiogenesis
Raw material for membrane synthesis and energy generation Unsaturated lipids increase cell membrane permeability and
nutrient intake
Possible implicationsResults and Discussion
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Microscopic observationFigure 3.9
Anti-tumour effect determined at 0.25mg/mL extract exposure -> could be clearly observed using microscopy at 2mg/mL extract exposure
Results and Discussion
2mg/mL vs31 stressed Ethanol
2mg/mL KK6 stressed Ethanol
Complete medium only
0.1% (v/v) DMSO only [safe dose]
KU812
2mg/mL vs88 stressed dH2O
2mg/mL KK6 stressed dH2O
Complete medium only
0.1% (v/v) DMSO only [safe dose]
MB231
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Anti-tumour test with Actinomycin-D The smaller the IC50 values, the greater the
potency of a particular compound or drug.
Gentler gradient= Wider therapeutic window=adverse drug event unlikely to be observed with subtle changes in drug concentration.
Steeper gradient= Narrower therapeutic windows adverse drug event likely to occur with subtle changes in drug concentration.
Results and Discussion
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Anti-tumour test with Actinomycin-D
Figure 3.11
MB231 exposed to..
Gradient IC50 value
Stressed vs31 dH2O crude extract
-3.72 %.mg-1.mL 9.38 x 1010 mg/mL
Actinomycin-D -20.8 %.mg-1.mL 3.81mg/mL
Wider therapeutic window, Less potent
Results and Discussion
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Anti-tumour test with Actinomycin-D
-D
-D
-D
-D
-D
-D
Non-stressed vs31 KU812 Hexane
Stressed vs31 KU812 Hexane
Non-stressed vs31 KU812 Ethanol
Non-stressed vs31 KU812 dH2O
Stressed vs31 KU812 Ethanol
Stressed vs31 KU812 dH2O
Figure 3.12
Results and Discussion
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Gradient of Actinomycin-D > microalgae crude extract
-D
-D
-D
-D
-D
Non-stressed vs31 KU812 Hexane
Stressed vs31 KU812 Hexane
Non-stressed vs31 KU812 Ethanol
Stressed vs31 KU812 Ethanol
Stressed vs31 KU812 dH2O
Figure 3.12
KU812 exposed to..
Gradient IC50 value
Non-stressed vs31 Hexane crude extract
-8.45 %.mg-1.mL
0.554mg/mL
Non-stressed vs31 Ethanol crude extract
-9.11%.mg-1.mL
93.8mg/mL
Stressed vs31 dH2O crude extract
-2.46%.mg-1.mL
1.00 x 1011mg/mL
Actinomycin-D
-14.8 %.mg-1.mL
13.9mg/mL
Narrowest therapeutic window
Most potent
Results and Discussion
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Gradient of Actinomycin-D < microalgae crude extract
-D
-D
Stressed vs31 KU812 Hexane
Stressed vs31 KU812 Ethanol
Figure 3.12
KU812 exposed to..
Gradient IC50 value
Stressed vs31 Hexane crude extract
-17.4 %.mg-1.mL
22.5mg/mL
Stressed vs31 Ethanol crude extract
-44.1%.mg-1.mL
0.812mg/mL
Actinomycin-D
-14.8 %.mg-1.mL
13.9mg/mL
Most potentNarrowest therapeutic window
Results and Discussion
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Anti-tumour test with Actinomycin-D
Non-stressed vs31 KU812 dH2O
KU812 exposed to..
Gradient IC50 value
Non-stressed vs31 dH2O crude extract
+16.9 %.mg-1.mL
3.35 x 10-2mg/mL
Actinomycin-D
-14.8 %.mg-1.mL
13.9mg/mL
Figure 3.12
• At 0.25mg/mL of the extract significantly depicted that it inhibited the proliferation of KU812
• However, a positive correlation observed between cell viability & concentration of the extract
• High amount of bioactive compounds which had promoted cellular proliferation
Results and Discussion
-D
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Anti-tumour test with Actinomycin-D
-D
-D
-D
-D
-D
-D
Non-stressed vs31 KU812 Hexane
Stressed vs31 KU812 Hexane
Non-stressed vs31 KU812 Ethanol
Non-stressed vs31 KU812 dH2O
Stressed vs31 KU812 Ethanol
Stressed vs31 KU812 dH2O
Figure 3.12
• 50% tested microalgae extracts on KU812 cell line (B, C and F) displayed higher IC50 values than Actinomycin-D Crude Concentration of anti-tumour metabolites smaller
per unit volume
• Other 50% of the tested microalgae extracts on KU812 cell line (A, D and E) revealed smaller IC50 values than Actinomycin-D Crude High concentration of anti-tumour metabolites
OR Small concentration of anti-tumour metabolites had
targeted a critical apoptotic cascade-> greater effect in inhibiting KU812 growth
Results and Discussion
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• Identity of components exhibiting growth inhibitory effects could have been conclusively verified using chromatography
• Prospective use of microalgae crude extracts as anti-tumour therapy in humans could have been substantiated by conducting cytotoxicity tests on non-transformed human cell lines
• Examining local microalgae species for anti-oxidant properties and potential to repress malignant transformation and cancer onset
Future Work
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Summary
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Investigating anti-tumour effects from microalgae isolated from Singapore’s water bodies
By Strain
KK6 (NIL)
vs31 inhibited basophil leukemic cells
vs38 & vs88 inhibited breast cancer cells
By Extraction Solvent
12/15 are ethanol & dH2O
Anti-tumour bioactive compounds = medium to high chemical polarity, with targeted modes of action
3/15 are hexane
Lipids
By Growth Condns
Stressed
Non-stressed
Lipids
Side Proje
ctVarying potencies & therapeutic windows as compared to those of Actinomycin-D
Possible clinical treatment of cancer with local
microalgae strains in future
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Thank you.
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Baenke, F., Peck, B., Miess, H., and Schulze, A., 2013. Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development. Disease models and mechanisms, 6(6), pp. 1353-1363.
Boopathy, N. S., and Kathiresan, K., 2010. Anticancer Drugs from Marine Flora: An Overview. Journal of Oncology, Volume 2010, pp. 1-18.
Huang, J.-h. J., and Cheung, C.-K. P., 2011. +UVA treatment increases the degree of unsaturation in microalgal fatty acids and total carotenoid content in Nitzchia closterium (Bacillariophyceae) ad Isochrysis zhangjiangensis (Chrysophyceae). Food Chemistry, Volume 129, pp. 783-791.
Ibrahim, A. M. M., Mostafa, M. H., El-Masry, M. H. and El-Naggar, M. M. A., 2005. Active biological materials inhibiting tumour initiation extracted from marine algae. Egyptian Journal of Aquatic Research, Volume 31(1). pp. 146-155.
Lin, H. C., Chou, A. T., Chuang, M. Y., Liao, T. Y., Tsai, W. S., and Chiu, T. H., 2012. The effects of Caulerpa microphysa enzyme-digested extracts on ACE-inhibitory activity and in vitro anti-tumour properties. Food Chemistry, Volume 134. pp. 2235-2241.
Mujtaba, G., Choi, W., Lee, C.-G., and Lee, K., 2012. Lipid production by Chlorella vulgaris after a shift from nutrient-rich to nitrogen starvation conditions. Bioresource Technology, Volume 123, pp. 279-283.
National Cancer Institute, 2014. Targeted Cancer Therapies. [Online] Available at: http://www.cancer.gov/cancertopics/factsheet/Therapy/targeted[Accessed 12 December 2014]. Singh, S., Kate, B. N., and Banerjee, U. C., 2005. Bioactive Compounds from Cyanobacteria and microalgae: An Overview. Critical Reviews in Biotechnology, Volume 25, pp. 73–95
Bibliography
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Cultivation & harvesting of microalgae strains Mass of dried extracts were recorded
Re-dissolve dried extracts in DMSO to achieve a Master stock (2000mg/mL) before serial dilutions to produce 4 more Master stocks:
2000mg/mL
1000mg/mL
500mg/mL
250mg/mL 62.5mg/mL
Materials and Methods
Microtube
Glass tube
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Microscopic observation
KU812 Comparison
MB231 Comparison
A B
G H
C D E
I J K
Figure 3.9
• Cells were viewed under 400x magnification using Olympus CK40 inverted microscopy
• Comparison of the cell morphology/cell confluency & presence of dark blue precipitate inside cells after incubation with MTT for~3hours
• C and I are negative controls (exposed to complete medium only)
• D and J are “Safe-DMSO” controls
• E and K are “Lethal-DMSO” controls
Effectiveness of microalgae extracts on cancer cells?
Cell type Effective IneffectiveKU812 A B
2mg/mL vs31 stressed Ethanol
2mg/mL KK6 stressed Ethanol
MB231 G H2mg/mL vs31 stressed dH2O
2mg/mL KK6 stressed dH2O
• Anti-tumour effect determined at 0.25mg/mL extract exposure -> could be clearly observed using microscopy at 2mg/mL extract exposure
Results and Discussion
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Microalgae strains vs31, vs38 and vs88 displayed anti-cancer properties, except for KK6
Anti- tumour bioactive compounds likely of medium to high chemical polarity, with targeted modes of action
Anti-tumour bioactive compounds unlikely to be lipids-based
The peculiar case of non-stressed vs31 distilled water against KU812 (Figure 3.12E)
Varying potencies & therapeutic windows as compared to
those of Actinomycin-D
Possible clinical treatment of cancer with local microalgae strains in future
Conclusions
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