Microsoft PowerPoint - Heme oxygenase 2012 Anti cancer effects of plant tetrapyrroles.pptKonKonííkovkováá R, VaR, Vakovkováá K, VanK, Vanííkovkováá J, J, Zelenka J, Zelenka J, SubhanovSubhanováá I, MuchovI, Muchováá L, L,
ZadinovZadinováá M, M, VVíítek Ltek L
11stst FacultyFaculty of of MedicineMedicine, , Charles University in PragueCharles University in Prague
Czech Czech RepublicRepublic
cancer and heme oxygenase activity
Effects of plant tetrapyrrolic compounds on pancreatic
cancer and heme oxygenase activity
Tetrapyrroles in blue-green/red algaeTetrapyrroles in blue-green/red algae
Spirulina platensis (Cyanophyta, phycocyanins) 3,5 billion years old!
Tetrapyrroles in red algae (Rhodophyta - Cyanidium caldarium, Porphyridium cruentum): phycoerythrins
Tetrapyrroles in red algae (Rhodophyta - Cyanidium caldarium, Porphyridium cruentum): phycoerythrins
Chlorophyllin
Phycoerythrobilin
Phycocyanobilin
AnticancerAnticancer effectseffects of bilirubin, of bilirubin, phycocyanobilinphycocyanobilin andand chlorophyllschlorophylls
Keshavan P et al. Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization. Int J Cancer 2004;112:433-45.
Ollinger R et al. Bilirubin inhibits tumor cell growth via activation of ERK. Cell Cycle 2007;6:3078-85.
Jiraskova A at al. Association of serum bilirubin and promoter variations in HMOX1 and UGT1A1 genes with sporadic colorectal cancer. Int J Cancer 2011 [Epub]
Ismail MF et al. Chemoprevention of rat liver toxicity and carcinogenesis by Spirulina. Int J Biol Sci 2009;5:377-87.
Castro DJ et al. Identifying efficacious approaches to chemoprevention with chlorophyllin, purified chlorophylls and freeze-dried spinach in a mouse model of transplacental carcinogenesis. Carcinogenesis 2009;30:315
Balder et al. Heme and chlorophyll intake and risk of colorectal cancer in the Netherlands cohort study. Cancer Epidemiol Biomarkers Prev 2006;15:717-25.
HypothesesHypotheses Does Spirulina platensis have antiproliferative effects on human pancreatic cancer? Can algeal tetrapyrroles account for these antiproliferative effects? Do algeal tetrapyrroles affect HMOX activity in a way analogous to metaloporphyrins?
Drummond and Kappas. Prevention of neonatal hyperbilirubinemia by tin protoporphyrin IX, a potent competitive inhibitor of heme oxidation. PNAS 1981;78:6466-70.
HypothesesHypotheses Does Spirulina platensis have antiproliferative effects on human pancreatic cancer? Can algeal tetrapyrroles account for these antiproliferative effects? Do algeal tetrapyrroles affect HMOX activity in a way analogous to metaloporphyrins? Do algeal tertrapyrroles effect mitochondrial membrane potential analogously to bilirubin?
MethodsMethods In vitro studies on pancreatic cancer cell lines PaTu-8902, BxPC-3, MIA PaCa-2 exposed to various tetrapyrrolic compounds.
Commercial tetrapyrroles used except for phycocyanobilin which was isolated from freeze-dried S. platensis (Martin Bauer, GmbH, Germany, isolation method adopted from Smith A.G., Witty M.: Heme, chlorophyll, and bilins - Method and protocols.
Humana Press 2002) and verified by MS and NMR spectra
HMOX activity (measured as CO production by GC) and HMOX1/BLVRA mRNA expressions were determined
The effect of tetrapyrroles on mitochondrial membrane potential determined by fluorescent dye (JC-1)
In vivo studies on athymic mice xenotransplanted with human pancreatic cancer treated with S. platensis (PaTu-8902)
Effect of S. platensis extract on viability of human pancreatic cancer cell lines
Effect of S. platensis extract on viability of human pancreatic cancer cell lines
Vliv extraktu ze Spiruliny platensis na viabilitu nádorových linií karcinomu pankreatu
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*p<0.05, Kruskal-Wallis test with post-hoc analysis Viability determined by MTT test
concentration [g/L]
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Effect of oral intake of S. platensis on pancreatic ca growth
Effect of oral intake of S. platensis on pancreatic ca growth
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Data analyzed by RM ANOVA on Ranks with post-hoc testing
12 athymic mice (CD1 strain) sc. transplanted with PaTu-8902 pancreatic ca cell line 6 mice treated daily with S. platensis (0.5 g/kg for 17 days)
Spirulina-treated placebo-treated
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PA-TU-8902 MIA PaCa-2 BxPC-3
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p<0.05, Kruskal-Wallis test with post-hoc analysis, viability determined by MTT test
Chlorophyllin
100% 167% 100% 92% 48% 94% 0%
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control UCB  10uM
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HMOX activity measured as CO production by GC-reduction gas detector (pmol CO/hr/mg protein), qPCR analyses performed on Applied Biosystems ViiA7 instrument
Inhibitory effects of chlorophyllin observed also in other pancreatic (MiaPaCa-2) and prostate ca cells (PC3) BLVRA expression was not affected by any of used substances
UCB: unconjugated bilirubin; PCB: phycocyanobilin
Plant tetrapyrroles and mitochondrial membrane potentialPlant tetrapyrroles and mitochondrial membrane potential 12 hr incubation of hepatoblastoma and pancreatic ca cells with therapeutics Addition of JC-1 fluorescent dye (Invitrogen, 20´) Determination of MMP as a relative proportion of precipitated and free dye
0.74PCB (10 uM)
1.29PCB (5 uM)
2.91PCB (1 uM)
3.29PCB (0.5 uM)
0.55chlorophyllin (30 uM)
1.34chlorophyllin (3 uM)
ConclusionsConclusions Oral administration of Spirulina platensis has potent antitumor effect on experimental human pancreatic cancer In vitro, both chlorophylls and phycocyanobilin demonstrated potent antiproliferative effects on pancreatic cancer cells Chlorophylls substantially modulated HMOX activity in cancer cells, which might contribute to their putative antiproliferative action Chlorophylls, as well as phycocyanobilin at low concentrations inhibited mitochondrial membrane potential, which might also account for their anticancer effects
AcknowledgementsAcknowledgements