heme oxygenase 2012 anti cancer effects of plant tetrapyrroles
TRANSCRIPT
KonKonííččkovkováá R, VaR, Vaňňkovkováá 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
Effects of plant tetrapyrroliccompounds on pancreatic
cancer and heme oxygenaseactivity
Effects of plant tetrapyrroliccompounds on pancreatic
cancer and heme oxygenaseactivity
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 Cycle2007;6:3078-85.
Jiraskova A at al. Association of serum bilirubin and promoter variations in HMOX1 andUGT1A1 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 transplacentalcarcinogenesis. 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.
HypothesesHypothesesDoes Spirulina platensis have antiproliferativeeffects 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.
HypothesesHypothesesDoes Spirulina platensis have antiproliferativeeffects 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 mitochondrialmembrane potential analogously to bilirubin?
MethodsMethodsIn vitro studies on pancreatic cancer cell lines PaTu-8902, BxPC-3, MIA PaCa-2 exposed to various tetrapyrroliccompounds.
Commercial tetrapyrroles used except for phycocyanobilinwhich 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) andHMOX1/BLVRA mRNA expressions were determined
The effect of tetrapyrroles on mitochondrial membranepotential determined by fluorescent dye (JC-1)
In vivo studies on athymic mice xenotransplanted with humanpancreatic 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
0
20
40
60
80
100
120
140
0,08 0,16 0,31 0,63 1,25 2,50koncentrace g/L
viab
ilita
buněk
v %
PA-TU-8902
MIA PaCa-2
BxPC-3
*p<0.05, Kruskal-Wallisovo srovnání s post-hoc testováním, viabilita určena na základě MTT eseje
*
*
*
*
*
**
*
*p<0.05, Kruskal-Wallis test with post-hoc analysisViability determined by MTT test
concentration [g/L]
Cell viability
[%]
*
*
Effect of oral intake of S. platensison pancreatic ca growth
Effect of oral intake of S. platensison pancreatic ca growth
0,15
0,25
0,35
0,45
0,55
0,65
0,75
0,85
0 4 7 10 14 17 20days
Tum
or s
ize
(cm
3 )
placebo
Spirulina
Data analyzed by RM ANOVA on Ranks with post-hoc testing
12 athymic mice (CD1 strain) sc. transplanted with PaTu-8902 pancreatic ca cell line6 mice treated daily with S. platensis (0.5 g/kg for 17 days)
Spirulina-treated placebo-treated
Are plant tetrapyrroles responsible forobserved antiproliferative effects?
Are plant tetrapyrroles responsible forobserved antiproliferative effects?
Vliv fykokyanobilinu na viabilitu nádorových linií karcinomu pankreatu
0
20
40
60
80
100
120
140
31,3 62,5 125 250 500
koncentrace μmol/L
viab
ilita
buněk
v %
PA-TU-8902MIA PaCa-2
BxPC-3
*p<0.05, Kruskal-Wallisovo srovnání s post-hoc testováním, viabilita určena na základě MTT eseje
*
*
*
*
Vliv bilirubinu na viabilitu nádorových linií karcinomu pankreatu
0
20
40
60
80
100
120
5 10 50 100koncentrace μmol/L
viab
ilita
bun
ěk v
%
PA-TU-8902
MIA PaCa-2
BxPC-3
*p<0.05, Kruskal-Wallisovo srovnání s post-hoc testováním, viabilita určena na základě MTT
**
* **
**
**
Phycocyanobilin Effect of chlorophyllin on pancreatic cell lines viability
020406080
100120140
31 63 125 250 500
concentration μmol/L
cell
viab
ility
%
PA-TU-8902MIA PaCa-2BxPC-3
*p<0.05, Kruskal-Wallis comparison w ith post-hoc testing, viability w as determinated by MTT assay
***
***
p<0.05, Kruskal-Wallis test with post-hoc analysis, viability determined by MTT test
Chlorophyllin
concentration [μmol/L] concentration [μmol/L]
concentration [μmol/L]
Cell viability
[%]
Cell viability
[%]
Cell viability
[%]
BilirubinB
Plant tetrapyrroles and heme oxygenasePlant tetrapyrroles and heme oxygenasePaTu‐8902 HMOX activity
100% 167% 100% 92% 48% 94%0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
control 1 hemin 30 uM
control UCB 10uM
chlorophyllin30uM
PCB30uM
HO activity (%
of control)
2
HMOX1 mRNA expression
HMOX activity measured as CO production by GC-reduction gasdetector (pmol CO/hr/mg protein), qPCR analyses performed on Applied Biosystems ViiA7instrument
Inhibitory effects of chlorophyllinobserved 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 potential12 hr incubation ofhepatoblastoma andpancreatic ca cellswith therapeuticsAddition of JC-1 fluorescent dye(Invitrogen, 20´)Determination ofMMP as a relativeproportion ofprecipitated and freedye
0.74PCB (10 uM)
1.29PCB (5 uM)
2.91PCB (1 uM)
3.29PCB (0.5 uM)
6.30controlPaTu‐8902
3.54S. platensis extrakt (0.3 mg/ml)
3.55S. platensis extract(0.1 mg/ml)
4.14S. platensis extract(0.03 mg/ml)
0.55chlorophyllin (30 uM)
1.34chlorophyllin (3 uM)
5.65controlHepG2
MMPInterventionCell line
MMP: mitochondrial membrane potential; PCB: phycocyanobilin
ConclusionsConclusionsOral administration of Spirulina platensis has potentantitumor effect on experimental human pancreatic cancerIn vitro, both chlorophylls and phycocyanobilindemonstrated potent antiproliferative effects on pancreatic cancer cellsChlorophylls substantially modulated HMOX activity in cancer cells, which might contribute to their putativeantiproliferative actionChlorophylls, as well as phycocyanobilin at lowconcentrations inhibited mitochondrial membranepotential, which might also account for their anticancereffects