C.I. M¨ uller et al. / Leukemia Research 30 (2006) 841–848
96h in 96-well plates (Flow Laboratories, Irvine, CA).Thereafter, 10
l of MTT solution (in 5mg/ml PBS,Roche) was added to each well and incubated for 4hin a humidiﬁed atmosphere at 37
C according to themanufacturer’s protocol. Consecutively, 50
l solubilizationsolution (20% SDS) was added into the wells and incubatedovernight (16h). After culture, cell number and viabilitywere evaluated by measuring the mitochondrial-dependentconversion of the yellow tetrazolium salt MTT to purpleformazan crystals by metabolic active cells. The resultingcolored solution was quantiﬁed at 540nm using an enzyme-linked immunoabsorbent assay reader (ELISA reader,Bio-Rad).
2.4. Cell cycle analysis
) were incubated for 72h either withor without
g/ml). They were collected,washed, suspended in cold 1
PBS, ﬁxed in 75% methanoland stained with propidium iodide (PI). Cell cycle analysiswas performed using the Becton Dickinson Flow Cytometer.
2.5. Assessment of apoptosis by Annexin V assay
Apoptotic cell death was examined by Annexin V-apoptosis detection kit (Pharmingen Inc., San Diego, CA).During early stages of apoptosis, phosphatidylserine (PS)becomes externalized on the outer plasma membrane. Inorder to be able to distinguish between apoptosis andnecrosis, cells were stained with FITC-labeled AnnexinV and propidium iodide (PI). Annexin V binds to theexternalized PS, whereas PI is able to penetrate theincreasingly permeable plasma membrane during necrosisor later stages of apoptosis and binds to cellular DNA.Cells were analyzed by ﬂuorescence activated cell sorting(FACS).Four hematological cell lines (HL-60, U937, Blin-1 andRPMI8226) were treated with
at four differentconcentrations(50,100,150and200
g/ml)for72h.Controlcells and treated cells were analyzed for staining by AnnexinV and PI.
2.6. Assessment of apoptosis by determination of mitochondrial membrane potential
Apoptosis was further investigated by analysis of the mitochondrial membrane potential (
) by JC-1assay according to the manufacturer’s protocol (CellTechnology, MN). JC-1 (5,5
-tetraethylbenzimidazolcarbocyanine iodide) is a lipophiliccationic dye which selectively incorporates into intactmitochondria and undergoes a reversible change in ﬂu-orescence emission from green to red as mitochondrialmembrane potential increases. During apoptosis, loss of themitochondrial membrane potential occurs. In cells with highmembrane potential, the formation of dimers of the dye ispromoted, resulting in red ﬂuorescence. In contrast, in cellswithlowmembranepotential,JC-1isinitsmonomericform,which then ﬂuoresces green. The red/green ﬂuorescenceintensity ratio is used to analyze whether a cell is apoptoticor not. A decrease in red/green ratio indicates an increasein apoptotic cells. Fluorescence of the cells is measuredby ﬂow cytometry. Formation of JC-1 aggregates withinthe mitochondria results in emission of red ﬂuorescencein the 590nm spectrum. If the mitochondriae collapseduring apoptosis, the ﬂuorescence shifts to the green 510nmspectrum.
2.7. Western blot analysis
Total cell lysate (25
g) was electrophoresed on 10–20%SDS-polyacrylamide gel (Bio-Rad, Hercules, CA) andtransferred by electroblotting to a polyvinylidene ﬂuoridemembrane (Pall Corporation, Pensacola, FL). The mem-brane was incubated overnight with the following anti-bodies: anti-p21
(SC-397, 1:500 dilution) and anti-p27
(SC-528, 1:500 dilution), both rabbit polyclonalantibodies from Santa Cruz Biotechnology (Santa Cruz,CA) followed by a secondary horseradish peroxidase-conjugated donkey anti-rabbit antibody (Amersham Bio-sciences). Detection was performed using the SuperSignal
chemiluminescence substrate (Pierce, Rockford, IL). Blotswere also subjected to overnight incubation with amurine monoclonal GAPDH antibody (RDI-TRK5G4-6C5, 1:10,000 dilution; Research Diagnostics, Flanders,NJ) followed by a secondary horseradish peroxidase-conjugated sheep anti-mouse antibody (Amersham Bio-sciences). Western blots were stripped between hybridiza-tionswithstrippingbuffer(10mMTris–HClpH2.3,150mMNaCl).
2.8. Cytospin preparations and staining
Cytospin preparations were performed for seven hemato-logic(NCI-H929,RPMI8226,Blin-1,Nalm-6,Daudi,U937,HL-60) and six solid tumor (ARO, BHP2-7, PC3, DU145,NCEB-1, MCF-7) cell lines after 96h treatment with
g/ml). Slides were consecutively stained withDiff Quik
(Dade Behring, Switzerland) staining solutionaccording to the manufacturer’s protocol.
3.1. HPLC analysis of G. lucidum extract
extract was standardized to 0.15% gan-oderic acid C2 content (Fig. 1a).Fig. 1bshows the HPLC
chromatogram of ganoderic acid C2 standard eluting at aretention time of 32.9min.Fig. 1cshows the
extract spiked with ganoderic acid C2 standard conﬁrmingits presence at the retention time of 32.9min.