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INTERNATIONAL JOURNAL OF Oncology 11: 437-448. 1997
Apoptosis and cell cycle effects induced by extracts
of the Chinese herbal preparation PC SPES
H. DOROTA HALICKA'. BARBARA ARDELT'. GLORIA JUAN'. ABRAHAM
MITTELMAN-'.
SOPHIE CHEN'. FRANK TRAGANOS 1 and ZBIGNIEW DARZYNKIEWICZ
'The Cancer Research Institute. New York Medical College. Valhalla. NY 10595: Zalmen A.
Arlin Cancer Institute.
Valhalla. NY 10595: International Medical Research. Inc.. Brea. CA 9262 1. USA
Received July 1. 1997: Accepted July 14. 1997
Abstract. A herbal preparation denoted PC SPES11 is available in 'natural food' or 'health food'
stores in the United States. This mixture (patent pending, US serial number 08/697,920) consists
of extracts from 8 different herbs, 7 originating from China and one from America, and is sold as
a dietary supplement. Although several components of this herbal mixture were reported to have
antiproliferative and/or antitumor activity little is known about the possible in vitro cytostatic or
cytotoxic properties of the formulation. Composition of PC SPES is standardized by HPLC; the
ethanol extract is characterized by the presence of 6 distinct components, reproducible from
batch to batch. This extract suppressed cell proliferation and reduced the clonogenicity of a
variety of human tumor cell lines, including PC-3 and LNCaP prostate carcinomas, MCF-7 and
T47-D breast carcinomas, SK-N-MC neuroepithelioma, Colo 38 melanoma, U937
histiomonocytic lymphoma, as well as HL-60 and MOLT-4 leukemias. The sensitivity to PC
SPES was different for particular cell lines, with MCF-7 cells being the most sensitive (IC50= 20
nl/ml) and Colo 38 the most resistant (IC50 = 430 nl/ml) in clonogenicity assays. The
___________________________________
Correspondence to: Dr Zbigniew Darzynkiewicz, The Cancer Research- institute, New York,
Medical College 100 Grasslands Road. Elmsford. NY 10523, USA
Abbreviations: HPLC, high performance liquid chromatography; DAPI, 4',6'diamidino-2-phenylindole;
PHA,
phytohemagglutinin; HBSS, Hanks' balanced salt solution; PI, propidium iodide; BSA, bovine
serum albumin; FBS, fetal bovine serum; PBS, phosphate buffered saline; FITC, fluorescein
isothiocyanate; ICO, inhibition of cell clonogenicity by 50%; TdT, terminal deoxynucleotidyl
transferase
Key words: Prostate cancer, Dendrantherma morifolium; Tzvel Ganoderma lucidium; Karst,
Glycyrrhiza glabra L, Isatis indigotica; Fort, Panax pseudo-ginseng, Robdosia rubescens,
Scutellaria baicalensis; Georgi, Serenoa repens
predominant cell cycle effect induced by PC SPES was the prolongation of G, phase. Apoptosis
was observed after exposure of tumor cells to PC SPES for 48 h and longer. PC SPES also
downregulated expression of bcl-2, the gene protecting cells against apoptosis (studied in U937
cells) and sensitized these cells to y-irradiation. The cell cycle progression of mitogen stimulated
human lymphocytes was not affected at PC SPES concentrations which induced cytotoxic and
cytostatic effects in tumor cells. The data indicate that PC SPES is cytostatic and cytotoxic for
different tumor cell lines and modulates the cell's propensity to undergo apoptosis.
Introduction
A variety of herbs with presumed antitumor activity are being sold in 'health food' or 'natural
food' stores. Although some of them are known to contain active, chemically identified,
components. little is known about the cytostatic or cytotoxic activity of the particular composite
herbal mixtures. Two herbal preparations are currently being sold as dietary supplements for
cancer patients by BotanicLab (Brea, CA), SPES (1) and PC SPES (2). The SPES and PC
SPES preparations each contain somewhat different mixtures of herbs. The antitumor and
analgesic properties of these herbal combinations are undergoing clinical testing for the treatment
of different tumors in several hospitals in China where there is anecdotal evidence that symptoms
associated with cancer progression are alleviated in some patients. In the present study we
investigated the effects of extracts of these herbs on a variety of cell lines in vitro. Our choice
of
these preparations for study was influenced by their quality control, namely, routine analysis of
the extracts by HPLC, assuring that each batch has reproducibly identifiable components (see
Results).
Initial studies demonstrated cytotoxic effects of the alcoholic extract of SPES on hepatoma cells
in vitro and suppression of growth of the Bel 7402 hepatoma in nude mice (1). There is also
evidence that many of the individual components of these two preparations have antiproliferative
and/or antitumor activity both in vitro and in vivo (see Discussion). The objective of the present
study was to explore the possible cytostatic or cytotoxic activity of PC
SPES on several tumor cell types in vitro. The data indicate that ethanol extracts of PC SPES
suppress cell proliferation and induce apoptosis. Most interestingly, PC SPES downregulates
expression of bcl-2, the gene regulating the cell's propensity to undergo apoptosis (3,4) and
appears to sensitize cells to radiation.
Materials and methods
PC SPES extracts. PC SPES preparations (obtained from BotanicLab, Brea, CA) are in the
form of gelatin capsules containing powdered herbal extracts consisting of the following herbs:
Dendrantherma morifolium, Tzvel; Ganodernia lucidiurn, Karst; Glycyrrhiza glabra L;
Isatis indigotica, Fort; Panax pseudo-ginseng, Wall; Robdosia rubescens; Scutellaria
baicalensis, Georgi and Serenoa repens (2). The content of a single capsule containing life
herbal mixture was transferred into a 5 ml polypropylene tube containing 1.0 ml of 95% ethanol.
The tube was transferred to a shaker water bath and was agitated for 60 min at 370C. The solid
particles were then sedimented by centrifugation (800 x 10 min), the extract solution filtered
through a 0.22 gm Millipore filter and distributed in 100 ul aliquots into Eppendorf tubes which
were kept at -20 C; the samples were frozen only once before use. The extracts were then
diluted in culture media to the appropriate final concentrations as indicated in the Results. The
solvent alone (95% ethanol) was included in control cultures to obtain a final ethanol
concentration equivalent to that in the respective cultures containing extracts of PC SPES.
HPLC of the 70% ethanol extract was performed on a Shimadsu SC 6A system. Two
solvent pumps containing water (A) and acetonitrile (B) were used to
generate a linear gradient. The components were separated on an Alltech preparatory C18
reverse phase column (250 min x 22 mm). A volume of 500 ul of the stock solution of the herbal
sample was injected into the HPLC system. The detection wavelength was 254 nm based on the
absorption spectrum. An elution flow rate of 4-8 ml/min was applied.
Cells. The following human cell lines were used in the present study: Promyelocytic
leukemic HL-60 (kindly provided by Dr H.A. Crissman of the Los Alamos National Laboratory,
Los Alamos, NM), T-cell leukemic MOLT-4, histiocytic lymphoma U937, breast
adenocarcinoma MCF-7, ductal breast carcinoma T47-D, prostate adenocarcinoma PC-3,
neuroepithelioma SK-N-MC (all obtained from American Type Tissue Collection, Rockville,
MD), melanoma Colo 38 (kindly provided by Dr S. Ferrone of the NY Medical College,
Valhalla, NY) and prostate adenocarcinoma LNCaP (kindly provided by Dr J.W. Chiao of the
NY Medical College, Valhalla, NY). All media, and supplements (FBS, penicillin, streptomycin,
L-glutamine) were obtained from Gibco BRL (Grand Island, NY). The suspension cultures were
passaged by dilution to a cell concentration 2x10^5 cells/ mi. and the experiments were
performed on asynchronous, exponentially growing cells in cultures at cell densities below 10^6
cells/mi. The cells from cultures which grow attached to the flasks' surface were collected by
trypsinization and re-seeded at low cell densities
to maintain continuous, exponential growth. The cultures were periodically tested for
mycoplasma infection by staining of cytocentrifuge preparations with the DNA fluorochrome
DAPI (Molecular Probes. Eugene, OR). Peripheral blood mononuclear cells obtained by
venipuncture from healthy donors were isolated by density gradient separation. The cells were
washed twice with phosphate buffered saline and resuspended at a concentration of' 10" cells/ml
in RPM1 1640 medium containing 1017c FBS and stimulated to proliferate by the addition of-
10 ;.w/ml of PHA (Sigma Chemical Co., St. Louis. NIO). Other details concerning culture
conditions are presented elsewhere (5-7).
Clonogenicity studies. The suppressive effect of PC SPES on the ability of cells to develop
colonies in vitro was studied using MCF-7 and T47-D breast cancer cells. LNCaP and PC-3
prostate cancer cells, Colo 38 melanoma cells and SK-N-MC neuroepithelioma cells. Two
hundred or 2.000 cells were plated per flask (25 ml Falcon flask; Fisher Scientific. Fairlawn, NJ)
and the flasks were incubated for 24 hr at 37 C in 5% CO2 in a humidified atmosphere to allow
the cells to attach. Various aliquots of PC SPES extract were then added into cultures and the
cultures were left undisturbed for an additional 7-8 days. Control cultures were treated with
aliquots of 95% ethanol equivalent to the highest volume of the herbal extract
used in clonogenicity studies. The cells were fixed in a 3:1 (vol/vol) mixture of ethanol and glacial
acetic acid and stained with a 1% solution of crystal violet. The cultures were run in triplicate.
Colonies were counted using a darkfield Quebec colony counter (American Optical. Buffalo,
NY).
Cell proliferation and viability. The individual cell lines were exposed for 24. 48 and 72 h
to
PC SPES extract at concentrations ranging from 0.01 to 4.0 ul per nil of culture medium. Cell
counts were performed manually at each time point by hemocytometer with viability determined
based on the trypan blue exclusion assay.
Effects of PC SPES on cell cycle progression. To determine if PC SPES effected cell
progression through the cell cycle cultures exposed to varying concentrations of the extract for
48 h were harvested, the cells washed with HBSS and fixed in ice-cold 70% ethanol. Aliquots of
fixed cells were rehydrated into HBSS and stained with 1.0 ug/ml DAPI and 10 ug/ml
sulforhodamine 101 (Eastman Kodak, Rochester, NY) dissolved in 10 mM piperazine-N,N-bis-2-ethanesulfonic
acid buffer (Calbiochem, La Jolla, CA) containing 100 mM NaCl, 2 mM
MgCl, and 0.1% Triton X-100 (Sigma) at pH 6.8 as previously described (7). The blue (DNA-specific) DAPI
fluorescence and the red (protein-specific) sulforhodamine fluorescence was
excited with UV light and collected with appropriate filters in an ICP-22 (Ortho Diagnostic,
Westwood, MA) flow cytometer. The data from 0.5 to I.OxIO5 cells were collected for each
treatment and the DNA histograms deconvoluted using Multicycle™ software developed by Dr
P. Rabinovitch of the University of Washington (Seattle, WA) and provided by Phoenix Flow
Systems (San Diego, CA).
Detection of apoptosis
DNA degradation detected by gel electrophoresis: A recently developed method for selective
extraction of degraded DNA from apoptotic cells was used (9). Briefly, untreated control and
PC SPES treated cells were collected by centrifugation and fixed in suspension overnight in 70%
ethanol. The cells were then centrifuged and the ethanol completely removed. The cell pellets (1-2x106
cells), in 0.5 ml Eppendorf tubes, were resuspended in 40 ul of phosphate citrate (PC)
buffer consisting of 192 parts of 0.2 M Na2HPO, and 8 parts 0. 1 M citric acid (pH 7.8) and
agitated on shaker for 30 min at room temperature. After centrifugation at 1000 x for 5 min. the
supernatant was transferred to new tubes and treated with 3 41 of 0.25% Nonidet NP-40
(Sigma) in distilled water followed by 3 ul of a solution of Rnase A
(Sigma: 1 mg/ml also in water). After 30 min incubation at 37,1C. 3 pi of a solution of proteinase
K (I mg/ml: Boehringer Mannheim, Indianapolis, IN) was added and the extract incubated for an
additional 30 min at 3711C. Subsequently, 12 ul of loading buffer (0.25% bromophenol blue,
0.25% xylene cyanol FF, 30% glycerol) was added and the entire contents of the tube
transferred to the gel. Horizontal 0.8% agarose gel electrophoresis was performed at 2 V/cm for
16 h. The DNA in the gels was visualized under UV light after staining with 5 ug/ml of ethidium
bromide (Polysciences Inc., Warrington, PA).
In situ DNA strand break labeling: The DNA breaks occurring as the result of apoptosis were
detected by using exogenous TdT to incorporate fluorochrome labeled dUTP into 3' OH termini
at the break sites (10). In the present study, the incorporation of BrdUTP was detected with
FITC conjugated anti-BrdUrd (APO-BRDUT" kit kindly provided by Phoenix Flow Systems),
as described before (5.11). Briefly, the cells were prefixed in ice-cold 1% methanol-free
formaldehyde in PBS for 15 min on ice. then rinsed in PBS and stored in ice-cold 70% ethanol.
The cells were then centrifuged rinsed in PBS and the pellet resuspended in 50 ul of a solution
containing: 10 ul of reaction buffer (5 x concentrated; I M sodium cacodylate, 125 mM HCI and
125 mg/ml bovine serum albumin), 2.0 ul of BrdUTP stock solution (100 nmoles in 50 ul of 50
mM Tris HCI. pH 7.5); 0.5 uI (12.5 units) of terminal deoxynucleotidyl transferase in storage
buffer (25 units in 1 ul); 5 ul of CoCl2 solution (10 mM stock) and 33.5 uI distilled H20. The
cells were incubated for 40 min at 37°C. Control samples were incubated in the absence of TdT.
The reaction was terminated by adding 1.5 ml of the rinsing buffer and cell centrifugation at 300 x
g for 5 min. The pellet was then resuspended in 100 ul of FITC conjugated anti-BrdUrd MoAb
solution and incubated for 1 h at room temperature. DNA was stained by addition of 1 ml of the
propidium iodide (PI) staining solution containing 5 ug/ ml PI and 100 4g/mI DNase-free RNase
in PBS for 30 min at room temperature in the dark. The percentage of apoptotic cells were
determined on a FACScan flow cytometer (Beckton Dickinson, San Jose,
CA) in which the green FITC fluorescence was proportional to the number of DNA strand
breaks and the red PI fluorescence to DNA content. The latter provided information pertaining
to the cell cycle phase position of the cells. Expression of bcl-2
Immunocytochemical detection of bcl-2:
Exponentially growing U937 lymphoma and MOLT-4 lymphocytic leukemia cells were exposed
to either 3.0 ul/ml PC SPES or the equivalent amount of ethanol for 48 hr. The cell cultures were
then fixed in 1% formaldehyde for 15 min on ice, washed twice and the cell pellet post-fixed in
ice cold 70% ethanol overnight. Following rehydration, the cells (1-2x1O') were stained for bcl-2 using
a 1: 100 dilution of the mouse antihuman bcl-2 antibody (type 124. Dako. Carpinteria,
CA) in 1% BSA in PBS overnight at 4 C. The cells were then centrifuged and the pellet
resuspended in 100 pl of 1% BSA in PBS containing goat anti-mouse FITC-labeled antibody
(Dako; FITC-conjugated (Fab'), fragment) at 1:30 titer. The cells were incubated for 30 min at
room temperature in the dark, then counterstained for DNA bv addition of 1 ml of P1 solution
(final PI concentration 10 ug-ml) containing 100 ug/ml of RNAse A. The bivariate green FITC
(bcl-2) versus red PI (DNA) fluorescence was measured on a FACScan flow cytometer
(Becton Dickinson).
Western blotting: Equal numbers of cells from each sample were washed in PBS, centrifuged
and the pellets frozen at -70 C until used. The pellets were lysed by passage through a 21 gauge
needle in PBS containing 1% NP-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl
sulphate supplemented with phenylmethylsulfonylfluoride (0.1 mg/ml), aprotinin (1 ug/ml) and
sodium orthovanadate (I mM). The lysates were centrifuged for 20 min at 15,000 x g at 4 C and
the supernatant collected. Proteins were separated on a 1 mm thick 12% polyacrylamide mini
gel. Electrophoresis was carried out at 100 V for 10 min and then 120 V for 50 min at room
temperature. The gels were then equilibrated in 25 mM Tris, 200 mM glycine. pH 8.8 and 20%
methanol for 10 min at 4,1C and the proteins transferred to nitrocellulose at 100 V for 60 min.
The filters were blocked with 5% non-fat dry milk in 100 mM Tris-HCI, pH 8.0, 150 mM NaCl
and 0.05% Tween 20 and reacted with bcl-2 antibody (Dako) at dilutions of 1:75 and 1:200.
The binding of the antibody was detected by ECL (Amersham Life Science, Arlington Heights,
IL) using peroxidase labeled secondary anti-mouse IgG antibody (at a dilution of 1:5.000).
Images of the immunoblots were captured and digitized by an HP ScanJet IIcx (Hewlett
Packard, Greeley, CO) using HP
DeskScan II software. Densitometric measures of the spots on the immunoblots were then made
with SigmaGel (Jandel Scientific, San Rafael, CA).
Combined treatment with PC SPES and radiation. U937 cells were grown for 24 h in the
presence of 2.0 id/ml of PC SPES at which time they received either 0, 1.5 or 5.0 Gy of
irradiation delivered at a dose rate of 12 Gy/min on a JBL 437C irradiator (Oris Corp.). The
cells were then returned to culture and grown for an additional 48 hr in the constant presence of
2.0 u1/ml of PC SPES. The cells were then harvested from culture, washed in HBSS and fixed
overnight in ice cold 70% ethanol. The cell aliquots were then rehydrated into HBSS and stained
with DAPI and sulforhodamine 101, measured by flow cytometry and analyzed as described
above.
Results
Components of PC SPES identified by HPLC. The HPLC profile of the batch of PC SPES used
in the present study is presented in Fig. 1. This herbal preparation is being standardized',
according to the manufacturer's assurance to reproduce the presence of six UV254 absorbing
peaks labeled A-F shown in Fig. 1. Indeed the manufacturer provided us with HPLC profiles of
three batches of PC SPES and each batch contained six peaks as shown in Fig. 1. The relative
heights of the peaks, however, were different from batch to batch (not shown). The chemical
structures of the components represented by these peaks are presently under study using nuclear
magnetic resonance and mass spectroscopy. Preliminary data suggest that these components are
of low molecular weights and belong to the class of alkaloids and polyphenols.
Effects of PC SPES on cell growth and proliferation. Cells were exposed to the PC SPES
extract at concentrations of 0.0 1 to 2.0 ul/ml of culture medium for 24. 48 or 72 h. Table I
illustrates the effects of such treatments on human leukemia and lymphoma cell lines growing in
suspension cultures. The lymphocytic and myelogenous leukemia cells, MOLT-4 and HL-60,
respectively, appeared to be the most sensitive while the U937 lymphoma the least sensitive to
the growth inhibitory effects of PC SPES at equivalent (2 ul/ml) concentration over the time
course of the experiment (Table 1).
When clonogenicity was compared among the adherent cell cultures, it was apparent that MCF-7 breast
carcinoma cells were the most sensitive followed by PC-3 prostate cancer and SK-N-MC neuroepithelioma
cells which, in turn, were more sensitive than LNCaP prostate and T47-D
breast carcinoma cells; Colo 38
melanoma cells were the least sensitive to the activity of PC SPES. The ID50 for these cell lines
were 20, 54, 70, 120, 220 and 430 nl/ml, respectively (Fig. 2).
Effects of PC SPES on cell cycle progression. The cell cycle distribution was estimated from the
DNA frequency histograms of different cell lines and of mitogen-stimulated human lymphocytes
after 48 h or 72 h of cell growth in the absence or presence of 2.0 ul/ml PC SPES (Table II; Fig.
3)t As is evident, PC SPES had no significant effect on the cell cycle distribution of PHA
stimulated lymphocytes. However, accumulation of cells in G, and a concomitant decrease in
proportion of cells in S phase were apparent in all tumor lines. The most dramatic effect was
seen in the case of MCF-7 cells, where the percentage of S phase cells was decreased nearly
40-fold, from 39 to 1%. Unlike other cell lines which were affected by PC SPES already after
48 h, the cell cycle of U937 cells was unchanged during the initial 48 hr of incubation with the
herbs. However, the proportion of U937 cells in G, was increased after 72 hr incubation with
2.0 ul/ml PC SPES.
The response of prostate cancer PC 3 line to PC SPES, in contrast to other lines, also
manifested an increased proportion of cells in G2M (Table II). However, this line grows at two
DNA ploidy levels (not shown), Thus, the location (DNA content) of the G2M cells of the lower
DNA
Figure 1. HPLC absorbance trace of a 70% ethanol extract of PC SPES batch used in the present experiments.
The components were separated on an Alltech preparatory C18 reverse phase column and a volume (500 ul)
of the stock solution of the herbal sample injected into (he HPLC system. Using an elution flow rate
of 4-8
ml/min. a linear gradient was generated with a two solvent system of water and acetonitrile and the
components detected at a wavelength of 254 nm. The presence of fractions labeled A-F was common to other
batches of PC SPES.
Table I. Cell growth in the continuous presence of PC SPES
Cell Line PC Spes Suppression of cell growth*
µl/ml 24 h 28 h 72 h
U937 2.0 5 36 50
HL-60 0.1 4 31 37
0.5 4 33 50
1.0 46 54 64
2.0 46 56 65
MOLT-4 2.0 44 72 ND
*The data are expressed as percent decrease in number of trypan blue excluding cells in PC
SPES treated cultures compared to parallel control cultures containing equivalent concentration
of the PC SPES solvent (ethanol) but no herb. ND, not determined.
ploidy cell population on the histograms, overlaps with that of the G cells of the higher ploidy
population. Therefore the cells represented by the G2/M peak consist of both, G2/M cells of the
lower DNA ploidy population as well as of G, tetraploid cells. It is likely, given the above, that
the observed increase in proportion of G2/M cells after PC SPES actually reflects the increase in
proportion of G, cells of the tetraploid population. In support of this explanation is the
observation of the lowered percentage of S and G2/M
Figure 2. The effect of PC SPES on clonogenicity of various adherent cell lines. Breast
(MCF-7 and T47-D),
prostate (LNCaP and PC-3) and other (Colo 38 melanoma and SK-N-MC neuroepithelioma) cells were seeded
at a density of 200 or 2000 cells/dish and grown in the absence or presence of varying concentrations
of PC
SPES for a period long enough (up to 8 days) for the cells to form colonies greater than 50 cells.
The colony
counts of PC SPES-treated
cultures (in triplicate) were compared to the percentage of colonies in cultures treated with diluent
(ethanol)
as present in the highest (2 µl/ml) PC SPES concentration. The data are presented as
percent of the control;
PC SPES extract concentration required to reduce colony formation by 50% (IC50) was
determined from the
respective plots.
Table II. The effect of PC SPES on cell cycle distribution.
Cell line PC SPES Cell cycle distributiona
µl/ml G1 S G2M Apoptosisb
PHA
Lymphocytes 0 62 31 7 3
2.0 62 30 8 7
MOLT-4 0 49 36 15 < 1
2.0 63 30 7 < 1
U937 0 48 40 12 < 1
2.0 61 32 7 17
PC 3 0 35 31 34 8
2.0 20 13 67 19
MCF-7 0 39 39 22 < 1
2.0 83 1 16 1
Colo 38 0 41 40 19 < 1
2.0 61 33 6 < 1
aCell cycle distribution was determined using the MulticyleTM computer program to deconvulate DNA content frequency distributions of DAPI
stained cells, measured by flow cytometry.
bApoptotic index was estimated from DNA content frequency histograms as the percentage of cells with
fractional DNA content, i.e. the cells
with DNA
content between 10 and 90% of cells represented by the G1 peak in untreated cultures (11).
cExposure to the herbal extract was for a
period of 72 h in the case of U937 while for all other cell lines exposure was 48 h.
cells at the tetraploid DNA level in the PC SPES treated cultures (not Shown)
Figure 3. The DNA content frequency distribution histograms of MCF-7 cells grown for 48 h in the absence
(control) and presence of 2 ul/ml of PC SPES. Accumulation of cells in G, and a corresponding drop in
the
percentage of cells in S and G2/M was observed in the presence of PC SPES.
Apoptosis. Fig. 4 illustrates the morphological changes associated with cell death of the cells
treated with PC SPES. Cells were grown in the absence (A, C and E) and presence (B, D and
F) of 2.0 ul/ml PC SPES for 48 h. In each instance, U937 (B) lymphoma, MCF-7 (D) breast
adenocarcinoma and PC-3 (F) prostate carcinoma cultures treated with PC SPES contained
cells with distinctly condensed chromatin, DNA hyperchromicity following staining with the
specific fluorochrome DAPI, loss of the structural nuclear framework, frequent nuclear
fragmentation and cell shrinkage. All these features are characteristic of the apoptotic mode of
cell death. The electrophoretic pattern of DNA extracted from HL-60 or U937 cells,
preincubated with 2.0 ul/ml of PC SPES for 48 h showed a characteristic 'laddering', indicative
of internucleosomal DNA cleavage which generates mono- and oligo-nucleosomal size DNA
fragments (Fig. 5).
Figure 4. The morphology of U937 lymphoma (A.B). MCF-7 breast adenocarcinoma (C.D) and PC-3 prostate
adenocarcinonia (E,F) grown for 48 hr in the absence (A,C,E) or presence (B,D,F) of 2 ul/ml of PC SPES.
The
cells were counterstained with the DNA specific fluorochrome DAPI and viewed under mixed illumination.
combining_UV light excitation and Nomarski interference contrast. Mitotic figures and apoptotic cells
(thick
arrows) are occasionally seen in the
untreated cultures (A,E). Numerous apoptotic cells characterized by highly condensed chromatin and nuclear
fragmentation are present in cultures treated with PC SPES (B, D, F).
A similar laddering pattern, considered to be a characteristic feature of DNA degradation during
apoptosis, was observed in the case of DNA extracted from HL-60 cells treated for 4 h with
0.15 µg/ml of CPT. Since it is well established that short term
Figure 5. Agarose gel electrophoresis of DNA extracted from HL-60 and U937 cells grown in the absence
and
presence of PC SPES and compared with DNA extracted from HL-60 cells exposed to the classical inducer
of
apoptosis CPT, which served as a positive control. HL-60 and U937 cells were grown in the presence of
O or
2.0 ul/ml PC SPES for48 h. or 150 nM CPT for 4 h. DNA was extracted as described before (9). Note that
DNA
extracted from cells exposed to PC SPIES was cleaved to the extent that it shows the 'laddering' pattern.
typical of apoptosis. similar to [hat induced by the DNA topoisomerasc inhibitor CPT.
exposure of HL-60 cells to CPT induces their apoptosis (9-11) these cells were presently used
as a positive control (Fig. 5).
Apoptotic cells are also characterized by fractional DNA content and therefore
can be
identified by flow cytometry based on cellular DNA content analysis (9). The increased
percentage of cells with fractional DNA content was observed after incubation of U937 and PC
3 lines with PC SPES (Table II).
Figure 6. Modulation of bcl-2 expression by PC SPES in U937 lymphoma and MOLT-4 cells.
Cells from both lines were exposed to either vehicle 9ethanol) alone (control) or 2 ul/ml PC
SPES for 48 hr. the presence of bcl-2 was detected in individual cells immunocytochemically:
KNA was counterstained with propidium iodie. Cells incubated with isotype IgG served as
control: the trapezoids describing the limits of the IgG background fluorescence are
superimposed on the cytograms of cells stained with the antibody to bcl-2. A decrease in bcl-2
expression in both U937 and MOLT -4 cells exposed to PC SPES was observed. The decrease
was reflected by both the decreased percentage of cells considered positive for the expression of
bcl-2 and the mean value of bcl-2 for this population.
Figure 7. Western blots of bcl-2 protein extracted from U937 cells exposed for 48 h to 0 or 2.0
VI/ml PC SPES as described in Fig. 6. The protein was isolated and subjected to
electrophoresis. Immunoblots were prepared using two dilutions of 1--75-aridJ 100iofthe-antibodyaagamst
bcl-2. Densitometric scanning of the bands demonstrated that at the higher
antibody dilution (1:200) PC SPES treatment resulted in a 90% reduction in band intensity while
at the higher antibody titre ( 1:75)~ the decrease was approximately 34% compared to control
cultures treated with vehicle (ethanol) alone.
Expression of bcl-2. U937 cells were exposed for 48 h to 2.0 ml/ml of PC SPES extract and
expression of bcl-2 was detected in individual cells immunocytochemically and measured by flow
cytometry (Fig. 6). Furthermore, expression of bcl-2 was detected by Western blotting (Fig. 7).
The percentage of cells with detectable level of bcl-2 (determined by comparing cells labeled
with the antibody specific for bcl-2 with an isotype control) decreased by 33%, from 95 +/- 4%
to 64 +/- 14% , in cultures treated with PC SPES. The mean expression of bcl-2 by the positive
cells decreased by 43%, from 86±24 to 49±10 arbitrary units of FITC fluorescence. These
results represent the means of four separate experiments. The changes
in bcl-2 expression in MOLT-4 cells were similar, but less pronounced compared to U037 cells.
The percentage of bcl-2 positive MOLT-4 cells was decreased by 12%, and the mean bcl-2
expression by 36% (from 59 to 3S arbitrary units), following 48 hr incubation with PC SPES.
This apparent decrease in bcl-2 expression in U917 cells was confirmed by gel electrophoresis.
Cells were treated for 48 h as above, the protein was isolated and subjected to
electrophoresis. Immunoblots were prepared using two dilutions 1:75 and 1:200 of the antibody
against bcl-2. The intensity of the bcl-2 band was minimal in the PC SPES treated cells at the
lower titer. Although at higher concentrations of the antibody, the band was distinct in the PC
SPES treated cells, it was of lower intensity compared to the band generated by proteins
extracted from the untreated, control cells (Fig. 7). Densitometry of the bands showed a 34%
and 90% reduction of the band intensity in PC SPES treated cells compared to the untreated
controls at 1:75 and 1:200 titer of MoAb, respectively.
Increased sensitivity of PC SPES treated cells to y-irradiation. Fig. 8 illustrates the effect of
preincubation of U937 cells with PC SPES on their subsequent sensitivity to y-irradiation in
terms of induction of apoptosis. There were 22% apoptotic cells in the cultures treated with PC
SPES alone compared to 6% in the control treated with an equivalent amount of ethanol vehicle.
The proportion of S phase cells was markedly reduced in the culture containing PC SPES, from
cells by 15%.
Figure 8. Increased sensitivity of U937 cells to y-irradiation following exposure to PC SPES.
Cells were either untreated or exposed to 2.0 ul/ml PC SPES for 48 h. to 1.5 or 5.0 Gy of y-radiation
alone. or were grown in the presence of 2.0 ul/ml PC SPES for 48 It followed by
exposure to 1.5 or 5.0 Gy of radiation. PC SPES alone caused a G t arrest and increased the
proportion of apoptotic cells. characterized by fractional DNA content (Ap). compared to
control cultures. Exposure to 1,5-Gyalone bad _MinM1.CffCCLMi.rijhrr
reU.y~ibUjivpcihrxcU.rucL%j4iAjihutino.rti~eJ 031 a near doubling of the apoptotic population.
However, when radiation was preceded by exposure to PC SPES there was an additional
increase in the proportion of cells undergoing apoptosis regardless of the level of radiation. When
PC SPES treatment was followed by a dose of 5.0 GN several peaks were evident in the
histograms. suggestive of the presence of cells with fractional DNA (Ap) content originating from
the G,M as well as the G populations.
Irradiation with 1.5 Gy alone did not increase the percent of apoptotic cells compared to the
control but decreased somewhat the proportion of cells in S (by 7%) and increased the
percentage (by 4%) of cells in G,M. However. as many as 32% of the cells showed reduced
DNA content typical of apoptosis in the cultures which were pretreated with PC SPES and then
irradiated with 1.5 Gy. Interestingly, the proportion of S phase cells was reduced to as little as
2%. while G,M cells increased to 15% in these cultures. An even higher proportion
of apoptotic cells (46%) was observed when the PC SPES pretreated cells were irradiated with
5.0 Gy. The proportion of S phase cells was reduced to 5% and the proportion in G,M
increased to 34% in these cultures.
Discussion
Cytostatic and cytotoxic effects of PC SPES extracts. Ethanol extracts of PC SPES showed
significant cytostatic and cytotoxic activity on several tumor lines. Decreased rate of cell
proliferation, reduced clonogenicity, increased proportion of cells in G, phase of the cycle,
induction of apoptosis and downregulation of bc1-2 expression were observed at different
concentrations of the extract. The cytostatic and cytotoxic effects of PC SPES were common to
all the tumor cell lines studied regardless of their lineage. Cell sensitivity to this herbal mixture,
however, varied between different lines, with MCF-7 cells being the most sensitive, and Colo 38
cells the most resistant, in the clonogenicity assay. The increased proportion of cells in G, phase
observed in tumor cell cultures treated with extracts of PC-SPES (Table II and Fig. 3) could
either be a result of their arrest in G, (or increased transit time through this phase) or be due to
a
selective cell death in S and G,/M. However, the initial increase in proportion of cells in G, was
apparent in some cell lines already after 24 hr. It and at a range of PC SPES concentrations
which did not induce significant cell death at that time; apoptosis occurred with a delay, generally
after 48 h. The early accumulation of cells in G1 in PC SPES treated cultures, thus, could not be
due to cell dell death in S and G/M but rather was reflecting a slowdown in cell progression
through G. Exposure of PC-3 cells to PC SPES led to an increased proportion of cells with 4C
and higher KNA content (Table II). Increased percentage of bi- and multinucleated cells as well
as cells with giant nuclei, indicative of endoreduplication, was observed in these cultures after 48
hr (not shown). In the case of PC-3 cells, thus, the herbal mixture in addition to blocking cells in
G1, appeared to perturb cytokinesis.
Apoptosis and suppression of bc1-2 expression. Coincident with a decrease in the percent of
live cells, an increased proportion of apoptotic cells was seen
in PC SPES treated cultures of all cell lines. Apoptotic cells were identified by flow cytometry as
the cells with a fractional DNA content ('sub-G, cells') after fixation with ethanol (Fig. 8) and as
cells with a large number of DNA strand breaks detected by their labeling with BrdUTP by
exogenous TdT (11; not shown). The apoptotic mode of cell death was additionally confirmed
by the presence of a 'laddering pattern' on DNA electrophoretograms (Fig. 5) and by
characteristic features of cell morphology (Fig. 4). The highest frequency of apoptotic cells was
seen in cultures of HL-60 and U937 cells treated with PC SPES. Expression of bc1-2 protein
declined in U937 cells exposed PC SPES. Because bc1-2 provides resistance to apoptosis,
these data suggested that PC SPES treatment may sensitize cells to antitumor agents Indeed,
more than an additive increase in the proportion of apoptotic cells, and a decreased number of
live cells, were observed in cultures of PC SPES treated cells subjected to y-irradiation,
compared to cultures incubated with PC SPES or exposed to y-irradiation alone (Fig. 8).
Additional studies are underway to reveal whether PC SPES affects expression of bcl-2 in other
cell lines and whether it sensitizes cells to antitumor modalities other than y-irradiation.
Active components of PC SPES. PC SPES is a herbal preparation which consists of eight
different plants listed in Material and methods. Several individual herbs in the PC SPES mixture
have been reported to be biologically active and may contribute to the presently observed
effects. Scuttelaria baicalensis contains baicalein, a flavonoid which has antiproliferative and
lipoxygenase-inhibitory activity (12). Baicalein and its analogues baicalin and wogonin also show
anti-inflammatory, and antibacterial activity against a wide range of microorganisms (13-17).
Exposure of three human hepatocellular carcinoma lines to baicalein resulted in inhibition of their
DNA topoisomerase 11 (top 2) activity and induction of apoptosis (18,19) while treatment of
other hepatoma lines with these same agents suppressed cell proliferation (20). Baicalein was
also shown to suppress proliferation of human T-lymphoid leukemia (CEM) cells and inhibit their
protein tyrosine kinase activity (12). Interestingly, another isoflavone, genistein, which like
baicalein, also is an inhibitor of top 2 and of protein tyrosine kinase, was shown to induce
apoptosis and exert similar cell cycle effects as PC SPES (21). Flavonoids isolated from
Scuttelaria, including baicalein, also inhibit 3', 5'-cyclic monophosphate (cAMP)
phosphodiesterase (22), and were shown to have both antimutagenic activity (23.24) and to
suppress skin tumor promotion in a mouse carcinogenesis model (25,26). Extracts from Chinese
mushroom Ganoderma lucidum contain numerous glycans, three of which have significant in vivo
antitumor activity against the murine S-180 sarcoma (27-29). Proteins isolated from this herb
exhibit immunomodulatory activity (30,31). Serenoa repens contains a phytoestrogen which
lowers endogenous estrogens in host animals (32). The lipido-sterol
extract of Serenoa (Permixon) inhibits proliferation of benign and prostate cancer cells most
likely by preventing binding of the dihydrosterone to the cytosolic androgen receptor (33.34).
Permixon is used, outside of the United States, in treatment of symptomatic benign prostatic
hyperplasia, where it shows an efficiency comparable to that of finasteride (Proscar) (35-38).
Glycerrhiza is most known for its antimutagenic (39-41), anti- inflammatory (42) and
antimicrobial (43.44) effects. Steroid analogues of Glycerrhiza also modulate steroid metabolism
by inhibition of the steroid dehydrogenases (45) while flavonoids inhibit the activity of cAMP
phosphodiesterase (46). Triterpenoids isolated from this herb strongly enhance the cytotoxicity of
daunomycin, doxorubicin and vinblastine on multidrug resistant P388 leukemic cells in vitro (47),
while pectic polysaccharides have mitogenic activity (48).
Polysaccharides isolated from Isatis indigotica appear to have immunostimulatory activity (14).
Another component of this herb, indirubin, is reported to have antineoplastic activity, and
appears to be widely used in China in the treatment of leukemias (49-53). The most biologically
active components contain varieties of Panax ginseng (reviewed in 14). Consumption of this herb
appears to lower the incidence of cancer (54, 55). Saponins (panaxosides, ginsenosides)
extracted from this herb, while they suppress proliferation of different tumor types in vitro and
have antitumor (human ovarian tumor in immunodeficient mice) effect in vivo (56.57), appear to
have modest stimulatory effects on normal fibroblasts or lymphocytes (58-60). Extracts from this
herb have also been shown to exert antimutagenic activity in different carcino- genicity model
systems (61, 62) induce cell differentiation (63) and inhibit tumor metastasis (64.65). Similar to
extracts from Glycyrrhiza, Panax extracts also increase the sensitivity of multidrug resistant tumor
cells to several antitumor drugs (66,67).
Possible antitumor activity of PC SPES. It is of obvious interest to discern whether the
presently observed in vitro effects of PC SPES extracts can be interpolated to its supposed in
vivo antitumor activity. Several observations suggest that such interpolation is possible. Thus, the
suppression of clonogenicity of some tumor lines was
observed at very low concentration of' the extract, namely at 0.1 u1/ml; which is equivalent to
0.01% of a single capsule. Assuming full absorption of the active components of the orally
administered preparation and their uniform distribution in tissues, the patients who take this herbal
preparation at the dose recommended by the manufacturer (9 capsules per day) may achieve a
concentration per body weight comparable to that observed to be effective ill vitro. Lacking any
pharmacokinetics data, however, these are very approximate and only theoretical assumptions.
The decrease in expression of bcl-2 in PC SPES treated cells and their enhanced sensitivity to y-irradiation,
suggest that this herbal preparation may predispose tumor cells to other antitumor
modalities. The cytostatic and cytotoxic effects of PC SPES presently observed, are not
incompatible with the possibility that this herbal combination may indeed exert antitumor
properties. It appears, however, that other cell lineages in addition to prostate carcinoma (e.g.
breast carcinoma MCF-7) are also sensitive to PC SPES. Animal studies, in particular on human
tumors xenografted on immunodeficient mice, are needed, to provide further evidence of the
possible utility of PC SPES as an antitumor agent alone, or in combination with radio- or
chemotherapy. It is difficult to explain why PC SPES at concentrations which induced cytostatic
and cytotoxic effects on several tumor cell lines failed to exert an effect on PHA stimulated
normal lymphocytes. The defect which is most common to all tumors appears to be at the level of
sequestration/release of the E2F family of proteins: this event involves phosphorylation of pRB
and manifests as a loss of function at the cyclin D restriction point (68-70). It is possible,
therefore, that among the target(s) of the active components of PC SPES may be kinases and/or
phosphatases whose inhibition restores the cyclin D restriction point in tumor cells.
Herbal mixture vs. individual components. As discussed above the PC SPES herbal concoction
has clearly enough active components to explain all the in vitro effects presently observed. It is
stressed, however, by the manufacturer (2) that it is the combination of herbs, rather than each of
them individually, which is responsible for the overall in vivo antineoplastic activity of this
preparation. Individual herbs, even at higher doses, apparently have no such activity PC SPES
thus, bears some similarity to 'sho-saiko-to', a herbal medicine consisting of a combination of
seven herbs (14, 71, 72) three of which
(Glycyrrhiza, Scuttellaria and Ginseng) are also found in PC-SPES. Sho-saiko-to herbal
medicine is very popular in Japan and China for the treatment of a variety of chronic liver
diseases (14). In addition, it was reported to inhibit the development of hepatocellular carcinoma
in patients with liver cirrhosis and to have antitumor activity (73). Interestingly, similar to the
presently observed effects of PC SPES, sho-saiko-to was shown to inhibit proliferation of
several tumor cell line by arresting cells in G, and inducing apoptosis while having little effect on
normal cells (71). The effects of individual, presumably active, components from sho-saiko-to
(baicalein. wogonin, glycyrrhizin, saikosaponins a, b and c, and ginsenosides Rb1 and Rg1), had
no significant effect on the growth of human hepatocellular carcinoma cell lines, while sho-saiko-to
suppressed growth by 65%; an exception was baicalin which inhibited growth by about 40%
(71). Another popular Chinese herbal mixture is 'juzentaihoto', containing 10 herbs, some of
which (Ginseng, Glycyrrhiza) are also found in PC SPES. This mixture was shown to enhance
the effectiveness of mitomycin-C in treatment of p-388 leukemias in mice (74). The strategy of
preparation of Chinese or Japanese herbal medicine thus, often relies on a combination of
different ingredients which may have either synergistic or new effects. The analysis of individual
active components of antitumor herbal mixtures such as PC SPES, therefore, may not reveal the
activity which may be responsible for the overall effect that are seen when all herbs are used in
combination. There is a growing body of evidence of redundancy of many regulatory molecules,
especially the molecules related to cell cycle progression and apoptosis. This is exemplified in the
survival of animals with key regulatory genes e.g. such as tumor suppressor gene p53 or cyclin
D1, deleted (75, 76). Surprisingly, many gene-deficient animals not only survive but they show
relatively minor effects of such gene deletion. Likewise, new cell lines having particular regulatory
genes deleted are constantly being developed in different laboratories. Clearly, deletion of a
regulatory molecule is often compensated by other molecules, providing a redundancy which
enhances the cell chances for survival. It may be expected, therefore, that a single active
component of a particular herb, or single antitumor drug, if it targets a pathway regulated by
molecules may not be fully effective. A combination of drugs containing components which affect
the redundant pathways, in such a case, may have either synergistic or entirely new
antiproliferative activities. The situation may be reminiscent of the recent therapeutic advances
against HIV infection, where a combination of active drugs is superior in delaying
development of' AIDS than any of- them alone. It is possible that the traditional Chinese herbal
concoctions were empirically developed via observation of new activities when herbs were
combined rather than administered alone. If synergism or new activities result from herbal
combinations, the identification of individual constituents responsible for the overall antitumor
activity may be a difficult task, especially in such complex herbal mixtures as
PC SPES (2) or SPES ( I ) .
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