Cycloprodigiosin Hydrochloride, a New H⫹/Cl⫺ Symporter,
Induces Apoptosis in Human and Rat Hepatocellular Cancer Cell
Lines In Vitro and Inhibits the Growth of Hepatocellular
Carcinoma Xenografts in Nude Mice
CHIZUKO YAMAMOTO,1 HIROTO TAKEMOTO,1 KENJI KUNO,1 DAIGO YAMAMOTO,2 AIRO TSUBURA,2 KEIKO KAMATA,3 HAJIME HIRATA,3
AKITSUGU YAMAMOTO,4 HARUHIKO KANO,1 TOSHIHITO SEKI,1 AND KYOICHI INOUE1
The effects of cycloprodigiosin hydrochloride (cPrG-
HCl), a new H⫹/Clⴚ symporter, were examined in liver
cancer cell lines in vitro and in vivo. In the in vitro MTT
assay, cPrG-HCl inhibited the growth of 6 liver cancer cell
lines (Huh-7, HCC-M, HCC-T, dRLh-84, and H-35, hepato-
cellular carcinoma; HepG2, hepatoblastoma) in a dose- and
time-dependent manner. The 50% inhibitory concentra-
tions (IC50) at 72 hours’ treatment for liver cancer cell lines
were 276 to 592 nmol/L, while that for isolated normal rat
hepatocyte was 8.4 ␮mol/L. The cPrG-HCl treatment of
Huh-7 cells induced apoptosis as confirmed by the appear-
ance of a subG1 population, intranucleosomal DNA fragmen-
tation, and chromatin condensation. cPrG-HCl raised the
pH of acidic organelles and lowered pHi (below pH 6.8). In
addition, the apoptosis in Huh-7 cells induced by cPrG-HCl
was strongly suppressed when the cells were cultured with
imidazole, a cell-permeable base. In the in vivo assay, nude
mice bearing subcutaneous xenografted Huh-7 cells re-
ceived 2 weeks of treatment with cPrG-HCl (1 or 10
mg/kg/d) subcutaneously. This treatment significantly inhib-
ited tumor growth compared with the control after 8 days.
The control mice were treated with 1% dimethylsulfoxide
(DMSO) in saline (vehicle). A histopathological examina-
tion using the terminal deoxynucleotidyl transferase medi-
ated dUTP biotin nick end labeling (TUNEL) method
showed apoptosis in the treated tumor cells. No pathologi-
cal changes were observed in any organs, and the serum
alanine transaminase levels remained within normal limits.
Abbreviations: pHi, intracellular pH; V-ATPase, vacuolar-type H⫹-ATPase; cPrG-HCl,
cycloprodigiosin hydrochloride; DMEM, Dulbecco’s modified Eagle minimum essential
medium; FBS, fetal bovine serum; DMSO, dimethylsulfoxide; PBS, phosphate-buffered
saline; MTT, 3-(4,5-dimethylthiazol-2-2-yl), 5 diphenyltetrazolium bromide; IC50, 50%
inhibitory concentration; BCECF-AM, 28,78-bis-(Carboxyethyl)-5(68)-carboxyfluores-
cein acetoxymethyl ester; TUNEL, terminal deoxynucleotidyl transferase mediated
dUTP biotin nick end labeling.
From the Departments of 1Internal Medicine III, 2Pathology II, and 4Physiology I,
Kansai Medical University, Moriguchi, Osaka, Japan; and 3Department of Life Science,
Faculty of Science, Himeji Institute of Technology, Hyogo, Japan.
Received March 22, 1999; accepted July 9, 1999.
Supported in part by the Grant-in-Aid from the Ministry of Education, Science, and
Culture, Japan, and Science Research Promotion Fund of the Japanese Private School
Promotion Foundation.
Address reprint requests to: Chizuko Yamamoto, M.D., Department of Internal
Medicine III, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan. E-mail:
yamamotd@takii.kmu.ac.jp; fax: 81-6-6997-5490.
Copyright r 1999 by the American Association for the Study of Liver Diseases.
0270-9139/99/3004-0012$3.00/0
894
These results suggest that cPrG-HCl may be useful for the
treatment of hepatocellular carcinoma. (HEPATOLOGY 1999;
30:894-902.)
Apoptosis is a form of programmed cell death character-
ized by nuclear chromatin condensation, cell shrinkage,
membrane blebbing, and DNA fragmentation.1-3 The relation-
ship between intracellular acidification and apoptosis has
been studied.4-9 In recent studies, the intracellular acidifica-
tion may lead to activation of endonucleases and induce
apoptosis in tumor cells.6,8,9
In addition, the role of intracellular pH (pHi) in apoptosis
and cell proliferation has been investigated.10,11 The pHi of
cancer cells has been reported to be more alkaline than that of
normal cells, and the maintenance of a neutral or slightly
alkaline pHi is required for cell growth, transformation, and
metabolism.10,11 Therefore, increasing the intracellular acid-
ity by inhibiting the pHi regulatory mechanisms is cytotoxic
and suggests that the pHi regulatory mechanisms may serve
as targets for tumor therapy.6,8,9
Prodigiosins (prodigiosin, metacycloprodigiosin, and pro-
digiosin 25-C) are red pigments produced as chromophores
by various bacteria including Serratia marcescens, Pseuodomo-
nas magnesiorubera, and others.12 Among the prodigiosin
family, prodigiosin 25-C inhibited H⫹ translocation by vacu-
olar-type H⫹-ATPase (V-ATPase) without any effect on its
ATP hydrolytic activity and suppressed the growth of neoplas-
tic Chinese hamster ovary cells.13 We previously reported that
cycloprodigiosin hydrochloride (cPrG-HCl), which is a mem-
ber of the prodigiosin family and is more pure and stable than
the others, inhibited H⫹ translocation by V-ATPase in the
same manner as other prodigiosins.14 Recently, it was found
that prodigiosins promote H⫹/Cl⫺ symport across vesicular
membranes, resulting in an uncoupling of V-ATPase.13,15,16
Therefore, these reports suggested that prodigiosins are
useful pH regulators and may be promising anticancer drugs.
To date, the nature of the interaction between the alteration
of pHi and apoptosis induced by cPrG-HCl has not been
investigated. Therefore, in this study, we have shown that
cPrG-HCl suppressed the cellular proliferation and induced
apoptosis as a result of a decrease of pHi on liver cancer cell
lines.
MATERIALS AND METHODS
Cell Culture. Six liver cancer cell lines (Huh-7, HCC-M, and
HCC-T, human hepatocellular carcinoma; HepG2, human hepato-
HEPATOLOGY Vol. 30, No. 4, 1999 YAMAMOTO ET AL. 895

TABLE 1. IC50 Values of cPrG-HCl Treatment for 72 Hours

Origin IC50 (nmol/L)

Huh-7 Human 276

HCC-M Human 313
HCC-T Human 532
HepG2 Human 592
dRLh-84 Rat 530
H-35 Rat 485
Hepatocyte Rat 8,395
NOTE. Growth inhibition was determined by the percent inhibition
compared with untreated control cells by MTT assay.

blastoma; dRLh-84 and H-35, rat hepatocellular carcinoma) were

used in this study. HCC-M and HCC-T were donated by Dr. H. Saito
of Keio University.17 Huh-7 and HepG2 were obtained from Riken
Cell Bank (Tsukuba, Japan). dRLh-84 was obtained from Health
Science Research Resources Bank (Osaka, Japan). H-35 was obtained
from American Type Culture Collection (Rockville, MD). The
origins and characteristics have been described elsewhere.17-22 All
cell lines were maintained in Dulbecco’s modified Eagle minimum
essential medium (DMEM) (Nissui, Tokyo, Japan) supplemented
with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and
streptomycin (100 µg/mL) and were grown in a 5% CO2/95% air
humidified atmosphere at 37°C. Trypsinized cells were frozen and
stored in liquid nitrogen until use. Rat primary hepatocyte cultures
were isolated and cultured as described previously.23,24 In brief,
hepatocytes were isolated by collagenase perfusion from male Wistar
strain rats and suspended in culture medium (Williams’ medium E
supplemented with 10% newborn calf serum, 5 mmol/L HEPES, 100
U/mL penicillin, 1 mg/mL streptomycin, 10 nmol/L dexamethasone,
and 10 nmol/L insulin). After 2 hours, the medium was replaced by
fresh serum-free medium and the cells were cultured for 24 hours
and then used for experiments.
Preparation of cPrG-HCl. cPrG·HCl (molecular weight: 357.9) was
isolated from the marine bacterium Pseudoalteromonas denitrificans
and stored at ⫺20°C.14 Crystalline cPrG-HCl was dissolved in
dimethylsulfoxide (DMSO) at a concentration of 5 mg/mL and
diluted with phosphate-buffered saline (PBS) (⫺) immediately
before use.
MTT Assay. To assess the direct effects of cPrG-HCl, cell growth
inhibition was evaluated by a colorimetric 3-(4,5-dimethylthiazol-2-
2-yl),5 diphenyltetrazolium bromide (MTT) assay, as described
previously.25 Initially, the cells were seeded at a plating density of
1.0 ⫻ 104 cells per well, then cultured for 24 hours to allow
adhesion to the plate. After preincubation, the culture medium was
changed to the experimental medium supplemented with cPrG-HCl
in the absence or presence of imidazole (WAKO, Osaka, Japan). The
final concentration of DMSO in the medium was adjusted to less
than 0.2% (vol/vol), which was found to have no antiproliferative
effect on the cells.
cPrG-HCl was added at concentrations between 0.01 µmol/L and
10 µmol/L, to span a 4-log range, respectively. After being cultured
with cPrG-HCl for the indicated period, 5 mg/mL of MTT (Sigma, St.
Louis, MO), diluted with PBS (⫺) (pH 7.6) was added to each well at
13 µL per well in a 96-well plate, followed by incubation at 37°C for
an additional 4 hours. The reaction was terminated by aspirating the
medium from each well. DMSO, at 150 µL per well, was added to the
96-well plate to dissolve the formazan crystals. All plates were then

=
FIG. 1. To assess the direct effects of cPrG-HCl, cell growth inhibition
was evaluated by a colorimetric MTT assay for 24 to 72 hours. Concentra-
tion- and time-dependency curves of the effects of cPrG-HCl treatments on
Huh-7cells (A) and hepatocyte cells (B) are shown. In addition, when the
cells were cultured with 10 mmol/L of imidazole, the effect in Huh-7 cells
induced by cPrG-HCl was examined (C). (䊊), 24 hours; (䉭), 48 hours; (䊐),
72 hours.
896 YAMAMOTO ET AL. HEPATOLOGY October 1999

placed on a plate shaker for 2 minutes. The 96-well plate samples

were immediately read at 540 nm on a scanning multiwell spectro-
photometer. The percentage of surviving cells exposed to cPrG-HCl
was determined after continuous incubation for 24 to 72 hours. The
data points represented the mean values of 8 wells. Additional
controls consisted of the culture medium alone. The IC50 was
determined as the concentration of drug that produced a 50%
reduction of absorbance at 540 nm. IC50 of mean values and their
standard deviation in each experiment were calculated from tripli-
cate experiments.
Flow Cytometric Analysis of DNA Contents. Huh-7 cells were exposed
to cPrG·HCl at 276.5 nmol/L, in the absence or presence of
imidazole (10 mmol/L) in DMEM supplemented with 10% FBS for
24 to 72 hours.6 Cultured cells remaining on the flask bottom and
floating cells in culture medium were mixed and washed in PBS (⫺),
followed by centrifugation, and fixed in 70% ethanol at 4°C for 4
hours. After centrifugation, the cells were treated with RNase and
diluted to 0.01% in PBS (⫺), followed by the addition of propidium
iodide (50 µg/mL) in PBS (⫺). The cells were analyzed with
FACScan (Becton Dickinson, San Jose, CA) using Modfit LT
software.
DNA Fragmentation. Huh-7 cells were exposed to cPrG·HCl at
276.5 nmol/L, in the absence or presence of imidazole (10 mmol/L).
The cells remaining on the flask bottom and floating cells were
harvested and DNA of the cells was isolated using the Sepa Gene kit
(SANKO, Tokyo, Japan), which is based on the guanidine thiocya-
nate extraction procedure, followed by electrophoresis on a horizon-
tal 0.8% agarose gel containing ethidium bromide (5 mg/mL) and
visualized under ultraviolet illumination.25
Electron Microscopy. Huh-7 cells remaining on the flask bottom
and floating cells were harvested, centrifuged, and then cells were
fixed in 2.5% glutaraldehyde, postfixed in 2% osmium tetroxide,
and embedded in Luveak-812 (Nacalai Tesque, Kyoto, Japan).
Ultrathin sections were stained with lead citrate and uranyl acetate,
and examined with a Hitachi-600 electron microscope (Hitachi,
Tokyo, Japan).25
Vital Fluorescence Microscopy. The living cultured cells were stained
with acridine orange (Sigma, St. Louis, MO) following the protocol
previously described.26 Huh-7 cells on a chamber slide were exposed
to cPrG-HCl in DMEM supplemented with 10% FBS and incubated
at 37°C. The cells were then incubated at 37°C for 10 minutes with 5
µg/mL acridine orange in Hanks’ solution. After washing with
Hanks’ solution, the chamber slides were examined by confocal laser
microscopy (Olympus, Tokyo, Japan).
Flow Cytometric Analysis of pHi. Huh-7 cells were exposed to
cPrG·HCl at 276.5 nmol/L, in the absence or presence of imidazole
(10 mmol/L). The cells remaining on the flask bottom and floating
cells were harvested, centrifuged, and then loaded with 2 µg/mL
28,78-bis-(Carboxyethyl)-5(68)-carboxyfluorescein acetoxymethyl es-
ter (BCECF-AM) (Molecular Probes, Calbiochem, San Diego, CA) at
37°C for 30 minutes in PBS (⫺). After centrifugation, the cells were
resuspended in PBS (⫺) and analyzed by FACS Vantage (Becton
Dickinson), with excitation at 488 nm and emission ratio analysis at
530 and 630 nm using Lysis II software.27 The emission ratios were
converted to pH values by comparison with the ratios observed for
cells treated with nigercin (Sigma) in a high-potassium buffer at a
defined pH.27
Animals. Female BALB/c nude mice, 5 weeks of age, were pur- FIG. 2. Huh-7 cells were exposed to cPrG-HCl at 276.5 nmol/L, in the
chased from Clea Japan. The mice were fed a commercial pellet diet absence or presence of imidazole (10 mmol/L) in DMEM supplemented with
(CMF; Oriental Yeast, Tokyo, Japan) and given tap water freely. The 10% FBS for 24 to 72 hours. Cultured cells remaining on the flask bottom
mice were acclimated for 1 week before the experiment and were and floating cells in culture medium were mixed and washed in PBS (⫺),
inoculated with tumor cells at 6 weeks of age. followed by centrifugation, and fixed in 70% ethanol at 4°C for 4 hours. After
Tumor Growth In Vivo. The semiconfluent Huh-7 cells grown in centrifugation, the cells were treated with RNase and diluted to 0.01% in PBS
(⫺), followed by the addition of propidium iodide (50 µg/mL) in PBS (⫺).
DMEM supplemented with 10% FBS at 37°C were trypsinized, and
Then, the cells were analyzed with FACScan. DNA histograms of Huh-7 cells
107 viable cells/0.2 mL of PBS (⫺) were injected into the back of 24 grown in the presence of (A) medium alone, (B) 276.5 nmol/L of cPrG-HCl,
mice with a 26-gauge needle.28 Ten days after transplantation, the and (C) 276.5 nmol/L of cPrG-HCl plus 10 mmol/L of imidazole for 72
tumors grew to a volume of approximately 75 mm3. The mice were hours.
then divided randomly into three groups (10-mg/kg cPrG-HCl–
HEPATOLOGY Vol. 30, No. 4, 1999 YAMAMOTO ET AL. 897

TABLE 2. Cell-Cycle Analysis With cPrG-HCl Treatment for 72 Hours was measured by the Biuret method, and albumin in serum was
Treatment G0/G1 S G2/M measured by the bromocresol green method. The blood urea
nitrogen in serum was measured by the urease-indophenol method,
Untreated control 59.3 ⫾ 2.5 20.2 ⫾ 0.6 20.5 ⫾ 1.2 and creatinine in serum was measured by the Jaffe method. The
cPrG-HCl 75.8 ⫾ 2.3*† 7.6 ⫾ 1.1*† 16.6 ⫾ 2.1* experiments were conducted following the Guidelines for Animal
cPrG-HCl plus imidazole 65.4 ⫾ 1.2‡ 18.1 ⫾ 1.8 16.5 ⫾ 1.5‡ Experimentation (Japanese Association for Laboratory Animal Sci-
*P ⬍ .01 vs. control, †P ⬍ .01 between cPrG-HCl and cPrG-HCl plus
ence, 1987).
TUNEL Method. The cell death in the tumor was further evaluated
imidazole, and ‡P ⬍ .01 between cPrG-HCl plus imidazole and untreated
control.
by the terminal deoxynucleotidyl transferase mediated dUTP biotin
nick end labeling (TUNEL) method.29 Apop Tag (in situ apoptosis
detection kit; Oncor, Gaithersburg, MD) was used according to the
treated group, 1-mg/kg cPrG-HCl–treated group, and cPrG-HCl– manufacturer’s instructions. For apoptotic indices, more than 2,000
untreated group) with 8 mice per group. cPrG-HCl was freshly
prepared and subcutaneously injected daily in 1% DMSO in saline
(vehicle). The mice were weighed before each cPrG-HCl injection
throughout the study. In the controls, the same volumes of vehicle
without cPrG-HCl were injected. The size and weight of the locally
growing tumor were then determined. Tumor volume was measured
with a caliper and calculated using a standard formula: width2 ⫻
length ⫻0.5. The tumor growth was assessed by monitoring the
mean tumor volume and tumor weight upon removal. Animal
weights at the time of autopsy were calculated by subtracting the
tumor weight from the animal weight immediately before autopsy.
At autopsy, the mice were anesthetized with diethyl ether, and blood,
for serum preparation, was collected by heart puncture. All organs
and tumors were then removed and processed for hematoxylin-
eosin histological examination. The alanine transaminase in serum
was measured by the ultraviolet rate method, total protein in serum

FIG. 3. The DNA of Huh-7 cells was isolated using the Sepa Gene kit,
which is based on the guanidine thiocyanate extraction procedure, followed
by electrophoresis on a horizontal 0.8% agarose gel containing ethidium
bromide (5 mg/mL) and visualized under ultraviolet illumination. Lane 1,
untreated control; lane 2, exposed to 276.5 nmol/L of cPrG-HCl for 72 hours;
lane 3, exposed to 276.5 nmol/L of cPrG-HCl plus 10 mmol/L of imidazole
for 72 hours. DNA fragmentation was seen in lane 2. m ⫽ marker.
FIG. 4. Huh-7 cells were fixed in 2.5% glutaraldehyde, postfixed in 2%
osmium tetroxide, and embedded in Luveak-812. Ultrathin sections were
stained with lead citrate and uranyl acetate, and examined with a Hitachi-600
electron microscope. Electron micrographs of (A) untreated cells (bar ⫽ 10
µm) and (B) treated cells with 276.5 nmol/L of cPrG-HCl for 48 hours (bar ⫽
4 µm). The cell shrinkage and nuclear chromatin condensation are shown
(B).
898 YAMAMOTO ET AL. HEPATOLOGY October 1999

cPrG-HCl strongly suppressed when the cells were cultured

with imidazole, a cell-permeable base (pK 6.93) (Fig. 1C).
Mode of Cell Death. Among the human liver cancer cell lines
tested, cPrG-HCl evoked the most marked cell growth
inhibition in Huh-7 cells. Thus, Huh-7 cells were chosen for
the following experiments. Huh-7 cells treated with cPrG-
HCl (at the IC50 value, 276.5 nmol/L) were analyzed by flow
cytometry. After 72 hours of treatment with cPrG-HCl, the
distribution of cells in different phases of the cell cycle was
determined and are shown in Fig. 2. While the sub-G1
fraction of untreated cells was undetectable (Fig. 2A), that of
cPrG-HCl–treated cells was 42.6% ⫾ 1.5% (Fig. 2B). The
sub-G1 fractions after 24 and 48 hours in culture supple-
mented with cPrG-HCl were 12.5% and 35.6%, respectively.
Furthermore, we examined the effect of imidazole on the

FIG. 5. The living cultured cells were stained with acridine orange.
Huh-7 cells on a chamber slide were exposed to cPrG-HCl in DMEM
supplemented with 10% FBS and incubated at 37°C for 1 hour. The cells were
then incubated at 37°C for 10 minutes with 5 µg/mL acridine orange in
Hanks’ solution. After washing with Hanks’ solution, the chamber slides
were examined by confocal laser microscopy. Acridine orange staining of (A)
untreated cells and (B) treated cells with 276.5 nmol/L of cPrG-HCl for 1
hour. Bar ⫽ 10 µm.

cells were counted in each section and were calculated from the
percentage of cells scored positive at 400⫻ magnification.
Data Analysis. Each experiment was performed at least three
times, and all results were expressed as the mean ⫾ SD. The
significance of differences was determined with the Student’s t test in
vitro. The data were analyzed using a Kruskal-Wallis nonparametric
ANOVA test, followed by comparison of groups using the Mann-
Whitney U test in vivo. The differences with probability value less
than 0.05 were considered significant.

RESULTS
Cell Growth Control by cPrG-HCl. To evaluate the effects of
cPrG-HCl on cell growth, MTT assays were performed, and
the IC50 values of cPrG-HCl treatment for 72 hours were
summarized (Table 1). cPrG-HCl exerted significant dose-
and time-dependent inhibition on all liver cancer cell lines,
and the IC50 values were less than 600 nmol/L, while the IC50
value of rat primary hepatocytes was 8.4 µmol/L (Fig. 1A, 1B,
Table 1). In addition, the effect in Huh-7 cells induced by
FIG. 6. Huh-7 cells were harvested, centrifuged, and then loaded with 2
µg/mL BCECF-AM at 37°C for 30 minutes in PBS (⫺). After centrifugation,
the cells were resuspended in PBS (⫺), and analyzed by FACS Vantage, with
excitation at 488 nm and emission ratio analysis at 530 and 630 nm using
Lysis II software. Representative data of flow cytometric pH analysis in
Huh-7 for 24 hours (A) and time-course pH changes (B) are shown. The
black line and (䊊) show the untreated control. The blue line and (䊉) show
cPrG-HCl ⫹ imidazole–treated cells. The red line and (䉭) show cPrG-HCl–
treated cells.
HEPATOLOGY Vol. 30, No. 4, 1999 YAMAMOTO ET AL. 899

sub-G1 fraction of cPrG-HCl–treated Huh-7 cells. The sub-G1

fraction (7.9% ⫾ 1.1%) was significantly decreased in the
presence of imidazole (Fig. 2C).
The effect of cPrG-HCl on the distribution of cells in the
cell cycle is shown in Table 2. Progressive increases in the pro-
portion of cells in the G0/G1 phase, and decreases in the
proportion of cells in the S and G2/M phases, were noted. The
cPrG-HCl ⫹ imidazole treatment resulted in a smaller G0/G1
fraction compared with cPrG-HCl treatment. Intranucleo-
somal DNA fragmentation appeared when Huh-7 cells were
exposed to cPrG-HCl for 24, 48 (data not shown), and 72
hours (Fig. 3, lane 2). However, DNA fragmentation was
absent in cells treated with cPrG-HCl ⫹ imidazole (lane 3).
Electron microscopically, compared with untreated cells (Fig.
4A), cell shrinkage and chromatin condensation were ob-
served following cPrG·HCl treatment for 48 hours and
thereafter (Fig. 4B).
Fluorescence Microscopy. The effect of cPrG-HCl on acidifica-
tion of lysosomes was tested by vital staining with acridine
orange. When Huh-7 cells were stained with acridine orange,
the nuclei, in particular, the nucleoli, and the cytoplasm
showed green fluorescence, whereas orange fluorescence was
observed in a granular pattern in the cytoplasm. This
distribution pattern suggests that the orange fluorescence is
caused by acidified lysosomes. While orange fluorescence
was observed in cPrG-HCl–untreated cells (Fig. 5A), it was
not seen in cPrG-HCl–treated cells for 1 hour (Fig. 5B) and
thereafter.
pHi. To examine the changes in pHi induced by cPrG-HCl,
we treated the cells for the indicated duration, loaded them
with BCECF-AM, and evaluated pHi by flow cytometry.
When Huh-7 cells were exposed to cPrG-HCl for 24 hours,
the mean pHi decreased from 7.3 to 6.8 (Fig. 6A and 6B).
However, the mean pHi in cPrG-HCl ⫹ imidazole–treated
cells slightly decreased from 7.3 to 7.05 in 24 hours (Fig. 6A
and 6B).
Effect of cPrG-HCl on Tumor Growth In Vivo. The effects of
cPrG-HCl at doses of 1 mg/kg and 10 mg/kg were compared
with the cPrG-HCl–untreated controls. Tumor growth curves,
determined by tumor volume, are shown in Fig. 7 and Table
3. In the 10-mg/kg cPrG-HCl–treated group, evaluated by the
T/C value, a remarkable effect of the treatment was observed:
there was up to 81.3% reduction in tumor weight relative to
the untreated controls.
Histopathological Examination of Tumors. Tumors in the cPrG-
HCl–untreated groups exhibited histopathological character-
istics of well-differentiated hepatocellular carcinoma (Fig. 8A
and 8B). However, a large number of tumor cells in both the
high-dose– and low-dose–treated groups showed signs of
apoptosis, with cell shrinkage and chromatin condensation
(Fig. 8D and 8E). These cells were also identified as apoptotic
by the TUNEL method, and the apoptotic index (TUNEL)
was increased in the cPrG-HCl–treated tumors (significantly
different from the controls) (Fig. 8C, 8F, Table 3).
Side Effects of cPrG-HCl. The cPrG-HCl–treated mice were
active throughout the course of the experiment, and there
were no differences in weight gain among the groups (Table
3). Serum alanine transaminase, total protein, albumin, blood
urea nitrogen, and creatinine remained within the normal
limits (Table 4), and histopathological examinations revealed
no changes among the groups in the liver, kidney, adrenal
FIG. 7. The Huh-7 cells were trypsinized, and 107 viable cells/0.2 mL of
PBS (⫺) were injected into the backs of 24 mice with a 26-gauge needle. The
tumors grew to a volume of approximately 75 mm3. The mice were then
divided randomly into three groups (10-mg/kg cPrG-HCl–treated group,
1-mg/kg cPrG-HCl–treated group, and cPrG-HCl–untreated group) with 8
mice per group. cPrG-HCl was freshly prepared and subcutaneously injected
daily in 1% DMSO in saline (vehicle). In the controls, the same volumes of
vehicle without cPrG-HCl were injected. Tumor volume was measured with a
caliper and calculated using a standard formula: width2 ⫻ length ⫻ 0.5. (䊊),
Untreated control; (䊐), 1-mg/kg cPrG-HCl–treated group; (䉭), 10-mg/kg
cPrG-HCl–treated group. *P ⬍ .01 compared with the untreated control.
gland, gastrointestinal tract, spleen, pancreas, lung, bladder,
uterus, ovary, and bone marrow.
DISCUSSION
In the present study, we examined the effect of a new
antibiotic compound, cPrG-HCl, on liver cancer cells both in
vitro and in vivo. cPrG-HCl inhibited the growth of 6 liver
cancer cell lines in vitro at concentrations of submicromolar
ranges, while it had little effect on normal rat hepatocytes.
Among the cell lines, the Huh-7 cells died by apoptosis
typified by distinctive morphological changes, DNA fragmen-
tation, as well as the appearance of a sub-G1 population of
cellular DNA histograms.
cPrG-HCl is a prodigiosin derivative that is suggested to be
a potent inhibitor of the proliferation of mouse T cells as well
as Jurkat cells.14 Although its biochemical target has not yet
been identified, cPrG-HCl uncouples H⫹ translocation of
vacuolar ATPase without affecting ATP hydrolysis resulting in
alkalinization of acidic compartments in the cells.13,14 Re-
cently, it has been reported that other prodigiosin derivatives
TABLE 3. Tumor Volume, Apoptotic Index, and Body Weight at Autopsy
Tumor
Drug Body Weight T/C Apoptotic
(dose, mg/kg) (g) mg (%)† Index (%)
Control 20.7 ⫾ 0.5 1,907 ⫾ 358 100 0.6 ⫾ 0.3
cPrG-HCl (1) 21.1 ⫾ 0.9 1,194 ⫾ 184* 62.6* 8.7 ⫾ 1.3*
cPrG-HCl (10) 20.8 ⫾ 0.8 357 ⫾ 124* 18.7* 17.6 ⫾ 3.5*
*Significantly different from control (P ⬍ .01).
†(Mean values of treated group/mean values of control group) ⫻ 100.
900 YAMAMOTO ET AL. HEPATOLOGY October 1999

FIG. 8. Histopathological examination of tumors. (A-C) Xenografted Huh-7 cell tumor in the control group and (D-F) the tumor with treatment of
cPrG-HCl (10 mg/kg). The treatment with cPrG-HCl produced tumor cells with signs of apoptosis including cell shrinkage and chromatin condensation
(arrowhead [E]) and positive staining by the TUNEL method (F). (Hematoxylin-eosin staining: [A, D] ⫻100; [B, E] ⫻400; TUNEL staining: [C, F] ⫻400.)
HEPATOLOGY Vol. 30, No. 4, 1999
TABLE 4. Serum Biochemical Values at Autopsy
Drug (dose, mg/kg) ALT (mU/ml) TP (g/dl)
Control 27.4 ⫾ 5.1 5.5 ⫾ 0.5
cPrG · HCl (1) 28.0 ⫾ 4.5 5.2 ⫾ 0.4
cPrG · HCl(10) 28.2 ⫾ 5.2 5.6 ⫾ 0.7
facilitate H⫹/Cl⫺ symport across membranes of lysosomes.15,16
We also observed that the acidification of plant vacuoles
driven by pyrophosphatase was potently inhibited by cPrG-
HCl in the presence of Cl⫺ ions.30,31 It is thus suggested that
the prodigiosin and its derivatives, including cPrG-HCl, act
as an H⫹/Cl⫺ symporter that facilitates H⫹ movement across
membranes following the downward gradient of Cl⫺ ions
directs (external ⬎ internal). In Huh-7 cells, cPrG-HCl
induced not only the alkalinization of lysosomes (Fig. 5B),
but also a marked decrease in pHi, from 7.3 to 6.8 (Fig. 6) and
leading to apoptosis (Figs. 2 and 3). In contrast, maintenance
of pHi at alkaline ranges by the addition of the membrane-
permeable weak base, imidazole, attenuated the cytotoxic
effects of cPrG-HCl (Figs. 1, 2, and Table 2). Taken together,
it is plausible that cPrG-HCl induces acidification of the
cytosol and thereby leads to apoptosis on Huh-7 cells.
The cytosolic pH in transformed or cancerous cells is
generally regulated at neutral or even slightly more alkaline
than normal cells32 by a variety of pHi homeostatic machin-
ery, including Na⫹/H⫹ exchanger, Na⫹-dependent and -inde-
pendent Cl⫺/HCO3⫺ exchangers, V-ATPase, and others.10
Drugs known to inhibit these types of reactions, such as
amylorides,33-35 4,4-diisothiocyanstilbene 2,2-disulphonic
acid,36,37 or bafilomycin A138, perturb the pHi homeostasis,
and thereby lead to apoptosis of cancer cells. Furthermore, an
ionophore, nigericin, which abolishes the pH gradient across
cell membranes by exchanging K⫹ ions with H⫹, also has
antitumor effects.33 Thus, the acidification of cytosol and/or
the disruption of the pH gradient across the cytoplasmic
membrane appears to be involved in triggering the apoptotic
process in cancer cells. In addition, it is of particular interest
that undecycloprodigiosin (prodigiosin 25-C) suppresses the
proliferation of human lymphocytes by blocking the cell
cycle at the mid to late G1 phase.39 Prodigiosin 25-C inhibits
the phosphorylation of the retinoblastoma protein and sup-
presses the induction of cyclin E, cyclin A, cyclin-dependent
kinase-2, and cyclin-dependent kinase-4. These observations
are consistent with the results obtained in this study: The cell
population of the G0/G1 phase increased, and that of the S
phase decreased, during 72 hours of incubation with cPrG-
HCl (Table 2).
We further investigated the effect of cPrG-HCl on the
growth of xenografted Huh-7 cells in nude mice. The
cPrG-HCl treatment at either low (1-mg/kg) or high (10-mg/
kg) doses effectively suppressed tumor growth (Fig. 7, Table
3). Histopathological examinations of the tumors showed the
appearance of apoptotic morphologies in cPrG-HCl–treated
mice. Remarkably, there were no signs of abnormality in body
weight, histopathological changes in any organs, and serum
enzymes among control and cPrG-HCl–treated mice, even
after daily administration of 10 mg/kg of cPrG-HCl for 14
days.
Although the molecular mechanisms underlying the sup-
pressive effect of cPrG-HCl on tumor cells must be studied
YAMAMOTO ET AL. 901
Alb (g/dl) BUN (mg/dl) Cr (mg/dl)
3.0 ⫾ 0.4 32.8 ⫾ 5.1 0.05 ⫾ 0.01
2.9 ⫾ 0.5 34.2 ⫾ 6.2 0.04 ⫾ 0.01
3.0 ⫾ 0.3 32.0 ⫾ 6.1 0.07 ⫾ 0.01
further, its high potency on liver cancer cells, and nominally
no toxicity on normal cells, raises the possibility of its
therapeutic use as an effective anticancer drug.
Acknowledgment: The authors are indebted Mr. H. Kuriki
and Mr. F. Ishida for technical assistance.
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