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From the * Department of Andrology, the
Department of Transfusion, and the
Department of Urology, Jinling Hospital,
Nanjing University Clinical School of Medicine, Nanjing, P. R. China.
| Correspondence to: X. J. Shang, Department of Andrology, Jinling Hospital, Nanjing University Clinical School of Medicine, Nanjing 210002, P. R. China (e-mail: shangxj{at}androl.cn); or G. Yao, Department of Transfusion, Jinling Hospital, Nanjing University Clinical School of Medicine, Nanjing 210002, P. R. China (e-mail: yaogenhong{at}yahoo.com). |
| Received for publication April 16, 2008; accepted for publication October 30, 2008. |
 |
Abstract |
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m) was analyzed by FCM with rhodamine 123 staining.
After 24 hours of treatment with 300 µg/mL procyanidin, the apoptosis rate
of PC-3 cells was 44.86%, and m was significantly decreased by
87.30%. With the extending of procyanidin treatment, the apoptosis rate
decreased whereas the necrosis rate increased. Procyanidin could induce
apoptosis and necrosis in PC-3 cells, which might be related to
down-regulation of m.
Key words: Grape seed extract, cell death, anticancer, mitochondrial membrane potential
Procyanidin, a polyphenol compound with strong bioactivity and pharmacologic activity, resides widely in grape seeds, hawthorn, and pine bark. A number of potential mechanisms of procyanidin have emerged, such as serving as an important in vivo antioxidant, decreasing blood pressure, reducing risk of cancer, inhibiting bacteria, and so on. In recent years, procyanidin has been well studied all over the world for its high performance, low toxicity, and high bioavailability (Cos et al, 2004). It is reported that procyanidin has shown pleiotropic anticancer effects on different cancer cells, such as cutaneous carcinoma, oral carcinoma, breast carcinoma, bronchogenic carcinoma, liver carcinoma, PCa, pancreatic carcinoma, gastric carcinoma, and so on, along with somatotrophic effects on normal cells (Ye et al, 1999). The anticancer effects of procyanidin were cytotoxic effects. However, there is no report on the effect of procyanidin on PCa and its mechanism. Therefore, in the present study, the effects of procyanidin on the induction of apoptosis in human hormone-resistant PC-3 cells were studied.
The mitochondrion, an important and distinct organelle in eukaryotic cells,
is the cell powerhouse, the depository of Ca2+, and the main
organelle producing reactive oxygen species in the cells. By converting energy
in the manner of peroxidation and phosphorylation, it provides essential
energy to maintain all kinds of vital movement. The mitochondrial membrane
potential (m), which is necessary in the process of the formation
and maintenance of oxidative phosphorylation, regulates the selectivity and
permeability of the mitochondrial membrane to all kinds of material. By this
means, it maintains the normal structure and function of the mitochondrial
membrane (Zamzami et al,
1996). In order to determine whether procyanidin-induced apoptosis
and necrosis of prostate cancer PC-3 cells is related to alteration of
mitochondrion, we used flow cytometry (FCM) to detect changes in
m.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cell Growth
PC-3 cells were cultured as a monolayer in RPMI 1640 medium containing 5%
calf serum, 100 U/mL penicillin, 100 U/mL streptomycin, 2 mmol/L
L-glutamine, and 10 mmol/L HEPES in a cell culture flask. The
culture was maintained in humidified incubator in an atmosphere of 5%
CO2 at 37°C. The medium was changed every 48 hours unless
specified otherwise. The cells were subcultured with 0.25% trypsin and split
at a 1:3 ratio.
m Analysis
To detect the alterations in m, FCM was performed using the
m-sensitive dye Rh123. PC-3 cells in log phase growth were plated
onto 6-well plates at a density of 1 x 105 cells/well and
allowed to attach for 24 hours. Then, the medium was replaced with an equal
volume of fresh medium containing different concentrations (100, 200, and 300
µg/mL) of procyanidin. PBS was used as the negative control. At the end of
24 hours of treatment, cultures were incubated with Rh123 (1 mg/mL in dimethyl
sulfoxide) at 37°C for 30 minutes, the final concentration of which was 5
µg/mL. Adherent cells were washed twice with PBS and harvested by a brief
trypsinization followed by another wash with PBS. The changes in m
were analyzed by FCM using Rh123 as the indicator, with the single beam at 488
nm excitation wavelength and 530 nm emission wavelengths.
Quantitative Apoptotic Cell Death Assay
To quantify procyanidin-induced apoptotic death of PC-3 cells, annexin V
and PI staining were performed followed by FCM. PC-3 cells in log phase growth
were plated onto 6-well plates at a density of 1 x 105
cells/well and allowed to attach for 24 hours. Then, the medium was replaced
with an equal volume of fresh medium containing different concentrations (100,
200, and 300 µg/mL) of procyanidin for desired periods of time (6 and 24
hours). After the treatment, the attached cells were collected by
trypsinization and washed twice with PBS, and the cell suspension was
subjected to 400 µl 1 x binding buffer, 5 µl annexin V, and 10
µl PI staining and then left in the dark for 15 minutes. Normal, apoptotic,
necrotic, and mechanically damaged cells were determined by FCM with the
single beam at 488 nm excitation wavelength. PBS was used as the negative
control.
|
±
SEM. Statistical significance between groups was analyzed by 1-way analysis of
variance followed by Student-Newman-Keuls multiple comparisons tests. A
P value of < .05 was considered significant. |
Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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mm were detected by FCM. With the increasing concentrations of
procyanidin, the numbers of PC-3 cells increased notably in the
hypofluorescent part, from 32.57% to 87.30%
(Figure 1). Especially with the
low concentration (100 µg/mL) of procyanidin, cells increased to 84.70% in
the hypofluorescent part compared with the control cells (32.57%). A decrease
in m was observed in PC-3 cells after procyanidin treatment. In
conclusion, procyanidin could decrease m, which in turn to the
reduction of fluorochrome entering PC-3 cells and hence the diminished
fluorescence.
Effects of PC on Apoptosis in PC-3 Cells
We assessed the dose- (100–300 µg/mL) and time-dependent (6 and 24
hours) apoptotic effects of procyanidin on PC-3 cells using annexin V and PI
staining, which is used to detect pristine apoptosis efficiently. As shown in
Figures 2 and
3, after the treatment of PC-3
cells, apoptotic cells were seen in the lower right part and necrotic cells
were seen in the upper right part. This indicated that procyanidin induced
strong apoptosis in both dose- and time-dependent manners. In our study, PC
treatment of different doses for 6 hours resulted in 2.33%, 6.42%, and 42.05%
apoptosis compared with the control (0.33%). The 24-hour treatment showed that
procyanidin led to 5.64%, 6.70%, and 44.86% apoptosis. Similar results were
shown on the necrotic PC-3 cells. After 6 hours of treatment, the necrotic
rates were 0.8%, 6.8%, 29.84%, and 39.33% in 0, 100, 200, and 300 µg/mL
procyanidin respectively. In 24 hours, the necrotic rates were 0.8%, 13.38%,
13.88%, and 50.81% in 0, 100, 200, and 300 µg/mL procyanidin respectively.
Procyanidin was shown to be capable of inducing apoptosis and necrosis in PC-3
cells in a dose-/time-dependent manner.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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m, the decrease of
adenosine triphosphate (ATP) produced by mitochondrion, and the reduction of
translation and transcription in mitochondrion, which result in apoptosis and
necrosis (Mayer and Oberbauer,
2003) Rh123 served to determine the alteration of mitochondrion
membrane potential. In the present study, we found that procyanidin treatment
significantly increased the percentage of PC-3 cells with hypofluorescence,
suggesting a remarkable decrease in m, which could damage the
function and integrity of mitochondrial membrane. The apoptosis was presumably
procyanidin induced. The distribution of membrane phospholipids in normal cells is known to be asymmetric. Phospholipids with negative electricity (like phosphatidylserine [PS]) are on the internal surface of membrane. The neutral phospholipids are on the superficies externa. During pristine apoptosis, PS on the internal surface removes to the superficies externa as extracellular phosphatidylserine, a hallmark of apoptotic cells. In the present study, the apoptotic and necrotic cells were detected by annexin V/PI double staining (Vermes et al, 1995; Yao et al, 2007). The results showed that with the increase in the procyanidin dose, the percentages of both cells increased respectively.
Our study showed that 24-hour procyanidin treatment induced dissipation of
m earlier than PS reversion, indicating that the former occurred
before the apoptosis and necrosis of PC-3 cells, which was the pristine
manifestation of corpuscular toxic action. The mitochondrion is one of the
most important organelles regulating the apoptosis of cells, and there is
consanguineous association between the dissipation of m and
apoptosis. We thought that the procyanidin induced free radicals and active
oxygen species, thereby resulting in the decrease of m. Thus, it
is deduced that the participation of mitochondria-related mechanism is one of
the factors resulting in procyanidin-induced apoptosis.
The alteration in the function of mitochondrion could result in diminished
production of ATP and degraded dehydrogenase action; affect the respiration,
metabolism, and energy provision of cells; and cause damage or even death to
cells (Green and Reed, 1998).
If cytochrome c or apoptosis inducer is released from mitochondria,
it can activate the caspase enzymatic system and act on DNA in cell nucleus
and cell skeleton, resulting in inconvertible apoptosis. If the oxygen free
radical from mitochondria increases, it can lead to the decrease of
m, the hindrance of ATP formation, and eventually the loss of
electron transfer function and necrosis of cells
(An et al, 2004). In
conclusion, our study shows that procyanidin can induce apoptosis and necrosis
of PC-3 cells in a mitochondrion-dependent manner. In activating the caspase
enzymatic system, more inducer could lead to necrosis whereas less inducer
could result in apoptosis (Lemasters et al,
1998). More studies are needed in the future to get more relevant
information to reinforce our findings.
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Acknowledgments |
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Footnotes |
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References |
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Green DR, Reed JC. Mitochondria and apoptosis.
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Hildeman DA, Mitchell T, Teague TK, Henson P, Day BJ, Kappler J, Marrack PC. Reactive oxygen species regulate activation-induced T cell apoptosis. Immunity. 1999; 10: 735 –744.[CrossRef][Medline]
Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics, 1998. CA Cancer J Clin. 1998; 48: 6 –29.[Abstract]
Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, Crowe RA, Cascio WE, Bradham CA, Brenner DA, Herman B. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta. 1998; 1366: 177 –196.[Medline]
Mayer B, Oberbauer R. Mitochondrial regulation of apoptosis. News Physiol Sci
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Yao G, Ling L, Luan J, Ye D, Zhu P. Nonylphenol induces apoptosis of Jurkats cells by a caspase-8 dependent mechanism. Int Immunopharmacol. 2007;7: 444 –453.[CrossRef][Medline]
Ye X, Krohn RL, Liu W, Joshi SS, Kuszynski CA, McGinn TR, Bagchi M, Preuss HG, Stohs SJ, Bagchi D. The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells. Mol Cell Biochem. 1999;196: 99 –108.[CrossRef][Medline]
Zamzami N, Marchetti P, Castedo M, Hirsch T, Susin SA, Masse B, Kroemer G. Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis. FEBS Lett. 1996;384: 53 –57.[CrossRef][Medline]
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