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ONCOLOGY LETTERS 6: 1123-1127, 2013 



Spica Prunellae extract inhibits the proliferation of human 
colon carcinoma cells via the regulation of the cell cycle 

WEI LIN 1 ' 2 , LIANGPU ZHENG 1 ' 2 , QUNCHUAN ZHUANG 1 ' 2 , ALING SHEN 1 ' 2 , LIYALIU 1 ' 2 , 
YOUQINCHEN 3 , THOMAS J. SFERRA 3 and JUNPENG 1 ' 2 

1 2 

Academy of Integrative Medicine; Fujian Key Laboratory of Integrative Medicine on Geriatrics, 

Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, P.R. China; 
"Rainbow Babies and Children's Hospital, Case Western Reserve University School of Medicine, 

Cleveland, OH 44106, USA 

Received May 7, 2013; Accepted July 24, 2013 
DOI: 10.3892/ol.2013.1512 



Abstract. Spica Prunellae has long been used as a signifi- 
cant component in numerous traditional Chinese medicine 
(TCM) formulas to clinically treat cancers. Previously, 
Spica Prunellae was shown to promote cancer cell apoptosis 
and inhibit angiogenesis in vivo and in vitro. To further 
elucidate the precise mechanism of its tumoricidal activity, 
the effect of the ethanol extract of Spica Prunellae (EESP) on 
the proliferation of human colon carcinoma HT-29 cells was 
elucidated and the underlying molecular mechanisms were 
investigated. The proliferation of HT-29 cells was evaluated 
using 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium 
bromide (MTT) and colony formation analyses. The cell cycle 
was determined using fluorescence-activated cell sorting 
(FACS) with propidium iodide (PI) staining. The mRNA 
and protein expression of cyclin-dependent kinase 4 (CDK4) 
and cyclin Dl was examined using RT-PCR and western 
blotting, respectively. EESP was observed to inhibit HT-29 
viability and survival in a dose- and time-dependent manner. 
Furthermore, EESP treatment blocked G,/S cell cycle progres- 
sion and reduced the expression of pro-proliferative cyclin 
Dl and CDK4 at the transcriptional and translational levels. 
Altogether, these data suggest that the inhibition of cell prolif- 



Correspondence to: Dr Jun Peng, Academy of Integrative 
Medicine, Fujian University of Traditional Chinese Medicine, 
1 Huatuo Road, Minhou Shangjie, Fuzhou, Fujian 350122, 
P.R. China 

E-mail: pjunlab@hotmail.com 

Abbreviations: CRC, colorectal cancer; TCM, traditional 
Chinese medicine; EESP, ethanol extract of Spica 
Prunellae; CDK4, cyclin dependent kinase 4; MTT, 
3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; 
DMSO, dimethyl sulfoxide 

Key words: Spica Prunellae, colorectal cancer, herbal medicine, 
proliferation, cell cycle 



eration via G,/S cell cycle arrest may be one of the mechanisms 
through which Spica Prunellae treats cancer. 

Introduction 

Colorectal carcinoma (CRC) is one of the most common 
cancers with over one million new cases worldwide every 
year (1,2). Although surgical resection and complete removal 
of the tumor offers the best prognosis for long-term survival, 
~20% of CRC patients present with metastatic disease at the 
time of the diagnosis, and surgery may not always extirpate 
the recurrence of advanced CRC (3). Therefore, chemotherapy 
remains one of the major non-surgical therapeutic approaches 
for patients with advanced CRC. Despite the steady progress 
that has been made in the field of chemotherapy and targeted 
therapy, the majority of patients that undergo chemotherapy 
experience severe, debilitating and lethal adverse drug events 
that considerably outweigh the benefits (3-5). In addition, the 
long-term administration of currently used chemotherapeutic 
agents usually generates drug resistance (6). These problems 
highlight the urgent requirement for the development of novel 
anticancer agents. 

Natural products, including traditional Chinese medicine 
(TCM), have received great interest as they have relatively 
few side-effects and have long been used clinically as a 
significant alternative remedy for a variety of cancers (7-14). 
Spica Prunellae, the fruit-spikes of the perennial plant, 
Prunella vulgaris L., is a medicinal herb that is widely 
distributed in Northeast Asia. As a well-known Chinese 
folk medicinal herb with pharmacological properties of 
heat-clearing and detoxification, Spica Prunellae is tradi- 
tionally used to treat poor vision, blood stasis, edema, 
acute conjunctivitis, lymphatic tuberculosis, scrofula, acute 
mastitis, mammary gland hyperplasia, thyromegaly and 
hypertension (15). Furthermore, Spica Prunellae has also 
been employed as a significant component in several TCM 
formulas for the clinical treatment of several types of cancer, 
including CRC (16,17). Although we previously reported 
that the extract of Spica Prunellae promotes the apoptosis of 
human colon carcinoma cells and displays anti-angiogenic 
activity in vitro (18,19), the mode of its anticancer action 



1124 



LIN et ah SPICA PRUNELLAE INHIBITS HT-29 CELL PROLIFERATION 



remains largely unknown. To further elucidate the mechanism 
of the tumoricidal activity of Spica Prunellae, the present 
study evaluated the effect of the ethanol extract of Spica 
Prunellae (EESP) on the proliferation of human colon carci- 
noma HT-29 cells and investigated the underlying molecular 
mechanisms. 

Methods 

Materials and reagents. Dulbecco's modified Eagle's medium 
(DMEM), fetal bovine serum (FBS), penicillin-streptomycin, 
trypsin-ethylenediaminetetraacetic acid (EDTA) and TRIzol 
reagent were purchased from Invitrogen Corporation (Carlsbad, 
CA, USA). Superscript II reverse transcriptase was provided 
by Promega Corporation (Madison, WI, USA). Cyclin Dl, 
cyclin-dependent kinase 4 (CDK4), p-actin antibodies 
and horseradish peroxidase (HRP)-conjugated secondary 
antibodies were obtained from Cell Signaling Technology 
(Danvers, MA, USA). All the other chemicals that were used, 
unless otherwise stated, were obtained from Sigma-Aldrich 
Corporation (St. Louis, MO, USA). 

Preparation of EESP. A total of 500 g Spica Prunellae was 
extracted with 5,000 ml 85% ethanol using a reflux method and 
filtered . The ethanol solvent was evaporated on a rotary evapo- 
rator (RE-2000; Shanghai Yarong Biochemical Instrument 
Factory, Shanghai, China) and concentrated to a relative 
density of 1.05. Dried powder EESP was obtained by spray 
desiccation using a spray dryer (B-290; Biichi Labortechnik 
AG, Flawil, Switzerland). The stock solution of EESP was 
prepared by dissolving the EESP powder in 50% dimethyl 
sulfoxide (DMSO) to a stock concentration of 500 mg/ml, and 
the working concentrations were made by diluting the stock 
solution in the cell culture medium. The final concentration of 
DMSO in the medium for all the cell experiments was <0.5%. 

Cell culture. Human colon carcinoma HT-29 cells were 
obtained from the Cell Bank of the Chinese Academy of 
Sciences (Shanghai, China). The cells were grown in DMEM 
containing 10% (v/v) FBS, 100 U/ml penicillin and 100 ^g/ml 
streptomycin, in a 37°C humidified incubator with 5% C0 2 . 
The cells were subcultured at 80-90% confluency. 

Cell viability evaluation. Cell viability was assessed using 
a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium 
bromide (MTT) colorimetric assay. The HT-29 cells were 
seeded into 96-well plates at a density of lxlO 4 cells/well in 
0.1 ml medium. The cells were treated with various concen- 
trations of EESP for different periods of time. At the end of 
the treatment, 100 fil MTT [0.5 mg/ml in phosphate-buffered 
saline (PBS)] was added to each well and the samples were 
incubated for an additional 4 h at 37°C. The purple-blue MTT 
formazan precipitate was dissolved in 100 yi\ DMSO. The 
absorbance was measured at 570 nm using an ELISA reader 
(ELX800; BioTek, Winooski, VT, USA). 

Colony formation . The HT-29 cells were seeded into 6-well 
plates at a density of 2xl0 5 cells/well in 2 ml medium. 
Following the treatment with various concentrations of EESP 
for 24 h, the cells were harvested and diluted in fresh medium 



in the absence of EESP and then reseeded into 6-well plates at 
a density of lxlO 3 cells/well. Following an eight-day incuba- 
tion period in a 37°C humidified incubator with 5% C0 2 , the 
formed colonies were fixed with 10% formaldehyde, stained 
with 0.01% crystal violet and counted. Cell survival was calcu- 
lated by normalizing the survival of the control cells as 100%. 

Cell cycle analysis. The cell cycle analysis was performed 
by flow cytometry using a fluorescence-activated cell sorting 
(FACS) caliber (Becton Dickinson, San Jose, CA, USA) 
and propidium iodide (PI) staining. Subsequent to being 
treated with various concentrations of EESP for 24 h, the 
HT-29 cells were harvested and adjusted to a concentration of 
lxlO 6 cells/ml, then fixed in 70% ethanol at 4°C overnight. The 
fixed cells were washed twice with cold PBS and then incu- 
bated for 30 min with RNase (8 /(g/ml) and PI (10 fig/ml). The 
fluorescent signal was detected through the FL2 channel and 
the proportion of DNA that was present in the various phases 
was analyzed using ModfitLT Version 3.0 (Verity Software 
House, Topsham, ME, USA). 

RNA extraction and RT-PCR analysis. The HT-29 cells were 
seeded into 6-well plates at a density of 2xl0 5 cells/well in 2 ml 
medium. The cells were treated with various concentrations of 
EESP for 24 h. Total RNA was isolated using TRIzol reagent. 
01igo(dT)-primed RNA (1 fig) was reverse transcribed with 
Superscript II reverse transcriptase according to the manufac- 
turer's instructions. The obtained cDNA was used to determine 
the amount of CDK4, cyclin Dl and glyceraldehyde 3-phos- 
phate dehydrogenase (GAPDH) mRNA using PCR with Taq 
DNA polymerase (Fermentas, Waltham, MA, USA). GAPDH 
was used as an internal control. The primers that were used for 
amplification of the CDK4, cyclin Dl and GAPDH transcripts 
were as follows: CDK4 forward, 5'-CAT GTA GAC CAG GAC 
CTA AGC-3' and reverse, 5'-AAC TGG CGC ATC AGA TCC 
TAG-3'; cyclin Dl forward, 5'-TGG ATG CTG GAG GTC 
TGC GAG GAA-3' and reverse, 5'-GGC TTC GAT CTG CTC 
CTG GCA GGC-3'; and GAPDH forward, 5'-GT CAT CCA 
TGA CAA CTT TGG-3' and reverse, 5'-GA GCT TGA CAA 
AGT GGT CGT-3'. 

Western blotting. The HT-29 cells were seeded into 25-cm 2 
flasks at a density of 2xl0 5 cells/well in 5 ml medium. The 
cells were treated with various concentrations of EESP 
for 24 h and then lysed with mammalian cell lysis buffer 
containing protease and phosphatase inhibitor cock- 
tails. The lysates were resolved in 12% sodium dodecyl 
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 
and electroblotted. The polyvinylidene difluoride (PVDF) 
membranes were blocked with 5% skimmed milk and probed 
with primary antibodies against cyclin Dl (monoclonal, 
mouse), CDK4 (monoclonal, mouse) and P-actin (polyclonal, 
rabbit) at 1:1,000 dilution overnight at 4°C and then with the 
appropriate HRP-conjugated secondary antibody followed 
by enhanced chemiluminescence detection. 

Statistical analysis. All the data are presented as the mean of 
three determinations. The data were analyzed using the SPSS 
package for Windows (version 11.5; SPSS, Inc., Chicago, IL, 
USA). The statistical analysis of the data was performed with 



ONCOLOGY LETTERS 6: 1123-1127, 2013 



1125 



120 




EESP (mg/ml) 



Figure 1. Effect of EESP on the viability of HT-29 cells. The cells were 
treated with various concentrations of EESP for the indicated time periods. 
Cell viability was determined using an MTT assay. The data were normal- 
ized to the viability of the control cells (100%, treated with 0.5% DMSO 
vehicle). The data are presented as the mean ± SD (error bars) from three 
independent experiments. P<0.05, vs. the control cells. EESP, ethanol extract 
of Spica Prunellae; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazo- 
lium bromide; DMSO, dimethyl sulfoxide. 



an ANOVA. P<0.05 was considered to indicate a statistically 
significant difference. 

Results 

EESP inhibits HT-29 cell proliferation. HT-29 cell viability 
was examined using an MTT assay to compare the relative 
number of cells in EESP-treated monolayers with untreated 
controls. As shown in Fig. 1, treatment with 0.5-2.0 mg/ml 
EESP for 12, 24 or 48 h, respectively, reduced cell viability 
by 6.5-49.6, 18.4-68.7 or 36.7-82.2% compared with the 
untreated control cells (P<0.05). To further verify these 
results, the effect of EESP on HT-29 cell survival was exam- 
ined using a colony formation assay. As shown in Fig. 2, 
EESP treatment dose-dependently reduced the cell survival 
rate by 28.8-89.8% compared with the untreated control 
cells (P<0.05). Collectively, these data indicate that EESP 
inhibited HT-29 cell growth and proliferation in a dose- and 
time-dependent manner. 

EESP prevents the G/S progression of HT-29 cells. To eluci- 
date the mechanism of the anti-proliferative activity of EESP, 
its effect on cell cycle progression was examined in HT-29 cells 
using FACS analysis with PI staining. As shown in Fig. 3, the 
percentage proportion of S -phase cells following treatment 
with 0, 0.5, 1 and 2 mg/ml EESP was 46.1±5.3, 29.5±3.3, 
22.5±3.0 and 14.7±2.1%, respectively (P<0.05), indicating that 
the inhibitory effect of EESP on HT-29 cell proliferation was 
mediated by G/S cell cycle arrest. 

EESP inhibits the expression of cyclin Dl and CDK4 in 
HT-29 cells. To further explore the mechanism by which 
EESP inhibited cell proliferation and G/S transition, RT-PCR 
and western blot analysis were performed to respectively 
examine the mRNA and protein expression of cyclin Dl and 
CDK4 in the HT-29 cells. As shown in Fig. 4A and C, EESP 
treatment significantly and dose-dependently reduced the 
mRNA expression of pro-proliferative cyclin Dl and CDK4 



A EESP Omg/ml 




EESP (mg/ml) 



Figure 2. Effect of EESP on cell survival in HT-29 cells. (A) Cell survival 
was determined using a colony formation analysis. The images are repre- 
sentative of three independent experiments. (B) The data were normalized 
to the control cells and are shown as the mean ± SD (error bars) from three 
independent experiments. *P<0.05, vs. the control cells. EESP, ethanol extract 
of Spica Prunellae. 



in the HT-29 cells. The results of the western blot analysis 
revealed that the protein expression patterns of cyclin Dl 
and CDK4 were similar to their respective mRNA levels 
(Fig 4B and D). 



1126 



LIN et ah SPICA PRUNELLAE INHIBITS HT-29 CELL PROLIFERATION 




Figure 3. Effect of EESP on cell cycle progression in HT-29 cells. The cells were treated with the indicated concentrations of EESP for 24 h, stained with PI 
and analyzed using FACS. The proportion of DNA in the S-phase was calculated using ModfitLT Version 3.0 Software. Data are shown as the mean ± SD 
(error bars) from three independent experiments. P<0.05, vs. the control cells. EESP, ethanol extract of Spica Prunellae; PI, propidium iodide; FACS, fluores- 
cence-activated cell sorting. 



EESP (mg/ml) D EESP (mg/ml) 




CDK4 CVclinDl CDK4 Cydin Dl 



Figure 4. Effect of EESP on CDK4 and cyclin Dl expression in HT-29 cells. The cells were treated with the indicated concentrations of EESP for 24 h. (A) The 
mRNA levels of CDK4 and cyclin Dl were determined using RT-PCR. (B) The protein expression levels of CDK4 and cyclin Dl were analyzed by western 
blotting. GAPDH and p-actin were used as the internal controls for the RT-PCR and western blotting assays, respectively. The data are representative of three 
independent experiments. (C and D) Densitometric analysis. The data were normalized to the mean mRNA or protein expression of the untreated control cells 
(100%).'P<0.05, vs. the control cells. EESP, ethanol extract of Spica Prunellae; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CDK4, cyclin-dependent 
kinase 4. 



Discussion 

Due to drug resistance and the adverse effects of the majority 
of currently used cancer chemotherapies, natural prod- 
ucts receive great interest since they have relatively fewer 
side-effects and have been used clinically for thousands of 
years as important alternative remedies for a variety of diseases, 



including cancer (7-14). One promising medicinal plant is 
Spica Prunellae, which is widely distributed in Northeast 
Asia. As a well-known traditional Chinese folk-medicine, 
it is traditionally used to treat poor vision, blood stasis, 
edema, acute conjunctivitis, lymphatic tuberculosis, scrofula, 
acute mastitis, mammary gland hyperplasia, thyromegaly 
and hypertention (15). In addition, Spica Prunellae has long 



ONCOLOGY LETTERS 6: 1123-1127, 2013 1127 



been employed for the clinical treatment of several types of 
cancer (16,17). Although we previously reported that Spica 
Prunellae promotes cancer cell apoptosis and inhibits tumor 
angiogenesis (18,19), the precise mechanism of its potential 
tumoricidal activity remains largely unclear. Therefore, prior 
to the development of Spica Prunellae as an anticancer agent, 
the mode of its anti-tumor action should be further elucidated. 

Cancer cells are characterized by an uncontrolled increase 
in cell proliferation (20). The presents study therefore inves- 
tigated the effect of Spica Prunellae on the proliferation of 
human colon carcinoma HT-29 cells. By using MTT and 
colony formation analyses, it was demonstrated that EESP 
dose- and time-dependently inhibited the proliferation of 
the HT-29 cells. Eukaryotic cell proliferation is primarily 
regulated by the cell cycle, which consists of four periods: 
The S phase (DNA synthesis phase), M phase (mitosis), G, 
phase and G 2 phase. G[/S transition is one of the two main 
checkpoints of the cell cycle (21), which is responsible for 
the initiation and completion of DNA replication. Using 
FACS analysis with PI staining the present study observed 
that the percentage proportion of S -phase cells was reduced 
by EESP treatment in a dose-dependent manner, indicating 
that the inhibitory effect of EESP on HT-29 cell proliferation 
was mediated by G,/S cell cycle arrest. Gj/S progression is 
strongly regulated by cyclin Dl, which exerts its function by 
forming an active complex with its major catalytic partners, 
including CDK4 (22-24). An unchecked or hyperactivated 
cyclin D1/CDK4 complex often leads to uncontrolled cell 
division and malignancy (25). By performing RT-PCR and 
western blot analyses, the present study identified that EESP 
treatment suppressed the expression of pro-proliferative 
cyclin Dl and CDK4 in the HT-29 cells at the transcriptional 
and translational levels. 

In conclusion, the present study demonstrated for the first 
time that Spica Prunellae inhibits the proliferation of cancer 
cells through G,/S cell cycle arrest, which may be one of the 
mechanisms through which Spica Prunellae exerts its anti- 
tumor activity. 

Acknowledgements 

This study was sponsored by the National Natural Science 
Foundations of China (grant nos. 81073097 and 81202790). 

References 

1 . Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: 
Global cancer statistics. CA Cancer J Clin 61: 69-90, 2011. 

2. Markowitz SD and Bertagnolli MM: Molecular origins of 
cancer: Molecular basis of colorectal cancer. N Engl J Med 361: 
2449-2460, 2009. 

3. Jiang WQ, Fu FF, Li YX, Wang WB, Wang HH, Jiang HP and 
Teng LS: Molecular biomarkers of colorectal cancer: prognostic 
and predictive tools for clinical practice. J Zhejiang Univ Sci 
B 13: 663-675,2012. 

4. Hanahan D and Weinberg RA: The hallmarks of cancer. Cell 100: 
57-70, 2000. 



5. Lippman SM: The dilemma and promise of cancer chemopre- 
vention. Nat Clin Pract Oncol 10: 523, 2006. 

6. Longley DB, Allen WL and Johnston PG: Drug resistance, 
predictive markers and pharmacogenomics in colorectal cancer. 
Biochim Biophys Acta 1766: 184-196, 2006. 

7. Gordaliza M: Natural products as leads to anticancer drugs. Clin 
Transl Oncol 9: 767-776, 2007. 

8. Jia L: Cancer complementary and alternative medicine research 
at the US National Cancer Institute. Chin J Integr Med 18: 
325-332,2012. 

9. Carmady B and Smith CA: Use of Chinese medicine by cancer 
patients: a review of surveys. Chin Med 9: 22, 201 1. 

10. Liu J, Li X, Liu J, Ma L, Li X and F0nneb0 V: Traditional Chinese 
medicine in cancer care: a review of case reports published in 
Chinese literature. Forsch Komplementmed 18: 257-263, 2011. 

11. Yang GY, Li X, Li XL, Wang L, Li J, Song X, Chen J, Guo Y, 
Sun X, Wang S, Zhang Z, Zhou X and Liu J: Traditional Chinese 
medicine in cancer care: a review of case series published in the 
Chinese literature. Evid Based Complement Alternat 2012: 1-8, 
2012. 

12. Newman DJ, Cragg GM and Snader KM: The influence of natural 
products upon drug discovery. Nat Prod Rep 17: 215-234, 2000. 

13. Taixiang W, Munro AJ and Guanjian L: Chinese medical herbs 
for chemotherapy side effects in colorectal cancer patients. 
Cochrane Database of Systematic Reviews, Issue 1, CD004540. 
DOI: 10 . 1002/ 1465 1858 .CD004540 .pub2 . 

14. Zhang M, Liu X, Li J, He L and Tripathy D: Chinese medicinal 
herbs to treat the side-effects of chemotherapy in breast cancer 
patients. Cochrane Database of Systematic Reviews, Issue 2, 
CD004921. DOI: 10.1002/14651858.CD004921.pub2. 

15. Chinese Pharmacopeia Commission. Pharmacopoeia of the 
People's Republic of China. Chinese Medical Science and 
Technology Press, Beijing, p263, 2010. 

16. Cao Z, Lin W, Huang Z, Chen X, Zhao J, Zheng L, Ye H, Liu Z, 
Liao L and Du J: Ethyl acetate extraction from a Chinese herbal 
formula, Jiedu Xiaozheng Yin, inhibits the proliferation of hepa- 
tocellular carcinoma cells via induction of G0/G1 phase arrest in 
vivo and in vitro. Int J Oncol 42: 202-210, 2013. 

17. Cao Z, Lin W, Huang Z, Chen X, Zhao J, Zheng L, Ye H, Liu Z, 
Liao L and Du J: Jiedu Xiaozheng Yin, a Chinese herbal formula, 
inhibits tumor angiogenesis via downregulation of VEGF-A 
and VEGFR-2 expression in vivo and in vitro. Oncol Rep 29: 
1080-1086,2013. 

18. Zheng LP, Chen YQ, Lin W, Zhuang QC, Chen XZ, Xu 
W, Liu XX, Peng J and Sferra TJ: Spica Prunellae extract 
promotes mitochondrion-dependent apoptosis in a human 
colon carcinoma cell line. Afr J Phar Pharmacol 5: 327-335, 
2011. 

19. Lin W, Zheng LP, Zhao JY, Zhuang QC, Hong ZF, Xu W, 
Chen YQ, Sferra TJ, and Peng J: Anti-angiogenic effect of Spica 
Prunellae extract in vivo and in vitro. Afr J Phar Pharmacol 24: 
2647-2654,2011. 

20. Evan GI and Vousden KH: Proliferation, cell cycle and apoptosis 
in cancer. Nature 411: 342-348, 2001. 

21 . Nurse P: Ordering S phase and M phase in the cell cycle. Cell 79: 
547-550, 1994. 

22. Morgan DO: Principles of CDK regulation. Nature 374: 131-134, 
1995. 

23. Chen Y, Robles AI, Martinez LA, Liu F, Gimenez-Conti IB and 
Conti CJ: Expression of Gl cyclins, cyclin-dependent kinases, 
and cyclin-dependent kinase inhibitors in androgen-induced 
prostate proliferation in castrated rats. Cell Growth Differ 7: 
1571-1578, 1996. 

24. Grana X and Reddy EP: Cell cycle control in mammalian 
cells: role of cyclins, cyclin dependent kinases (CDKs), growth 
suppressor genes and cyclin-dependent kinase inhibitors (CKIs). 
Oncogene 11: 211-219, 1995. 

25. Zafonte BT, Hulit J, Amanatullah DF, Albanese C, Wang 
C, Rosen E, Reutens A, Sparano JA, Lisanti MP and Pestell 
RG: Cell-cycle dysregulation in breast cancer: breast cancer 
therapies targeting the cell cycle. Front Biosci 5: D938-D961, 
2000.