Abstract Introduction Chinese Medicinal Herb Scutellaria barbata Modulates Apoptosis in TRAMP-C1 Cell Line And Tumor Development in TRAMP Mice

TRAMP Mouse

Methods and Materials

Brian Y.Y. Wong, Hannah Wong, Amy Shepherd, & Alessandro Cavalcante,

Department of Biology, Union College, Lincoln, NE 68506

Scutellaria barbata (SB) has been used in traditional Chinese medicine for treating

various cancers such as liver, lung and rectal tumors. Prostate cancer is the second leading cause of deaths due to cancer among men in America.

adenocarcinoma of the mouse prostate (TRAMP) mimics Transgenic heterogeneic tumor progression in human prostate cancer, providing a relevant pre-clinical model for identifying important pathways in tumorigenesis, androgen independence, and metastasis of prostate cancer.

Detection of tumor by MRI and palpation are valid surrogate measures of tumor progression in this model. These mice are shown to be excellent models for screening of novel drugs and chemopreventive regimens for potential human use. TRAMP C-1 is a cell line established from the mouse. In this study, we determined the extent of apoptosis and necrosis by labeling the DNA strand breaks of TRAMP C-1 cancer cells on chamber slides after DNase I digestion of the non-treatment control group and incubation with 1mg/ml aqueous extract of SB in the experimental groups for 2 and 8 hours separately using terminal deoxynucleotidyl transferase (TdT) TUNEL assay. Daily oral feeding of male TRAMP mice (9 weeks old) in random groups with sterile water as placebo and experimental groups with 8mg and 16mg of sterile aqueous extracts of SB respectively were also performed. Efficacy was evaluated by the absence of palpable tumor formation. Significant time-dependent apoptotic and necrotic effects of SB on the cells were observed. After culturing with SB for 2 hours, the induction of apoptosis in TRAMP-C1 was 90.3 + 2.9%; while with 8-hour incubation, significant lower percentage of cells undergone apoptosis as compared to the control with positive DNA strands breaks staining (71.7 + 3.6% < 88.5 + 3.5%, p<0.05). The amount of necrotic cells also increased significantly from 4.8% to 23.8% between 2 and 8 hours of incubation, p<0.05. These shows that 2 hours is the optimum incubation time for SB to induce apoptosis in TRAMP-C1. In the placebo group of the oral feeding experiment, palpable tumors developed initially presented at 19 weeks of age and by 32 weeks, all of the mice had palpable tumors; while at this time 20% and 30% of the mice in the 8 mg/day SB group and 16 mg/day SB group were free of tumor respectively.

In both of the low-dose and high-dose SB groups, there were a lag of 2 and 4 weeks before the detection of palpable tumors separately. At 27 weeks, less than 30% of the placebo animals was free of palpable tumors; while in the low-dose and high-dose groups, there were 50% and 70% tumor free mice respectively. The period of time at which 50% of the mice had tumors was 24.5 weeks in the placebo group, 27 weeks in the low-dose group, and 28.5 weeks in the high-dose group. There was difference between the placebo and high-dose groups, p<0.05. Our data demonstrates SB contain phytochemicals that modulate apoptosis of the TRAMP mouse prostate cancer cells in vitro and delay tumor development

in

vivo,

thus suggesting its potential chemopreventive effects.

TRAMP-C1 cells (100x)

Cells stained with crystal violet after the Cytotoxicity Assay

Induction of Apoptosis of TRAMP-C1 Cells by SB

Apoptotic Cells Necrotic Cells Normal Cells

Results

Positive control

88.5 ± 3.5

9.0 ± 5.0

2.5 ± 1.5

TRAMP-C1 cells (250x)

TUNEL Assay. Cells (2 hr. SB treatment) counterstained with DAB substrate after

2hr Incubation

90.3 ± 2.9

4.8 ± 1.3

5.0 ± 1.8

8hr Incubation

*71.7 ± 3.6

*23.8 ± 2.0

4.5 ± 0.5

Table 1 above shows the percentage of each type cell present in each experimental group, compared to the positive control where the DNA was digested by DNAse. These values represent the average % of cell populations from two separate experiments.

* Indicates that the percentage of cells in a particular population is significantly different from the control (p<0.05) .

Scutellaria barbata

Preparation of herbal extract

Crude SB was purchased from Nuherbs Co.,Oakland, CA. It was grounded into fine powder. 10g of powder was suspended into 100ml of distilled water and heated for 1hr at 56 °C.

The suspension was then centrifuged at 2000rpm for 10min, filtered and supernatant centrifuged again at 3000rpm for 10min. The supernatant was then filter-sterilized, lyopholized and the dry weight was determined. All concentrations used in experiment were based on dry weight of extract

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Cell Culture Preparation

TRAMP-C1 cells were obtained from Dr.Greenberg, Baylor College of Medicine. Cells were received frozen in cryo-tubes and thawed by placing in a 37 °C water bath. Cells were triturated with a 3ml, 18 gage needle and placed in a 60mm cell culture dish with 5ml warm media. Cells were then incubated at 37 °C in 5% CO 2 incubator for 24 hrs. After 24hrs, cells were split by addition of 3ml of trypsin. Cells were washed with 6ml Hanks buffer and centrifuged at 2500 rpm for 5 minutes. Supernatant was discarded, and 6ml of Hanks buffer added to pellet and centrifuged for another 5min at 2500rpm. Concentration of cells was then determined by staining with trypan blue and scored with hemocytometer chamber.

Cytotoxicity and LD 50

TRAMP-C1 cells (50,000/plate) were seeded into 60 mm plates in triplicates and treated with varying log concentrations of SB (0, 0.015, 0.15, 1.5 mg/ml) for 24hours. Cells were then washed with Hanks buffer allowed to grow for 5 days. After 5 days cells were washed with PBS buffer and fixed with 5ml of methanol for 3 minutes, air-dried, and stained with crystal violet. Cell colonies were then counted. Percent of survival cells was calculated and semi-log survival curve was plotted and LD 50 was determined as 1 mg/ml.

TUNEL Assay

Protocol for the TUNEL Assay was based on the

In Situ

cell Death Detection Kit, POD 2001 Manual from Roche Applied Science

6

. Cells were cultured in chamber slides and treated with SB. Two experimental groups (2hr and 8hr) and a negative control and a positive control were used. After treatment, samples were dried and fixed with Fixation solution for 1hr at 15-25 °C. Cells were then incubated at 15-25°C for 10min with Blocking Solution. After blocking solution was removed, Permeabilization solution added. Positive Control was incubated with DNase, Grade I, while 50 µl of TUNEL reaction mixture was added to each sample and incubated in a humidified, dark chamber. Signal conversion was then conducted by addition of 50 µl Converter-POD followed by DAB Substrate as illustrated in the diagram below: Fixed cells with Fluorescein-labeled DNA strand breaks Anti-fluorescein Antibody conjugated With POD (Roche Applied Science 2001) DAB Substrate for POD

TRAMP Mice Herbal Feeding & Prostate Tumor Development

PB-Tag transgenic C57BL/6 mice were cross-bred with FVB wild-type mice. The hybrid litters were screened by PCR for the presence of the PB-Tag transgene

5

. Positive males were fed daily with 0.2 to 0.4 ml of sterile water or aqueous SB extract via a stomach tube connected to a 1ml syringe. The various feeding dosages were tested before the research began and were found to be non-toxic to the mice. Daily oral feeding of male TRAMP mice in randomized groups with sterile water as placebo (n=21) and experimental groups with 8mg (n=10, one died) and 16mg (n=10) of sterile aqueous extracts of SB respectively were started when the animals were 8 weeks old.

Efficacy SB to divert or slow down tumor development was evaluated by the absence of palpable tumor formation.

Scutellaria barbata

(SB) (Chinese name Pan-chih-lien of the Labiatae family) is a 12-35cm tall annual herb, found along fields and ditches in southern China. It is a species related to mint. SB has a mildly bitter taste and is commonly used in Chinese Medicine for the treatment of a variety of ailments such as appendicitis, hepatitis, snake bites, lung, liver and rectal cancer

2

. Previous research has found that SB may also possess anti-mutagenic and chemopreventative properties

7

.

Our recent study further reveals SB treatment elevated H 2 O 2 and hydroxyl radical production in the macrophage-like RAW 264.7 mouse peritoneal cell line. These data indicate that SB modulates Cox-2, and iNOS protein expression, as well as PGE2 and NOx to the formation of reactive oxygen species

3

.

in vitro

and that this may be related Prostate Cancer is the most common cancer in American men. It was predicted to have killed as many as 30,200 people by the end of 2002. The incidence of prostate cancer has found to be affected by race, geography, diet and genetics. There is yet no known cure for prostate cancer

1

.

Transgenic Adenocarcinoma Mouse Prostate Model (TRAMP) is a spontaneous autochthonous transgenic mouse model. It was created by incorporation of the PB SV40T antigen transgene into the syngeneic C57B1/6 host. This antigen transgene contains the rat probasin (PB) gene and the SV40 T antigen (Tag). SV40 is a papovavirus which produces tumors in animals. Animals with these gene develop severe hyperplasia and cribriform structures by 12 weeks and 100% display tumors by 24-30 weeks. Transgenic adenocarcinoma of the mouse prostate (TRAMP) mimics heterogeneic tumor progression in human prostate cancer, providing a relevant pre-clinical model for identifying important pathways in tumorigenesis, androgen independence, and metastasis of prostate cancer. Detection of tumor by MRI and palpation are valid surrogate measures of tumor progression in this model. These mice are shown to be excellent models for screening of novel drugs and chemopreventive regimens for potential human use. TRAMP- C1 line was established from the prostate adenocarcinoma of a 32 week old C57B1/6 male. Two other lines –C2, -C3 were established and differ in their growth rates and morphology

4

.

100 90 80 70 60 50 40 30 20 10 0

Chemopreventive Effects of SB in TRAMP Mice

Placebo (n=21) SB 8mg/day (n=9) SB 16mg/day (n=10)

18 19 20 21 22 23 24 25 26 27 Mice Age (weeks) 28 29 30 31 32 33 34 35

Conclusion

The TUNEL results of this research suggest that SB was most effective at inducing apoptosis in TRAMP-C1 with 2 hours of treatment.

Previous study demonstrated that oral feeding with the same SB extract (4 mg/day) significantly inhibited the growth of renal cell carcinoma (Renca) in Balb/c mice with the transplanted cancer cells

8

.

These

in vitro

plus animal data further illustrates that SB contains phytochemicals which modulate apoptosis of mouse prostate cancer cells and delay tumor development in TRAMP mice, suggesting potential chemopreventive effect in human prostate cancer development.

References

1. American Cancer Society. Cancer Facts and Figures (2003).

2. Chong S.C. and L.H. Lee. Chinese Med. Herbs of H.K., Vol. 2 (1988).

3.

Harris G.K.,

et al

. AACR 94 th Annual Meeting Proceedings 44:1419 (2003).

4.

Gingrich J.R. and N.M. Greenberg, Toxicologic Pathology 24:502-504 (1996).

5. Raghow S., et al. Cancer Res 60:4093-4097 (2000).

6. Roche Applied Science.

In situ

Cell Death Detection Kit (POD) (2002).

7.

Wong B.Y.

et al

. Mutation Res. 279:209-216 (1992).

8. Wong B.Y.

et al

. Cancer Biother Radiopharm 11:51-56 (1996).

Acknowledgements

1. The Nebraska Academy of Sciences, Inc. for student research award.

2. Dr.

N.M. Greenberg, Baylor Medical College for TRAMP mice & cells.

3. Stephen Nazario, Benjamin Wong, & Stephen Yen for pictures & diagrams.