Journal of Undergraduate Research
Volume 8, Issue 2
2 - November/December 2006

Investigation of Apoptosis in Human Prostate Carcinoma PC-3 Cells Treated with Gamma Irradiation, Vitamin E, and Novasoy Isoflavon

B. Mauldin, K. T. Shiverick, L. Rice, T. Medrano

ABSTRACT

Sensitizing PC-3 human prostate carcinoma cells with isoflavone and antioxidants before irradiation could lead to a greater induction of apoptosis and provide a greater understanding of how to induce PC-3 cancerous cells into programmed death. Instead of using only high doses of irradiation, patients could take a supplement that would prime their cancerous cells for apoptosis then undergo a lower than standard dose of irradiation. This would improve the safety and quality of life for patients afflicted with prostate cancer. The initial goal of the research was to perform qualitative analysis of the PC-3 cells after various treatments and combinational treatments with isoflavone, vitamin E, and gamma irradiation and use biochemical assays to quantify the degree of apoptosis. However, after initial studies with Hoechst stain and PARP antibody immunoblots, it was discovered that the PC-3 cell line does not induce apoptosis. Instead, results indicate this cell line undergoes a multinucleated cell death.

INTRODUCTION

Carcinoma of the prostate is the most common malignant tumor in men, with more than 180,400 newly diagnosed cases and 31,000 deaths in the United States each year1. Radiation therapy is one of the major treatment modalities in the management of human cancer. X- or y- irradiation is commonly used in fractional doses; however, radio-immunotherapy and heavy particle radiation such as neutrons or protons are also occasionally used2. Statistical studies have shown that Japanese and Chinese men have the lowest occurrence of prostate carcinoma in the world1. This has stimulated interest in Genistein, an isoflavone metabolite found in soy, an anticancer agent with the ability to induce apoptosis in human prostate carcinoma cells (PC-3)1. Apoptosis, the genetically programmed mode of cell death, is important in the response of cells to cancer therapy3. Apoptosis has emerged as an appropriate therapeutic target for the effective elimination of prostate cancer cells in androgen-dependent and –independent prostatic tumors3.

Homeostasis in multicellular organisms depends on a regulatory balance among cell proliferation, differentiation and apoptosis. A disturbance in this balance results in numerous pathological conditions, including cancers, autoimmune disorders and degenerative diseases4. Apoptosis is induced by cytochrome c followed by a Caspase cascade which in turn signals the cell to “commit suicide” and form a dense body of nucleic and proteinacious matter. The cascade is usually induced after abnormal microtubule formation or aberrant attachment to the kinetochore in mitosis4. This serves as a checkpoint for both cell cycle arrest and for the apoptotic pathway.

Previous studies involving induction of apoptosis in human prostate cancer by irradiation have indicated high levels of radiation induced DNA fragmentation and rapid cell death in DU-145 human prostate carcinoma. These results suggest that when the cells are left to grow after 72-144 hours, the main mechanism of apoptosis is apparent using an immunoflourescence assay that is specific for 3’-OH in cellular DNA5.  Other studies with apoptosis have focused on the use of antioxidants to induce apoptosis in PC-3 cells. In one study, the active component of vitamin E, gamma-Tocopherol, was found to inhibit proliferation of prostate cancer cells (LNCaP and PC-3), while having no morphological or biochemical manifestation on normal prostate epithelial cells6. Such studies present the possibility of a conjunction between irradiation and ingested activators of apoptosis in androgen-independent PC-3 cells.

Currently, there are several techniques used to identify cells undergoing apoptosis. One of the most widely used techniques is the use of a PARP antibody in congruence with western blot assays. The cleavage of the poly-ADP-ribose polymerase is a relatively straightforward indication of early apoptosis7. The goal of this research is to identify a method for inducing apoptosis in PC-3 cells through a combination treatment of antioxidants and gamma irradiation. Apoptosis will be qualitatively determined by western blot analysis and fluorescence microscopy. 

MATERIALS AND METHODS

Cell Culture and Treatments.

The human prostate cancer cell line, PC-3, was purchased from the American Type Culture Collection (ATCC) in Rockville, MD. The PC-3 cells were proliferated and stored in F12K HAM medium obtained from Sigma-Aldrich Inc. in St. Louis, MO. Ten percent fetal bovine serum, 100 units/ml penicillin-streptomycin and 2 mM 1-glutamine was added to the medium. The cells were stored and grown in a humidified 5% CO2 incubator at 37°C.

Irradiation and Treatment of Cells.

PC-3 cells were plated out onto chamber slides and allowed to grow before being treated with Novasoy, vitamin E, irradiation or a combination of the three treatments. Before platting, cell viability was checked by the cells' ability to exude 0.1% trypan blue. Equal number of cells were added to the slides and allowed to grow for 12 hours. Then they were treated and allowed to grow 48 hours before being collected. A control group was subjected to 0Gy irradiation, while a second group was treated with 6Gy gamma irradiation.

Hoechst Stain and the Fluorescent Microscope.

Hoechst 33342 (H342) is the dye that was used to stain the cells after they had been treated, but while they were still in the 24-well plates. The stain allowed for observation of the cells under the fluorescence microscope. The dye is excited by ultraviolet light at around 350 nm and was originally suspended in dH20 at a 1mM concentration. The dye was added to the cell suspension from the stock solution to make a final concentration of 10 µM. The cells were incubated for 1 hour at 37°C. After this point the cells were taken to the microscope where they were observed for morphological changes. Apoptotic bodies and mitotic cells were observed and counted.

Western immunoblots.

PC-3 lysates that had been treated with irradiation were used to perform a western blot. Equal amounts of protein were electrophoresed on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked in TBS-Tween20 containing 5% nonfat milk powder for one hour at room temperature. They were then incubated for one hour with the PARP antibody (Santa Cruz Inc., Santa Cruz, CA). Membranes were treated for 1 hour with horseradish peroxidase-conjugated anti-mouse secondary antibody (Bio-Rad Laboratories, Hercules, CA). The protein bands were detected using enhanced chemoilluminescent detection system (Amersham Pharmacia Biotech UK Ltd.).

RESULTS AND DISCUSSION

Figure 1. PARP cleavage detected by 12% PAGE western blot.

Figure 1. PARP cleavage detected by 12% PAGE western blot.

Figure 2. PC-3 cells treated with 6Gy gamma irradiation, stained with Hoechst stain, and excited with UV light at 350nm.

Figure 2. PC-3 cells treated with 6Gy gamma irradiation, stained with Hoechst stain, and excited with UV light at 350nm. Mitosis shown in upper right corner.

Figure 3. PC-3 cells treated with DMSO, stained with Hoechst stain, and excited with UV light at 350 nm.

Figure 3. PC-3 cells treated with DMSO, stained with Hoechst stain, and excited with UV light at 350 nm. Mitosis bottom right, cell death left corner.

Figure 4. PC-3 cells treated with vitamin E and isoflavone, stained with Hoechst stain, and excited with UV light at 350 nm.

Figure 4. PC-3 cells treated with vitamin E and isoflavone, stained with Hoechst stain, and excited with UV light at 350 nm. Cell death fragments apparent throughout central area of the slide. 

Figure 5. PC-3 cells treated with vitamin E, stained with Hoechst stain, and excited with UV light at

Figure 5. PC-3 cells treated with vitamin E, stained with Hoechst stain, and excited with UV light at 350 nm. Multinucleated cell in bottom left section of the slide.

PARP cleavage is an early indicator of apoptosis. The western blot analysis as shown in Figure 1 gave no indication of cleaved PARP in a dose-related manner. Other experiments performed with PARP antibody detection also presented a lack of PARP cleavage. The lack of cleavage suggests that apoptosis was not being induced. After numerous studies with the fluorescence microscope, results like those shown in Figures 2-5 were obtained. Microscope images were taken and then scored according to the appearance of apoptotic bodies, mitosis or cellular fragments. This method of identifying indicators of apoptosis proved inadequate. The results could not be analyzed quantitatively because there was not possible to determine if cell fragments like those shown in figure 4 resulted from apoptosis or another form of cell death. Bromfield theorized that PC-3 cells are not killed by apoptosis but instead die through a post-mitotic cell death8. The multinucleated cell at the bottom right of Figure 5 appears to be a post-mitotic cell like those described in Bromfield’s research. Despite being unsuccessful at identifying apoptosis in PC-3 cells, this research did show that isoflavone, vitamin E, and gamma irradiation did have an affect in activating the cell death of PC-3 cells. It also gave support to the research presented by Bromfield. Future research into understanding the exact pathway of cell death in PC-3 cells would be beneficial to developing better treatments for prostate cancer.

REFERENCES

  1. Hilman, G. G., Forman, J., Kucuk, O., Yudelev, M., Rubio, Johanna, Layer, A., Tekyi-Mensah, S., Abrams, J., Sarkar, Fazlul, “Genistein Potentiates the Radiation Effect on Prostate Carcinoma Cells,” Clinical Cancer Research,Vol. 7, pp. 382-390, 2001.
  2. Kypranou, Natasha, King, Edward D., Bradbury, David, Rhee, Juong G., “bcl-2 Over-Expression Delays Radiation-Induced Apoptosis without Affecting the Clonogenic Survival of Human Prostate Cancer Cells,” International Journal of Cancer, Vol. 70, pp. 341-348, 1997.
  3. Prasad, K. N., Cole, W.C., Kumar, B., Prasad, K. C. “Pros and Cons of Antioxidant use during Radiation Therapy,” Cancer Treatment Reviews, Vol. 28, pp. 79-91, 2002.
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  5. Algan, O., Stobbe CC, Helt AM, Hanks GE, Chapman JD. “Radiation Inactivation of Human Prostate Cancer Cells: the role of Apoptosis.” Radiation Res, Vol. 146, pp. 267-75, 1996.
  6. Jiang, Q., Wong, J., Fryst, H., Saba, JD, Ames, BN. “Gamma-Tocopherol or Combinations of vitamin E forms induce Cell Death in Human Prostate Cancer Cells by Interrupting Sphingolipid Synthesis,” Proc National Academy of Science U.S.A., Vol. 101,pp. 17825-30, 2004.
  7. Chaobin Hu, Steven Smith, Liyi Pang, Yoel Sadovsky, Michael Nelson. “The Influence of Differentiation on the expression of p53 and Bcl-2 Family Proteins in Cultured Term Human Trophoblasts,” Department of Obstetrics and Gynecology Washington School of Medicine.
  8. Bromfield, G.P., Meng, A.,  Warde, P., and R G Bristow, “Cell death in irradiated prostate epithelial cells: role of apoptotic and clonogenic cell kill,” Prostate Cancer and Prostatic Diseases, Vol. 6, pp. 73-85, 2003.

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