Moreover, the study will use genetic and pharmacology approach to carry out the experiment. Typically, the autophagy pharmacological inhibition is to use TR induced cell death to detect its effect on PC3 cells. To detect the presence of autophagic, it is critical to use TEM (transmission electron microscopy) which is the widely used technique to monitor the presence of autophagy. The genetic inhibition will involve reducing the shATG7 while increasing shLAMP2-expressing cells. The extent of knockdown will be estimated to be > 50% for ATG7 in C4-2 cells and PC3 cells.
The proposal will use the following materials to carry out the experiment:
Plasmids and Reagents
The proposal will use the recombinant dulanermin (Apo2L/TRAIL)) for the experiment. The following regents will also be used to carry out the experiment:
3-MA (3-methyladenine, and Sigma-Aldrich (propidium iodide).
Moreover, the following plasmids will be used to carry out the experiment:
pCL10 and GFP-mCherry-LC3B plasmids.
dr 8.7 and pVSVG lentiviral packaging plasmids pLKO.1 -- puro vector control, shATG7, and shLAMP2.
Fluorescein Conjugate (control siRNA),
The study will also use the following antibodies:
LAMP2, and BECN1,
HRP secondary anti-rabbit,
HRP secondary anti-mouse.
Treatments and Cell culture
The PCa cells lines LNCaP, DU145, and PC3 will be maintained in RPMI 1640 media and the media will be supplement with:
10% fetal bovine serum,
L-glutamine, and penicillin-streptomycin (100 unit/ml).
The cells will be grown in a humidified incubator at 5% CO2 and 37°C Cell will be treated with 100 ml/ng TRAIL / Apo2L and 10 mM of 3-MA.
Human prostate tissue will be collected from patients suffering from prostate cancer. The tissue will be collected based on the pathological approved protocol.
Electron microscopic analyses
Cells will be fixed in:
2.5% glutaraldehyde, pH 7.3 for 24 h.
dehydration with ethanol.
After dehydration and staining with ethanol, the samples will embedded with 12 medium eponate. The diamond knife will be used to cut thin section and stained with lead nitrate and citrate. The analysis will be carried out using a Philips electron microscope (CM12) operated at 60 kV. Cells with more than 10 vacuoles will be scored as autophagy positive. However, 10 cells per sample will be used for quantification.
The study will use quantitative technique to analyze all data collected. The statistical analysis will be using Pearson's correlation analysis. The data will also be presented in graphs, tables, charts to carry out the analysis. The Microsoft Excel 2007 will be used to present the t-test and P. values.
The researcher will secure approval from appropriate authorities before carrying the research. The study will also protect all the information of all the human subjects that will be used for the experiment to achieve 100% privacy.
The paper carries out the research proposal on prostate cancer. In both developed and developing countries, prostate cancer is one of the leading causes of death among older men aged 50-year and above. The study reviews previous studies to enhance a greater understanding on the prostate cancer. Moreover, the study will use experimental research to complete the proposal, and the quantitative statistical technique will be used to carry out data analysis using correlation techniques. The research method and data analysis will assist in achieving the following research aims:
1-To understand the mechanism how autophagy inhibition targets directly the ubiquitination of γH2AX.
2-Test whether shATG7 knockdown induced LNCap cells could influence γH2AX level following radio sensitivity by suppressing DNA repair.
3-To determine the interacting partner of USP14 in autophagy deficient cells.
This study will provide several contributions:
First, the study will assist in understanding mechanism of prostate cancer, which will assist in arriving at the solution for the management of prostate cancer.
Moreover, the research outcome will assist in understanding interaction of USP14 in the autophagy deficient cells.
More importantly, the study will assist in understanding the shATG7 knockdown and its influence on γH2AX level following radio sensitivity by suppressing DNA repair.
The study will contribute to the body of knowledge of academic and medical communities.
Kroemer, G.L, Marino G, Levine, B. (2009). Autophagy and the integrated stress response. Cell Death Differ. 16(1):21-30.
Li, N.S.L. Hsieh, W.L. Rapic-Otrin, C. et al. (2014). Ubiquitin-Specific Protease 5 Is Required for the Efficient Repair of DNA Double-Strand Breaks: e84899. PLoS One. 14;9(1).
Lock., R. Roy., S. Kenific., C.M. (2010). Autophagy facilitates glycolysis during Ras-mediated oncogenic transformation. Mol. Biol. Cell. 22(2): 165-178.
Siegel., R, (2011). Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J. Clin, 61 (4): 212 -- 236.
Sippl, W. Collura, V. Colland, F.(2011). Ubiquitin-specific proteases as cancer drug targets. Health and Wellness Resources Center.
Singh, K. (2011). Molecular Basis of Autophagy-Mediated Resistance to Radiation and Apo2L/TRAIL Therapy in Prostate Cancer Cells. U.S. Army Medical Research and Materiel Command, Maryland.
Singh, K. Sharma, S. Mir, M.C. et al. (2014). Autophagic flux determines cell death and survival in response to Apo2L/TRAIL (dulanermin). Mol Cancer: 13: 70.
Subramaniam, P. Michela, P. Dennis, S.H. et al. (2013). Compensatory increase in USP14 activity accompanies impaired proteasomal proteolysis during aging. Pub Med Central.
Schwickart M. Huang X. Lill, J.R. et al.(2010) Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival. Nature, 463:103-107.
Yang, Z.J.Chee, C.E. Huang, S. et al. (2011). The Role of Autophagy in Cancer: Therapeutic Implications. Mol Cancer Ther; 10:1533-1541.