Analysis of tumor necrosis factor α-induced and nuclear factor κB-silenced LNCaP prostate cancer cells by RT-qPCR.
Prostate cancer is the second leading cause of morbidity and mortality in males in the Western world. In the present study, LNCaP, which is an androgen receptor-positive and androgen-responsive prostate cancer cell line derived from lymph node metastasis, and DU145, which is an androgen receptor-negative prostate cancer cell line derived from brain metastasis, were investigated. TNFα treatment decreased p105 and p50 expression and R1881 treatment slightly decreased p105 expression but increased p50 expression with or without TNFα induction. As an aggressive prostate cancer cell line, DU145 transfected with six transmembrane protein of prostate (STAMP)1 or STAMP2 was also exposed to TNFα. Western blotting indicated that transfection with either STAMP gene caused a significant increase in NFκB expression following TNFα induction. In addition, following the treatment of LNCaP cells with TNFα, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed with a panel of apoptosis-related gene primers. The apoptosis-related genes p53, p73, caspase 7 and caspase 9 showed statistically significant increases in expression levels while the expression levels of MDM2 and STAMP1 decreased following TNFα induction. Furthermore, LNCaP cells were transfected with a small interfering NFκB (siNFκB) construct for 1 and 4 days and induced with TNFα for the final 24 h. RT-qPCR amplifications were performed with apoptosis-related gene primers, including p53, caspases and STAMPs. However, no changes in the level of STAMP2 were observed between cells in the presence or absence of TNFα induction or between those transfected or not transfected with siNFκB; however, the level of STAMP1 was significantly decreased by TNFα induction, and significantly increased with siNFκB transfection. Silencing of the survival gene NFκB caused anti-apoptotic STAMP1 expression to increase, which repressed p53, together with MDM2. NFκB silencing had varying effects on a panel of cancer regulatory genes. Therefore, the effective inhibition of NFκB may be critical in providing a targeted pathway for prostate cancer prevention.
Department of Pharmacology, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey.
Ceren
C
Gonen-Korkmaz
Department of Pharmacology, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey.
Gulnur
G
Sevin
Department of Pharmacology, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey.
Goksel
G
Gokce
Department of Pharmacology, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey.
Mehmet Zuhuri
MZ
Arun
Department of Pharmacology, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey.
Gokce
G
Yildirim
Department of Pharmacology, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey.
Buket
B
Reel
Department of Pharmacology, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey.
Aysegul
A
Kaymak
Department of Pharmacology, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey.
Deniz
D
Ogut
NFκB; STAMP genes; TNFα; cell culture; gene silencing; p53; prostate cancer