Silencing of Lactotransferrin expression by methylation in prostate cancer progression.
BACKGROUND
Cancer cells gain selection advantages by the coordinated silencing of protective and by the activation of cell proliferation/cell survival genes. Evaluations of epithelial cell transcriptome of benign and malignant prostate glands by laser capture microdissection (LCM) identified Lactotransferrin (LTF) as the most significantly downregulated gene in prostate cancer (CaP) cells (p < 10(-6)). Frequent downregulation, significant association of LTF with PSA recurrence-free survival in CaP patients and the established anti-tumorigenic effects of LTF in experimental cancer models have provided impetus to evaluate LTF expression features and mechanisms in CaP specimens.


METHODS
LTF mRNA expression analysis was performed in LCM derived benign and malignant prostate epithelial cells by using Affymetrix GeneChip and QRT-PCR. LTF protein expression was assessed in tissue specimens by immunohistochemistry and in serum samples from CaP patients compared to healthy male control by using ELISA. Mechanism of LTF downregulation was analyzed in 5-azadeoxycytidine treated LNCaP and LAPC4 cells using MALDI-TOF MS. Proliferation and cell cycle analysis of CaP cells by FACS flow cytrometry was assessed in LNCaP cell cultures.


RESULTS
Quantitative analysis of LTF mRNA expression in tumor cells revealed marked downregulation of LTF with significant associations to decreased PSA recurrence-free survival of CaP patients (n = 100, p < or = 0.0322). Moreover, low levels of LTF protein expression was observed in tumor tissues as well as in sera from CaP patients (p < or = 0.0001). LTF promoter downstream CpG island methylation was found in LNCaP and LAPC4 cells. Furthermore, replenishing of LTF by supplementing growth media with LTF protein resulted in reduced cell growth. Cell cycle analysis revealed robust increases in apoptosis in response to LTF treatment.


CONCLUSIONS
This study highlights the potential for LTF in chemoprevention and to become a biologically relevant prognostic marker of CaP, suggesting that silencing of the LTF gene may be causally linked to CaP progression.
Shaheduzzaman
Syed
S
Center for Prostate Disease Research, Department of Surgery, Uniformed Services University, Rockville, Maryland 20852, USA.
Vishwanath
Anu
A
Furusato
Bungo
B
Cullen
Jennifer
J
Chen
Yongmei
Y
Bañez
Lionel
L
Nau
Martin
M
Ravindranath
Lakshmi
L
Kim
Kee-Hong
KH
Mohammed
Ahmed
A
Chen
Yidong
Y
Ehrich
Mathias
M
Srikantan
Vasantha
V
Sesterhenn
Isabell A
IA
McLeod
David
D
Vahey
Maryanne
M
Petrovics
Gyorgy
G
Dobi
Albert
A
Srivastava
Shiv
S
D017209:Apoptosis; D019175:DNA Methylation; D018450:Disease Progression; D016193:G1 Phase; D020868:Gene Silencing; D006801:Humans; D007781:Lactoferrin / Q000032:analysis / Q000235:genetics*; D008297:Male; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis
D012333:RNA, Messenger; D007781:Lactoferrin