Overexpression of Thymosin .BETA.4 Increases Pseudopodia Formation in LNCaP Prostate Cancer Cells

Abstract

Most patients with advanced prostate cancer are candidates for androgen ablation therapy because growth and progression of prostate cancer cells are initially androgen dependent.1,2) Although androgen ablation therapy initially causes tumor regression in Ͼ80% of cases, prostate cancer eventually progresses from an androgen-dependent to an aggressive androgen-independent state after the therapy, and the prostate cancer in this state is often difficult to cure. 2,3) One of the reasons why prostate cancer cells after androgen ablation therapy acquire aggressive phenotype is changes occur in the expression of androgen-regulated genes after the therapy. 4,5) In our recent study, we found that thymosin b4 expression in androgen-sensitive prostate cancer LNCaP cells is elevated in androgen-deprived culture condition. 6)Thymosin b4 is a small acidic 4.9-kDa polypeptide widely distributed in human tissues. Thymosin b4 functions as a major G-actin sequestering factor that modulates dynamic changes of actin cytoskeleton. 7) In addition, thymosin b4 is involved in a variety of physiologic and pathologic processes. For example, thymosin b4 is known to promote wound healing, tumor metastasis, and angiogenesis. [8][9][10] Overexpression of thymosin b4 is detected highly in metastatic cancer cells in human and animal models. [11][12][13] In addition, thymosin b4 stimulates cardiac cell migration and survival related to cardiac repair.14) In the present study, we found that thymosin b4 expression increases pseudopodia formation related to cell migration and invasion in human prostate LNCaP cells. MATERIALS AND METHODS MaterialsWortmannin was purchased from SigmaAldrich Japan K.K. (Tokyo, Japan). Toxin B from Clostridium difficile was from List Biological Laboratories (Campbell, CA, U.S.A.). U0126 was purchased from Wako Pure Chemical (Osaka, Japan). Rapamycin was from Merck Ltd. (Tokyo, Japan).Cell Culture Human prostate cancer LNCaP cells were from American Type Culture Collection (Rockville, MD, U.S.A.). LNCaP cells were cultured in RPMI-1640 medium containing 10% fetal calf serum (FCS) under a humidified atmosphere with 5% CO 2 at 37°C. Plasmid ConstructionsTotal RNA from LNCaP cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.) then first-strand complementary DNA was synthesized from the total RNA using SuperScript III (Invitrogen) according to the manufacturer's instructions. The cDNA coding region of human thymosin b4 was amplified by polymerase chain reaction (PCR) from the cDNA using primers containing restriction sites for XbaI and HindIII. The sequences of the primers were, sense: 5Ј-GCTCTAGAAT-GTCTGACAAACCCGATATG-3Ј; antisense: 5Ј-CCAAGC-TTTTACGATTCGCCTGCTTGCTT-3Ј. PCR was performed using PrimeSTAR HS DNA Polymerase (Takara Bio Inc., Otsu, Japan) under the following conditions: 32 cycles of 30 s at 98°C, 30 s at 58°C, and 30 s at 72°C. The resulting PCR product was digested with XbaI and HindIII and inserted into the XbaI and HindIII sites of a pRK5 mammalian expression vector. The plasmid vect...

Immunoreactivity of thymosin beta 4 in human foetal and adult genitourinary tract

“…19 Presence of Tβ 4 has been reported during the formation of actin-based pseudopodia in tumoral cells, facilitating cell migration and prostate cancer progression. 20 Our research has also shown high Tβ 4 presence in tumour infiltrating mast cells. 21 Overall, our research suggests that Tβ 4 has an important role in the development of different embryonic and adult organs.…”

Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression

“…Filamin-A mutant lacking the actin-binding domain is unable to repress the promoter activity, suggesting that filamin-A mediates its suppressive function to rRNA transcription together with actin. 72 Also ABPs such as Thymosin-β4 73 and α-actinin, 74 which are both targets of Rho GTPase signaling, 75,76 have been found in the nucleus. However their nuclear function has not been determined.…”

Effects of thymosin β4 on wound healing of rat palatal mucosa

“…Upregulation of Tβ 4 by cDNA-mediated transfection was found to cause actin depolymerization in fibroblast cells (1), which demonstrated that the modulation of Tβ 4 affected the polymerization state of actin cytoskeleton, as well as other cell processes associated with the organization of actin cytoskeleton, such as cell migration. Ito et al (2) showed that overexpression of Tβ 4 enhanced the formation of actin-based pseudopodia and cell motility in prostate cancer cells, providing direct evidence that Tβ 4 plays a role in cell migration. Colon cancer cells overexpressing Tβ 4 also exhibited enhanced cell migration, due in part to the activation of the Rac1 signaling pathway (3).…”