Androgen receptor down regulation by small interference RNA induces cell growth inhibition in androgen sensitive as well as in androgen independent prostate cancer cells

We investigated the effects of androgen receptor (AR) down regulation with a small interference RNA molecule (siRNA_AR(start)) on androgen sensitive LNCaP and androgen independent LNCaPabl prostate cancer cells, the latter representing an in vitro model for the development of therapy resistance in prostate cancer. Although LNCaPabl cells express increased levels of AR in comparison with androgen sensitive LNCaP cells, the protein was significantly down regulated in response to siRNA_AR(start) treatment. This AR down regulation resulted in a marked cell growth inhibition in both cell lines. By contrast, DU-145 prostate cancer cells, which lack AR expression, were not inhibited by the siRNA_AR(start). In consequence to AR down regulation, both cell lines, LNCaP and LNCaPabl, shared a highly similar gene expression profile in terms of major changes in cell cycle regulatory genes. The cell cycle inhibitor p21(Waf1/Cip1) as well as cyclin D1 were significantly up regulated by siRNA_AR(start) treatment, considering a switch in cyclin expression towards cell cycle retardation. Control molecules had moderate effects on cell proliferation and gene expression, respectively. In summary, we found that AR inhibition with siRNA induces cell growth retardation in androgen sensitive as well as in androgen independent prostate cancer cells and thus may represent an interesting approach to combat hormone-refractory prostate cancer.     

Androgen Suppresses the Proliferation of Androgen Receptor-Positive Castration-Resistant Prostate Cancer Cells via Inhibition of Cdk2, CyclinA,and Skp2

The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3^AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3^AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27^Kip1; and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3^AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3^AR cells partially blocked accumulation of p27^Kip1 and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.     

Regulation of fibroblast growth factor-like protein(s) in the androgen-responsive human prostate carcinoma cell line LNCaP

Growth of the normal and malignant prostate is known to be regulated by androgens. Part of their effect has been suggested to be mediated through coordinated regulation of secreted growth factors with autocrine function. We now examine the biological role of preferentially paracrine acting factors in growth control of prostate cancer, i.e. fibroblast growth factor(s) (FGF). Coculture experiments using the androgen-responsive human prostate carcinoma cell line LNCaP as feeder cells and the FGF-dependent human adrenal carcinoma SW-13 cell line as target cells show that (i) LNCaP cells induce growth of SW-13 cells, (ii) even higher stimulation of SW-13 cells is seen in the presence of androgen treated LNCaP cells and (iii) a specific anti-bFGF antibody inhibits growth of SW-13 cells induced by androgen treated LNCaP cells; no proliferation of SW-13 cells occurs in the absence of LNCaP cells. Partial purification of the secretory products of LNCaP cells was performed by affinity chromatography using a heparin sepharose column. Fractions were tested for biological activity in a soft agar assay with SW-13 cells. Several activities could be detected, the main activity was eluted with about 1.5 M NaCl. These data suggest that androgen treatment of LNCaP cells leads to enhanced secretion of proteins which belong to the FGF-family.     

Autophagy protects LNCaP cells under androgen deprivation conditions

Androgen plays a critical role in the development and progression of prostate cancer. However, the regulatory role of androgen in the autophagic process and the function of the increased autophagosomes following androgen deprivation remain poorly understood. We found that autophagosomes, which were induced upon serum deprivation in LNCaP cells, can be significantly suppressed by dihydrotestosterone (DHT). Pharmacological inhibition of autophagy by 3-methyladenine led to increased apoptosis of LNCaP cells in serum-free medium compared to the medium with DHT or serum. Additionally, depletion of Beclin 1 to inhibit autophagy by small interfering RNA resulted in a slower proliferation of LNCaP cells in the medium depleted of serum than in the medium with DHT. Altogether, these findings suggested that LNCaP cells can resort to the autophagic pathway to survive under androgen deprivation conditions, which can be a novel mechanism involved in the transition of prostate cancer cells from an androgen-dependent to an androgen-independent cell type.     

Growth factors in human prostate cancer cells: implications for an improved treatment of prostate cancer

It has been previously shown that estrogens may exert their action on human breast cancer cells through coordinated control of secreted growth factors which act in an autocrine and paracrine fashion. Growth stimulation of the androgen receptor negative prostate carcinoma cell line DU-145 by dihydrotestosterone in the presence of the androgen-responsive human prostate carcinoma cell line LNCaP now indicates that androgens may regulate growth of prostate carcinoma cells through related mechanisms. A variety of androgen-regulated growth modulatory activities with autocrine and paracrine potential can be detected in conditioned media from LNCaP cells partially purified by ion exchange chromatography. Androgen-induced growth of LNCaP cells is partially inhibited by the polyanions suramin and dextran sulfates which antagonize growth factor action. These data suggest the existence of at least two different mechanisms of growth regulation by androgen which can be distinguished by their different sensitivity to growth factor inhibitory agents. We conclude that the combination of antipeptidergic substances and androgen withdrawal would represent a new and promising strategy for treatment of human prostate cancer.     

Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals

Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.     

Differential inhibition of Smad6 and Smad7 on bone morphogenetic protein- and activin-mediated growth arrest and apoptosis in B cells

Smad6 and Smad7 prevent ligand-induced activation of signal-transducing Smad proteins in the transforming growth factor-beta family. Here we demonstrate that both Smad6 and Smad7 are human bone morphogenetic protein-2 (hBMP-2)-inducible antagonists of hBMP-2-induced growth arrest and apoptosis in mouse B cell hybridoma HS-72 cells. Moreover, we confirmed that the ectopic expressions of Smad6 and Smad7 inhibited the hBMP-2-induced Smad1/Smad5 phosphorylation. We previously reported that Smad7 is an activin A-inducible antagonist of activin A-induced growth arrest and apoptosis in HS-72 cells. Interestingly, although mRNA expression of Smad6 was induced by activin A in HS-72 cells, Smad6 showed no antagonistic effect on activin A-induced growth arrest and apoptosis. Moreover, we found that the ectopic expression of Smad7, but not Smad6, inhibited the activin A-induced Smad2 phosphorylation in HS-72 cells. Thus, Smad6 and Smad7 exhibit differential inhibitory effects in bone morphogenetic protein-2- and activin A-mediated signaling in B lineage cells.     

Somatostatin Derivative (smsDX) Targets Cellular Metabolism in Prostate Cancer Cells after Androgen Deprivation Therapy

Cancer cell metabolism responsive to androgen deprivation therapy (ADT) may be involved in the development and progression of prostate cancer and the ultimate failure of androgen-deprivation therapy. To investigate the metabolism regulation effects on androgen-independent growth of prostate cancer, an established LNCaP-s cell model that resembles the clinical scenario of castration-resistant prostate cancer (CRPC), was used in this current study. This cell line was cultured from androgen-sensitive LNCaP parental cells, in an androgen-reduced condition, resembling clinical androgen deprivation therapy. To assess the effects of smsDX on the invasiveness of prostate cancer cells we used wound healing assay and Matrigel™ invasion assay. We evaluated differentially expressed proteins of the parental LNCaP cells and LNCaP-s cells after ADT by means of two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF mass spectrometric analysis. The covered area in the wound and the number of cells invading through a Matrigel chamber were significantly smaller for cells treated with smsDX than they were for control cells treated with vehicle. 56 proteins were found differentially expressed in LNCaP-s cells compared to LNCaP cells, majority of them were down-regulated after ADT treatment. 104 proteins of LNCaP cells and 86 in LNCaP-s cells, separately, were found differentially expressed after treatment with smsDX, When we explored these protein functions within the website UniProtKB/Swiss-Prot, surprisingly, most of the proteins were found to be involved in the cellular metabolism and mitochondrial function regulation. LNCaP-s as potential metastatic androgen-independent cancer cells, its metabolism and mitochondrial functions could be altered by a new somatostatin derivative smsDX, the smsDX regulatory effects on metabolism in LNCaP-s deliver more therapeutic information with the treatment of CRPC.     

EGFR signaling pathway negatively regulates PSA expression and secretion via the PI3K-Akt pathway in LNCaP prostate cancer cells

Epidermal growth factor (EGF) and its receptor (EGFR) are involved in hormone-refractory growth and poor prognosis of a subgroup of human prostate cancer. In this communication, we investigated the regulation of PSA by the EGFR signaling pathway using LNCaP C-81 prostate cancer cells. Administration of EGF stimulated the growth of LNCaP C-81 cells, however, PSA expression and secretion were suppressed. An EGFR inhibitor, AG1478, abrogated the PSA suppression effect by EGF, in concurrence with the suppression of tyro-phosphorylation levels of EGFR. Interestingly, the AR level was also decreased in EGF-treated LNCaP C-81 cells. Moreover, LY294002, but not PD98059, inhibited the PSA and AR suppression effect by EGF in concurrence with the suppression of phosphorylation levels of Akt. In conclusion, our results strongly suggest the existence of a novel androgen-independent PSA regulatory mechanism, i.e., the EGFR signaling pathway negatively regulates PSA expression which may be induced by the alteration of AR expression via the PI3K-Akt pathway in LNCaP C-81 cells.     

Growth inhibition of androgen-responsive prostate cancer cells with brefeldin A targeting cell cycle and androgen receptor

Background  

Androgen ablation is one of the viable therapeutic options for patients with primary hormone (androgen)-dependent prostate cancer. However, an antibiotic brefeldin A (BFA) has been shown to exhibit the growth inhibitory effect on human cancer cells. We thus investigated if BFA might inhibit proliferation of androgen-responsive prostate cancer LNCaP cells and also explored how it would be carried out, focusing on cell cycle and androgen receptor (AR).     

Vitamin E succinate inhibits human prostate cancer cell growth via modulating cell cycle regulatory machinery

Several epidemiological studies have demonstrated that vitamin E is a chemopreventative agent for prostate cancer. alpha-Tocopheryl succinate (VES), a derivative of vitamin E, effectively modulates prostate cancer cell growth. However, little is known about the mechanisms regarding this action. Here we show that VES causes human prostate cancer cell LNCaP arrest at G1 phase. This effect is accomplished through VES significantly decreasing expression of the cell cycle regulatory proteins cyclin D1, D3, and E, cdk2 and 4, but not cdk6. Furthermore, VES reduces cdk4 kinase activity, Rb phosphorylation, and cyclin E mRNA expression. Recently there is increasing interest in the protective effect of the VES and selenium combination on prostate cancer. Here we show that VES and selenium work through different mechanisms to exert their inhibitory effects on prostate cancer cells. Taken together, our studies suggest that VES-mediated prostate cancer cell G1/S arrest is a consequence of the regulation of multiple molecules of the cell cycle regulatory machinery.