Cytotoxic effects induced by a combination of cyclopamine and gefitinib, the selective hedgehog and epidermal growth factor receptor signaling inhibitors, in prostate cancer cells

Although the blockade of the hedgehog cascade by using cyclopamine has been reported to inhibit the growth of some cancer cell types, few studies on the mechanism by which this drug alone or in combination with other cytotoxic agents induces its cytotoxic effect have been reported. In our study, we evaluate, for the first time, the antiproliferative and cytotoxic effects induced by a combination of selective SMO inhibitor, cyclopamine and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib on metastatic prostate cancer (PC) cells. The results revealed that cyclopamine, alone or at a lower concentration in combination with gefitinib, inhibited the growth of sonic hedgehog- (SHH), epidermal growth factor- (EGF) and serum-stimulated androgen-sensitive LNCaP-C33 and LNCaP-LN3 and androgen-independent LNCaP-C81, DU145 and PC3 cells. The antiproliferative effect of cyclopamine and gefitinib, alone or in combination, was mediated via a blockade of the PC3 cells in the G1 phase of the cell cycle. Importantly, the combined cyclopamine and gefitinib also caused a higher rate of apoptotic death of PC cells compared to single agents. The cytotoxic effect induced by these drugs in PC3 cells appears to be mediated at least, in part, via the mitochondrial pathway through the depolarization of the mitochondrial membrane and the release of cytochrome c and reactive oxygen species into the cytosol. This was also accompanied by the activation of caspase cascades, PARP cleavage and DNA fragmentation. Additionally, the combined cyclopamine and gefitinib were more effective at suppressing the invasiveness of PC3 cells through matrigel in vitro as the drugs alone. These findings indicate that the simultaneous blockade of SHH-GLI-1 and EGF-EGFR signaling, which results in the growth arrest and massive rate of apoptotic cell death, represents a promising strategy for a more effective treatment of metastatic PC forms.     

Synergistic antiproliferative and apoptotic effects induced by epidermal growth factor receptor and protein kinase a inhibitors in human prostatic cancer cell lines

Our results revealed that the blockade of epidermal growth factor receptor (EGFR) tyrosine kinase and protein kinase A (PKA) signaling pathways by specific inhibitors (PD153035 and Rp-cAMPs) leads to a synergistic inhibition of EGF- and serum-stimulated growth of human prostatic cancer cells (LNCaP, DU145 and PC3) concomitant with an arrest in the G1 phase of cellular cycle. Of particular interest, the combination of PD153035 and Rp-cAMPs also caused a more substantial apoptotic/necrotic death of these prostatic cancer cells as compared to drugs alone. Moreover, we observed that the inhibition of acidic sphingomyelinase and caspase cascades results in a marked reduction of DNA fragmentation and apoptotic death induced by PD153035, alone or in combination with Rp-cAMPs, in EGF stimulated PC3 cells. This suggests that these agents might mediate their cytotoxic effects at least in part via the ceramide generation and activation of caspase signaling pathways. N-oleoylethanolamine (OE), an inhibitor of acidic ceramidase, consistently potentiated the apoptotic effects of PD153035 in all the prostatic cancer cell lines tested. Additionally, the cellular ceramide content estimated for PC3 cells was increased after treatment with PD153035, alone or in combination, at a lower dose with OE and Rp-cAMPs.The synergistic apoptotic effect of PD153035 plus Rp-cAMPs induced in PC3 was also accompanied by a significant rate of mitochondrial membrane depolarization and release of cytochrome c into cytosol as compared to drugs alone. Combined, the results indicated that the simultaneous inhibition of EGFR and PKA signaling cascades might lead to a more massive apoptotic death of metastatic prostatic cancer cells by increasing ceramide accumulation and activating of caspase cascade of a mitochondrial dependent manner.     

Additive antitumor effects of the epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib (Iressa), and the nonsteroidal antiandrogen, bicalutamide (Casodex), in prostate cancer cells in vitro

Progression from an androgen-dependent to an androgen-independent state often occurs in patients with prostate cancer (PCa) who undergo hormonal therapy. We have investigated whether inhibition of the epidermal growth factor receptor (EGFR) signaling pathway affects the antitumor effect of a nonsteroidal antiandrogen. Gefitinib (Iressa), an EGFR tyrosine kinase inhibitor, and bicalutamide (Casodex), a nonsteroidal antiandrogen [androgen receptor (AR) antagonist], were administered alone and in combination to AR-positive human PCa cell lines. FACS analysis showed lower EGFR expression levels on AR-positive cells (LNCaP, CWR22, CWR22R 2152 and AR-transfected DU145 cell lines) compared with AR-negative cells (DU145, PC3 and TSU-Pr1). Moreover, in AR-transfected DU145 cells, chronic treatment with bicalutamide increased EGFR expression to levels similar to androgen-independent DU145 cells. All AR-positive PCa cell lines were sensitive to gefitinib (IC50 = 0.1-0.6 microM), whereas higher concentrations of bicalutamide were needed to reduce AR-positive PCa cell line proliferation (IC50 = 0.8-2.0 microM). Low doses of gefitinib increased the antitumor effects of bicalutamide by strongly reducing the IC50 of bicalutamide (approximately 10-fold). Similarly, bicalutamide increased the antiproliferative effects of gefitinib by reducing the IC50 of gefitinib (approximately 5-fold). Taken together, our data suggest that in androgen-dependent cell lines, addition of gefitinib in combination with bicalutamide results in concurrent dual inhibition of AR and EGFR/HER2 pathways. This causes a significant delay in the onset of EGFR-driven androgen independence.     

Identification of genes linked to gefitinib treatment in prostate cancer cell lines with or without resistance to androgen: a clue to application of gefitinib to hormone-resistant prostate cancer

Understanding the molecular action of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, might allow us to perform more effective therapies for hormone-independent advanced prostate cancer. A DNA microarray study was undertaken to comprehensively analyze the alteration of levels of 1,081 genes after gefitinib treatment in androgen-independent PC3 and DU145 cells and androgen-dependent LNCaP cells. The proliferation of PC3, DU145 and LNCaP cells was significantly inhibited by 50.2%, 83.8% and 55.2%, respectively, 6 days after 10 microM gefitinib administration. Of the above 1,081 genes, we identified 23, 13 and 33 genes with significantly different expression in PC3, DU145 and LNCaP cells, respectively, 24 h after 10 microM-gefitinib exposure. Among the identified genes, only Quiescin Q6, a negative cell cycle regulator, was increased after gefitinib treatment in all three cell lines regardless of gefitinib sensitivity. Except for Quiescin Q6, there were no overlapping genes between PC3 and DU145 cells. However, levels of several oncogenes or proliferation-related genes were changed after gefitinib treatment in the 2 androgen-independent cell lines. We also identified 7 unique genes [glycyl-tRNA synthetase, interferon, alpha-inducible protein, stratifin, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, dual specificity phosphatase 9, guanine nucleotide binding protein (G protein) beta polypeptide 2, neural retina leucine zipper] whose levels were altered exclusively after gefitinib administration in gefitinib-resistant PC3 and LNCaP cells, but not in DU145 cells, suggesting that these 7 genes could be targets for overcoming gefitinib resistance. Collectively, our molecular profiling data will serve as a framework for understanding the molecular action of gefitinib for prostate cancer.     

Methylseleninic acid potentiates apoptosis induced by chemotherapeutic drugs in androgen-independent prostate cancer cells.

PURPOSE: To test whether and how selenium enhances the apoptosis potency of selected chemotherapeutic drugs in prostate cancer (PCA) cells. EXPERIMENTAL DESIGN: DU145 and PC3 human androgen-independent PCA cells were exposed to minimal apoptotic doses of selenium and/or the topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin (SN38), the topoisomerase II inhibitor etoposide or the microtubule inhibitor paclitaxel/taxol. Apoptosis was measured by ELISA for histone-associated DNA fragments, by flow cytometric analysis of sub-G(1) fraction, and by immunoblot analysis of cleaved poly(ADP-ribose)polymerase. Pharmacologic inhibitors were used to manipulate caspases and c-Jun-NH(2)-terminal kinases (JNK). RESULTS: The methylselenol precursor methylseleninic acid (MSeA) increased the apoptosis potency of SN38, etoposide, or paclitaxel by several folds higher than the expected sum of the apoptosis induced by MSeA and each drug alone. The combination treatment did not further enhance JNK1/2 phosphorylation that was induced by each drug in DU145 cells. The JNK inhibitor SP600125 substantially decreased the activation of caspases and apoptosis induced by MSeA combination with SN38 or etoposide and completely blocked these events induced by MSeA/paclitaxel. The caspase-8 inhibitor zIETDfmk completely abolished apoptosis and caspase-9 and caspase-3 cleavage, whereas the caspase-9 inhibitor zLEHDfmk significantly decreased caspase-3 cleavage and apoptosis but had no effect on caspase-8 cleavage. None of these caspase inhibitors abolished JNK1/2 phosphorylation. A JNK-independent suppression of survivin by SN38 and etoposide, but not by paclitaxel, was also observed. In contrast to MSeA, selenite did not show any enhancing effect on the apoptosis induced by these drugs. CONCLUSIONS: MSeA enhanced apoptosis induced by cancer therapeutic drugs in androgen-independent PCA cells. In DU145 cells, the enhancing effect was primarily through interactions between MSeA and JNK-dependent targets to amplify the caspase-8-initiated activation cascades. The results suggest a novel use of methyl selenium for improving the chemotherapy of PCA.     

Alterations in the focal adhesion kinase/Src signal transduction pathway correlate with increased migratory capacity of prostate carcinoma cells

Focal adhesion kinase (FAK) has been implicated in the regulation of cell migration. In addition, FAK expression is increased in a number of highly metastatic tumor cell lines. Therefore, we investigated the role of FAK in regulating migration of prostate carcinoma cell lines with increasing metastatic potential. We show that highly tumorigenic PC3 and DU145 cells exhibit intrinsic migratory capacity, while poorly tumorigenic LNCaP cells require a stimulus to migrate. Increased metastatic potential of PC3 and DU145 cells correlates with increased FAK expression, overall tyrosine phosphorylation and activity, as measured by autophosphorylation of tyrosine 397. However, in PC3 and DU145 cells, FAK autophosphorylation is adhesion dependent whereas a second site of tyrosine phosphorylation, tyrosine 861, a Src specific site, is uncoupled from adhesion-dependent signaling events. Finally, inhibiting the FAK/Src signal transduction pathway by over expressing FRNK (Focal adhesion kinase-Related Non-Kinase), an inhibitor of FAK activation, or treatment with PP2, a Src family kinase inhibitor, significantly inhibited migration of prostate carcinoma cell lines, demonstrating that tumor cell migration continues to be dependent on signals emanating from this pathway.     

Imaging agents for in vivo molecular profiling of disseminated prostate cancer--targeting EGFR receptors in prostate cancer: comparison of cellular processing of [111In]-labeled affibody molecule Z(EGFR:2377) and cetuximab

Expression of receptor tyrosine-kinase (RTK) EGFR is low in normal prostate, but increases in prostate cancer. This receptor is significantly up-regulated as tumors progress into higher grade, androgen-insensitive and metastatic lesions. The up-regulated receptors could serve as targets for novel selective anti-cancer drugs, e.g. antibodies and tyrosine kinase inhibitors. Radionuclide imaging of RTK can facilitate patient stratification and monitoring of anti-RTK therapy of prostate cancer. The goal of the study was to evaluate binding and cellar processing of radiolabeled EGFR-targeting conjugates by prostate cancer cell lines. Receptor expression of EGFR was studied in three prostate cancer cell lines: DU145 (brain metastasis of PC, hormone insensitive), PC3 (bone metastasis of PC) and LNCaP (lymph node metastasis of PC, androgen and estrogen receptor positive). Uptake and internalization of anti-EGFR mAbs (cetuximab) and affibody molecule (Z2377) labeled with indium-111 was investigated. EGFR expression on prostate cancer cell lines was clearly demonstrated. Both labelled conjugates 111In-Z2377 and 111In-cetuximab bound to prostate cancer cells in the receptor mediated model. Expression levels were modest but correlate with degree of hormone independence. Internalization of Affibody molecules was relatively slow in all cell lines. Internalization of mAbs was more rapid. The level of EGFR expression in these cell lines is sufficient for in vivo molecular imaging. Slow internalization indicates possibility of the use of non-residualizing labels for affibody molecules.     

Human chorionic gonadotropin beta (HCGbeta) down-regulates E-cadherin and promotes human prostate carcinoma cell migration and invasion

BACKGROUND: Membrane-associated human chorionic gonadotropin beta (HCGbeta) is correlated with a poor prognosis in localized prostate adenocarcinoma. The relationship between HCGbeta and metastasis, however, is unclear. METHODS: To shed some light on the issue, two stable prostate carcinoma cell lines overexpressing HCGbeta, designated DU145 HCGbeta and PC3 HCGbeta, were created and compared with empty vector stably transfected DU145 and PC3 cells (control cells). RESULTS: HCGbeta expression resulted in a change in morphology; the cells were more elongated and had multiple pseudopodia, while the control cells were more rounded. This change in morphology was duplicated by incubating control cells in conditioned medium from the DU145 HCGbeta or PC3 HCGbeta cells, or by adding purified HCGbeta to control medium. The DU145 HCGbeta and PC3 HCGbeta cells were also less adherent than the controls, as assessed by the ease with which trypsin-EDTA could remove them from culture plates. Reduced adherence could be duplicated by incubation of control cells with either conditioned medium or purified HCGbeta. Western blot analysis showed that DU145 HCGbeta and PC3 HCGbeta cells expressed less E-cadherin than control cells and that a change of medium increased expression of E-cadherin. Addition of conditioned medium, or purified HCGbeta, to control cells down-regulated E-cadherin. Cell migration and invasion assays showed that DU145 HCGbeta and PC3 HCGbeta cells were more migratory and invasive than controls and that treatment of control cells with either conditioned medium or purified HCGbeta increased their migratory/invasive capacity. CONCLUSION: The data indicate that HCGbeta is directly responsible for changes in prostate carcinoma cells associated with an increased metastatic phenotype.     

Down?regulation of E?cadherin enhances prostate cancer chemoresistance via Notch signaling

Background: The chemoresistance of prostate cancer (PCa) is invariably associated with the aggressiveness and metastasis of this disease. New emerging evidence indicates that the epithelial-to-mesenchymal transition (EMT) may play pivotal roles in the development of chemoresistance and metastasis. As a hallmark of EMT, E-cadherin is suggested to be a key marker in the development of chemoresistance. However, the molecular mechanisms underlying PCa chemoresistance remain unclear. The current study aimed to explore the association between EMT and chemoresistance in PCa as well as whether changing the expression of E-cadherin would affect PCa chemoresistance.Methods: Parental PC3 and DU145 cells and their chemoresistant PC3-TxR and DU145-TxR cells were analyzed. PC3-TxR and DU145-TxR cells were transfected with E-cadherin-expressing lentivirus to overexpress E-cadherin; PC3 and DU145 cells were transfected with small interfering RNA to silence E-cadherin. Changes of EMT phenotype-related markers and signaling pathways were assessed by Western blotting and quantitative real-time polymerase chain reaction. Tumor cell migration, invasion, and colony formation were then evaluated by wound healing, transwell, and colony formation assays, respectively. The drug sensitivity was evaluated using MTS assay.Results: Chemoresistant PC3-TxR and DU145-TxR cells exhibited an invasive and metastatic phenotype that associated with EMT, including the down-regulation of E-cadherin and up-regulation of Vimentin, Snail, and N-cadherin,comparing with that of parental PC3 and DU145 cells. When E-cadherin was overexpressed in PC3-TxR and DU145-TxR cells, the expression of Vimentin and Claudin-1 was down-regulated, and tumor cell migration and invasion were inhibited. In particular, the sensitivity to paclitaxel was reactivated in E-cadherin-overexpressing PC3-TxR and DU145-TxR cells. When E-cadherin expression was silenced in parental PC3 and DU145 cells, the expression of Vimentin and Snail was up-regulated, and, particularly, the sensitivity to paclitaxel was decreased. Interestingly, Notch-1 expression was up-regulated in PC3-TxR and DU145-TxR cells, whereas the E-cadherin expression was down-regulated in these cells comparing with their parental cells. The use of γ-secretase inhibitor, a Notch signaling pathway inhibitor, significantly increased the sensitivity of chemoresistant cells to paclitaxel.Conclusion: The down-regulation of E-cadherin enhances PCa chemoresistance via Notch signaling, and inhibiting the Notch signaling pathway may reverse PCa chemoresistance     

Enzastaurin inhibits tumours sensitive and resistant to anti-EGFR drugs

We investigated the antitumour effect and ability to overcome the resistance to anti-EGFR drugs of enzastaurin, an inhibitor of VEGFR-dependent PKCbeta signalling. Enzastaurin was evaluated alone and in combination with the EGFR inhibitor gefitinib, on growth and signalling protein expression in human cancer cells sensitive and resistant to anti-EGFR drugs, both in vitro and in nude mice. We demonstrated the marked inhibitory activity of enzastaurin against GEO colon and PC3 prostate cancer cells and their gefitinib-resistant counterparts GEO-GR and PC3-GR, accompanied by inhibition of pAkt and its effector pp70S6K, pGSK3beta and VEGF expression and secretion. Moreover, enzastaurin showed a cooperative effect with gefitinib in parental and in gefitinib-resistant cells. Remarkably, these results were confirmed in vivo, where enzastaurin showed antitumour activity and cooperativity with gefitinib in mice grafted with GEO and GEO-GR tumours, incrementing their median survival and inhibiting the aforesaid protein expression and secretion in tumour specimens. In conclusion, enzastaurin by interfering with signalling proteins implicated in EGFR drug resistance markedly cooperates with gefitinib in sensitive and gefitinib-resistant tumours, thus overcoming and reverting such resistance and providing a rational basis for its development in patients resistant to anti-EGFR drugs.     

Differential efficacy of combined therapy with radiation and AEE788 in high and low EGFR-expressing androgen-independent prostate tumor models

PURPOSE: To determine the efficacy of combining radiation (XRT) with a dual epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor inhibitor, AEE788, in prostate cancer models with different levels of EGFR expression. METHODS AND MATERIALS: Immunoblotting was performed for EGFR, phosphorylated-EGFR, and phosphorylated-AKT in prostate cancer cells. Clonogenic assays were performed on DU145, PC-3, and human umbilical vein endothelial cells treated with XRT +/- AEE788. Tumor xenografts were established for DU145 and PC-3 on hind limbs of athymic nude mice assigned to four treatment groups: (1) control, (2) AEE788, (3) XRT, and (4) AEE788 + XRT. Tumor blood flow and growth measurements were performed using immunohistochemistry and imaging. RESULTS: AEE788 effectively decreased phosphorylated-EGFR and phosphorylated-AKT levels in DU145 and PC-3 cells. Clonogenic assays showed no radiosensitization for DU145 and PC-3 colonies treated with AEE788 + XRT. However, AEE788 caused decreased proliferation in DU145 cells. AEE788 showed a radiosensitization effect in human umbilical vein endothelial cells and increased apoptosis susceptibility. Concurrent AEE788 + XRT compared with either alone led to significant tumor growth delay in DU145 tumors. Conversely, PC-3 tumors derived no added benefit from combined-modality therapy. In DU145 tumors, a significant decrease in tumor blood flow with combination therapy was shown by using power Doppler sonography and tumor blood vessel destruction on immunohistochemistry. Maldi-spectrometry (MS) imaging showed that AEE788 is bioavailable and heterogeneously distributed in DU145 tumors undergoing therapy. CONCLUSIONS: AEE788 + XRT showed efficacy in vitro/in vivo with DU145-based cell models, whereas PC-3-based models were adequately treated with XRT alone without added benefit from combination therapy. These findings correlated with differences in EGFR expression and showed effects on both tumor cell proliferation and vascular destruction.     

Analysis of cabazitaxel‐resistant mechanism in human castration‐resistant prostate cancer

Cabazitaxel (CBZ) is approved for docetaxel‐resistant castration‐resistant prostate cancer (CRPC). However, efficacy of CBZ for CRPC is limited and there are no effective treatments for CBZ‐resistant CRPC. In order to investigate the CBZ‐resistant mechanism, the establishment of a CBZ‐resistant cell line is urgently needed. We established CBZ‐resistant CRPC cell lines DU145CR and PC3CR by incubating DU145 and PC3 cells with gradually increasing concentrations of CBZ for approximately 2 years. We analyzed the gene expression profiles and cell cycle changes using microarray and flow cytometry. Pathway analysis revealed DU145CR cells had enhanced gene clusters of cell division and mitotic nuclear division. Enhancement of ERK signaling was detected in DU145CR cells. DU145CR cells had resistance to G₂/M arrest induced by CBZ through ERK signaling activation. The MEK inhibitor PD184352 significantly inhibited cell proliferation of DU145CR. In contrast to DU145CR, PC3CR cells had enhancement of PI3K/AKT signaling. The PI3K/mTOR inhibitor NVP‐BEZ 235 had a significant antitumor effect in PC3CR cells. Cabazitaxel ‐resistant CRPC cells established in our laboratory had enhancement of cell cycle progression signals and resistance to G₂/M arrest induced by CBZ. Enhancement of ERK signaling or PI3K/AKT signaling were detected in the cell lines, so ERK or PI3K/AKT could be therapeutic targets for CBZ‐resistant CRPC.     

PX-478, an inhibitor of hypoxia-inducible factor-1alpha, enhances radiosensitivity of prostate carcinoma cells

Overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) in human tumors is associated with poor prognosis and poor outcome to radiation therapy. Inhibition of HIF-1alpha is considered as a promising approach in cancer therapy. The purpose of this study was to test the efficacy of a novel HIF-1alpha inhibitor PX-478 as a radiosensitizer under normoxic and hypoxic conditions in vitro. PC3 and DU 145 prostate carcinoma cells were treated with PX-478 for 20 hr, and HIF-1alpha protein level and clonogenic cell survival were determined under normoxia and hypoxia. Effects of PX-478 on cell cycle distribution and phosphorylation of H2AX histone were evaluated. PX-478 decreased HIF-1alpha protein in PC3 and DU 145 cells. PX-478 produced cytotoxicity in both cell lines with enhanced toxicity under hypoxia for DU-145. PX-478 (20 mumol/L) enhanced the radiosensitivity of PC3 cells irradiated under normoxic and hypoxic condition with enhancement factor (EF) 1.4 and 1.56, respectively. The drug was less effective in inhibiting HIF-1alpha and enhancing radiosensitivity of DU 145 cells compared to PC3 cells with EF 1.13 (normoxia) and 1.25 (hypoxia) at 50 mumol/L concentration. PX-478 induced S/G2M arrest in PC3 but not in DU 145 cells. Treatment of PC3 and DU 145 cells with the drug resulted in phosphorylation of H2AX histone and prolongation of gammaH2AX expression in the irradiated cells. PX-478 is now undergoing Phase I clinical trials as an oral agent. Although the precise mechanism of enhancement of radiosensitivity remains to be identified, this study suggests a potential role for PX-478 as a clinical radiation enhancer.     

Expression and function of the complement membrane attack complex inhibitor protectin (CD59) in human prostate cancer

Protectin (CD59) inhibits homologous complement-mediated cytolysis by preventing formation of the membrane attack complex at the point of insertion and polymerization of C9 into cell membranes. The present study investigated the expression and function of CD59 on human prostatic tumor cells in situ and on 5 human prostate cell lines in vitro originating from either metastatic tumors or benign prostate hypertrophy epithelial cells. Immunohistochemical staining of prostate carcinoma tissue with monoclonal antibody (MAb) MEM43 revealed weak to moderately strong expression of CD59 by prostate glandular epithelial cells. Flow cytometry with MEM43 demonstrated that the 5 prostate cell lines expressed different relative quantities of CD59. Indirect immunofluorescence analysis revealed uniform membrane staining of DU145 and PC3 cell lines with no membranous granularity in the staining pattern. Western immunoblots with MAb BRIC 229 showed that PC3 and DU145 cells express CD59 with a m.w. of 18-25 kDa. Treatment of DU145 and PC3 cells with phosphatidylinositol-specific phospholipase C caused a significant decrease of CD59 expression indicating that the CD59 expressed by prostate cancer cells is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage. PC3 and DU145 cells were completely resistant to human complement-mediated cytolysis but became sensitive to killing in the presence of the CD59-neutralizing MAb YTH53.1. We conclude that malignant and benign human prostate cells express CD59 that is GPI-linked to the cell surface and that CD59 may regulate the immunological response to cancerous prostate cells by protecting the cells from the cytolytic activity of complement. Int. J. Cancer 71: 1049-1055, 1997.© 1997 Wiley-Liss Inc.     

Resistance to growth inhibitory and apoptotic effects of phorbol ester and UCN-01 in aggressive cancer cell lines

7-Hydroxystaurosporine (UCN-01), a non-selective inhibitor of protein kinase C (PKC), and phorbol ester (PMA), a PKC activator, are undergoing clinical evaluations. We investigated the effects of UCN-01 and PMA on a panel of prostate cancer cell lines. While PMA induced p21WAF1/CIP1 and arrest growth of LNCaP cancer cells (IC50 = 0.5-1 nM), aggressive cancer cell lines (DU145, PC3, and PC3M) were resistant to PMA (IC50 >5000 nM). Low concentrations (25-50 nM) of UCN-01 abrogated PMA-induced p21 and growth arrest in LNCaP cells. These low doses of UCN-01 however did not inhibit proliferation of any prostate cancer cell line. PMA-sensitive LNCaP cells were resistant to clinically relevant concentrations of UCN-01 (IC50 = 1.2 microM), but UCN-01 inhibited growth of DU145 and PC3/3M with an IC50 of 200-400 nM. For comparison, PMA-sensitive HL60 leukemia cells were sensitive to UCN-01 due to rapid apoptosis caused by UCN-01. In PMA-resistant prostate cancer cells, UCN-01 downregulated cyclin D1, induced p21, caused morphological differentiation, and G1-phase arrest leading to slow cell death without caspase activation. Importantly, normal prostate epithelial cells (PrEC) were very sensitive to both PMA (IC50 = 0.2 nM) and UCN-01. In PrEC, UCN-01 downregulated cyclin D1 and arrest growth with an IC50 less than 100 nM. We conclude that loss of sensitivity to either UCN-01 or PMA accompanies progression of prostate cancer.     

Androgen-independent prostate cancer cells circumvent EGFR inhibition by overexpression of alternative HER receptors and ligands

The deregulation of the epidermal growth factor receptor (EGFR) pathway plays a major role in the pathogenesis of prostate cancer (PCa). However, therapies targeting EGFR have demonstrated limited effectiveness in PCa. A potential mechanism to overcome EGFR blockade in cancer cells is the autocrine activation of alternative receptors of the human EGFR (HER) family through the overexpression of the HER receptors and ligands. In the present study, we were interested in analyzing if this intrinsic resistance mechanism might contribute to the inefficacy of EGFR inhibitors in PCa. To this end, we selected two androgen-independent human prostate carcinoma cell lines (DU145 and PC3) and established DU145 erlotinib-resistant cells (DUErR). Cells were treated with three EGFR inhibitors (cetuximab, gefinitib and erlotinib) and the sensitivity to each treatment was assessed. The gene expression of the four EGFR/HER receptors and seven ligands of the HER family was analyzed by real-time PCR prior to and after each treatment. The receptors expression and activation were further characterized by flow cytometry and western blot analysis. EGFR inhibition rapidly induced enhanced gene expression of the EGF, betacellulin and neuregulin-1 ligands along with HER2, HER3 and HER4 receptors in the DU145 cells. In contrast, slight changes were observed in the PC3 cells, which are defective in the phosphatase and tensin homolog (PTEN) tumor suppressor gene. In the erlotinib-resistant DUErR cells, the expression of HER2 and HER3 was increased at mRNA and protein levels together with neuregulin-1, leading to enhanced HER3 phosphorylation and the activation of the downstream PI3K/Akt survival pathway. HER3 blockage by a monoclonal antibody restored the cytostatic activity of erlotinib in DUErR cells. Our results confirm that the overexpression and autocrine activation of HER3 play a key role in mediating the resistance to EGFR inhibitors in androgen-independent PCa cells.     

Gefitinib-trastuzumab combination on hormone-refractory prostate cancer xenograft

New drugs and new combinations of drugs have recently shown promising clinical activity in hormone refractory prostate cancer. We studied the association of gefitinib with trastuzumab on the androgen-refractory prostate cancer cell line DU145 expressing both epidermal growth factor receptor (EGFR) and HER-2. Drug combinations with radiotherapy (RT) were considered along with the analysis of factors linked to cell proliferation and apoptosis. The antitumour effects of gefitinib were more pronounced than those observed with trastuzumab. In mice receiving the gefitinib-trastuzumab combination, reduction in tumour volume was inferior to that predicted by the observed impact of the agents alone. The presence of trastuzumab markedly attenuated the relative increase on p27 expression and the Bax:Bcl2 ratio induced by gefitinib. The combination gefitinib-RT had similar antitumour effects as those predicted by the impact of the individual treatments, whereas the effect of the trastuzumab-RT combination was inferior to that predicted by the individual effects. The present data should be borne in mind when designing new clinical schedules for treatment of hormone-refractory prostate cancer including the use of HER inhibitors.