Tau oligomers accumulation sensitizes prostate cancer cells to docetaxel treatment.

PURPOSE: Human tau is a highly dynamic, multifunctional protein expressed in different isoforms and conformers, known to modulate microtubule turnover. Tau oligomers are considered pathologic forms of the protein able to initiate specific protein accumulation diseases, called tauopathies. In our study, we investigated the potential association between autophagy and tau oligomers accumulation and its role in the response of prostate cancer cells to docetaxel. METHODS: We evaluated in vitro the expression of tau oligomers in prostate cancer cell lines, PC3 and DU145, in presence of autophagy inhibitors and investigated the role of tau oligomers accumulation in resistance to docetaxel treatment. RESULTS: Tau protein was basally expressed in prostate cancer lines as several monomeric and oligomeric forms. The pharmacologic inhibition of autophagy induced in cancer cells the accumulation of tau protein, with a prevalent expression of oligomeric forms. Immunofluorescence analysis of untreated cells revealed that tau was visible mainly in dividing cells where it was localized on the mitotic spindle. Inhibition of autophagy determined an evident upregulation of tau signal in dividing cells and the presence of aberrant monoastral mitotic spindles. The accumulation of tau oligomers was associated with DNA DSB and increased cytotoxic effect by docetaxel. CONCLUSIONS: Our data indicate that autophagy could exert a promoting role in cancer growth and during chemotherapy facilitating degradation of tau protein and thus blocking the antimitotic effect of accumulated tau oligomers. Thus, therapeutic strategies aimed at stimulating tau oligomers formation, such as autophagy inhibition, could be an effective adjuvant in cancer therapy.

Serum 25(OH)D seasonality in urologic patients from central Italy.

Hypovitaminosis D is increasingly recognized as a cofactor in several diseases. In addition to bone homeostasis, vitamin D status influences immune system, muscle activity and cell differentiation in different tissues. Vitamin D is produced in the skin upon exposure to UVB rays, and sufficient levels of serum 25(OH)D are dependent mostly on adequate sun exposure, and then on specific physiologic variables, including skin type, age and Body Mass Index (BMI). In contrast with common belief, epidemiologic data are demonstrating that hypovitaminosis D must be a clinical concern not only in northern Countries. In our study, we investigated vitamin D status in a male population enrolled in a urology clinic of central Italy. In addition, we evaluated the correlation between vitamin D status and UVB irradiance measured in our region. The two principal pathologies in the 95 enrolled patients (mean age 66years) were benign prostate hypertrophy and prostate carcinoma. >50% of patients had serum 25(OH)D values in the deficient range (<20ng/mL), and only 16% of cases had serum vitamin D concentration higher than 30ng/mL (optimal range). The seasonal stratification of vitamin D concentrations revealed an evident trend with the minimum mean value recorded in April and a maximum mean value obtained in September. UVB irradiance measured by pyranometer in our region (Abruzzo, central Italy) revealed a large difference during the year, with winter months characterized by an UV irradiance about tenfold lower than summer months. Then we applied a mathematical model in order to evaluate the expected vitamin D production according to the standard erythemal dose measured in the different seasons. In winter months, the low available UVB radiation and the small exposed skin area resulted not sufficient to obtain the recommended serum doses of vitamin D. Although in summer months UVB irradiance was largely in excess to produce vitamin D in the skin, serum vitamin D resulted sufficient in September only in those patients who declared an outdoor time of at least 3h per day in the previous summer. In conclusion, hypovitaminosis D is largely represented in elderly persons in our region. Seasonal fluctuation in serum 25(OH)D was explained by a reduced availability of UVB in winter and by insufficient solar exposure in summer. The relatively high outdoor time that emerged to be correlated with sufficient serum 25(OH)D in autumn warrants further studies to individuate potential risk co-variables for hypovitaminosis D in elderly men.

Leptin contributes to long-term stabilization of HIF-1α in cancer cells subjected to oxygen limiting conditions.

Leptin, a cytokine produced by the adipose tissue in response to food intake, is a key player in the regulation of energy balance and body weight control. Physiological action of leptin in modulating the metabolic adaptation of different peripheral tissues supports the hypothesis that it could also exert a direct effect on cancer cells. In vitro, treatment with leptin up-regulated HIF-1α and stimulated adhesion and invasion of prostate cancer cells cultured in hypoxia. Leptin action was effective in both low and high glycolytic cancer cell lines, and determined the up-regulation of lactate exporter MCT4 and its associated protein CD147. HIF-1α stabilization was oligomycin-independent and was associated with an important modulation of mitochondrial homeostasis. In fact, leptin treatment produced mitochondrial biogenesis, stabilization of mitochondrial membrane potential and increased uncoupled respiration through the up-regulation of UCP2. Furthermore, leptin counteracted the downmodulation of SIRT1 induced by hypoxia, and persistent high levels of SIRT1 were directly involved in HIF-1α stabilization. Leptin can sustain cancer progression in hypoxic environment and when mitochondrial respiration is impaired. Leptin signaling axis, including the new proposed intermediate SIRT1, could represent a new diagnostic and therapeutic target in prostate cancer.

Tumor-stroma metabolic relationship based on lactate shuttle can sustain prostate cancer progression.

BACKGROUND: Cancer cell adopts peculiar metabolic strategies aimed to sustain the continuous proliferation in an environment characterized by relevant fluctuations in oxygen and nutrient levels. Monocarboxylate transporters MCT1 and MCT4 can drive such adaptation permitting the transport across plasma membrane of different monocarboxylic acids involved in energy metabolism. METHODS: Role of MCTs in tumor-stroma metabolic relationship was investigated in vitro and in vivo using transformed prostate epithelial cells, carcinoma cell lines and normal fibroblasts. Moreover prostate tissues from carcinoma and benign hypertrophy cases were analyzed for individuating clinical-pathological implications of MCT1 and MCT4 expression. RESULTS: Transformed prostate epithelial (TPE) and prostate cancer (PCa) cells express both MCT1 and MCT4 and demonstrated variable dependence on aerobic glycolysis for maintaining their proliferative rate. In glucose-restriction the presence of L-lactate determined, after 24 h of treatment, in PCa cells the up-regulation of MCT1 and of cytochrome c oxidase subunit I (COX1), and reduced the activation of AMP-activated protein kinase respect to untreated cells. The blockade of MCT1 function, performed by si RNA silencing, determined an appreciable antiproliferative effect when L-lactate was utilized as energetic fuel. Accordingly L-lactate released by high glycolytic human diploid fibroblasts WI-38 sustained survival and growth of TPE and PCa cells in low glucose culture medium. In parallel, the treatment with conditioned medium from PCa cells was sufficient to induce glycolytic metabolism in WI-38 cells, with upregulation of HIF-1a and MCT4. Co-injection of PCa cells with high glycolytic WI-38 fibroblasts determined an impressive increase in tumor growth rate in a xenograft model that was abrogated by MCT1 silencing in PCa cells. The possible interplay based on L-lactate shuttle between tumor and stroma was confirmed also in human PCa tissue where we observed a positive correlation between stromal MCT4 and tumor MCT1 expression. CONCLUSIONS: Our data demonstrated that PCa progression may benefit of MCT1 expression in tumor cells and of MCT4 in tumor-associated stromal cells. Therefore, MCTs may result promising therapeutic targets in different phases of neoplastic transformation according to a strategy aimed to contrast the energy metabolic adaptation of PCa cells to stressful environments.

Increased expression of a set of genes enriched in oxygen binding function discloses a predisposition of breast cancer bone metastases to generate metastasis spread in multiple organs.

Bone is the preferential site of distant metastasis in breast carcinoma (BrCa). Patients with metastasis restricted to bone (BO) usually show a longer overall survival compared to patients who rapidly develop multiple metastases also involving liver and lung. Hence, molecular predisposition to generate bone and visceral metastases (BV) represents a clear indication of poor clinical outcome. We performed microarray analysis with two different chip platforms, Affymetrix and Agilent, on bone metastasis samples from BO and BV patients. The unsupervised hierarchical clustering of the resulting transcriptomes correlated with the clinical progression, segregating the BO from the BV profiles. Matching the twofold significantly regulated genes from Affymetrix and Agilent chips resulted in a 15-gene signature with 13 upregulated and two downregulated genes in BV versus BO bone metastasis samples. In order to validate the resulting signature, we isolated different MDA-MB-231 clonal subpopulations that metastasize only in the bone (MDA-BO) or in bone and visceral tissues (MDA-BV). Six of the signature genes were also significantly upregulated in MDA-BV compared to MDA-BO clones. A group of upregulated genes, including Hemoglobin B (HBB), were involved in oxygen metabolism, and in vitro functional analysis of HBB revealed that its expression in the MDA subpopulations was associated with a reduced production of hydrogen peroxide. Expression of HBB was detected in primary BrCa tissue but not in normal breast epithelial cells. Metastatic lymph nodes were frequently more positive for HBB compared to the corresponding primary tumors, whereas BO metastases had a lower expression than BV metastases, suggesting a positive correlation between HBB and ability of bone metastasis to rapidly spread to other organs. We propose that HBB, along with other genes involved in oxygen metabolism, confers a more aggressive metastatic phenotype in BrCa cells disseminated to bone.

Identification of potent c-Src inhibitors strongly affecting the proliferation of human neuroblastoma cells.

Neuroblastoma (NB) represents the most common extracranial paediatric solid tumor for which no specific FDA-approved treatment is currently available. The tyrosine kinase c-Src has been reported to play an important role in the differentiation, cell-adhesion and survival of NB cells. Starting from dual Src/Abl inhibitors previously found active in NB cell lines (1-3), small modification of the original structures almost abolished the Abl activity with a contemporary improvement of affinity and specificity for c-Src. Among the synthesized compounds, the most potent c-Src inhibitor (10a) showed a very interesting antiproliferative activity in SH-SY5Y cells with an IC(50) of 80 nM and a favourable ADME profile. A 3D SAR analysis was also attempted and may guide the design of more potent c-Src inhibitors as potential agents for NB treatment.

Tissue print of prostate biopsy: a novel tool in the diagnostic procedure of prostate cancer.

BACKGROUND: Nowadays, the histological examination of prostate core needle biopsies is still regarded as the gold standard in the diagnosis of prostate cancer (PCa). We investigated if the tissue print of core needle biopsy (biopsy print) could be used as adjunctive molecular investigative procedures in conjunction with routine histological examination of biopsy to improve PCa diagnosis. METHODS: The direct contact of PCa core biopsy to nitrocellulose membrane resulted in the release of a cellular micropeel that was used for downstream analytical procedures. RESULTS: By zymogram print-phoresis we demonstrated that matrix metalloproteases MMP-2 and MMP-9 could be visualized in biopsy prints and that the gelatinolytic activity was positively correlated with immunohistochemistry analysis of the same markers in matched bioptic specimens. Moreover, we compared the ability to detect the PCa-associated hypermethylation of GSTP1 promoter in DNA extracted from biopsy prints with those of the corresponding core needle biopsies. Biopsy prints demonstrated the same specificity of biopsies in detecting PCa (50%) while the sensitivity and the positive predictive value were lower than biopsies (56% vs 78% and 63% vs 70%, respectively). CONCLUSIONS: Biopsy print, combining a molecular point of view to the routinely hystopathological analysis of prostate biopsies, should be a useful tool to improve the diagnosis of PCa.

EphA2 induces metastatic growth regulating amoeboid motility and clonogenic potential in prostate carcinoma cells.

EphA2 kinase regulates cell shape, adhesion, and motility and is frequently overexpressed in several cancers, including melanoma, prostate, breast, and colon cancers and lung carcinoma. Although a function in both tumor onset and metastasis has been proposed, the role played by EphA2 in tumor progression is still debated. In melanoma, EphA2 has been reported to affect cell migration and invasiveness allowing cells to move by a proteolysis-independent strategy, commonly referred as amoeboid motility. With the aim to understand the role of EphA2 in prostate cancer metastatic spreading, we stably silenced EphA2 expression in a model of aggressive metastatic prostate carcinoma. Our results show that EphA2 drives the metastatic program of prostate carcinoma, although its involvement greatly differs among metastatic steps. Indeed, EphA2 expression (i) greatly affects prostate carcinoma cell motility style, guiding an amoeboid movement based on Rho-mediated cell rounding and independent from metalloprotases, (ii) is ineffective on transendothelial migration, adhesion onto extracellular matrix proteins, and on resistance to anoikis, (iii) regulates clonogenic potential of prostate carcinoma, thereby increasing anchorage-independent growth and self-renewal, prostasphere formation, tumor onset, dissemination to bone, and growth of metastatic colonies. Our finding indicate that EphA2-overexpressing prostate carcinoma cells gain an invasive benefit from their amoeboid motility style to escape from primary tumors and then, enhancing their clonogenic potential successfully target bone and grow metastases, thereby acknowledging EphA2 as a target for antimetastatic therapy of aggressive prostate cancers.

Suberoylanilide hydroxamic acid partly reverses resistance to paclitaxel in human ovarian cancer cell lines.

OBJECTIVES: The purpose of this study was to determine whether the addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) to paclitaxel (PTX) can sensitize PTX-resistant human ovarian cancer cell lines (CABA-PTX and IGROV-PTX) in vitro. METHODS: SAHA was studied in combination with paclitaxel in PTX-sensitive and PTX-resistant human ovarian cancer cell lines. Using cell proliferation analysis, immunofluorescence, and flow cytometric assays, we can determine whether the resistance was partly removed when the cells were treated with a combination of SAHA and PTX. Cells were also assayed for cytochrome c release. The levels of acetylated tubulin, ß-tubulin, and HDAC6 were quantified by Western blots. RESULTS: SAHA in combination with PTX led to a more pronounced inhibition of cell growth compared with PTX alone. In addition, the combined exposure to PTX and SAHA resulted in a marked arrest in the G2/M phase of the cell cycle and in a significant increase in the percentage of apoptotic cells. The expression of acetylated tubulin was dramatically increased by exposure to the combination of PTX and SAHA. These data paralleled the findings of an increased expression of HDAC6 in the presence of PTX in PTX-resistant cell lines. CONCLUSIONS: The results of this study suggest the existence of a novel resistance mechanism based upon the upregulation of HDAC6 and that the histone deacetylase inhibitor SAHA holds promise to overcome PTX resistance in ovarian cancer cell lines.

Receptor activator of NF-kappaB ligand enhances breast cancer-induced osteolytic lesions through upregulation of extracellular matrix metalloproteinase inducer/CD147.

Breast cancer shows a strong predilection to metastasize to bone. Cell surface glycoprotein extracellular matrix metalloproteinase inducer (EMMPRIN)/CD147 induces metalloproteinases (MMP) and vascular endothelial growth factor (VEGF), which may support osteoclastic activity and increased incidence of breast cancer bone metastases. In support of this hypothesis, we observed that MDA-MB-231 human breast tumor cells engineered to overexpress EMMPRIN strongly induced osteolytic lesions in immunodeficient mice, which was blunted by in vivo treatment with an EMMPRIN blocking antibody. Similarly, these cells exhibited increased expression of MMP-9 and VEGF relative to control cells. Treatment of MDA-MB-231 cells with the osteoclastogenic cytokine receptor activator of NF-kappaB ligand (RANKL) upregulated EMMPRIN expression with a parallel increase of MMP-9 and VEGF. Conditioned medium from osteoblasts similarly increased EMMPRIN, MMP-9, and VEGF expression in cells. Osteoblast treatment with the RANKL decoy receptor osteoprotegerin abolished this effect. EMMPRIN overexpression stimulated MDA-MB-231 cell invasion but not proliferation. Conversely, small interfering RNA-mediated knockdown of EMMPRIN downregulated MMP-9 and VEGF basal expression and RANKL-stimulated expression, and reduced cell invasion. Our results argue that EMMPRIN drives breast cancer-induced osteolytic lesions and that activation of the RANKL pathway increases EMMPRIN in osteotropic tumor cells, in turn enhancing tumor-induced bone resorption.

New pyrazolo-[3,4-d]-pyrimidine derivative Src kinase inhibitors lead to cell cycle arrest and tumor growth reduction of human medulloblastoma cells.

Medulloblastoma is the most common malignant brain tumor in children, and despite improvements in the overall survival rate, it still lacks an effective treatment. Src plays an important role in cancer, and recently high Src activity was documented in medulloblastoma. In this report, we examined the effects of novel pyrazolo-[3,4-d]-pyrimidine derivative Src inhibitors in medulloblastoma. By MTS assay, we showed that the pyrimidine derivatives indicated as S7, S29, and SI163 greatly reduce the growth rate of medulloblastoma cells by inhibiting Src phosphorylation, compared with HT22 non-neoplastic nerve cells. These compounds also halt cells in the G(2)/M phase, and this effect likely occurs through the regulation of cdc2 and CDC25C phosphorylation, as shown by Western blot. Moreover, the exposure to pyrimidine derivatives induces apoptosis, assayed by the supravital propidium iodide assay, through modulation of the apoptotic proteins Bax and Bcl2, and inhibits tumor growth in vivo in a mouse model. Notably, S7, S29, and SI163 show major inhibitory effects on medulloblastoma cell growth compared with the chemotherapeutic agents cisplatin and etoposide. In conclusion, our results suggest that S7, S29, and SI163 could be novel attractive candidates for the treatment of medulloblastoma or tumors characterized by high Src activity.

Kinase-dependent and -independent roles of EphA2 in the regulation of prostate cancer invasion and metastasis.

Ligand-activated Eph tyrosine kinases regulate cellular repulsion, morphology, adhesion, and motility. EphA2 kinase is frequently up-regulated in several different types of cancers, including prostate, breast, colon, and lung carcinomas, as well as in melanoma. The existing data do not clarify whether EphA2 receptor phosphorylation or its simple overexpression, which likely leads to Eph kinase-independent responses, plays a role in the progression of malignant prostate cancer. In this study, we address the role of EphA2 tyrosine phosphorylation in prostate carcinoma cell adhesion, motility, invasion, and formation of metastases. Tumor cells expressing kinase-deficient EphA2 mutants, as well as an EphA2 variant lacking the cytoplasmic domain, are defective in ephrinA1-mediated cell rounding, retraction fiber formation, de-adhesion from the extracellular matrix, RhoA and Rac1 GTPase regulation, three-dimensional matrix invasion, and in vivo metastasis, suggesting a key role for EphA2 kinase activity. Nevertheless, EphA2 regulation of cell motility and invasion, as well as the formation of bone and visceral tumor colonies, reveals a component of both EphA2 kinase-dependent and -independent features. These results uncover a differential requirement for EphA2 kinase activity in the regulation of prostate carcinoma metastasis outcome, suggesting that although the kinase activity of EphA2 is required for the regulation of cell adhesion and cytoskeletal rearrangement, some distinct kinase-dependent and -independent pathways likely cooperate to drive cancer cell migration, invasion, and metastasis outcome.

Bicalutamide demonstrates biologic effectiveness in prostate cancer cell lines and tumor primary cultures irrespective of Her2/neu expression levels.

OBJECTIVES: To evaluate the role of Her2/neu as a molecular marker predictive of the treatment response to bicalutamide in prostate cancer (PCa). METHODS: Four PCa cell lines with graded Her2/neu expression levels and 63 primary tumor cultures derived from men with PCa and selected according to Her2/neu tumor levels were used. Primary tumor cultures and PCa cell lines were treated with bicalutamide (0.1-10 microM) in the presence of dehydrotestosterone (10(-12) M) for 4 days. The presence of a significant correlation between Her2/nue expression and drug efficacy was used to define the role of Her2/neu as molecular predictor of bicalutamide effectiveness. As an indicator of drug effectiveness we used the concentration that inhibits 50% values determined after bicalutamide treatment. RESULTS: After bicalutamide treatment, no significant differences in the concentration that inhibits 50% were found among the different tumor cell lines (P = .081). In this experimental model, the correlation analysis suggested that the effectiveness of this drug was not influenced by Her2/neu levels (r = 0.053, P = .823). In primary cultures with high Her2/neu levels (43 tumor cultures), the mean value of the concentration that inhibits 50% for bicalutamide was 0.43 +/- 0.13 microM, and in cultures with low Her2/neu levels (20 tumor cultures), the same parameter was 0.5 +/- 0.16 microM (P = .14). The correlation analysis suggested that the effectiveness of this drug was not influenced by Her2/neu levels (r = 0.33, P = .101). CONCLUSIONS: Our biologic data seem to indicate that the antitumor effect of bicalutamide is independent of Her2/neu levels in preclinical models of PCa. Bicalutamide could be configured as a pharmacologic option to treat patients with high or low levels of Her2/neu.

Azacitidine improves antitumor effects of docetaxel and cisplatin in aggressive prostate cancer models.

One of the major obstacles in the treatment of hormone-refractory prostate cancer (HRPC) is the development of chemoresistant tumors. The aim of this study is to evaluate the role of azacitidine as chemosensitizing agent in association with docetaxel (DTX) and cisplatin using two models of aggressive prostate cancer, the 22rv1, and PC3 cell lines. Azacitidine shows antiproliferative effects associated with increased proportion of cells in G0/G1 and evident apoptosis in 22rv1 cells and increased proportion of cells in G2/M phase with the absence of acute cell killing in PC3 cells. In vivo, azacitidine (0.8 mg/kg i.p.) reduced tumor proliferation and induced apoptosis in both xenografts upmodulating the expression of p16INKA, Bax, Bak, p21/WAF1, and p27/KIP1, and inhibiting the activation of Akt activity and the expression of cyclin D1, Bcl-2, and Bcl-XL. In vitro treatments with azacitidine lead to upregulation of cleaved caspase 3 and PARP. BCl2 antagonists, such as HA-14-1, enhanced the effects of azacitidine in these two prostate cancer models. In addition, azacitidine showed synergistic effects with both DTX and cisplatin. In vivo this agent caused tumor growth delay without complete regression in xenograft systems. Azacitidine sensitized PC3 and 22rv1 xenografts to DTX and cisplatin treatments. These combinations were also tolerable in mice and superior to either agent alone. As DTX is the standard first-line chemotherapy for HRPC, the development of DTX-based combination therapies is of great interest in this disease stage. Our results provide a rationale for clinical trials on combination treatments with azacitidine in patients with hormone-refractory and chemoresistant prostate tumors.

Indolyl-pyrrolone as a new scaffold for Pim1 inhibitors.

Pim1 belongs to a family of serine/threonine kinases, which is involved in the control of cell growth, differentiation, and apoptosis. Pim1 plays a pivotal role in cytokine signaling and is implicated in the development of a large number of tumors, representing a very attractive target for anticancer therapy. In this work, we applied a virtual screening protocol aimed at identifying small molecules able to inhibit Pim1 activity. The search of novel inhibitors was performed through a structure-based molecular modeling approach, taking advantage of the availability of the three-dimensional crystal structure of inhibitors bound to Pim1. Starting from the knowledge of protein-ligand complexes, the software LigandScout was used to generate pharmacophoric models, in turn used as queries to perform a virtual screening of databases, followed by docking experiments. As a result, a restricted set of candidates for biological testing was identified. Finally, among the six compounds selected as potential inhibitors of Pim1, two candidates endowed with a significant activity against Pim1 emerged. Interestingly, one of these compounds has a chemical scaffold different from inhibitors previously identified.

Her2 crosstalks with TrkA in a subset of prostate cancer cells: rationale for a guided dual treatment.

BACKGROUND: To date, no effective therapeutic treatment prevents prostate cancer (PCa) progression to more advanced and invasive disease forms. It has been demonstrated that the simultaneous high expression of p185(HER2) and TrkA might confer a proliferative advantage to PCa cells. METHODS: In this work we verified the crosstalk between TrkA and Her2 signaling pathways and the effects of a combined treatment with Her2 and TrkA inhibitors. RESULTS: NGF induced TrkA activation and stimulated cell proliferation of PCa cells. NGF induced also tyrosine phosphorylation of p185(HER2). This event was only partially inhibited by the pan Trk inhibitor, CEP-701 but was strongly blocked by pertuzumab, a humanized antibody blocking Her2 heterodimerization. In presence of NGF, TrkA and Her2 co-precipitated and this was dependent to the relative high cellular levels of TrkA since when cell lysates were immunoprecipitated with an antibody against Her2 the amount of TrkA were proportional to the cellular levels of this receptor. On the contrary when we immunoprecipitated using an antibody against TrkA the amount of Her2 seemed independent to cellular levels of Her2. So, combined treatment between CEP-701 and pertuzumab showed supra-additive effects in cells with higher levels of TrkA and Her2 suggesting once again that this was indicative of a higher response to treatment. CONCLUSIONS: Our data suggest that the dual inhibition of TrkA and Her2 may be useful in a subset of patients in which TrkA and Her2 are overexpressed and in which the possibility of TrkA and Her2 protein-binding is elevated.

Downmodulation of dimethyl transferase activity enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in prostate cancer cells.

One of the major obstacles in curing prostate cancer is the development of drug resistance. It is not only imperative to discover the molecular basis of resistance but also to find therapeutic agents that can disrupt the resistant pathways. Tumor necrosis factor TNF-related apoptosis-inducing ligand TRAIL-like ligands or agonist TRAIL-receptor monoclonal antibodies have entered phase I and II clinical trials with a very limited cytotoxic profile when used systemically in a variety of cancers. Therefore, TRAIL-receptor agonists are new proapoptotic pharmaceutical agents with great potential as new cancer therapeutic agents. Although many cancer cells undergo TRAIL-mediated apoptosis, some are resistant to TRAIL. Therefore, we have been investigating mechanisms to overcome TRAIL resistance in cancer cells so that TRAIL-associated compounds can be used effectively in clinical trials. Epigenetic inactivation of proapoptotic genes, or activation of survival signaling, can cause cross-resistance to several anti-tumor therapies and to immune cytotoxic lymphocytes. We hypothesize that 5-aza-2 deoxycytidine aza-dCR, decitabine may render TRAIL-resistant prostate cancer cells sensitive to caspase-8-mediated apoptosis and may, therefore, be therapeutically efficient. We evaluated the antiproliferative effects of decitabine on the following four prostate cancer cell lines: well-differentiated AR positive LnCaP p53(+), PTEN- and 22rv1 p53(+) and PTEN(+)]; poorly-differentiated AR negative PC3 p53-, PTEN- and DU145 p53 mutant, PTEN(+). Here, we provide evidence that treatment with sub-optimal concentrations of decitabine are additive to TRAIL effects in well-differentiated PCa cells whereas the same treatment shows synergistic effects in poorly-differentiated PCa cells through increased caspase-8 expression, down-modulation of Akt activation and through the expression of certain anti-apoptotic molecules including FLIP, PED/PEA-15, survivin and c-IAP-1. Our findings demonstrate that decitabine at relatively low concentrations restores caspase-8 expression and sensitises resistant PCa cells to TRAIL-induced apoptosis leading to important implications in novel therapeutic strategies targeting defective apoptosis pathways in advanced prostate tumors.

Effects of dutasteride on prostate carcinoma primary cultures: a comparative study with finasteride and MK386.

PURPOSE: The profound decrease in serum dihydrotestosterone observed with the dual 5alpha-reductase inhibitor dutasteride makes it an attractive agent for prostate cancer therapy. To our knowledge we compared for the first time the antitumor effect of dutasteride with that of the specific 5alpha-reductase-1 inhibitor MK386 and the specific 5alpha-reductase-2 inhibitor finasteride in human prostate primary cultures. MATERIALS AND METHODS: Biochemical markers of the cellular response to 5alpha-reductase inhibitors were evaluated in primary cultures of prostate epithelial cancer cells from 54 patients with prostate carcinoma. RESULTS: In our cohort of 54 patients prostate cancer cell growth decreased with dutasteride in 42 (about 78%), whereas in 21 (39%) it decreased with finasteride or MK386 alone. We observed a relationship between the levels of 5alpha-reductase enzymes in cell culture extracts and those revealed by immunohistochemistry in sections of samples from which we established primary cultures. Finasteride effects depended on 5alpha-reductase-2 levels and they were higher when the 5alpha-reductase-1:2 ratio was low. However, dutasteride effects were related to 5alpha-reductase-1 and 2 levels, and were not influenced by the 5alpha-reductase-1:2 ratio. Conversely the effects of MK386 were related to 5alpha-reductase-1 levels and they were higher when the 5alpha-reductase-1:2 ratio was high. CONCLUSIONS: Our data may provide a rationale for the use of a dual 5alpha-reductase inhibitor rather than a mono specific inhibitor for the prevention or treatment of early prostate cancer. This finding appears to confirm some preliminary clinical results and it could be due to the simultaneous presence of each 5alpha-reductase isoenzyme in prostate tumor cells.

Akt down-modulation induces apoptosis of human prostate cancer cells and synergizes with EGFR tyrosine kinase inhibitors.

BACKGROUND: PTEN is a well-characterized tumor suppressor that negatively regulates cell growth and survival through the modulation of PI3K/Akt pathway. METHODS: In this paper, we investigated the effects of an PI3K/Akt inhibitor, perifosine, in human prostate cancer (PCa) cells analyzing cell proliferation, apoptosis, and the synergy with EGFR inhibitors. RESULTS: Clinically achievable concentrations of perifosine, as well as Akt gene knockdown, induced a G0/G1 arrest and apoptosis in PTEN defective PCa cells. Although PTEN introduction was able to restore the control of Akt activity and to reduce cell proliferation, the manipulation of PTEN gene was not able alone to influence apoptosis. Perifosine induced apoptotic program also in PTEN positive cells when Akt activity was augmented by EGF suggesting the possibility that this drug could be used in combination with EGFR inhibitors. The combination treatment between erlotinib and pharmacological or molecular Akt knockdown, indeed, showed synergistic effects. This is the first demonstration that a pharmacological compound against Akt activity can restore the efficacy against EGFR inhibitors in PCa and has important therapeutic fallout since EGFR inhibitors have demonstrated very low effectiveness in PCa patients. CONCLUSIONS: Taken together our data have an important clinical relevance in the treatment of advanced prostate tumors. However, further studies in the setting of combination therapies in advanced PCas are necessary.

Chronic azacitidine treatment results in differentiating effects, sensitizes against bicalutamide in androgen-independent prostate cancer cells.

BACKGROUND: About 20-30% of hormone-independent PCa are characterized by the extensive loss of AR expression that appears to occur at the transcriptional level. Treatment of AR-negative PCa cells with demethylating agents (Aza-CR) leads to expression of AR mRNA and protein. Here, we investigate the effect of Aza-CR administered both acutely and chronically on AR expression, PSA expression, cell survival, and proliferation in androgen-independent/AR-negative PCa cells. We also studied whether epigenetically reactivated AR is a target for bicalutamide therapy. METHODS: The in vitro effect of Aza-CR as single agent and its ability to induce AR expression and to augment the efficacy to bicalutamide were assessed using two androgen-independent and AR-negative cell lines (PC3 and DU145). RESULTS: Our results show that acute treatment (4 days) with Aza-CR results in a relatively low decrease in cell proliferation with G2 cell cycle arrest and no significant evidence of apoptosis or AR expression. Interestingly, when Aza-CR was chronically administered (20 days), this treatment resulted in marked decrease in tumor cell proliferation with significant increase in AR and PSA protein levels. Furthermore, following Aza-CR chronic treatment the formerly androgen-independent PC3 and DU145 cells increase their susceptibility to the apoptotic effects of bicalutamide. CONCLUSIONS: Aza-CR acute treatment has modest effects on androgen-independent and AR-negative PCa cell survival and proliferation, but chronic administration results in profound decrease in proliferation and in sensitization to antiandrogen agents. All these effects seem, in some measure, dependent on a partial restoration of androgen regulation.