Anders Ullén

Additive/synergistic antitumoral effects on prostate cancer cells in vitro following treatment with a combination of docetaxel and zoledronic acid

Once bone metastasized and androgen independent, prostate cancer is often associated with skeletal morbidity and disability. New treatment modalities that can palliate symptoms from the skeleton and inhibit further progression are warranted. In this study, the antitumoral effects following treatment with a combination of docetaxel and the new generation bisphosphonate, zoledronic acid, were investigated on two hormone-refractory prostate cancer cell lines: PC3 and DU145. The prostate cancer cells were treated with increasing concentrations of zoledronic acid in the absence or presence of docetaxel. Toxicity was measured using fluorometric microculture cytotoxic assay technique. A concentration of 25 µM, zoledronic acid reduced the viable cell number to 68% and 98% for PC3 and DU145 cells respectively. Docetaxel, on the other hand, at a concentration of 0.1 ng/ml, had no effect on the viability. However, a combination of zoledronic acid and docetaxel reduced the cell number to 60% and 81% respectively. Furthermore, zoledronic acid in the concentration range 12.5 µM–50 µM enhanced the antitumoral effects of docetaxel (0.01–1 ng/ml) in an additive and/or synergistic manner for both cell lines. These data support the hypothesis that zoledronic acid, in addition to having bone resorption inhibiting properties, also exhibits anti-tumoral effects. It also appears that combined treatment with docetaxel causes additive and/or synergistic cytostatic effects on prostate cancer cells.

Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells

Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles called prostasomes that belong to the exosome family. Prostate cancer cells have preserved this ability to generate and export exosomes to the extracellular space. We previously demonstrated that human prostasomes have an ATP-forming capacity. In this study, we compared the capacity of extracellular vesicles (EVs) to generate ATP between normal seminal prostasomes and exosomes secreted by PC3 cells (PC3 exosomes), a prostate cancer cell line. Proteomic analyses identified enzymes of the glycolytic chain in both prostasomes and PC3 exosomes, and we found that both of them were capable of generating ATP when supplied with substrates. Notably, the net production of extracellular ATP was low for prostasomes due to a high ATPase activity contrary to an elevated net ATP level for PC3 exosomes because of their low ATPase activity. The uptake of the 2 types of EVs by normal prostate epithelial cells (CRL2221) and prostate cancer cells (PC3) was visualized and measured, demonstrating differential kinetics. Interestingly, this uptake was dependent upon an ongoing glycolytic flux involving extracellular ATP formation by EVs and/or intracellular ATP produced from the recipient cells. We conclude that the internalization of EVs into recipient cells is an energy-requiring process also demanding an active V-ATPase and the capacity of EVs to generate extracellular ATP may play a role in this process.

Chemotherapy-induced bladder cancer.

The use of chemotherapy in the management of cancer started in the 1940s when the cytotoxic effect of nitrogen mustard was first explored. The therapeutic concept was based on warfare experience of the toxic effects of nitrogen mustard on the lymphatic system. A large number of chemotherapeutic drugs have been developed since then, and by the year 2000 more than 50 drugs were available for clinical use. The chemotherapy regimens usually consist of drug combinations with the aim of overcoming tumour cell resistance. Chemotherapy is now one of the cornerstones for curative and palliative treatment of malignant diseases. However, essentially all chemotherapeutic drugs also induce short-term and/or long-term side-effects. This article highlights the risk of inducing secondary bladder cancer. The main treatment focus over the years has been on cyclophosphamide and derivatives thereof. This drug belongs to the oxaphosphorin group and is chemically related to nitrogen mustard. Cyclophosphamide is a prodrug that needs bioactivation by cytochrome P450 enzymes to exert its cytotoxic effects. Of the three main DNA-binding components (phosphoramide mustard, nornitrogen mustard and acrolein) phosphoramide is the main active cytotoxic component. The presumed mechanism of action is alkylation of DNA resulting in DNA cleavage, and cross-linkage of DNA strands and DNA proteins. This, in turn, affects DNA replication and induces apoptosis. Cyclophosphamide is currently used primarily in the management of malignant diseases such as Hodgkin’s and non-Hodgkin’s lymphomas, leukaemias, breast cancer, ovarian cancer, small cell lung cancer and neuroblastomas. The drug is also used as immunosuppressive therapy in benign diseases such as Wegener’s granulomatosis, a necrotizing granulomatous vasculitis, and in Goodpasture’s syndrome. The first human case of urinary bladder cancer attributed to cyclophosphamide exposure was reported in the early 1970s [1]. Since then numerous reports have been published on this phenomenon [2 8]. Travis et al. quantified the risk of bladder cancer following cyclophosphamide therapy in a large cohort (n 6171) of 2-year survivors of non-Hodgkin’s lymphoma (NHL) [3]. Forty-eight patients with secondary cancer of the urinary tract were identified and matched to 136 control subjects with NHL who did not develop a second malignancy. Detailed information on chemotherapeutic drugs and cumulative dose received was collected for all subjects. Some of the patients in the cohort also received radiation therapy. Dose to the target organ was estimated from individual radiotherapy records. Evaluations of the risk of secondary cancer as a result of treatment with cyclophosphamide alone, radiation alone or both therapies were made relative to those

Prostate cancer cell lines lack amplification : Overexpression of HER2

The potential overexpression of HER2 in prostate cancer cells has attended significant interest during the past few years, both as potential target for HER2 pathway focused therapy and as a mechanism involved in the progression to androgen independence. Conflicting results have been reported concerning HER2 status on clinical material, differences which generally have been attributed to methodological differences. Nevertheless, HER2 has been utilized for targeted therapy of prostate cancer in a number of preclinical studies and is still regarded as an exciting target molecule. In this study, the HER2 status of three widely used prostate cancer cell lines and corresponding xenografts has been analysed. By use of validated and FDA approved analytical staining techniques none of these cell lines or xenografts were shown to overexpress/amplify HER2, as demonstrated by immunohistochemistry and fluorescense in situ hybridization. These findings are important for the interpretation and understanding of the therapeutic effects when developing drugs targeting HER2 in prostate cancer cell lines and also emphasize the importance of using broad and validated analytical techniques.

Vascular endothelial growth factor receptor 2, but not S100A4 or S100A6, correlates with prolonged survival in advanced urothelial carcinoma

OBJECTIVE A major challenge in muscle-invasive urothelial carcinoma (UC) is to identify biomarkers that can predict disease prognosis and treatment response after cystectomy. Therefore, we analyzed the potential prognostic value of the proteins vascular endothelial growth factor receptor 2 (VEGFR2), S100A4, and S100A6 in UC. METHODS Retrospective outcome data and tumor specimens from 83 cystectomy patients with histologically confirmed invasive UC were included. Expression levels of VEGFR2 (also called flk-1 and KDR), S100A4, and S100A6 were analyzed in primary tumor tissue by immunohistochemistry. RESULTS Immunohistochemical staining and analysis of VEGFR2, S100A4, and S100A6 showed localization mainly in tumor cell cytoplasm. High VEGFR2 expression and low tumor category were independent variables associated with longer overall survival (OS) and disease-free survival, revealed by a bivariate Cox proportional hazards regression model (both P<0.001). In addition, the univariate log-rank test and the Cox model demonstrated that OS beyond 2 years was significantly greater among patients with low S100A6 expression than in those with high S100A6 expression (P = 0.017 and 0.022, respectively). Differences in tumor expression of S100A4 were not significantly associated with outcome. CONCLUSION In this study, VEGFR2 expression was significantly correlated with risk of disease relapse and OS in a defined cohort of patients with UC of the bladder treated by cystectomy.

Melphalan-flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma.

Background: Chemotherapy options in advanced urothelial carcinoma (UC) remain limited. Here we evaluated the peptide‐based alkylating agent melphalan‐flufenamide (mel‐flufen) for UC.

Long-term health-related quality of life after curative treatment for prostate cancer: a regional cross-sectional comparison of two standard treatment modalities.

Effects on long-term health-related quality of life (HRQoL) were evaluated in patients treated for localized prostate cancer by two standard modalities: radical retropubic prostatectomy (RP) and external beam radiotherapy combined with a high-dose-rate brachytherapy boost (HDRBT-EBRT). The HRQoL data were compared with age-adjusted normative data. Men diagnosed with localized prostate cancer and treated with curative intent in Gothenburg, Sweden, 1988-1997 were included. HRQoL was measured in October 2000 using the EORTC QLQ-C30 and EORTC QLQ-PR25 questionnaires, with a response rate of 82% (n=347). No differences in patient characteristics were found between the two treatment groups, except regarding tumor stage and PSA recurrence at the time of the questionnaires. In the RP group, 42% had T1 and 6% had T3-4 tumors; corresponding proportions in the HDRBT-EBRT group were 29% and 13% (p=0.01). PSA recurrence was detected in 44% of RP patients and 9% of HDRBT-EBRT patients. In most domains, mean HRQoL scores were high and similar to the scores for the age-adjusted normative sample. However, patients reported better role and physical function compared to the normal population. We also observed more sleeping disturbances but less pain among patients than in the normal population. The disease-specific questionnaires showed statistically significant higher levels of bowel and urinary problems in the irradiated group than in the RP group, and the absolute difference between the groups was small and had minor clinical significance. We conclude that overall the general quality of life was rated high by the patients irrespective of curative treatment modality and in agreement with age-adjusted normative data. Statistically significant differences in bowel and urinary symptoms were found between the two treatment groups in favor of the RP group, but the clinical significance was small.

Management of advanced prostate cancer – new drugs

Androgen deprivation therapy by chemical or surgical castration remains the cornerstone in the management of advanced disease. Although initially effective, the effect of currently available androgen deprivation therapies is transient and most patients develop progressive disease despite low levels of testosterone. It is generally believed that tumour progression is associated with continued signalling via the androgen receptor pathway through mechanisms such as androgen receptor mutation, androgen receptor amplifi cation, ligand-independent androgen receptor activation or enhanced local production of androgens. The recognition of the antiandrogen withdrawal response, which demonstrates the continuous importance of the androgen receptor signalling pathway in castrate resistant prostate cancer, has stimulated the development of new exciting androgen targeting therapies. MDV3100, which is a nonsteroidal compound and RD162, are examples of two new orally available interesting agents that target the androgen receptor with higher affi nity than bicalutamide [1]. MDV3100 has shown promising phase I/II activity [2] and is currently evaluated in a placebo-controlled randomised phase III trial in patients progressive on docetaxel therapy. EPI-001 represents from a mechanistic point-of-view, a conceptually new and interesting compound. It binds to the N-terminal domain of the androgen receptor and has a new mode of action as it targets the transactivation of the androgen receptor, regardless of the presence of androgen [3,4]. Other efforts are made to further improve the blockage of testosterone production. Abiraterone acetate is an interesting, potent small molecule that irreversibly inhibits cytochrome p17 which catalyzes two key reactions in androgen biosynthesis [5]. This compound has been evaluated in a phase III trial in

Cell-free DNA profiling of metastatic prostate cancer reveals microsatellite instability, structural rearrangements and clonal hematopoiesis

Background There are multiple existing and emerging therapeutic avenues for metastatic prostate cancer, with a common denominator, which is the need for predictive biomarkers. Circulating tumor DNA (ctDNA) has the potential to cost-efficiently accelerate precision medicine trials to improve clinical efficacy and diminish costs and toxicity. However, comprehensive ctDNA profiling in metastatic prostate cancer to date has been limited. Methods A combination of targeted- and low-pass whole genome sequencing was performed on plasma cell-free DNA and matched white blood cell germline DNA in 364 blood samples from 217 metastatic prostate cancer patients. Results ctDNA was detected in 85.9% of baseline samples, correlated to line of therapy and was mirrored by circulating tumor cell enumeration of synchronous blood samples. Comprehensive profiling of the androgen receptor (AR) revealed a continuous increase in the fraction of patients with intra-AR structural variation, from 15.4% during first line mCRPC therapy to 45.2% in fourth line, indicating a continuous evolution of AR during the course of the disease. Patients displayed frequent alterations in DNA repair deficiency genes (18.0%). Additionally, the microsatellite instability phenotype was identified in 3.81% of eligible samples (≥0.1 ctDNA fraction). Sequencing of non-repetitive intronic- and exonic regions of PTEN, RB1 and TP53 detected biallelic inactivation in 47.5%, 20.3% and 44.1% of samples with ≥0.2 ctDNA fraction, respectively. Only one patient carried a clonal high-impact variant without a detectable second hit. Intronic high-impact structural variation was twice as common as exonic mutations in PTEN and RB1. Finally, 14.6% of patients presented false positive variants due to clonal hematopoiesis, commonly ignored in commercially available assays. Conclusions ctDNA profiles appear to mirror the genomic landscape of metastatic prostate cancer tissue and may cost-efficiently provide somatic information in clinical trials designed to identify predictive biomarkers. However, intronic sequencing of the interrogated tumor suppressors challenge the ubiquitous focus on coding regions and is vital, together with profiling of synchronous white blood cells, to minimize erroneous assignments which in turn may confound results and impede true associations in clinical trials.

Effect on prostate volume following neoadjuvant treatment with an androgen receptor inhibitor monotherapy versus castration plus an androgen receptor inhibitor in prostate cancer patients intended for curative radiation therapy: A randomised study

To avoid pubic arch interference, prostate cancer patients are treated with neoadjuvant androgen deprivation therapy (ADT) to achieve prostate volume (PV) reduction prior to radiation treatment. The aim of the present randomised study was to compare the effects on PV of two regimens of ADT, an androgen receptor inhibitor monotherapy vs. castration plus an androgen receptor inhibitor. Consecutive patients with non-metastatic prostate cancer were included in a randomised neoadjuvant study, comparing an androgen receptor inhibitor monotherapy vs. castration plus an androgen receptor inhibitor. PV was assessed prior to the start of endocrine neoadjuvant treatment and prior to the start of radiation therapy (RT). PV assessment was performed by transrectal ultrasound. A total of 110 patients were included. Final sample constituted 88 (80%) patients due to lack of PV information. Castration plus an androgen receptor inhibitor was more effective in PV reduction compared with an androgen receptor inhibitor alone (P<0.001). Planning target volume decreased in the combination arm. There was no significant difference in clinical or demographic or length of neoadjuvant hormonal treatment between the groups. Overall, a significantly larger PV reduction was achieved by castration plus androgen receptor inhibitor, as compared with androgen receptor inhibitor monotherapy. The PV reduction, however, appeared not to translate into better health associated quality of life during the subsequently given curative intended combined EBRT and HDR-brachytherapy. Potential differences between these two treatments regarding anti-tumor effects on micro metastatic disease and radiation potentiating effect remains to be addressed in future prospective trials.