The Key Role of Wettability and Boundary Layer in Dissolution Rate Test.

BACKGROUND/OBJECTIVES: The present work proposes a mathematical model able to describe the dissolution of poly-disperse drug spherical particles in a solution (Dissolution Rate Test-DRT). DRT is a pivotal test performed in the pharmaceutical field to qualitatively assess drug bioavailability. METHODS: The proposed mathematical model relies on the key hallmarks of DRT, such as particle size distribution, solubility, wettability, hydrodynamic conditions in the dissolving liquid of finite dimensions, and possible re-crystallization during the dissolution process. The spherical shape of the drug particles was the only cue simplification applied. Two model drugs were considered to check model robustness: theophylline (both soluble and wettable) and praziquantel (both poorly soluble and wettable). RESULTS: The DRT data analysis within the proposed model allows us to understand that for theophylline, the main resistance to dissolution is due to the boundary layer surrounding drug particles, whereas wettability plays a negligible role. Conversely, the effect of low wettability cannot be neglected for praziquantel. These results are validated by the determination of drug wettability performed while measuring the solid-liquid contact angle on four liquids with decreasing polarities. Moreover, the percentage of drug polarity was determined. CONCLUSIONS: The proposed mathematical model confirms the importance of the different physical phenomena leading the dissolution of poly-disperse solid drug particles in a solution. Although a comprehensive mathematical model was proposed and applied, the DRT data of theophylline and praziquantel was successfully fitted by means of just two fitting parameters.

Elucidating the unexpected cell adhesive properties of agarose substrates. The effect of mechanics, fetal bovine serum and specific peptide sequences.

2D agarose substrates have recently been surprisingly shown to be permissive for cell adhesion, depending on their mechanics and the use of the adhesive proteins of fetal bovine serum (FBS) in the cell culture medium. Here, we elucidate how the cells exhibit two anchoring mechanisms depending on the amount of FBS. Under low FBS conditions, the cells recognize the surface-coupled adhesive sequences of fibronectin via the binding of the heterodimer α5ß1 integrin. Functionality of the actomyosin axis and mechanoactivation of focal adhesion kinase (FAK) are essential for the stretching of the protein, thereby accessing the "synergy" PPSRN site and enhancing cell adhesion in combination with the downstream RGD motif. Under high FBS conditions, the specific peptide sequences are much less relevant as the adsorbed serum proteins conceal the coupled fibronectin and the cells recognize the adhesive protein vitronectin, which is constitutively present in FBS, via the binding of the heterodimer αvß3 integrin. Similarly, the intracellular tension and FAK activity are decisive, which collectively indicate that the cells stretch the partially cryptic RGD site of vitronectin and thus make it more accessible for integrin binding. Both anchoring mechanisms only work properly if the agarose substrate is mechanically compliant in terms of linear stress-strain response, unraveling a critical balance between the mechanics of the agarose substrate and the presentation of the adhesive peptides. STATEMENT OF SIGNIFICANCE: In the context of biomaterial design, agarose hydrogels are known to lack intrinsic cell-adhesive peptide motifs and are therefore commonly used for the development of non-permissive 2D substrates. However, we unexpectedly found that agarose hydrogels can become permissive substrates for cell adhesion, depending on a compliant mechanical response of the substrate and the use of fetal bovine serum (FBS) as protein reservoir in the cell culture medium. We describe here two anchoring mechanisms that cells harness to adhere to agarose substrates, depending on the amount of FBS. Our results will have a major impact on the field of mechanobiology and shed light on the central role of FBS as a natural source of adhesive proteins that could promote cell anchoring.

New challenges in hepatocellular carcinoma: A role for PIWI-interacting RNAs?

Hepatocellular carcinoma (HCC) is the most common and deadliest subtype of liver cancer worldwide and, therefore, poses an enormous threat to global health. Understanding the molecular mechanisms underlying the development and progression of HCC is central to improving our clinical approaches. PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that bind to PIWI family proteins to regulate gene expression at transcriptional and post-transcriptional levels. A growing body of work shows that the dysregulation of piRNAs plays a crucial role in the progression of various human cancers. In this editorial, we report on the current knowledge of HCC-associated piRNAs and their potential clinical utility. Based on the editorial by Papadopoulos and Trifylli, on the role and clinical evaluation of exosomal circular RNAs in HCC, we highlight this other emerging class of non-coding RNAs.

In Vitro and In Vivo Evaluation of the Effects of Drug 2c and Derivatives on Ovarian Cancer Cells.

BACKGROUND: The identification of novel therapeutic strategies for ovarian cancer (OC), the most lethal gynecological neoplasm, is of utmost urgency. Here, we have tested the effectiveness of the compound 2c (4-hydroxy-2,6-bis(4-nitrobenzylidene)cyclohexanone 2). 2c interferes with the cysteine-dependent deubiquitinating enzyme (DUB) UCHL5, thus affecting the ubiquitin-proteasome-dependent degradation of proteins. METHODS: 2c phenotypic/molecular effects were studied in two OC 2D/3D culture models and in a mouse xenograft model. Furthermore, we propose an in silico model of 2c interaction with DUB-UCHL5. Finally, we have tested the effect of 2c conjugated to several linkers to generate 2c/derivatives usable for improved drug delivery. RESULTS: 2c effectively impairs the OC cell line and primary tumor cell viability in both 2D and 3D conditions. The effectiveness is confirmed in a xenograft mouse model of OC. We show that 2c impairs proteasome activity and triggers apoptosis, most likely by interacting with DUB-UCHL5. We also propose a mechanism for the interaction with DUB-UCHL5 via an in silico evaluation of the enzyme-inhibitor complex. 2c also reduces cell growth by down-regulating the level of the transcription factor E2F1. Eventually, 2c activity is often retained after the conjugation with linkers. CONCLUSION: Our data strongly support the potential therapeutic value of 2c/derivatives in OC.

Plasma Circular RNAs as Biomarkers for Breast Cancer.

Breast cancer (BC) is currently the most common neoplasm, the second leading cause of cancer death in women worldwide, and is a major health problem. The discovery of new biomarkers is crucial to improve our knowledge of breast cancer and strengthen our clinical approaches to diagnosis, prognosis, and follow-up. In recent decades, there has been increasing interest in circulating RNA (circRNA) as modulators of gene expression involved in tumor development and progression. The study of circulating circRNAs (ccircRNAs) in plasma may provide new non-invasive diagnostic, prognostic, and predictive biomarkers for BC. This review describes the latest findings on BC-associated ccircRNAs in plasma and their clinical utility. Several ccircRNAs in plasma have shown great potential as BC biomarkers, especially from a diagnostic point of view. Mechanistically, most of the reported BC-associated ccircRNAs are involved in the regulation of cell survival, proliferation, and invasion, mainly via MAPK/AKT signaling pathways. However, the study of circRNAs is a relatively new area of research, and a larger number of studies will be crucial to confirm their potential as plasma biomarkers and to understand their involvement in BC.

Mucus Structure, Viscoelastic Properties, and Composition in Chronic Respiratory Diseases.

The respiratory mucus, a viscoelastic gel, effectuates a primary line of the airway defense when operated by the mucociliary clearance. In chronic respiratory diseases (CRDs), such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF), the mucus is overproduced and its solid content augments, changing its structure and viscoelastic properties and determining a derangement of essential defense mechanisms against opportunistic microbial (virus and bacteria) pathogens. This ensues in damaging of the airways, leading to a vicious cycle of obstruction and infection responsible for the harsh clinical evolution of these CRDs. Here, we review the essential features of normal and pathological mucus (i.e., sputum in CF, COPD, and asthma), i.e., mucin content, structure (mesh size), micro/macro-rheology, pH, and osmotic pressure, ending with the awareness that sputum biomarkers (mucins, inflammatory proteins and peptides, and metabolites) might serve to indicate acute exacerbation and response to therapies. There are some indications that old and novel treatments may change the structure, viscoelastic properties, and biomarker content of sputum; however, a wealth of work is still needed to embrace these measures as correlates of disease severity in association with (or even as substitutes of) pulmonary functional tests.

Agarose Cryogels: Production Process Modeling and Structural Characterization.

A cryogel is a cross-linked polymer network with different properties that are determined by its manufacturing technique. The formation of a cryogel occurs at low temperatures and results in a porous structure whose pore size is affected by thermal conditions. The adjustable pore sizes of cryogels make them attractive for diverse applications. In this study, the influence of the external operational temperature, which affects the cooling and freezing rates, on the production of cryogels with 2% w/w agarose is investigated. Moreover, a mathematical model is developed to simulate the cryogel production process and provide an initial estimate of the pore size within the structure. The predictions of the model, supported by qualitative light microscopy images, demonstrate that cryogels produced at higher process temperatures exhibit larger pore sizes. Moreover, the existence of pore size distribution within the gel structure is confirmed. Finally, stress relaxation tests, coupled with an image analysis, validates that cryogels produced at lower temperatures possess a higher stiffness and slower water release rates.

BCG-Unresponsive Non-Muscle-Invasive Bladder Cancer: Current Treatment Landscape and Novel Emerging Molecular Targets.

Urothelial carcinoma (UC), the sixth most common cancer in Western countries, includes upper tract urothelial carcinoma (UTUC) and bladder carcinoma (BC) as the most common cancers among UCs (90-95%). BC is the most common cancer and can be a highly heterogeneous disease, including both non-muscle-invasive (NMIBC) and muscle-invasive (MIBC) forms with different oncologic outcomes. Approximately 80% of new BC diagnoses are classified as NMIBC after the initial transurethral resection of the bladder tumor (TURBt). In this setting, intravesical instillation of Bacillus Calmette-Guerin (BCG) is the current standard treatment for intermediate- and high-risk patients. Unfortunately, recurrence occurs in 30% to 40% of patients despite adequate BCG treatment. Radical cystectomy (RC) is currently considered the standard treatment for NMIBC that does not respond to BCG. However, RC is a complex surgical procedure with a recognized high perioperative morbidity that is dependent on the patient, disease behaviors, and surgical factors and is associated with a significant impact on quality of life. Therefore, there is an unmet clinical need for alternative bladder-preserving treatments for patients who desire a bladder-sparing approach or are too frail for major surgery. In this review, we aim to present the strategies in BCG-unresponsive NMIBC, focusing on novel molecular therapeutic targets.

Next-Generation Sequencing and Triple-Negative Breast Cancer: Insights and Applications.

The poor survival of triple-negative breast cancer (TNBC) is due to its aggressive behavior, large heterogeneity, and high risk of recurrence. A comprehensive molecular investigation of this type of breast cancer using high-throughput next-generation sequencing (NGS) methods may help to elucidate its potential progression and discover biomarkers related to patient survival. In this review, the NGS applications in TNBC research are described. Many NGS studies point to TP53 mutations, immunocheckpoint response genes, and aberrations in the PIK3CA and DNA repair pathways as recurrent pathogenic alterations in TNBC. Beyond their diagnostic and predictive/prognostic value, these findings suggest potential personalized treatments in PD -L1-positive TNBC or in TNBC with a homologous recombination deficit. Moreover, the comprehensive sequencing of large genomes with NGS has enabled the identification of novel markers with clinical value in TNBC, such as AURKA, MYC, and JARID2 mutations. In addition, NGS investigations to explore ethnicity-specific alterations have pointed to EZH2 overexpression, BRCA1 alterations, and a BRCA2-delaAAGA mutation as possible molecular signatures of African and African American TNBC. Finally, the development of long-read sequencing methods and their combination with optimized short-read techniques promise to improve the efficiency of NGS approaches for future massive clinical use.

Zebrafish as an Experimental Model for Human Disease.

Belonging to the family of Cyprinidae, the zebrafish is a small freshwater fish present in the rivers of Bangladesh, Northern India and Southern Nepal [...].

A Case-Control Study by ddPCR of ALU 260/111 and LINE-1 266/97 Copy Number Ratio in Circulating Cell-Free DNA in Plasma Revealed LINE-1 266/97 as a Potential Biomarker for Early Breast Cancer Detection.

BACKGROUND: In Western countries, breast cancer (BC) is the most common cancer in women. Early detection has a positive impact on survival, quality of life, and public health costs. Mammography screening programs have increased early detection rates, but new approaches to more personalized surveillance could further improve diagnosis. Circulating cell-free DNA (cfDNA) in blood could provide a potential tool for early diagnosis by analyzing cfDNA quantity, circulating tumor DNA mutations, or cfDNA integrity (cfDI). METHODS: Plasma was obtained from the blood of 106 breast cancer patients (cases) and 103 healthy women (controls). Digital droplet PCR was used for the determination of ALU 260/111 bp and LINE-1 266/97 bp copy number ratio and cfDI. cfDNA abundance was calculated using copies of the EEF1A2 gene. The accuracy of biomarker discrimination was analyzed with receiver operating characteristic curve (ROC). Sensitivity analyses were performed to account for age as a potential confounder. RESULTS: Cases had significantly lower ALU 260/111 or LINE-1 266/97 copy number ratios (median; ALU 260/111 = 0.08, LINE-1 266/97 = 0.20), compared with control (median; ALU 260/111 = 0.10, LINE-1 266/97 = 0.28) (p < 0.001). ROC analysis showed that copy number ratio discriminated cases from controls (area under the curve, AUC = 0.69, 95% CI: 0.62-0.76 for ALU and 0.80, 95% CI: 0.73-0.86 for LINE-1). ROC from cfDI confirmed the better diagnostic performance of LINE-1 compared with ALU. CONCLUSIONS: Analysis of LINE-1 266/97 copy number ratio or cfDI by ddPCR appears to be a useful noninvasive test that could aid in early BC detection. Further studies in a large cohort are needed to validate the biomarker.

Targeting Non-Coding RNAs for the Development of Novel Hepatocellular Carcinoma Therapeutic Approaches.

Hepatocellular carcinoma (HCC) remains a global health challenge, representing the third leading cause of cancer deaths worldwide. Although therapeutic advances have been made in the few last years, the prognosis remains poor. Thus, there is a dire need to develop novel therapeutic strategies. In this regard, two approaches can be considered: (1) the identification of tumor-targeted delivery systems and (2) the targeting of molecule(s) whose aberrant expression is confined to tumor cells. In this work, we focused on the second approach. Among the different kinds of possible target molecules, we discuss the potential therapeutic value of targeting non-coding RNAs (ncRNAs), which include micro interfering RNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). These molecules represent the most significant RNA transcripts in cells and can regulate many HCC features, including proliferation, apoptosis, invasion and metastasis. In the first part of the review, the main characteristics of HCC and ncRNAs are described. The involvement of ncRNAs in HCC is then presented over five sections: (a) miRNAs, (b) lncRNAs, (c) circRNAs, (d) ncRNAs and drug resistance and (e) ncRNAs and liver fibrosis. Overall, this work provides the reader with the most recent state-of-the-art approaches in this field, highlighting key trends and opportunities for more advanced and efficacious HCC treatments.

Nanomechanical Characterization of Ovarian Cancer Cell Lines as a Marker of Response to 2c Treatment.

Epithelial ovarian cancers (EOCs) are a heterogeneous group of tumors with different molecular and clinical features. In past decades, few improvements have been achieved in terms of EOC management and treatment efficacy, such that the 5-year survival rate of patients remained almost unchanged. A better characterization of EOCs' heterogeneity is needed to identify cancer vulnerabilities, stratify patients and adopt proper therapies. The mechanical features of malignant cells are emerging as new biomarkers of cancer invasiveness and drug resistance that can further improve our knowledge of EOC biology and allow the identification of new molecular targets. In this study, we determined the inter and intra-mechanical heterogeneity of eight ovarian cancer cell lines and their association with tumor invasiveness and resistance to an anti-tumoral drug with cytoskeleton depolymerization activity (2c).

Autophagy Inhibitor Chloroquine Downmodulates Hepatic Stellate Cell Activation and Liver Damage in Bile-Duct-Ligated Mice.

Hepatic stellate cell (HSC) activation via the autophagy pathway is a critical factor in liver fibrogenesis. This study tests the hypothesis that chloroquine (CQ) treatment can prevent autophagy and HSC activation in vitro and in vivo in bile-duct-ligated (BDL) mice. Sham-operated and BDL mice were treated with either PBS or CQ in two 60 mg/kg doses the day (D) before and after surgery. On day 2 (2D), HSCs were isolated, and their biological activities were evaluated by measuring intracellular lipid content, α-sma/collagen, and expression of autophagy lc3, sqstm1/p62 markers. The treatment efficacy on liver function was evaluated with serum albumin, transaminases (AST/ALT), and hepatic histology. Primary HSCs were treated in vitro for 24 h with CQ at 0, 2.5, 5, 10, 30, and 50 µM. Autophagy and HSC activation were assessed after 2D of treatment. CQ treatment improved serum AST/ALT, albumin, and bile duct proliferation in 2D BDL mice. This is associated with a suppression of HSC activation, shown by higher HSC lipid content and collagen I staining, along with the blockage of HSC autophagy indicated by an increase in p62 level and reduction in lc3 staining. CQ 5 µM inhibited autophagy in primary HSCs in vitro by increasing p62 and lc3 accumulation, thereby suppressing their in vitro activation. The autophagy inhibitor CQ reduced HSC activation in vitro and in vivo. CQ improved liver function and reduced liver injury in BDL mice.

Rheological and low field NMR characterization of hydrophobically-modified PEG hydrogels for drug delivery.

The focus of this work is on the characterization of hydrophobically-modified polyethylene glycol hydrogels, to be used as drug delivery systems, by means of the combined used of rheology and low field Nuclear Magnetic Resonance. Indeed, these two techniques allowed understanding how the transient physical bonds deriving from hydrophobic association superimpose to the pre-existing covalent bonds. We found that the improvement of physical bonds can be achieved not only by increasing the content of hydrophobic segments but also by using thermal treatments after hydrogel preparation. Moreover, we proved the reliability of an overall interpretative model linking the dependence of the shear modulus and the average magnetic relaxation time. Finally, we proposed a new mathematical approach for the determination of the magnetic relaxation spectrum. This approach reduced the computational heaviness of the procedure and allowed to easily discern the different contributes nested in the overall magnetic relaxation spectrum, an aspect that the traditional approach cannot provide directly.

An Overview of Circulating Cell-Free Nucleic Acids in Diagnosis and Prognosis of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer due to its molecular heterogeneity and poor clinical outcomes. Analysis of circulating cell-free tumor nucleic acids (ctNAs) can improve our understanding of TNBC and provide efficient and non-invasive clinical biomarkers that may be representative of tumor heterogeneity. In this review, we summarize the potential of ctNAs to aid TNBC diagnosis and prognosis. For example, tumor fraction of circulating cell-free DNA (TFx) may be useful for molecular prognosis of TNBC: high TFx levels after neoadjuvant chemotherapy have been associated with shorter progression-free survival and relapse-free survival. Mutations and copy number variations of TP53 and PIK3CA/AKT genes in plasma may be important markers of TNBC onset, progression, metastasis, and for clinical follow-up. In contrast, the expression profile of circulating cell-free tumor non-coding RNAs (ctncRNAs) can be predictive of molecular subtypes of breast cancer and thus aid in the identification of TBNC. Finally, dysregulation of some circulating cell-free tumor miRNAs (miR17, miR19a, miR19b, miR25, miR93, miR105, miR199a) may have a predictive value for chemotherapy resistance. In conclusion, a growing number of efforts are highlighting the potential of ctNAs for future clinical applications in the diagnosis, prognosis, and follow-up of TNBC.

Potential Application of Small Interfering RNA in Gastro-Intestinal Tumors.

Despite the progress made in the diagnoses and therapy of gastrointestinal cancers, these diseases are still plagued by a high mortality. Thus, novel therapeutic approaches are urgently required. In this regard, small interfering RNA (siRNA), double-stranded RNA molecules able to specifically target the mRNA of pathological genes, have the potential to be of therapeutic value. To be effective in the human body, siRNAs need to be protected against degradation. Additionally, they need to target the tumor, leaving the normal tissue untouched in an effort to preserve organ function. To accomplish these tasks, siRNAs have been formulated with smart delivery systems such has polymers and lipids. While siRNA protection is not particularly difficult to achieve, their targeting of tumor cells remains problematic. Here, after introducing the general features of gastrointestinal cancers, we describe siRNA characteristics together with representative delivery systems developed for gastrointestinal cancers. Afterward, we present a selection of research papers employing siRNAs against upper- and lower- gastrointestinal cancers. For the liver, we also consider papers using siRNAs to combat liver cirrhosis, a relevant risk factor for liver cancer development. Finally, we present a brief description of clinical trials employing siRNAs for gastrointestinal cancers.

Modelling drug diffusion through unstirred water layers allows real-time quantification of free/loaded drug fractions and release kinetics from colloidal-based formulations.

The correlation between in vivo and in vitro data is yet not sufficiently optimized to allow a significant reduction and replacement of animal testing in pharmaceutical development. One of the main reasons for this lies in the poor mechanistic understanding and interpretation of the physical mechanisms enabling formulation rely on for deploying the drug. One mechanism that still lacks a proper interpretation is the kinetics of drug release from nanocarriers. In this work, we investigate two different types of classical enabling formulations - i) cyclodextrin solutions and ii) liposomal dispersions - by a combination of an experimental method (i.e. UV-Vis localized spectroscopy) and mathematical modelling/numerical data fitting. With this approach, we are able to discriminate precisely between the amount of drug bound to nanocarriers or freely dissolved at any time point; in addition, we can precisely estimate the binding and diffusivity constants of all chemical species (free drug/bound drug). The results obtained should serve as the first milestone for the further development of reliable in vitro/in silico models for the prediction of in vivo drug bioavailability when enabling formulations are used.

An Overview of siRNA Delivery Strategies for Urological Cancers.

The treatment of urological cancers has been significantly improved in recent years. However, for the advanced stages of these cancers and/or for those developing resistance, novel therapeutic options need to be developed. Among the innovative strategies, the use of small interfering RNA (siRNA) seems to be of great therapeutic interest. siRNAs are double-stranded RNA molecules which can specifically target virtually any mRNA of pathological genes. For this reason, siRNAs have a great therapeutic potential for human diseases including urological cancers. However, the fragile nature of siRNAs in the biological environment imposes the development of appropriate delivery systems to protect them. Thus, ensuring siRNA reaches its deep tissue target while maintaining structural and functional integrity represents one of the major challenges. To reach this goal, siRNA-based therapies require the development of fine, tailor-made delivery systems. Polymeric nanoparticles, lipid nanoparticles, nanobubbles and magnetic nanoparticles are among nano-delivery systems studied recently to meet this demand. In this review, after an introduction about the main features of urological tumors, we describe siRNA characteristics together with representative delivery systems developed for urology applications; the examples reported are subdivided on the basis of the different delivery materials and on the different urological cancers.

High eEF1A1 Protein Levels Mark Aggressive Prostate Cancers and the In Vitro Targeting of eEF1A1 Reveals the eEF1A1-actin Complex as a New Potential Target for Therapy.

Although the eukaryotic elongation factor eEF1A1 plays a role in various tumours, there is little information on its prognosis/therapeutic value in prostate carcinoma. In high-grade and castration-resistant prostate carcinoma (CRPC), the identification of novel therapeutic markers/targets remains a priority. The expression of eEF1A1 protein was determined in formalin-fixed, paraffin-embedded prostate cancer and hyperplasia tissue by IHC. The role of eEF1A1 was investigated in a cellular model using a DNA aptamer (GT75) we previously developed. We used the aggressive CRPC cancer PC-3 and non-tumourigenic PZHPV-7 lines. Cytotoxicity was measured by the MTS assay and eEF1A1 protein levels by in-cell Western assays. The mRNA levels of eEF1A1 were measured by qPCR and ddPCR. Higher expression of eEF1A1 was found in Gleason 7-8 compared with 4-6 tissues (Gleason ≥ 7, 87% versus Gleason ≤ 6, 54%; p = 0.033). Patients with a high expression of eEF1A1 had a worse clinical outcome. In PC-3, but not in PZHPV-7, GT75 decreased cell viability and increased autophagy and cell detachment. In PC-3 cells, but not in PZHPV-7, GT75 mainly co-localised with the fraction of eEF1A1 bound to actin. Overexpression of the eEF1A1 protein can identify aggressive forms of prostate cancer. The targeting of eEF1A1 by GT75 impaired cell viability in PC-3 cancer cells but not in PZHPV-7 non-tumourigenic cells, indicating a specific role for the protein in cancer survival. The eEF1A1-actin complexes appear to be critical for the viability of PC-3 cancer cells, suggesting that eEF1A1 may be an attractive target for therapeutic strategies in advanced forms of prostate cancer.