A Comparison of Currently Approved Small Interfering RNA (siRNA) Medications to Alternative Treatments by Costs, Indications, and Medicaid Coverage.

Small interfering RNA (siRNA)-based medications offer the ability to target previously undruggable targets and have now received FDA approval in five instances for orphan or uncommon diseases. The current siRNA "-sirans" are directed towards hepatic molecular targets. Because they are not conventional drug formulae, their ultimate clinical success will require overcoming multiple barriers beyond their pharmacology. The minimal patient numbers leave fewer patients to bear the costs of R&D and manufacture; therefore, the cost of these drugs, questionable third-party reimbursement, and competition from other drug classes for the same low number of patients are impediments to patient access. The parenteral route of administration, as well as emerging safety restrictions, are also drawbacks to siRNA. With this review, we document currently approved siRNA drugs by condition, approval date, administration route and frequencies. We have estimated the available patient populations for siran therapies using the U.S. Medicaid and Medicare populations and sought to identify the frequency with which large Medicaid formularies list siRNA drugs. Current comparative costs between the siRNA drugs and alternatives have been presented, and the review summarizes current adverse events as reported to the FDA's Adverse Event Reporting System. Our review and data indicate that sirans are extremely expensive and seldom recognized in posted Medicaid formularies. However, alternative treatments for these conditions are no less costly, usually do not have significantly different adverse events, and are often less convenient for the patient.

Review of adult gender transition medications: mechanisms, efficacy measures, and pharmacogenomic considerations.

Gender dysphoria is the imparity between a person's experienced gender and their birth-assigned gender. Gender transition is the process of adapting a person's sexual characteristics to match their experienced gender. The number of adults receiving sex hormone therapy for gender dysphoria is increasingly and these pharmacotherapies are increasing being prescribed in a general practice setting. The role of hormone therapy is to reverse or reduce physical sexual characteristics of the birth-assigned gender and enhance and build characteristics aligning to the expressed gender and these therapies apply to both transgender and gender nonconforming patients. Recognizing the options and interpreting the effects of gender transition therapies are fundamental to the discussion and treatment of gender dysphoria. This review summarizes pharmacodynamics, comparative dosing, adverse effects, monitoring, and potential pharmacogenetic influence of current pharmacotherapy. These include the use of 17-beta-estradiol, spironolactone, testosterone, GnRH agonists as well as adjunctive phosphodiesterase-5 inhibitors. The article also addresses gaps within the published literature including optimal routes of administration for individual patients, risks of malignancy and dosing reductions as transgender patients age.

ISCCM Guidelines for the Use of Non-invasive Ventilation in Acute Respiratory Failure in Adult ICUs.

A. ACUTE HYPERCAPNIC RESPIRATORY FAILURE A1. Acute Exacerbation of COPD: Recommendations: NIV should be used in management of acute exacerbation of COPD in patients with acute or acute-on-chronic respiratory acidosis (pH = 7.25-7.35). (1A) NIV should be attempted in patients with acute exacerbation of COPD (pH <7.25 & PaCO2 ≥ 45) before initiating invasive mechanical ventilation (IMV) except in patients requiring immediate intubation. (2A). Lower the pH higher the chance of failure of NIV. (2B) NIV should not to be used routinely in normo- or mildly hyper-capneic patients with acute exacerbation of COPD, without acidosis (pH > 7.35). (2B) A2. NIV in ARF due to Chest wall deformities/Neuromuscular diseases: Recommendations: NIV may be used in patients of ARF due to chest wall deformity/Neuromuscular diseases. (PaCO2 ≥ 45) (UPP) A3. NIV in ARF due to Obesity hypoventilation syndrome (OHS): Recommendations: NIV may be used in AHRF in OHS patients when they present with acute hypercapnic or acute on chronic respiratory failure (pH 45). (3B) NIV/CPAP may be used in obese, hypercapnic patients with OHS and/or right heart failure in the absence of acidosis. (UPP) B. NIV IN ACUTE HYPOXEMIC RESPIRATORY FAILURE: B1. NIV in Acute Cardiogenic Pulmonary Oedema: Recommendations: NIV is recommended in hospital patients with ARF, due to Cardiogenic pulmonary edema. (1A). NIV should be used in patients with acute heart failure/ cardiogenic pulmonary edema, right from emergency department itself. (1B) Both CPAP and BiPAP modes are safe and effective in patients with cardiogenic pulmonary edema. (1A). However, BPAP (NIV-PS) should be preferred in cardiogenic pulmonary edema with hypercapnia. (3A) B2. NIV in acute hypoxemic respiratory failure: Recommendations: NIV may be used over conventional oxygen therapy in mild early acute hypoxemic respiratory failure (P/F ratio <300 and >200 mmHg), under close supervision. (2B) We strongly recommend against a trial of NIV in patients with acute hypoxemic failure with P/F ratio <150. (2A) B3. NIV in ARF due to Chest Trauma: Recommendations: NIV may be used in traumatic flail chest along with adequate pain relief. (3B) B4. NIV in Immunocompromised Host: Recommendations: In Immunocompromised patients with early ARF, we may consider NIV over conventional oxygen. (2B). B5. NIV in Palliative Care: Recommendations: We strongly recommend use of NIV for reducing dyspnea in palliative care setting. (2A) B6. NIV in post-operative cases: Recommendations: NIV should be used in patients with post-operative acute respiratory failure. (2A) B6a. NIV in abdominal surgery: Recommendations: NIV may be used in patients with ARF following abdominal surgeries. (2A) B6b. NIV in bariatric surgery: Recommendations: NIV may be used in post-bariatric surgery patients with pre-existent OSA or OHS. (3A) B6c. NIV in Thoracic surgery: Recommendations: In cardiothoracic surgeries, use of NIV is recommended post operatively for acute respiratory failure to improve oxygenation and reduce chance of reintubation. (2A) NIV should not be used in patients undergoing esophageal surgery. (UPP) B6d. NIV in post lung transplant: Recommendations: NIV may be used for shortening weaning time and to avoid re-intubation following lung transplantation. (2B) B7. NIV during Procedures (ETI/Bronchoscopy/TEE/Endoscopy): Recommendations: NIV may be used for pre-oxygenation before intubation. (2B) NIV with appropriate interface may be used in patients of ARF during Bronchoscopy/Endoscopy to improve oxygenation. (3B) B8. NIV in Viral Pneumonitis ARDS: Recommendations: NIV cannot be considered as a treatment of choice for patients with acute respiratory failure with H1N1 pneumonia. However, it may be reasonable to use NIV in selected patients with single organ involvement, in a strictly controlled environment with close monitoring. (2B) B9. NIV and Acute exacerbation of Pulmonary Tuberculosis: Recommendations: Careful use of NIV in patients with acute Tuberculosis may be considered, with effective infection control precautions to prevent air-borne transmission. (3B) B10. NIV after planned extubation in high risk patients: Recommendation: We recommend that NIV may be used to wean high risk patients from invasive mechanical ventilation as it reduces re-intubation rate. (2B) B11. NIV for respiratory distress post extubation: Recommendations: We recommend that NIV therapy should not be used to manage respiratory distress post-extubation in high risk patients. (2B) C. APPLICATION OF NIV: Recommendation: Choice of mode should be mainly decided by factors like disease etiology and severity, the breathing effort by the patient and the operator familiarity and experience. (UPP) We suggest using flow trigger over pressure triggering in assisted modes, as it provides better patient ventilator synchrony. Especially in COPD patients, flow triggering has been found to benefit auto PEEP. (3B) D. MANAGEMENT OF PATIENT ON NIV: D1. Sedation: Recommendations: A non-pharmacological approach to calm the patient (Reassuring the patient, proper environment) should always be tried before administrating sedatives. (UPP) In patients on NIV, sedation may be used with extremely close monitoring and only in an ICU setting with lookout for signs of NIV failure. (UPP) E. EQUIPMENT: Recommendations: We recommend that portable bilevel ventilators or specifically designed ICU ventilators with non-invasive mode should be used for delivering Non-invasive ventilation in critically ill patients. (UPP) Both critical care ventilators with leak compensation and bi-level ventilators have been equally effective in decreasing the WOB, RR, and PaCO2. (3B) Currently, Oronasal mask is the most preferred interface for non-invasive ventilation for acute respiratory failure. (3B) F. WEANING: Recommendations: We recommend that weaning from NIV may be done by a standardized protocol driven approach of the unit. (2B) How to cite this article: Chawla R, Dixit SB, Zirpe KG, Chaudhry D, Khilnani GC, Mehta Y, et al. ISCCM Guidelines for the Use of Non-invasive Ventilation in Acute Respiratory Failure in Adult ICUs. Indian J Crit Care Med 2020;24(Suppl 1):S61-S81.

A Veterinary Comparative Counseling Elective Featuring Web-based, Student-created, Client Information Sheets.

OBJECTIVE: To design and implement a course in Companion Animal Comparative Counseling that would expose students (N=38) to essential elements of veterinary therapeutics and provide them with the opportunity to apply their knowledge by writing and posting client information sheets (CIS) on an open web site. DESIGN: The elective course was limited to companion animals. Nine different topics were covered over the semester. Class sessions included a didactic component, trivia questions, and field trips. There were 4 major graded assessments: an examination on foundation knowledge, followed by two comparative counseling assessments and evaluation of group-composed CIS. Attendance and participation were also considered. ASSESSMENT: The class learned comparative disease states, how to counsel on common pet prescriptions, where to access informatics about specific veterinary drugs, and how to create their own CIS. CONCLUSION: As pharmacists, these students may have improved their training in veterinary comparative pharmacy.

Use of SkypeTM sessions with top cancer trial physicians to bring Interprofessional Education (IPE) into a didactic oncology course.

OBJECTIVE: To incorporate meaningful Interprofessional Education (IPE) into a core didactic oncology course by bringing into the classroom a series of cancer specialist physicians, from around the United States, who instructed from and discussed their own clinical trials. METHODS: The instructor recruited physicians from selected nationwide cancer centers as well as from more regional institutions. Prior to each IPE session, students pre-read relevant trials or other materials. At an arranged time, an approximately 30-minute Skype™ session began which included student questions. Assessments of student learning included individual examinations and a group trial. Survey data was also collected on student views of interprofessionalism, cancer trial informatics and the role of pharmacists on trial teams. RESULTS: Group clinical trials showed substantial student creativity and achievement of new skills in trial design. Essays indicated that students learned as individuals. Survey data showed that prior to the interprofessional sessions, students felt uncomfortable discussing trial designs/results with graduates of other health professions. Following the sessions and group trial assignment, students gained significant confidence discussing trials with physicians and felt increased recognition for the pharmacist's role in the trial team and increased appreciation for the perspective of trial physicians. Students reported that they enjoyed the opportunity to talk with top cancer physicians. CONCLUSIONS: Didactic core courses can be modified to accommodate meaningful interprofessional team education. Considerable time was required to arrange external Skype™ speakers; however, this IPE oncology exercise was very highly rated by students indicating that this was preparatory time well-invested.

Toxicologic assessment of a commercial decolorized whole leaf aloe vera juice, lily of the desert filtered whole leaf juice with aloesorb.

Aloe vera, a common ingredient in cosmetics, is increasingly being consumed as a beverage supplement. Although consumer interest in aloe likely stems from its association with several health benefits, a concern has also been raised by a National Toxicology Program Report that a nondecolorized whole leaf aloe vera extract taken internally by rats was associated with intestinal mucosal hyperplasia and ultimately malignancy. We tested a decolorized whole leaf (DCWL) aloe vera, treated with activated charcoal to remove the latex portion of the plant, for genotoxicity in bacteria, acute/subacute toxicity in B6C3F1 mice, and subchronic toxicity in F344 rats. We found this DCWL aloe vera juice to be nongenotoxic in histidine reversion and DNA repair assays. Following acute administration, mice exhibited no adverse signs at 3- or 14-day evaluation periods. When fed to male and female F344 rats over 13 weeks, DCWL aloe led to no toxicity as assessed by behavior, stools, weight gain, feed consumption, organ weights, and hematologic or clinical chemistry profiles. These rats had intestinal mucosal morphologies-examined grossly and microscopically-that were similar to controls. Our studies show that oral administration of this DCWL aloe juice has a different toxicology profile than that of the untreated aloe juice at exposures up to 13 weeks.

An in vitro and in vivo toxicologic evaluation of a stabilized aloe vera gel supplement drink in mice.

Aloe vera gel is increasingly consumed as a beverage dietary supplement. The purpose of this study was to determine potential toxicity of a stabilized aloe vera gel derived from the inner gel fillet and marketed as a drink. The gel juice was assessed through assays of genotoxicity in vivo and acute and subchronic toxicity in B6C3F1 mice. Aloe vera did not increase the SOS DNA repair response in Escherichia coli and at 1× and 0.25× it did not increase mutagenesis of Salmonella TA100 resulting in histidine biosynthesis. At 3 and 14days following acute exposure, male and female mice gavaged with the stabilized aloe gel had daily appearances, total body weight gain, selected organ weights, necropsy and hematology tests similar to control mice gavaged with water. After a 13-week aloe gel feed study, male and female mice evaluated by the same criteria as the acute study plus feed consumption and serum chemistry tests were found to be equivalent to control groups. These data indicate that a commercial stabilized aloe gel consumed as a beverage was not genotoxic or toxic in vivo. These results contrast with those obtained using preparations containing aloe latex phenolic compounds such as anthraquinones.

Synthesis and biological investigations of a ZnPc-antiCEA bioconjugate for imaging of colorectal cancer.

Two zinc(II) phthalocyanines (ZnPcs) were conjugated with a monoclonal antibody (MAb) directed against carcinoembryonic antigen (CEA), using an in situ activated carboxylic acid on the ZnPcs. The bioconjugate with the highest ZnPc/MAb ratio of 3 was investigated in vitro for its ability to target and fluorescently label human colorectal HT-29 cells. The ZnPc-CEA MAb 2 was observed to efficiently target HT-29 cells, about 37 times more than unconjugated ZnPc. Furthermore, in the presence of a 4-fold excess of unlabelled anti-CEA antibody, the fluorescence signal of 2 was reduced by ~90% showing that the targeting is CEA-mediated. These studies further confirm the high specificity of Pc-antibody conjugates for antigens over-expressed on tumor cells and warrant further investigations of these immunoconjugates and their derivatives for imaging of colorectal cancer.

Phthalocyanine-peptide conjugates for epidermal growth factor receptor targeting.

Four phthalocyanine (Pc)-peptide conjugates designed to target the epidermal growth factor receptor (EGFR) were synthesized and evaluated in vitro using four cell lines: human carcinoma A431 and HEp2, human colorectal HT-29, and kidney Vero (negative control) cells. Two peptide ligands for EGFR were investigated: EGFR-L1 and -L2, bearing 6 and 13 amino acid residues, respectively. The peptides and Pc-conjugates were shown to bind to EGFR using both theoretical (Autodock) and experimental (SPR) investigations. The Pc-EGFR-L1 conjugates 5a and 5b efficiently targeted EGFR and were internalized, in part due to their cationic charge, whereas the uncharged Pc-EGFR-L2 conjugates 4b and 6a poorly targeted EGFR maybe due to their low aqueous solubility. All conjugates were nontoxic (IC(50) > 100 µM) to HT-29 cells, both in the dark and upon light activation (1 J/cm(2)). Intravenous (iv) administration of conjugate 5b into nude mice bearing A431 and HT-29 human tumor xenografts resulted in a near-IR fluorescence signal at ca. 700 nm, 24 h after administration. Our studies show that Pc-EGFR-L1 conjugates are promising near-IR fluorescent contrast agents for CRC and potentially other EGFR overexpressing cancers.

Structure-based development of small molecule PFKFB3 inhibitors: a framework for potential cancer therapeutic agents targeting the Warburg effect.

Cancer cells adopt glycolysis as the major source of metabolic energy production for fast cell growth. The HIF-1-induced PFKFB3 plays a key role in this adaptation by elevating the concentration of Fru-2,6-BP, the most potent glycolysis stimulator. As this metabolic conversion has been suggested to be a hallmark of cancer, PFKFB3 has emerged as a novel target for cancer chemotherapy. Here, we report that a small molecular inhibitor, N4A, was identified as an initial lead compound for PFKFB3 inhibitor with therapeutic potential. In an attempt to improve its potency, we determined the crystal structure of the PFKFB3•N4A complex to 2.4 Šresolution and, exploiting the resulting molecular information, attained the more potent YN1. When tested on cultured cancer cells, both N4A and YN1 inhibited PFKFB3, suppressing the Fru-2,6-BP level, which in turn suppressed glycolysis and, ultimately, led to cell death. This study validates PFKFB3 as a target for new cancer therapies and provides a framework for future development efforts.

Oncolytic herpes simplex virus 1 encoding 15-prostaglandin dehydrogenase mitigates immune suppression and reduces ectopic primary and metastatic breast cancer in mice.

Oncolytic herpes simplex virus 1 (HSV-1) viruses armed with immunomodulatory transgenes have shown potential for enhanced antitumor therapy by overcoming tumor-based immune suppression and promoting antitumor effector cell development. Previously, we reported that the new oncolytic HSV-1 virus, OncSyn (OS), engineered to fuse tumor cells, prevented tumor growth and metastasis to distal organs in the 4T1/BALB/c immunocompetent breast cancer mouse model, suggesting the elicitation of antitumor immune responses (Israyelyan et al., Hum. Gen. Ther. 18:5, 2007, and Israyelyan et al., Virol. J. 5:68, 2008). The OSV virus was constructed by deleting the OS viral host shutoff gene (vhs; UL41) to further attenuate the virus and permit dendritic cell activation and antigen presentation. Subsequently, the OSVP virus was constructed by inserting into the OSV viral genome a murine 15-prostaglandin dehydrogenase (15-PGDH) expression cassette, designed to constitutively express 15-PGDH upon infection. 15-PGDH is a tumor suppressor protein and the primary enzyme responsible for the degradation of prostaglandin E2 (PGE2), which is known to promote tumor development. OSVP, OSV, and OS treatment of 4T1 tumors in BALB/c mice effectively reduced primary tumor growth and inhibited metastatic development of secondary tumors. OSVP was able to significantly inhibit the development and accumulation of 4T1 metastatic tumor cells in the lungs of treated mice. Ex vivo analysis of immune cells following treatment showed increased inflammatory cytokine production and the presence of mature dendritic cells for the OSVP, OSV, and OS viruses. A statistically significant decrease in splenic myeloid-derived suppressor cells (MDSC) was observed only for OSVP-treated mice. These results show that intratumoral oncolytic herpes is highly immunogenic and suggest that 15-PGDH expression by OSVP enhanced the antitumor immune response initiated by viral infection of primary tumor cells, leading to reduced development of pulmonary metastases. The availability of the OSVP genome as a bacterial artificial chromosome allows for the rapid insertion of additional immunomodulatory genes that could further assist in the induction of potent antitumor immune responses against primary and metastatic tumors.

Photoinduced cytotoxicity and biodistribution of prostate cancer cell-targeted porphyrins.

A series of five porphyrin-peptide conjugates bearing one or two sequences containing a cell penetrating peptide (CPP), a nuclear localization signal (NLS), or a bifunctional CPP-NLS or NLS-CPP sequences were synthesized and investigated in vitro using PC-3M human prostate cancer cells, in comparison with FDA-approved purified hematoporphyrin derivative (Porfimer Sodium) and mTHPC. The most promising porphyrin-HIV-1 Tat (48-60) conjugate 2 [lowest dark cytotoxicity (IC50 = 38.0 microM), highest phototoxicity (IC50 = 0.40 microM at 1 J/cm2)] was further evaluated in an in vivo biodistribution study using SCID mice bearing PC-3M tumors, in comparison with purified hematoporphyrin derivative. Porphyrin conjugate 2 was more tumor selective than the hematoporphyrin derivative and accumulated to a significantly greater extent in tumors. Our results show that effective photodynamic cytotoxicity can be induced in human prostate cancer cells with minimal dark toxicity and that selective accumulation in prostate tumors can be achieved in vivo with porphyrin-targeted photosensitizers.

Effective treatment of human breast tumor in a mouse xenograft model with herpes simplex virus type 1 specifying the NV1020 genomic deletion and the gBsyn3 syncytial mutation enabling high viral replication and spread in breast cancer cells.

A new oncolytic and fusogenic herpes simplex virus type 1 (HSV-1) was constructed on the basis of the wildtype HSV-1(F) strain. To provide for safety and tumor selectivity, the virus carried a large deletion including one of the two alpha4, gamma(1)34.5, alpha0 genes and the latency-associated transcript region. The gamma(1)34.5 gene, a major neurovirulence factor, was replaced by a gene cassette constitutively expressing the red fluorescent protein gene. Homologous recombination was used to transfer the fusogenic gBsyn3 mutation to the viral genome to produce the OncSyn virus. OncSyn causes extensive virus-induced cell fusion (syncytia) and replicates to higher titers than the parental Onc and HSV-1(F) strains in breast cancer cells. Biochemical analysis revealed that the OncSyn virus retains a stable genome and expresses all major viral glycoproteins. A xenograft mouse model system using MDA-MB-435S-luc (MM4L) human breast cancer cells constitutively expressing the luciferase gene implanted within the interscapular region of animals was used to test the ability of the virus to inactivate breast tumor cells in vivo. Seventy-two mice bearing MM4L breast cancer xenografts were randomly divided into three groups and given two rounds of three consecutive intratumoral injections of OncSyn, inactivated OncSyn, or phosphate-buffered saline 3 days apart. A single round of virus injections resulted in a drastic reduction of tumor sizes (p

A doxycycline-inducible urokinase receptor (uPAR) upregulates uPAR activities including resistance to anoikis in human prostate cancer cell lines.

BACKGROUND: The urokinase receptor (uPAR) mediates a diverse array of cellular processes including several events involved in prostate cancer metastasis. Many of these activities are initiated or enhanced by uPAR binding to its proteolytic ligand, urokinase (uPA). Our objective in this study was to generate and test an inducible lentiviral system capable of expressing uPAR and DsRed fluorescent protein in human prostate cancer cell lines. RESULTS: A DsRed-uPAR fusion construct was inserted into a lentiviral vector. Transduction of human prostate cancer cell lines with this virus and with a virus containing a reverse-tetracycline transactivator (rt-TA) resulted in a stable transgene which induced both uPAR and DsRed proteins in a dose-responsive fashion upon stimulation with doxycycline. Immunoblots and immunofluorescence studies indicated no detectable uPAR expression in non-induced prostate cancer cell lines. Cells with induced-uPAR demonstrated increased cellular adhesion to the matrix substrate vitronectin and increased net cell proliferation compared to uninduced cells. Finally, induced uPAR-expressing prostate cancer cells were resistant to anoikis over an extended time period when grown in suspension. CONCLUSION: This doxycycline-inducible lentivirus system produces titerable levels of biologically active uPAR in vitro. This tool can be used to dissect cellular events following induction of uPAR in prostate cancer cells.

Immunohistochemical staining of urokinase plasminogen activator-like and urokinase plasminogen activator receptor-like proteins in the urinary tract of healthy dogs.

OBJECTIVE: To determine distribution of urokinase plasminogen activator-like protein and urokinase plasminogen activator receptor-like protein in urinary tract tissues of healthy dogs. ANIMALS: 11 healthy dogs. PROCEDURES: Necropsy specimens from kidney, ureter, bladder, urethra, prostate, and testis were obtained from 4 sexually intact female dogs, 5 sexually intact males, and 2 castrated males; dogs ranged in age from juvenile to adult. Urokinase plasminogen activator-like protein and urokinase plasminogen activator receptor-like protein in tissue lysates from kidney, prostate, and testis were identified by use of SDS-PAGE, western blot analysis, and immunoprecipitation. Urokinase plasminogen activator-like protein and urokinase plasminogen activator receptor-like protein in kidney, ureter, urinary bladder, urethra, prostate, and testis were identified by use of immunohistochemical staining of tissue sections. RESULTS: Urokinase plasminogen activator-like protein and urokinase plasminogen activator receptor-like protein in the molecular-weight range published for urokinase and urokinase receptor (53 and 33 kd for urokinase and 60 to 65 kd for urokinase receptor) were identified. Distribution of the proteins identified by use of immunohistochemical staining was comparable with published information for humans and mice for the urinary tract. Staining of these proteins was detected in more tissue types than reported in healthy humans. CONCLUSIONS AND CLINICAL RELEVANCE: Urokinase plasminogen activator-like protein and urokinase plasminogen activator receptor-like protein were detected in the urinary tract of healthy dogs. This information is important for further evaluation of the functions of urokinase and urokinase receptor in the canine urinary tract and the pathophysiologic features of urinary tract disease.

Prostate cancer cells show elevated urokinase receptor in a mouse model of metastasis.

BACKGROUND: The urokinase receptor (uPAR) governs several functions necessary during invasion and metastasis such as motility, degradation of the extracellular matrix and adhesion. This receptor has been recently associated with clinical prostate cancer progression. Experimentally, inhibition of uPAR reduces colonization of extra-prostatic sites in animal models. Our objective in this study was to compare uPAR expression in orthotopic vs. metastatic foci in vivo and to examine at the cellular level how uPAR might promote early stages of metastasis. RESULTS: We show that uPAR staining is significantly greater in regional lymph node metastases than in the intraprostatic tumor mass. Using transient over-expression, we found that uPAR increases in vitro motility and chemotactic invasion. Finally, we demonstrate that uPAR is up-regulated by a significant subpopulation prostate cancer cells following matrix detachment and maintenance in suspension and we provide evidence that prostate cancer cells with elevations in uPAR have an enhanced resistance to anoikis. CONCLUSION: These data provide new evidence that uPAR can be induced by cancer cells during metastasis in vivo and that this elevated uPAR enhances resistance to anoikis in vitro.

The superoxide scavenger TEMPOL induces urokinase receptor (uPAR) expression in human prostate cancer cells.

There is little understanding of the effect that reactive oxygen metabolites have on cellular behavior during the processes of invasion and metastasis. These oxygen metabolites could interact with a number of targets modulating their function such as enzymes involved in basement membrane dissolution, adhesion molecules involved in motility or receptors involved in proliferation. We investigated the effect of increased scavenging of superoxide anions on the expression of the urokinase receptor (uPAR) in PC-3M human prostate cancer cells. Urokinase receptor is a GPI-linked cell surface molecule which mediates multiple functions including adhesion, proliferation and pericellular proteolysis. Addition of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) to PC-3M cultures stimulated expression of uPAR protein peaking between 48 and 72 hours. Cell surface expression of the uPAR was also increased. Surprisingly, uPAR transcript levels increased only slightly and this mild increase did not coincide with the striking degree of protein increase. This disparity indicates that the TEMPOL effect on uPAR occurs through a post-transcriptional mechanism. TEMPOL presence in PC-3M cultures reduced intracellular superoxide-type species by 75% as assayed by NBT dye conversion; however this reduction significantly diminished within hours following TEMPOL removal. The time gap between TEMPOL treatment and peak uPAR protein expression suggests that reduction of reactive oxygen metabolites in prostate cancer cells initiates a multistep pathway which requires several hours to culminate in uPAR induction. These findings reveal a novel pathway for uPAR regulation involving reactive oxygens such as superoxide anion.

Aryl hydrocarbon exposure induces expression of MMP-9 in human prostate cancer cell lines.

Although aromatic hydrocarbons have been extensively studied with regard to tumor formation, there has been little investigation into effects of these environmental chemicals on regulation of genes involved in tumor invasion. We investigated effects of three arylhydrocarbons on expression of MMP-9 in PC-3 and DU145 human prostate cancer cells. TCDD exposure lead to dose and time dependent increases in MMP-9 expression. Benzo(a)pyrene and a PAH-containing soot (BDS) also induced this MMP. These hydrocarbons also stimulated MMP-9 protein secretion. Our data demonstrate that aryl hydrocarbons can stimulate the production of MMP-9 in human prostate cancer cells.