Development of a Bladder Cancer-on-a-Chip Model to Assess Bladder Cancer Cell Invasiveness.

We have developed a bladder cancer-on-a-chip model which supports the 3D growth of cells and can be used to assess and quantify bladder cancer cell invasiveness in a physiologically appropriate environment. Three bladder cancer cell lines (T24, J82, and RT4) were resuspended in 50% Matrigel® and grown within a multi-channel organ-on-a-chip system. The ability of live cells to invade across into an adjacent 50% Matrigel®-only channel was assessed over a 2-day period. Cell lines isolated from patients with high-grade bladder cancer (T24 and J82) invaded across into the Matrigel®-only channel at a much higher frequency compared to cells isolated from a patient with low-grade cancer (RT4) (p < 0.001). The T24 and J82 cells also invaded further distances into the Matrigel®-only channel compared to the RT4 cells (p < 0.001). The cell phenotype within the model was maintained as assessed by cell morphology and immunohistochemical analysis of E-cadherin. Treatment with ATN-161, an α5ß1 integrin inhibitor and well-known migrastatic drug, caused a dose-dependent decrease in the invasiveness of the J82 cells (p < 0.01). The combined data demonstrate that our bladder cancer-on-a-chip model supports the retention of the bladder cancer cell phenotype and can be used to reproducibly assess and quantify the invasiveness of live bladder cancer cells.

Impact of high-stakes exams, incentives, and preparation on student performance: Insights from the pharmacy curriculum outcomes assessment.

INTRODUCTION: Efforts to ensure success of pharmacy students in passing pharmacy standardized exams require substantial investments. Engaging students effectively can be a challenge when there are no consequences for non-participation or poor performance. This study examined how engagement reinforcement, including high-stake exam requirements, instructional strategies, and incentives, impacted student performance on the Pharmacy Curriculum Outcomes Assessment (PCOA). METHODS: PCOA scores, milestone exams, grade point averages (GPAs), and PCOA preparedness assessments for cohorts (Co) that received high-stakes exams, incentives, and preparation (Co2019, Co2020, and Co2021) was compared with those that did not receive these interventions (Co2017 and Co2018). Students' perceptions regarding reinforcement, incentive, and preparedness were evaluated using an anonymous survey. RESULTS: Analyzing data from 545 students over five years, mandated PCOA preparedness, high-stakes PCOA requirements, and incentives for Co2019, Co2020, and Co2021 improved scores by 11% to 18% compared to Co2017 and Co2018. This corresponded to a rise in performance from the 12th to 27th percentile for Co2017 and Co2018 to the 39th to 49th percentile for Co2019, Co2020, and Co2021. In these later cohorts, PCOA scores consistently correlated with the school's milestone exams and students' cumulative GPAs (correlation coefficients 0.47-0.70, P < .001), while no such correlation was observed in Co2017 and Co2018. Faculty-led PCOA preparation yielded better results (48.2% in Co2020, 45.8% in Co2021) than self-learning (42% in Co2019). Students using faculty-prepared assessments reported increased confidence in biomedical and pharmaceutical sciences. CONCLUSIONS: This study highlights the importance of high-stakes requirements, incentives, and thorough preparation in improving PCOA results.

A pharmacist-led community-based survey study: Determining the impact of the Covid-19 pandemic on actionable factors associated with worse cancer outcomes and cancer health disparities.

Purpose: The goals of this cross-sectional community-based survey study were to assess the impact of the Covid-19 pandemic on actionable factors which are known to contribute to worse cancer outcomes, and to determine whether race and ethnicity-based differences exist. Methods: A survey study which captured demographic information and changes in cancer outcomes-related factors since the start of the Covid-19 pandemic, was conducted at a public Covid-19 vaccination clinic over a period of 10 days during March 2021. Surveys were administered in multiple languages. Chi-square tests and ANOVA followed by post-hoc Dunnett testing assessed for race and ethnicity-based differences. Results: A total of 949 people participated (61.6% participation rate). Ninety-three surveys were removed based on inclusion criteria giving a final participant number of 856. Many participants reported postponing cancer screenings (17.8%) and cancellation of medical appointments (22.8% and 25.8% reported cancelled appointments by providers or themselves, respectively) due to the pandemic. Participants also reported decreased physical activity (44.7%) and increased tobacco and/or marijuana usage (7.0%). Conversely, participants reported consuming more fruits and vegetables (21.4%) and decreasing alcohol consumption (21.4%). Several race-related differences but no ethnicity-related differences were observed. Conclusion: Our data can be used to help guide pharmacist-led targeted outreach in our community which will help mitigate Covid-19 pandemic-driven changes in behaviors associated with worse cancer outcomes and exacerbation of cancer health disparities. To our knowledge, this is the first cancer outcomes-related study to be conducted at a public Covid-19 vaccination site and is the first pharmacist-led study in this area.

Effectiveness of an Advanced Naloxone Training, Simulation, and Assessment of Second-Year Pharmacy Students.

Background: Opioid overdoses continue to be one of the most urgent public health priorities. In 2020, reported overdose deaths in the United States reached a high of over 93,000 cases. As the COVID-19 pandemic and opioid crisis continues to be addressed, life-saving agents must be more widely accessible to those with a high overdose risk. An essential step to increasing access is to train student pharmacists to dispense naloxone. Once licensed, the number of personnel authorized to dispense naloxone can increase. Objectives: To design a training program to educate second-year pharmacy (P2) students on furnishing naloxone under a state protocol. Methods: A multi-phased curriculum-based naloxone training program was delivered to P2 students and included lecture-based education, team-based learning (TBL) applications, case-based scenarios, and summative assessments to improve student knowledge and confidence in furnishing naloxone. Students were surveyed on their knowledge and confidence with naloxone prior to training, after the in-class training and TBL applications and after three assessments. Assessments included simulated patient counseling, case-based scenarios, and proper dispensing of naloxone in a community pharmacy simulation lab. Results: A total of 185 student pharmacists completed the naloxone training program and 68 completed all three surveys. Average scores for naloxone assessments were 83% for the APPS lab patient case, 90.5% for the prescription label typed for the naloxone product, and 88.5% for patient counseling. Statistically significant increases in knowledge-based quiz-like scores (42.1% after training vs. 7.2% after assessment) and in the proportion of students affirmatively answering survey questions after training and assessment was observed. Conclusion: Multi-phase curriculum-based naloxone training program improved pharmacy student knowledge and confidence in furnishing naloxone under a state BOP protocol.

Exploring the therapeutic potential of Neem (Azadirachta Indica) for the treatment of prostate cancer: a literature review.

Background and Objective: Multiple studies have demonstrated the medical potency of plant extracts and specific phytochemicals as therapeutics for prostate cancer (PCa) patients. Of note, the Neem plant known for its role as an antibiotic and anti-inflammatory is underexplored with an untapped potential for further development. This review focuses on extracts and phytochemicals derived from the Neem tree (Latin name; Azadirachta indica), commonly used throughout Southeast Asia for the prevention and treatment of a wide array of diseases including cancer. To date, there are more than 130 biologically active compounds that have been isolated from the Neem tree including azadirachtin, nimbolinin, nimbin, nimbidin, nimbidol, which have demonstrated a wide range of biological activities including anti-microbial, anti-fertility, anti-inflammatory, anti-arthritic, hepatoprotective, anti-diabetic, anti-ulcer, and anti-cancer effects. Very few scientific reports focus on the benefits of Neem in PCa, even though this herb has been used to prevent the disease and its progression for years in complementary and alternative medicine. Methods: We used the search engines like PubMed, InCommon and Google using the key words: "Neem", "Cancer", "Prostate Cancer" and related words to find the information and data within the time frame from 1980-2022 for our article study. Key Content and Findings: Here, we provide an overview of Neem extracts and phytochemical derivatives with a focus on their known potential and ability to inhibit specific cellular signaling pathways and processes which drive PCa incidence and progression. Conclusions: The information presented here indicate that Neem and its derivatives have a therapeutic potential for the treatment of PCa when used as a single agent or in combination with conventional chemotherapeutics.

COVID-19-Driven Improvements and Innovations in Pharmacy Education: A Scoping Review.

The COVID-19 pandemic led to many colleges of pharmacy having to make major changes relating to their infrastructure and delivery of their curriculum within a very short time frame, including the transition of many components to an online setting. This scoping review sought to summarize what is known about the impact of COVID-19 on pharmacy education and the effectiveness of adaptation strategies which were put in place. PubMed, Web of Science, OVID Medline, and MedEdPortal were searched to identify pharmacy education-related articles published since the beginning of the COVID-19 pandemic. For article inclusion, the following criteria had to be met: described original research, related directly to PharmD or PharmBS education, related to the impact of COVID-19 on pharmacy education, and was available in English. Out of a total of 813 articles, 50 primary research articles were selected for inclusion. Our review of these identified four domains relating to the impact of COVID-19 on pharmacy education and/or effectiveness of adaptation strategies: (1) lab-based courses and activities (including interprofessional education activities), (2) experiential education, (3) didactic education, and (4) student well-being. The key research findings are summarized and discussed. While the COVID-19 pandemic has clearly brought many challenges to pharmacy education, it has also led to key improvements and innovations.

Use of RNA-Seq and a Transgenic Mouse Model to Identify Genes Which May Contribute to Mutant p53-Driven Prostate Cancer Initiation.

We previously demonstrated that the Trp53-R270H mutation can drive prostate cancer (CaP) initiation using the FVB.129S4 (Trp53tm3Tyj/wt); FVB.129S (Nkx3-1tm3(cre)Mmswt) genetically engineered mouse model (GEM). We now validate this finding in a different model (B6.129S4-Trp53tm3.1Tyj/J mice) and use RNA-sequencing (RNA-Seq) to identify genes which may contribute to Trp53 R270H-mediated prostate carcinogenesis. Wildtype (Trp53WT/WT), heterozygous (Trp53R270H/WT), and homozygous mice (Trp53R270H/R270H) were exposed to 5 Gy irradiation to activate and stabilize p53, and thereby enhance our ability to identify differences in transcriptional activity between the three groups of mice. Mouse prostates were harvested 6 h post-irradiation and processed for histological/immunohistochemistry (IHC) analysis or were snap-frozen for RNA extraction and transcriptome profiling. IHC analyses determined that presence of the Trp53-R270H mutation impacts apoptosis (lower caspase 3 activity) but not cell proliferation (Ki67). RNA-Seq analysis identified 1378 differentially expressed genes, including wildtype p53 target genes (E.g., Cdkn1a, Bax, Bcl2, Kras, Mdm2), p53 gain-of-function (GOF)-related genes (Mgmt, Id4), and CaP-related genes (Cav-1, Raf1, Kras). Further understanding the mechanisms which contribute to prostate carcinogenesis could allow for the development of improved preventive methods, diagnostics, and treatments for CaP.

Androgen Receptor-Mediated Nuclear Transport of NRDP1 in Prostate Cancer Cells Is Associated with Worse Patient Outcomes.

To our knowledge, our group is the first to demonstrate that NRDP1 is located in the nucleus as well as the cytoplasm of CaP cells. Subcellular fractionation, immunohistochemistry, and immunofluorescence analysis combined with confocal microscopy were used to validate this finding. Subcellular fractionation followed by western blot analysis revealed a strong association between AR and NRDP1 localization when AR expression and/or cellular localization was manipulated via treatment with R1881, AR-specific siRNA, or enzalutamide. Transfection of LNCaP with various NRDP1 and AR constructs followed by immunoprecipitation confirmed binding of NRDP1 to AR is possible and determined that binding requires the hinge region of AR. Co-transfection with NRDP1 constructs and HA-ubiquitin followed by subcellular fractionation confirmed that nuclear NRDP1 retains its ubiquitin ligase activity. We also show that increased nuclear NRDP1 is associated with PSA recurrence in CaP patients (n = 162, odds ratio; 1.238, p = 0.007) and that higher levels of nuclear NRDP1 are found in castration resistant cell lines (CWR22Rv1 and PC3) compared to androgen sensitive cell lines (LNCaP and MDA-PCa-3B). The combined data indicate that NRDP1 plays a role in mediating CaP progression and supports further investigation of both the mechanism by which nuclear transport occurs and the identification of specific nuclear targets.

Use of Team-Based Learning Pedagogy to Prepare for a Pharmacy School Accreditation Self-Study.

Ensuring adequate engagement and preparation of all stakeholders in an accreditation self-study can be challenging for many reasons, including lack of motivation and inadequate understanding of expectations and procedures. The goal of this exploratory study was to determine whether using team-based learning (TBL) pedagogy to deliver an accreditation preparation workshop could effectively prepare and engage participants. A Likert-scale questionnaire was administered to workshop attendees (n = 52) to determine whether they found TBL-based training helpful and whether it promoted engagement. Twenty-four attendees completed the survey (46%). More than 80% of participants strongly agreed or agreed with 12 statements relating to perceptions of self and participant engagement within team activities and the usefulness of team activities. More than 65% of participants strongly agreed or agreed with statements relating to the helpfulness of the TBL approach in preparing for the self-study (five questions). Subgroup analysis showed no significant difference in responses based on whether on not participants had previously been involved in an accreditation self study. Our data indicate that a TBL approach can be an effective way to engage and prepare stakeholders for an accreditation self-study, and that TBL pedagogy has utility outside of the classroom setting.

Use of Concept Mapping to Identify Expectations of Pharmacy Students Selecting Elective Courses.

The objective of this study was to demonstrate the use of concept mapping as a method for analyzing pharmacy students' qualitative perceptions of their expectations of elective courses and to thus help guide delivery methods and course content. A survey containing demographic, Likert scale, and open-ended questions was administered to second-year pharmacy students prior to the start of elective courses and an innovative methodology, concept mapping, was used to identify major themes relating to student expectations. The association between preferred class delivery method (online versus in person) with student gender and English-as-a-second-language status (ESL) was also assessed. Note that this study was conducted prior to the COVID-19 pandemic. Ninety-eight out of 133 students (74%) completed the survey. Overall, 56% students stated that they preferred online delivery of courses (68% of these students were female, 36% were male). ESL status did not impact preference. The most common themes relating to student course expectations were the desire to learn about the elective course topic as well "real-world" utility. Our combined data indicate that delivery method is a key factor contributing to students' choice of elective course and that concept mapping is an effective and efficient way to help identify student expectations of elective courses.

Use of End-of-Class Quizzes to Promote Pharmacy Student Self-Reflection, Motivate Students to Improve Study Habits, and to Improve Performance on Summative Examinations.

Underperforming students are often unaware of deficiencies requiring improvement until after poor performance on summative exams. The goal of the current study was to determine whether inclusion of individual end-of-class formative quizzes, which comprise of higher level Bloom's questions, could encourage students to reflect on and address deficiencies and improve academic performance. Ninety-seven out of 123 first-year pharmacy students (79%) enrolled in a Biochemistry and Cell & Molecular Biology course participated in a single-blinded, randomized, controlled, crossover study. Paired t-test analyses demonstrated that that implementation of individual end-of-class formative quizzes resulted in significantly higher summative exam scores for below average students (p = 0.029). Notably, inclusion of quizzes significantly improved performance on higher Bloom's questions for these students (p = 0.006). Analysis of surveys completed by students prior to summative exam indicate that the formative end-of-class quizzes helped students identify deficiencies (89%) and making them feel compelled to study more (83%) and attend review sessions (61%). Many students indicated that quizzes increased stress levels (45%). Our collective data indicate that quizzes can improve summative exam performance for below average first year pharmacy students, and improve self-reflection and student motivation to study. However, the impact on student stress levels should be considered.

Genistein Combined Polysaccharide (GCP) Can Inhibit Intracrine Androgen Synthesis in Prostate Cancer Cells.

Our group and others have previously shown that genistein combined polysaccharide (GCP), an aglycone isoflavone-rich extract with high bioavailability and low toxicity, can inhibit prostate cancer (CaP) cell growth and survival as well as androgen receptor (AR) activity. We now elucidate the mechanism by which this may occur using LNCaP and PC-346C CaP cell lines; GCP can inhibit intracrine androgen synthesis in CaP cells. UPLC-MS/MS and qPCR analyses demonstrated that GCP can mediate a ~3-fold decrease in testosterone levels (p < 0.001) and cause decreased expression of intracrine androgen synthesis pathway enzymes (~2.5-fold decrease of 3ßHSD (p < 0.001), 17ßHSD (p < 0.001), CYP17A (p < 0.01), SRB1 (p < 0.0001), and StAR (p < 0.01)), respectively. Reverse-phase HPLC fractionation and bioassay identified three active GCP fractions. Subsequent NMR and LC-MS analysis of the fraction with the highest level of activity, fraction 40, identified genistein as the primary active component of GCP responsible for its anti-proliferative, pro-apoptotic, and anti-AR activity. GCP, fraction 40, and genistein all mediated at least a ~2-fold change in these biological activities relative to vehicle control (p < 0.001). Genistein caused similar decreases in the expression of 17ßHSD and CYP17A (2.5-fold (p < 0.001) and 1.5-fold decrease (p < 0.01), respectively) compared to GCP, however it did not cause altered expression of the other intracrine androgen synthesis pathway enzymes; 3ßHSD, SRB1, and StAR. Our combined data indicate that GCP and/or genistein may have clinical utility and that further pre-clinical studies are warranted.

Impact of Completion of a Pre-Pharmacy Biochemistry Course and Competency Levels in Pre-Pharmacy Courses on Pharmacy Student Performance.

Poor performance in foundational science courses, which are usually taken during the first or second year of pharmacy school, can have several negative consequences including increases in student drop-out rates and increases in the number of dismissals and remediating students. The primary goal of the current study was to determine whether completion of a pre-pharmacy biochemistry course and/or performance on a biochemistry competency test (administered at the beginning of the pharmacy program) are associated with pharmacy student performance in foundational science courses and overall academic performance. A secondary goal was to determine whether performance in pre-pharmacy courses and/or student demographics are associated with pharmacy student performance. Prospective univariate analyses (n = 75) determined that completion of a pre-pharmacy biochemistry course is not associated with pharmacy student performance. However, performance on a biochemistry competency test was associated with performance in Biochemistry and Cell&Molecular Biology (p = 0.002). Furthermore, post-hoc analyses determined that pre-pharmacy cumulative chemistry GPA correlates with performance in both the Biochemistry and Cell&Molecular Biology and Medicinal Chemistry foundational science courses (p = 0.002 and p = 0.04, respectively) and can predict first year GPA (p = 0.002). The combined data indicate that further assessment of the impact of pre-pharmacy competency in biochemistry and chemistry on pharmacy student success is warranted.

Obatoclax, a BH3 Mimetic, Enhances Cisplatin-Induced Apoptosis and Decreases the Clonogenicity of Muscle Invasive Bladder Cancer Cells via Mechanisms That Involve the Inhibition of Pro-Survival Molecules as Well as Cell Cycle Regulators.

Several studies by our group and others have determined that expression levels of Bcl-2 and/or Bcl-xL, pro-survival molecules which are associated with chemoresistance, are elevated in patients with muscle invasive bladder cancer (MI-BC). The goal of this study was to determine whether combining Obatoclax, a BH3 mimetic which inhibits pro-survival Bcl-2 family members, can improve responses to cisplatin chemotherapy, the standard of care treatment for MI-BC. Three MI-BC cell lines (T24, TCCSuP, 5637) were treated with Obatoclax alone or in combination with cisplatin and/or pre-miR-34a, a molecule which we have previously shown to inhibit MI-BC cell proliferation via decreasing Cdk6 expression. Proliferation, clonogenic, and apoptosis assays confirmed that Obatoclax can decrease cell proliferation and promote apoptosis in a dose-dependent manner. Combination treatment experiments identified Obatoclax + cisplatin as the most effective treatment. Immunoprecipitation and Western analyses indicate that, in addition to being able to inhibit Bcl-2 and Bcl-xL, Obatoclax can also decrease cyclin D1 and Cdk4/6 expression levels. This has not previously been reported. The combined data demonstrate that Obatoclax can inhibit cell proliferation, promote apoptosis, and significantly enhance the effectiveness of cisplatin in MI-BC cells via mechanisms that likely involve the inhibition of both pro-survival molecules and cell cycle regulators.

XPO1 inhibition by selinexor induces potent cytotoxicity against high grade bladder malignancies.

Treatment options for high grade urothelial cancers are limited and have remained largely unchanged for several decades. Selinexor (KPT-330), a first in class small molecule that inhibits the nuclear export protein XPO1, has shown efficacy as a single agent treatment for numerous different malignancies, but its efficacy in limiting bladder malignancies has not been tested. In this study we assessed selinexor-dependent cytotoxicity in several bladder tumor cells and report that selinexor effectively reduced XPO1 expression and limited cell viability in a dose dependent manner. The decrease in cell viability was due to an induction of apoptosis and cell cycle arrest. These results were recapitulated in in vivo studies where selinexor decreased tumor growth. Tumors treated with selinexor expressed lower levels of XPO1, cyclin A, cyclin B, and CDK2 and increased levels of RB and CDK inhibitor p27, a result that is consistent with growth arrest. Cells expressing wildtype RB, a potent tumor suppressor that promotes growth arrest and apoptosis, were most susceptible to selinexor. Cell fractionation and immunofluorescence studies showed that selinexor treatment increased nuclear RB levels and mechanistic studies revealed that RB ablation curtailed the response to the drug. Conversely, limiting CDK4/6 dependent RB phosphorylation by palbociclib was additive with selinexor in reducing bladder tumor cell viability, confirming that RB activity has a role in the response to XPO1 inhibition. These results provide a rationale for XPO1 inhibition as a novel strategy for the treatment of bladder malignancies.

MicroRNA profiling of dogs with transitional cell carcinoma of the bladder using blood and urine samples.

BACKGROUND: Early signs of canine transitional cell carcinoma (TCC) are frequently assumed to be caused by other lower urinary tract diseases (LUTD) such as urinary tract infections, resulting in late diagnosis of TCC which could be fatal. The development of a non-invasive clinical test for TCC could dramatically reduce mortality. To determine whether microRNAs (miRNAs) can be used as non-invasive diagnostic biomarkers, we assessed miRNA expression in blood and/or urine from dogs with clinically normal bladders (n = 28), LUTD (n = 25), and TCC (n = 17). Expression levels of 5 miRNA associated with TCC pathophysiology (miR-34a, let-7c, miR-16, miR-103b, and miR-106b) were assessed by quantitative real-time PCR. RESULTS: Statistical analyses using ranked ANOVA identified significant differences in miR-103b and miR-16 levels between urine samples from LUTD and TCC patients (miR-103b, p = 0.002; and miR-16, p = 0.016). No statistically significant differences in miRNA levels were observed between blood samples from LUTD versus TCC patients. Expression levels of miR-34a trended with miR-16, let-7c, and miR-103b levels in individual normal urine samples, however, this coordination was completely lost in TCC urine samples. In contrast, co-ordination of miR-34a, miR-16, let-7c, and miR-103b expression levels was maintained in blood samples from TCC patients. CONCLUSIONS: Our combined data indicate a potential role for miR-103b and miR-16 as diagnostic urine biomarkers for TCC, and that further investigation of miR-103b and miR-16 in the dysregulation of coordinated miRNA expression in bladder carcinogenesis is warranted.

Decreased expression of let-7c is associated with non-response of muscle-invasive bladder cancer patients to neoadjuvant chemotherapy.

The identification and development of biomarkers which predict response of muscle invasive bladder cancer (MIBC) patients to neoadjuvant chemotherapy would likely increase usage of this treatment option and thereby improve patient survival rates. MiRNA array and qRT-PCR validation was used to identify miRNA which are associated with response to neoadjuvant chemotherapy. RNA was extracted from a total of 41 archival, fully annotated, MIBC patient diagnostic biopsies (20 chemo-responders and 21 non-responders (response is defined as > 5 year survival rate and being pT0 post-chemotherapy)). Microarray and qPCR identified let-7c as being differentially expressed in chemo-responder versus non-responder patients. Patients with higher let-7c expression levels had significantly higher odds of responding to chemotherapy (p = 0.023, OR 2.493, 95% CI 1.121, 5.546), and assessment of let-7c levels allowed for prediction of patient response (AUC 0.72, positive predictive value 59%). Decreased let-7c was associated with MIBC incidence (p < 0.001), and significantly correlated with other related miRNA including those that were not differentially expressed between responders and non-responders. The combined data indicate let-7c plays a role in mediating chemoresistance to neoadjuvant chemotherapy in MIBC patients, and is a modest, yet clinically meaningful, predictor of patient response.

Molecular Dissection of Induced Platinum Resistance through Functional and Gene Expression Analysis in a Cell Culture Model of Bladder Cancer.

We report herein the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formation and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.

The role of EGFR family inhibitors in muscle invasive bladder cancer: a review of clinical data and molecular evidence.

PURPOSE: Conventional platinum based chemotherapy for advanced urothelial carcinoma is plagued by common resistance to this regimen. Several studies implicate the EGFR family of RTKs in urothelial carcinoma progression and chemoresistance. Many groups have investigated the effects of inhibitors of this family in patients with urothelial carcinoma. This review focuses on the underlying molecular pathways that lead to urothelial carcinoma resistance to EGFR family inhibitors. MATERIALS AND METHODS: We performed a PubMed® search for peer reviewed literature on bladder cancer development, EGFR family expression, clinical trials of EGFR family inhibitors and molecular bypass pathways. Research articles deemed to be relevant were examined and a summary of original data was created. Meta-analysis of expression profiles was also performed for each EGFR family member based on data sets accessible via Oncomine®. RESULTS: Many clinical trials using inhibitors of EGFR family RTKs have been done or are under way. Those that have concluded with results published to date do not show an added benefit over standard of care chemotherapy in an adjuvant or second line setting. However, a neoadjuvant study using erlotinib before radical cystectomy demonstrated promising results. CONCLUSIONS: Clinical and preclinical studies show that for reasons not currently clear prior treatment with chemotherapeutic agents rendered patients with urothelial carcinoma with muscle invasive bladder cancer resistant to EGFR family inhibitors as well. However, EGFR family inhibitors may be of use in patients with no prior chemotherapy in whom EGFR or ERBB2 is over expressed.

Enhancing the effectiveness of androgen deprivation in prostate cancer by inducing Filamin A nuclear localization.

As prostate cancer (CaP) is regulated by androgen receptor (AR) activity, metastatic CaP is treated with androgen deprivation therapy (ADT). Despite initial response, patients on ADT eventually progress to castration-resistant CaP (CRPC), which is currently incurable. We previously showed that cleavage of the 280 kDa structural protein Filamin A (FlnA) to a 90 kDa fragment, and nuclear localization of the cleaved product, sensitized CRPC cells to ADT. Hence, treatment promoting FlnA nuclear localization would enhance androgen responsiveness. Here, we show that FlnA nuclear localization induced apoptosis in CRPC cells during ADT, identifying it as a treatment tool in advanced CaP. Significantly, the natural product genistein combined polysaccharide (GCP) had a similar effect. Investigation of the mechanism of GCP-induced apoptosis showed that GCP induced FlnA cleavage and nuclear localization and that apoptosis resulting from GCP treatment was mediated by FlnA nuclear localization. Two main components of GCP are genistein and daidzein: the ability of GCP to induce G2 arrest was due to genistein whereas sensitivity to ADT stemmed from daidzein; hence, both were needed to mediate GCP's effects. FlnA cleavage is regulated by its phosphorylation; we show that ADT enhanced FlnA phosphorylation, which prevented its cleavage, whereas GCP inhibited FlnA phosphorylation, thereby sensitizing CaP cells to ADT. In a mouse model of CaP recurrence, GCP, but not vehicle, impeded relapse following castration, indicating that GCP, when administered with ADT, interrupted the development of CRPC. These results demonstrate the efficacy of GCP in promoting FlnA nuclear localization and enhancing androgen responsiveness in CaP.