Physician stress in the era of COVID-19 vaccine disparity: a multi-institutional survey.

CONTEXT: Healthcare workers are at a high risk of infection during infectious disease outbreaks, such as the COVID-19 pandemic. Despite the availability of several vaccines against COVID-19, the absence of vaccination in patients and colleagues remains a continuous source of stress in healthcare workers. We conducted a survey of physician preceptors, both MDs and DOs, to explore the impact of differences in the patients' and colleagues' vaccination status on their well-being, stress, and burnout. OBJECTIVES: The objective of this study is to determine whether exposure to unvaccinated patients and/or colleagues increases stress and burnout in physician preceptors by utilizing a self-reported survey. METHODS: This multi-institutional study was carried out in the United States in 2022. An online survey questionnaire was utilized to collect data from physicians working as preceptors for multiple academic institutions. The anonymous Qualtrics® survey utilized a modified version of the questionnaire from the expanded Physician Well-being Index (ePWBI) designed by MedEd Web Solutions (MEWS). Statistical analysis on both descriptive and qualitative data were performed. Utilizing a threshold of p≤0.05, data analysis revealed many statistically significant relationships between the variables. RESULTS: A total of 218 physician preceptors completed the survey. The survey results showed that physicians overwhelmingly (p < 0.001) felt that all patients (and healthcare workers) should be vaccinated. The results also indicated that physicians experienced more stress when working with unvaccinated patients (p<0.001), and these stressors were often associated with the physician's gender and age. Furthermore, physicians stated that both their assessment and treatment plans were significantly different for vaccinated vs unvaccinated patients (p=0.039 and p=0.0167, respectively). Most importantly, stress levels (p<0.001) and burnout characteristics (p=0.024) were noted by physicians, both in themselves and in their colleagues. CONCLUSIONS: Findings suggest that physician stress and burnout is a common theme due to the differences in vaccination status of patients admitted to COVID-19 clinics. Due to a more rapid progression of COVID-19 in unvaccinated patients, treatment plans for vaccinated vs unvaccinated patients were also considerably different.

Osteopathic Manipulative Treatment Decreases Hospital Stay and Healthcare Cost in the Neonatal Intensive Care Unit.

Osteopathic manipulative treatment (OMT) is used in both inpatient and outpatient settings. Evidence suggests that OMT can reduce both patients' recovery time and the financial cost of their acute medical treatment and rehabilitation. Multiple studies from neonatal intensive care units (NICUs) are presented in this article that demonstrate infants treated with OMT recover faster, are discharged earlier, and have lower healthcare costs than their non-OMT-treated counterparts. Data clearly show that adjunctive OMT facilitates feeding coordination in newborns, such as latching, suckling, swallowing, and breathing, and increases long-term weight gain and maintenance, which reduces hospital length of stay (LOS). Osteopathic techniques, such as soft tissue manipulation, balanced ligamentous tension, myofascial release, and osteopathic cranial manipulation (OCM), can reduce regurgitation, vomiting, milky bilious, or bloody discharge and decrease the need for constipation treatment. OMT can also be effective in reducing the complications of pneumonia in premature babies. Studies show the use of OCM and lymphatic pump technique (LPT) reduces the occurrence of both aspiration and environmentally acquired pneumonia, resulting in significantly lower morbidity and mortality in infants. Based on published findings, it is determined that OMT is clinically effective, cost efficient, a less invasive alternative to surgery, and a less toxic choice to pharmacologic drugs. Therefore, routine incorporation of OMT in the NICU can be of great benefit in infants with multiple disorders. Future OMT research should aim to initiate clinical trial designs that include randomized controlled trials with larger cohorts of infants admitted to the NICU. Furthermore, a streamlined and concerted effort to elucidate the underlying molecular mechanisms associated with the beneficial effects of OMT will aid in understanding the significant value of incorporating OMT into optimal patient care.

Osteopathic Manipulative Medicine: A Brief Review of the Hands-On Treatment Approaches and Their Therapeutic Uses.

Osteopathic manipulative medicine (OMM) is an emerging practice in the healthcare field with increasing popularity and evidence-based therapy. Osteopathic manipulative treatments (OMT) include hands-on manipulations of different body structures to increase systemic homeostasis and total patient well-being. Indeed, this new realm of the whole patient-based approach is being taught in osteopathic schools around the country, and the osteopathic principles of a mind-body-spirit-based treatment are being instilled in many new Doctor of Osteopathy (D.O.) students. However, despite their proven therapeutic value, there are still many individuals, both in and outside the medical profession, who are unaware (or misinformed) of the therapeutic uses and potential benefits of OMT. Here, we provide a brief introduction to this osteopathic therapeutic approach, focusing on the hands-on techniques that are regularly implemented in the clinical setting. It is becoming increasingly evident that different OMTs can be implemented to enhance patient recovery, both alone and in conjunction with the targeted therapies used in allopathic regimens. Therefore, it may be beneficial to inform the general medical community and educate the public and those associated with the healthcare field about the benefits of using OMT as a treatment modality. OMT is lower-cost, noninvasive, and highly effective in promoting full-body healing by targeting the nervous, lymphatic, immune, and vascular systems. There is a growing body of literature related to osteopathic research and the possible molecular pathways involved in the healing process, and this burgeoning field of medicine is expected to increase in value in the healthcare field. This brief review article explains the frequently utilized OMT modalities and their recognized therapeutic benefits, which underscore the need to understand the possible molecular mechanisms and circulating biomarkers linked to the systemic benefits of osteopathic medicine.

Pre-Exposure to Stress-Inducing Agents Increase the Anticancer Efficacy of Focused Ultrasound against Aggressive Prostate Cancer Cells.

Despite the initial success in treatment of localized prostate cancer (PCa) using surgery, radiation or hormonal therapy, recurrence of aggressive tumors dictates morbidity and mortality. Focused ultrasound (FUS) is being tested as a targeted, noninvasive approach to eliminate the localized PCa foci, and strategies to enhance the anticancer potential of FUS have a high translational value. Since aggressive cancer cells utilize oxidative stress (Ox-stress) and endoplasmic reticulum stress (ER-stress) pathways for their survival and recurrence, we hypothesized that pre-treatment with drugs that disrupt stress-signaling pathways in tumor cells may increase FUS efficacy. Using four different PCa cell lines, i.e., LNCaP, C4-2B, 22Rv1 and DU145, we tested the in vitro effects of FUS, alone and in combination with two clinically tested drugs that increase Ox-stress (i.e., CDDO-me) or ER-stress (i.e., nelfinavir). As compared to standalone FUS, significant (p < 0.05) suppressions in both survival and recurrence of PCa cells were observed following pre-sensitization with low-dose CDDO-me (100 nM) and/or nelfinavir (2 µM). In drug pre-sensitized cells, significant anticancer effects were evident at a FUS intensity of as low as 0.7 kW/cm2. This combined mechanochemical disruption (MCD) approach decreased cell proliferation, migration and clonogenic ability and increased apoptosis/necrosis and reactive oxygen species (ROS) production. Furthermore, although activated in cells that survived standalone FUS, pre-sensitization with CDDO-me and/or nelfinavir suppressed both total and activated (phosphorylated) NF-κB and Akt protein levels. Thus, a combined MCD therapy may be a safe and effective approach towards the targeted elimination of aggressive PCa cells.

Latent HIV-Exosomes Induce Mitochondrial Hyperfusion Due to Loss of Phosphorylated Dynamin-Related Protein 1 in Brain Endothelium.

Damage to the cerebral vascular endothelium is a critical initiating event in the development of HIV-1-associated neurocognitive disorders. To study the role of mitochondria in cerebral endothelial dysfunction, we investigated how exosomes, isolated from both cell lines with integrated provirus and HIV-1 infected primary cells (HIV-exosomes), accelerate the dysfunction of primary human brain microvascular endothelial cells (HBMVECs) by inducing mitochondrial hyperfusion, and reducing the expression of phosphorylated endothelial nitric oxide synthase (p-eNOS). The quantitative analysis of the extracellular vesicles (EVs) indicates that the isolated EVs were predominantly exosomes. It was further supported by the detection of exosomal markers, and the absence of large EV-related protein in the isolated EVs. The exosomes were readily taken up by primary HBMVECs. HIV-exosomes induce cellular and mitochondrial superoxide production but reduce mitochondrial membrane potential in HBMVECs. HIV-exosomes increase mitochondrial hyperfusion, possibly due to loss of phosphorylated dynamin-related protein 1 (p-DRP1). HIV-exosomes, containing the HIV-Tat protein, and viral Tat protein reduce the expression of p-DRP1 and p-eNOS, and accelerate brain endothelial dysfunction. Finally, exosomes isolated from HIV-1 infected primary human peripheral blood mononuclear cells (hPBMCs) produce more exosomes than uninfected controls and reduce both p-DRP1 and p-eNOS expressions in primary HBMVECs. Our novel findings reveal the significant role of HIV-exosomes on dysregulation of mitochondrial function, which induces adverse changes in the function of the brain microvascular endothelium.

Oxidative stress and redox signaling in CRPC progression: therapeutic potential of clinically-tested Nrf2-activators.

Androgen deprivation therapy (ADT) is the mainstay regimen in patients with androgen-dependent prostate cancer (PCa). However, the selection of androgen-independent cancer cells leads to castrate resistant prostate cancer (CRPC). The aggressive phenotype of CRPC cells underscores the need to elucidate mechanisms and therapeutic strategies to suppress CRPC outgrowth. Despite ADT, the activation of androgen receptor (AR) transcription factor continues via crosstalk with parallel signaling pathways. Understanding of how these signaling cascades are initiated and amplified post-ADT is lacking. Hormone deprivation can increase oxidative stress and the resultant reactive oxygen species (ROS) may activate both AR and non-AR signaling. Moreover, ROS-induced inflammatory cytokines may further amplify these redox signaling pathways to augment AR function. However, clinical trials using ROS quenching small molecule antioxidants have not suppressed CRPC progression, suggesting that more potent and persistent suppression of redox signaling in CRPC cells will be needed. The transcription factor Nrf2 increases the expression of numerous antioxidant enzymes and downregulates the function of inflammatory transcription factors, e.g., nuclear factor kappa B. We documented that Nrf2 overexpression can suppress AR-mediated transcription in CRPC cell lines. Furthermore, two Nrf2 activating agents, sulforaphane (a phytochemical) and bardoxolone-methyl (a drug in clinical trial) suppress AR levels and sensitize CRPC cells to anti-androgens. These observations implicate the benefits of potent Nrf2-activators to suppress the lethal signaling cascades that lead to CRPC outgrowth. This review article will address the redox signaling networks that augment AR signaling during PCa progression to CRPC, and the possible utility of Nrf2-activating agents as an adjunct to ADT.

A New Humanized Mouse Model Mimics Humans in Lacking α-Gal Epitopes and Secreting Anti-Gal Antibodies.

Mice have been used as accepted tools for investigating complex human diseases and new drug therapies because of their shared genetics and anatomical characteristics with humans. However, the tissues in mice are different from humans in that human cells have a natural mutation in the α1,3 galactosyltransferase (α1,3GT) gene and lack α-Gal epitopes on glycosylated proteins, whereas mice and other nonprimate mammals express this epitope. The lack of α-Gal epitopes in humans results in the loss of immune tolerance to this epitope and production of abundant natural anti-Gal Abs. These natural anti-Gal Abs can be used as an adjuvant to enhance processing of vaccine epitopes to APCs. However, wild-type mice and all existing humanized mouse models cannot be used to test the efficacy of vaccines expressing α-Gal epitopes because they express α-Gal epitopes and lack anti-Gal Abs. Therefore, in an effort to bridge the gap between the mouse models and humans, we developed a new humanized mouse model that mimics humans in that it lacks α-Gal epitopes and secretes human anti-Gal Abs. The new humanized mouse model (Hu-NSG/α-Galnull) is designed to be used for preclinical evaluations of viral and tumor vaccines based on α-Gal epitopes, human-specific immune responses, xenotransplantation studies, and in vivo biomaterials evaluation. To our knowledge, our new Hu-NSG/α-Galnull is the first available humanized mouse model with such features.

Bardoxolone-Methyl (CDDO-Me) Suppresses Androgen Receptor and Its Splice-Variant AR-V7 and Enhances Efficacy of Enzalutamide in Prostate Cancer Cells.

Androgen receptor (AR) signaling is fundamental to prostate cancer (PC) progression, and hence, androgen deprivation therapy (ADT) remains a mainstay of treatment. However, augmented AR signaling via both full length AR (AR-FL) and constitutively active AR splice variants, especially AR-V7, is associated with the recurrence of castration resistant prostate cancer (CRPC). Oxidative stress also plays a crucial role in anti-androgen resistance and CRPC outgrowth. We examined whether a triterpenoid antioxidant drug, Bardoxolone-methyl, known as CDDO-Me or RTA 402, can decrease AR-FL and AR-V7 expression in PC cells. Nanomolar (nM) concentrations of CDDO-Me rapidly downregulated AR-FL in LNCaP and C4-2B cells, and both AR-FL and AR-V7 in CWR22Rv1 (22Rv1) cells. The AR-suppressive effect of CDDO-Me was evident at both the mRNA and protein levels. Mechanistically, acute exposure (2 h) to CDDO-Me increased and long-term exposure (24 h) decreased reactive oxygen species (ROS) levels in cells. This was concomitant with an increase in the anti-oxidant transcription factor, Nrf2. The anti-oxidant N-acetyl cysteine (NAC) could overcome this AR-suppressive effect of CDDO-Me. Co-exposure of PC cells to CDDO-Me enhanced the efficacy of a clinically approved anti-androgen, enzalutamide (ENZ), as evident by decreased cell-viability along with migration and colony forming ability of PC cells. Thus, CDDO-Me which is in several late-stage clinical trials, may be used as an adjunct to ADT in PC patients.

The Membrane-Active Phytopeptide Cycloviolacin O2 Simultaneously Targets HIV-1-infected Cells and Infectious Viral Particles to Potentiate the Efficacy of Antiretroviral Drugs.

Background: Novel strategies to increase the efficacy of antiretroviral (ARV) drugs will be of crucial importance. We hypothesize that membranes of HIV-1-infected cells and enveloped HIV-1 particles may be preferentially targeted by the phytopeptide, cycloviolacin O2 (CyO2) to significantly enhance ARV efficacy. Methods: Physiologically safe concentrations of CyO2 were determined via red blood cell (RBC) hemolysis. SYTOX-green dye-uptake and radiolabeled saquinavir (³H-SQV) uptake assays were used to measure pore-formation and drug uptake, respectively. ELISA, reporter assays and ultracentrifugation were conducted to analyze the antiviral efficacy of HIV-1 protease and fusion inhibitors alone and co-exposed to CyO2. Results: CyO2 concentrations below 0.5 µM did not show substantial hemolytic activity, yet these concentrations enabled rapid pore-formation in HIV-infected T-cells and monocytes and increased drug uptake. ELISA for HIV-1 p24 indicated that CyO2 enhances the antiviral efficacy of both SQV and nelfinavir. CyO2 (< 0.5 µM) alone decreases HIV-1 p24 production, but it did not affect the transcription regulatory function of the HIV-1 long terminal repeat (LTR). Ultracentrifugation studies clearly showed that CyO2 exposure disrupted viral integrity and decreased the p24 content of viral particles. Furthermore, direct HIV-1 inactivation by CyO2 enhanced the efficacy of enfuvirtide. Conclusions: The membrane-active properties of CyO2 may help suppress viral load and augment antiretroviral drug efficacy.

Mesenchymal stem cells are attracted to latent HIV-1-infected cells and enable virus reactivation via a non-canonical PI3K-NFκB signaling pathway.

Persistence of latent HIV-1 in macrophages (MACs) and T-helper lymphocytes (THLs) remain a major therapeutic challenge. Currently available latency reversing agents (LRAs) are not very effective in vivo. Therefore, understanding of physiologic mechanisms that dictate HIV-1 latency/reactivation in reservoirs is clearly needed. Mesenchymal stromal/stem cells (MSCs) regulate the function of immune cells; however, their role in regulating virus production from latently-infected MACs & THLs is not known. We documented that exposure to MSCs or their conditioned media (MSC-CM) rapidly increased HIV-1 p24 production from the latently-infected U1 (MAC) & ACH2 (THL) cell lines. Exposure to MSCs also increased HIV-1 long terminal repeat (LTR) directed gene expression in the MAC and THL reporter lines, U937-VRX and J-Lat (9.2), respectively. MSCs exposed to CM from U1 cells (U1-CM) showed enhanced migratory ability towards latently-infected cells and retained their latency-reactivation potential. Molecular studies showed that MSC-mediated latency-reactivation was dependent upon both the phosphatidyl inositol-3-kinase (PI3K) and nuclear factor-κB (NFκB) signaling pathways. The pre-clinically tested inhibitors of PI3K (PX-866) and NFκB (CDDO-Me) suppressed MSC-mediated HIV-1 reactivation. Furthermore, coexposure to MSC-CM enhanced the latency-reactivation efficacy of the approved LRAs, vorinostat and panobinostat. Our findings on MSC-mediated latency-reactivation may provide novel strategies against persistent HIV-1 reservoirs.

High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer.

Targeting exosome biogenesis and release may have potential clinical implications for cancer therapy. Herein, we have optimized a quantitative high throughput screen (qHTS) assay to identify compounds that modulate exosome biogenesis and/or release by aggressive prostate cancer (PCa) CD63-GFP-expressing C4-2B cells. A total of 4,580 compounds were screened from the LOPAC library (a collection of 1,280 pharmacologically active compounds) and the NPC library (NCGC collection of 3,300 compounds approved for clinical use). Twenty-two compounds were found to be either potent activators or inhibitors of intracellular GFP signal in the CD63-GFP-expressing C4-2B cells. The activity of lead compounds in modulating the secretion of exosomes was validated by a tunable resistive pulse sensing (TRPS) system (qNano-IZON) and flow cytometry. The mechanism of action of the lead compounds in modulating exosome biogenesis and/or secretion were delineated by immunoblot analysis of protein markers of the endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways. The lead compounds tipifarnib, neticonazole, climbazole, ketoconazole, and triademenol were validated as potent inhibitors and sitafloxacin, forskolin, SB218795, fenoterol, nitrefazole and pentetrazol as activators of exosome biogenesis and/or secretion in PC cells. Our findings implicate the potential utility of drug-repurposing as novel adjunct therapeutic strategies in advanced cancer.

The Antiretroviral Agent Nelfinavir Mesylate: A Potential Therapy for Systemic Sclerosis.

OBJECTIVE: Transforming growth factor ß1 (TGFß1) is considered a key factor in fibrogenesis, and blocking TGFß1 signaling pathways diminishes fibrogenesis in animal models. The objective of this study was to determine whether nelfinavir mesylate (NFV), a drug approved by the Food and Drug Administration (FDA) for treating HIV infection, could be repurposed to treat pulmonary fibrosis in patients with systemic sclerosis (SSc). METHODS: Normal human lung, ventricular, and skin fibroblasts as well as lung fibroblasts from SSc patients were used to determine the effects of NFV on fibroblast-to-myofibroblast differentiation mediated by TGFß1. The efficacy of NFV was also evaluated in an animal model of SSc (bleomycin-induced pulmonary fibrosis). In addition, in silico analysis was performed to determine novel off-target effects of NFV. RESULTS: NFV inhibited TGFß1-mediated fibroblast-to-myofibroblast differentiation in lung fibroblasts through inhibition of the TGFß1 canonical pathway. NFV also inhibited differentiation of skin and ventricular fibroblasts and adipocyte precursors into myofibroblasts. Activation of the TGFß1/mechanistic target of rapamycin pathway inhibited autophagy in lung fibroblasts, favoring collagen deposition, and NFV counteracted this effect in a dose-dependent manner. Moreover, NFV significantly reduced lung injury and collagen deposition in an animal model of SSc. In silico analysis of NFV binding proteins revealed new putative beneficial mechanisms of action, consistent with known common pathways in fibrogenesis. CONCLUSION: NFV abrogates TGFß1-mediated fibroblast-to-myofibroblast differentiation and pulmonary fibrosis through off-target protein binding, a finding that supports consideration of this FDA-approved medication as an antifibrotic agent.

PRL­3 increases the aggressive phenotype of prostate cancer cells in vitro and its expression correlates with high-grade prostate tumors in patients.

The increased expression of phosphatase of regenerating liver-3 (PRL­3) has been shown to be associated with the aggressive and metastatic phenotype of different solid tumors. However, it is not known whether PRL­3 plays a similar role in the progression of prostate cancer (PCa). In this study, immunoblot analysis of androgen receptor (AR)-positive PCa lines (LNCaP and LNCaP­SF) revealed the constitutive cytoplasmic expression of PRL­3, and stimulation with R1881 (AR agonist) rapidly increased the nuclear translocation of PRL­3. The AR-negative cell lines exhibited negligible PRL­3 expression, and the ectopic overexpression of PRL­3 increased both the proliferative and invasive potential of PC3 and DU145 cells. In addition, we measured PRL­3 protein expression in human prostate tumor sections. A high-density prostate tumor microarray (TMA) was immunostained to assess whether PRL­3 expression and its subcellular localization (cytoplasmic and nuclear levels) is associated with the Gleason score (GS), Gleason grade (GG) and tumor stage (T-stage). Digital image analysis (DIA) revealed that PRL­3 expression was significantly higher in the malignant cores, as compared to the non­malignant areas. Increases in both total and nuclear PRL­3 levels were also associated with a higher GS and GG. Metastatic tumors (T4­stage) had lower cytoplasmic, but higher nuclear PRL­3 levels. Furthermore, the nuclear/cytoplasmic ratio for PRL­3 in the tumors graded as GS7 could effectively distinguish between indolent (3+4) and aggressive (4+3) disease. Thus, our experiments using PCa lines suggested that PRL­3 is an AR-regulated gene and its androgen-induced nuclear localization may increase the aggressive behavior of PCa cells. Furthermore, the digital analysis of immunostained tumor sections suggested that PRL­3 may be an effective biomarker of high-grade PCa, and its nuclear/cytoplasmic ratio may be used to distinguish between indolent vs. aggressive tumors.

Estradiol-ERß2 signaling axis confers growth and migration of CRPC cells through TMPRSS2-ETV5 gene fusion.

Estrogen receptor beta (ERß) splice variants are implicated in prostate cancer (PC) progression; however their underlying mechanisms remain elusive. We report that non-canonical activation of estradiol (E2)-ERß2 signaling axis primes growth, colony-forming ability and migration of the androgen receptor (AR)-null castration-resistant PC (CRPC) cells under androgen-deprived conditions (ADC). The non-classical E2-ERß2 mediates phosphorylation and activation of Src-IGF-1R complex, which in turn triggers p65-dependent transcriptional upregulation of the androgen-regulated serine protease TMPRSS2:ETV5a/TMPRSS2:ETV5b gene fusions under ADC. siRNA silencing of TMPRSS2 and/or ETV5 suggests that TMPRSS2:ETV5 fusions facilitates the E2-ERß induced growth and migration effects via NF-κB-dependent induction of cyclin D1 and MMP2 and MMP9 in PC-3 cells. Collectively, our results unravel the functional significance of oncogenic TMPRSS2:ETV5 fusions in mediating growth and migration of E2-ERß2 signaling axis in CRPC cells. E2-ERß2 signaling axis may have significant therapeutic and prognostic implications in patients with CRPC.

Multimodal actions of the phytochemical sulforaphane suppress both AR and AR-V7 in 22Rv1 cells: Advocating a potent pharmaceutical combination against castration-resistant prostate cancer.

Prostate cancer (PCa) cells expressing full-length androgen receptor (AR-FL) are susceptible to androgen deprivation therapy (ADT). However, outgrowth of castration-resistant prostate cancer (CRPC) can occur due to the expression of constitutively active (ligand-independent) AR splice variants, particularly AR-V7. We previously demonstrated that sulforaphane (SFN), an isothiocyanate phytochemical, can decrease AR-FL levels in the PCa cell lines, LNCaP and C4-2B. Here, we examined the efficacy of SFN in targeting both AR-FL and AR-V7 in the CRPC cell line, CWR22Rv1 (22Rv1). MTT cell viability, wound-heal assay, and colony forming unit (CFU) measurements revealed that 22Rv1 cells are resistant to the anti-androgen, enzalutamide (ENZ). However, co-exposure to SFN sensitized these cells to the potent anticancer effects of ENZ (P<0.05). Immunoblot analyses showed that SFN (5-20 µM) rapidly decreases both AR-FL and AR-V7 levels, and immunofluorescence microscopy (IFM) depicted decreased AR in both cytoplasm and nucleus with SFN treatment. SFN increased both ubiquitination and proteasomal activity in 22Rv1 cells. Studies using a protein synthesis inhibitor (cycloheximide) or a proteasomal inhibitor (MG132) indicated that SFN increases both ubiquitin-mediated aggregation and subsequent proteasomal-degradation of AR proteins. Previous studies reported that SFN inhibits the chaperone activity of heat-shock protein 90 (Hsp90) and induces the nuclear factor erythroid-2-like 2 (Nrf2) transcription factor. Therefore, we investigated whether the Hsp90 inhibitor, ganetespib (G) or the Nrf2 activator, bardoxolone methyl (BM) can similarly suppress AR levels in 22Rv1 cells. Low doses of G and BM, alone or in combination, decreased both AR-FL and AR-V7 levels, and combined exposure to G+BM sensitized 22Rv1 cells to ENZ. Therefore, adjunct treatment with the phytochemical SFN or a safe pharmaceutical combination of G+BM may be effective against CRPC cells, especially those expressing AR-V7.

Manumycin A suppresses exosome biogenesis and secretion via targeted inhibition of Ras/Raf/ERK1/2 signaling and hnRNP H1 in castration-resistant prostate cancer cells.

Emerging evidence links exosomes to cancer progression by the trafficking of oncogenic factors and neoplastic reprogramming of stem cells. This necessitates identification and integration of functionally validated exosome-targeting therapeutics into current cancer management regimens. We employed quantitative high throughput screen on two libraries to identify exosome-targeting drugs; a commercially available collection of 1280 pharmacologically active compounds and a collection of 3300 clinically approved compounds. Manumycin-A (MA), a natural microbial metabolite, was identified as an inhibitor of exosome biogenesis and secretion by castration-resistant prostate cancer (CRPC) C4-2B, but not the normal RWPE-1, cells. While no effect was observed on cell growth, MA attenuated ESCRT-0 proteins Hrs, ALIX and Rab27a and exosome biogenesis and secretion by CRPC cells. The MA inhibitory effect is primarily mediated via targeted inhibition of the Ras/Raf/ERK1/2 signaling. The Ras-dependent MA suppression of exosome biogenesis and secretion is partly mediated by ERK-dependent inhibition of the oncogenic splicing factor hnRNP H1. Our findings suggest that MA is a potential drug candidate to suppress exosome biogenesis and secretion by CRPC cells.

An Ex Vivo Tissue Culture Model for Anti-angiogenic Drug Testing.

Angiogenesis, defined as the growth of new blood vessels from existing ones, plays a key role in development, growth, and tissue repair. Its necessary role in tumor growth and metastasis has led to the creation of a new category of anti-angiogenic cancer therapies. Preclinical development and evaluation of potential drug candidates require models that mimic real microvascular networks. Here, we describe the rat mesentery culture model as a simple ex vivo assay that offers time-lapse imaging of intact microvascular network remodeling and demonstrate its application for anti-angiogenic drug testing.

Sulforaphane increases the efficacy of anti-androgens by rapidly decreasing androgen receptor levels in prostate cancer cells.

Prostate cancer (PCa) cells utilize androgen for their growth. Hence, androgen deprivation therapy (ADT) using anti-androgens, e.g. bicalutamide (BIC) and enzalutamide (ENZ), is a mainstay of treatment. However, the outgrowth of castration resistant PCa (CRPC) cells remains a significant problem. These CRPC cells express androgen receptor (AR) and utilize the intratumoral androgen towards their continued growth and invasion. Sulforaphane (SFN), a naturally occurring isothiocyanate found in cruciferous vegetables, can decrease AR protein levels. In the present study, we tested the combined efficacy of anti-androgens and SFN in suppressing PCa cell growth, motility and clonogenic ability. Both androgen-dependent (LNCaP) and androgen-independent (C4-2B) cells were used to monitor the effects of BIC and ENZ, alone and in combination with SFN. Co-exposure to SFN significantly (p<0.005) enhanced the anti-proliferative effects of anti-androgens and downregulated expression of the AR-responsive gene, prostate specific antigen (PSA) (p<0.05). Exposure to SFN decreased AR protein levels in a time- and dose-dependent manner with almost no AR detected at 24 h with 15 µM SFN (p<0.005). This rapid and potent AR suppression by SFN occurred by both AR protein degradation, as suggested by cycloheximide (CHX) co-exposure studies, and by suppression of AR gene expression, as evident from quantitative RT-PCR experiments. Pre-exposure to SFN also reduced R1881-stimulated nuclear localization of AR, and combined treatment with SFN and anti-androgens abrogated the mitogenic effects of this AR-agonist (p<0.005). Wound-healing assays revealed that co-exposure to SFN and anti-androgens can significantly (p<0.005) reduce PCa cell migration. In addition, long-term exposures (14 days) to much lower concentrations of these agents, SFN (0.2 µM), BIC (1 µM) and/or ENZ (0.4 µM) significantly (p<0.005) decreased the number of colony forming units (CFUs). These findings clearly suggest that SFN may be used as a promising adjunct agent to augment the efficacy of anti-androgens against aggressive PCa cells.

Tripping on TRIB3 at the junction of health, metabolic dysfunction and cancer.

Metabolic diseases like obesity, atherosclerosis and diabetes are frequently associated with increased risk of aggressive cancers. Although metabolic dysfunctions in normal cells are manifested due to defective signaling networks that control cellular homeostasis, malignant cells utilize these signaling networks for their increased survival, growth and metastasis. Despite decades of research, a common mechanistic link between these chronic pathologies is still not well delineated. Evidences show that the unfolded protein response (UPR) and the endoplasmic reticulum stress (ERS) pathways are often dysregulated in both metabolic diseases and cancer. The UPR also triggers coordinated signaling with both PI3K/AKT/mTOR and Autophagy pathways in order to promote stress-adaptive mechanisms. Whereas, uncontrolled UPR and the resultant ERS escalates cells towards metabolic dysfunctions and ultimately cell death. In this review, we will discuss findings that implicate a crucial role for the multifunctional ERS-induced protein, TRIB3. The 'pseudokinase' function of TRIB3 facilitates the inactivation of multiple transcription factors and signaling proteins. The MEK1 binding domain of TRIB3 enables it to deactivate multiple MAP-kinases. In addition, the COP1 motif of TRIB3 assists ubiquitination and proteasomal degradation of numerous TRIB3 associated proteins. The most well studied action of TRIB3 has been on the PI3K/AKT/mTOR pathway, where TRIB3-mediated inhibition of AKT phosphorylation decreases insulin signaling and cell survival. Conversely, cancer cells can either upregulate the AKT survival pathway by suppressing TRIB3 expression or alter TRIB3 localization to degrade differentiation inducing nuclear transcription factors such as C/EBPα and PPARγ. The gain-of-function Q84R polymorphism in TRIB3 is associated with increased risk of diabetes and atherosclerosis. TRIB3 acts as a crucial 'stress adjusting switch' that links homeostasis, metabolic disease and cancer; and is being actively investigated as a disease biomarker and therapeutic target.

Nelfinavir targets multiple drug resistance mechanisms to increase the efficacy of doxorubicin in MCF-7/Dox breast cancer cells.

Development of multidrug resistance (MDR) remains a significant problem in cancer chemotherapy and underscores the importance of using chemosensitizers. Well known MDR mechanisms include: (i) upregulation of drug-efflux; (ii) increased signaling via AKT; and (iii) decreased apoptosis. Therefore, chemosensitizers should target multiple resistance mechanisms. We investigated the efficacy of nelfinavir (NFV), a clinically approved anti-HIV drug, in increasing doxorubicin (DOX) toxicity in a MDR breast cancer cell line, MCF-7/Dox. As compared to parental MCF-7 cells, the MCF-7/Dox were 15-20 fold more resistant to DOX-induced cytotoxicity at 48 h post-exposure (DOX IC50 = 1.8 µM vs. 32.4 µM). Coexposures to NFV could significantly (p < 0.05) decrease DOX-IC50 in MCF-7/Dox cells. Multiple exposures to physiologic concentrations of NFV (2.25 µM or 6.75 µM) decreased DOX-IC50 by 21-fold and 50-fold, respectively. Interestingly, although single exposure to NFV transiently induced P-glycoprotein (P-gp) levels, multiple treatments with NFV inhibited both P-gp expression and efflux function, which increased intracellular DOX concentrations. Single exposure to NFV augmented the markers of cell-survival (AKT) and autophagy (LC3-II), whereas multiple exposures enabled suppression of both total AKT (t-AKT) and insulin like growth factor-1 (IGF-1)-induced phosphorylated AKT (p-AKT) levels. Multiple exposures to NFV also resulted in increased unfolded protein response (UPR) transducers, e.g. Grp78, p-PERK, p-eIF2α, and ATF-4; and endoplasmic reticulum (ER) stress induced death sensors, e.g. CHOP & TRIB-3. Multiple exposures to NFV also abrogated the mitogenic effects of IGF-1. In mice carrying MCF-7/Dox tumor xenografts, intraperitoneal (i.p.) injection of NFV (20 mg/kg/day) and DOX (2 mg/kg/twice/wk) decreased tumor growth more significantly (p < 0.01) than either agent alone. Immunohistochemical (IHC) analysis revealed decreased p-AKT and Ki-67 levels. Thus, NFV overcomes MDR in breast cancer cells and should be tested as an adjunct to chemotherapy.