Development and evaluation of a low-cost, portable, LED-based device for PDT treatment of early-stage oral cancer in resource-limited settings.

BACKGROUND: Photodynamic therapy (PDT) using δ-aminolevulinic acid (ALA) photosensitization has shown promise in clinical studies for the treatment of early-stage oral malignancies with fewer potential side effects than traditional therapies. Light delivery to oral lesions can also carried out with limited medical infrastructure suggesting the potential for implementation of PDT in global health settings. OBJECTIVES: We sought to develop a cost-effective, battery-powered, fiber-coupled PDT system suitable for intraoral light delivery enabled by smartphone interface and embedded electronics for ease of operation. METHODS: Device performance was assessed in measurements of optical power output, spectral stability, and preclinical assessment of PDT response in ALA-photosensitized squamous carcinoma cell cultures and murine subcutaneous tumor xenografts. RESULTS: The system achieves target optoelectronic performance with a stable battery-powered output of approximately 180 mW from the fiber tip within the desired spectral window for PpIX activation. The device has a compact configuration, user friendly operation and flexible light delivery for the oral cavity. In cell culture, we show that the overall dose-response is consistent with established light sources and complete cell death of ALA photosensitized cells can be achieved in the irradiated zone. In vivo PDT response (reduction in tumor volume) is comparable with a commercial 635 nm laser. CONCLUSIONS: We developed a low-cost, LED-based, fiber-coupled PDT light delivery source that has stable output on battery power and suitable form factor for deployment in rural and/or resource-limited settings. Lasers Surg. Med. 9999:1-7, 2018. © 2018 Wiley Periodicals, Inc.

The mutational landscape of lethal castration-resistant prostate cancer.

Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains and losses, including ETS gene family fusions, PTEN loss and androgen receptor (AR) amplification, which drive prostate cancer development and progression to lethal, metastatic castration-resistant prostate cancer (CRPC). However, less is known about the role of mutations. Here we sequenced the exomes of 50 lethal, heavily pre-treated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment-naive, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPCs (2.00 per megabase) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1 that define a subtype of ETS gene family fusion-negative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in approximately one-third of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Furthermore, we identified recurrent mutations in multiple chromatin- and histone-modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with the AR, which is required for AR-mediated signalling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signalling and increases tumour growth. Proteins that physically interact with the AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX (also known as KDM6A) and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signalling deregulated in prostate cancer, and prioritize candidates for future study.

Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression.

Multiple, complex molecular events characterize cancer development and progression. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of critical biomarkers for cancer invasion and disease aggressiveness. Although gene and protein expression have been extensively profiled in human tumours, little is known about the global metabolomic alterations that characterize neoplastic progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we profiled more than 1,126 metabolites across 262 clinical samples related to prostate cancer (42 tissues and 110 each of urine and plasma). These unbiased metabolomic profiles were able to distinguish benign prostate, clinically localized prostate cancer and metastatic disease. Sarcosine, an N-methyl derivative of the amino acid glycine, was identified as a differential metabolite that was highly increased during prostate cancer progression to metastasis and can be detected non-invasively in urine. Sarcosine levels were also increased in invasive prostate cancer cell lines relative to benign prostate epithelial cells. Knockdown of glycine-N-methyl transferase, the enzyme that generates sarcosine from glycine, attenuated prostate cancer invasion. Addition of exogenous sarcosine or knockdown of the enzyme that leads to sarcosine degradation, sarcosine dehydrogenase, induced an invasive phenotype in benign prostate epithelial cells. Androgen receptor and the ERG gene fusion product coordinately regulate components of the sarcosine pathway. Here, by profiling the metabolomic alterations of prostate cancer progression, we reveal sarcosine as a potentially important metabolic intermediary of cancer cell invasion and aggressivity.

Molecular imaging of Akt kinase activity.

The serine/threonine kinase Akt mediates mitogenic and anti-apoptotic responses that result from activation of multiple signaling cascades. It is considered a key determinant of tumor aggressiveness and is a major target for anticancer drug development. Here, we describe a new reporter molecule whose bioluminescence activity within live cells and in mice can be used to measure Akt activity. Akt activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to activation or inhibition of receptor tyrosine kinase, inhibition of phosphoinositide 3-kinase, or direct inhibition of Akt. The results provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate Akt activity, revealing the usefulness of this reporter for rapid dose and schedule optimization in the drug development process.

Clinical assessment of a low-cost, hand-held, smartphone-attached intraoral imaging probe for 5-aminolevulinic acid photodynamic therapy monitoring and guidance.

Significance: India has one of the highest rates of oral squamous cell carcinoma (OSCC) in the world, with an incidence of 15 per 100,000 and more than 70,000 deaths per year. The problem is exacerbated by a lack of medical infrastructure and routine screening, especially in rural areas. New technologies for oral cancer detection and timely treatment at the point of care are urgently needed. Aim: Our study aimed to use a hand-held smartphone-coupled intraoral imaging device, previously investigated for autofluorescence (auto-FL) diagnostics adapted here for treatment guidance and monitoring photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence (FL). Approach: A total of 12 patients with 14 buccal mucosal lesions having moderately/well-differentiated micro-invasive OSCC lesions (<2 cm diameter and <5 mm depth) were systemically (in oral solution) administered three doses of 20 mg/kg ALA (total 60 mg/kg). Lesion site PpIX and auto-FL were imaged using the multichannel FL and polarized white-light oral cancer imaging probe before/after ALA administration and after light delivery (fractionated, total 100 J/cm2 of 635 nm red LED light). Results: The handheld device was conducive for access to lesion site images in the oral cavity. Segmentation of ratiometric images in which PpIX FL is mapped relative to auto-FL enabled improved demarcation of lesion boundaries relative to PpIX alone. A relative FL (R-value) threshold of 1.4 was found to segment lesion site PpIX production among the patients with mild to severe dysplasia malignancy. The segmented lesion size is well correlated with ultrasound findings. Lesions for which R-value was >1.65 at the time of treatment were associated with successful outcomes. Conclusion: These results indicate the utility of a low-cost, handheld intraoral imaging probe for image-guided PDT and treatment monitoring while also laying the groundwork for an integrated approach, combining cancer screening and treatment with the same hardware.

Variable reference alignment: an improved peak alignment protocol for NMR spectral data with large intersample variation.

In an effort to address the variable correspondence problem across large sample cohorts common in metabolomic/metabonomic studies, we have developed a prealignment protocol that aims to generate spectral segments sharing a common target spectrum. Under the assumption that a single reference spectrum will not correctly represent all spectra of a data set, the goal of this approach is to perform local alignment corrections on spectral regions which share a common "most similar" spectrum. A natural beneficial outcome of this procedure is the automatic definition of spectral segments, a feature that is not common to all alignment methods. This protocol is shown to specifically improve the quality of alignment in (1)H NMR data sets exhibiting large intersample compositional variation (e.g., pH, ionic strength). As a proof-of-principle demonstration, we have utilized two recently developed alignment algorithms specific to NMR data, recursive segment-wise peak alignment and interval correlated shifting, and applied them to two data sets composed of 15 aqueous cell line extract and 20 human urine (1)H NMR profiles. Application of this protocol represents a fundamental shift from current alignment methodologies that seek to correct misalignments utilizing a single representative spectrum, with the added benefit that it can be appended to any alignment algorithm.

Quantitative proteomic profiling of prostate cancer reveals a role for miR-128 in prostate cancer.

Multiple, complex molecular events characterize cancer development and progression. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of biomarkers of cancer invasion and disease aggressiveness. Although alterations in gene expression have been extensively quantified during neoplastic progression, complementary analyses of proteomic changes have been limited. Here we interrogate the proteomic alterations in a cohort of 15 prostate-derived tissues that included five each from adjacent benign prostate, clinically localized prostate cancer, and metastatic disease from distant sites. The experimental strategy couples isobaric tags for relative and absolute quantitation with multidimensional liquid phase peptide fractionation followed by tandem mass spectrometry. Over 1000 proteins were quantified across the specimens and delineated into clinically localized and metastatic prostate cancer-specific signatures. Included in these class-specific profiles were both proteins that were known to be dysregulated during prostate cancer progression and new ones defined by this study. Enrichment analysis of the prostate cancer-specific proteomic signature, to gain insight into the functional consequences of these alterations, revealed involvement of miR-128-a/b regulation during prostate cancer progression. This finding was validated using real time PCR analysis for microRNA transcript levels in an independent set of 15 clinical specimens. miR-128 levels were elevated in benign prostate epithelial cell lines compared with invasive prostate cancer cells. Knockdown of miR-128 induced invasion in benign prostate epithelial cells, whereas its overexpression attenuated invasion in prostate cancer cells. Taken together, our profiles of the proteomic alterations of prostate cancer progression revealed miR-128 as a potentially important negative regulator of prostate cancer cell invasion.

Clinical evaluation of a mobile, low-cost system for fluorescence guided photodynamic therapy of early oral cancer in India.

BACKGROUND: Morbidity and mortality due to oral cancer in India are exacerbated by a lack of access to effective treatments amongst medically underserved populations. We developed a user-friendly low-cost, portable fibre-coupled LED system for photodynamic therapy (PDT) of early oral lesions, using a smartphone fluorescence imaging device for treatment guidance, and 3D printed fibreoptic attachments for ergonomic intraoral light delivery. METHODS: 30 patients with T1N0M0 buccal mucosal cancer were recruited from the JN Medical College clinics, Aligarh, and rural screening camps. Tumour limits were defined by external ultrasound (US), white light photos and increased tumour fluorescence after oral administration of the photosensitising agent ALA (60 mg/kg, divided doses), monitored by a smartphone fluorescence imaging device. 100 J/cm2 LED light (635 nm peak) was delivered followed by repeat fluorescence to assess photobleaching. US and biopsy were repeated after 7-17 days. This trial is registered with ClinicalTrials.gov, NCT03638622, and the study has been completed. FINDINGS: There were no significant complications or discomfort. No sedation was required. No residual disease was detected in 22 out of 30 patients who completed the study (26 of 34 lesions, 76% complete tumour response, 50 weeks median follow-up) with up to 7.2 mm depth of necrosis. Treatment failures were attributed to large tumour size and/or inadequate light delivery (documented by limited photobleaching). Moderately differentiated lesions were more responsive than well-differentiated cancers. INTERPRETATION: This simple and low-cost adaptation of fluorescenceguided PDT is effective for treatment of early-stage malignant oral lesions and may have implications in global health.

Molecular imaging of epidermal growth factor receptor kinase activity.

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is commonly altered in different tumor types, leading to abnormally regulated kinase activity and excessive activation of downstream signaling cascades, including cell proliferation, differentiation, and migration. To investigate the EGFR signaling events in real time and in living cells and animals, here we describe a multidomain chimeric reporter whose bioluminescence can be used as a surrogate for EGFR kinase activity. This luciferase-based reporter was developed in squamous cell carcinoma cells (UMSCC1) to generate a cancer therapy model for imaging EGFR. The reporter is designed to act as a phosphorylated substrate of EGFR and reconstitutes luciferase activity when it is not phosphorylated, thereby providing a robust indication of EGFR inhibition. We validated the reporter in vitro and demonstrated that its activity could be differentially modulated by EGFR tyrosine kinase inhibition with erlotonib or receptor activation with epidermal growth factor. Further experiments in vivo demonstrated quantitative and dynamic monitoring of EGFR tyrosine kinase activity in xenograft. Results obtained from these studies provide unique insight into pharmacokinetics and pharmacodynamics of agents that modulate EGFR activity, revealing the usefulness of this reporter in evaluating drug availability and cell targeting in both living cells and mouse models.

Development of selected reaction monitoring-MS methodology to measure peptide biomarkers in prostate cancer.

Prostate cancer is a leading cause of cancer-related death. The current modality of diagnosis, the measurement of serum PSA, not only suffers from lack of specificity, but does not distinguish clinical cases in which current treatment measures would be most successful, i.e. aggressive, life-threatening tumors. A multiplexed MS methodology, selected reaction monitoring-MS/MS coupled with stable isotope dilution (SID), was developed and tested in both cells lines and clinical tissue samples. Standard curves were generated for two peptides representing PSA and one peptide from each of two additional orthogonally validated biomarkers, AMACR and EZH2. The standard curves show high reproducibility, sensitivity, and good linearity. All four peptides were then measured in six clinically relevant cell lines and are in agreement with the biochemical characteristics of each individual cell line. The SID selected reaction monitoring-MS/MS methodology was then transferred to tissue samples, in which the assay shows potential to differentiate benign disease from localized cancer and localized cancer from aggressive metastatic disease. These results establish the preliminary development of a rational targeted MS platform that strives to bridge the gap between discovery and validation of biomarkers for the detection of prostate cancer.

Clinical evaluation of smartphone-based fluorescence imaging for guidance and monitoring of ALA-PDT treatment of early oral cancer.

SIGNIFICANCE: India has one of the highest rates of oral cancer incidence in the world, accounting for 30% of reported cancers. In rural areas, a lack of adequate medical infrastructure contributes to unchecked disease progression and dismal mortality rates. Photodynamic therapy (PDT) has emerged as an effective modality with potential for treating early stage disease in resource-limited settings, while photosensitizer fluorescence can be leveraged for treatment guidance. AIM: Our aim was to assess the capability of a simple smartphone-based device for imaging 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence for treatment guidance and monitoring as part of an ongoing clinical study evaluating low-cost technology for ALA-based PDT treatment of early oral cancer. APPROACH: A total of 29 subjects with <2 cm diameter moderately/well-differentiated microinvasive ( < 5 mm depth) oral squamous cell carcinoma lesions (33 lesions total, mean area ∼1.23 cm2) were administered 60 mg / kg ALA in oral solution and imaged before and after delivery of 100 J / cm2 total light dose to the lesion surface. Smartphone-based fluorescence and white light (WL) images were analyzed and compared with ultrasound (US) imaging of the same lesions. RESULTS: We present a comparative analysis of pre- and post-treatment fluorescence, WL, and US images of oral lesions. There was no significant difference in the distribution of lesion widths measured by fluorescence and US (mean widths of 14.5 and 15.3 mm, respectively) and linear regression shows good agreement (R2 = 0.91). In general, PpIX fluorescence images obtained prior to therapeutic light delivery are able to resolve lesion margins while dramatic photobleaching (∼42 % ) is visible post-treatment. Segmentation of the photobleached area confirms the boundaries of the irradiated zone. CONCLUSIONS: A simple smartphone-based approach for imaging oral lesions is shown to agree in most cases with US, suggesting that this approach may be a useful tool to aid in PDT treatment guidance and monitoring photobleaching as part of a low-cost platform for intraoral PDT.

Flow-induced Shear Stress Confers Resistance to Carboplatin in an Adherent Three-Dimensional Model for Ovarian Cancer: A Role for EGFR-Targeted Photoimmunotherapy Informed by Physical Stress.

A key reason for the persistently grim statistics associated with metastatic ovarian cancer is resistance to conventional agents, including platinum-based chemotherapies. A major source of treatment failure is the high degree of genetic and molecular heterogeneity, which results from significant underlying genomic instability, as well as stromal and physical cues in the microenvironment. Ovarian cancer commonly disseminates via transcoelomic routes to distant sites, which is associated with the frequent production of malignant ascites, as well as the poorest prognosis. In addition to providing a cell and protein-rich environment for cancer growth and progression, ascitic fluid also confers physical stress on tumors. An understudied area in ovarian cancer research is the impact of fluid shear stress on treatment failure. Here, we investigate the effect of fluid shear stress on response to platinum-based chemotherapy and the modulation of molecular pathways associated with aggressive disease in a perfusion model for adherent 3D ovarian cancer nodules. Resistance to carboplatin is observed under flow with a concomitant increase in the expression and activation of the epidermal growth factor receptor (EGFR) as well as downstream signaling members mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK). The uptake of platinum by the 3D ovarian cancer nodules was significantly higher in flow cultures compared to static cultures. A downregulation of phospho-focal adhesion kinase (p-FAK), vinculin, and phospho-paxillin was observed following carboplatin treatment in both flow and static cultures. Interestingly, low-dose anti-EGFR photoimmunotherapy (PIT), a targeted photochemical modality, was found to be equally effective in ovarian tumors grown under flow and static conditions. These findings highlight the need to further develop PIT-based combinations that target the EGFR, and sensitize ovarian cancers to chemotherapy in the context of flow-induced shear stress.

Platform for ergonomic intraoral photodynamic therapy using low-cost, modular 3D-printed components: Design, comfort and clinical evaluation.

Oral cancer prevalence is increasing at an alarming rate worldwide, especially in developing countries which lack the medical infrastructure to manage it. For example, the oral cancer burden in India has been identified as a public health crisis. The high expense and logistical barriers to obtaining treatment with surgery, radiotherapy and chemotherapy often result in progression to unmanageable late stage disease with high morbidity. Even when curative, these approaches can be cosmetically and functionally disfiguring with extensive side effects. An alternate effective therapy for oral cancer is a light based spatially-targeted cytotoxic therapy called photodynamic therapy (PDT). Despite excellent healing of the oral mucosa in PDT, a lack of robust enabling technology for intraoral light delivery has limited its broader implementation. Leveraging advances in 3D printing, we have developed an intraoral light delivery system consisting of modular 3D printed light applicators with pre-calibrated dosimetry and mouth props that can be utilized to perform PDT in conscious subjects without the need of extensive infrastructure or manual positioning of an optical fiber. To evaluate the stability of the light applicators, we utilized an endoscope in lieu of the optical fiber to monitor motion in the fiducial markers. Here we showcase the stability (less than 2 mm deviation in both horizontal and vertical axis) and ergonomics of our applicators in delivering light precisely to the target location in ten healthy volunteers. We also demonstrate in five subjects with T1N0M0 oral lesions that our applicators coupled with a low-cost fiber coupled LED-based light source served as a complete platform for intraoral light delivery achieving complete tumor response with no residual disease at initial histopathology follow up in these patients. Overall, our approach potentiates PDT as a viable therapeutic option for early stage oral lesions that can be delivered in low resource settings.

Noninvasive molecular imaging sheds light on the synergy between 5-fluorouracil and TRAIL/Apo2L for cancer therapy.

PURPOSE: In a previous report, a recombinant luciferase reporter, activated during apoptosis via caspase-3 cleavage, was developed for imaging of apoptosis using bioluminescence. The ability to noninvasively image apoptosis in vivo could dramatically benefit the preclinical development of therapeutics targeting the apoptotic pathway. In this study, we examined the use of 5-fluorouracil (5-FU) for sensitizing D54 tumors to tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL) therapy by monitoring apoptotic activity in vivo using bioluminescence imaging. EXPERIMENTAL DESIGN: Using our apoptosis imaging platform and diffusion magnetic resonance imaging (MRI), we monitored the antitumor effects of 5-FU, TRAIL, and 5-FU + TRAIL using D54 xenografts. Additionally, volumetric and histologic analyses were done for correlation with findings from bioluminescence imaging and diffusion MRI. RESULTS: Bioluminescence imaging showed that therapy with TRAIL alone produced an initial 400% increase in apoptotic activity that rapidly diminished during the 10-day treatment period despite continued therapy. In contrast, concomitant 5-FU and TRAIL therapy elicited an apoptotic response that was sustained throughout the entire therapeutic course. Using diffusion MRI, an enhanced tumor response was detected when concomitant therapy was given versus TRAIL-alone therapy. Last, concomitant therapy resulted in a prolonged growth delay ( approximately 9 days) compared with TRAIL alone ( approximately 4 days). CONCLUSION: We showed that concomitant 5-FU and TRAIL therapy indeed enhanced apoptotic activity in vivo, which translated into greater tumor control. Moreover, this technique sheds light on the synergy of 5-FU and TRAIL as evidenced by differences in the temporal activation of caspase-3 resulting from the different therapeutic regimens.

Prospective early response imaging biomarker for neoadjuvant breast cancer chemotherapy.

PURPOSE: The American Cancer Society estimates that in 2006, 212,920 women will be diagnosed with breast cancer and that 40,970 women will die from the disease. The development of more efficacious chemotherapies has improved outcomes, but the rapid assessment of clinical benefit from these agents remains challenging. In breast cancer patients receiving neoadjuvant chemotherapy, treatment response is traditionally assessed by physical examination and volumetric-based measurements, which are subjective and require macroscopic changes in tumor morphology. In this study, we evaluate the feasibility of using diffusion magnetic resonance imaging (MRI) as a reliable and quantitative measure for the early assessment of response in a breast cancer model. EXPERIMENTAL DESIGN: Mice implanted with human breast cancer (MX-1) were treated with cyclophosphamide and evaluated using diffusion MRI and growth kinetics. Histologic analyses using terminal nucleotidyl transferase-mediated nick end labeling and H&E were done on tumor samples for correlation with imaging results. RESULTS: Cyclophosphamide treatment resulted in a significant reduction in tumor volumes compared with controls. The mean apparent diffusion change for treated tumors at days 4 and 7 posttreatment was 44 +/- 5% and 94 +/- 7%, respectively, which was statistically greater (P < 0.05) than the control tumors at the same time intervals. The median time-to-progression for control and treated groups was 11 and 32 days, respectively (P < 0.05). CONCLUSION: Diffusion MRI was shown to detect early changes in the tumor microenvironment, which correlated with standard measures of tumor response as well as overall outcome. Moreover, these findings show the feasibility of using diffusion MRI for assessing treatment response of a breast tumor model in a neoadjuvant setting.

Targeting the MLL complex in castration-resistant prostate cancer.

Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration-resistant prostate cancer (CRPC). Although prior work has focused on targeting AR directly, co-activators of AR signaling, which may represent new therapeutic targets, are relatively underexplored. Here we demonstrate that the mixed-lineage leukemia protein (MLL) complex, a well-known driver of MLL fusion-positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via the menin-MLL subunit. Menin expression is higher in CRPC than in both hormone-naive prostate cancer and benign prostate tissue, and high menin expression correlates with poor overall survival of individuals diagnosed with prostate cancer. Treatment with a small-molecule inhibitor of menin-MLL interaction blocks AR signaling and inhibits the growth of castration-resistant tumors in vivo in mice. Taken together, this work identifies the MLL complex as a crucial co-activator of AR and a potential therapeutic target in advanced prostate cancer.

Variations in angiotensin-converting enzyme gene insertion/deletion polymorphism in Indian populations of different ethnic origins.

The pattern of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism in the Indian population is poorly known. In order to determine the status of the polymorphism, young unrelated male army recruits were screened. The population had cultural and linguistic differences and lived in an environment that varied significantly from one region to another. Analysis of the genotype, showed higher frequency of the insertion allele in four of the five groups i.e. I allele frequency was significantly higher (P < 0.05) in Dogras, Assamese and Kumaonese. The deletion allele frequency was comparatively higher in the fifth group that belonged to Punjab. A correlation was observed between the genotype and enzyme activity. Involvement of a single D allele in the genotype enhanced the activity up to 37.56 3.13%. The results suggested ethnic heterogeneity with a significant gene cline with higher insertion allele frequency. Such population-based data on various polymorphisms can ultimately be exploited in pharmacogenomics.

Renin angiotensin aldosterone system and ACE I/D gene polymorphism in high-altitude pulmonary edema.

INTRODUCTION: People who visit high altitude are exposed to a stressful environment, and many of them suffer from altitude-induced conditions, including high altitude pulmonary edema (HAPE). We investigated the renin angiotensin aldosterone system (RAAS) and the possible association of angiotensin converting enzyme (ACE) insertion/deletion (I/D) gene polymorphism in the development of HAPE in Indian men. METHODS: Subjects were all low-altitude natives: 19 men who developed HAPE within 1-3 d of arrival at 3000 to 3800 m (patients); and 20 age-matched men who did not develop HAPE during a period of a month or more at > or = 3500 m (controls). We recorded the arterial oxygen saturation (Sao2), heart rate (HR), and blood pressure (BP) of both groups and measured their levels of plasma renin activity (PRA), ACE, aldosterone, and serum electrolytes. Polymerase chain reaction was used to investigate a 287 base pair alu repeat sequence I/D polymorphism in the ACE gene. RESULTS: Compared with controls, patients showed a significantly lower Sao2 and a higher HR. They also had significantly higher plasma PRA, aldosterone, ACE, and serum sodium (Na+) and potassium (K+). No significant difference was observed in ACE I/D allele frequencies. DISCUSSION: The results suggested that RAAS is involved in the development of HAPE in low-altitude natives, but there is no association of ACE I/D gene polymorphism with HAPE.

The role of sarcosine metabolism in prostate cancer progression.

Metabolomic profiling of prostate cancer (PCa) progression identified markedly elevated levels of sarcosine (N-methyl glycine) in metastatic PCa and modest but significant elevation of the metabolite in PCa urine. Here, we examine the role of key enzymes associated with sarcosine metabolism in PCa progression. Consistent with our earlier report, sarcosine levels were significantly elevated in PCa urine sediments compared to controls, with a modest area under the receiver operating characteristic curve of 0.71. In addition, the expression of sarcosine biosynthetic enzyme, glycine N-methyltransferase (GNMT), was elevated in PCa tissues, while sarcosine dehydrogenase (SARDH) and pipecolic acid oxidase (PIPOX), which metabolize sarcosine, were reduced in prostate tumors. Consistent with this, GNMT promoted the oncogenic potential of prostate cells by facilitating sarcosine production, while SARDH and PIPOX reduced the oncogenic potential of prostate cells by metabolizing sarcosine. Accordingly, addition of sarcosine, but not glycine or alanine, induced invasion and intravasation in an in vivo PCa model. In contrast, GNMT knockdown or SARDH overexpression in PCa xenografts inhibited tumor growth. Taken together, these studies substantiate the role of sarcosine in PCa progression.

A novel kinase inhibitor of FADD phosphorylation chemosensitizes through the inhibition of NF-κB.

Fas-associated protein with death domain (FADD) is a cytosolic adapter protein essential for mediating death receptor-induced apoptosis. It has also been implicated in a number of nonapoptotic activities including embryogenesis, cell-cycle progression, cell proliferation, and tumorigenesis. Our recent studies have shown that high levels of phosphorylated FADD (p-FADD) in tumor cells correlate with increased activation of the antiapoptotic transcription factor NF-κB and is a biomarker for aggressive disease and poor clinical outcome. These findings suggest that inhibition of FADD phosphorylation is a viable target for cancer therapy. A high-throughput screen using a cell-based assay for monitoring FADD-kinase activity identified NSC 47147 as a small molecule inhibitor of FADD phosphorylation. The compound was evaluated in live cells and mouse tumors for its efficacy as an inhibitor of FADD-kinase activity through the inhibition of casein kinase 1α. NSC 47147 was shown to decrease levels of p-FADD and NF-κB activity such that combination therapy leads to greater induction of apoptosis and enhanced tumor control than either agent alone. The studies described here show the utility of bioluminescent cell-based assays for the identification of active compounds and the validation of drug-target interaction in a living subject. In addition, the presented results provide proof-of-principle studies as to the validity of targeting FADD-kinase activity as a novel cancer therapy strategy.