Publications by authors named "Vyomesh Patel"

106 Publications

Comparative Profiling of Four Lignans (Phyllanthin, Hypophyllanthin, Nirtetralin, and Niranthin) in Nine Phyllanthus Species from India Using a Validated Reversed Phase HPLC-PDA Detection Method.

J AOAC Int 2020 Sep 30. Epub 2020 Sep 30.

ICAR-Directorate of Medicinal and Aromatic Plants Research, Anand, 387310, Gujarat, India.

Background: Phyllanthus species exhibit a wide range of in vitro and in vivo pharmacological activities; however, little is known about the compounds present in the extracts that are responsible for such actions.

Objective: Development and validation of a simple reversed phase HPLC-PDA method for profiling of phyllanthin, hypophyllanthin, nirtetralin, and niranthin in extracts of Phyllanthus species was carried out.

Methods: Separation was achieved using an XBridge column® (150 × 4.6 mm, 5.0 µm id) in an isocratic elution mode with mobile phase comprising of a mixture of acetonitrile and water with TFA (0.05%, v/v, pH = 2.15) at ambient temperature with a flow rate of 1 mL/min.

Results: Phyllanthin, hypophyllanthin, nirtetralin, and niranthin were eluted at mean retention times of 10.47, 11.10, 13.67, and 14.53 min, respectively. LOD and LOQ for all four analytes were 0.75 and 3.00 μg/mL, respectively. RSDr values for intraday and interday precision for phyllanthin, hypophyllanthin, nirtetralin, and niranthin were 0.38-1.32 and 0.45-1.77%; 0.22-3.69 and 0.24-3.04%, 0.73-2.37 and 0.09-0.31%, and 1.56-2.77 and 0.12-0.68%, respectively.

Conclusions: The developed and validated HPLC-PDA method was applied for identification and quantification of phyllanthin, hypophyllanthin, nirtetralin, and niranthin in extracts of different plant parts of selected Phyllanthus species. The outcome of the present investigation could be useful for selection of best species to promote its commercial cultivation and suitable extraction solvent for preparation of lignan-enriched fractions. This HPLC-PDA method could be useful for quality control of herbal formulations containing plants from Phyllanthus species.
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http://dx.doi.org/10.1093/jaoacint/qsaa133DOI Listing
September 2020

Cigalike electronic nicotine delivery systems e-liquids contain variable levels of metals.

Sci Rep 2020 07 17;10(1):11907. Epub 2020 Jul 17.

Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, USA.

Electronic nicotine delivery systems (ENDS) are prefilled, battery-operated products intended to deliver nicotine to the user via an inhaled complex aerosol formed by heating a liquid composed of propylene glycol and glycerol, also referred to as vegetable glycerin and collectively called e-liquid, that contains nicotine and various flavor ingredients. Since their introduction in 2006, the number of ENDS on the market has increased exponentially. Despite their growing ubiquity, the possible health risks associated with ENDS use remain poorly understood. One potential concern is the presence of toxic metals in the e-liquid and aerosol. Herein, we report the evaluation of the metal content in the e-liquids from a series of commercially available cigalike ENDS brands (various flavors) determined using inductively coupled plasma mass spectrometry (ICP-MS) following e-liquid extraction. Each brand of cigalike ENDS was purchased at least three times at retail outlets in the Baltimore, Maryland metropolitan region over a period of six months (September 2017 to February 2018). This allowed for comparison of batch-to-batch variability. Several potentially toxic metals, including lead, chromium, copper, and nickel were detected in the e-liquids. In addition, high variability in metal concentrations within and between brands and flavors was observed . The internal assembled parts of each cartridge were analyzed by X-ray imaging, before dissembling so that the materials used to manufacture each cartridge could be evaluated to determine the metals they contained. Following washing to remove traces of e-liquid, lead, chromium, copper and nickel were all detected in the cigalike ENDS prefilled cartridges, suggesting one potential source for the metals found in the e-liquids. Collectively, these findings can inform further evaluation of product design and manufacturing processes, including quantification of metal concentrations in e-liquids over foreseeable storage times, safeguards against high concentrations of metals in the e-liquid before and after aerosolization (by contact with a metal heating coil), and control over batch-to-batch variability.
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http://dx.doi.org/10.1038/s41598-020-67789-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368082PMC
July 2020

Current Perspectives on Nasopharyngeal Carcinoma.

Adv Exp Med Biol 2019 ;1164:11-34

Cancer Research Malaysia, Subang Jaya, Selangor, Malaysia.

Of the ~129,079 new cases of nasopharyngeal carcinoma (NPC) and 72,987 associated deaths estimated for 2018, the majority will be geographically localized to South East Asia, and likely to show an upward trend annually. It is thought that disparities in dietary habits, lifestyle, and exposures to harmful environmental factors are likely the root cause of NPC incidence rates to differ geographically. Genetic differences due to ethnicity and the Epstein Barr virus (EBV) are likely contributing factors. Pertinently, NPC is associated with poor prognosis which is largely attributed to lack of awareness of the salient symptoms of NPC. These include nose hemorrhage and headaches and coupled with detection and the limited therapeutic options. Treatment options include radiotherapy or chemotherapy or combination of both. Surgical excision is generally the last option considered for advanced and metastatic disease, given the close proximity of nasopharynx to brain stem cell area, major blood vessels, and nerves. To improve outcome of NPC patients, novel cellular and in vivo systems are needed to allow an understanding of the underling molecular events causal for NPC pathogenesis and for identifying novel therapeutic targets and effective therapies. While challenges and gaps in current NPC research are noted, some advances in targeted therapies and immunotherapies targeting EBV NPCs are discussed in this chapter, which may offer improvements in outcome of NPC patients.
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http://dx.doi.org/10.1007/978-3-030-22254-3_2DOI Listing
October 2019

Effects of palbociclib on oral squamous cell carcinoma and the role of in conferring resistance.

Cancer Biol Med 2019 May;16(2):264-275

Head and Neck Team, Cancer Research Malaysia, Selangor 47500, Malaysia.

Objective: Lack of effective therapies remains a problem in the treatment of oral squamous cell carcinoma (OSCC), especially in patients with advanced tumors. OSCC development is driven by multiple aberrancies within the cell cycle pathway, including amplification of cyclin D1 and loss of p16. Hence, cell cycle inhibitors of the CDK4/6-cyclin D axis are appealing targets for OSCC treatment. Here, we determined the potency of palbociclib and identified genetic features that are associated with the response of palbociclib in OSCC.

Methods: The effect of palbociclib was evaluated in a panel of well-characterized OSCC cell lines by cell proliferation assays and further confirmed by evaluation in xenograft models. -mutant isogenic cell lines were used to investigate the effect of mutation towards palbociclib response.

Results: We demonstrated that 80% of OSCC cell lines are sensitive to palbociclib at sub-micromolar concentrations. Consistently, palbociclib was effective in controlling tumor growth in mice. We identified that palbociclib-resistant cells harbored mutations in . Using isogenic cell lines, we showed that mutant cells are less responsive to palbociclib as compared to wild-type cells with concurrent upregulation of CDK2 and cyclin E1 protein levels. We further demonstrated that the combination of a PI3K/mTOR inhibitor (PF-04691502) and palbociclib completely controlled tumor growth in mice.

Conclusions: This study demonstrated the potency of palbociclib in OSCC models and provides a rationale for the inclusion of testing in the clinical evaluation of CDK4/6 inhibitors and suggests combination approaches for further clinical studies.
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http://dx.doi.org/10.20892/j.issn.2095-3941.2018.0257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713638PMC
May 2019

An Oncogenic Role for Four-Jointed Box 1 (FJX1) in Nasopharyngeal Carcinoma.

Dis Markers 2019 19;2019:3857853. Epub 2019 May 19.

Cancer Research Malaysia, No. 1, Jalan SS12/1A, Subang Jaya, Selangor, Malaysia.

Nasopharyngeal carcinoma (NPC) is a highly metastatic cancer prevalent in Southern China and Southeast Asia. The current knowledge on the molecular pathogenesis of NPC is still inadequate to improve disease management. Using gene expression microarrays, we have identified the () gene to be upregulated in primary NPC tissues relative to nonmalignant tissues. An orthologue of human , the gene in Drosophila and in mouse, has reported to be associated with cancer progression pathways. However, the exact function of FJX1 in human is not well characterized. The overexpression of FJX1 mRNA was validated in primary NPC tissue samples, and the level of FJX1 protein was significantly higher in a subset of NPC tissues (42%) compared to the normal epithelium, where no expression of FJX1 was observed ( = 0.01). FJX1 is also found to be overexpressed in microarray datasets and TCGA datasets of other cancers including head and neck cancer, colorectal, and ovarian cancer. Both siRNA knockdown and overexpression experiments in NPC cell lines showed that FJX1 promotes cell proliferation, anchorage-dependent growth, and cellular invasion. Cyclin D1 and E1 mRNA levels were increased following FJX1 expression indicating that FJX1 enhances proliferation by regulating key proteins governing the cell cycle. Our data suggest that the overexpression of FJX1 contributes to a more aggressive phenotype of NPC cells and further investigations into FJX1 as a potential therapeutic target for NPC are warranted. The evaluation of FJX1 as an immunotherapy target for NPC and other cancers is currently ongoing.
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http://dx.doi.org/10.1155/2019/3857853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545767PMC
December 2019

IFITM3 knockdown reduces the expression of CCND1 and CDK4 and suppresses the growth of oral squamous cell carcinoma cells.

Cell Oncol (Dordr) 2019 Aug 4;42(4):477-490. Epub 2019 Apr 4.

Head and Neck Cancer Research Team, Cancer Research Malaysia, 2nd Floor, Outpatient Centre, Subang Jaya Medical Centre, 47500, Subang Jaya, Selangor, Malaysia.

Purpose: Oral squamous cell carcinoma (OSCC) is a challenging disease to treat. Up to 50% of OSCC patients with advanced disease develop recurrences. Elucidation of key molecular mechanisms underlying OSCC development may provide opportunities to target specific genes and, thus, to improve patient survival. In this study, we examined the expression and functional role of interferon transmembrane protein 3 (IFITM3) in OSCC development.

Methods: The expression of IFITM3 in OSCC and normal oral mucosal tissues was assessed by qRT-PCR and immunohistochemistry. The role of IFITM3 in driving OSCC cell proliferation and survival was examined using siRNA-mediated gene knockdown, and the role of IFITM3 in driving cell cycle regulators was examined using Western blotting.

Results: We found that IFITM3 is overexpressed in more than 79% of primary OSCCs. We also found that IFITM3 knockdown led to impaired OSCC cell growth through inhibition of cell proliferation, induction of cell cycle arrest, senescence and apoptosis. In addition, we found that IFITM3 knockdown led to reduced expressions of CCND1 and CDK4 and reduced RB phosphorylation, leading to inhibition of OSCC cell growth. This information may be instrumental for the design of novel targeted therapeutic strategies.

Conclusions: From our data we conclude that IFITM3 is overexpressed in OSCC and may regulate the CCND1-CDK4/6-pRB axis to mediate OSCC cell growth.
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http://dx.doi.org/10.1007/s13402-019-00437-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771307PMC
August 2019

Novel 2-Benzoyl-6-(2,3-Dimethoxybenzylidene)-Cyclohexenol Confers Selectivity toward Human MLH1 Defective Cancer Cells through Synthetic Lethality.

SLAS Discov 2019 06 21;24(5):548-562. Epub 2019 Mar 21.

1 Cancer Research Malaysia, Subang Jaya, Selangor, Malaysia.

DNA mismatch repair (MMR) deficiency has been associated with a higher risk of developing colorectal, endometrial, and ovarian cancer, and confers resistance in conventional chemotherapy. In addition to the lack of treatment options that work efficaciously on these MMR-deficient cancer patients, there is a great need to discover new drug leads for this purpose. In this study, we screened through a library of commercial and semisynthetic natural compounds to identify potential synthetic lethal drugs that may selectively target MLH1 mutants using MLH1 isogenic colorectal cancer cell lines and various cancer cell lines with known MLH1 status. We identified a novel diarylpentanoid analogue, 2-benzoyl-6-(2,3-dimethoxybenzylidene)-cyclohexenol, coded as AS13, that demonstrated selective toxicity toward MLH1-deficient cancer cells. Subsequent analysis suggested AS13 induced elevated levels of oxidative stress, resulting in DNA damage where only the proficient MLH1 cells were able to be repaired and hence escaping cellular death. While AS13 is modest in potency and selectivity, this discovery has the potential to lead to further drug development that may offer better treatment options for cancer patients with MLH1 deficiency.
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http://dx.doi.org/10.1177/2472555219831405DOI Listing
June 2019

Synergistic Growth Inhibition by Afatinib and Trametinib in Preclinical Oral Squamous Cell Carcinoma Models.

Target Oncol 2019 04;14(2):223-235

Head and Neck Cancer Research Team, Cancer Research Malaysia, No. 1, Jalan SS12/1A, 47500, Subang Jaya, Selangor, Malaysia.

Background: Given that aberrant activation of epidermal growth factor receptor family receptors (ErbB) is a common event in oral squamous cell carcinoma, and that high expression of these receptor proteins is often associated with poor prognosis, this rationalizes the approach of targeting ErbB signaling pathways to improve the survival of patients with oral squamous cell carcinoma. However, monotherapy with the ErbB blocker afatinib has shown limited survival benefits.

Objectives: This study was performed to identify mechanisms of afatinib resistance and to explore potential afatinib-based combination treatments with other targeted inhibitors in oral squamous cell carcinoma.

Methods: We determined the anti-proliferative effects of afatinib on a panel of oral squamous cell carcinoma cell lines using a crystal violet-growth inhibition assay, click-iT 5-ethynyl-2'-deoxyuridine staining, and cell-cycle analysis. Biochemical assays were performed to study the underlying mechanism of drug treatment as a single agent or in combination with the MEK inhibitor trametinib. We further evaluated and compared the anti-tumor effects of single agent and combined treatment by using oral squamous cell carcinoma xenograft models.

Results: In this study, we showed that afatinib inhibited oral squamous cell carcinoma cell proliferation via cell-cycle arrest at the G/G phase, and inhibited tumor growth in xenograft mouse models. Interestingly, we demonstrated reactivation of the mitogen-activated protein kinase (ERK1/2) pathway in vitro, which possibly reduced the effects of ErbB inhibition. Concomitant treatment of oral squamous cell carcinoma cells with afatinib and trametinib synergized the anti-tumor effects in oral squamous cell carcinoma-bearing mouse models.

Conclusions: Our findings provide insight into the molecular mechanism of resistance to afatinib and support further clinical evaluation into the combination of afatinib and MEK inhibition in the treatment of oral squamous cell carcinoma.
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http://dx.doi.org/10.1007/s11523-019-00626-8DOI Listing
April 2019

The 4717C > G polymorphism in periplakin modulates sensitivity to EGFR inhibitors.

Sci Rep 2019 02 20;9(1):2357. Epub 2019 Feb 20.

Head and Neck Cancer Research Team, Cancer Research Malaysia, No. 1, Jalan SS12/1A, 47500, Subang Jaya, Selangor, Malaysia.

The use of EGFR inhibitors on oral squamous cell carcinoma (OSCC) as monotherapy yielded modest clinical outcomes and therefore would benefit from biomarkers that could predict which patient subsets are likely to respond. Here, we determined the efficacy of erlotinib in OSCC cell lines, and by comparing sensitive and resistant lines to identify potential biomarkers. We focused on the 4717C > G polymorphism in periplakin (PPL) where the CC genotype was associated with erlotinib resistance. To validate this, erlotinib-resistant cell lines harbouring CC genotype were engineered to overexpress the GG genotype and vice versa. Isogenic cell lines were then studied for their response to erlotinib treatment. We demonstrated that overexpression of the GG genotype in erlotinib-resistant lines sensitized them to erlotinib and inhibition of AKT phosphorylation. Similarly, the expression of the CC genotype conferred resistance to erlotinib with a concomitant increase in AKT phosphorylation. We also demonstrated that cell lines with the CC genotype generally are more resistant to other EGFR inhibitors than those with the GG genotype. Overall, we showed that a specific polymorphism in the PPL gene could confer resistance to erlotinib and other EGFR inhibitors and further work to evaluate these as biomarkers of response is warranted.
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http://dx.doi.org/10.1038/s41598-019-38742-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382785PMC
February 2019

Urinary concentrations of monohydroxylated polycyclic aromatic hydrocarbons in adults from the U.S. Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014).

Environ Int 2019 02 6;123:201-208. Epub 2018 Dec 6.

Centers for Disease Control and Prevention, National Center for Environmental Health, 4770 Buford Hwy NE, Atlanta, GA 30341, USA. Electronic address:

Background: Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants formed from incomplete combustion of organic matter; some PAHs are carcinogens. Smoking, diet, and other activities contribute to exposure to PAHs. Exposure data to PAHs among combustible tobacco product users (e.g. cigarette smokers) exist; however, among non-combustible tobacco products users (e.g., e-cigarette users), such data are rather limited.

Objectives: We sought to evaluate exposure to PAHs among participants in Wave 1 (2013-2014) of the Population Assessment of Tobacco and Health (PATH) Study based on the type of tobacco product (combustible vs non-combustible), and frequency and intensity of product use.

Methods: We quantified seven PAH urinary biomarkers in 11,519 PATH Study participants. From self-reported information, we categorized 8327 participants based on their use of tobacco products as never-tobacco user (never user, n = 1700), exclusive current established combustible products user (combustible products user, n = 5767), and exclusive current established non-combustible products user (non-combustible products user, n = 860). We further classified tobacco users as exclusive cigarette user (cigarette user, n = 3964), exclusive smokeless product user (SLT user, n = 509), and exclusive e-cigarette user (e-cigarette user, n = 280). Last, we categorized frequency of product use (everyday vs some days) and time since use (last hour, within 3 days, over 3 days). We calculated geometric mean (GM) concentrations, and evaluated associations between tobacco product user categories and PAH biomarkers concentrations.

Results: Combustible products users had significantly higher GMs of all biomarkers than non-combustible products users and never users; non-combustible products users had significantly higher GMs than never users for four of seven biomarkers. For all biomarkers examined, cigarette users had the highest GMs compared to other tobacco-product users. Interestingly, GMs of 2-hydroxyfluorene, 3-hydroxyfluorene and ∑2,3-hydroxyphenanthrene were significantly higher in SLT users than in e-cigarette users; 3-hydroxyfluorene and 1-hydroxypyrene were also significantly higher in e-cigarette and SLT users than in never users. Everyday cigarette and SLT users had significantly higher GMs for most biomarkers than some days' users; cigarette and SLT users who used the product in the last hour had significantly higher GMs of most biomarkers than other occasional cigarette or SLT users respectively. By contrast, everyday e-cigarette users' GMs of most biomarkers did not differ significantly from those in some days' e-cigarette users; we did not observe clear trends by time of last use among e-cigarette users.

Conclusions: Users of tobacco products had higher PAH urinary biomarker concentrations compared to never users, and concentrations differed by type and frequency of tobacco product use.
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http://dx.doi.org/10.1016/j.envint.2018.11.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331224PMC
February 2019

In vitro evaluation of dual-antigenic PV1 peptide vaccine in head and neck cancer patients.

Hum Vaccin Immunother 2019 12;15(1):167-178. Epub 2018 Oct 12.

a Cancer Research Malaysia , Subang Jaya , Selangor , Malaysia.

Peptide vaccines derived from tumour-associated antigens have been used as an immunotherapeutic approach to induce specific cytotoxic immune response against tumour. We previously identified that MAGED4B and FJX1 proteins are overexpressed in HNSCC patients; and further demonstrated that two HLA-A2-restricted 9-11 amino acid peptides derived from these proteins were able to induce anti-tumour immune responses in vitro independently using PBMCs isolated from these patients. In this study, we evaluated the immunogenicity and efficacy of a dual-antigenic peptide vaccine (PV1), comprised of MAGED4B and FJX1 peptides in HNSCC patients. We first demonstrated that 94.8% of HNSCC patients expressed MAGED4B and/or FJX1 by immunohistochemistry, suggesting that PV1 could benefit the majority of HNSCC patients. The presence of pre-existing MAGED4B and FJX1-specific T-cells was detected using a HLA-A2 dimer assay and efficacy of PV1 to induce T-cell to secrete cytotoxic cytokine was evaluated using ELISPOT assay. Pre-existing PV1-specific T-cells were detected in all patients. Notably, we demonstrated that patients' T-cells were able to secrete cytotoxic cytokines upon exposure to target cells expressing the respective antigen post PV1 stimulation. Furthermore, patients with high expression of MAGED4B and FJX1 in their tumours were more responsive to PV1 stimulation, demonstrating the specificity of the PV1 peptide vaccine. Additionally, we also demonstrated the expression of MAGED4B and FJX1 in breast, lung, colon, prostate and rectal cancer suggesting the potential use of PV1 in these cancers. In summary, PV1 could be a good vaccine candidate for the treatment of HNSCC patients and other cancers expressing these antigens.
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http://dx.doi.org/10.1080/21645515.2018.1520584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363157PMC
February 2020

Interference Potential of Tannins and Chlorophylls in Zebrafish Phenotypic-Based Assays.

Assay Drug Dev Technol 2018 10 9;16(7):408-419. Epub 2018 Jul 9.

Cancer Research Malaysia , Subang Jaya, Selangor, Malaysia .

Natural products are prolific producers of diverse chemical scaffolds, which have yielded several clinically useful drugs. However, the complex features of natural products present challenges for identifying bioactive molecules using high-throughput screens. For most assays, measured endpoints are either colorimetric or luminescence based. Thus, the presence of the major metabolites, tannins, and chlorophylls, in natural products could potentially interfere with these measurements to give either false-positive or false-negative hits. In this context, zebrafish phenotypic assays provide an alternative approach to bioprospect naturally occurring bioactive compounds. Whether tannins and/or chlorophylls interfere in zebrafish phenotypic assays, is unclear. In this study, we evaluated the interference potential of tannins and chlorophylls against efficacy of known small-molecule inhibitors that are known to cause phenotypic abnormalities in developing zebrafish embryos. First, we fractionated tannin-enriched fraction (TEF) and chlorophyll-enriched fraction (CEF) from Camellia sinensis and cotreated them with PD0325901 [mitogen-activated protein kinase-kinase (MEK) inhibitor] and sunitinib malate (SM; anti-[lymph]angiogenic drug). While TEF and CEF did not interfere with phenotypic or molecular endpoints of PD0325901, TEF at 100 μg/mL partially masked the antiangiogenic effect of SM. On the other hand, CEF (100 μg/mL) was toxic when treated up to 6 dpf. Furthermore, CEF at 100 μg/mL potentially enhanced the activity of γ-secretase inhibitors, resulting in toxicity of treated embryos. Our study provides evidence that the presence of tannin and/or chlorophyll in natural products do interfere with zebrafish phenotype assays used for identifying potential hits. However, this may be target/assay dependent and thus requiring additional optimization steps to assess interference potential of tannins and chlorophylls before performing any screening assay.
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http://dx.doi.org/10.1089/adt.2017.833DOI Listing
October 2018

Zerumbone targets the CXCR4-RhoA and PI3K-mTOR signaling axis to reduce motility and proliferation of oral cancer cells.

Phytomedicine 2018 Jan 9;39:33-41. Epub 2017 Dec 9.

Cancer Research Malaysia, No. 1, Jalan SS12/1A, 47500 Subang Jaya, Selangor, Malaysia; Dept of Oral & Maxillofacial Clinical Sciences, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia. Electronic address:

Background: The CXCR4-RhoA and PI3K-mTOR signaling pathways play crucial roles in the dissemination and tumorigenesis of oral squamous cell carcinoma (OSCC). Activation of these pathways have made them promising molecular targets in the treatment of OSCC. Zerumbone, a bioactive monocyclic sesquiterpene isolated from the rhizomes of tropical ginger, Zingiber zerumbet (L.) Roscoe ex Sm. has displayed promising anticancer properties with the ability to modulate multiple molecular targets involved in carcinogenesis. While the anticancer activities of zerumbone have been well explored across different types of cancer, the molecular mechanism of action of zerumbone in OSCC remains largely unknown.

Purpose: Here, we investigated whether OSCC cells were sensitive towards zerumbone treatment and further determined the molecular pathways involved in the mechanism of action.

Methods: Cytotoxicity, anti-proliferative, anti-migratory and anti-invasive effects of zerumbone were tested on a panel of OSCC cell lines. The mechanism of action of zerumbone was investigated by analysing the effects on the CXCR4-RhoA and PI3K-mTOR pathways by western blotting.

Results: Our panel of OSCC cells was broadly sensitive towards zerumbone with IC values of less than 5 µM whereas normal keratinocyte cells were less responsive with IC values of more than 25 µM. Representative OSCC cells revealed that zerumbone inhibited OSCC proliferation and induced cell cycle arrest and apoptosis. In addition, zerumbone treatment inhibited migration and invasion of OSCC cells, with concurrent suppression of endogenous CXCR4 protein expression in a time and dose-dependent manner. RhoA-pull down assay showed reduction in the expression of RhoA-GTP, suggesting the inactivation of RhoA by zerumbone. In association with this, zerumbone also inhibited the PI3K-mTOR pathway through the inactivation of Akt and S6 proteins.

Conclusion: We provide evidence that zerumbone could inhibit the activation of CXCR4-RhoA and PI3K-mTOR signaling pathways leading to the reduced cell viability of OSCC cells. Our results suggest that zerumbone is a promising phytoagent for development of new therapeutics for OSCC treatment.
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http://dx.doi.org/10.1016/j.phymed.2017.12.011DOI Listing
January 2018

Alternative splicing promotes tumour aggressiveness and drug resistance in African American prostate cancer.

Nat Commun 2017 06 30;8:15921. Epub 2017 Jun 30.

Department of Pharmacology and Physiology, School of Medicine and Health Sciences, The George Washington University, Washington, District Of Columbia 20037, USA.

Clinical challenges exist in reducing prostate cancer (PCa) disparities. The RNA splicing landscape of PCa across racial populations has not been fully explored as a potential molecular mechanism contributing to race-related tumour aggressiveness. Here, we identify novel genome-wide, race-specific RNA splicing events as critical drivers of PCa aggressiveness and therapeutic resistance in African American (AA) men. AA-enriched splice variants of PIK3CD, FGFR3, TSC2 and RASGRP2 contribute to greater oncogenic potential compared with corresponding European American (EA)-expressing variants. Ectopic overexpression of the newly cloned AA-enriched variant, PIK3CD-S, in EA PCa cell lines enhances AKT/mTOR signalling and increases proliferative and invasive capacity in vitro and confers resistance to selective PI3Kδ inhibitor, CAL-101 (idelalisib), in mouse xenograft models. High PIK3CD-S expression in PCa specimens associates with poor survival. These results highlight the potential of RNA splice variants to serve as novel biomarkers and molecular targets for developmental therapeutics in aggressive PCa.
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http://dx.doi.org/10.1038/ncomms15921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497057PMC
June 2017

Canthin-6-one Isolated from Bruceajavanica Root Blocks Cancer Cells in the G2/M phase and Synergizes with Cisplatin.

Nat Prod Commun 2017 May;12(5):771-778

Poor prognosis of most cancer patients is in part, due to limited therapeutic options. Furthermore, as chemotherapy remains the standard-of-care for several cancers, partial or lack of response remains a concern and compounding this are the adverse side effects of the treatment that severely impacts the quality of life and survival. In pursuit of improving treatment options, we have opted to investigate the unique chemical skeleton of natural compounds as anticancer therapies. In this study, from an initial screen of 31 crude methanol extracts from -15 plant species using HL60 cells, the root extract of Bruceajavanica (L.) Merr indicated the presence of bioactive compounds. Subsequent bioassay-guided purification on the root extract yielded two alkaloids canthin-6-one (1) and bruceolline J (2), which were further investigated for their bioactivity in representative human cancer lines and normal phenotypic counterparts. MTT assay demonstrated ED50 values from 34.7-72.9 gM for 1 and 16.0-54.0 gM for 2 for the cancer cell lines panel. NP69 cells also demonstrated sensitivity to. both compounds (9.3 piM and 4.5 pM). As amount of 2 isolated were limiting, we focused on 1 to further identify novel anticancer properties in PC3 and HeLa cancer lines. We observed at 30 gM, I induced a G2/M phase arrest coinciding with decreased cell proliferation. Furthermore, I was able to synergize the cytotoxic effect of cisplatin when used in combination, suggesting the potential of combination therapy for those less responsive lesions to standard chemotherapy.
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May 2017

Exome Sequencing Identifies Potentially Druggable Mutations in Nasopharyngeal Carcinoma.

Sci Rep 2017 03 3;7:42980. Epub 2017 Mar 3.

Cancer Research Malaysia, 47500 Subang Jaya, Selangor, Malaysia.

In this study, we first performed whole exome sequencing of DNA from 10 untreated and clinically annotated fresh frozen nasopharyngeal carcinoma (NPC) biopsies and matched bloods to identify somatically mutated genes that may be amenable to targeted therapeutic strategies. We identified a total of 323 mutations which were either non-synonymous (n = 238) or synonymous (n = 85). Furthermore, our analysis revealed genes in key cancer pathways (DNA repair, cell cycle regulation, apoptosis, immune response, lipid signaling) were mutated, of which those in the lipid-signaling pathway were the most enriched. We next extended our analysis on a prioritized sub-set of 37 mutated genes plus top 5 mutated cancer genes listed in COSMIC using a custom designed HaloPlex target enrichment panel with an additional 88 NPC samples. Our analysis identified 160 additional non-synonymous mutations in 37/42 genes in 66/88 samples. Of these, 99/160 mutations within potentially druggable pathways were further selected for validation. Sanger sequencing revealed that 77/99 variants were true positives, giving an accuracy of 78%. Taken together, our study indicated that ~72% (n = 71/98) of NPC samples harbored mutations in one of the four cancer pathways (EGFR-PI3K-Akt-mTOR, NOTCH, NF-κB, DNA repair) which may be potentially useful as predictive biomarkers of response to matched targeted therapies.
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http://dx.doi.org/10.1038/srep42980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5335658PMC
March 2017

Zebrafish phenotypic screen identifies novel Notch antagonists.

Invest New Drugs 2017 04 5;35(2):166-179. Epub 2017 Jan 5.

Cancer Research Malaysia, 12A, Jalan TP5, Taman Perindustrian UEP, 47600, Subang Jaya, Malaysia.

Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G/G cell cycle arrest of ORL-150 cells through inducing p27. Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent.
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http://dx.doi.org/10.1007/s10637-016-0423-yDOI Listing
April 2017

Copper supplementation amplifies the anti-tumor effect of curcumin in oral cancer cells.

Phytomedicine 2016 Nov 9;23(12):1535-1544. Epub 2016 Sep 9.

School of Science, Monash University Malaysia, Selangor 46150, Malaysia. Electronic address:

Background: Oral cancer is the sixth most common cancer worldwide and 90% of oral malignancies are caused by oral squamous cell carcinoma (OSCC). Curcumin, a phytocompound derived from turmeric (Curcuma longa) was observed to have anti-cancer activity which can be developed as an alternative treatment option for OSCC. However, OSCC cells with various clinical-pathological features respond differentially to curcumin treatment.

Hypothesis: Intracellular copper levels have been reported to correlate with tumor pathogenesis and affect the sensitivity of cancer cells to cytotoxic chemotherapy. We hypothesized that intracellular copper levels may affect the sensitivity of oral cancer cells to curcumin.

Methods: We analysed the correlation between intracellular copper levels and response to curcumin treatment in a panel of OSCC cell lines derived from oral cancer patients. Exogenous copper was supplemented in curcumin insensitive cell lines to observe the effect of copper on curcumin-mediated inhibition of cell viability and migration, as well as induction of oxidative stress and apoptosis. Protein markers of cell migration and oxidative stress were also analysed using Western blotting.

Results: Concentrations of curcumin which inhibited 50% OSCC cell viability (IC) was reduced up to 5 times in the presence of 250 µM copper. Increased copper level in curcumin-treated OSCC cells was accompanied by the induction of intracellular ROS and increased level of Nrf2 which regulates oxidative stress responses in cells. Supplemental copper also inhibited migration of curcumin-treated cells with enhanced level of E-cadherin and decreased vimentin, indications of suppressed epithelial-mesenchymal transition. Early apoptosis was observed in combined treatment but not in treatment with curcumin or copper alone.

Conclusion: Supplement of copper significantly enhanced the inhibitory effect of curcumin treatment on migration and viability of oral cancer cells. Together, these findings provide molecular insight into the role of copper in overcoming insensitivity of oral cancer cells to curcumin treatment, suggesting a new strategy for cancer therapy.
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http://dx.doi.org/10.1016/j.phymed.2016.09.005DOI Listing
November 2016

Utilizing Zebrafish to Identify Anti-(Lymph)Angiogenic Compounds for Cancer Treatment: Promise and Future Challenges.

Microcirculation 2016 08;23(6):389-405

Drug Discovery, Cancer Research Malaysia, Subang Jaya, Selangor, Malaysia.

Cancer metastasis which predominantly occurs through blood and lymphatic vessels, is the leading cause of death in cancer patients. Consequently, several anti-angiogenic agents have been approved as therapeutic agents for human cancers such as metastatic renal cell carcinoma. Also, anti-lymphangiogenic drugs such as monoclonal antibodies VGX-100 and IMC-3C5 have undergone phase I clinical trials for advanced and metastatic solid tumors. Although anti-tumor-associated angiogenesis has proven to be a promising therapeutic strategy for human cancers, this approach is fraught with toxicities and development of drug resistance. This emphasizes the need for alternative anti-(lymph)angiogenic drugs. The use of zebrafish has become accepted as an established model for high-throughput screening, vascular biology, and cancer research. Importantly, various zebrafish transgenic lines have now been generated that can readily discriminate different vascular compartments. This now enables detailed in vivo studies that are relevant to both human physiological and tumor (lymph)angiogenesis to be conducted in zebrafish. This review highlights recent advancements in the zebrafish anti-vascular screening platform and showcases promising new anti-(lymph)angiogenic compounds that have been derived from this model. In addition, this review discusses the promises and challenges of the zebrafish model in the context of anti-(lymph)angiogenic compound discovery for cancer treatment.
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http://dx.doi.org/10.1111/micc.12289DOI Listing
August 2016

Genetically-defined novel oral squamous cell carcinoma cell lines for the development of molecular therapies.

Oncotarget 2016 May;7(19):27802-18

Cancer Research Malaysia, Subang Jaya, Selangor, Malaysia.

Emerging biological and translational insights from large sequencing efforts underscore the need for genetically-relevant cell lines to study the relationships between genomic alterations of tumors, and therapeutic dependencies. Here, we report a detailed characterization of a novel panel of clinically annotated oral squamous cell carcinoma (OSCC) cell lines, derived from patients with diverse ethnicity and risk habits. Molecular analysis by RNAseq and copy number alterations (CNA) identified that the cell lines harbour CNA that have been previously reported in OSCC, for example focal amplications in 3q, 7p, 8q, 11q, 20q and deletions in 3p, 5q, 8p, 18q. Similarly, our analysis identified the same cohort of frequently mutated genes previously reported in OSCC including TP53, CDKN2A, EPHA2, FAT1, NOTCH1, CASP8 and PIK3CA. Notably, we identified mutations (MLL4, USP9X, ARID2) in cell lines derived from betel quid users that may be associated with this specific risk factor. Gene expression profiles of the ORL lines also aligned with those reported for OSCC. By focusing on those gene expression signatures that are predictive of chemotherapeutic response, we observed that the ORL lines broadly clustered into three groups (cell cycle, xenobiotic metabolism, others). The ORL lines noted to be enriched in cell cycle genes responded preferentially to the CDK1 inhibitor RO3306, by MTT cell viability assay. Overall, our in-depth characterization of clinically annotated ORL lines provides new insight into the molecular alterations synonymous with OSCC, which can facilitate in the identification of biomarkers that can be used to guide diagnosis, prognosis, and treatment of OSCC.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053689PMC
http://dx.doi.org/10.18632/oncotarget.8533DOI Listing
May 2016

Sprouting Buds of Zebrafish Research in Malaysia: First Malaysia Zebrafish Disease Model Workshop.

Zebrafish 2016 Apr 15;13(2):138-41. Epub 2016 Jan 15.

Drug Discovery Team, Cancer Research Malaysia , Subang Jaya, Malaysia .

Zebrafish is gaining prominence as an important vertebrate model for investigating various human diseases. Zebrafish provides unique advantages such as optical clarity of embryos, high fecundity rate, and low cost of maintenance, making it a perfect complement to the murine model equivalent in biomedical research. Due to these advantages, researchers in Malaysia are starting to take notice and incorporate the zebrafish model into their research activities. However, zebrafish research in Malaysia is still in its infancy stage and many researchers still remain unaware of the full potential of the zebrafish model or have limited access to related tools and techniques that are widely utilized in many zebrafish laboratories worldwide. To overcome this, we organized the First Malaysia Zebrafish Disease Model Workshop in Malaysia that took place on 11th and 12th of November 2015. In this workshop, we showcased how the zebrafish model is being utilized in the biomedical field in international settings as well as in Malaysia. For this, notable international speakers and those from local universities known to be carrying out impactful research using zebrafish were invited to share some of the cutting edge techniques that are used in their laboratories that may one day be incorporated in the Malaysian scientific community.
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http://dx.doi.org/10.1089/zeb.2015.1203DOI Listing
April 2016

Rapid label-free profiling of oral cancer biomarker proteins using nano-UPLC-Q-TOF ion mobility mass spectrometry.

Proteomics Clin Appl 2016 Mar 25;10(3):280-9. Epub 2016 Jan 25.

Department of Chemistry, University of Connecticut, Storrs, CT, USA.

Purpose: It has become quite clear that single cancer biomarkers cannot in general provide high sensitivity and specificity for reliable clinical cancer diagnostics. This paper explores the feasibility of rapid detection of multiple biomarker proteins in model oral cancer samples using label-free protein relative quantitation.

Experimental Design: MS-based label-free quantitative proteomics offer a rapid alternative that bypasses the need for stable isotope containing compounds to chemically bind and label proteins. Total protein content in oral cancer cell culture conditioned media was precipitated, subjected to proteolytic digestion, and then analyzed using a nano-UPLC (where UPLC is ultra-performance liquid chromatography) coupled to a hybrid Q-Tof ion-mobility mass spectrometry (MS).

Results: Rapid, simultaneous identification and quantification of multiple possible cancer biomarker proteins was achieved. In a comparative study between cancer and noncancer samples, approximately 952 proteins were identified using a high-throughput 1D ion mobility assisted data independent acquisition (IM-DIA) approach. As we previously demonstrated that interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A) were readily detected in oral cancer cell conditioned media(1), we targeted these biomarker proteins to validate our approach. Target biomarker protein IL-8 was found between 3.5 and 8.8 fmol, while VEGF-A was found at 1.45 fmol in the cancer cell media.

Conclusions And Clinical Relevance: Overall, our data suggest that the nano-UPLC-IM-DIA bioassay is a feasible approach to identify and quantify proteins in complex samples without the need for stable isotope labeling. These results have significant implications for rapid tumor diagnostics and prognostics by monitoring proteins such as IL-8 and VEGF-A implicated in cancer development and progression. The analysis in tissue or plasma is not possible at this time, but the subsequent work would be needed for validation.
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http://dx.doi.org/10.1002/prca.201500025DOI Listing
March 2016

Identification of Four-Jointed Box 1 (FJX1)-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma.

PLoS One 2015 4;10(11):e0130464. Epub 2015 Nov 4.

Cancer Research Initiatives Foundation (CARIF), Sime Darby Medical Centre, Subang Jaya, Selangor, Malaysia.

Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9-20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130464PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633155PMC
June 2016

Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation.

Cancer Sci 2015 Oct 25;106(10):1333-40. Epub 2015 Sep 25.

Human Genetics Research Group, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.

Human papillomavirus (HPV) oncoproteins drive distinctive promoter methylation patterns in cancer. However, the underlying mechanism remains to be elucidated. Cyclin A1 (CCNA1) promoter methylation is strongly associated with HPV-associated cancer. CCNA1 methylation is found in HPV-associated cervical cancers, as well as in head and neck squamous cell cancer. Numerous pieces of evidence suggest that E7 may drive CCNA1 methylation. First, the CCNA1 promoter is methylated in HPV-positive epithelial lesions after transformation. Second, the CCNA1 promoter is methylated at a high level when HPV is integrated into the human genome. Finally, E7 has been shown to interact with DNA methyltransferase 1 (Dnmt1). Here, we sought to determine the mechanism by which E7 increases methylation in cervical cancer by using CCNA1 as a gene model. We investigated whether E7 induces CCNA1 promoter methylation, resulting in the loss of expression. Using both E7 knockdown and overexpression approaches in SiHa and C33a cells, our data showed that CCNA1 promoter methylation decreases with a corresponding increase in expression in E7 siRNA-transfected cells. By contrast, CCNA1 promoter methylation was augmented with a corresponding reduction in expression in E7-overexpressing cells. To confirm whether the binding of the E7-Dnmt1 complex to the CCNA1 promoter induced methylation and loss of expression, ChIP assays were carried out in E7-, del CR3-E7 and vector control-overexpressing C33a cells. The data showed that E7 induced CCNA1 methylation by forming a complex with Dnmt1 at the CCNA1 promoter, resulting in the subsequent reduction of expression in cancers. It is interesting to further explore the genome-wide mechanism of E7 oncoprotein-mediated DNA methylation.
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http://dx.doi.org/10.1111/cas.12761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4638020PMC
October 2015

Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling.

Sci Rep 2015 Jun 22;5:11541. Epub 2015 Jun 22.

Department of Pharmacology and Physiology, The George Washington University, Washington, DC.

Functional expression of voltage-gated Na(+) channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.
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http://dx.doi.org/10.1038/srep11541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476109PMC
June 2015

Identification and Functional Validation of Reciprocal microRNA-mRNA Pairings in African American Prostate Cancer Disparities.

Clin Cancer Res 2015 Nov 18;21(21):4970-84. Epub 2015 Jun 18.

Department of Pharmacology and Physiology, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia.

Purpose: African Americans (AA) exhibit higher rates of prostate cancer incidence and mortality compared with European American (EA) men. In addition to socioeconomic influences, biologic factors are believed to play a critical role in prostate cancer disparities. We investigated whether population-specific and -enriched miRNA-mRNA interactions might contribute to prostate cancer disparities.

Experimental Design: Integrative genomics was used, combining miRNA and mRNA profiling, miRNA target prediction, pathway analysis, and functional validation, to map miRNA-mRNA interactions associated with prostate cancer disparities.

Results: We identified 22 AA-specific and 18 EA-specific miRNAs in prostate cancer versus patient-matched normal prostate, and 10 "AA-enriched/-depleted" miRNAs in AA prostate cancer versus EA prostate cancer comparisons. Many of these population-specific/-enriched miRNAs could be paired with target mRNAs that exhibited an inverse pattern of differential expression. Pathway analysis revealed EGFR (or ERBB) signaling as a critical pathway significantly regulated by AA-specific/-enriched mRNAs and miRNA-mRNA pairings. Novel miRNA-mRNA pairings were validated by qRT-PCR, Western blot, and/or IHC analyses in prostate cancer specimens. Loss/gain of function assays performed in population-specific prostate cancer cell lines confirmed miR-133a/MCL1, miR-513c/STAT1, miR-96/FOXO3A, miR-145/ITPR2, and miR-34a/PPP2R2A as critical miRNA-mRNA pairings driving oncogenesis. Manipulating the balance of these pairings resulted in decreased proliferation and invasion, and enhanced sensitization to docetaxel-induced cytotoxicity in AA prostate cancer cells.

Conclusions: Our data suggest that AA-specific/-enriched miRNA-mRNA pairings may play a critical role in the activation of oncogenic pathways in AA prostate cancer. Our findings also suggest that miR-133a/MCL1, miR-513c/STAT1, and miR-96/FOXO3A may have clinical significance in the development of novel strategies for treating aggressive prostate cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-1566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631799PMC
November 2015

P53 polymorphism at codon 72 is associated with keratocystic odontogenic tumors in the Thai population.

Asian Pac J Cancer Prev 2015 ;16(5):1997-2001

Human Genetics Research Group, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok, Thailand E-mail :

Objective: To clarify the association between the p53 polymorphism at codon 72 and susceptibility to the sporadic keratocystic odontogenic tumor (KCOT).

Design: One hundred KCOTs and 160 match-group healthy controls were genotyped to ascertain the frequency of the p53 codon 72 polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), confirmed by direct sequencing.

Results: The frequencies of the Pro/Pro, Arg/Pro, and Arg/Arg genotypes were 23.8%, 49.4%, and 26.9%, respectively, in the controls, while the KCOT cohort demonstrated 43.0%, 39.0%, and 18.0%, respectively. Further analysis suggested that p53 Pro could be a KCOT-susceptible allele (OR (95%CI)=1.77 (1.22 to 2.59), p=0.0024), with a sex-adjusted OR (95%CI) of 1.71 (1.17-2.50), p=0.0046. Moreover, the results indicated that p53 codon 72 Pro homozygous was associated with a two-fold risk of developing KCOT (adjusted OR (95%CI) =2.17(1.23-3.84), p=0.0062).

Conclusions: The C/C genotype of P53 gene codon 72 increases the risk of developing sporadic KCOT and may be a useful tool for screening and diagnostic purposes.
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http://dx.doi.org/10.7314/apjcp.2015.16.5.1997DOI Listing
December 2015

Fluorescent, bioactive protein nanoparticles (prodots) for rapid, improved cellular uptake.

Bioconjug Chem 2015 Mar 2;26(3):396-404. Epub 2015 Feb 2.

†Department of Chemistry and Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3060, United States.

A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (Prodots) and their facile uptake into the cytoplasm of cells is described here. Prodots made from bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) were found to be 15-50 nm wide and have been characterized by gel electrophoresis, transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and optical microscopic methods. Data showed that the secondary structure of the protein in Prodots is retained to a significant extent and specific activities of nGO, nHRP, nCatalase, and nLipase were 80%, 70%, 65%, and 50% of their respective unmodified enzyme activities. Calorimetric studies indicated that the denaturation temperatures of nGO and nBSA increased while those of other Prodots remained nearly unchanged, and accelerated storage half-lives of Prodots at 60 °C increased by 4- to 8-fold. Exposure of nGO and nBSA+ nGO to cells indicated rapid uptake within 1-3 h, accompanied by significant blebbing of the plasma membrane, but no uptake has been noted in the absence of nGO. The presence of nGO/glucose in the media facilitated the uptake, and hydrogen peroxide induced membrane permeability could be responsible for this rapid uptake of Prodots. In control studies, FITC alone did not enter the cell, BSA-FITC was not internalized even in the presence of nGO, and there has been no uptake of nBSA-FITC in the absence of nGO. These are the very first examples of very rapid cellular uptake of fluorescent nanoparticles into cells, particularly nanoparticles made from pure proteins. The current approach is a simple and efficient method for the preparation of bioactive, fluorescent protein nanoparticles of controllable size for cellular imaging, and cell uptake is under the control of two separate chemical triggers.
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http://dx.doi.org/10.1021/bc500621hDOI Listing
March 2015