Molecular characterization of Hepatitis C virus 3a in Peshawar

Background The purpose of this study was to explore molecular epidemiology of HCV genotype 3a in Peshawar based on sequencing and phylogenetic analysis of Core region of HCV genome. Methods Chronically infected Hepatitis C virus infected patients enrolled under the Prime Minister Hepatitis C control program at three Tertiary care units of Peshawar [Khyber Teaching Hospital Peshawar, Lady Reading Hospital Peshawar, Hayat Abad Medical Complex Peshawar] were included in this cross sectional observational study. Qualitative detection of HCV and HCV genotyping was carried out by a modified reverse transcription-polymerase chain reaction (RT-PCR) and type specific genotyping assay. The Core gene of HCV genotype 3a was amplified, cloned and sequenced. The sequences obtained were used for phylogenetic analysis using MEGA 6 software. Results Among the 422 (82.75 %) PCR positive samples, 192 (45.5 %) were identified as having HCV genotype 3a infection. HCV Core gene sequencing was carried out randomly for the characterization of HCV 3a. Nucleotide sequence analysis of the obtained viral genomic sequences based on partial HCV 3a Core gene sequences with reference sequences from different countries showed that our sequences clustered with some local and regional sequences with high bootstrap values. Conclusion HCV 3a is highly prevalent in Peshawar, Pakistan and its phylogenetics based on Core gene sequences indicate the prevalence of different lineages of HCV 3a in Peshawar which may have consequences for disease management strategies causing more economic pressure on the impoverished population due to possible antiviral resistance.

Background
Hepatitis C virus (HCV), first identified in 1989 is the primary cause of chronic liver diseases including cirrhosis (60-85 %) and hepatocellular carcinoma [1], with an annual mortality rate of 3.5-5.5 million due to complications of end stage liver diseases [2]. An estimated 185 million people (3 % of the world's population) are infected worldwide with relatively high prevalence rates in developing countries [3,4]. In Pakistan, approximately 10-17 million people are chronically infected with HCV [5,6] with an overall prevalence rate of 5 % [7]. Frequency of HCV infection differs among the four provinces with an estimated 1.1 million people infected in KPK [8,9].
HCV is an enveloped, positive sense ssRNA genome [10] in the family Flaviviridae consisting of an oral reading frame (ORF) approximately of 9.6 kb in length [11]. HCV genome shows substantial nucleotide sequence variability in both the structural and nonstructural coding regions, with different isolates of HCV showing as much as 30 % nucleotide sequence divergence over the entire genome which is sufficient to alter the antigenic and biological characteristics of seven major genotypes of HCV [12]. These genotypes have distinct geographical distributions. Although HCV genotypes 1, 2 and 3 appear to have a worldwide distribution, their relative prevalence varies from one geographic area to another [13]. Identification of HCV genotype is extremely important as different genotypes are relevant to the epidemiology and clinical management of chronic HCV infection and is the strongest predictive parameter for sustained virological response [14].
The primary means to identify and classify new genotypes is either by phylogenetic analysis of sequences or by measures of pairwise sequence similarity [15]. Although it is generally true that longer sequences are more informative for classification, it is usually possible to identify genotypes by sequence comparison of relatively short sub genomic regions of HCV [16]. While region encoding the 5′ non-coding region (5′ NCR) is too conserved for HCV subtypes identification, sequencing and phylogenetic analysis of nucleotide sequences amplified in the region of the genome encoding the Core protein is frequently used for classification of HCV into genotypes and subtypes [17][18][19]. HCV Core gene has sufficient genetic diversity and can produce topologically identical trees to those obtained upon analysis of complete genome sequences [20].
Molecular epidemiological studies previously conducted in Peshawar, Pakistan have indicated that the most prevalent HCV subtype is 3a accounting for 70-90 % of HCV infections based solely on type specific PCR based genotyping methods developed 15 to 20 years ago [21,22]. At present, there is little information about the genetic history and evolution of HCV genotype 3a in Peshawar which needs to be investigated for several reasons including increasing antiviral resistance in the case of HCV 3a in Peshawar, KP province and its evolutionary relationship with other regional or global isolates. As the prevalence of HCV 3a has been reported to be very high among the general population of Peshawar, therefore, we embarked on the current study to investigate it at molecular level by Type-specific assay, sequencing of the Core gene and phylogenetic analysis in order to have a clear epidemiological picture of the prevalent HCV 3a isolates.

Sampling
Chronically infected Hepatitis C virus infected patients enrolled under the Prime minister Hepatitis C control program at three Tertiary care units [Khyber Teaching Hospital Peshawar, Lady Reading Hospital Peshawar, Hayat Abad Medical Complex Peshawar] willingly participated in this observational study. A non-probability convenient sampling technique was used to collect data. Written consent was obtained from all the eligible study participants for participation and publication of the data and the Institutional Ethics Committee (Khyber Medical University Ethics Board) approved the study. Blood samples were collected from the patients in sterile vacutainers and sera extraction was carried out at the Institute of Biotechnology and Genetic Engineering Peshawar.

RNA Extraction
Total RNA was extracted from each sample using Favor-Gen RNA isolation Kit (Favorgen Biotech corp, Taiwan, CAT No FAVNK 001) according to the manufacturer's instructions. The isolated RNA was stored either stored at-80C or immediately used for RT PCR.

HCV Genotyping
Reverse Transcription PCR followed by Type-specific PCR for identification of HCV subtypes was carried out essentially according to [23] with modifications in the Universal and Type-specific primers according to the latest HCV sequencing data ( Table 1).

Amplification of HCV 3a Core gene
A representative number of samples (58/192), which turned out positive for HCV 3a by the Type-specific assay described earlier, were used for a second round of RNA extraction, RT PCR and subsequent regular nested PCR for the amplification of Core gene using genespecific primer [24].
Cloning and sequencing of the Core gene The Core gene products were eluted from agarose gel using Pure Link™ Quick Gel Extraction Kit (https:// www.thermofisher.com/pk/en/home.html). The eluted products were cloned in pGEMT-Easy vector and transformed in DH5-alfa strain of E.coli (Promega). Sequencing PCR of the Core gene was carried out using vector-specific sense and antisense primers in a sequencing PCR (Big Dye Deoxy Terminator method). Bidirectional sequencing run was performed using ABI PRISM 310 DNA sequencer [Applied Biosystems].

Blast analysis
In case of each isolate, three independent clones were sequenced. The respective sequences were aligned and

Phylogenetic analysis
HCV Core gene sequences from various geographical regions of the world were retrieved from the online sequence repositories and Phylogenetic tree was constructed by using Mega6 [25].

Statistical analysis
SPSS version 20 was used for data analysis. The qualitative variables were described using percentage and the quantitative variables were described using the mean, median and standard deviation.

Results
For identification of HCV genotype 3a infection among the actively infected HCV samples, RT PCR followed by Type specific nested PCR based genotyping assay was carried out.

Discussion
Accumulation of nucleotide substitutions in the HCV genome results in diversification and evolution into seven major genotypes and a series of subtypes [26]. The classification of HCV by viral genotype is not only important for appropriate treatment regimen and assessment of global viral evolution but their epidemiological data can reveal transmission activity and migration movement of infected individuals from the endemic area [27][28][29]. These viral types and subtypes show differing distribution in different geographic regions which have provided investigators with an epidemiologic marker that can be used to trace the source of HCV infection in a given population [30]. Sequencing and phylogenetic analysis of HCV Core gene nucleotide sequences has earlier been used for identification and classification of HCV isolates into genotypes and subtypes [19]. HCV genotype 3a is the most abundant in Pakistan and it has earlier been documented that HCV genotype 3a is relatively more responsive to therapy as compared to genotype 1 or 4 [28,29,31]. However, in Pakistan resistance is being observed in case of relatively responsive and easy to treat HCV genotype 3a [32]. The present study is the first report describing the genotypic and evolutionary analysis of HCV 3a isolated from chronic HCV infected patients of Peshawar, based on a modified genotyping assay and subsequent sequencing and phylogenetic analysis. Investigation of the different circulating genotypes and their evolution is not only crucial for epidemiological and clinical analysis but might be helpful for the improvement of diagnostic tests and treatment regimens [33]. HCV infection is highly prevalent in Pakistan with an overall prevalence rate of 5 % [34]. The estimated HCV prevalence in KP province is 1.1 % [8]. In this study, out of total 510 seroactive patients investigated, 82.75 % were actively infected with HCV having high proportion of HCV genotype 3a (45.50 %) which is in agreement with most recent studies in Peshawar and Pakistan reporting HCV 3a to be the most prevalent genotype [35,36], however the percentage prevalence is lower than the previous reports claiming much higher prevalence of HCV genotype 3a (60-74 %) [22,37]. One possible explanation for this changing HCV genotype 3a landscape could be the change in epidemiological pattern over times as a result of people migration [38] or the inadequate sensitivity of old genotyping assays to correctly type the circulating viral strains due to substantial genetic heterogeneity inherent to RNA viruses [26]. Moreover the emerging 69 Fig. 1 Phylogenetic tree of HCV 3a Core gene sequences constructed by Maximum Likelihood algorithm (MEGA 6 Software) with Bootstrap values shown on the branches. Tree shows the phylogenetic relationship of twelve newly reported sequences, marked in red with 36 Core gene sequences from different geographical regions. The isolate/country/Accession no of the sequences are shown in figure resistance to interferon therapy experienced in case of HCV genotype 3a [32] might possibly be due to the less prevalent genotypes other than HCV 3a or variants of HCV 3a evolved overtimes which can cause substantial economic and health burden over the infected population. Internationally accepted guidelines for the treatment of hepatitis C are rarely followed in KPK and people undergo successive therapies once the initial response of antiviral drugs is negative [32]. Due to lack of awareness, the practice mentioned above is not only causing economic and health related losses but is also contributing towards the evolution of more resistant HCV 3a types.
Molecular evolutionary analysis of the obtained viral genome sequences revealed that some HCV 3a isolates of Peshawar clustered closer to local isolates ( Fig. 1) indicating the previous existence of similar types in other parts of Pakistan, which may have spread to Peshawar, KPK province via various transmission routes. Some of the HCV 3a isolates grouped with European, south Asian, Iranian and Chinese HCV 3a isolates (Fig. 1). Peshawar city is located in the northwestern region of Pakistan. It shares international border with the Afghanistan which has been home to conflicts for the past 40 years resulting in migration of various ethnic groups into and out of Afghanistan. These migrations have changed epidemiological patterns of various pathogens overtimes including HCV. Phylogenetic analysis indicate that foreign presence in Afghanistan and migration of Afghanis to Peshawar might have contributed towards the spread of isolates which are genetically closer to European, Indian, Chinese and Iranian isolates as HCV genotype 3a is also highly prevalent in neighboring countries including China [38], India [39] and Iran [40].
This study has some limitations. Due to convenient sampling, selection bias might have occurred. Moreover we have characterized HCV 3a entirely on the bases of partial core gene sequences. To elucidate the epidemiology of emerging HCV 3a in Peshawar and to further improve the accuracy of diagnostic assays and treatment regimens, there is a need to analyze complete coding sequences of more diverse regions of HCV genome on a comparatively large scale.

Conclusion
This study concludes that HCV 3a is highly prevalent in Peshawar, Pakistan and its phylogenetics based on Core gene sequences indicate the prevalence of different lineages of HCV 3a in Peshawar which may have consequences for disease management strategies causing more economic pressure on the impoverished population due to possible antiviral resistance.

Availability of data and materials
The datasets supporting the conclusions of this article are available in the GenBank [National Center for Biotechnol- ogy

Competing interests
None of the authors have any competing interests in the manuscript.
Authors' contributions IA and JA designed the study. AG, IA, FZ and NZ carried out the experimental work. JA, IA and IAK helped with data analysis, writing the MS and reviewing it thoroughly. All authors read and approved the final MS.
Authors' information IA is a Fulbrighter working at CIIT Islamabad as Associate Professor and Leader of the Virology group. JA is a professor and director of the Institute of Basic Medical Sciences, Khyber Medical University Peshawar. His major interest is microbiology. AG is a PhD scholar while FZ and NZ are graduate students who have worked in IA's lab. IAK is a professor at the University of Agriculture Peshawar and his major interest is entomology and microbiology.


extfieldsofstudy: Biology Medicine

text:
HOXA9 is Underexpressed in Cervical Cancer Cells and its Restoration Decreases Proliferation, Migration and Expression of Epithelial-to-Mesenchymal Transition Genes

HOX transcription factors are evolutionarily conserved in many different species and are involved in important cellular processes such as morphogenesis, differentiation, and proliferation. They have also recently been implicated in carcinogenesis, but their precise role in cancer, especially in cervical cancer (CC), remains unclear. In this work, using microarray assays followed by the quantitative polymerase chain reaction (qPCR), we found that the expression of 25 HOX genes was downregulated in CC derived cell lines compared with non-tumorigenic keratinocytes. In particular, the expression of HOXA9 was observed as down-modulated in CC-derived cell lines. The expression of HOXA9 has not been previously reported in CC, or in normal keratinocytes of the cervix. We found that normal CC from women without cervical lesions express HOXA9 ; in contrast, CC cell lines and samples of biopsies from women with CC showed significantly diminished HOXA9 expression. Furthermore, we found that methylation at the first exon of HOXA9 could play an important role in modulating the expression of this gene. Exogenous restoration of HOXA9 expression in CC cell lines decreased cell proliferation and migration, and induced an epithelial-like phenotype. Interestingly, the silencing of human papilloma virus (HPV) E6 and E7 oncogenes induced expression of HOXA9 . In conclusion, controlling HOXA9 expression appears to be a necessary step during CC development. Further studies are needed to delineate the role of HOXA9 during malignant progression and to afford more insights into the relationship between downmodulation of HOXA9 and viral HPV oncoprotein expression during cercical cancer development.

Introduction
Cervical Cancer (CC) is the fourth most common cancer in women, and the seventh overall, with an estimated 528,000 new cases in 2012 (Ferlay et al., 2015). Development of this disease is closely associated with human papilloma virus (HPV) infection (Bosch and Munoz, 2002); however, it is estimated that only 0.03% of women who become infected with HPV proceed to develop CC (Sasagawa et al., 2012). Therefore, viral infection per se is not considered sufficient to generate a neoplastic process; it is, rather, a first alteration that predisposes cells to subsequent changes; if those do occur, the neoplasm is generated (Perez-Plasencia et al., 2008). It is evident that additional factors, such as genomic instability caused probably by viral oncogenes (E6 and E7), environmental factors, or the genetic background itself, are necessary for cervical tumorigenesis (Hyland et al., 2011). Alterations in developmental signaling pathways or transcription factors that regulate the ontogeny, such HOX genes, have recently been proposed as alternative secondary modifications that could lead to malignant transformation (Karamboulas and Ailles, 2013).
HOX genes in humans comprise a highly conserved family of 39 transcription factors that are grouped into four clusters: HOXA; B; C, and D, each group consisting of 13 paralog genes, with 9-11 members, distributed on the basis of sequence similarity and relative position within the group (Acampora et al., 1989). HOX genes play an important role in the delicate balance between cell proliferation and differentiation that is essential for normal fetal development during embryogenesis; the abnormal function of these transcription factors has been implicated in human diseases, particularly in neoplastic processes (Castelli-Gair, 1998;Lappin et al., 2006). Thus, in recent years, much effort has been devoted to the study of HOX genes and proteins and their link with cancer (Shah and Sukumar, 2010). In cervix, despite some evidences of HOX member modulation (Alami et al., 1999;Hung et al., 2003;Lopez et al., 2006b;Gonzalez-Herrera et al., 2015), the participation of these transcription factors in cervical carcinogenesis has not been explored in depth. In this work, we evaluated HOX gene deregulation in CC; in particular, we focused on HOXA9 modulation and whether this phenomenon results in an advantage for the CC cell.

Cell culture
HeLa, SiHa, and C-33A cell lines derived from CC, as well as the non-tumorigenic keratinocyte HaCaT cell line, were obtained from the generosity of the German Cancer Research Center (DKFZ, Heidelberg, Germany). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing GlutaMAXTM and supplemented with 10% Fetal Bovine Serum (FBS), 100 U/ml Penicillin, and 100 µg/ml Streptomycin, at 37°C with an atmosphere of 5% CO2 and 90% relative humidity (all of the previously mentioned products were acquired from GIBCO®, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were propagated according to the methods recommended by the suppliers.

Cervical samples and HPV detection
Informed consent was obtained from all individual participants included in the study (Prot.R-2012-785-090). Patients were recruited at the Western Medical Center of the Mexican Institute of Social Security (IMSS) and Cervical scrapes of clinically healthy women without HPV infection were collected with a cytobrush during gynecological examination and placed into PreservCyt transport medium (Hologic, Bedford, MA, USA). Cervical biopsies of patients diagnosed with Squamous Cell Carcinoma (SCC) and with HPV infection were collected, frozen, and stored until processing. HPV detection was performed as follows: genomic DNA was extracted from all samples and screened by conventional PCR utilizing the PGMY 09/11 primers, as described elsewhere (Gravitt et al., 2000), and samples that were positive to HPV were further analyzed using the Linear Array HPV Genotyping Test (Roche Applied Science, Penzberg, Germany) (Coutlee et al., 2006).

Isolation of primary keratinocytes
Primary Keratinocytes (Kers) were obtained from the normal cervixes of women who underwent hysterectomy, and the samples were taken under signed informed consent at the Western Medical Center -IMSS. Biopsies were collected in EPILIFE CF medium (Thermo Fisher Scientific, Inc.) with 100 U/ml Penicillin and 100 μg/ ml Streptomycin. After removal of connective and fatty tissue, the biopsy was chopped into 0.5-cm2 fragments and treated with 25 U/ml collagenase (Sigma-Aldrich Quimica, S. A. de R.L. de C.V, Toluca, México) and 25 U/ml dispase (Thermo Fisher Scientific, Inc.) overnight at 4oC with constant movement. Then, the epidermis was removed from connective tissue with tweezers. Epithelial cells were disrupted by incubation for 30 min at 37 o C in 2 ml of 0.25% trypsin-EDTA solution (Thermo Fisher Scientific, Inc.). Trypsin was neutralized with FBS and the cells were collected by centrifugation and resuspended in 13 ml of selective medium for keratinocytes EPILIFE CF supplemented with Human Keratinocyte Growth Supplement containing: 0.2% v/v pituitary gland extract (BPE), 5 μg/ml bovine insulin, 0.18 μg/ml hydrocortisone, 5 μg/ml bovine transferrin, 0.2 ng/ml human Epidermal Growth Factor (EGF), and Gentamicin/Amphotericin (all from Thermo Fisher Scientific, Inc.). The cultures were maintained at 37 o C in a humidified atmosphere with 5% CO 2 .

RNA isolation and expression microarrays
Total RNA from the different cell lines, cervical scrapes, and biopsies was extracted with the NucleoSpin RNA Kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer's instructions. RNA was quantified by absorbance at 260/280 nm. For microarray analysis, RNA quality was determined using an Agilent 2100 Bioanalyzer and the RNA 6000 NanoChip Kit (Agilent Technologies, Santa Clara, CA, USA). Double stranded complementary DNA (cDNA) was generated from 10µg of RNA using the cDNA Synthesis Kit System (Roche Applied Science) and purified with the GenEluteTM PCR Clean-Up Kit (Sigma-Aldrich Quimica, S. A. de R.L. de C.V.); thereafter, the cDNA was labeled with Cy3 and hybridized in a Human Gene Expression Array 12x135K (Roche Applied Science). Microarrays were scanned on the MS200 Scanner and the data obtained was processed using DEVA ver 1.2 software (Roche Applied Science). Fluorescence intensities were normalized using the RMA algorithm and the information was subsequently analyzed with CLC Main WorkBench ver. 7.0.3 software. Genes that were differentially expressed with a fold-change >1.5 and that exhibited a permutation p-value <0.05 were selected.

RT-PCR and qPCR assays
cDNA synthesis was performed from 5 µg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science) primed with Oligo dT. The cDNA obtained was employed in a quantitative Polymerase Chain Reaction (qPCR) to evaluate gene expression levels utilizing the LightCycler® FastStart DNA Master PLUS SYBR Green I Kit with 2.0 LightCycler technology (Roche Applied Science) under conditions specified by the manufacturer. The gene-specific primers (Supplementary  Table 1) were designed with Oligo Primer Analysis ver. 6.0 software (Molecular Biology Insights, Inc., Colorado Springs, CO, USA) from sequences obtained at the Entrez Nucleotide Database of the National Center of Biotechnology Information (NCBI). Quantification of PCR products was calculated employing Light Cycler ver. 4.1 software and was calculated using the following reference genes: GAPDH; ACTB; RPLP0, and RP18S.
Two types of analysis were used: 2ΔΔCP and ΔCP. A melting curve analysis was also performed for each gene to determine the specificity of the reaction.

CpG methylation detection assay
To determine the presence of CpG islands, analysis of 1,400-bp upstream and 800-bp downstream of translation start was performed using the MethPrimer online tool (Li and Dahiya, 2002). Additionally, localization of restriction sites for MspI and HpaII restriction enzymes was determined utilizing CLC Main Workbench ver. 7.0 software (Qiagen, Germantown, MD, USA). Specific primer pairs were designed using Oligo Primer Analysis ver. 6.0 software (Molecular Biology Insights, Inc.) to amplify five different zones (Z1-Z5), which cover the different CpG islands found (Supplementary Table 2, Figure 3a). Genomic DNA from cell lines and primary keratinocytes was extracted with the QIAamp DNA Mini Kit (Qiagen) and digested with MspI restriction enzyme, and its isoschizomer non-methylation-sensitive HpaII (New England BioLabs, Inc., Ipswich, MA, USA) at 37 o C for 1 h. The digested DNA was used as template for PCR amplification reactions utilizing the Expand High Fidelity enzyme (Sigma-Aldrich Quimica S. de R.L. de C.V.) as recommended by the manufacturer. The amplification products were separated by electrophoresis in 2% agarose gels, visualized, and digitalized for analysis employing a DigiDoc-It system program (UVP, LLC. Upland, CA, USA).

Immunohistochemistry
Histologically normal cervical epithelium (35) and CC (21) samples that were paraffin-embedded were utilized to construct a Tissue MicroArray (TMA) using the Tissue Microarray ATA 100 (Chemicon, Temecula, CA, USA). TMA slices were used for automated immunodetection assays utilizing the Ventana BenchMark System (Roche Applied Science), the anti-HOXA9 antibody (cat. Ab922565; Abcam, Cambridge, MA, USA), and the ultraView Universal DAB Detection Kit (Roche Applied Science). HOXA9 expression was evaluated by a Pathologist and classified according to level of positivity as low, medium, or high.

Clonogenicity assays and colony formation
Cell culture plates (p60) were seeded with 5 x 10 4 cells in 3 ml of medium; after 24 h of culture, the plates were infected with 2 x 10 s Inclusion-Forming Unit (IFU) of supernatant containing viral particles (LVX-Puro-HOXA9 and LVX-Puro empty virus); as control, one plate remained uninfected. At 48-h post-infection, Puromycin was added to cultures at a final concentration of 1 µg/ml. Five days later, the cells were fixed with 3.7% paraformaldehyde, stained with 0.05% crystal violet, and photo documented. Assays were performed in triplicate. This infection scheme was also evaluated by using the xCELLigence system (Roche Applied Science).

Cell proliferation and viability assays
The cell proliferation of HOXA9-expressing HeLa cells (in the presence of Doxycycline) was assessed with the xCELLigence system platform (Roche Applied Science). Briefly, 2.5 x 10 3 cells per well were seeded on a 96-plate E-Plate wells and introduced into the xCELLigence reading station. After 24 h, Doxycycline was added to the culture cells to induce HOXA9 expression; proliferation was assessed in real time every 30 min during 72 h. Three independent experiments were performed with four duplicates in each case. Cell viability was analyzed by the cleavage of tetrazolium salt Water Soluble Tetrazolium Salts (WST-1) to formazan (Roche Applied Science) by reading the absorbance at 440 nm on a microtiter plate reader (Synergy™ HT Multi-Mode Microplate Reader; Biotek Instruments, Inc., Winooski, VT, USA). The value of HOXA9 uninfected cells (HeLa TET) was employed as 100% cell survival.

Migration assays
Hela-LVX and HeLa-LVX-HOXA9 cells were seeded onto a p60 plate (5 x 10 4 cells in 3 ml of medium); after 24 h of culture, the surface of each plate was gently scratched with a cell scraper; cells were washed with PBS, and 3 ml of fresh DMEM medium was added. Photographs were taken at 0, 24, and 48 h to evaluate the scratches. The photographs were analyzed with ImageJ ver. 1.49 software and the Wound Healing Tool plug-in, which evaluates the wound area (implying the area not covered by cells). The data obtained were graphed employing Microsoft Excel.

Apoptosis detection
Apoptosis detection was performed by Flow Cytometry (FC) using Annexin-V-FLUOS/Propidium iodide (Roche Applied Science) as markers of cell death. In brief, cells were treated with cold PBS/EDTA and harvested by scraping, packed by centrifugation and washed twice with PBS. Apoptotic cells were incubated for 15 min at room temperature with Annexin-V-FLUOS in combination with Propidium Iodide (PI). The percentage of positive cells was determined by FC using an Attune Cytometer (Thermo Fisher Scientific, Inc).

Statistical analysis
All experiments were performed in duplicate and repeated independently at least twice. The results are expressed as means ± Standard Deviation (SD). For differences between groups, analysis was performed using an unpaired, two-tailed Student t -test and statistical significance at set at p<0.05.

Altered HOX Gene Expression Between Non-tumorigenic Keratinocytes (HaCaT) and CC-derived Cells (HeLa)
With the aim of determining HOX genes differentially expressed in CC-derived cells, a genetic expression comparison between HeLa (CC-derived cell line) and HaCaT (non-tumorigenic keratinocyte derived cell line) using a microarray-based platform (see Materials and Methods) was performed. Variations in the expression levels of HOX genes were calculated taking the corresponding expression in HaCaT cells as reference and applying a Student t -test to discriminate differences in expression with a p value of <0.05. From this analysis, it was observed that of the 39 human HOX genes, the expression of 26 members remain unchanged in both cell lines; however HOXA3, HOXA9, HOXB4, HOXC12, HOXC13, HOXD10, and HOXD11 were observed with lower expression in HeLa cells compared with HaCaT, and for HOXA11, HOXB3, HOXB5, HOXB6, HOXB9, and HOXB13, increased expression was observed ( Figure  1a). To validate the observations of the microarray experiments, differentially expressed genes were evaluated by quantitative real time PCR in HaCaT and HeLa cells. For these assays, three reference genes (GAPDH, PGK1, and RPLP0) were used for normalization, and expression of HaCaT cells was utilized as calibrator (set as 1). With the exception of HOXD11, the expression of all analyzed genes was confirmed by qPCR ( Figure 1b). The downmodulation of HOXA9 is noteworthy, with a relative expression value of 0.09. This gene is involved in the regulation of endothelial cell proliferation and has been related with a tumorigenic suppression in some epithelial models. In order to evaluate the possible participation of HOXA9 in cervical carcinogenesis, we analyzed this gene further.

HOXA9 mRNA Expression Is Diminished in CC Cell Lines
With the purpose of knowing whether downregulation of HOXA9 is a common characteristic of CC cells the expression of this gene was analyzed by qPCR in additional CC-derived cell lines: SiHa (positive to HPV16), and C-33A (negative to HPV infection). Because SYBR green-based detection was used for the qPCR, the melting curves of the amplicons were determined to discard unspecific amplifications, and the expected Molecular Weights (MW) of qPCR products were also confirmed on agarose gels (Figure 2a). The relative expression of HOXA9 was determined in HeLa, SiHa, and C-33A cells using the expression of HaCaT as calibrator. A significant decrease in HOXA9 levels was observed in the three tumorigenic cell lines, with C-33A the cell line showing lowest expression (0.0099-fold), followed by HeLa (0.06-fold), and SiHa (0.38-fold) (Figure 2a).

CC biopsies showed lower expression of HOXA9 mRNA than control cervical cells
To determine whether the diminished expression of HOXA9 observed in CC-derived cell lines is also present in CC samples, nine samples of biopsies from patients with CC and 18 cervical scrapes from women without obvious cervical lesions were recruited ( Figure 2b). HOXA9 mRNA levels were analyzed and expression values were calculated for each patient by ΔCp analysis, that is, the Cp of the HOXA9 gene minus the expression of a reference gene from the same patient; from this analysis, it was observed that controls have lower ΔCp values, indicating HaCaT cells determined by microarray analysis (statistically significant, p ≤0.005). In the right panel, overexpressed or underexpressed HOX genes in HeLa cells are shown, including gene accession number, official symbol, official full name, and fold -change. (b) Validation using qPCR of over-and underexpressed genes selected from microarray expression assays; relative expression is shown as fold change (2ΔΔCp). Values obtained were normalized to three reference genes: GAPDH; PGK1, and RPLP0, and HaCaT cells were employed as calibrator (set as 1). Means ± SD from at least three independent experiments are shown Asian Pacific Journal of Cancer Prevention, Vol 17, 2016 1041 DOI:http://dx.doi.org/10.7314/APJCP.2016.17.3.1037 HOXA9 is Underexpressed in Cervical Cancer Cells andits Restoration Decreases Proliferation andMigration that HOXA9 expression is higher in controls than in patients with CC. These differences were statistically significant and correlated with our previous results.

The HOXA9 Promotor Is Methylated in CC Cell Lines, But Not in Keratinocytes and HaCaT Cells
To determine whether the expression of HOXA9 is reduced in tumor cells due to hypermethylation, as was previously described in other tissues, the methylation status of five different zones belonging to four CpG islands located within the promoter and gene sequence of HOXA9 (-1,400pb to +800pb) was evaluated by restriction with methylation-sensitive and -insensitive restriction enzymes (HpaII and MspI, respectively), followed by PCR ( Figure  3a, see Materials and Methods). As depicted in Figure 3b, HaCaT cells demonstrated the methylation of Zones 1 and 5, localized at the ends of the analyzed area, but Zones 2, 3, and 4, which correspond to the near promoter region and to the first exon, were not methylated. In contrast, HeLa and C-33A cells exhibited methylation in all analyzed zones (Z1-Z5). Interestingly, SiHa cells have a pattern similar to that of HaCaT cells (Z1-and Z5-methylated) with some methylation in Z2, which corresponds to the promoter region. As control, the methylation status of normal cervical primary keratinocytes isolated from a patient without cervical lesions and without HPV infection, was also included; normal keratinocytes demonstrated nearly no methylation in all analyzed zones.

HPV18 E6 and E7 Oncoproteins Regulates HOXA9 Expression
Our previous results indicated that HOXA9 was methylated in some CC-derived cell lines, particularly in the HPV18-infected HeLa cell line. To address the possible participation of HPV18 oncoproteins in methylation status, shRNA designed to target the E6 and E7 of HPV18 were transduced in HeLa cells (see Material and Methods). After this, expression of E6 and E7 at the mRNA level  Representative image of the sequence: -1,400bp to +800bp from the transcription start of the HOXA9 gene, predicted CpG islands, PCR-amplified regions (Z1-Z5), and restriction sites (MspI/ HpaII) are depicted as dark boxes, horizontal lines, or vertical light-grey lines, respectively. (b) Gel electrophoresis of the amplification products for Z1-Z5 after restriction with MspI or HpaII for HaCaT, HeLa, SiHa, and C-33A cell lines and normal primary Keratinocytes (Kers). (c) Relative HOXA9 expression in HeLa cells transduced with shRNA coding virus directed to E6/E7 of HPV18 (shE6/E7-1 and shE6/E7-2), and irrelevant shRNA was also used as control (shIrre). Expression values of E6/E7 and HOXA9 were calculated relative to the expression observed in HeLa-shIrre. For normalization, reference genes RPL32, RP18S, and RPLP0 were used   was measured by qPCR. Because two different shRNA directed to E6 and E7 genes were used, the silenced HeLa cells were denominated shE6/E7-1 and shE6/E7-2. As illustrated in Fig 3c, expression of the oncogenes diminished from 1-to 0.63-fold for shE6/E7-1 and from 1-to 0.58-fold for shE6/E7-2. Utilizing these models, the HOXA9 expression was also evaluated by qPCR. As can be observed in Figure 3d, HOXA9 expression increases in both models of E6/E7 silenced cells, indicating possible participation of HPV oncogenes in the regulation of HOXA9.

HOXA9 Protein Expression Was Diminished in CC Tissues
To corroborate these previous observations, HOXA9 expression was evaluated by immunohistochemistry in biopsies from patients with CC, and also in healthy cervical samples as control. The HOXA9 signal was evaluated by a Pathologist and classified as negative, low (+), medium (++), and high (+++), according to the positivity. The percentages of different degrees of HOXA9 expression were calculated and represented graphically (Figure 4). It was found that 8.57% of normal cervicaltissue samples have high HOXA9 expression, 62.8% medium expression, and 28.57% low expression; instead, in CC samples, high HOXA9 expression decreases to 4.76%, medium expression decreases to 57.14%, and low expression increases to 38.09%. These results indicate that protein expression of HOXA9 has a tendency to decrease in CC when compared with normal cervical tissues.

Exogenous HOXA9 Expression Induced Lower Colony Formation in CC-derived Cell Lines and Modified the Cellular Phenotype
The questions that arose from the previous observations including the following: why was the expression of HOXA9 diminished in CC-derived cell lines and in CC biopsies, and was diminution of HOXA9 an advantage to cancer-cell establishment. To determine whether the presence of HOXA9 is compatible with cell growth and proliferation, expression of HOXA9 was exogenously restored in CC-derived cells. For this, lentiviral particles carrying a Puromycin-resistant gene (LVX) and the Puromycin-resistant plus HOXA9 reading frame (LVX-HOXA9) were produced; thus, only infected cells would be able to survive and form colonies when cultured in the presence of Puromycin. As observed in Figure 5a, cells that were not infected did not survive and no colony formation was observed; instead, cells infected with viral particles carrying only the Puromycin-resistant gene (LVX) formed  . Values were normalized using GAPDH and ACTB as reference genes a great number of colonies. However, cells infected with viral particles carrying the Puromycin-resistant gene and HOXA9 (LVX-HOXA9) formed low numbers of colonies (Figure 5b), indicating that the presence of HOXA9 affects the growth of CC-derived cells.
In addition, it was observed that infection with exogenous HOXA9 not only decreased the number of colonies formed, but also changed cellular morphology, that is, HeLa cells, which normally grow isolated with no visible cell junctions and with mesenchymal morphology, in presence of HOXA9 cells have larger and irregular borders with indicia of cell junctions and epithelial morphology (Figure 5c). Due to the observation of such changes, we decided to evaluate modifications in the expression of genes involved in the epithelialto-mesenchymal transition (CDH1, GRHL2, TCF4, TWIST1, WNT5A, and WNT5B) after restoration of HOXA9 (Figure 5d). We found that genes such as GRHL2, which is normally present in epithelial, and not in mesenchymal cells, increase their expression when HOXA9 is restored. Likewise, the TCF4 and TWIST1 genes, which are related with the mesenchymal phenotype, decrease their expression 0.75-fold and 0.79-fold, respectively ( Figure 5e).

Restoration of HOXA9 Expression Decreases Cell Proliferation and Cell Metabolism, But Does Not Induce Apoptosis in CC-derived Cell Lines.
The results of clonogenic assays suggest a modulation of cell proliferation and viability by HOXA9. This hypothesis was further evaluated by measuring the cell growth of HeLa cells infected with viral particles carrying the Puromycin-resistant gene alone or with HOXA9. Cell growth was evaluated in real time using the Real-Time Cell Analyzer (RTCA) xCELLigence System. Cells were seeded, infected with viral particles and, after 72 h, Puromycin was added to the culture. As presented in Figure 6a, uninfected HeLa cells were not able to survive and died at 120 h of culture. Cells infected with lentiviral particles carrying the Puromycin-resistant gene only (LVX) exhibited growth curves that indicated more surviving cells; in turn, cells infected with the Puromycinresistant gene and HOXA9 (LVX-HOXA9) displayed growth curves indicating a lower number of surviving cells in comparison with those shown with LVX alone. These results corroborate the negative effect that HOXA9 exert on the growth of the CC-derived cell line. Employing another strategy, a conditional model (TETracyclinecontrolled Transcriptional Activation [TET] system) was additionally developed, in which HOXA9 expression is controlled by the presence of Doxycycline in the culture; two different clones were obtained. Using this system, cell proliferation was also evaluated with and without the inducer of HOXA9 expression. As displayed in Figure 6b, induction of HOXA9 reduced cell proliferation in both clones.
Taking advantage of the previously developed conditional model, cell metabolism was also evaluated by using WST-1. As depicted in Figure 6c, we found that HOXA9 expression strongly decreases cell metabolism; similar results were also obtained using the SiHa cell line (data not shown).

Exogenous HOXA9 Expression Decreases the Migration Rate of HeLa Cells
Another feature of cancer cells is an increased ability to migrate. In order to test whether exogenous HOXA9 expression modulated CC-cell migration, scratch assays were performed on cultures of HeLa cells infected with LVX-Puro (viral particles only with the Puromycin gene) and with LVX-HOXA9 cells (viral particles with the HOXA9 and the Puromycin gene). Time for woundclosure was evaluated at 0 h, 24 h, and 48 h. As can be observed in Figure 6d, cells expressing HOXA9 migrate more slowly than cells without it. Images of cell growth were analyzed by the "ImageJ" computer program to calculate the uncovered area of the wound. These analyses showed that, although initially (0 h), the wound area of HeLa-LVX-Puro cells was slightly larger, it decreased faster than the wound area of HeLa-LVX-HOXA9.
Because HOXA9 decreases colony formation, cell proliferation, cell migration, and cell metabolism, it was in our interest to evaluate whether HOXA9 also modulates the apoptosis process. The percentage of apoptotic cells in the presence or not of HOXA9 was evaluated and no differences between early and late apoptosis, comparing Hela-LVX vs. Hela-LVX-HOXA9 and HeLa-TET vs. HeLa-TET-HOXA9 were observed (data not shown).

Discussion
HOX genes encode transcription factors that regulate and determine different cell types during embryonic development (Castelli-Gair, 1998). They are considered as master regulators involved in fundamental processes during the development of an organism (Svingen and Tonissen, 2006). In recent years, abnormal expression of these genes has been linked with the emergence of various, solid and hematopoietic malignancies (16)(17)(18)(19)(20). In CC, there are evidences concerning the overexpression or downregulation of HOX members, indicating its possible role during cervical carcinogenesis (Lopez et al., 2006b;Gonzalez-Herrera et al., 2015). In order to underscore differences in HOX genes expression in CC and to corroborate and complement previous findings, we performed a comparison by microarray of the mRNA expression profile of two cell lines: one derived from CC (HeLa), and the other, from non-tumorigenic keratinocytes (HaCaT). A differential expression of HOXA3, HOXA9, and HOXA11 genes in HeLa compared with HaCaT cells was found. The HOXA cluster has been related with normal development of urogenital tracts (Burel et al., 2006); in particular, HOXA9 and -11 are important in controlling the embryonic development of Müller ducts (Hsieh-Li et al., 1995). With regard to HOXA9, this gene has been found overexpressed in hematological malignancies (Milne et al., 2010){Zhao, 2014 #203}, but in solid tumors, especially epithelial tumors, it has been observed as downregulated (Gilbert et al., 2010;Chen et al., 2012;Hwang et al., 2015). These observations were in agreement with our observations. We found that HOXA11 was slightly upregulated in Hela cells compared with HaCaT. The expression of HOXA11 was found unchanged in normal and malignant cervical cells (Lopez et al., 2006b). A report from Bai et al. indicates that HOXA11 is regulated by methylation and that the expression of this gene decreases in gastric cancer; moreover, these authors observed that silencing of HOXA11 induced cell proliferation of tumor cells (Bai et al., 2014).
Concerning the HOX B locus, we observed a modulation of HOXB3, -4, -5, -6, -9, and -13, and of these, only HOXB4 was found to be downregulated, while the remaining genes were overexpressed. According to our results, overexpression of HOXB genes has been observed in CC (Lopez et al., 2006a). Lopez et al. found the expression of HOXB1, -B3, -B5, -B6, -B7, -B8, and -B9 genes in normal cervix epithelia and in SCC. Moreover, the authors found the expression of HOXB2, -B4, and -B13 only in tumor tissues. We did not find variations in the expression of HOXB2 and observed downregulation of HOXB4, perhaps due to the different approach and the targets measured (we measured mRNA and Lopez et al., protein). Despite these dissimilarities, we also found upregulation of HOXB13. This phenomenon has also been observed in other works (Lopez et al., 2006b;Gonzalez-Herrera et al., 2015) {Gonzalez-Herrera, 2014 #204}, and in other cancer types (Zhu et al., 2014;Barresi et al., 2015){Long, 2013 #205}{Zhu, 2011 #206}. Regarding the HOXC cluster, we observed reduced expression of HOXC12 and -C13 in the CC-derived cell line HeLa. HOXC12 was recently proposed as a tumor suppressor gene downregulated by methylation in head and neck SCC (Guerrero-Preston et al., 2014). HOXC13 regulates the expression of keratins, and abnormal expression of this gene has been observed in malignant pilomatricoma (Jave-Suarez et al., 2002;Cribier et al., 2006); in SCC, expression of HOXC13 is augmented and modulated by histone methylation (Marcinkiewicz and Gudas, 2014b; Marcinkiewicz and Gudas, 2014a). Overexpression of HOXC13 was additionally observed in melanoma (Cantile et al., 2012), and functional studies indicate that overexpression of HOXC13 induces proliferation and tumorigenesis; conversely, silencing of this gene induces apoptosis and cell cycle arrest (Kasiri et al., 2013).
HOXD10 was observed with lower expression in HeLa cells compared with HaCaT, and the expression of HOXD11 remains unchanged in both cell lines. Posterior HOXD and -A (9-11) genes have redundant functions in reproductive organ development, and mutations in these genes induce changes in the epithelia, from columnar to squamous, and promote the migration of myeloid suppressor cells (Raines et al., 2013). In this regard, downmodulation of HOXD10 and HOXA9 could be involved in the maintenance of the epithelial phenotype and in the modulation of the immune response. The discrepancies between our data and those previously reported in relation to HOXB4, HOXC12, and HOXC13 may be due to the different techniques used, as well as to the new isoforms recently described (Brunskill and Potter, 2012).
Particularly with HOXA9, we observed that the proximal sequence (-200 bases) at the transcription start and the following (+150) bases after this, comprises an area of critical importance for the regulation of HOXA9 mRNA, and that it is a principal area of methylation. Our observations are in agreement with those shown in other models of epithelial malignancies; methylation and HOXA9 loss of expression have been reported in breast, ovarian, bladder, oral, and lung cancer (Rauch et al., 2007;Wu et al., 2007;Guerrero-Preston et al., 2011;Kim et al., 2013;Conway et al., 2014). All of these observations Indicate a possible role of HOXA9 as a tumor suppressor gene in epithelial cells; unlike its well described oncogenic potential in hematopoietic malignancies (Collins and Hess, 2015).
The tumor suppressor function of HOXA9 has been extensively investigated in breast cancer, where it has been shown that HOXA9 directly regulates BRCA1 and other genes involved in invasion, growth, and metastasis (Gilbert et al., 2010). Uchida et al. observed that high methylation levels of HOXA9 are associated with a high risk of metastatic tumors; in addition, the authors found that reduced HOXA9 expression confers an advantage on cell growth (Uchida et al., 2014); these observations are consistent with our results in CC-derived cells. We observed that restoration of HOXA9 expression in CCcells lines infected with HPV 18 significantly decreases cell growth, colony formation, metabolism, and cell migration. Contrary to our results, Sun et al. observed high expression of HOXA9 in laryngeal SCC compared with normal mucosal cells; also, using liver hepatocellular cells, these authors demonstrated that downregulation of HOXA9 inhibits cell growth (Sun et al., 2013). Conversely and in agreement with our results, Uchida et al. observed that re-expression of HOXA9 in oral cancer cell lines inhibits cell growth (Uchida et al., 2014). The different tissues employed in those experiments could explain the controversial observations; hepatocellular cells are different from oral cancer cells, which derive from squamous epithelial tissue, as do CC cells.
We also observed that HOXA9 restoration induces morphological changes that resemble the epithelial phenotype. Because HOXA9 regulates the expression of cadherins (VE-Cadherin and P-Cadherin) (Rossig et al., 2005;Ko and Naora, 2014), we evaluated the expression of E-cadherin (CDH1), together with other important markers of epithelial-to-mesenchymal transition. The expression of CDH1, TCF4, and TWIST1 was slightly modified by the restoration of HOXA9; however, GRHL2 was dramatically overexpressed, i.e, >14 times compared with the control. GRHL2 is a novel suppressor of epithelial to mesenchymal transition that is expressed in normal cervical epithelium and with reduced expression in CC cells (Torres-Reyes et al., 2014;Mehrazarin et al., 2015). In agreement to these results, variations in the expression of HOXA9 were previously observed by changes in epithelial and mesenchymal interactions in the intestine, suggesting a role of this gene in epithelial and mesenchymal communication (Duluc et al., 1997).
An important observation was the upregulation of HOXA9 when HPV oncogenes were silenced. There is no previous evidence of HOXA9 regulation by HPV infection, or of HOX genes regulation by the E6 or E7 oncoproteins. More studies are needed in this regard, and our group is currently working on this. Although our study reinforces the role of HOXA9 as tumor suppressor in the cervical epithelia, many questions remain concerning HOXA9 that have arisen from our and other observations. It is important to emphasize that the dual behavior of HOXA9 as an oncogene or tumor suppressor highlights the importance of tissue context in its function.
In summary, HOXA9 expression is downmodulated in some types of CC cells, and that could depend on the HPV type that it infects. HOXA9 downmodulation represents an advantage for HPV18-infected CC cells, which in that condition are more proliferative and have a high migration rate. The expression of HOXA9 is regulated by methylation and by the HPV oncogenes, and the restoration of HOXA9 induces lowers proliferation and the markers of ephitelialto-mesenchymal transition.
Conflict of interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Ethical approval: "All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards."


s2fieldsofstudy: Biology Engineering

text:
Improved production of cytotoxic thailanstatins A and D through metabolic engineering of Burkholderia thailandensis MSMB43 and pilot scale fermentation.

Thailanstatin A (TST-A) is a potent antiproliferative natural product discovered by our group from Burkholderia thailandensis MSMB43 through a genome-guided approach. The limited supply of TST-A, due to its low titer in bacterial fermentation, modest stability and very low recovery rate during purification, has hindered the investigations of TST-A as an anticancer drug candidate. Here we report the significant yield improvement of TST-A and its direct precursor, thailanstatin D (TST-D), through metabolic engineering of the thailanstatin biosynthetic pathway in MSMB43. Deletion of tstP, which encodes a dioxygenase involved in converting TST-A to downstream products including FR901464 (FR), resulted in 58% increase of the TST-A titer to 144.7 ± 2.3 mg/L and 132% increase of the TST-D titer to 14.6 ± 0.5 mg/L in the fermentation broth, respectively. Deletion of tstR, which encodes a cytochrome P450 involved in converting TST-D to TST-A, resulted in more than 7-fold increase of the TST-D titer to 53.2 ± 12.1 mg/L in the fermentation broth. An execution of 90 L pilot-scale fed-batch fermentation of the tstP deletion mutant in a 120-L fermentor led to the preparation of 714 mg of TST-A with greater than 98.5% purity. The half-life of TST-D in a phosphate buffer was found to be at least 202 h, significantly longer than that of TST-A or FR, suggesting superior stability. However, the IC50 values of TST-D against representative human cancer cell lines were determined to be greater than those of TST-A, indicating weaker antiproliferative activity. This work enabled us to prepare sufficient quantities of TST-A and TST-D for our ongoing translational research.

Introduction
Cancer has the second highest mortality rate only next to cardiovascular diseases and is usually associated with specific genetic and epigenetic changes in cellular processes. [1][2][3] Pre-mRNA splicing inhibitors, including FR901464 (FR; Fig. 1), 4-6 spliceostatins, 7,8 thailanstatins, 9 pladienolides, 10 FD-895, 11 and herboxidiene (GEX1), [12][13][14] are a promising class of bacterial natural products or their derivatives for cancer drug discovery and development. FR demonstrated not only potent antiproliferative activity against an array of human cancer cell lines with IC50 values in the low nanomolar (nM) range but also the ability to prolong the life of tumor-bearing mice. 5 However, due to its instability as well as unacceptable levels of toxicity, FR has been abandoned from a drug development program. 15 Mechanistically FR and spliceostatin A (SSA) inhibit pre-mRNA splicing by binding to SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein in the spliceosome. 16 FR was discovered by Nakajima et al. from Pseudomonas sp. No. 2663 (recently re-classified as Burkholderia sp. FERM BP-3421 17 ) through cell-based screenings; 4 thailanstatin A (TST-A; Fig. 1) was discovered by us from Burkholderia thailandensis MSMB43 through genome mining. 9 TST-A biosynthesis in MSMB43 and FR biosynthesis in FERM BP-3421 appear to use the same biosynthetic logic, 9,17,18 raising the possibility that these two strains are either identical or very closely related. Three oxygenase activities, including a flavindependent monooxygenase (FMO) domain encoded by fr9H/tstGH, a cytochrome P450 encoded by fr9R/tstR and a Fe(II)/α-ketoglutaratedependent dioxygenase encoded by fr9P/tstP, were proposed in the formation of a heavily decorated tetrahydropyran ring (Fig. 1). Specifically, the Fr9P/TstP dioxygenase was shown to convert a C1 acetic acid group in TST-A into a hemiketal group in FR through oxidative decarboxylation. As such, TST-A is a precursor of FR. The carboxylic acid moiety in TST-A is beneficial for drug development for the following reasons. First, this carboxylic acid moiety enables TST-A to be more stable than the hemiketal-containing FR. 9 Second, when the carboxylic acid group of TST-A is modified to an ester linkage, which is more lipophilic and enhances the cell membrane permeability of the derived compound, the cytotoxicity of the compound is significantly increased. 8 Our investigation of TST-A as an anticancer drug candidate encountered a shortage of compound supply, primarily due to its low titer in bacterial fermentation, modest stability and a very low recovery rate during purification. 9 Here we report the significant yield improvement of TST-A through metabolic engineering of the thailanstatin biosynthetic pathway in MSMB43 and pilot scale fermentation. During the course of this research, a direct TST-A precursor was isolated and named as thailanstatin D in 2013 (TST-D; Fig. 1), which turned out to be identical to the recently reported spliceostatin C (SSC). 17 The antiproliferative activity and stability of TST-D were assessed and reported as well.

Strain, plasmid and media
All strains and plasmids used in this study are listed in Table 1. Luria-Bertani (LB) agar or medium was used with appropriate concentration of antibiotics for routine cultivation of B. thailandensis MSMB43 strains or Escherichia coli strains. A 2S4G medium 17 was used for bacterial fermentation in flasks; a slightly modified 2S4G medium composed of 40 g/L glycerol, 12.5 g/L HySoy soypeptone, 2 g/L (NH4)2SO4, 0.1 g/L MgSO4·7H2O and 2 g/L CaCO3 (pH 7.0) was used for fed-batch bacterial fermentation in a fermentor. The concentrated feed medium contained 400 g/L glycerol, 20 g/L (NH4)2SO4 and 1 g/L MgSO4·7H2O (pH 7.0).

Construction of gene deletion mutants of B. thailandensis MSMB43
A multiplex PCR method 20 was used to create gene deletion mutant strains of MSMB43 (Fig. S1), as we routinely did in previous works. 19,22 PCR primers used for mutagenesis and for verification of mutant genotypes are listed in Table S1.

Bacterial fermentation, extraction and chromatographic purification of TST-A and TST-D
Bacterial fermentation, extraction, isolation and purification were performed according to schemes and associated description given in the Supplementary material (Figs. S2 and S3). TST-D was identified to be identical to spliceostatin C (SSC) 17 with extensive UV, IR, HR-MS and NMR analyses (Figs. S4-S15).

Titer determination by LC-MS analysis
The titers of TST-A and TST-D in fermentation broth were quantified with an Agilent 6400 Series Triple Quadrupole LC-MS system equipped with an Agilent Eclipse Plus C18 column (φ 4.6 × 100 mm, 3.5 μm) and a UV detector. Briefly, each 0.5 mL of fermentation broth was sampled at various time points and was extracted twice with equal volume of ethyl acetate. Two extracts were combined, dried in a refrigerated CentriVap centrifugal vacuum concentrator (Labconco) and subsequently re-suspended in 0.5 mL of acetonitrile and filtered through a 0.22 μm filter. Two microliters of such acetonitrile solution was injected into the LC-MS system. The LC solvents included buffer A (water with 0.1% formic acid, FA) and buffer B (acetonitrile with 0.1% FA). The column was eluted with a linear gradient from 15% to 55% buffer B in 35 min, monitored at 235 nm and with a flow rate of 0.5 mL/min. MS signals were collected in positive mode under the following conditions: N2 gas temperature, 325°C; gas flow, 10 L/min; nebulizer pressure, 20 psi;  sheath gas temperature, 400°C; sheath N2 gas flow, 12 L/min; capillary voltage, 4000 V; nozzle voltage, 500 V. TST-A and TST-D were eluted at 24 min and 31 min, respectively. The extracted ion species [M + H] + for TST-A and TST-D were 536 m/z and 520 m/z, respectively. For the generation of standard curves, TST-A and TST-D were separately dissolved in phosphate buffer saline (PBS, pH 7.4) at a concentration series of 0.02, 0.01, 0.005, 0.0025, 0.00125, 0.000625 and 0.0003125 mg/mL. Then 0.5 mL of each sample of TST-A or TST-D was extracted twice with equal volume of ethyl acetate. The extracts were combined, tried under vacuum and re-suspended in 0.5 mL of acetonitrile, and 2 μL of which was applied into the LC-MS system. A standard curve was created by plotting the compound concentrations against the extracted ion peak areas and showed linearity within the range of concentrations tested (Figs. S16 and S17).
The concentration of a sample was calculated using the equation generated from the standard curve.
For simple identification of TST-A and TST-D, the analysis was performed under the same LC-MS condition but with a different elution procedure: 0-9 min, from 15% to 100% buffer B; 9-12 min, in 100% buffer B; 12-13 min, from 100% to 15% buffer B; 13-18 min, in 15% buffer B. TST-A and TST-D were eluted at 8.2 and 9.2 min, respectively.

Stability and cytotoxicity assays
The stability and cytotoxicity of TST-D were assayed according to our previously described methods 9 to determine the IC50 value and half-life (t1/2), using FR901464 as a reference.  (Table 1). Those genetic events were all verified by PCR diagnosis (Fig. S1).

Quantitative LC-MS analysis of samples from a 4-day flask fermentation showed that the titers of TST-A and TST-D produced by
BthΔtstP strain increased 58% to 144.7 ± 2.3 mg/L and 132.0% to 14.6 ± 0.5 mg/L, respectively, and the titer of TST-D produced by BthΔtstR strain increased more than 7-fold to 53.2 ± 12.1 mg/L, all compared to the titers produced by BthWT strain. Meanwhile, deletion of tstP abolished the production of FR and deletion of tstR abolished the production of both TST-A and FR (Fig. 2).
Time-course monitoring of the production of TST-A and TST-D by BthΔtstP strain during pilot scale fed-batch fermentation demonstrated that both compounds reached their highest titers (181.9 mg/L for TST-A and 19.3 mg/L for TST-D) at 96 h, after which the titers declined (Fig. 3).

Recovery, isolation and purification of TST-A and TST-D
Totally, 236 g of crude extract containing an estimated 11.7 g of TST-A and 1.0 g of TST-D was obtained from about 90 L of fedbatch fermentation broth of BthΔtstP. A quantitative comparison between the compound titers in the fermentation broth harvested at 120 h (titers 129.6 mg/L for TST-A and 11.4 mg/L for TST-D) and after one round of 2% HP-20 resin absorption in the spent broth indicated that at least 96% of TST-A and 98% of TST-D were recovered by HP-20 absorption (Fig. S18). After one round of low pressure silica gel column separation, two rounds of flash  chromatography separation and one round of preparative HPLC, 714 mg of TST-A with greater than 98.5% purity was obtained (Figs. S19-S22), reflecting an overall 6% recovery rate for TST-A from the fermentation broth. By using a similar purification protocol, 61.5 mg of TST-D was purified from 54 L of BthΔtstR fermentation in flasks, reflection a very low recovery rate of 2% (Figs. S23 and S24), mainly due to the interference of FK228, a known compound coproduced by the MSMB43 strains 23 and having a similar retention time with that of TST-D.

TST-D is less cytotoxic but more stable than FR or TST-A
In vitro assay showed that TST-D possesses potent antiproliferative activities with IC50 values in the single nM range, yet it is a weaker compound compared to FR or TST-A (Table 2; Fig. S25). Interestingly, TST-D demonstrated a half-life in phosphate buffer (pH 7.4) at 37°C greater than 202 h, significantly longer than that of FR (~7.8 h) (Fig. 4) or TST-A (~78 h). 9

Discussion
Pre-mRNA splicing inhibitors have emerged as a promising class of anticancer drug candidates. 24 However, the development of FR, a prototypic compound of splicing inhibitors, has suffered a setback due to its instability as well as toxicity. 15 TST-A, a natural analog of FR discovered by our group, exhibited comparable activity as FR but much greater stability than FR. 9 Therefore, we have been engaging TST-A in translational research as a cancer drug candidate. This current work was designed to solve the supply issue of TST-A.
During the course of this research, TST-D was discovered as the direct precursor of TST-A and was shown to have even greater stability than TST-A. We also observed several physicochemical factors that affect the production and/or purification of TST-A and/or TST-D, and learned valuable lessons. First, in the presence of high concentrations of sodium chloride in a fermentation medium (such as M8 medium used previously 9 ), significant portion of the epoxidecontaining TST-A is spontaneously converted to the chlorohydrincontaining TST-B at ambient temperature, confirming an independent assessment. 8 We learned to omit sodium chloride in the fermentation medium (i.e. 2S4G medium 17 ) and from reagents or solvents used the downstream purification steps for TST-A. Second, TST-A appeared unstable in the crude extract if let unprocessed at room temperature for a period of days. Empirically we found that TST-A can be stabilized in the crude extract by mixing with silica gel immediately after having been concentrated with a rotary evaporator. Third, formic acid (0.1%, v/v) in chromatographic solvents is necessary for effective separation of TST-A in ODS columns. Fourth, as noted above, MSMB43 strains also produce high levels of FK228, 23 and TST-D and FK228 showed similar retention times and overlapping peaks in ODS chromatography, which hampered the separation of TST-D from FK228 and results in a very low recovery rate of TST-D. Fortunately, FK228 can be crystallized from the semi-purified FPLC fractions. We learned to insert a crystallization step to effectively remove the majority of FK228 from the semi-purified TST-D fractions prior to ODS column HPLC separation (Fig. S3). Lastly, final concentrating of large volume of preparative HPLC fractions containing purified TST-A or TST-D in acetonitrile-water should be achieved through a short period of rotary evaporation at 35°C to remove acetonitrile followed by lyophilization to remove water completely.
Considering that both TST-A and TST-D contain a stabilizing C17carboxylic acid group, that a C4-hydroxyl group is the only difference between TST-A and TST-D, and that a C3-epoxide group is known to be critical for bioactivity 9 and is present in all three compounds FR, TST-A and TST-D (Fig. 1), the observed results suggest a bipolar contribution of the C4-hydroxyl group to the structure-activity relationship of those compounds. On the one hand, this C4-hydroxyl group appeared to synergize with the C3-epoxide group in FR and TST-A for their superb antiproliferative activities; on the other hand, this C4-hydroxyl group is also a labile factor for FR and TST-A.
In conclusion, we provided a successful scale-up production method for the preparation of adequate quantity of TST-A and TST-D for translational studies, employing engineered strains of B. thailandensis MSMB43 and a pilot-scale fed-batch fermentation system. We also observed that TST-D is more stable than TST-A in a physiologically relevant phosphate buffer but is less potent than TST-A against several cancer cell lines tested.

Acknowledgments
This work was supported in part by a grant from the US National Institute of Health/National Cancer Institute (R01 CA152212) and a pilot grant from the US National Institute of Health/National Center for Advancing Translational Sciences through the University of Texas Southwestern Medical Center (award number 5UL1TR001105).

Appendix: Supplementary material
Supplementary data to this article can be found online at doi:10.1016/j.synbio.2016.02.002. * IC50, half-maximal growth inhibition concentration provided as the average of triplicate well results with standard deviation. ** Historical data from our previous publication. 9 Fig. 4. Stability of TST-D and FR in a phosphate buffer.


Punicalagin Induce the Production of Nitric Oxide and Inhibit Angiotensin Converting Enzyme in Endothelial Cell

Introduction: punicalagin, a hydrolysable tannin polyphenol from pomegranate, reported to have a protective effect against many diseases due to its high antioxidant and free radical scavenging activities. Aim, this study investigated the potential antihypertensive activity of punicalagin in human derived EA.hy926 endothelial cell model, via two mechanisms. In first mechanism, punicalagin enhance the nitric oxide production through scavenging reactive oxygen specious (ROS) and activating the endothelial nitric oxide synthase enzyme (eNOS). In second mechanism, punicalagin showed effect activity by inhibiting the angiotensin converting enzyme (ACE). Methods: The effect of different concentrations (1-100 μM) of punicalagin on EA.hy926 cells was measured using MTT assay. Induction of ROS was done using Ang II and scavenging activity of punicalagin was assessed by flow cytometer and fluorimeter. NO production was measured in order to determine the effective dose of punicalagin followed by measuring the ability of punicalagin to induce eNOS activity and the enzyme expression by Western blotting cellular Ca concentration and ACE inhibition were also determined. Results: Punicalagin (1-60 μM) reduced ROS production in EA.hy926, which induced by added angiotensin II, as shown by flow cytometry and by fluorometry. In addition, at the same concentration the nitric oxide production was increased in a dose-dependent manner due to increased eNOS activation. The activation of eNOS enzyme was promoted by an increase of cellular calcium concentration at the tested concentrations. Examined punicalagin concentrations significantly inhibited ACE activity, possibly due to zinc binding. Conclusion: punicalagin clearly exhibits the potential for reducing hypertensive activity by a dual mechanism of nitric oxide synthase induction by increasing nitric oxide levels, and ACE inhibition.

Introduction
Cardiovascular disease affects the circulatory system (heart, artery and blood vessels). This disease is a leading cause of death all over the world [1]. In the USA, according to the American Heart Association report in 2013, cardiovascular disease (CVD) deaths were 21.42% [2]. According to the World Health Organization (WHO), in the United Kingdom, CVD was one of the main causes of death in 2011 reaching about 19.77%, while in Saudi Arabia the death rate from CVD was 23.98% in the same year [3,4].
High blood pressure is a major risk factor for some chronic diseases such as stroke, renal disease, and cardiovascular disease [5]. The regulation of blood pressure is maintained by various physiological systems including the kinin-nitric oxide system (KNOS) and renin-angiotensin system.
High and unregulated production of reactive oxygen species (ROS) is another risk factor for heart disease [6,7]. In endothelial cells, enzyme systems involving NADPH oxidase, xanthine oxidase, and the mitochondrial respiratory chain are responsible for ROS production [8]. ROS production needs to be regulated or adverse effects like oxidation may damage cell macronutrients such as proteins, lipids, and nucleic acids. Balance in ROS concentration can be achieved by the antioxidant system that exists in the human body and by taking antioxidant supplements [9]. Moreover, ROS has the ability to oxidize nitric oxide (NO) produced from endothelial cells, leading to endothelial dysfunction and the initiation and development of cardiac disease [10].
Nitric oxide (NO) is known as a relaxing factor because it acts as a vasodilator, increases blood flow, and inhibits platelet aggregation and adhesion [11]. Calcium-dependent endothelial NO synthase (eNOS) is one of the important factors responsible for the production of NO in endothelial cells. Increased levels of NO in the endothelial cells are often due to the increased protein expression of the eNOS enzyme or by scavenging the ROS produced within the cells [12,13].
In renin-angiotensin system, angiotensin II is produced from angiotensin I by the catalytic effect of angiotensin converting enzyme; this is higher than normal levels in patients with hypertension [14,15]. Angiotensin II acts as vasoconstrictor, increasing blood pressure. For this reason, inhibition of ACE activity is a pharmacological target for the treatment of hypertension [16]. The ACE enzyme has a Zn 2+ ion in its two active sites [17]. Substrate binding and catalysis of ACE indicate the mechanism of Zn 2+ ion [18]. One of the mechanisms of ACE inhibitors is the ability to bind the Zn 2+ ion [19].
Several research groups have reported a significant interaction between biological systems and dietary polyphenols from vegetables and fruits, that may lead to beneficial anticancer [20,21], anti-inflammatory [22], antioxidant (ROS scavenging or metal chelating) [23], and antibacterial properties [24]. Pomegranate contains a high quantity of different polyphenols e.g. tannins, ellagic tannins, anthocyanins, catechins, gallic, and ellagic acid [25,26] including punicalagin (of the ellagitannin class). There are very few studies on punicalagin to date. Therefore, the effect and mechanism of punicalagin as an anti-hypertensive compound was investigated in the EA.hy926 cell line for two pathways.

Materials
The human endothelial-like immortalized cell line Ea.hy926 was kindly donated by Dr. Bodman-Smith University of Surrey, UK. Ethylene glycol tetraacetic acid (EGTA), hippuryl-histidyl-leucine (H-H-L) and angiotensin II were obtained from Sigma-Aldrich Chemical Co, Poole, UK.

Cell Viability and Nitric Oxide (NO) Release
The EA.hy926 cell line was seeded in DMEM with high glucose content (4.5 g/L), and supplemented with 10% FBS and 5% penicillin. Different punicalagin concentrations (1-100 µM) were tested on Ea.hy926 cells to measure the cell viability and nitric oxide (NO) production. Briefly, Ea.hy926 cells were seeded into 96 well tissue culture plates at a density of 1 × 10 4 cells/200 µl DMEM serum media. Once the cells reached 60-70% confluence, they were treated with punicalagin for 12, 24, or 48 hours. Different incubation times were tested to determine the optimal time for nitrite production, as a marker of nitric oxide production and cell viability. Nitrite levels in the treated cell supernatant were measured by the Griess kit according to the Griess kit instructions and cell viability was measured by MTT assay, at each experimental time. Cells pretreated with different concentrations of punicalagin were incubated with 20 µl of MTT dye (5 mg/ml PBS) at 37 °C and 5% CO 2 to measure cell viability after different incubation times. At the end of each experimental time, the culture medium was aspirated and 100 µl of DMSO was added to each well for 30 seconds at room temperature to dissolve the formazan crystals. The purple colour produced was measured at 492 nm using a plate reader (Boehring CO, Marburg, Germany).

Measurement of ROS by Flow Cytometry
Dichlorofluorescein dye (non-fluorescent CM-H 2 DCFDA) has the ability to diffuse through the cell membrane [27]. The fluorescence intensity is proportional to the ROS content [27]. The ROS level in the Ea.hy926 cell line was determined by flow cytometry, using CM-H 2 DCFDA dye after incubation with 1, 20, 40, and 60 µM punicalagin. Ea.hy926 cells 1x10 6 cells/ml were exposed to different punicalagin concentrations for 24 hours after reach (60-70%) confluence. Half of pre-treated cells were exposed to 10 nM angiotensin II for 1 hour, while the other half were left untreated. The cells were trypsinised, washed and in re-suspended 2 ml HBSS. Cells were then incubated with 5 µM CM-H 2 DCFDA (prepared in DMSO) for 30 minutes at 37 °C and 5% CO 2 . DCFDA florescence was then measured using a BD FACSCanto Flow cytometer (California, USA). At least 10,000 events were acquired in the gated regions using an emission wavelength of 520 nm.

Measurement of ROS by Fluorometry
The florescent dye, dihydroethidium can penetrate the cell membrane and is oxidized by superoxide radicals to form 2-hydroxyethidium; a red fluorescent product [28]. This Punicalagin Induce the Production of Nitric Oxide and Inhibit Angiotensin Converting Enzyme in Endothelial Cell Line EA.hy926 interacts with DNA to enhance intracellular fluorescence [29,30]. Ea.hy926 cells 1x10 6 cells/ml was exposed to different punicalagin concentrations (1, 20, 40 and 60 µM) for 24 hours after reach 60-70% confluence. After 24 hours, one group of treated cells was incubated with angiotensin II (10 nM) for 1 hour and another group was left untreated (control). All cells were then washed with HBSS buffer. Cells were covered with 3 ml HBSS buffer and incubated with 25 µM of DHE dye for 30 minutes at 37 °C and 5% CO 2 . HBSS buffer was discarded and the cells were scraped from the flask with 1 ml cold methanol. The cell suspension was sonicated and filtered through a 0.22 µM membrane filter. The 2-hydroxyethidium was detected by fluorimetry (Varian, USA) with excitation and emission wavelengths of 480 nm and 580 nm, respectively.

Determination of Cellular Calcium Concentration by Fluorescence Method
Intracellular calcium concentrations were measured by fluorometry after loading the cells with fura-2/AM dye. Free intracellular calcium will bind with the membrane diffusible fluorescent dye fura-2/AM [31]. Briefly, Ea.hy926 cells were cultured in 25cm 2 flasks at 1x10 6 cells/ml and exposed to different concentration(1, 20, 40 and 60 µM) after they reached 60-70% confluence. Following 24 hours incubation, cells were loaded with 5 µM fura-2/AM dye and incubated for 45 minutes at 37 °C and 5% CO 2 . Consequently, the dye was removed and the cells were washed and scraped from the flask with 2 ml HBSS buffer. The calcium concentration was detected by fluorometry (Varian, USA) with excitation and emission wavelengths of 340 nm and 510 nm, respectively. Ethyleneglycoltetraacetic acid (EGTA) at 1 mM was used as negative control instead of punicalagin.

Determination of Endothelial Nitric Oxide Synthase
Enzyme (eNOS) Activity in the Ea.hy926 Cell Line Ea,hy926 Cells treated with different concentrations of punicalagin (1, 20, 40 and 60 µM) for 24 hours, were lysed as follows. After this incubation, the cells were trypsinized and the resulting cell suspension centrifuged at 1500 × g for 3 minutes with 5 ml PBS to wash. Supernatants were removed and the cell pellets were lysed by adding 300 µl of lysis buffer (Sigma Aldrich Chemical Company). The lysis cell was kept it on ice for 20 minutes and then stored them at -20°C until eNOS activity was measured. EGTA (1 mM) was used as a negative control in place of punicalagin. eNOS synthase activity was measured using a nitric oxide synthase assay colorimetric kit (Bioassay System, ENOS-100) According to the manufacturer's instructions.

Assessment of eNOS Expression by Western Blot
Ea,hy926 cells were treated with 1, 20, 40 and 60 µM punicalagin and incubated for 24 hours and lysed lysis buffer (Sigma Aldrich Chemical Company)as described in 2.2.5 and stored at -80 °C until protein determination and western blot experiment were carried out. The protein concentration for each sample was measured by Bradford method using BioRad assay [32] following the manufacturer's instructions. An Invitrogen NuPAGE 4-12% Bis-Tris gel was used for protein electrophoresis. The proteins were then transferred to a polyvinyl difluoride (PVDF). The results were visualized by chemiluminescence using Amersham film.
2.2.7. Measuring ACE Activity in Ea.hy926 Angiotensin converting enzyme activity was measured in the Ea.hy926 cell line after 24 hours of exposure to different concentrations of punicalagin (1, 20, 40 and 60 µM) using a modified fluorometric method [33]. Briefly, Ea.hy926 cells were seeded to confluence in 25cm 2 flasks (1x10 6 cells /ml) and then incubated with punicalagin (1, 20, 40 and 60 µM) for 24 hours. Treated cells were washed three times with 3 ml HBSS buffer. The cells were scraped from the flask with 1 ml HBSS and frozen at -20 °C until assayed. For the assay, frozen cells were thawed and sonicated then 20 µl samples were added to 80 µl of H-H-L (5 mM prepared in HBSS buffer) and incubated at 37 °C for 3 hours. The incubated cells were then mixed with 1.4 ml NaOH (0.5 N) to stop the reaction. The fluorescent dye, O-phthaldialdehyde (100 µl of 10 mg/ml in methanol) was used to detect the histidyl-leucine reaction product. The reagents were incubated for 5 minutes at room temperature followed by the addition of 250 µl of HCl (6 N). A was used to measure the fluorescence of the samples was measured by fluorometry (Varian, USA), using an excitation wavelength of 365 nm and an emission wavelength of 495 nm. Captopril (1 µM) was used as a positive control.

Statistical Analyses
All experiments were performed at least in triplicate. For the 96-well microtiter tissue culture plates, 4 replicate wells were used per category. The data were analyzed by Graphpad Prism version 6. For significant differences between control and experimental values, the P-value between groups was determined by one-way analysis of variance followed by Bonferroni test. The significance level was set at P ≤0.05.

Cell Viability and NO Production
Cell viability and nitric oxide production was measured over a time course to determine the optimal experimental exposure time. The data in (Figure 1 A) show that cell viability increased significantly (p ≤ 0.05) after treatment with 1, 20, 40, and 60 µM punicalagin for 12 hours, but that NO production was similar compared with the untreated control (Figure 1 B). In contrast, there was a significant decrease in cell viability at 80 and 100 µM punicalagin concentrations (p < 0.0001) with a corresponding significant decrease in NO production at 12 hours (p < 0.0001). After 24 hours incubation time with 1-60 µM punicalagin, cell viability and NO production were significantly increased (p ≤ 0.05) compared with untreated cells (control) as shown in (Figures 1 C and D). As for the 12-hours incubation, 80 and 100 µM punicalagin caused a significant decrease in cell viability and NO production compared with untreated cells (p ≤ 0.05).
After 48 hours incubation significant decreases in cell viability (p ≤ 0.05) and NO production (p < 0.001) were observed at all punicalagin concentrations (1, 20, 40, 60, 80 and 100 µM) (Figures 1 E and F). Based on the above cell viability and NO production results, punicalagin concentrations in the range 1 -60 µM were selected for exposure to cells for 24 hours in future experiments because higher punicalagin concentrations 80-100 µM produced a toxic effect on Ea.hy926 cells. The significantly enhanced release of NO due to the activation of eNOS enzyme is described below in section 3.3.

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Punicalagin Induce the Production of Nitric Oxide and Inhibit Angiotensin Converting Enzyme in Endothelial Cell Line EA.hy926

Measurement of ROS by Flow Cytometry and Fluorometry
Angiotensin (Ang) II has the ability to stimulate ROS production in endothelial cells through activation of a redox-sensitive signaling system. In an endothelial cell line, NADPH oxidase was considered as a source of ROS, responding to Ang II with the donation of an electron to reduce a molecule of oxygen to produce the superoxide anion, O 2 •− [34]. ROS production in the Ea.hy926 cell line, treated with different concentrations of punicalagin for 24 hours, in the absence of Ang II stimulation, was not significantly different to ROS production in cells not treated with punicalagin. This was true using either flow cytometry (Figure 2 A) or fluorimetry (Figure 3 A) as the ROS determination method.
The ability of different concentrations of punicalagin added to Ea.hy296 cells for 24 hours to reduce ROS production was assessed by flow cytometry after treating cells with 10 nM Ang II for 1 hour (Figure 2  The results were confirmed by a second method, measuring the conversion of DHE 2-hydroxyethidium in the presence of ROS, by fluorimetry (Figure 3 B). The fluorescence intensity in Ea.hy926 cells incubated for 24 hours with punicalagin (1, 20, 40 and 60 µM) and then treated with 10 nM Ang II also decreased in a dose-dependent manner compared with cells treated with Ang II only (positive control). The fluorescence intensity was 0.83, 0.59, 0.54, 0.48 and 0.46 for positive control, and 1, 20, 40 and 60 µM punicalagin, respectively. These findings are in contrast with cells not stimulated with Ang II, in which no significant difference was found in ROS levels between punicalagin-treated cells and control by flow cytometry or fluorescence methods (Figure 2 A and 3 A, respectively). These results illustrate that punicalagin has the ability to scavenge ROS and thereby protect cellular macromolecules from ROS-mediated damage.   1-60 µM); B) pre-treated cells with punicalagin incubated with 10 nM angiotensin II for 1 hour. Values are mean ± SD, n=3. Comparisons of means were made using a one-way ANOVA followed by Bonferroni's test (ns= non significant, **= p < 0.001 and ***P < 0.0001 when compared with control). Protein was extracted from Ea.hy926 cells after treatment with 1-60 μM punicalagin for 24 hours. EGTA 1 mM incubated for 3 hours was used as a negative control. Data represent mean ± SD of more than three experiments. Comparisons of means were made using a one-way ANOVA followed by Bonferroni's test (*= p < 0.05; **=p < 0.001; and *** = p < 0.0001).

Determination of Cellular Calcium Concentration, eNOS Activity and Expression
Different concentrations of punicalagin (1, 20, 40 and 60 µM) exposed to Ea.hy926 cell line for 24 hours caused a significant dose-dependent increase in intracellular Ca 2+ concentration p < 0.001 (Figure 4 A). Since eNOS activity is dependent on Ca 2+ , the cytoplasmic Ca 2+ concentration was investigated in response to different punicalagin concentrations. EGTA (a Ca 2+ inhibitor) was used as the negative control.
This study demonstrates that the punicalagin from pomegranate (1-60 µM) produced a significant increase in calcium concentration in EA.hy926 cell line after incubation for 24 hours. Polyphenols can affect intracellular Ca 2+ store and release it or increase the entrance of Ca 2+ through the cell membrane [35]. This suggestion could explain the significant induction of Ca 2+ concentration by different punicalagin concentrations in the endothelial cell line (p < 0.001).
The effects of punicalagin at different concentrations (1,20,40, and 60 µM) on the activation of the eNOS enzyme were determined in Ea.hy926 cells after 24 hours exposure time. As shown in Figure 4 B, punicalagin caused a significant increase in eNOS enzyme activity that was dose-dependent p ≤ 0.05. EGTA (1 mM) was used as the negative control.
Western blot analysis using a specific antibody against eNOS revealed no change in eNOS protein expression in Ea.hy926 cells treated with punicalagin compared with untreated control cells (Figure 4 C). The calcium concentration for 1 mM EGTA (negative control) was significantly decreased (p ≤ 0.05); therefore, the protein expression level of eNOS enzyme was down regulated and eNOS enzyme activity decreased. Punicalagin may increase eNOS activity via eNOS phosphorylation, which is enhanced by the activation of redox-sensitive phosphatidylinositol-3 (PI3)/ protein kinase B (AKT) pathways [36,37].

Measuring ACE Activity in Ea.hy926 Cells
In this study, incubation of Ea.hy926 with different concentrations of punicalagin (1, 20,   . ACE inhibition activity in Ea.hy926 cell exposed to punicalagin (1-60 µM) for 24 hours. Data represent mean ±SD of more than three experiments. Comparisons of means were made using a one-way ANOVA followed by Bonferroni's test (*=p< 0.05, **p<0.001 and *** = p < 0.0001).
Captopril was used as a positive control.

Discussion
Several studies have shown the effect of polyphenols to produce NO in agreement with our findings. EA.hy296 cell line incubated with red wine polyphenol extract (100-600 µg/ml for 18 hours produced a significant increase in NO production [38]. Dihydrocaffeic acid (caffeic acid metabolite) at different concentrations (0-200 µM) significantly increased the production of NO after being added to EA.hy926 cells for 18 hours in a dose-dependent manner [39]. Another study was performed on NO production after incubating the bovine pulmonary artery endothelial cell line with 50 and 100 µM pomegranate juice for 24 and 48 hours. The observed result showed an increased NO production [40]. Punicalagin and o-galloyl punicalagin extracted from Terminalia calamansanai plant leaves (50 µM each) produced a significant increase in NO production in bovine aortic cell line [41]. NO production was also significantly increased in a dose-dependent manner after incubating the EA.hy926 cell line for 24 hours with different polyphenols (resveratrol, epicatechin gallate and epigallocatechin gallate) [42]. Different concentrations (0.1-10 µM) of polyphenols extracted with 70 % acetone from black currant were exposed to EA.hy926 cell line for 10 minutes and produced a significant increase in NO productionin a dose-dependent manner [36].
Numerous studies have been conducted on endothelial cell lines to examine the effect of polyphenols as ACE inhibitors. Black tea, green tea and rooibos tea, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate were incubated with HUVEC cell line at different concentrations for 10 minutes. All experimental components inhibited the activity of ACE at all concentrations except rooibos tea. Rooibos tea did not contain any catechin compounds [43]. In 2009, another study was performed on HUVEC cell line to investigate the inhibition of ACE enzyme after exposure to aqueous phenolic extract from Vaccinium myrtillus (bilberry). The bilberry extract contained several polyphenols such as quercetin, stillbene, resveratrol, ferulic acid and coumaric acid; these components (0.000625-0.1 mg/ml) significantly inhibited ACE activity in a dose dependent manner in HUVEC cell line treated for 10 minutes [16]. Aviram and Dornfeld (2001) found that serum ACE enzyme activity was significantly inhibited by 36% in seven-hypertensive patients who consumed pomegranate juice for 2 weeks. Each treated patient's serum was incubated with pomegranate juice (50-350 µM) for 15 minutes at 37 °C. The enzyme activity was significantly inhibited in a dose dependent manner [14]. Thus, all the previous research results show similar effects to punicalagin in the present study as an inhibitor for ACE activity on EA.hy926 cell line which is a new finding. The ACE inhibitory activity may be due to metal chelation by punicalagin as zinc ion is present in the active site of ACE.
As reported previously from several in vitro studies, the presence of the OH group in phenolic compounds plays an important role in scavenging ROS [44,45,46]. The Chinese medicine, Seabuckthorn, contains different flavonoids e.g. quercetin, isorhamnetin and kaempherol. Seabuckthorn at different concentrations (9.38-37.5 µg/ml) and 15 µg/ml quercetin was incubated with EA.hy926 cell line for 20 minutes separately before adding 100 µg/ml oxidized LDL as a ROS inducer for a 24 hours incubation time. A protective effect against superoxide anion in EA.hy926 cells was found [47]. ROS production was significantly decreased in liver cell line (HepG2) exposed to different concentrations of quercetin and rutin (1-100 µM) for 24 hours followed by incubation with 200 µM H 2 O 2 for 3 hours [48]. Pomegranate juice, concord juice and blueberry juice antioxidant activities were examined and showed scavenging of superoxide anion and protection of NO destruction from ROS action. Pomegranate juice, diluted 6-fold and using a small volume of 3 µl, showed very high antioxidant activity while the same effect was only possible from 300 µl undiluted blueberry or 1000 µl undiluted concord grape [40]. In this study, punicalagin concentrations (1-60 µM) showed a significant reduction of ROS levels in Ea.hy926 cell line after stimulation by Ang II (p < 0.001).
This study demonstrates that the punicalagin from pomegranate (1-60 µM) produced a significant increase in calcium concentration in EA.hy926 cell line after incubation for 24 hours. Polyphenols can affect intracellular Ca 2+ store and release it or increase the entrance of Ca 2+ through the cell membrane [35]. This suggestion could explain the significant induction of Ca 2+ concentration by different punicalagin concentrations in the endothelial cell line (p < 0.001).
As eNOS enzyme is calcium-dependent, several researchers measured the calcium concentration in EA.hy926 cell line after incubation with different polyphenols. EA.hy926 cell line was incubated for 20 hours with 100-600 µg/ml red wine polyphenols water extract. The experimental dose showed a significant increase in calcium concentration in EA.hy926 cell line, which increased the eNOS protein, level, and showed a significant release of NO [38]. Another study was performed on calcium concentrations in EA.hy926 after incubation with punicalagin and o-galloylpunicalagin extracted from Terminalia Calamansanai plant leaves; EA.hy926 cell line, exposed to 50 µM of each component for 12 hours produced a significant increase in calcium concentration [41].
Similar to this research outcome, pomegranate juice (50 and 100 µM) was examined on bovine pulmonary artery endothelial cell line for 24 and 48 hours. The treatment did not show any significant effect on protein expression on eNOS enzyme at any concentration [40]. Chen et al., (2008) examined the effect of punicalagin and o-galloylpunicalagin extracted from Terminalia Calamansanai leaves on eNOS expression. Protein expression of eNOS enzyme in bovine aorteic cell line was not affected after incubation for 12 hours with 50 µM of punicalagin and o-galloylpunicalagin extracted from Terminalia Calamansanai leaves [41].
In contrast, Leikert et al., (2002) found that red wine polyphenols water extract (100-600 µg/ml) had increased eNOS protein levels after incubation in EA.hy926 cell line for 20 hours [38]. Huang et al., (2004) found a significant increase in protein expression and activity of eNOS enzyme in EA.hy926 cell line after incubation with dihydrocaffeic aicd (caffeic acid metabolite) at different concentrations (0-200 µM) for 18 hours. The effect of resveratrol, epigallocatechingallate and epicatechingallate on eNOS enzyme was investigated on EA.hy926 cell line at different concentrations (0-100 µM) for 24 hours. The protein level of eNOS enzyme was significantly increased in a dose dependent manner [42]. Edirisinghe et al., (2011) studied the black currant polyphenols 70% acetone extract on protein expression of eNOS. Different extract concentrations (0.1-10 µM) were applied on EA.hy926 cell line for 10 minutes. The treated cells produced a significant increase in eNOS enzyme expression associated with the concentration increase [36]. Different concentration from Hesperetin, type of flavanone present in citrus fruits, (0.01-10 µM) and incubated for 1-30 minutes significantly stimulates phosphorylation of Akt, AMPK, and eNOS to produce NO in a concentration-and time-dependent manner in vascular endothelial cells [49].

Conclusions
All results presented in this study support the proposition that punicalagin is a type of polyphenol that could play a role in reducing the risk of cardiovascular disease. As observed previously, punicalagin incubated with EA.hy926 cells at different concentrations for 24 hours produced a significant inhibition of ACE enzyme activity and increased NO production. Increased NO production was via increased eNOS activity due to an increase in calcium concentration. There was no induction of eNOS enzyme expression observed. This NO level is protected from destruction by ROS through punicalagin scavenging activity. The above findings indicate that punicalagin may be helpful for lowering blood pressure; this could be achieved through dietary intervention or by the production of new anti-hypertensive treatments from a natural product.