CYP1A1 MspI Polymorphism and Cervical Carcinoma Risk in the Multi-Ethnic Population of Malaysia: a Case-Control Study.

BACKGROUND: Tobacco smoking is considered a risk factor for cervical cancer development due to the presence of tobacco based carcinogenic metabolites in cervical cells of female smokers. In this study, we investigated the role of the T3801C (MspI) polymorphism of CYP1A1, a gene encoding an enzyme necessary for the initiation of tobacco based carcinogen metabolism, on cervical cancer risk. The T to C substitution may alter CYP1A1 activities, potentially elevating cervical cancer risk. Since results of gene-disease association studies vary according to the study population, the multi-ethnic population of Malaysia provides an excellent representative cohort for identifying and comparing the cervical cancer risk among the 3 major ethnics in Southeast Asia in relation to CYP1A1 MspI polymorphism. MATERIALS AND METHODS: A total of 195 Thin Prep Pap smear samples from HPV negative and cancer free females were randomly selected as controls while 106 formalin fixed paraffin embedded samples from females with invasive cervical cancer were randomly selected for the cases group. The polymorphisms were identified using restriction fragment length polymorphism (RFLP) PCR. RESULTS: We found no significant associations between CYP1A1 MspI polymorphism and cervical cancer in the general Malaysian female population. However, upon ethnic stratification, the variant C/C genotype was significantly associated with a 4.66-fold increase in cervical cancer risk in Malay females (95% CI= 1.21-17.9; p=0.03). No significant association was observed in the Chinese and Indian females. Additionally, there were no significant associations in the dominant model and allele frequency model analysis in both the general and ethnically stratified female population of Malaysia. CONCLUSIONS: Our findings suggest that the C/C genotype of CYP1A1 MspI polymorphism is associated with the development of cervical carcinoma in the Malay females of Malaysia.

NCI in vitro and in silico anticancer screen, cell cycle pertubation and apoptosis-inducing potential of new acylated, benzylidene and isopropylidene derivatives of andrographolide.

Andrographolide (AGP) is the main bioactive constituent isolated from the traditional medicinal, Andrographis paniculata which contributes towards its various biological activities, including anticancer property. In this study, a series of new AGP derivatives were semi-synthesised and screened against the NCI in vitro 60 cell lines. From the screening results, we had identified SRS07 as the most potent AGP derivative, against breast and colon cancer cell lines. Subsequently, SRS07 was tested for its capability to induce cell cycle arrest and apoptosis in MCF-7 and HCT116 cancer cells. SRS07 effectively induced G1 cell cycle arrest in both cell lines and ultimately apoptosis by inducing DNA fragmentation in HCT116 cells. The apoptotic cell death induced by SRS07 was confirmed via FITC Annexin-V double staining. Western blot analysis of SRS07-treated HCT116 cells revealed that the compound induced apoptosis be activating caspase 8 which in turn cleaved Bid to t-Bid to initiate cell death cascade. Prediction of the possible mode of action of SRS07 by utilising NCI COMPARE analysis failed to reveal a distinct mechanism category. Hence, it is speculated that SRS07 possesses novel mechanism of action. In conclusion, SRS07 demonstrated superior in vitro anticancer profiles and emerged as a potential lead anticancer candidate.

In vitro 3D colon tumor penetrability of SRJ09, a new anti-cancer andrographolide analog.

Limited tumor penetrability of anti-cancer drugs is recognized as one of the major factors that lead to poor anti-tumor activity. SRJ09 (3,19-(2-bromobenzylidene) andrographolide) has been identified as a lead anti-cancer agent for colon cancer. Recently, this compound was shown by us to be a mutant K-Ras binder. In this present study, the penetrability of SRJ09 through the DLD-1 colon cancer multicell layer (MCL) was evaluated. The amount of SRJ09 that penetrated through the MCL was quantitated by utilizing high performance liquid chromatography (HPLC). Histopathological staining was used to visualize the morphology of MCL. A chemosensitivity assay was performed to assess the anti-cancer activity of SRJ09 in DLD-1 cells. SRJ09 was able to penetrate through DLD-1 MCL and is inversely proportional with the MCL thickness. The flow rates for SRJ09 through MCL were 0.90 ± 0.20 µM/min/cm(2) and 0.56 ± 0.06 µM/min/cm(2) for days 1 and 5, respectively, which are better than doxorubicin. Histopathological examination revealed that the integrity of the DLD-1 MCL was retained and no visible damage was inflicted on the cell membrane, confirming the penetration of SRJ09 was by diffusion. Short term exposure (1 h) in DLD-1 cells demonstrated SRJ09 had IC50 of 41 µM which was approximately 4-folds lower than andrographolide, the parent compound of SRJ09. In conclusion, SRJ09 successfully penetrated through DLD-1 MCL by diffusion and emerged as a potential candidate to be developed as a clinically viable anti-colon cancer drug.

Molecular targets in the discovery and development of novel antimetastatic agents: current progress and future prospects.

Tumour invasion and metastasis have been recognized as major causal factors in the morbidity and mortality among cancer patients. Many advances in the knowledge of cancer metastasis have yielded an impressive array of attractive drug targets, including enzymes, receptors and multiple signalling pathways. The present review summarizes the molecular pathogenesis of metastasis and the identification of novel molecular targets used in the discovery of antimetastatic agents. Several promising targets have been highlighted, including receptor tyrosine kinases, effector molecules involved in angiogenesis, matrix metalloproteinases (MMPs), urokinase plasminogen activator, adhesion molecules and their receptors, signalling pathways (e.g. phosphatidylinositol 3-kinase, phospholipase Cγ1, mitogen-activated protein kinases, c-Src kinase, c-Met kinases and heat shock protein. The discovery and development of potential novel therapeutics for each of the targets are also discussed in this review. Among these, the most promising agents that have shown remarkable clinical outcome are anti-angiogenic agents (e.g. bevacizumab). Newer agents, such as c-Met kinase inhibitors, are still undergoing preclinical studies and are yet to have their clinical efficacy proven. Some therapeutics, such as first-generation MMP inhibitors (MMPIs; e.g. marimastat) and more selective versions of them (e.g. prinomastat, tanomastat), have undergone clinical trials. Unfortunately, these drugs produced serious adverse effects that led to the premature termination of their development. In the future, third-generation MMPIs and inhibitors of signalling pathways and adhesion molecules could form valuable novel classes of drugs in the anticancer armamentarium to combat metastasis.

Barrientosiimonas humi gen. nov., sp. nov., an actinobacterium of the family Dermacoccaceae.

Three novel actinobacteria, strains 39(T), 40 and 41, were isolated from soil collected from Barrientos Island in the Antarctic. The taxonomic status of these strains was determined using a polyphasic approach. Comparison of 16S rRNA gene sequences revealed that strain 39(T) represented a novel lineage within the family Dermacoccaceae and was most closely related to members of the genera Demetria (96.9 % 16S rRNA gene sequence similarity), Branchiibius (95.7 %), Dermacoccus (94.4-95.3 %), Calidifontibacter (94.6 %), Luteipulveratus (94.3 %), Yimella (94.2 %) and Kytococcus (93.1 %). Cells were irregular cocci and short rods. The peptidoglycan type was A4α with an L-Lys-L-Ser-D-Asp interpeptide bridge. The cell-wall sugars were galactose and glucose. The major menaquinone was MK-8(H(4)). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphoglycolipid, two glycolipids and one unknown phospholipid. The acyl type of the cell-wall polysaccharide was N-acetyl. The major cellular fatty acids were anteiso-C(17 : 0) (41.97 %), anteiso-C(17 : 1)ω9c (32.16 %) and iso-C(16 : 0) (7.68 %). The DNA G+C content of strain 39(T) was 68.4 mol%. On the basis of phylogenetic and phenotypic differences from other genera of the family Dermacoccaceae, a novel genus and species, Barrientosiimonas humi gen. nov., sp. nov., is proposed; the type strain of the type species is 39(T) (=CGMCC 4.6864(T) = DSM 24617(T)).

Molecular characterization of Antarctic actinobacteria and screening for antimicrobial metabolite production.

The present study aimed to isolate actinobacteria from soil samples and characterized them using molecular tools and screened their secondary metabolites for antimicrobial activities. Thirty-nine strains from four different location of Barrientos Island, Antarctica using 12 types of isolation media was isolated. The isolates were preceded to screening of secondary metabolites for antimicrobial and antifungal activities. Using high-throughput screening methods, 38% (15/39) of isolates produced bioactive metabolites. Approximately 18% (7/39), 18% (7/39), 10% (4/39) and 2.5% (1/39) of isolates inhibited growth of Candida albicans ATCC 10231(T), Staphylococcus aurues ATCC 51650(T), methicillin-resistant Staphylococcus aurues (MRSA) ATCC BAA-44(T) and Pseudomonas aeruginosa ATCC 10145(T), respectively. Molecular characterization techniques like 16S rRNA analysis, Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), Random amplified polymorphic DNA (RAPD) and composite analyses were used to characterize the actinobacteria strains. Analysis of 16S rRNA sequences is still one of the most powerful methods to determine higher taxonomic relationships of Actinobacteria. Both RAPD and ERIC-PCR fingerprinting have shown good discriminatory capability but RAPD proved to be better in discriminatory power than ERIC-PCR. Our results demonstrated that composite analysis of both fingerprinting generally increased the discrimination ability and generated best clustering for actinobacteria strains in this study.

p53 codon 72 polymorphisms and random amplified polymorphic DNA analysis of non-melanoma skin cancer through archival formalin-fixed paraffin-embedded tissue.

Non-melanoma skin cancer (NMSC) is classified among the ten most frequent cancers in Malaysia. A common polymorphism at codon 72 of the p53 tumor suppressor gene and its influence on cancer risk has been studied for different types of cancer with mixed and inconsistent results with limited published data on the Malaysian population so far. In the present study, the frequency of p53 codon 72 polymorphism in 60 patients with NMSC was investigated from archival formalin-fixed paraffin-embedded (FFPE) tissue obtained from Hospital Universiti Kebangsaan Malaysia (HUKM). Additionally, random amplified polymorhic DNA -polymorphic chain reaction (RAPD-PCR) was employed for preliminary biomarker development. NMSC FFPE samples (70%) possess Arg/Arg, 20% with Pro/Pro and 10% with Arg/Pro. In total, there was no significant difference in the p53 codon 72 genotypes between histological types of NMSC, gender, race, tumor location and age group. However, there was an apparent age-associated increase in the Arg/Arg genotype but did not reach statistical significance (P=0.235). NMSC types and demographic characteristics did not influence genotype distribution. On the other hand, BCC and SCC distributions are influenced by age group, race and tumor location.

Comparison between allicin and fluconazole in Candida albicans biofilm inhibition and in suppression of HWP1 gene expression.

Candida albicans is an opportunistic human pathogen with the ability to differentiate and grow in filamentous forms and exist as biofilms. The biofilms are a barrier to treatment as they are often resistant to the antifungal drugs. In this study, we investigated the antifungal activity of allicin, an active compound of garlic on various isolates of C. albicans. The effect of allicin on biofilm production in C. albicans as compared to fluconazole, an antifungal drug, was investigated using the tetrazolium (XTT) reduction-dependent growth and crystal violet assays as well as scanning electron microscopy (SEM). Allicin-treated cells exhibited significant reduction in biofilm growth (p<0.05) compared to fluconazole-treated and also growth control cells. Moreover, observation by SEM of allicin and fluconazole-treated cells confirmed a dose-dependent membrane disruption and decreased production of organisms. Finally, the expression of selected genes involved in biofilm formation such as HWP1 was evaluated by semi-quantitative RT-PCR and relative real time RT-PCR. Allicin was shown to down-regulate the expression of HWP1.

Myeloid sarcoma of the urinary bladder with cutaneous tumour seeding after percutaneous suprapubic catheterization.

Myeloid sarcoma (MS) is a rare extramedullary myeloid tumour. It has been reported in various sites, including lymph node, bone, skin, soft tissue, various organs and the CNS. It may precede or occur concurrently with acute myeloid leukemia. Urinary bladder involvement is extremely uncommon. We report a 70-year-old female who had MS of the urinary bladder, presented with frank and persistent hematuria associated with lower abdominal pain. She subsequently had tumour seeding in the abdominal skin via percutaneous suprapubic catheter. Tumours from both the urinary bladder and skin showed immature cells that were immunoreactive toward LCA (focal), MPO (strong), CD99 (weak) and CD117 (weak). Summary of cases in the literature is presented. The potential of its misdiagnosis and the useful markers for the diagnosis of MS are discussed.

Comparison between efficacy of allicin and fluconazole against Candida albicans in vitro and in a systemic candidiasis mouse model.

The efficacy of allicin compared with fluconazole in alleviating systemic Candida albicans infections was evaluated both in vitro and in vivo through a systemic candidiasis mouse model. Determination of in vitro minimum inhibitory concentrations (MICs) for different C. albicans isolates revealed that both allicin and fluconazole showed different MICs that ranged from 0.05 to 12.5 µg mL(-1) and 0.25 to 16 µg mL(-1) , respectively. A time-kill study showed a significant effect of allicin (P<0.01) against C. albicans, comparable to that of fluconazole. Scanning electron microscopy observation revealed that, similar to fluconazole, allicin produced structural destruction of C. albicans cell surface at low MIC and lysis or puncture at high MIC concentrations. Treatment of BALB/c mice systemically infected with C. albicans showed that although the allicin treatment (at 5 mg kg(-1) day(-1) ) was slightly less efficacious than fluconazole treatment in terms of the fungal load reduction and host survival time, it was still effective against C. albicans in terms of mean survival time, which increased from 8.4 to 15.8 days. These results demonstrate the efficacy of anticandidal effects of allicin both in vitro and in an animal model of candidiasis and affirm the potential of allicin as an adjuvant therapy to fluconazole.

Expression analysis of SIR2 and SAPs1-4 gene expression in Candida albicans treated with allicin compared to fluconazole.

One of the main factors for virulence of fungus such as Candida albicans is the ability to change its morphology from yeast to hyphae. Allicin, one of the volatile sulfur-oil compounds from freshly crushed garlic, has a variety of antifungal activities. In this study, the effect of allicin on growth and hyphae production in C. albicans as compared to fluconazole, an antifungal drug was investigated using survival time in vitro and microscopic image at different time intervals. Additionally, the expression of selected genes involved in hyphae formation and development such as SIR2 and SAP1-4 was evaluated by semi-quantitative RT-PCR and relative real time RT-PCR. Allicin was shown to down-regulate the expression of SIR2 (5.54 fold), similar to fluconazole (3.48 fold) at 2x MIC concentrations. Interestingly, allicin had no effect on SAPs1-4 expression, whereas fluconazole was able to suppress SAP4 expression. Our findings showed that allicin was effective in suppressing hyphae development of C. albicans to an extent that is sometimes equal or more than fluconazole. Moreover, allicin and fluconazole seemed to share a common anti-Candida mechanism through inhibition of SIR2 gene, while fluconazole appeared to also exert its fungistatic effect through another pathway that involved SAP4 suppression.

Myeloid sarcoma of the urinary bladder with cutaneous tumour seeding after percutaneous suprapubic catheterization

Myeloid sarcoma (MS) is a rare extramedullary myeloid tumour. It has been reported in various sites, including lymph node, bone, skin, soft tissue, various organs and the CNS. It may precede or occur concurrently with acute myeloid leukemia. Urinary bladder involvement is extremely uncommon. We report a 70-year-old female who had MS of the urinary bladder, presented with frank and persistent hematuria associated with lower abdominal pain. She subsequently had tumour seeding in the abdominal skin via percutaneous suprapubic catheter. Tumours from both the urinary bladder and skin showed immature cells that were immunoreactive toward LCA (focal), MPO (strong), CD99 (weak) and CD117 (weak). Summary of cases in the literature is presented. The potential of its misdiagnosis and the useful markers for the diagnosis of MS are discussed

A modified in situ RT-PCR method for localizing fungal-specific gene expression in Candida-infected mice renal cells.

In situ Reverse Transcriptase PCR (in situ RT-PCR) can amplify mRNA and localize gene expression in cells. However, this method is not feasible in fungi as the thick fungal cell wall constitutes a barrier to this procedure. We developed a two step in situ RT-PCR procedure which enabled the detection and localization of Candida tropicalis mRNA expression in formalin-fixed, paraffin-embedded (FFPE) mouse kidney sections. This in situ hybridization study revealed the first direct evidence for deposition of Candida tropicalis secreted aspartic proteinase 2 (CtSAP2) in the tip of pseudohyphae and its involvement in acute systemic candidiasis. We conclude that in situ RT-PCR can be successfully applied to FFPE tissues and will offer new perspectives in studying gene expression in Candida species.

Virulotyping of Salmonella enterica subsp. enterica isolated from indigenous vegetables and poultry meat in Malaysia using multiplex-PCR.

The increased occurrence of Salmonella occurrence in local indigenous vegetables and poultry meat can be a potential health hazards. This study is aimed to detect the prevalence of twenty different virulence factors among Salmonella enterica strains isolated from poultry and local indigenous vegetables in Malaysia via an optimized, rapid and specific multiplex PCR assay. The assay encompasses a total of 19 Salmonella pathogenicity islands genes and a quorum sensing gene (sdiA) in three multiplex reaction sets. A total of 114 Salmonella enterica isolates belonging to 38 different serovars were tested. Each isolate in under this study was found to possess up to 70% of the virulence genes tested and exhibited variable pathogenicity gene patterns. Reproducibility of the multiplex PCR assay was found to be 100% and the detection limit of the optimized multiplex PCR was tested with lowest detectable concentration of DNA 0.8 pg microl(-1). This study demonstrated various Salmonella pathogenicity island virulence gene patterns even within the same serovar. This sets of multiplex PCR system provide a fast and reliable typing approach based on Salmonella pathogenicity islands, thus enabling an effective monitoring of emerging pathogenic Salmonella strains as an additional tool in Salmonella surveillance studies.