Effects of Root Extracts of Eurycoma longifolia Jack on Corpus Cavernosum of Rat.

OBJECTIVE: This study was conducted to investigate the mechanisms of action of Eurycoma longifolia in rat corpus cavernosum. MATERIALS AND METHODS: Tincture of the roots was concentrated to dryness by evaporating the ethanol in vacuo. This ethanolic extract was partitioned into 5 fractions sequentially with hexane, dichloromethane (DCM), ethyl acetate, butanol, and water. The corpus cavernosum relaxant activity of each fraction was investigated. The DCM fraction which showed the highest potency in relaxing phenylephrine-precontracted corpora cavernosa was purified by column chromatography. The effects of the most potent DCM subfraction in relaxing phenylephrine-precontracted corpora cavernosa, DCM-I, on angiotensin I- or angiotensin II-induced contractions in corpora cavernosa were investigated. The effects of DCM-I pretreatment on the responses of phenylephrine-precontracted corpora cavernosa to angiotensin II or bradykinin were also studied. An in vitro assay was conducted to evaluate the effect of DCM-I on angiotensin-converting enzyme activity. RESULTS: Fraction DCM-I decreased the maximal contractions (100%) evoked by angiotensin I and angiotensin II to 30 ± 14% and 26 ± 16% (p < 0.001), respectively. In phenylephrine-precontracted corpora cavernosa, DCM-I pretreatment caused angiotensin II to induce 82 ± 27% relaxation of maximal contraction (p < 0.01) and enhanced (p < 0.001) bradykinin-induced relaxations from 47 ± 8% to 100 ± 5%. In vitro, DCM-I was able to reduce (p < 0.001) the maximal angiotensin-converting enzyme activity to 78 ± 0.24%. CONCLUSION: Fraction DCM-I was able to antagonize angiotensin II-induced contraction to cause corpus cavernosum relaxation via inhibition of angiotensin II type 1 receptor and enhance bradykinin-induced relaxation through inhibition of angiotensin-converting enzyme.

King cobra (Ophiophagus hannah) venom L-amino acid oxidase induces apoptosis in PC-3 cells and suppresses PC-3 solid tumor growth in a tumor xenograft mouse model.

King cobra (Ophiophagus hannah) venom L-amino acid oxidase (OH-LAAO), a heat stable enzyme, has been shown to exhibit very potent anti-proliferative activity against human breast and lung tumorigenic cells but not in their non-tumorigenic counterparts. We further examine its in vitro and in vivo anti-tumor activity in a human prostate adenocarcinoma (PC-3) model. OH-LAAO demonstrated potent cytotoxicity against PC-3 cells with IC50 of 0.05 µg/mL after 72 h incubation in vitro. It induced apoptosis as evidenced with an increase in caspase-3/7 cleavages and an increase in annexin V-stained cells. To examine its in vivo anti-tumor activity, we treated PC-3 tumor xenograft implanted subcutaneously in immunodeficient NU/NU (nude) mice with 1 µg/g OH-LAAO given intraperitoneally (i.p.). After 8 weeks of treatment, OH-LAAO treated PC-3 tumors were markedly inhibited, when compared to the control group (P <0.05). TUNEL staining analysis on the tumor sections showed a significantly increase of apoptotic cells in the LAAO-treated animals. Histological examinations of the vital organs in these two groups showed no significant differences with normal tissues, indicating no obvious tissue damage. The treatment also did not cause any significant changes on the body weight of the mice during the duration of the study. These observations suggest that OH-LAAO cytotoxic effects may be specific to tumor xenografts and less to normal organs. Given its potent anti-tumor activities shown in vitro as well as in vivo, the king cobra venom LAAO can potentially be developed to treat prostate cancer and other solid tumors.

Development of an indirect enzyme immunoassay using monoclonal antibodies for the measurement of 17alpha-hydroxyprogesterone in human serum.

An indirect enzyme immunoassay for the measurement of total 17alpha-hydroxyprogesterone (17OHP) in serum using monoclonal antibodies generated in our laboratory was developed. Here, (a) instead of extraction with solvents, serum was heated to free protein-bound 17OHP and assay was performed at pH 9.6, (b) to ensure uniform assay conditions for both standards and samples, buffer for standards contained charcoal-stripped pre-heated pooled cord serum. Assays were done in 96-well EIA microplates pre-coated with 17alpha-hydroxyprogesterone-3-(o-carboxymethyl)oxime: bovine serum albumin. Secondary antibody was horseradish peroxidase-linked sheep anti-mouse IgG polyclonal antibody. The method was accurate and suitable for screening for congenital adrenal hyperplasia.

LMTK3 confers chemo-resistance in breast cancer.

Lemur tyrosine kinase 3 (LMTK3) is an oncogenic kinase that is involved in different types of cancer (breast, lung, gastric, colorectal) and biological processes including proliferation, invasion, migration, chromatin remodeling as well as innate and acquired endocrine resistance. However, the role of LMTK3 in response to cytotoxic chemotherapy has not been investigated thus far. Using both 2D and 3D tissue culture models, we found that overexpression of LMTK3 decreased the sensitivity of breast cancer cell lines to cytotoxic (doxorubicin) treatment. In a mouse model we showed that ectopic overexpression of LMTK3 decreases the efficacy of doxorubicin in reducing tumor growth. Interestingly, breast cancer cells overexpressing LMTK3 delayed the generation of double strand breaks (DSBs) after exposure to doxorubicin, as measured by the formation of γH2AX foci. This effect was at least partly mediated by decreased activity of ataxia-telangiectasia mutated kinase (ATM) as indicated by its reduced phosphorylation levels. In addition, our RNA-seq analyses showed that doxorubicin differentially regulated the expression of over 700 genes depending on LMTK3 protein expression levels. Furthermore, these genes were found to promote DNA repair, cell viability and tumorigenesis processes / pathways in LMTK3-overexpressing MCF7 cells. In human cancers, immunohistochemistry staining of LMTK3 in pre- and post-chemotherapy breast tumor pairs from four separate clinical cohorts revealed a significant increase of LMTK3 following both doxorubicin and docetaxel based chemotherapy. In aggregate, our findings show for the first time a contribution of LMTK3 in cytotoxic drug resistance in breast cancer.

Heterogeneity in cancer cells: variation in drug response in different primary and secondary colorectal cancer cell lines in vitro.

Tumor heterogeneity may give rise to differential responses to chemotherapy drugs. Therefore, unraveling tumor heterogeneity has an implication for biomarker discovery and cancer therapeutics. To test this phenomenon, we investigated the differential responses of three secondary colorectal cancer cell lines of different origins (HCT116, HT29, and SW620 cells) and four novel primary cell lines obtained from different colorectal cancer patients to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP) and explored the differences in gene expression among the primary cell lines in response to exposure to cytotoxic drugs. Cells were exposed to different doses of 5-FU and L-OHP separately or in combinations of equitoxic drug or equimolar drug ratios (median effect of Chou-Talalay principle). Cell viability was assessed using MTT assay and the respective IC50 values were determined. Changes in gene expression in primary cell lines after exposure to the same drug doses were compared using real-time PCR array. The sensitivities (IC50) of different cell lines, both secondary and primary, to 5-FU and L-OHP were significantly different, whether in monotherapy or combined treatment. Primary cell lines needed higher doses to reach IC50. There were variations in gene expression among the primary cell lines of different chemosensitivities to the challenge of the same combined dose of 5-FU and L-OHP. The results confirm the heterogeneous nature of colorectal cancer cells from different patient tumors. Studies using primary cancer cells established from patient's tumors rather than secondary cell lines will more closely reflect the actual character of the disease.

First Report of Eurycoma longifolia Jack Root Extract Causing Relaxation of Aortic Rings in Rats.

Although Eurycoma longifolia has been studied for erectile function, the blood pressure- (BP-) lowering effect has yet to be verified. Hence, this study aims at investigating the BP-lowering properties of the plant with a view to develop an antihypertensive agent that could also preserve erectile function. Ethanolic root extract was partitioned by hexane, dichloromethane (DCM), ethyl acetate, butanol, and water. The DCM fraction, found to be potent in relaxing phenylephrine- (PE-) precontracted rat aortic rings, was further purified by column chromatography. Subfraction DCM-II, being the most active in relaxing aortae, was studied for effects on the renin-angiotensin and kallikrein-kinin systems in aortic rings. The effect of DCM-II on angiotensin-converting enzyme (ACE) activity was also evaluated in vitro. Results showed that DCM-II reduced (p < 0.05) the contractions evoked by angiotensin I and angiotensin II (Ang II). In PE-precontracted rings treated with DCM-II, the Ang II-induced contraction was attenuated (p < 0.05) while bradykinin- (BK-) induced relaxation enhanced (p < 0.001). In vitro, DCM-II inhibited (p < 0.001) the activity of ACE. These data demonstrate that the vasodilatory effect of DCM-II appears to be mediated via inhibition of Ang II type 1 receptor and ACE as well as enhancement of Ang II type 2 receptor activation and BK activity.

Stable expression of H1C2 monoclonal antibody in NS0 and CHO cells using pFUSE and UCOE expression system.

From our recent publications, it was found that the deimmunization method (Dharshanan et al. (2012) Sci Res Essays 7:2288-2299) should be applied for the development of humanized anti-C2 monoclonal antibody (H1C2 mAb). However, the overlapping-PCR mutagenesis procedure used to insert the variable regions into cloning vectors was laborious and time-consuming. Additionally, the expression of H1C2 mAb in NS0 cells was low in static culture vessels. Therefore H1C2 mAb was redeveloped by deimmunization method with the following modifications in order to optimize the production of H1C2 mAb. First, instead of the overlapping-PCR mutagenesis procedure, synthetic DNA coding the variable regions were used to express the mAb. Second, two expression vectors, pFUSE and UCOE, were used to express H1C2 mAb in NS0 cells and CHO cells in order to investigate the combination that gave the highest number of high producing stable clones. This will provide the highest chance of finding clones with the requisite high productivity and stability required for manufacturing. We found that transfection of UCOE in CHO cells generated the highest number of high producing stable clones. To our knowledge, this is the first time that H1C2 mAb has been expressed in CHO cells.

Culture of low passage colorectal cancer cells and demonstration of variation in selected tumour marker expression.

There is increasing evidence that a tumour comprises of heterogeneous population of cells. Thus, studying homogenous cell lines in vitro may yield results that are not reflective of the true situation in a tumour and studying low passage cell lines maintained in a heterogeneous population before they transform away from the original state may provide a more complete picture of colorectal cancer. A method was developed to isolate and establish low passage colorectal cancer cell lines from tumour biopsies. The media contents, combination of antimicrobials and specimen collection and transport conditions employed, successfully eliminated microbial contamination which is frequently present in samples obtained from the gastrointestinal tract. A variety of growth forms indicating a heterogeneous mixture of cells was seen in the initial cultures. Using fluorescence immunocytochemistry, primary tumour cultures were shown to variably express selected tumour markers, carcinoembryonic antigen and C2 antigen. These low passage cell lines growing in a heterogeneous environment would more closely reflect the characteristics of the cells of the original tumour.

Pigmentation and dermal conservative effects of the astonishing algae Sargassum polycystum and Padina tenuis on guinea pigs, human epidermal melanocytes (HEM) and Chang cells.

BACKGROUND: The preference for a fairer skin-tone has become a common trend among both men and women around the world. In this study, seaweeds Sargassum polycystum and Padina tenuis were investigated for their in vitro and in vivo potentials in working as skin whitening agents. Seaweed has been used as a revolutionary skin repairing agent in both traditional and modern preparations. The high antioxidant content is one of the prime reasons for its potent action. It has been employed in traditional Chinese and Japanese medicine. For centuries, most medical practitioners in the Asian cultures have known seaweed as an organic source of vitamins, minerals, fatty acids like omega-3 and omega-6 and antioxidants. The present objective of the study was to evaluate the potent dermal protective effect of the two seaweeds Sargassum polycystum and Padina tenuis on human cell lines and guinea pigs. MATERIAL AND METHODS: Seaweeds were extracted with ethanol and further fractionated with hexane, ethyl acetate and water. The extracts were tested for mushroom tyrosinase inhibitory activity, cytotoxicity in human epidermal melanocyte (HEM), and Chang cells. Extracts with potent melanocytotoxicity were formulated into cosmetic cream and tested on guinea pigs in dermal irritation tests and de-pigmentation assessments. RESULTS: Both Sargassum polycystum and Padina tenuis seaweeds showed significant inhibitory effect on mushroom tyrosinase in the concentration tested. SPEt showed most potent cytotoxicity on HEM (IC50 of 36µg/ml), followed by SPHF (65µg/ml), and PTHF (78.5µg/ml). SPHF and SPEt reduced melanin content in skin of guinea pigs when assessed histologically. CONCLUSION: SPEt, SPHF and PTHF were able to inhibit HEM proliferation in vitro, with SPHF being most potent and did not cause any dermal irritation in guinea pigs. The results obtained indicate that SPHF is a promising pharmacological or cosmetic agent.

Development and evaluation of polymerase chain reaction assay to detect Burkholderia genus and to differentiate the species in clinical specimens.

Molecular-based techniques are becoming desirable as tools for identification of infectious diseases. Amongst the Burkholderia spp., there is a need to differentiate Burkholderia pseudomallei from Burkholderia cepacia, as misidentification could lead to false treatment of patients. In this study, conventional PCR assay targeting three genes was developed. Primers were designed for the amplification of Burkholderia genus-specific groEL gene, B. pseudomallei-specific mprA gene and B. cepacia-specific zmpA gene. The specificity and sensitivity of the assay was tested with 15 negative control strains and 71 Burkholderia spp. isolates including positive controls B. pseudomallei K96243 and ATCC B. cepacia strain. All B. pseudomallei strains were positive for groEL (139 bp) and mprA (162 bp), indicating a sensitivity of 100%. All B. cepacia strains produced amplicons for detection of groEL and zmpA (147 bp). Specificity using negative strains was 100%. In this study, a PCR assay specific for the detection of Burkholderia spp. and differentiation of the genus B. pseudomallei and B. cepacia was developed. The conventional assay has to be performed separately for each species due to the similar size of the PCR products amplified. This format may therefore be recommended for use as a diagnostic tool in laboratories where real-time PCR machines are not available. However, the real-time PCR was able to detect and differentiate the genus and species in single duplex assay.

Production and characterization of monoclonal antibodies against 17alpha-hydroxyprogesterone.

Hybridomas secreting monoclonal antibodies (MAbs) against 17alpha-hydroxyprogesterone (17OHP) have been generated. These MAbs are highly specific and have an affinity of 7-12 x 10(7) M(1). The hybridomas were obtained by fusion of spleen cells from immunized mice with mouse myeloma P3X63 Ag8.653 cells. The antigen used for immunization was 17OHP conjugated to bovine serum albumin (17OHP:BSA). Fused cells were plated and cloned in 96-well microtiter plates. Wells containing hybridomas were screened simultaneously for specific gamma globulin (IgG) and anti-17OHP activity using an enzyme-linked immunosorbent assay (ELISA)-based method, which is faster than the conventional radioimmunoassay (RIA) screening procedure. Limiting dilution methods were used to obtain single hybridoma clones producing MAb. The stable hybridomas secreting anti-17OHP MAbs were expanded into bioreactors or ascites fluid for large-scale production of the required antibodies. These MAbs will be used in the formulation of a 17OHP assay kit to screen for congenital adrenal hyperplasia (CAH) in local newborn human population.

A Study of the in vitro cytotoxic activity of Gelsemium elegans using human ovarian and breast cancer cell lines

The crude methanol extracts of Gelsemium elegans leaves were assessed for their cytotoxic activity using the microculture 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cellular viability. This study utilized two different types of human cancer cell lines, CaOV-3 (human ovarian cancer cells) and MDA-MB-231 (human estrogen receptor negative breast cancer cells), allowing for comparison of toxicity of G. elegans against these two cancer cells lines. Our results showed that the methanol extract of G. elegans exhibited high cytotoxicity against the human ovarian cancer cell line CaOV-3 with an IC50 value of 5microg/ml after 96 h incubation. However, G. elegans displayed discernibly less toxicity against the MDA-MB-231 cells with an IC50 value 40microg/ml after 96 h incubation and this effect was dose- and time-dependent, up to 72h and 20-30 microg/ml. In conclusion, our results demonstrated that G. elegans is potently cytotoxic against the human ovarian cancer cell line CaOV-3 and to a lesser extend towards the human breast carcinoma cancer MDA-MB-231 cells, suggesting that the extract is selective towards CaOV-3 cells and may have a chemotherapeutic role for ovarian cancer treatment in the future.