Abstract: Alpha (α)-tomatine, a major saponin found in tomato has been shown to inhibit the growth of androgen-independent prostate cancer PC-3 cells. The effects of α-tomatine in combination with the chemotherapeutic agent paclitaxel against PC-3 cells were investigated in the present study. Combined treatment with a sub-toxic dose of α-tomatine and paclitaxel significantly decreased cell viability with concomitant increase in the percentage of apoptotic PC-3 cells. The combined treatment, however, had no cytotoxic effect on the non-neoplastic prostate RWPE-1 cells. Apoptosis of PC-3 cells was accompanied by the inhibition of PI3K/Akt pro-survival signaling, an increase in the expression of the pro-apoptotic protein BAD but a decrease in the expressions of anti-apoptotic proteins, Bcl-2 and Bcl-xL. Results from a mouse xenograft model showed the combined treatment completely suppressed subcutaneous tumor growth without significant side effects. Consistent with its in vitro anti-cancer effects, tumor materials from mice showed increased apoptosis of tumor cells with reduced protein expression of activated PI3K/Akt. These results suggest that the synergistic anti-cancer effects of paclitaxel and α-tomatine may be beneficial for refractory prostate cancer treatment.
Abstract: Nuclear factor-kappa B (NF-κB) plays a role in prostate cancer and agents that suppress its activation may inhibit development or progression of this malignancy. Alpha (α)-tomatine is the major saponin present in tomato (Lycopersicon esculentum) and we have previously reported that it suppresses tumor necrosis factor-alpha (TNF-α)-induced nuclear translocation of nuclear factor-kappa B (NF-κB) in androgen-independent prostate cancer PC-3 cells and also potently induces apoptosis of these cells. However, the precise mechanism by which α-tomatine suppresses NF-κB nuclear translocation is yet to be elucidated and the anti-tumor activity of this agent in vivo has not been examined.
Abstract: MicroRNAs (miRNAs) regulate various cellular processes. While several genes associated with replicative senescence have been described in endothelial cells, miRNAs that regulate these genes remain largely unknown. The present study was designed to identify miRNAs associated with replicative senescence and their target genes in human umbilical vein endothelial cells (HUVECs). An integrated miRNA and gene profiling approach revealed that hsa-miR-299-3p is upregulated in senescent HUVECs compared with the young cells, and one of its target genes could be IGF1. IGF1 was upregulated in senescent compared with young HUVECs, and knockdown of hsa-miR-299-3p dose-dependently increased the mRNA expression of IGF1, more significantly observed in the presenescent cells (passage 19) compared with the senescent cells (passage 25). Knockdown of hsa-miR-299-3p also resulted in significant reduction in the percentage of cells positively stained for senescence-associated β-galactosidase and increases in cell viability measured by MTT assay but marginal increases in cell proliferation and cell migration capacity measured by real-time growth kinetics analysis. Moreover, knockdown of hsa-miR-299-3p also increased proliferation of cells treated with H2O2 to induce senescence. These findings suggest that hsa-miR-299-3p may delay or protect against replicative senescence by improving the metabolic activity of the senesced cells but does not stimulate growth of the remaining cells in senescent cultures. Hence, these findings provide an early insight into the role of hsa-miR-299-3p in the modulation of replicative senescence in HUVECs.
Abstract: Alarin is an alternative-splicing form of GALP (galanin-like peptide). It shares only 5 conserved amino acids at the N-terminal region with GALP which is involved in a diverse range of normal brain functions. This study seeks to investigate whether alarin has additional functions due to its differences from GALP. Here, we have shown using a radial diffusion assay that alarin but not GALP inhibited the growth of Escherichia coli (strain ML-35). The conserved N-terminal region, however, remained essential for the antimicrobial activity of alarin as truncated peptides showed reduced killing effect. Moreover, alarin inhibited the growth of E. coli in a similar potency as human cathelicidin LL-37, a well-studied antimicrobial peptide. Electron microscopy further showed that alarin induced bacterial membrane blebbing but unlike LL-37, it did not cause hemolysis of erythrocytes. In addition, alarin is only active against the gram-negative bacteria, E. coli but not the gram-positive bacteria, Staphylococcus aureus. Thus, these data suggest that alarin has potentials as an antimicrobial and should be considered for the development in human therapeutics.
Abstract: We have identified a novel 7-azaindole series of anaplastic lymphoma kinase (ALK) inhibitors. Compounds 7b, 7 m and 7 n demonstrate excellent potencies in biochemical and cellular assays. X-ray crystal structure of one of the compounds (7 k) revealed a unique binding mode with the benzyl group occupying the back pocket, explaining its potency towards ALK and selectivity over tested kinases particularly Aurora-A. This binding mode is in contrast to that of known ALK inhibitors such as Crizotinib and NVP-TAE684 which occupy the ribose binding pocket, close to DFG motif.
Abstract: This study investigates the effects of 2-phenyl-1-benzopyran-4-one (flavone) on DENV-2 infectivity in Vero cells. Virus adsorption and attachment and intracellular virus replication were investigated using a foci forming unit assay (FFUA) and quantitative RT-PCR, respectively. Addition of flavone (100 μg/mL) significantly increased the number of DENV-2 foci by 35.66% ± 1.52 and 49.66% ± 2.51 when added during and after virus adsorption to the Vero cells, respectively. The average foci size after 4 days of infection increased by 33% ± 2.11 and 89% ± 2.13. The DENV-2 specific RNA copy number in the flavone-treated infected cells increased by 6.41- and 23.1-fold when compared to the mock-treated infected cells. Flavone (100 μg/mL) did not promote or inhibit Vero cell proliferation. The CC₅₀ value of flavone against Vero cells was 446 µg/mL. These results suggest that flavone might enhance dengue virus replication by acting antagonistically towards flavonoids known to inhibit dengue virus replication.
Abstract: Targeting angiogenesis has emerged as an attractive and promising strategy in anti-cancer therapeutic development. The present study investigates the anti-angiogenic potential of Panduratin A (PA), a natural chalcone isolated from Boesenbergia rotunda by using both in vitro and in vivo assays.
Abstract: Dengue is a serious arboviral disease currently with no effective antiviral therapy or approved vaccine available. Therefore, finding the effective compound against dengue virus (DENV) replication is very important. Among the natural compounds, bioflavonoids derived mainly from plants are of interest because of their biological and medicinal benefits.
Abstract: Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 μg/ml. The concentration that inhibited 50% of cell growth (GI(50)) was 5.1 μg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p<0.0001, T-test). At 8 μg/ml (GI(70)), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p<0.05, T-test, n=8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 μg/ml to 15 μg/ml with a GI(50) of 0.58 μg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p<0.05, T-test, n=9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 μg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.
Abstract: Alpha-tomatine (α-tomatine) is the major saponin in tomato (Lycopersicon esculentum). This study investigates the chemopreventive potential of α-tomatine on androgen-independent human prostatic adenocarcinoma PC-3 cells.
Abstract: The graft-versus-myeloma (GVM) effect represents a powerful form of immune attack exerted by alloreactive T cells against multiple myeloma cells, which leads to clinical responses in multiple myeloma transplant recipients. Whether myeloma cells are themselves able to induce alloreactive T cells capable of the GVM effect is not defined. Using adoptive transfer of T naive cells into myeloma-bearing mice (established by transplantation of human RPMI8226-TGL myeloma cells into CD122(+) cell-depleted NOD/SCID hosts), we found that myeloma cells induced alloreactive T cells that suppressed myeloma growth and prolonged survival of T cell recipients. Myeloma-induced alloreactive T cells arising in the myeloma-infiltrated bones exerted cytotoxic activity against resident myeloma cells, but limited activity against control myeloma cells obtained from myeloma-bearing mice that did not receive T naive cells. These myeloma-induced alloreactive T cells were derived through multiple CD8(+) T cell divisions and enriched in double-positive (DP) T cells coexpressing the CD8αα and CD4 coreceptors. MHC class I expression on myeloma cells and contact with T cells were required for CD8(+) T cell divisions and DP-T cell development. DP-T cells present in myeloma-infiltrated bones contained a higher proportion of cells expressing cytotoxic mediators IFN-γ and/or perforin compared with single-positive CD8(+) T cells, acquired the capacity to degranulate as measured by CD107 expression, and contributed to an elevated perforin level seen in the myeloma-infiltrated bones. These observations suggest that myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones are enriched with DP-T cells equipped with cytotoxic effector functions that are likely to be involved in the GVM effect.
Abstract: Dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. Effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2) in Vero cell was evaluated. Anti-dengue activity of these compounds was determined at different stages of DENV-2 infection and replication cycle. DENV replication was measured by Foci Forming Unit Reduction Assay (FFURA) and quantitative RT-PCR. Selectivity Index value (SI) was determined as the ratio of cytotoxic concentration 50 (CC50) to inhibitory concentration 50 (IC50) for each compound.
Abstract: Gangliosides are target receptors for bacterial entry, yet those present in human milk exhibit a protective role against bacterial infection. Here, we show that treatment with ganglioside mixture at a concentration of 100 microg/mL resulted in significant inhibition of the vacuole formation activity of Helicobacter pylori vacuolating cytotoxin (VacA) in gastric epithelial cancer AZ-521 cells. All gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1b) examined showed good neutralizing capacity against VacA. A pull-down assay was performed using lyso-GM1 coupled to Sepharose as the tagged polysaccharide polymer to capture VacA from H. pylori culture supernatant. GM1-VacA complexes were successfully precipitated, suggesting that GM1 binds directly to VacA. The hydrodynamic binding of lyso-GM1 and VacA measured by fluorescence correlation spectroscopy had a K(d) value of 190 nM. VacA also bound to lyso-GM1 at pH 2 corresponding to the physiological pH of human stomach. Collectively, these results showed that direct binding of H. pylori VacA to free gangliosides neutralizes the toxin activity of VacA. These findings offer an alternative insight into the role of gangliosides in VacA toxicity and the pathogenesis of H. pylori.
Abstract: The normally high concentration of zinc in normal prostate gland is significantly reduced in malignant prostate tissues, but its precise role in prostate tumorigenesis remains unclear. The present study investigates the growth and transcriptional responses of LNCaP prostate cancer cells to prolonged high Zn2+ treatment. Restoration of high intracellular Zn2+ to LNCaP cells significantly reduced the cell proliferation rate by 42.2+/-7.4% at the exponential growth phase and the efficiency of colony formation on soft agar by 87.2+/-2.5% at week 5 post-treatment. At least 161 LNCaP cell genes responded to the high intracellular Zn2+, including approximately 10.6% genes that negatively regulate cell growth and approximately 16.1% genes that promote cancer cell proliferation. Inhibition of cell growth was transient as normal proliferation rate and colony formation efficiency were restored later even in the continuous presence of high intracellular Zn2+. RT-qPCR showed constitutively higher expression levels of FBL, CD164 and STEAP1 in LNCaP cells. FBL and CD164 were responsive to the treatment with Zn2+ in PNT2 prostate normal cells and were further overexpressed in the prolonged Zn2+-treated LNCaP cells. These observations suggest that in general high Zn2+ has suppressive effects on prostate cancer cell growth but continuous exposure to an environment of high Zn2+ can lead to the overexpression of cancer promoting genes such as FBL and CD164. This could be the antagonistic mechanism used to overcome the initial cell growth inhibitory effects of high Zn2+. These findings support a potential detrimental role of Zn2+ in prostate cancer.
Abstract: Malignant prostate tissues have markedly reduced zinc (Zn(2+)) contents in comparison to non-malignant tissues. In this study, we restored a high intracellular Zn(2+) level to LNCaP prostate cancer cells by culturing the cells in a growth medium supplemented with a supraphysiological concentration of Zn(2+) (10 microg/ml) over 5 weeks. The intracellular Zn(2+) level increased in the Zn(2+)-treated cells, and there was a marked increase in the presence of zincosomes, a Zn(2+)-specific intracellular organelle. The proliferation rate of the Zn(2+)-treated cells was markedly reduced. There was also a significant increase (36.6% +/- 6.4%) in the total tyrosine phosphorylated proteins. Vaccinia H1-related (VHR) phosphatase, zeta chain-associated protein-70 (ZAP-70) kinase and phosphorylated extracellular signal-regulated protein kinase 1 and 2 (p-ERK 1 and 2) were also present in higher abundance. Treatment with TPEN, which chelates Zn(2+), reduced the abundance of VHR phosphatase and ZAP-70 kinase, but increased the abundance of p-ERK 1. However, the TPEN treatment restored the Zn(2+)-treated LNCaP cell proliferation to a rate comparable to that of the non Zn(2+)-treated cells. These results highlight the importance of a high intracellular Zn(2+) content and the VHR/ZAP-70-associated pathways in the modulation of LNCaP prostate cancer cell growth.
Abstract: Prostate cancer is an age-related disease that is linked to the inability of prostate cells to accumulate zinc following transformation. It is shown in the present study that the basal percentage of normal prostate cells expressing senescence-associated beta-galactosidase (SA-beta-gal) is higher than that of the cancer cells. In the presence of high zinc in the cell culture medium, the percentage of normal prostate cells expressing the SA-beta-gal increased but not that of the cancer cells. Increased intracellular zinc occurs in the prostate cancer cells treated with supraphysiologic concentration of zinc but it does not induce senescence or decrease the telomerase activities in these cells. Senescence, however, occurred when the prostate cancer cells DNA is damaged by irradiation. These findings suggest that prostate cancer cells are insensitive to the senescence-inducing effects of zinc but the cancer cells retain the capacity to undergo senescence through other pathways.
Abstract: Chikungunya virus infection recently reemerged in Malaysia after 7 years of nondetection. Genomic sequences of recovered isolates were highly similar to those of Malaysian isolates from the 1998 outbreak. The reemergence of the infection is not part of the epidemics in other Indian Ocean countries but raises the possibility that chikungunya virus is endemic in Malaysia.
Abstract: The first flavivirus chimera encoding dengue 4 virus (D4) PrM and E structural proteins in a Japanese encephalitis virus (JEV) backbone was successfully generated using the long-PCR based cDNA-fragment stitching (LPCRcFS) technique, demonstrating the technique's applicability for rapid preparation of flavivirus chimeras. The JEV/D4 chimera multiplied at levels equal to JEV and D4 in the mosquito cell line C6/36, while in a mouse neuronal cell line (N2a) JEV replicated efficiently, but JEV/D4 and D4 did not. In mouse challenge experiments, JEV/D4 showed a lack of neuroinvasiveness similar to D4 when inoculated intraperitoneally, but demonstrated attenuated neurovirulence (LD50=3.17 x 10(4) f.f.u.) when inoculated intracranially. It was also noted that mice receiving intraperitoneal challenge with JEV/D4 possessed D4-specific neutralization antibody and in addition clearly showed resistance to JEV intraperitoneal challenge (at 100 x LD50). This suggests that immunity to anti-JEV non-structural protein(s) offers protection against JEV infection in vivo. Dengue secondary infection was also simulated by challenging mice pre-immunized with dengue 2 virus, with D4 or JEV/D4. Mice showed higher secondary antibody response to challenge with JEV/D4 than to D4, at 210,000 and 37,000 averaged ELISA units, respectively. Taken together, aside from demonstrating the LPCRcFS technique, it could be concluded that the PrM and E proteins are the major determinant of neuroinvasiveness for JEV. It is also expected that the JEV/D4 chimera with its pathogenicity in mice and atypical immune profile, could have applications in dengue prophylactic research, in vivo efficacy assessment of dengue vaccines and development of animal research on models of dengue secondary infection.
Abstract: Phylogenetic analyses of the envelope (E) gene sequence of five recently isolated dengue virus type 4 (DENV-4) suggested the emergence of a distinct geographical and temporal DENV-4 subgenotype IIA in Malaysia. Four of the isolates had direct ancestral lineage with DENV-4 Indonesia 1973 and showed evidence of intra-serotypic recombination with the other recently isolated DENV-4, MY01-22713. The E gene of isolate MY01-22713 had strong evidence of an earlier recombination involving DENV-4 genotype II Indonesia 1976 and genotype I Malaysia 1969. These results suggest that intra-serotypic recombination amongst DENV-4 from independent ancestral lineages may have contributed to the emergence of DENV-4 subgenotype IIA in Malaysia.
