Curcuma zedoaria also known as Temu putih is traditionally used in food preparations and treatment of various ailments including cancer. The cytotoxic activity of hexane, dichloromethane, ethyl acetate, methanol, and the methanol-soxhlet extracts of Curcuma zedoaria rhizomes was tested on two human cancer cell lines (Ca Ski and MCF-7) and a noncancer cell line (HUVEC) using MTT assay. Investigation on the chemical components in the hexane and dichloromethane fractions gave 19 compounds, namely, labda-8(17),12 diene-15,16 dial (1), dehydrocurdione (2), curcumenone (3), comosone II (4), curcumenol (5), procurcumenol (6), germacrone (7), zerumbone epoxide (8), zederone (9), 9-isopropylidene-2,6-dimethyl-11-oxatricyclo[6.2.1.0(1,5)]undec-6-en-8-ol (10), furanodiene (11), germacrone-4,5-epoxide (12), calcaratarin A (13), isoprocurcumenol (14), germacrone-1,10-epoxide (15), zerumin A (16), curcumanolide A (17), curcuzedoalide (18), and gweicurculactone (19). Compounds (1-19) were evaluated for their antiproliferative effect using MTT assay against four cancer cell lines (Ca Ski, MCF-7, PC-3, and HT-29). Curcumenone (3) and curcumenol (5) displayed strong antiproliferative activity (IC50 = 8.3 ± 1.0 and 9.3 ± 0.3 μg/mL, resp.) and were found to induce apoptotic cell death on MCF-7 cells using phase contrast and Hoechst 33342/PI double-staining assay. Thus, the present study provides basis for the ethnomedical application of Curcuma zedoaria in the treatment of breast cancer.
Plasmodium falciparum mitogen-activated protein (MAP) kinases, a family of enzymes central to signal transduction processes including inflammatory responses, are a promising target for antimalarial drug development. Our study shows for the first time that the P. falciparum specific MAP kinase 2 (PfMAP2) is colocalized in the nucleus of all of the asexual erythrocytic stages of P. falciparum and is particularly elevated in its phosphorylated form. It was also discovered that PfMAP2 is expressed in its highest quantity during the early trophozoite (ring form) stage and significantly reduced in the mature trophozoite and schizont stages. Although the phosphorylated form of the kinase is always more prevalent, its ratio relative to the nonphosphorylated form remained constant irrespective of the parasites' developmental stage. We have also shown that the TSH motif specifically renders PfMAP2 genetically divergent from the other plasmodial MAP kinase activation sites using Neighbour Joining analysis. Furthermore, TSH motif-specific designed antibody is crucial in determining the location of the expression of the PfMAP2 protein. However, by using immunoelectron microscopy, PPfMAP2 were detected ubiquitously in the parasitized erythrocytes. In summary, PfMAP2 may play a far more important role than previously thought and is a worthy candidate for research as an antimalarial.
Two new synthesized and characterized quinazoline Schiff bases 1 and 2 were investigated for anticancer activity against MCF-7 human breast cancer cell line. Compounds 1 and 2 demonstrated a remarkable antiproliferative effect, with an IC50 value of 6.246×10(-6) mol/L and 5.910×10(-6) mol/L, respectively, after 72 hours of treatment. Most apoptosis morphological features in treated MCF-7 cells were observed by AO/PI staining. The results of cell cycle analysis indicate that compounds did not induce S and M phase arrest in cell after 24 hours of treatment. Furthermore, MCF-7 cells treated with 1 and 2 subjected to apoptosis death, as exhibited by perturbation of mitochondrial membrane potential and cytochrome c release as well as increase in ROS formation. We also found activation of caspases-3/7, -8, and -9 in compounds 1 and 2. Moreover, inhibition of NF-κB translocation in MCF-7 cells treated by compound 1 significantly exhibited the association of extrinsic apoptosis pathway. Acute toxicity results demonstrated the nontoxic nature of the compounds in mice. Our results showed significant activity towards MCF-7 cells via either intrinsic or extrinsic mitochondrial pathway and are potential candidate for further in vivo and clinical breast cancer studies.
Electrophiles such as methylmercury (MeHg) affect cellular functions by covalent modification with endogenous thiols. Reactive persulfide species were recently reported to mediate antioxidant responses and redox signaling because of their strong nucleophilicity. In this study, we used MeHg as an environmental electrophile and found that exposure of cells to the exogenous electrophile elevated intracellular concentrations of the endogenous electrophilic molecule 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), accompanied by depletion of reactive persulfide species and 8-SH-cGMP which is a metabolite of 8-nitro-cGMP. Exposure to MeHg also induced S-guanylation and activation of H-Ras followed by injury to cerebellar granule neurons. The electrophile-induced activation of redox signaling and the consequent cell damage were attenuated by pretreatment with a reactive persulfide species donor. In conclusion, exogenous electrophiles such as MeHg with strong electrophilicity impair the redox signaling regulatory mechanism, particularly of intracellular reactive persulfide species and therefore lead to cellular pathogenesis. Our results suggest that reactive persulfide species may be potential therapeutic targets for attenuating cell injury by electrophiles.
Pathophysiological changes in diabetes like hyperglycemia, oxidative stress, insulin resistance and compensatory hyperinsulinemia predispose cells to malignant transformation and damage DNA repair mechanism. This study was designed to explore the potential synergistic toxic effects of anti-diabetic drug (Metformin), and an analgesic drug (Celecoxib) at cellular level. MTT assay run on Vero cell line revealed that the combinations of Metformin and Celecoxib augment the anti-proliferative effects, whereas Single cell gel electrophoresis spotlighted that Metformin produce non-significant DNA damage with the threshold concentration of 400μg/ml in peripheral blood mononuclear cells (lymphocytes and monocytes), while Celecoxib produced significant (P<0.05) DNA damage (class III comets) above the concentration of 75μg/ml, however the DNA damage or DNA tail protrusions by combinations of both drugs were less than what was observed with Celecoxib alone. Metformin or Celecoxib did not appear mutagenic against any mutant strains (TA 100 and TA 98) but their combination exhibited slight mutagenicity at much higher concentration. The results obtained at concentrations higher than the therapeutic level of drugs and reflect that Metformin in combination with Celecoxib synergistically inhibits the cell proliferation in a concentration dependent pattern. Since, this increase in cytotoxicity did not confer an increase in DNA damage; this combination could be adopted to inhibit the growth of malignant cell without producing any genotoxic or mutagenic effects at cellular level.
The current study investigated the cytotoxic effect of 3-(5-chloro-2-hydroxybenzylideneamino)-2-(5-chloro-2-hydroxyphenyl)-2,3-dihydroquinazolin-41(H)-one (A) and 3-(5-nitro-2-hydroxybenzylideneamino)-2-(5-nitro-2-hydroxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (B) on MCF-7, MDA-MB-231, MCF-10A and WRL-68 cells. The mechanism involved in apoptosis was assessed to evaluate the possible pathways induced by compound A and B. MTT assay results using A and B showed significant inhibition of MCF-7 cell viability, with IC50 values of 3. 27 ± 0.171 and 4.36 ± 0.219 μg/mL, respectively, after a 72 hour treatment period. Compound A and B did not demonstrate significant cytotoxic effects towards MDA-MB-231, WRL-68 and MCF-10A cells. Acute toxicity tests also revealed an absence of toxic effects on mice. Fluorescent microscopic studies confirmed distinct morphological changes (membrane blebbing and chromosome condensation) corresponding to typical apoptotic features in treated MCF-7 cells. Using Cellomics High Content Screening (HCS), we found that compound A and B could trigger the release of cytochrome c from mitochondria to the cytosol. The release of cytochrome c activated the expression of caspases-9 and then stimulated downstream executioner caspase-3/7. In addition, caspase-8 showed remarkable activity, followed by inhibition of NF-κB activation in A-and B-treated MCF-7 cells. The results indicated that A and B could induce apoptosis via a mechanism that involves either extrinsic or intrinsic pathways.
The current study investigated the anticancer properties of gold nanoparticles (SG-stabilized AuNPs) synthesized using water extracts of the brown seaweed Sargassum glaucescens (SG). SG-stabilized AuNPs were characterized by ultraviolet-visible spectroscopy, transmission and scanning electron microscopy, and energy dispersive X-ray fluorescence spectrometry. The SG-stabilized AuNPs were stable and small at 3.65 ± 1.69 nm in size. The in vitro anticancer effect of SG-stabilized AuNPs was determined on cervical (HeLa), liver (HepG2), breast (MDA-MB-231) and leukemia (CEM-ss) cell lines using fluorescence microscopy, flow cytometry, caspase activity determination, and MTT assays. After 72 h treatment, SG-stabilized AuNPs was shown to be significant (p < 0.05) cytotoxic to the cancer cells in a dose- and time-dependent manner. The IC50 values of SG-stabilized AuNPs on the HeLa, HepG2, CEM-ss, MDA-MB-231 cell lines were 4.75 ± 1.23, 7.14 ± 1.45, 10.32 ± 1.5, and 11.82 ± 0.9 μg/mL, respectively. On the other hand, SG-stabilized AuNPs showed no cytotoxic effect towards the normal human mammary epithelial cells (MCF-10A). SG-stabilized AuNPs significantly (p < 0.05) arrest HeLa cell cycle at G2/M phase and significantly (p < 0.05) activated caspases-3 and -9 activities. The anticancer effect of SG-stabilized AuNPs is via the intrinsic apoptotic pathway. The study showed that SG-stabilized AuNPs is a good candidate to be developed into a chemotherapeutic compound for the treatment of cancers especially cervical cancer.
Hendra virus (HeV) and Nipah virus (NiV) belong to the genus Henipavirus in the family Paramyxoviridae. Henipavirus infections were first reported in the 1990's causing severe and often fatal outbreaks in domestic animals and humans in Southeast Asia and Australia. NiV infections were observed in humans in Bangladesh, India and in the first outbreak in Malaysia, where pigs were also infected. HeV infections occurred in horses in the North-Eastern regions of Australia, with singular transmission events to humans. Bats of the genus Pteropus have been identified as the reservoir hosts for henipaviruses. Molecular and serological indications for the presence of henipa-like viruses in African fruit bats, pigs and humans have been published recently. In our study, truncated forms of HeV and NiV attachment (G) proteins as well as the full-length NiV nucleocapsid (N) protein were expressed using different expression systems. Based on these recombinant proteins, Enzyme-linked Immunosorbent Assays (ELISA) were developed for the detection of HeV or NiV specific antibodies in porcine serum samples. We used the NiV N ELISA for initial serum screening considering the general reactivity against henipaviruses. The G protein based ELISAs enabled the differentiation between HeV and NiV infections, since as expected, the sera displayed higher reactivity with the respective homologous antigens. In the future, these assays will present valuable tools for serosurveillance of swine and possibly other livestock or wildlife species in affected areas. Such studies will help assessing the potential risk for human and animal health worldwide by elucidating the distribution of henipaviruses.
Three main reproductive strategies have been described among insects: most common oviparity, ovoviviparity and viviparity. In the latter strategy, the embryonic development takes place within the body of the mother which provides gas exchange and nutrients for embryos. Here we present the results of histological and EM analyses of the female reproductive system of the viviparous earwig, Arixenia esau, focusing on all the modifications related to the viviparity. We show that in the studied species the embryonic development consists of two "physiological phases" that take place in two clearly disparate compartments, i.e. the terminal ovarian follicle and the uterus. In both compartments the embryos are associated with synthetically active epithelial cells. We suggest that these cells are involved in the nourishment of the embryo. Our results indicate that viviparity in arixeniids is more complex than previously considered. We propose the new term "pseudoplacento-uterotrophic viviparity" for this unique two-phase reproductive strategy.
This contribution features a small molecule that binds TrkC (tropomyosin receptor kinase C) receptor that tends to be overexpressed in metastatic breast cancer cells but not in other breast cancer cells. A sensitizer for (1)O2 production conjugated to this structure gives 1-PDT for photodynamic therapy. Isomeric 2-PDT does not bind TrkC and was used as a control throughout; similarly, TrkC- cancer cells were used to calibrate enhanced killing of TrkC+ cells. Ex vivo, 1- and 2-PDT where only cytotoxic when illuminated, and 1-PDT, gave higher cell death for TrkC+ breast cancer cells. A 1 h administration-to-illumination delay gave optimal TrkC+/TrkC--photocytotoxicity, and distribution studies showed the same delay was appropriate in vivo. In Balb/c mice, a maximum tolerated dose of 20 mg/kg was determined for 1-PDT. 1- and 2-PDT (single, 2 or 10 mg/kg doses and one illumination, throughout) had similar effects on implanted TrkC- tumors, and like those of 2-PDT on TrkC+ tumors. In contrast, 1-PDT caused dramatic TrkC+ tumor volume reduction (96% from initial) relative to the TrkC- tumors or 2-PDT in TrkC+ models. Moreover, 71% of the mice treated with 10 mg/kg 1-PDT (n = 7) showed full tumor remission and survived until 90 days with no metastasis to key organs.
Previous studies have shown that apoptosis-like features are observed in Blastocystis spp., an intestinal protozoan parasite, when exposed to the cytotoxic drug metronidazole (MTZ). This study reports that among the four subtypes of Blastocystis spp. investigated for rate of apoptosis when treated with MTZ, subtype 3 showed the highest significant increase after 72h of in vitro culture when treated with MTZ at 0.1mg/ml (79%; p<0.01) and 0.0001mg/ml (89%; p<0.001). The close correlation between viable cells and apoptotic cells for both dosages implies that the pathogenic potential of these isolates has been enhanced when treated with MTZ. This suggests that there is a mechanism in Blastocystis spp. that actually regulates the apoptotic process to produce higher number of viable cells when treated. Apoptosis may not just be programmed cell death but instead a mechanism to increase the number of viable cells to ensure survival during stressed conditions. The findings of the present study have an important contribution to influence chemotherapeutic approaches when developing drugs against the emerging Blastocystis spp. infections.
Fluorescence in situ hybridization (FISH) is increasingly gaining importance in clinical diagnostics settings. Due to the ability of the technique to detect chromosomal abnormalities in samples with low cellularity or containing a mixed population of cells even at a single-cell level, it has become more popular in cancer research and diagnosis. Here, we describe the FISH technique for detection of PAX8-PPARγ translocation in follicular thyroid neoplasms, and the optimal protocol for the detection of this fusion gene using in archival formalin-fixed paraffin-embedded (FFPE) thyroid tissue sections.
Blastocystis sp., an intestinal organism is known to cause diarrhea with metronidazole regarded as the first line of treatment despite reports of its resistance. The conflicting reports of variation in drug treatment have been ascribed to subtype differences. The present study evaluated in vitro responses due to metronidazole on ST3 isolated from three symptomatic and asymptomatic patients, respectively. Symptomatic isolates were obtained from clinical patients who showed symptoms such as diarrhea and abdominal bloating. Asymptomatic isolates from a stool survey carried out in a rural area. These patients had no other pathogens other than Blastocystis. Ultrastructural studies using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed drug-treated ST3 from symptomatic patients were irregular and amoebic with surface showing high-convoluted folding when treated with metronidazole. These organisms had higher number of mitochondrion-like organelle (MLO) with prominent cristae. However, the drug-treated ST3 from asymptomatic persons remained spherical in shape. Asymptomatic ST3 showed increase in the size of its central body with the MLO located at the periphery.
A novel cationic polymer, dextran-spermine (D-SPM), has been found to mediate gene expression in a wide variety of cell lines and in vivo through systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA) in cell lines and in the lungs of BALB/c mice via instillation delivery. In vitro studies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range.
The terbium trinitrate.trihydrate.18-crown ether-6, Tb(NO3)3(OH2)3.(18C6) complex has been characterized by elemental analysis, photoluminescence and single X-ray diffraction. The IC50 values were determined based on MTT assay while light and fluorescence microscopy imaging were employed to evaluate the cellular morphological changes. Alkaline comet assay was performed to analyze the DNA damage. The photoluminescence spectrum of the Tb complex excited at 325 nm displayed seven luminescence peaks corresponding to the (5)D4→(7)F(0, 1, 2, 3, 4, 5, 6) transitions. The cytotoxicity and genotoxicity studies indicated that the Tb(NO3)3(OH2)3.(18C6) complex and its salt form as well as the 18C6 molecule have excellent anti-amoebic activity with very low IC50 values are 7, 2.6 and 1.2 μg/mL, respectively, with significant decrease (p<0.05) in Acanthamoeba viability when the concentration was increased from 0 to 30 μg/mL. The mode of cell death in Acanthamoeba cells following treatment with the Tb complex was apoptosis. This is in contrast to the Tb(NO3)3.6H2O salt- and 18C6 molecule-treated Acanthamoeba, which exhibited necrotic type cells. The percentage of DNA damage following treatment with all the compounds at the IC25 values showed high percentage of type 1 with the % nuclei damage are 14.15±2.4; 46.00±4.2; 36.36±2.4; 45.16±0.6%, respectively for untreated, treated with Tb complex, Tb salt and 18C6 molecule. The work features promising potential of Tb(NO3)3(OH2)3.(18C6) complex as anti-amoebic agent, representing a therapeutic option for Acanthamoeba keratitis infection.
Crude methanol extracts of a marine sponge, Aaptos aaptos, collected from three different localities namely Kapas, Perhentian and Redang Islands, Terengganu, Malaysia, were tested in vitro on a pathogenic Acanthamoeba castellanii (IMR isolate) to examine their anti-amoebic potential. The examination of anti-Acanthamoebic activity of the extracts was conducted in 24 well plates for 72 h at 30 °C. All extracts possessed anti-amoebic activity with their IC(50) values ranging from 0.615 to 0.876 mg/mL. The effect of the methanol extracts on the surface morphology of A. castellanii was analysed under scanning electron microscopy. The ability of the extracts to disrupt the amoeba cell membrane was indicated by extensive cell's blebbing, changes in the surface morphology, reduced in cell size and with cystic appearance of extract-treated Acanthamoeba. Number of acanthapodia and food cup was also reduced in this Acanthamoeba. Morphological criteria of apoptosis in Acanthamoeba following treatment with the sponge's extracts was determined by acridine orange-propidium iodide staining and observed by fluorescence microscopy. By this technique, apoptotic and necrotic cells can be visualized and quantified. The genotoxic potential of the methanol extracts was performed by the alkaline comet assay. All methanol extracts used were significantly induced DNA damage compared to untreated Acanthamoeba by having high percentage of scores 1, 2, and 3 of the DNA damage. Results from cytotoxicity and genotoxicity studies carried out in the present study suggest that all methanol extracts of A. aaptos have anti-amoebic properties against A. castellanii.
Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in cell responses to mechanical stimuli that depend on the microscopic approach and the thresholding methods used and may result in contradictory interpretations.
Biphasic Calcium Phosphate (BCP) with a ratio of 20/80 Hydroxyapatite (HA)/Beta-tricalcium phosphate (β-TCP) promotes the differentiation of human dental pulp cells (HDPCs). In the current study, the genotoxicity of locally produced BCP of modified porosity (65%) with a mean pore size of 300micrometer (μm) was assessed using Comet and Ames assays. HDPCs were treated with BCP extract at three different inhibitory concentrations which were obtained based on cytotoxicity test conducted with concurrent negative and positive controls. The tail moment of HDPCs treated with BCP extract at all three concentrations showed no significant difference compared to negative control (p>0.05), indicating that BCP did not induce DNA damage to HDPCs. The BCP was evaluated using five tester strains of Salmonella typhimurium TA98, TA100, TA102, TA1537 and TA1538. Each strain was incubated with BCP extract with five different concentrations in the presence and absence of metabolic activation system (S9) mix. Concurrently, negative and positive controls were included. The average number of revertant colonies per plate treated with the BCP extract was less than double as compared to the number of revertant colonies in negative control plate and no dose-related increase was observed. Results from both assays suggested that the BCP of modified porosity did not exhibit any genotoxic effect under the present test conditions.
To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.