Flavokawain A Induces Apoptosis in MCF-7 and MDA-MB231 and Inhibits the Metastatic Process In Vitro

Introduction

The kava-kava plant (Piper methsyticum) is traditionally known as the pacific elixir by the pacific islanders for its role in a wide range of biological activities. The extract of the roots of this plant contains a variety of interesting molecules including Flavokawain A and this molecule is known to have anti-cancer properties. Breast cancer is still one of the leading diagnosed cancers in women today. The metastatic process is also very pertinent in the progression of tumorigenesis.

Methods

MCF-7 and MDA-MB231 cells were treated with several concentrations of FKA. The apoptotic analysis was done through the MTT assay, BrdU assay, Annexin V analysis, cell cycle analysis, JC-1 mitochondrial dye, AO/PI dual staining, caspase 8/9 fluorometric assay, quantitative real time PCR and western blot. For the metastatic assays, the in vitro scratch assay, trans-well migration/invasion assay, HUVEC tube formation assay, ex vivo rat aortic ring assay, quantitative real time PCR and western blot were employed.

Results

We have investigated the effects of FKA on the apoptotic and metastatic process in two breast cancer cell lines. FKA induces apoptosis in both MCF-7 and MDA-MB231 in a dose dependent manner through the intrinsic mitochondrial pathway. Additionally, FKA selectively induces a G2/M arrest in the cell cycle machinery of MDA-MB231 and G1 arrest in MCF-7. This suggests that FKA''s anti-cancer activity is dependent on the p53 status. Moreover, FKA also halted the migration and invasion process in MDA-MB231. The similar effects can be seen in the inhibition of the angiogenesis process as well.

Conclusions

FKA managed to induce apoptosis and inhibit the metastatic process in two breast cancer cell lines, in vitro. Overall, FKA may serve as a promising candidate in the search of a new anti-cancer drug especially in halting the metastatic process but further in vivo evidence is needed.     

Induction of Apoptosis in MCF-7 Cells via Oxidative Stress Generation,Mitochondria-Dependent and Caspase-Independent Pathway by Ethyl Acetate Extract of Dillenia suffruticosa and Its Chemical Profile

Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic acid, gallic acid and β-sitosterol-3-O-β-D-glucopyranoside. The cytotoxicity of the isolated compounds was determined using MTT assay. Gallic acid was found to be most cytotoxic against MCF-7 cell line compared to others, with IC₅₀ of 36 ± 1.7 μg/mL (P<0.05). In summary, EADs generated oxidative stress, induced cell cycle arrest and apoptosis in MCF-7 cells by regulating numerous genes and proteins that are involved in the apoptotic signal transduction pathway. Therefore, EADs has the potential to be developed as an anti-cancer agent against breast cancer.     

BaxΔ2 Is a Novel Bax Isoform Unique to Microsatellite Unstable Tumors

The pro-death Bcl-2 family protein and tumor suppressor Bax is frequently mutated in tumors with microsatellite instability (MSI). The mutation often results in a “Bax negative” phenotype and therefore is generally thought to be beneficial to the development of the tumor. Here, we report the identification of a novel Bax isoform, BaxΔ2, which is unique to microsatellite unstable tumors. BaxΔ2 is generated by a unique combination of a microsatellite deletion in Bax exon 3 and alternative splicing of Bax exon 2. Consistently, BaxΔ2 is only detected in MSI cell lines and primary tumors. BaxΔ2 is a potent cell death inducer but does not directly target mitochondria. In addition, BaxΔ2 sensitizes certain MSI tumor cells to a subset of chemotherapeutic agents, such as adriamycin. Thus, our data provide evidence that mutation and alternative splicing of tumor suppressors such as Bax are not always beneficial to tumor development but can be detrimental instead.     

Effect of calcium ions on the density of lecithin and its effective molecular volume in lecithin-water dispersions

As the packing structure of lipid molecules in the liposomes will vary in the presence of ions, it is expected that the density of lipid and the effective volume of lipid molecules in the dispersions will also vary, albeit minutely. Density measurements of lipid-water dispersions with the addition of Ca(2+) ions were determined accurately. The effect of Ca(2+) ions on the molecular packing structure of the liposomes was elucidated from the results obtained. The results for the density of the lecithin in the dispersions with and without the addition of Ca(2+) ions are, respectively, 1.0782 and 1.0579 g cm(-3) at 25 degrees C; and 1.0048 and 0.9961 g cm(-3) at 50 degrees C. The average values of the effective molecular volume of lecithin in the dispersions with and without the addition of Ca(2+) ions are, respectively, 1.131E-21 and 1.152E-21 cm(3) at 25 degrees C; and 1.213E-21 and 1.224E-21 cm(3) at 50 degrees C.     

Robust two-stage estimation in hierarchical nonlinear models

Hierarchical models encompass two sources of variation, namely within and among individuals in the population; thus, it is important to identify outliers that may arise at each sampling level. A two-stage approach to analyzing nonlinear repeated measurements naturally allows parametric modeling of the respective variance structure for the intraindividual random errors and interindividual random effects. We propose a robust two-stage procedure based on Huber's (1981, Robust Statistics) theory of M-estimation to accommodate separately aberrant responses within an experimental unit and subjects deviating from the study population when the usual assumptions of normality are violated. A toxicology study of chronic ozone exposure in rats illustrates the impact of outliers on the population inference and hence the advantage of adopting the robust methodology. The robust weights generated by the two-stage M-estimation process also serve as diagnostics for gauging the relative influence of outliers at each level of the hierarchical model. A practical appeal of our proposal is the computational simplicity since the estimation algorithm may be implemented using standard statistical software with a nonlinear least squares routine and iterative capability.     

Isolation of three naturally occurring O-β-glucopyranosides of procyanidin polymers

Three O-β-glucosides of procyanidin polymers (condensed tannins) have been isolated from the ripe fruits of Cydonia oblonga (quince) and the barks of Pinus brutia and Picea abies.     

Phylloflavan,a characteristic constituent of Phyllocladus species

Selective visualization of 2D TLC of extracts of Phyllocladus species with vanillin—hydrochloric acid showed a characteristic pattern of constituents attributable to catechin, epicatechin and phylloflavan, a new flavanoid compound. Optical rotation measurements showed that while catechin and epicatechin are of the normal type with the 2R configurations, the flavan moiety in phylloflavan is of the opposite 2S configuration. Spectroscopic data of the new compound and its hydrolysis products showed phylloflavan to be ent-epicatechin-3-δ-(3,4-dihydroxyphenyl)-β-hydroxypentanoate.     

Mitochondrial DNA variants help monitor the dynamics of Wolbachia invasion into host populations

Wolbachia is the most widespread endosymbiotic bacterium of insects and other arthropods that can rapidly invade host populations. Deliberate releases of Wolbachia into natural populations of the dengue fever mosquito, Aedes aegypti, are used as a novel biocontrol strategy for dengue suppression. Invasion of Wolbachia through the host population relies on factors such as high fidelity of the endosymbiont transmission and limited immigration of uninfected individuals, but these factors can be difficult to measure. One way of acquiring relevant information is to consider mitochondrial DNA (mtDNA) variation alongside Wolbachia in field-caught mosquitoes. Here we used diagnostic mtDNA markers to differentiate infection-associated mtDNA haplotypes from those of the uninfected mosquitoes at release sites. Unique haplotypes associated with Wolbachia were found at locations outside Australia. We also performed mathematical and qualitative analyses including modelling the expected dynamics of the Wolbachia and mtDNA variants during and after a release. Our analyses identified key features in haplotype frequency patterns to infer the presence of imperfect maternal transmission of Wolbachia, presence of immigration and possibly incomplete cytoplasmic incompatibility. We demonstrate that ongoing screening of the mtDNA variants should provide information on maternal leakage and immigration, particularly in releases outside Australia. As we demonstrate in a case study, our models to track the Wolbachia dynamics can be successfully applied to temporal studies in natural populations or Wolbachia release programs, as long as there is co-occurring mtDNA variation that differentiates infected and uninfected populations.