Multiple SNPs Downregulate Gene Expression of Matrix Metallopeptidase 2 in MCF7 Breast Cancer Cells

@#Introduction: On a global scale, breast cancer contributes the highest cancer-related deaths in women due to metastasis which renders the treatments ineffective and non-targeted. The members of Matrix Metallopeptidases, particularly Matrix Metallopeptidase 2 (MMP2), are among the key players in breast cancer metastasis. In most cases, MMP2 was markedly upregulated and linked to poor prognosis. In a previous study, in silico analyses revealed that several coding single nucleotide polymorphisms (SNPs) of MMP2 were shown to reduce gene expression and mRNA stability of MMP2 in Malaysian breast cancer patients. Therefore, to validate the in silico predictions, the objective of this study was to determine the effects of multiple coding SNPs of MMP2 on the gene expression and mRNA stability of MMP2 in breast cancer cells. Methods: In the current study, breast adenocarcinoma MCF7 cells were transfected with MMP2 wild type and variant containing the coding SNPs. After confirmation of transfection by DNA sequencing, the gene expression level of MMP2 was evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) whereas mRNA stability of MMP2 was determined following treatment with actinomycin D. Results: MMP2 wild type and variant were successfully transfected in MCF7 cells based on sequencing and PCR analysis. It was found that the presence of coding SNPs lowered the gene expression level of MMP2, but not the stability of MMP2 mRNA. Conclusion: This study supports the in silico effects of MMP2 coding SNPs on its gene expression in an in vitro model.

Detection of Calreticulin as a Candidate Prognostic Biomarker in Invasive Breast Carcinoma from a Biological Scaffold-Based 3D Co-culture System

@#Introduction: Breast cancer is the most common cancer in women and the world’s second leading cause of death in women, after lung cancer. Calreticulin (CRT), an endoplasmic reticulum (ER) multipurpose protein, has been proposed as a potential biomarker for breast cancer. However, reports on the correlation between CRT expression and cell invasiveness in breast cancer micro-tissues are scarce. Thus, in the current study, we analyzed the potential correlation between CRT and invasiveness of breast cancer in a biological scaffold-based 3D co-culture system. Methods: MCF7, MDA-MB-231 and MCF-10A breast cell lines were co-cultured in a 3-dimensional (3D) system with MRC-5 lung fibroblast cell line in the cell density ratio of 3:1. Thereafter, calreticulin gene and protein expression levels were determined based on quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively. Moreover, via RT-qPCR analysis, the gene expression levels of calreticulin-related candidate metastasis genes in breast cancer micro-tissues were carried out. Results: The results showed occasional foci of lumen-like morphology in the non-cancerous breast micro-tissues and the formation of solid clusters for breast cancer micro-tissues. Moreover, immunohistochemistry results revealed protein expression of calreticulin in non-cancerous and cancerous breast micro-tissues with cytoplasmic and nucleic acid localizations. It was found that PCMT1 and ER-α genes were significantly downregulated (p < 0.01) in invasive breast cancer micro-tissues. Conclusion: This study suggests that CRT and CRT-related candidate metastasis genes may potentially serve as prognostic biomarkers in invasive breast carcinoma.

The Potentiality of Citral in Targeting Breast Cancer Multicellular Tumour Spheroids (MTS)

@#Introduction: As the high incidence of breast cancer has a profound impact on a global scale, there is a critical need to improve the clinical outcome of the patients, including efforts to utilize bioactive natural products as treatment or preventive measures. Citral, the essential oil of lemongrass has been reported to possess cytotoxicity in breast cancer cell line . The aim of present study was to determine the capability of citral in targeting aldehyde dehydrogenase-positive (ALDH+) cells in breast cancer cells. Methods: Both MCF-7 and MDA-MB-231 cells were cultured in serum-free media to generate multicellular tumour spheroids for the evaluation of citral as an antiproliferative agent. The cells were treated with identified IC50 (50±4.30 µM and 56±3.17 µM of citral, respectively) to investigate the cytotoxicity of citral. Staining using Propidium Iodide (PI) and Hoechst 33342 was carried out to determine cell proliferation and viability. Finally, ALDH+ cells were quantified via ALDEFLUOR assay. Analysis of differences was carried out by analysis of variance (ANOVA) and independent t-test with p<0.05 considered statistically significant. Results: The size of spheroids in both cancer cell lines were reduced after treatment with the citral. PI and Hoechst 33342 staining also revealed that citral gave rise to a mixture of cells that are normal and undergoing apoptosis and necrosis. ALDEFLUOR assay analysis revealed citral significantly (p <0.05 ) inhibited the population of ALDH+ cells in MCF7 cells. Conclusion: It was demonstrated that citral reduced the ALDH+ cell population in MCF7 breast cancer spheroids by inhibiting the ALDH activity.

Optimized Aqueous Extraction Conditions for Maximal Phenolics, Flavonoids and Antioxidant Capacity from Artocarpus heterophyllus (Jackfruit) Leaves by Response Surface Methodology (RSM)

@#Introduction: There are numerous studies on the therapeutic properties of Artocarpus heterophyllus. However, studies on the aqueous extraction of A. heterophyllus leaves are limited. This present study was conducted to optimize the extraction conditions of A. heterophyllus leaves to yield the highest phenolic, flavonoids and antioxidant contents. Methods: Response surface methodology (RSM) was employed to obtain a higher phenolic extraction parameter(s) of A. heterophyllus leaves using Central Composite Design (CCD). The antioxidant activity was then determined via ABTS (2,29-azinobis (3 ethylbenzothiazoline-6-sulfonic acid)) and DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay and analysis of the individual phenolics was performed by high performance liquid chromatography (HPLC). Results: The optimum extraction conditions with higher phenolics content and antioxidant activity was achieved at 81°C, 100 min and 40 mL/g sample with a good desirability value of 0.87. Under these optimized parameters, total phenolics and flavonoids were 174.48 ± 4.05 mg GAE/g sample and 21.44 ± 0.05 mg RE/g sample, respectively. Meanwhile, antioxidant activity via ABTS and DPPH assays were 90.88% ± 0.09 and 87.22% ± 0.62, respectively. Finally, under optimal extraction conditions revealed 4 compounds identified as chlorogenic acid, quercetin, rutin and kaempferol. Conclusion: The optimisation are promising to improve phenolic yield and antioxidant activity in A. heterophyllus leaves. It also proved that A. heterophyllus leaves can be used as an alternative natural antioxidant especially in medicinal applications since all identified compound possess significant biological activities for human health.

Enhancement of Phenolics and Antioxidant Activity via Heat Assisted Extraction From Moringa oleifera Using Response Surface Methodology and Its Potential Bioactive Constituents

@#Introduction: Moringa oleifera Lam. is a miracle tree that has been widely utilised in folklore medicine due to its immense amount of phenolic constituents that could treat various ailments. Different techniques have been implemented to extract the phenolic but the parameters may not be optimised to further enhance the amount of phenolic extracted. Thus, the work aimed to enhance phenolic content and antioxidant activity of M. oleifera through RSM methodology, which is rapid and convenience. Methods: At first, antioxidant activity of different parts of M. oleifera (leaves, stem, pod and seed) were investigated. The plant part with the highest antioxidant activity was selected for the optimisation of extraction condition using RSM. In RSM, temperature (XA), extraction time (XB) and solid-liquid ratio (XC) were employed to study the effects on yield, total phenolics, flavonoids and antioxidant activity. Then, the optimum extraction condition obtained via RSM was utilised in LC-MS and HPLC analysis to determine the potential bioactive constituents. Results: The leaves of M. oleifera displayed the highest antioxidant activity as compared to other plant parts. The optimum extraction condition obtained for the leaves extract was: temperature (XA): 82°C, extraction time (XB): 48 min and solid-liquid ratio (XC): 1:30 g/mL (w/v). Meanwhile, LC-MS revealed the presence of gallic acid, chlorogenic acid, quercetin, kaempferol and 3-O-glucoside kaempferol. HPLC analysis detected six compounds; gallic acid, epicatechin gallate, chlorogenic acid, myricetin, quercetin and kaempferol. Conclusion: The optimisation are promising to improve yield and antioxidant activity in M. oleifera as compared to non-conventional extractions.

Germinated brown rice (GBR) reduces the incidence of aberrant crypt foci with the involvement of beta-catenin and COX-2 in azoxymethane-induced colon cancer in rats.

Chemoprevention has become an important area in cancer research due to the failure of current therapeutic modalities. Epidemiological and preclinical studies have demonstrated that nutrition plays a vital role in the etiology of cancer. This study was conducted to determine the chemopreventive effects of germinated brown rice (GBR) in rats induced with colon cancer. GBR is brown rice that has been claimed to be richer in nutrients compared to the common white rice. The male Sprague Dawley rats (6 weeks of age) were randomly divided into 5 groups: (G1) positive control (with colon cancer, unfed with GBR), (G2) fed with 2.5 g/kg of GBR (GBR (g)/weight of rat (kg)), (G3) fed with 5 g/kg of GBR, (G4) fed with 10 g/kg of GBR and (G5) negative control (without colon cancer, unfed with GBR). GBR was administered orally once daily via gavage after injection of 15 mg/kg of body weight of azoxymethane (AOM) once a week for two weeks, intraperitonially. After 8 weeks of treatment, animals were sacrificed and colons were removed. Colonic aberrant crypt foci (ACF) were evaluated histopathologically. Total number of ACF and AC, and multicrypt of ACF, and the expression of beta-catenin and COX-2 reduced significantly (p < 0.05) in all the groups treated with GBR (G2, G3 and G4) compared to the control group (G1). Spearman rank correlation test showed significant positive linear relationship between total beta-catenin and COX-2 score (Spearman's rho = 0.616, p = 0.0001). It is demonstrated that GBR inhibits the development of total number of ACF and AC, and multicrypt of ACF, reduces the expression of beta-catenin and COX-2, and thus can be a promising dietary supplement in prevention of colon cancer.