Natural Gallic Acid and Methyl Gallate Induces Apoptosis in Hela Cells through Regulation of Intrinsic and Extrinsic Protein Expression.

Induction of apoptosis is one of the targeted approaches in cancer therapies. As previously reported, natural products can induce apoptosis in in vitro cancer treatments. However, the underlying mechanisms of cancer cell death are poorly understood. The present study aimed to elucidate cell death mechanisms of gallic acid (GA) and methyl gallate (MG) from Quercus infectoria toward human cervical cancer cell lines (HeLa). The antiproliferative activity of GA and MG was characterised by an inhibitory concentration using 50% cell populations (IC50) by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Cervical cancer cells, HeLa, were treated with GA and MG for 72 h and calculated for IC50 values. The IC50 concentration of both compounds was used to elucidate the apoptotic mechanism using acridine orange/propidium iodide (AO/PI) staining, cell cycle analysis, the Annexin-V FITC dual staining assay, apoptotic proteins expressions (p53, Bax and Bcl-2) and caspase activation analysis. GA and MG inhibited the growth of HeLa cells with an IC50 value of 10.00 ± 0.67 µg/mL and 11.00 ± 0.58 µg/mL, respectively. AO/PI staining revealed incremental apoptotic cells. Cell cycle analysis revealed an accumulation of cells at the sub-G1 phase. The Annexin-V FITC assay showed that cell populations shifted from the viable to apoptotic quadrant. Moreover, p53 and Bax were upregulated, whereas Bcl-2 was markedly downregulated. Activation of caspase 8 and 9 showed an ultimate apoptotic event in HeLa cells treated with GA and MG. In conclusion, GA and MG significantly inhibited HeLa cell growth through apoptosis induction by the activation of the cell death mechanism via extrinsic and extrinsic pathways.

Immunomodulatory Effects of Asiaticoside Against Shigella flexneri-Infected Macrophages.

Macrophages provide the first line of defense against Shigella flexneri infection in the gastrointestinal tract by inducing a variety of inflammatory and antimicrobial responses. Secondary metabolites of plants are used as drugs against infections that are resistant to common antibiotics. In this study, the innate effects of asiaticoside on the proinflammatory activity of mouse macrophages infected with S. flexneri were investigated. The viability of the infected mouse macrophages were examined using viability assay, while the pro-inflammatory cytokines productions were determined using the enzyme-linked immunosorbent assay (ELISA) for determination of IL-1ß, IL-12 p40 and TNF-α levels. The production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) protein were determined using the Griess assay and western blot, respectively. Statistical analyses were performed using the Statistical Package of Social Sciences (SPSS) software, version 20. The data obtained from independent experiments (n = 3) were presented as the mean ± standard error of mean (SEM). The results showed that, asiaticoside stimulated the infected macrophages by stimulating increased production of TNF-α, IL-12 p40 and NO as well as increased expression of iNOS in a dose-dependent manner. In contrast the viability of the cells and the production of IL-1ß and were reduced also in a dose-dependent manner when compared to untreated cells. These results indicate that asiaticoside has immunomodulatory effects on the innate immune function of infected macrophages, showing the potential use of this compound to reduce the clinical symptoms of the infections.

Makrofaj bertindak sebagai sistem pertahanan awal terhadap jangkitan Shigella flexneri di dalam saluran gastrousus (saluran pencernaan) dengan mengaktifkan gerak balas inflamatori dan anti-mikrob. Banyak metabolit sekunder daripada tumbuhan digunakan sebagai agen untuk merawat penyakit yang rintang terhadap antibiotik. Dalam kajian ini, kesan gerak balas semulajadi asiatikosid terhadap aktiviti pro-inflamasi makrofaj mencit yang telah dijangkiti S. flexneri telah dikaji. Kedayahidupan makrofaj mencit dianalisis menggunakan asai kedayahidupan, manakala penghasilan sitokin pro-inflamasi IL-1ß, IL-12 p40 dan TNF-α dianalisis menggunakan esei imunoserapan terangkai enzim (ELISA). Penghasilan nitrik oksida (NO) dan pengekspresian protein sintase niktrik oksida teraruh (iNOS) dianalisis menggunakan asai Griess dan analisis pemblotan Western. Analisis statistik dijalankan menggunakan perisian 'Statistical Package of Social Sciences (SPSS)' versi 20. Data yang diperolehi daripada eksperimen tak bersandar (n = 3) dibentangkan sebagai min ± min ralat piawai (SEM). Hasil kajian menunjukkan, asiatikosid merangsang pengaktifan makrofaj mencit yang telah dijangkiti melalui peningkatan penghasilan TNF-α, IL12 p40 dan NO serta meningkatkan penghasilan iNOS bergantung kepada dos asiatikosid yang diberikan. Namun begitu, kedayahidupan sel dan penghasilan IL-1ß menurun seiring dengan dos yang diberikan dalam sel yang dirawat berbanding dengan sel yang tidak dirawat. Hasil kajian ini menunjukkan bahawa asiatikosid mempunyai kesan imunomodulatori terhadap fungsi imun semulajadi makrofaj yang dijangkiti, yang memberi gambaran potensinya untuk digunakan bagi mengurangkan simptom klinikal jangkitan S. flexneri.

Apoptosis Activity of the Mouse Macrophage Cell Line J774A.1 Infected with a Recombinant BCG consisting the C-Terminus of Merozoite Surface Protein-1 of Plasmodium falciparum.

Macrophage apoptosis exerts an efficient mechanism in controlling intracellular infection during innate immune response against various pathogens including malaria parasites. This study was carried out to determine the apoptosis activity in mouse macrophage cell line J774A.1 infected with a Mycobacterium bovis bacille Calmette-Guerin (BCG) clone and a recombinant BCG clone expressing the C-terminus of merozoite surface protein-1 (BCG-MSP1C) of Plasmodium falciparum for 48 h. In this study, a parent BCG cells was used as a control. The nuclear staining with Hoechst 33342 showed that the BCG-MSP1C cells was capable of increasing the nuclear condensation and morphological stages of apoptosis in the infected cells compared to the BCG-infected cells and the lipopolysaccharide (LPS)-stimulated cells. The flow cytometric analysis using Annexin-V and Propidium iodide (PI) staining confirmed that the BCG-MSP1C cells significantly increased the percentage of early apoptotic activity in the infected macrophage higher than the one stimulated by the parent BCG cells and LPS. This apoptotic response corresponded with the reduction of the anti-apoptotic Bcl-2 protein expression and higher p53 expression. The colorimetric assay demonstrated that the BCG cells capable of stimulating higher production of caspase-1, -3, -8 and -9 while the BCG-MSP1C cells stimulated the expression of caspase-1 and -9 in the infected macrophages, suggesting the involvement of mitochondrial-mediated (intrinsic) pathway of apoptosis. In conclusion, both the BCG and BCG-MSP1C cells are capable of inducing macrophage apoptosis activity in the mouse macrophage cell line J774A.1. This mechanism is important for the elimination of pathogens such as malaria parasite during the phagocytosis activity of macrophage. However, the BCG-MSP1C cells showed higher apoptosis activity than those produced by the parent BCG cells.