26 research outputs found
Anti-Allergic Cromones Inhibit Histamine and Eicosanoid Release from Activated Human and Murine Mast Cells by Releasing Annexin A1
No full textPMCID: PMC3601088This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Studies of the Anti-Cancer Effects of Flavokawin B Onhuman Breast Cancer Cell Lines, Mcf7 And Mda-Mb-231
No full textA natural compound, Flavokawin B, isolated and purified from extract of
Alpinia zerumbet was investigated for its anti-cancer properties on breast
cancer cell lines, estrogen dependant MCF-7 and estrogen non-dependant
MDA-MB-23. Tamoxifen, a non-steroidal anti-estrogen, primarily exploited
as a drug against hormone-dependent breast cancer, acts as the positive
control for this study. MCF-IOA, mammary epithelial cells serve as the
negative control. The cytotoxicities of Flavokawin B and Tamoxifen on
human breast cells were investigated using the MTT assay. The results
showed that the ICSo @ S.E.M) value of Flavokawin B on MCF-7 cell line
was determined to be 1 1.5 Ifi 0.01 5 pM/ml whilst the ICSo with Tarnoxifen
was at 10.2 + 0.012 W m l . The ICS0 value of Flavokawin B on MDA-MB-
23 1 cell line was determined to be 17.5 + 0.0 19 pM/ml whilst the ICS0 value
of Tamoxifen was at 32.5 2 4.2 pM/ml. The MTT assay results on normal
epithelial cell line, MCF-1 OA treated with Flavokawin B demonstrated that
the ICS0 value was 38.0 0.032 pM/ml whereas MCF-1OA treated with
Tamoxifen had an ICSo value of 28 + 0.021 pM/ml. All values were
statistically significant (p<0.05), as analysed using one sample T-test. The
breast cancer cell lines treated at ICS0 concentration of both compounds
before proceeding using confocal microscopy. There were no significant
changes observed in the untreated cells. However, apoptotic features were
that include membrane blebbing and nucleus condensation were evident at
24 hours. At 48 and 72 hours post treatment, convolution of nuclear
membrane, destruction of nuclear membrane and fragmentation of the
nucleus were observed. The TUNEL assay is designed to specifically detect
and quantify apoptotic cells within a cell population, which primarily
consists of both apoptotic and non-apoptotic cells. The TUNEL assay
conducted showed that Flavokawin B induces more apoptosis on MCF-7
and MDA-MB-23 1 compared to Tamoxifen. In contrast, Flavokawin B has
lesser lethal effects on MCF-1OA as compared to Tamoxifen. The levels of
IL-6 secretion in MDA-MB-231 cell line decreased significantly after
treatment with Flavokawin B. Immunofluorescence studies demonstrated
that the levels of IL-6 secretion commensurate with the presence of
membrane bound IL-6r when proliferation of the breast cells was inhibited
during treatment with both the compounds. The MCF-7 and MDA-MB-23 1
cell lines were arrested at G1 phase when treated with both Flavokawin B
and Tamoxifen. This shows that both the treatment follows similar
mechanism to induce cell phase arrest. In conclusion, it could be confirmed
that the pure compound Flavokawin B induces apoptosis in MCF-7 and
MDA-MB-23 1 breast cancer cell lines contributing to the discovery of new
alternative treatment strategy for breast cancer
Annexin A1 as an endogenous regulator of mast cell degranulation.
No full textPhDAnnexin A1 (Anx-A1) is a 37kDa protein that is secreted by some cells
in response to glucocorticoids (GCs) and which mediates several of their
acute anti-inflammatory effects. In addition to GCs, ‘mast cell stabilising’
cromones such as nedocromil also mobilise Anx-A1 by promoting its
phosphorylation by protein kinase C (PKC) and hence its secretion, which
explains their acute efficacy as anti-allergic agents. This thesis addresses a
fundamental aspect of Anx-A1 in the actions of anti-allergic drugs. In this
study, anti-allergic drugs such as H1 antagonists, mast cell stabilisers and
‘dual action’ drugs were first tested for their ability to enhance Anx-A1
phosphorylation in a model system using U937 cells.
Biochemical and immuno-fluorescent techniques were used to study
the mechanisms by which these drugs suppress mediator release from cord
blood derived mast cells (CDMCs) and murine bone-marrow derived mast
cells (BMDMCs) from wild type and Anx-A1 null-mice. This thesis suggest
that PKC activation is crucial for Anx-A1 export in mast cells and nedocromil
in the presence of dexamethasone, prolongs the duration of PKC activation
and subsequently phosphorylation, externalisation and release of Anx-A1
from CDMCs. The ability of nedocromil to inhibit β-hexosaminidase, tryptase,
histamine and PGD2 release are dependent on Anx-A1 in CDMCs.
Interestingly, ketotifen, a ‘dual action’ drug possesses a similar
pharmacological profile to nedocromil, but not promethazine, which does not
act through the Anx-A1 release. Strong evidence supports the notion that the
mechanisms of action of nedocromil are modulated by Anx-A1, thus the
possibility that FPR2 might be involved in the acute actions of nedocromil was
tested. Nedocromil inhibits the release of PGD2 through the activation of
FPR2 but not the inhibition of histamine release. A possible explanation for
this finding could be that Anx-A1 might be interacting with other FPR family
members to exert the histamine inhibitory effects. Although only a small
subset of the downstream intracellular signaling pathway of MAPK was
tested, the results indicate that Anx-A1 differentially regulates the activation of
p38 and JNK in CDMCs treated with nedocromil.
These findings indicate a novel model system in which Anx-A1
mediates the pharmacological actions of anti-allergic drugs and thus has an
important role in preventing the mast cell degranulation.Ministry of Science, Technology and Innovation (MOSTI) Malaysi
Endogenous Annexin-A1 Negatively Regulates Mast Cell-Mediated Allergic Reactions
No full textMast cell stabilizers like cromoglycate and nedocromil are mainstream treatments for ocular allergy. Biochemical studies in vitro suggest that these drugs prevent mast cell degranulation through the release of Annexin-A1 (Anx-A1) protein. However, the direct effect of Anx-A1 gene deletion on mast cell function in vitro and in vivo is yet to be fully investigated. Hence, we aim to elucidate the role of Anx-A1 in mast cell function, both in vivo and in vitro, using a transgenic mouse model where the Anx-A1 gene has been deleted. Bone marrow-derived mast cells (BMDMCs) were cultured from wild-type animals and compared throughout their development to BMDMCs obtained from mice lacking the Anx-A1 gene. The mast cell differentiation, maturity, mediator, and cytokine release were explored using multiple biochemical techniques, such as Western blots, ELISA, and flow cytometry analysis. Electron microscopy was used to identify metachromatic granules content of cells. For in vivo studies, Balb/C wild-type and Anx-A1-deficient mice were divided into the following groups: group 1, a control receiving only saline, and group 2, which had been sensitized by prior exposure to short ragweed (SRW) pollen by topical contact with the conjunctival mucosae. Allergic conjunctivitis was evaluated blind after 24 h by trained observers scoring clinical signs. Electron micrographs of BMDMCs from Anx-A1-null mice revealed more vacuoles overall and more fused vacuoles than wild-type cells, suggesting enhanced secretory activity. Congruent with these observations, BMDMCs lacking the Anx-A1 gene released significantly increased amounts of histamine both spontaneously as well as in response to Ig-E-FcεRI cross-linking compared to those from wild-type mice. Interestingly, the spontaneous release of IL-5, IL-6, IL-9, and monocyte chemoattractant protein-1 (MCP-1) were also markedly increased with a greater production observed upon IgE cross-linking. This latter finding is congruent with augmented calcium mobilization in BMDMCs lacking the Anx-A1 gene. In vivo, when compared to wild-type animals, Anx-A1-deficient mice exposed to SRW pollen displayed exacerbated signs and symptoms of allergic conjunctivitis. Taken together, these results suggest Anx-A1 is an important non-redundant regulator of mast cell reactivity and particularly in allergen mediated allergic reactions. © 2019 Sinniah, Yazid, Bena, Oliani, Perretti and Flower. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms
Synergistic Effects of 5-Fluorouracil in Combination with Diosmetin in Colorectal Cancer Cells
No full textColorectal cancer (CRC) is among the most commonly occurring cancers. The management of CRC includes laparoscopic surgery, radiotherapy, chemotherapies and neoadjuvant treatment. However, conventional chemotherapies have poor impact on combating CRC and are associated with severe toxic effects and high rates of relapse. Therefore, searching for a new combination regimen is a favorable consideration. The aim of this study was to elucidate the synergistic effect of 5-fluorouracil (5-FU) and diosmetin in an in vitro model on colorectal cancer cells. An MTT assay was conducted on HCT-116 cancer cells and they were treated with a concentration gradient of 5-FU and diosmetin individually and in combination. The combination index (CI) and dose reduction index (DRI) were calculated using CompuSyn software. Isobologram analysis and synergism determination were performed using the Combenefit software tool and the synergy score was calculated using the SynergyFinder 2.0 software tool. The apoptotic features of the cells were determined via an AO/PI double staining assay and an annexin V assay using a fluorescent microscope and the flow cytometry technique, respectively. The findings showed that the DRI of 5-FU was three-fold lower in the combination with a CI value of less than one, which indicates that there was a synergistic effect. The AO/PI microscopic results revealed signs of apoptosis and dead cells after 72 h of treatment. Flow cytometry analysis confirmed that the apoptotic effect of the combination was more prominent compared to 5-FU alone. The findings of this study offer a potential strategy for reducing the cytotoxicity and enhancing the efficacy of 5-FU on colorectal cancer cells through a synergistic study model
From NSAIDs to Glucocorticoids and Beyond
No full textOur interest in inflammation and its treatment stems from ancient times. Hippocrates used willow bark to treat inflammation, and many centuries later, salicylic acid and its derivative aspirin’s ability to inhibit cyclooxygenase enzymes was discovered. Glucocorticoids (GC) ushered in a new era of treatment for both chronic and acute inflammatory disease, but their potentially dangerous side effects led the pharmaceutical industry to seek other, safer, synthetic GC drugs. The discovery of the GC-inducible endogenous anti-inflammatory protein annexin A1 (AnxA1) and other endogenous proresolving mediators has opened a new era of anti-inflammatory therapy. This review aims to recapitulate the last four decades of research on NSAIDs, GCs, and AnxA1 and their anti-inflammatory effects
The Anti-allergic Cromones: Past, Present, and Future
No full textThe anti-allergic cromones were originally synthesized in the 1960s by Fisons Plc, and the first drug to emerge from this program, disodium cromoglycate was subsequently marketed for the treatment of asthma and other allergic conditions. Whilst early studies demonstrated that the ability of the cromones to prevent allergic reactions was due to their ‘mast cell stabilizing’ properties, the exact pharmacological mechanism by which this occurred, remained a mystery. Here, we briefly review the history of these drugs, recount some aspects of their pharmacology, and discuss two new explanations for their unique actions. We further suggest how these findings could be used to predict further uses for the cromones
Diosmetin Exerts Synergistic Effects in Combination with 5-Fluorouracil in Colorectal Cancer Cells
No full text5-Fluorouracil (5-FU) is a chemotherapeutic medication commonly used to treat colorectal cancer (CRC); however, the drug-associated adverse effects and toxicity have greatly affected its clinical use. Exploring another therapeutic strategy that lowers the toxicity of 5-FU while having a synergistic effect against CRC is thus a viable option. Diosmetin, a natural flavonoid, has been shown to inhibit the proliferation of many cancer cells, including CRC cells. This study aims to investigate the synergistic effect of diosmetin and 5-FU on HCT116 and HT29 colorectal cancer cells and to explore the apoptotic activity of this combination. The MTT assay was used to assess the viability of cells treated with monotherapy and combination therapy. The combination index (CI) and dose reduction index (DRI) were calculated using the CompuSyn software (version 1.0). The SynergyFinder 2.0 software was used to calculate the synergy score, while the Combenefit software was employed to perform isobologram analysis and synergism determination. The AO/PI double staining technique was used to detect the apoptotic characteristics of cells, whereas the flow cytometry technique was used to investigate the apoptosis induction and cell cycle arrest in cells. The combination of 5-FU and diosmetin showed a synergistic effect in HCT116 cells with a mean CI value of 0.66 ± 0.4, and an additive effect in HT29 cells with a CI value of 1.0 ± 0.2. The DRI of 5-FU in HCT116 cells was three times lower in the combination therapy compared to monotherapy of 5-FU. AO/PI microscopic examination and Annexin V analysis revealed that the combination-treated cells had more apoptotic cells than the monotherapy-treated cells, which was activated mainly through intrinsic apoptosis pathway. HCT116 cell death was confirmed by mitotic arrest in the G2/M phase. Our findings suggest that 5-FU/diosmetin combination exhibits synergistic effect against HCT116 cancer cells, and potentially reduces the unfavorable adverse effect of 5-FU while enhancing the anticancer efficacy by inducing apoptosis and interrupting mitosis. Further research studies are needed to validate the combination’s anti-tumorigenic activities in a xenograft animal model
Punicalagin Regulates Apoptosis-Autophagy Switch via Modulation of Annexin A1 in Colorectal Cancer
No full textPunicalagin (PU), a polyphenol extracted from pomegranate (Punica granatum) husk is proven to have anti-cancer effects on different types of cancer including colorectal cancer (CRC). Its role in modulating endogenous protein as a means of eliciting its anti-cancer effects, however, has not been explored to date. Hence, this study aimed to investigate the role of PU in modulating the interplay between apoptosis and autophagy by regulating Annexin A1 (Anx-A1) expression in HCT 116 colorectal adenocarcinoma cells. In the study, selective cytotoxicity, pro-apoptotic, autophagic and Anx-A1 downregulating properties of PU were shown which indicate therapeutic potential that this polyphenol has against CRC. Autophagy flux analysis via flow cytometry showed significant autophagosomes degradation in treated cells, proving the involvement of autophagy. Proteome profiling of 35 different proteins in the presence and absence of Anx-A1 antagonists in PU-treated cells demonstrated a complex interplay that happens between apoptosis and autophagy that suggests the possible simultaneous induction and inhibition of these two cell death mechanisms by PU. Overall, this study suggests that PU induces autophagy while maintaining basal level of apoptosis as the main mechanisms of cytotoxicity via the modulation of Anx-A1 expression in HCT 116 cells, and thus has a promising translational potential
Evaluation of Anti-Tumorigenic Effects of Diosmetin against Human Colon Cancer Xenografts in Athymic Nude Mice
No full textColon cancer is the third most common type of cancer in the world. Diosmetin (Dis), a natural O-methylated flavone, has been reported to have anti-cancer effects against different types of cancer. Although the mechanisms of action of Dis against several cancer cell lines are well reported, in vivo anti-tumorigenesis properties of this compound are still obscure. Therefore, this study aimed to investigate the anti-tumorigenesis properties of Dis against HCT-116 colon cancer xenografts in nude mice. HCT-116 colon cancer cells were injected in NCr nu/nu nude mice and treatment with Dis was initiated after the tumor volumes reached 100 mm3 and continued for four weeks. On the sacrificing date nude mice treated with 100 mg/kg of Dis showed significant lower tumor volume (264 ± 238.3 mm3) as compared to the untreated group (1428.8 ± 459.6 mm3). Anti-apoptotic Bcl-2 protein was significantly downregulated, while apoptotic protein (Bax) was significantly overexpressed in nude mice treated with 100 mg/kg Dis as compared to untreated mice. In conclusion, our in vivo results indicate that Dis significantly reduces tumor growth rate of HCT-116 colon cancer cells in nude mice at a dose of 100 mg/kg, and has no toxic effects in ICR mice up to 2000 mg/kg
