Chithan Kandaswami

Effects of flavonoids on immune and inflammatory cell functions

No doubt can remain that the flavonoids have profound effects on the function of immune and inflammatory cells as determined by a large number and variety of in vitro and some in vivo observations. That these ubiquitous dietary chemicals may have significant in vivo effects on homeostasis within the immune system and on the behavior of secondary cell systems comprising the inflammatory response seems highly likely but more work is required to strengthen this hypothesis. Ample evidence indicates that selected flavonoids, depending on structure, can affect (usually inhibit) secretory processes, mitogenesis, and cell-cell interactions including possible effects on adhesion molecule expression and function. The possible action of flavonoids on the function of cytoskeletal elements is suggested by their effects on secretory processes. Moreover, evidence indicates that certain flavonoids may affect gene expression and the elaboration and effects of cytokines and cytokine receptors. How all of these effects are mediated is not yet clear but one important mechanism may be the capacity of flavonoids to stimulate or inhibit protein phosphorylation and thereby regulate cell function. Perhaps the counterbalancing effect of cellular protein tyrosine phosphatases will also be found to be affected by flavonoids. Some flavonoid effects can certainly be attributed to their recognized antioxidant and radical scavenging properties. A potential mechanism of action that requires scrutiny, particularly in relation to enzyme inhibition, is the redox activity of appropriately configured flavonoids. Finally, in a number of cell systems it seems that resting cells are not affected significantly by flavonoids but once a cell becomes activated by a physiological stimulus a flavonoid-sensitive substance is generated and interaction of flavonoids with that substance dramatically alters the outcome of the activation process.

Free radical scavenging and antioxidant activity of plant flavonoids.

The occurrence of reactive oxygen species (ROS), termed as prooxidants, is a characteristic of normal aerobic organisms. The term “reactive oxygen species” collectively denotes oxygen-centered radicals such as superoxide (O2·-)and hydroxyl (·OH), as well as nonradical species derived from oxygen, such as hydrogen peroxide (H2O2), singlet oxygen (1ΔgO2) and hypochlorous acid (HOC1). Radical reactions are central to the maintenance of homeostasis in biological systems. Radical species perform a cardinal role in many physiological processes such as cytochrome P450-mediated oxidative transformation reactions, a plethora of enzymic oxidation reactions, oxidative phosphorylation, regulation of the tone of smooth muscle, and killing of microorganisms.1–3 Excessive generation of free radicals can have deleterious biological consequences.4–6 Organisms are equipped with an armamentarium of defense systems, termed antioxidants in order to safeguard them against the onslaught of ROS.1–3,7 When the generation of prooxidants overwhelms the capacity of antioxidant defense systems oxidative stress ensues. This can cause tissue damage leading to pathophysiological events. ROS play a pivotal role in the action of numerous foreign compounds (xenobiotics). Their increased production seems to accompany most forms of tissue injury.4,5 Whether sustained and increased production of ROS is a primary event in human disease progression or a secondary consequence of tissue injury has been discussed.5,6 Whatever may be the case, the formation of free radicals has been implicated in a multitude of disease states ranging from inflammatory/immune injury to myocardial infarction and cancer.

The Flavonoid Quercetin Inhibits Proinflammatory Cytokine (Tumor Necrosis Factor Alpha) Gene Expression in Normal Peripheral Blood Mononuclear Cells via Modulation of the NF-κβ System

ABSTRACT The flavonoids comprise a large class of low-molecular-weight plant metabolites ubiquitously distributed in food plants. These dietary antioxidants exert significant antitumor, antiallergic, and anti-inflammatory effects. The molecular mechanisms of their biological effects remain to be clearly understood. We investigated the anti-inflammatory potentials of a safe, common dietary flavonoid component, quercetin, for its ability to modulate the production and gene expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) by human peripheral blood mononuclear cells (PBMC). Our results showed that quercetin significantly inhibited TNF-α production and gene expression in a dose-dependent manner. Our results provide direct evidence of the anti-inflammatory effects of quercetin by PBMC, which are mediated by the inhibition of the proinflammatory cytokine TNF-α via modulation of NF-κβ1 and Iκβ.

Effects of luteolin and quercetin, inhibitors of tyrosine kinase, on cell growth and metastasis‐associated properties in A431 cells overexpressing epidermal growth factor receptor

Flavonoids display a wide range of pharmacological properties including anti‐inflammatory. Anti‐mutagenic, anti‐carcinogenic and anti‐cancer effects. Here, we evaluated the effects of eight flavonoids on the tumour cell proliferation, cellular protein phosphorylation, and matrix metalloproteinase (MMPs) secretion. Of the flavonoids examined, luteolin (Lu) and quercetin (Qu) were the two most potent agents, and significantly inhibited A431 cell proliferation with IC50 values of 19 and 21 μM, respectively. The epidermal growth factor (EGF) (10 nM) promoted growth of A431 cells (+25±4.6%) and mediated epidermal growth factor receptor (EGFR) tyrosine kinase activity and autophosphorylation of EGFR were inhibited by Lu and Qu. At concentration of 20 μM, both Lu and Qu markedly decreased the levels of phosphorylation of A431 cellular proteins, including EGFR. A431 cells treated with Lu or Qu exhibited protuberant cytoplasmic blebs and progressive shrinkage morphology. Lu and Qu also time‐dependently induced the appearance of a ladder pattern of DNA fragmentation, and this effect was abolished by EGF treatment. The addition of EGF only marginally diminished the inhibitory effect of luteolin and quercetin on the growth rate of A431 cells, treatment of cellular proteins with EGF and luteolin or quercetin greatly reduced protein phosphorylation, indicating Lu and Qu may act effectively to inhibit a wide range of protein kinases, including EGFR tyrosine kinase. EGF increased the levels of matrix metalloproteinase‐2 (MMP‐2) and matrix metalloproteinase‐9 (MMP‐9), while Lu and Qu appeared to suppress the secretion of these two MMPs in A431 cells. Examination of the relationship between the chemical structure and inhibitory effects of eight flavonoids reveal that the double bond between C2 and C3 in ring C and the OH groups on C3′ and C4′ in ring B are critical for the biological activities. This study demonstrates that the inhibitory effects of Lu and Qu, and the stimulatory effects of EGF, on tumour cell proliferation, cellular protein phosphorylation, and MMP secretion may be mediated at least partly through EGFR. This study supports the idea that Lu and Qu may have potential as anti‐cancer and anti‐metastasis agents.

Antitproliferative effects of citrus flavonoids on a human squamous cell carcinoma in vitro

We examined the effects of four plant flavonoids (quercetin, taxifolin, nobiletin and tangeretin) on the in vitro growth of a human squamous cell carcinoma cell line (HTB43). Cell cultures were treated with each flavonoid (2-8 micrograms/ml) for 3-7 days. Cell viability, as determined by counting cells, correlated well with that obtained from a colorimetric assay for cellular growth utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The polymethoxylated flavonoids, nobiletin and tangeretin, markedly inhibited cell growth at all concentrations tested on days 5 and 7. On day 3, the inhibition observed was 70-72% at 8 micrograms/ml, while on day 5, it ranged from 61-88% at 2-4 micrograms/ml. Quercetin and taxifolin exhibited no significant inhibition at any of the concentrations tested. This difference in activity may be due to the relatively greater membrane uptake of the polymethoxylated flavonoids since methoxylation of the phenolic groups decreases hydrophilicity of the flavonoid.

Matrix metalloproteinase-9 cooperates with transcription factor Snail to induce epithelial-mesenchymal transition.

One of the most fundamental biological processes in tumor metastasis is the process of epithelial–mesenchymal transition (EMT). During EMT, zinc‐finger‐family of transcription factors such as Snail, Slug and Twist, and matrix metalloproteinases (MMPs) are upregulated, and this correlates with increased tumor cell invasion and motility. We previously obtained a highly invasive A431‐III tumor subline, which is a rich source of MMP‐9 and observed a plausible link between MMP levels and the promotion of EMT. To gain further understanding of EMT, we investigated the contribution of distinct MMPs to the induction of EMT. Exposing A431, cervical carcinoma parental cells, to MMP‐9 stimulated a phenotypic alteration and cells became spindle‐like as shown for A431‐III cells. In the present communication, we document changes in gene expression profiles of A431‐P and A431‐III cells, including those of genes involved in cell adhesion, cytoskeleton reorganization, polarity, migration and transcription. Treatment of both A431‐P and A431‐III cells with GM6001, a broad spectrum MMP inhibitor, resulted in the diminution of vimentin and fibronectin, indicating a role for MMP‐9 in the induction of EMT. Abrogation of MMP‐9‐mediated cell–cell contact in both A431‐P and A431‐III cells using MMP‐9 siRNA resulted in decreased cell invasion, motility and altered cytoskeleton arrangement together with a reduction in Snail expression. Knockdown of Snail resulted in similar changes along with diminished MMP‐9 expression. These data suggest a higher capacity of MMP‐9 than that of Snail in eliciting the development of EMT in A431 cells. Based on these findings, we speculate that the overexpression of MMP‐9 in A431‐III cells might directly induce (or stimulate) EMT and that the transcriptional factor, Snail, could cooperatively engage in this phenomenon. (Cancer Sci 2011; 102: 815–827)

The flavonoid, quercetin, differentially regulates Th-1 (IFNγ) and Th-2 (IL4) cytokine gene expression by normal peripheral blood mononuclear cells

Flavonoids are plant metabolites that are dietary antioxidants and exert significant anti-tumor, anti-allergic, anti-inflammatory and anti-viral effects. It is generally accepted that Th-1 derived cytokines such as IL-2, IFNgamma and IL-12 promote cellular immunity while Th-2 derived cytokines such as IL-4, IL-5, IL-6 exert negative immunoregulatory effects on cellular immunity while upregulating humoral immunity. The molecular mechanisms underlying the biological activities of flavonoids have not been elucidated. We hypothesize that the flavonoid, quercetin, exert significant anti-viral and anti-tumor effects possibly by modulating the production of Th-1 and Th-2 derived cytokines. Peripheral blood mononuclear cells (PBMC, 1 x 10(6) cells/ml) from normal subjects were cultured with different concentrations of quercetin (0.5-50 microM) for 24-72 h and supernates were quantitated for IFN-gamma and IL-4 by ELISA and antiviral activity of IFNgamma by bioassay. FACS analysis was done to determine the number of IFN-gamma and IL-4 positive cells and RT-PCR was done to quantitate gene expression. Quercetin significantly induces the gene expression as well as the production of Th-1 derived IFNgamma and the downregulates Th-2 derived IL-4 by normal PBMC. Further, quercetin treatment increased the phenotypic expression of IFNgamma cells and decreased IL-4 positive cells by FACS analysis, which corroborate with protein secretion and gene expression studies. These results suggest that the beneficial immuno-stimulatory effects of quercetin may be mediated through the induction of Th-1 derived cytokine, IFNgamma, and inhibition of Th-2 derived cytokine, IL-4.

The effects of the bioflavonoid quercetin on squamous cell carcinoma of head and neck origin

Quercetin exhibits antitumor activity. We investigated the effect of quercetin on the in vitro and in vivo growth of two squamous cell carcinoma cell lines and a normal human lung fibroblast-like cell line. The in vivo effect was evaluated using implantable cell growth chambers implanted subcutaneously in immunocompetent rats. Quercetin was injected intraperitoneally, and multiple dosages were tested. Cells were counted on days 1, 3, 5, and 7, and growth curves were constructed. Quercetin caused inhibition of growth in both squamous cell carcinoma lines. Effect on the fibroblast-like human lung cells was noted only at the maximum concentration. Significant growth inhibition of squamous cell carcinoma was observed in implantable cell growth chambers retrieved 3 days after quercetin treatment. Quercetin appears to possess a cytotoxic effect on squamous cell carcinoma of head and neck origin both in vivo and in vitro. The inhibitory effect on malignant cells appears to be selective and dose-dependent.

Differential inhibition of proliferation of human squamous cell carcinoma, gliosarcoma and embryonic fibroblast-like lung cells in culture by plant flavonoids

We investigated the antiproliferative effect of two polyhydroxylated (quercetin and taxifolin) and two polymethoxylated (nobiletin and tangeretin) flavonoids against three cell lines in tissue culture. Tangeretin and nobiletin markedly inhibited the proliferation of a squamous cell carcinoma (HTB 43) and a gliosarcoma (9L) cell line at 2–8 μg/ml concentrations. Quercetin displayed no effect on 9L cell growth at these concentrations, while at 8 μg/ml it inhibited HTB 43 cell growth. Taxifolin slightly inhibited HTB 43 cell growth at 8 fig/ml, while moderately inhibiting HTB 43 cell growth at 2–8 μg/ml. The proliferation of a human lung fibroblast-like cell line (CCL 135) was relatively insensitive to low concentrations of the above flavonoids.

Ascorbic acid-enhanced antiproliferative effect of flavonoids on squamous cell carcinoma in vitro.

We examined the effects of flavone and two polyhydroxylated plant flavonoids (quercetin and fisetin), either singly or in combination with ascorbic acid, on the growth of a human squamous cell carcinoma cell line (HTB 43) in vitro. Fisetin and quercetin significantly impaired cell growth in the presence of ascorbic acid. Exposure of cells to ascorbic acid (2 µg/ml) and 2 µg/ml of either fisetin or quercetin resulted in 61 and 45% inhibition of cell growth, respectively, in 72 h, while treatment with ascorbic acid alone had no effect on cellular proliferation. Flavone and ascorbic acid, either as single agents or in combination, exhibited no significant inhibition at any of the concentrations tested. The enhancement of the antiproliferative effect of the above flavonoids by ascorbic acid may be due to its ability to protect these compounds against oxidative degradation.