Chrysin-Induced Regression of Angiogenesis via an Induction of DNA Damage Response and Oxidative Stress in In Vitro and In Vivo Models of Melanoma.

Despite the progress made in treatments, melanoma is one of the cancers for which its incidence and mortality have increased during recent decades. In the research of new therapeutic strategies, natural polyphenols such as chrysin could be good candidates owing to their capacities to modulate the different fundamental aspects of tumorigenesis and resistance mechanisms, such as oxidative stress and neoangiogenesis. In the present study, we sought to determine whether chrysin could exert antitumoral effects via the modulation of angiogenesis by acting on oxidative stress and associated DNA damage. For the first time, we show a link between chrysin-induced antiproliferative effects, the activation of the DNA damage pathway, and its ability to limit angiogenesis. More specifically, herein, we show that chrysin induces single- and double-stranded DNA breaks via the activation of the DNA damage response pathway: ATM (ataxia-telangiectasia-mutated)/Chk2 (checkpoint kinase 2) and ATR (ataxia telangiectasia and Rad3-related)/Chk1 (checkpoint kinase 1) pathways. Strong activation of this DNA damage response was found to be partly involved in the ability of chrysin to limit angiogenesis and may partly involve a direct interaction between the polyphenol and DNA G-quadruplex structures responsible for the replication fork collapse. Moreover, these events were associated with a marked reduction in melanoma cells' capacity to secrete proangiogenic factor VEGF-A. The disruption of these key protein actors in tumor growth by chrysin was also confirmed in a syngeneic model of B16 melanoma. This last point is of importance to further consider the use of chrysin as a new therapeutic strategy in melanoma treatment.

Protective Effect of Chrysin, a Dietary Flavone against Genotoxic and Oxidative Damage Induced by Mitomycin C in Balb/C Mice.

Anticancer drugs, such as Mitomycin C (MMC), can interact with biological molecules and cause genetic damage in normal cells. In this respect, we investigated the potential of chrysin, a flavone known as a potent scavenger of free radicals generated by anticancer agents, to protect mice against MMC-induced genotoxicity. The amount of DNA damage in the liver, kidney and bone marrow cells, in Balb/C mice treated with MMC (6 mg/kg, i.p) and the frequency of chromosomal aberrations indicated the genotoxic effect of MMC. Besides, a significant increase in the activities of antioxidant enzymes (SOD, CAT, GPx, GST) and lipid peroxidation is revealed. On the other hand, we noticed a regression of the genotoxic effect when studying the same parameters in Balb/C mice treated with chrysin (40 mg/kg b. wt., i.p) 24 h prior to MMC (6 mg/kg, i.p) injection. This study concluded that the protective effect of chrysin against genotoxicity of MMC results partly from its antioxidant effect.

Heat treatment improves the immunomodulatory and cellular antioxidant behavior of a natural flavanone: Eriodictyol.

Plants and natural molecules are generally consumed not in raw state but after different processing conditions (heating, mechanical agitation or cooking). The understanding of the chemistry and biological outcome of thermal treatment is still scarce. In the current study, Eriodictyol, a natural flavanone, has undergone heat treatment, generating hence three different products ((3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid, (3-(3,4-dihydroxyphenyl) propanal) and an unidentified component). The consequences of aforementioned treatment on the immunomodulatory behavior of resulted molecules were evaluated. The amount of nitric oxide production and the lysosomal enzyme activity were determined in vitro on mouse peritoneal macrophages. The kinetic of cellular antioxidant activity in splenocytes and macrophages was measured. The present investigation demonstrates that heat-processed eriodictyol significantly enhanced the proliferation of lymphocytes B and T compared to native eriodictyol. Indeed, this compound showed an important improvement on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities. In addition, the production of nitric oxide (NO) and suppression of phagocytic activity of activated macrophages have been increasingly important after thermal processing. Furthermore, it was also revealed that heat-treated Erio in comparison with the native (non heat-treated) molecule has a highest cellular anti-oxidant activity in splenocytes and macrophages cells. These findings highlight the importance of heat-process as feasible and effective strategy to improve the immunomodulatory and the antioxidant efficiency of an known flavanone Eriodictyol.

Antitumoral Potency by Immunomodulation of Chloroform Extract from Leaves of Nitraria retusa, Tunisian Medicinal Plant, via its Major Compounds ß-sitosterol and Palmitic Acid in BALB/c Mice Bearing Induced Tumor.

This study evaluated the antitumoral effect of Chloroform extract from Nitraria retusa leaves, via its major compounds ß-sitosterols and palmitic acid. BALB/c mice were subcutaneously inoculated with B16-F10 cells, then treated intra-peritoneally after 7 days with the chloroform extract for 21 days. They were then euthanized, and the tumors were weighed. Lung parenchyma was analyzed. Lymphocyte and macrophages proliferation, cytotoxic T lymphocyte (CTL) activities were evaluated using the MTT assay. Macrophage phagocytosis was evaluated by measuring the lysosomal activity and nitric oxide production. Antioxidant activity was studied by cellular antioxidant activity on macrophage and splenocytes and by lipid peroxidation inhibitory activity in liver cells, kidney, and serum. ß-sitosterols and palmitic acid, major compounds of chloroform extract, impeded remarkably the expansion of the transplantable tumor, protected the lung parenchyma, and increased splenocytes proliferation and both CTL activities in tumor-bearing mice. ß-sitosterols and palmitic acid were also seen to have enhanced lysosomal activity of host macrophages and antioxidant cellular activity. Also, they showed an inhibitory effect of lipid peroxidation. Our results suggest that antitumoral effect of ß-sitosterols and palmitic acid from chloroform extract is related with its immunomodulatory activity, and opens the way for a nutrition application and coprocessing phytotherapy against cancer.

Chrysin, a natural and biologically active flavonoid suppresses tumor growth of mouse B16F10 melanoma cells: In vitro and In vivo study.

Chrysin (5,7-dihydroxyflavone) is a natural and biologically active compound which has many biological activities as an anticancer agent. The current report is aimed at finding out whether the antitumor potential of chrysin, evidenced in vitro and in vivo, is linked or not to its effect on immunological mechanisms of melanoma-bearing mice. Chrysin-treated B16F10 cells were analyzed for their metabolic rate and apoptotic potentials. In vivo, BALB/c mice received a subcutaneous injection of B16F10 melanoma cells prior to antitumor treatments with chrysin (50 mg/kg b.w) for 14 days and 21 days. The results showed that chrysin inhibited cancer cell growth at a dose-dependent manner by inducing apoptosis and cell cycle arrest at G2/M phase. Moreover, chrysin suppressed melanoma tumor growth at an average of 60% (after 14 days of treatment) and 71% (after 21 days of treatment) compared to the tumor-bearing group. Furthermore, chrysin treatment increased the cytotoxic activity of NK, CTL and macrophages. The findings showed that chrysin antitumor action on the murine melanoma model was very promising, suggesting that chrysin could be a potentially good candidate for future use in alternative anti-melanoma treatments.

Immunomodulatory potential of hesperetin and chrysin through the cellular and humoral response.

Flavonoids are polyphenols frequently consumed in the diet they have been suggested to exert a number of beneficial actions on human health, including anti-inflammatory activity. This study investigated the immunomodulatory effects of two flavonoids, Chrysin and Hesperetin. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. We report for the first time that both tested flavonoids enhance lymphocyte proliferation at 3.12µM. Chrysin significantly inhibited lipopolysaccharide (LPS) and lectin stimulated splenocyte proliferation. Whereas, hesperetin enhanced LPS and lectin stimulated splenocyte proliferation. In addition, both flavonoids significantly enhance NK cell and CTL activities. Furthermore, our study demonstrated that depending on the concentrations, flavonoid molecules affect macrophage functions by modulating their lysosomal activity and nitric oxide (NO) release, suggesting a potential anti-inflammatory effect. We conclude that flavonoids such as chrysin and hesperetin may be potentially useful for modulating immune cell functions in physiological and pathological conditions and thus a good candidate as food addition component.

Investigation of immunomodulatory and anti-inflammatory effects of eriodictyol through its cellular anti-oxidant activity.

Many studies have been performed to assess the potential utility of natural products as immunomodulatory agents to enhance host responses against infection or to ameliorate immune-based pathologies. To determine whether eriodictyol has immunomodulatory effects and clarify which types of immune effector cells are stimulated in vitro, we investigated the stimulatory effect of eriodictyol on spleen cells isolated from BALB/c mice. Eriodictyol significantly stimulated splenocyte proliferation. However, only B lymphocytes (not T lymphocytes) could be stimulated by eriodictyol in a dose-related manner. Studies assessing potential effect of eriodictyol on innate immunity reported that eriodictyol enhanced significantly the killing activity of natural killer (NK) cells, T lymphocytes, and macrophages. We also demonstrated that eriodictyol inhibited nitric oxide (NO) production and lysosomal enzyme activity in murine peritoneal macrophages cultured ex-vivo, suggesting a potential anti-inflammatory effect in situ. Eriodictyol revealed also a cellular anti-oxidant activity in splenocytes and macrophages. Furthermore, eriodictyol increased catalase activity in spleen cells. From this data, it can be concluded that eriodictyol exhibited an immunomodulatory effect that could be ascribed in part to a cytoprotective effect related to its anti-oxidant activity.

Oligoesculin fraction induces anti-tumor effects and promotes immune responses on B16-F10 mice melanoma.

Laccase was used to enzymatically polymerize esculin. Oligoesculin fraction was obtained after ultrafiltration through a 5-kDa membrane. Several studies have been carried out to prove the effectiveness of natural substances such as immunomodulators to promote the anti-cancer activity in situ. The purpose of our report was to explore whether the anti-tumor potential of the oligoesculin fraction in vitro and in vivo is linked to its immunological mechanisms in melanoma-bearing mice. We revealed that oligoesculin fraction reduced B16-F10 proliferation and migration in vitro in a dose-related manner. Moreover, melanin synthesis and tyrosinase activity were inhibited in these melanoma cells in a concentration-dependent way. The anti-tumor potential of oligoesculin fraction was also assessed in vivo. Our results showed that intraperitoneal administration of oligoesculin fraction, at 50 mg/kg body weight (b.w.) for 21 days, reduced tumor size and weight with percentages of inhibition of 94 and 87 %, respectively. Oligoesculin fraction was effective in promoting lysosomal activity and nitric oxide (NO) production by peritoneal macrophages in tumor-implanted mice. In addition, the activities of natural killer (NK), cytotoxic T lymphocytes, and macrophages were significantly enhanced by oligoesculin fraction. These findings suggested that this polymer with its anti-tumor and immunomodulatory properties could be used for the treatment of melanoma.

Assessment in vitro of the genotoxicity, antigenotoxicity and antioxidant of Ceratonia siliqua L. extracts in murine leukaemia cells L1210 by comet assay.

Genotoxicity of Ceratonia siliqua extracts, was investigated by assessing their capacity to induce nucleus DNA degradation of murine leukaemia cells L1210, using the "Comet assay". The ability of total oligomer flavonoids (TOF) and aqueous extracts to protect cell DNA against oxidative stress induced by H2O2, was performed by pre- co or post-treatment of cells with the before mentioned extracts for different periods preceding exposure to H2O2 stress. No significant genotoxic effect was detected at different exposure times, except at the lowest concentration of TOF extract (16.25 µg/ml). It appears that extracts decreased DNA damage, induced by H2O2. Both of TOF and aqueous extracts exhibited cellular antioxidant capacity, with EC50 values of respectively <16.25 and < 35 µg/ml, as well as, a protective capacity against lipidperoxidation inducing using L1210 cells line as a cellular model. MDA inhibition percentages reached 88.43% and 90.52% with respectively 35.5 µg/ml of TOF extract and 70 µg/ml of aqueous extract. Antioxidant properties of carob leaf extracts revealed by our study make a good antioxidant protection and thus a good candidate as food addition component.

Genoprotective and neuroprotective effects of Daphne gnidium leaf methanol extract, tested on male mice.

Methanol extract of Daphne gnidium leaves was assessed for its antigenotoxic and neuroprotective effects through antioxidant and antibutyrylcholinesterase activities. Antigenotoxic activity was evaluated against methyl methanesulfonate injected intraperitoneally to mice, using the comet assay. The protective effect of D. gnidium reached 99.12%, at the lowest tested dose (44 mg/kg b.w.) in kidney cells, and 92.16% at the dose of 88 mg/kg b.w. in blood cells. The extract was dissolved in water and administrated to mice by intraperitoneal injection. Antioxidant activity was tested against DPPH radicals. It reached a maximum of 74.52% with an IC50 value of 45 µg/ml. Anticholinesterase activity was determined against butyrylcholinesterase, an enzyme linked to Alzheimer disease. The extract exhibited antibutyrylcholinestrase effect with an inhibition percentage of 35.82% at the lowest tested dose (44 mg/kg b.w.).

In vitro and in vivo anti-melanoma effects of Daphne gnidium aqueous extract via activation of the immune system.

The purpose of this study was to assess the antitumor and immunomodulatory effects of the aqueous extract from Daphne gnidium in mice-bearing melanoma tumor. Balb/C mice were subcutaneously implanted with B16-F10 cells and treated intraperitoneally with the aqueous extract at 200 mg/Kg b.w for 21 days. After euthanization on day 22, the tumors were weighed; lymphocyte proliferation, cytotoxic T lymphocyte (CTL), and natural killer (NK) cell activities were evaluated using the MTT assay. Macrophage phagocytosis was studied by measuring the lysosomal activity. In addition to its potential to inhibit the growth of the transplantable tumor, the aqueous extract remarkably induced splenocyte proliferation and both NK and CTL activities in tumor-bearing mice. The aqueous extract was also seen to have promoted lysosomal activity of host macrophages.

Effect of apigenin-7-glucoside, genkwanin and naringenin on tyrosinase activity and melanin synthesis in B16F10 melanoma cells.

AIMS: In this study, we have investigated the effects of apigenin-7-glucoside, genkwanin and naringenin, on mouse melanoma B16F10 cell proliferation. Influence of these natural products on percentage cell distribution in cycle phases and melanogenesis was also studied. MAIN METHODS: Cell viability was determined at various periods using the MTT assay, whereas effects of tested compounds on progression through the cell cycle were analyzed by flow cytometry. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Besides, the mechanism involved on the death route induced by the tested molecules was evaluated using the bis-benzimide trihydrochloride coloration method (Hoechst 33258). KEY FINDINGS: Apigenin-7-glucoside, genkwanin and naringenin exhibited significant anti-proliferative activity against B16F10 melanoma cells after 24 and 48 h of incubation. Furthermore, apigenin-7-glucoside, genkwanin and naringenin provoked an increase of subG0/G1, S and G2/M phase cell proportion with a significant decrease of cell proportion in G0/G1 phases. The results evaluated using Hoechst 33,258, confirm that the percentage of B16F10 cells observed in the sub G0/G1 phase were undergoing apoptosis. Moreover, apigenin-7-glucoside and naringenin revealed an ability to enhance melanogenesis synthesis and tyrosinase activity of B16F10 melanoma cells. Whereas genkwanin induces a decrease of melanin synthesis by inhibiting tyrosinase activity. SIGNIFICANCE: Our results promote the introduction of genkwanin in cosmetic preparations, as skin whitening agent, whereas apigenin-7-glucoside and naringenin should be introduced into cosmetic products as natural tanning agents.

Immunomodulatory and cellular antioxidant activities of pure compounds from Teucrium ramosissimum Desf.

Evaluation of the immunomodulatory activity of plant compounds is an interesting and growing area of research. Teucrium ramosissimum Desf. is a native and endemic medicinal plant from the South of Tunisia traditionally used for the treatment of many diseases. The anti-inflammatory activity of apigenin-7-glucoside, genkwanin, and naringenin isolated from T. ramosissimum were assayed. The phagocytic activities of macrophage and lymphocyte proliferation were investigated in the absence and presence of mitogens (lipopolysaccharide [LPS] or lectin). Depending on the concentrations, the compounds affect macrophage functions by modulating their lysosomal enzyme activity and nitric oxide (NO) release. The tested compounds enhance significantly splenocyte proliferation, either with or without mitogen stimulation. In studies to assess any potential effects of apigenin-7-glucoside, genkwanin, and naringenin on innate immunity, the results showed that these compounds significantly enhanced the killing activity of natural killer (NK) cells and cytotoxic activity of the T lymphocyte (CTL) isolated from splenocytes. These results suggest that T. ramosissimum compounds such as apigenin-7-glucoside, genkwanin, and naringenin may be potentially useful for modulating immune cell functions in physiological and pathological conditions.

Immunomodulatory potencies of isolated compounds from Crataegus azarolus through their antioxidant activities.

The search of natural immunomodulatory agents has become an area of great interest in order to reduce damage to the human body. In this study, the immunomodulatory potential of Crataegus azarolus and its isolated hyperoside on mouse lymphocytes and macrophages in vitro was assessed. The effect of C. azarolus natural compounds on splenocytes proliferation, natural killer (NK) and cytotoxic T lymphocytes (CTL) activities, and on macrophage-mediated cytotoxicity were assessed by MTT test. Phagocytic activity and inhibition of nitric oxide (NO) release by macrophages were also evaluated. The antioxidant capacity of these products was evaluated by determining their cellular antioxidant activity (CAA) in splenocytes and macrophages. Depending on the concentrations, both ethyl acetate (EA) extract and hyperoside (Hyp) from C. azarolus affect macrophage functions by modulating their lysosomal enzyme activity and nitric oxide release. Whereas, the above-mentioned products significantly promote LPS and lectin-stimulated splenocyte proliferation, implying a potential activation of lymphocytes B and T enhancing humoral and cellular immune responses. Moreover, EA extract and Hyp could enhance the activity of NK and T lymphocytes cells, as well as the macrophages-mediated cytotoxicity against B16F10 cells. The anti-inflammatory activity was concomitant with the cellular antioxidant effect of the tested compounds against macrophages and splenocytes. Collectively, C. azarolus and its isolated hyperoside exhibited an immunomodulatory effect through their antioxidant activity. These findings suggest that C. azarolus should be explored as a novel potential immunomodulatory agent for the treatment of inflammatory diseases.