Effects of Humulus lupulus extract or its Components on Viability, Lipid Peroxidation, and expression of Vascular Endothelial Growth Factor in Melanoma Cells and Fibroblasts

Cancer and aging are associated with altered cell viability and angiogenesis, which is mediated by vascular endothelial growth factor (VEGF). These alterations have been associated with cellular oxidative stress. Humuluslupulus (HOP) extract, and its components, which include alpha-acid, beta-acid, xanthoflavonoids, xanthohumunol, and isoxanthohumunol, exhibit antioxidant activity. This research examined the effects of HOP extract or its components on direct antioxidant activity, and on cell viability, lipid peroxidation, and expression of VEGF in melanoma cells, and dermal fibroblasts. The HOP extract, and its components exhibited direct antioxidant activity. In melanoma cells, HOP extract, and its components significantly inhibited cell viability and stimulated extracellular lipid hydroperoxides at all examined concentrations; and with few exceptions did not significantly alter intracellular lipid peroxidation or the expression of VEGF. In dermal fibroblasts, HOP extract and its components significantly inhibited cell viability and intracellular lipid peroxidation, and stimulated the expression at VEGF at the highest examined concentration; and with few exceptions did not significantly alter extracellular lipid peroxidation. It is inferred that HOP extract, and its components differentially and beneficially regulate the cellular biochemistry of melanoma cells, and fibroblasts for the management of cancer, and aging.

The mechanisms of the effects of HOP extract or its components on melanoma, or on dermal fibroblasts have not been reported. The goal of this research was to determine the direct antioxidant activity of HOP extract or its components, and its effects on cell viability, extracellular and intracellular lipid peroxidation, and the expression of VEGF in melanoma cells, and dermal fibroblasts.

Antioxidant Activity
The direct antioxidant activity of HOP extract or its components was determined by ABTS ® (2, 2'-azino-di-[3ethylbenzthiazoline sulphonate]) assay, based on the inhibition of the oxidation of ABTS ® to ABTS ® radical by metmyoglobin by antioxidants (Cayman Chemical Antioxidant Assay kit) [1]. HOP extract or its components (alpha-acid, beta-acid, xanthoflavonoids, xanthohumunol and isoxanthohumunol) at 0, 0.01, 0.04, 0.02 or 1% of respective 50mg/ml stock solutions, was incubated with ABTS and metmyoglobin and the inhibition of ABTS oxidation was measured spectrophotometrically at 405 nm. The HOP extract and its components were obtained from S. S. Steiner, Inc

Cell Culture
Melanoma cells (CRL-1619, American Tissue Cell Culture), and human neonatal dermal fibroblasts (Cascade Biologics) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum and 1% penicillin/streptomycin (Life Technologies) [1,[6][7][8]. Melanoma or fibroblasts cells were incubated with 0, 0.01, 0.04 or 0.02 % HOP extract or its components in experimental media composed of DMEM supplemented with 3% heat inactivated fetal bovine serum for 24 hours. The cells were examined for cell viability and intracellular lipid hydro peroxides. The extracellular media were examined for extracellular hydro peroxides and VEGF protein.

Lipid Peroxidation
The hydroperoxides, formed as a result of lipid peroxidation, were measured using PeroxiDetect kit (Sigma), which is based on the oxidation of ferrous to ferric ion by peroxides and the subsequent reaction of ferric ion with xylenol orange ("3,3′-bis[N,N-bis(carboxymethyl)aminomethyl] o-cresolsulfonephthalein, sodium salt") to a blue colored product (Sigma). Aliquots of cell lysates or media were incubated with ferrous/xylene orange solution and the reaction was measured spectrophotmetrically at 560 nm (Sigma).

VEGF Protein Levels
The VEGF protein levels were determined by indirect ELISA (Kirkguaard and Perry Laboratories Inc, Stressgen [1,6,7,8]. The media were incubated with coating buffer in 96 well immunosorbent plates for 24 hours at 4°C. The wells were blocked with bovine serum albumin, incubated with VEGF antibody for 24 hours at 4°C, washed, incubated with secondary antibody for 1 hour at room temperature, washed and incubated with peroxidase substrate till color formation, which was read spectrophotometrically at 405nm (Kirkguaard and Perry Laboratories Inc, Stressgen).

Data Analysis
The effects of each of the concentrations of HOP extract or its components were determined relative to control (without HOP extract or its components) at 100%.The significant effects of each of the concentrations of the HOP extract or its components in each of the cells (melanoma or fibroblasts) were determined relative to control (without HOP extract or components) by ANOVA and student t-tests at 95% confidence interval.

Regulation of melanoma and fibroblast intracellular hydroperoxides by HOP extract, and its components
The HOP extract or its components did not significantly alter intracellular lipid hydroperoxides in melanoma cells (except xantho-flavonoid and xanthohumol at 0.2%); and significantly inhibited it in fibroblast cells at the highest concentration, and in addition the second highest concentration for xantho-flavonoid, and xanthohumol (p<0.05) (Figure 4). In melanoma cells, 0.2% xantho-flavonoid and xanthohumol significantly stimulated intracellular hydroperoxides to 216% and 220% of control, respectively (p<0.05) ( Figure 4A).

Regulation of melanoma and fibroblast VEGF expression by HOP extract, and its components
The HOP extract or its components did not significantly alter VEGF expression in melanoma cells (except xanthoflavonoid); and significantly stimulated it in fibroblast cells at the highest concentration (p<0.05) ( Figure 5). In melanoma cells, 0.04 and 0.2% of xantho-flavonoid significantly inhibited VEGF expression to 60% and 53% of control, respectively (p<0.05) ( Figure 5A).

Discussion
Carcinogenesis and aging are associated with altered cellular redox balance due to exposure to environmental pollutants or ultraviolet radiation, and genetics [1][2][3][4][5]. Phenolic compounds with their antioxidant property have potential to modulate the cellular redox environment and thereby carcinogenesis or aging [1][2][3][4][5]. The differential beneficial effects of HOP extract or its extracts in cancer versus normal cells are from their pro-oxidant versus antioxidant effects, respectively, in these cells. The potential for differential effects, in cancer versus normal cells, may be from differential cellular environment in cancer versus normal cells, such as the concentrations of metals that catalyze Fenton or Haber-Weiss reactions [10,11]. While the HOP extract, and its components exhibited direct antioxidant activity, they had differential beneficial effects in melanoma cells, and dermal fibroblasts.
Carcinogenesis is associated with increased cell proliferation, and angiogenesis [1][2][3][4][5]. The HOP extract or its components, from the smallest to the highest concentration examined, significantly inhibited melanoma cell viability and Madridge J Clin Res. ISSN: 2638-3535 stimulated extracellular hydroperoxides, suggesting prooxidant mechanism in the inhibition of melanoma cell viability. In addition, xantho-flavonoid significantly inhibited the expression of VEGF in melanoma cells, suggesting antiangiogenic potential.
Aging is associated with loss of cell viability, and reduced angiogenesis [1][2][3][4][5]. The HOP extract or its components, at the highest concentration examined, significantly inhibited fibroblast cell viability and stimulated extracellular hydroperoxides, suggesting pro-oxidant mechanism. However, the HOP extract or its components significantly inhibited intracellular lipid peroxidation in fibroblast cells, suggesting cellular antioxidant activity; and significantly stimulated the expression of VEGF, suggesting angiogenic potential.

Conclusion
Carcinogenesis is associated with increased cell proliferation and angiogenesis, whereas aging is associated with reduced cell viability and angiogenesis. The melanoma cell viability was inhibited through pro-oxidant mechanism, at all concentrations of the HOP extract or its components; without antioxidant cellular effect or increased expression of VEGF. The fibroblasts cell viability was inhibited by prooxidant mechanism, at the highest concentration of HOP extract or its components, and was associated with increased cellular antioxidant activity and expression of VEGF.