Research SectionAntiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines
Introduction
Cancer in various forms is one of the leading causes of death in the human population. Chemoprevention and chemotherapy are employed in reducing human mortality due to cancer or in the control of cancer. There are several natural products of plant origin (phytochemicals) that are of potential value as chemopreventive or therapeutic agents. As therapeutic agents, the phytochemicals kill the cancer cells or stop their growth, leading to cancer remission. Some of the new cancer chemotherapeutic agents derived from plants in current clinical use are paclitaxel (Taxol), vincristine (Oncovin), podophyllotoxin and captothecin (Pezutto, 1997).
A screening test using human cancer cell lines is usually performed as an initial step in the evaluation of chemicals for cancer prevention and treatment (Skehan et al., 1990). Those compounds that are found to be potent in killing cancer cells or inhibiting cell growth are then further screened for their ability to suppress the growth of tumours in animal models. One group of compounds with potential antitumour activities are flavonoids, which are either synthetic or natural constituents of foods or drink (tea and fruit juices) consumed by humans. Flavonoids are composed of different chemical classes such as flavones (7,8-benzoflavone or α-naphthoflavone), isoflavones (genistein), flavonols (quercetin), flavanols [(+)-catechin], flavanones (naringenin) and chalcones (xanthohumol). These compounds differ in the level of oxidation of the flavane nucleus and in the number and position of hydroxyl and methoxyl substituents (Lee et al., 1994). Flavanones lack the double bond in the 2,3-position of the C ring present in the flavane nucleus. Opening of the C-ring of naringenin yields the chalcone, chalconaringenin. Chalcones are open C-ring flavonoids in which the two aromatic rings are joined by a three-carbon α,β-unsaturated carbonyl system (Bohm, 1994). Chalcones are converted to flavanones chemically by treatment of the former with sodium acetate in methanolic solution or enzymatically by chalcone isomerase (Stevens et al., 1997, Stevens et al., 1998). Flavonoids, including chalcones and flavanones, have been shown to inhibit the proliferation of cancer cells and inhibit tumour growth (Anto et al., 1995; De Vincenzo et al., 1995; Middleton and Kandaswami, 1994; Satomi, 1993; Seo et al., 1997; Wattenberg et al., 1994; Yit and Das, 1994).
The biological effects of the chalcones depend on their chemical structure. For example, methyl and hydroxy substituted chalcones are cytotoxic in Ehrlich ascites cells and Dalton's lymphoma ascites cells, whereas only hydroxy substituted chalcones reduce ascites tumours in mice (Anto et al., 1995). Of several chalcones studied, 3′-methyl-3-hydroxy-chalcone showed the strongest antitumour and antitumour-promoting activity (Nishino et al., 1993; Satomi, 1993; Shibata, 1994). This compound was found to inhibit the proliferation of various kinds of human malignant tumour cells representing gastric cancer, cervical cancer, pancreatic cancer and neuroblastoma (Satomi, 1993). Thus, substitutions on the chalcone structure have a profound influence on the anticarcinogenic effects of chalcone compounds.
Recently, six chalcones (xanthohumol, 2′,4′,6′,4-tetrahydroxy-3′-prenylchalcone, 2′,4′,6′,4-tetrahydroxy-3′-geranylchalcone, 5′-prenylxanthohumol, dehydrocycloxanthohumol and dehydrocycloxanthohumol hydrate) and three flavanones (isoxanthohumol, 6-prenylnaringenin and 8-prenylnaringenin) have been isolated from hops by Stevens et al. (1997). Four of the hop flavonoids with prenyl groups (xanthohumol, isoxanthohumol, 6-prenylnaringenin and 8-prenylnaringenin) are found in hopped beers (Stevens et al., 1998, Stevens et al., 1999). Xanthohumol is the most abundant prenylated flavonoid in hops whereas isoxanthohumol is the most abundant flavonoid in all types of beer tested (Stevens et al., 1998, 1999). Isoxanthohumol in beer is produced by the isomerization of xanthohumol during the brewing process (Stevens et al., 1999). The potential of the major prenylated hop compounds, xanthohumol and isoxanthohumol, as antineoplastic agents is not known. Other prenylated flavonoids display cytotoxic activity against several human cancer cell lines (Seo et al., 1997). It is possible that the prenylated flavonoids from hops and beer may have the ability to inhibit the proliferation of cancer cells, making them potentially useful as cancer chemopreventive agents. Thus, the objective of this research project was to examine the antiproliferative and cytotoxic activities of prenylated flavonoids from hops (Humulus lupulus) in human breast cancer (MCF-7), colon cancer (HT-29) and ovarian cancer (A-2780) cells in vitro. The cytotoxicity of these hop compounds in primary cultures of rat hepatocytes was also evaluated in an effort to determine their potential to cause liver toxicity in the intact animal. Hepatic toxicity is one of the early issues that need to be addressed in drug development and is therefore pertinent in our investigation on the overall evaluation of hop flavonoids as potential preventive agents for cancer in humans.
Section snippets
Chemicals
The chalcone compounds (Fig. 1) tested for their growth-inhibitory activities were xanthohumol (XN), 2′,4′,6′,4-tetrahydroxy-3′-prenylchalcone (TP), 2′,4′,6′,4-tetrahydroxy-3′-geranylchalcone (TG), dehydrocycloxanthohumol (DX), and dehydrocycloxanthohumol hydrate (DH). The other chalcone, 5′-prenylxanthohumol, isolated from hops was not available in sufficient quantities for this study. Isoxanthohumol (IX), a flavanone derivative of XN which is present in hops (Stevens et al., 1997), was also
Sulforhodamine assay
The SRB assay is an indirect measure of cell density or number of live cells attached to the culture plate (Skehan et al., 1990). As shown by Skehan et al. (1990), the SRB results are linear with the number of viable cells and protein content in the wells at densities from sparse subconfluence to multilayered supraconfluence. In agreement with these results, we found that dead cells detached from the plate and were removed with the culture media during the SRB assay. Microscopic examination of
Discussion
Previous studies have shown that certain synthesized or naturally-occurring chalcone compounds (not found in hops) exhibit antiproliferative activities in human cancer cell lines (De Mello et al., 1974; Ramanathan et al., 1993; Satomi, 1993; Yit and Das, 1994). The potency of the chalcones (butein, 2′-hydroxychalcone, 2-hydroxychalcone, 2′,6′-dihydroxy-4′-methoxychalcone and 2′,4-dihydroxychalcone) as inhibitors of growth of human colon adenocarcinoma cell line, CCL 220.1, was dependent on the
Acknowledgements
This work was supported by the Hop Research Council, Medical Research Foundation of Oregon, and NIEHS grant ES00210. This manuscript was issued by the Oregon State University Agricultural Experiment Station as Technical Paper No. 11318. The authors thank Sam Bradford and Chelsea Johnston for technical assistance.
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