Description of the Prior Art In general, malignant tumors contain a lot of hypoxic cells due to an inadequate vasculature (see: Moulder and Rockwell, Cancer Metastasis Rev., 5: 313-341, 1987; Vaupel et al., Cancer Res., 49: 6449-6465,1989) or changes in a supply of red blood cells in intratumoral microvessels (see: Kimura et al., Cancer Res., 56: 5522-5528, 1996). Although some of these hypoxic tumor cells may be converted into normoxic cells by reoxygenation attainable by reopening of temporally closed or clogged microvessels not to inhibit tumor cell growth (see: Brown, J. M., Br. J.

Radiol., 52: 650-656,1979) or by inactivation of tumor cells with fractionated radiation therapy (see: Kallman, R.

F., Radiology, 105: 135-142,1972), it has been known that, compare to normoxic cells, hypoxic cells are generally more resistant to radiation therapies or conventional chemotherapies (see: Teicher et al., Cancer Res., 41: 73-81, 1981 ; Gatenby et al., Int. J. Radiat. Oncol. Biol. Phys., 14: 831-838,1988; Teicher et al., Cancer Metastasis Rev., 13: 139-168,1994).

Although adaptation mechanism of tumor cells to a stress of low oxygen tension has not been understood clearly, hypoxia has been known to affect the patterns of gene expression in tumor cells (see: Brown and Giaccia, Int.

J. Radiat. Biol., 65: 95-102,1994) and it has been reported that stress reaction of normal cells is induced by low-oxygen environment and, in consequence, syntheses of stress proteins are induced in vivo and in vitro (see: Guttman et al., Cell, 22: 229-307,1980 ; Heacock and Sutherland, Br. J. Cancer, 62: 217-225,1990; Iwaki et al., Circulation, 87: 2023-2032,1993). For example, Baek et al. demonstrated that heat shock proteins such as hsp70 and hsp25 are upregulated in mouse radiation induced fibrosarcoma (RIF) cells by hypoxia, and hypoxic tumor cells with increased level of heat shock proteins are more resistant to hypoxia than normoxic cells (see : Baek et al., J. Biochem. & Mol. Biol., 32: 112-118,1999).

It has been demonstrated that growth factors such as VEGF (see: Stein et al., Mol. Cell. Biol., 15: 5363- 5368,1995), EPO (see: Wang and Semenza, Blood, 82: 3610- 3615,1993) and TGF 13-1 (transforming growth factor 1) (see: Brown et al., EXS., 79: 233-269,1997) required for angiogenesis which is an essential process for progression and metastasis of hypoxic tumor cells described above can be upregulated by hypoxia. Hence, cooperative induction of stress protein and angiogenesis factor genes are understood to render tumor cells adaptable to low oxygen stress, which allows the progression of tumor cells toward more malignant phenotype.

In order to overcome problems caused by hypoxic tumor cells, the following three methods are mostly employed in the art: i) oxygenation of tumor cells, ii) attenuation of hypoxic cells with radiation or chemotherapy, iii) induction of hypoxic cell death using the cytotoxin obtained from hypoxic cells (see: Brown and Koong, J. Intl. Cancer Inst., 83: 178-185,1991). However, since hypoxic tumor cells are resistant to both radiation

and chemotherapy, there is a continuing need to increase curative efficiency for tumor cells by inhibiting protein syntheses required for tumor cell survival and angiogenesis using the methods other than described above.

Summary of the Invention The present inventors have made an effort to increase curative efficiency for tumor cells, and discovered that treatment of hypoxic tumor cells with quercetin, which has been known to have inhibitory effect on expression of heat shock proteins, is able to inhibit expression of angionenesis-associated factors, VEGF and EGF, subsequently, increasing curative efficiency for tumor cells by inhibiting angiogenesis.

The primary object of the present invention is, therefore, to provide a method for inhibiting expression of VEGF and EPO genes by quercetin.

BRIEF DESCRIPTION OF THE DRAWINGS The above and the other objects and features of the present invention will become apparent from the following descriptions given in conjunction with the accompanying drawings, in which: Figure 1 is a graph showing protein synthesis rate with culture time.

Figure 2a is a graph showing the expression of VEGF gene.

Figure 2b is a graph showing the expression of EPO gene.

Figure 3a is a photograph of western blot of VEGF protein.

Figure 3b is a photograph of western blot of EPO protein.

Figure 4 is a graph showing the inhibition of angiogenesis in fertilized eggs depending on quercetin concentration.

Figure 5 is a photograph of X-ray film representing HIF-1 gene activity.

Figure 6a is a photograph of western blot representing expression of PKC 5 in cytoplasmic fraction.

Figure 6b is a photograph of western blot representing expression of PKC 5 in particulate fraction.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the method for inhibiting expression of VEGF and EPO genes of the invention, the expression of angiogenesis-associated factors is inhibited by treating hypoxic tumor cells with quercetin. That is, treatment of the tumor cells with the quercetin inhibits activity of protein kinase C delta (hereinafter referred to as'PKC 5'), subsequently, the activity of hypoxia inducible factor-1 (hereinafter referred to as'HIF-1') which is controlled by the PKC 5 becomes inhibited, and finally, the expression of genes for angiogenesis- associated factors, i. e., VEGF and EPO of which expression is regulated by the HIF-1 is inhibited.

The present invention is further illustrated as follows.

Based on the knowledge that quercetin which inhibits heat shock protein expression in tumor cells can be applied to the treatment of cancer, to find out alternative mechanism, mouse tumor cells-were treated with quercetin, hypoxia was induced by incubating the cells in a hypoxic chamber, total RNA and protein were isolated from the cells and the gene expression pattern of

angiogenesis relating factors, VEGF and EPO was examined.

As a result, it has been proved that quercetin plays a role in angiogenesis by verifying inhibition of the expression of angiogenesis relating genes in quercetin- treated tumor cells. Furthermore, to find out whether the inhibition of VEGF and EPO gene expression affects angiogenesis directly, angiogenesis was assayed employing CAM assay method in fertilized eggs with or without quercetin treatment, and the angiogenesis on the chorioallantoic membrane with quercetin treatment decreased compare to that without quercetin treatment.

Furthermore, to find out the mechanism of inhibitory effect of quercetin on VEGF and EPO gene expression, gene activity of HIF-1 which is known to control the VEGF and EPO gene expression was assayed employing EMSA (Electrophoretic Mobility Shift Assay). As a result, it has been found that HIF-1 gene activity in quercetin treated cells was decreased. Thus, to which enzyme of the upper level of gene regulation hierarchy is related to the decrease in HIF-1 activity, the expression of PKC 5 which is known to influence signal transfer in hypoxic cells and relate to HIF-1 activity was examined. As a result, PKC 5 was found not to be expressed in the cell membrane of tumor cells, indicating that PKC 5 is the upper level regulatory factor for underregulating expression of VEGF and EPO genes.

The present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention.

Example 1 : Induction of hypoxia in tumor cells In order to induce hypoxia in vitro which is a physiological characteristic of tumor cells, the tumor cells were cultured under a condition of oxygen-glucose depletion in a hypoxic chamber (Forma Scientific, U. S. A.)

at 37°C. To deplete intracellular oxygen and glucose, RIF tumor cells grown in a glucose-supplemented medium were incubated in a glucose-depleted medium (GIBCO-BRL, U. S. A.) with three times of medium changes which was preequilibrated with a low-oxygen gas mixture of 5% CO2- 85% N2-10% H2 at 37°C to maintain partial oxygen pressure at 0.02%. Hypoxia in RIF cells was discontinued by feeding the cells with a glucose-supplemented medium and incubating in a normoxic incubator.

Example 2: Determination of effective concentration of quercetin on tumor cells To determine the concentration of quercetin effective on RIF cells, RIF cells in a logarithmic phase were treated with quercetin at the concentrations of 0.05, 0.1 and 0.2mM at 37°C for 0,3,6,9,12 and 15 hours, and then cell viability was determined with trypan blue exclusion method. Quercetin dissolved in DMSO (dimethyl sulfoxide) was added to a medium to a final concentration of 0. 1% (v/v) or less. In this experiment, it has been found that the effective concentration of quercetin affecting cell viability was 0.05mM and optimal incubation time was 6 hours.

Example 3: Determination of exposure time of tumor cells to hypoxic condition To determine exposure time of tumor cells to hypoxic condition, protein biosynthesis rate was assessed by measuring uptake of 35S-labelled amino acids (see: Anderson et al., Mol. Cell. Biol., 9: 3509-3516,1989). Tumor cells were exposed to a hypoxic condition for 0, 2, 4,8 and 16 hours which were followed by incubation in a medium containing 35S-methionine (specific activity>140Ci/mM, Amersham, U. S. A.) respectively, and then protein biosynthesis rate was assessed by determination of

radioactivity incorporated into the cells (see: Fig. 1).

Figure 1 is a graph showing the protein biosynthesis rate with time, where protein biosynthesis rate is defined as radioactivity incorporated into lug of protein. As shown in Figure 1, when the cells were exposed for 2 hours, protein synthesis was inhibited to 67% of that of the cells under normoxic condition and the inhibition was maintained up to 8 hours. However, after 8 hours exposure, viable cell counts began to reduce due to cell death caused by hypoxia, thus, for further experiments, the cells were exposed to a hypoxic condition for 4 hours at which the cells show normoxic characteristics of viability and morphology.

Example 4: Effect of quercetin on transcription of VEGF and EPO genes To determine the rate of transcription of VEGF and EPO genes with or without quercetin treatment, RT-PCR was performed on the said genes. RIF cells with or without quercetin treatment were exposed to a hypoxic condition for 0,1,4 and 8 hours and then total RNA was isolated from the cells respectively. One microgram of total RNA, 500 ng of random hexamer, 0.5mM each of dNTP, 10X reaction buffer and 400 unit of MMLV (Moloney murine leukemia virus) reverse transcriptase were mixed and the mixture was incubated at 37°C for 1 hour. Using 2ul each of the above reaction mixture as a template, PCR was performed in a mixture containing 0.2mM each of VEGF primer 1: 5'- tgcactggaccctggcttta-3' (SEQ ID NO : 1) and primer 2: 5'- tttgcaggaacatttacacg-3' (SEQ ID NO : 2) or EPO primer 3: 5'- agccctgcgtctaatgtttc-3' (SEQ ID NO : 3) and primer 4: 5'- cgaccaccagagacccttca-3' (SEQ ID NO : 4), 10X PCR buffer solution (50mM KC1, 1. 5mM MgClz, 0.01% gelatin, lOmM Tris, pH 8.3) and AmpliTaq DNA polymerase (Perkin Elmer, U. S. A.).

The reaction product was subjected to a gel electrophoresis and each PCR product was quantitated

employing ImageQuant Software (Moloecular Dynamics, U. S. A.) respectively (see: Figs. 2a and 2b). Figures 2a and 2b are graphs showing the expression of VEGF gene and EPO gene, respectively. As shown in Figures 2a and 2b, the mRNA expression of VEGF and EPO genes was inhibited in quercetin-treated cells compare to that in quercetin- untreated cells.

Example 5: Effect of quercetin on the expression of VEGF and EPO genes RIF cells with or without quercetin treatment were exposed to a hypoxic condition for 0,1,4 and 8 hours, washed with cold phosphate buffer solution three times, resuspended in a solution of SDS-gel sample buffer (0.1% (w/v) bromophenol blue, 20% glycerol (v/v), 4% sodium dodecyl sulfate (w/v), 10% ß-mercaptoethanol (v/v)) and then heated at 100°C for 5 minutes. Then, 30ug ailquot of the protein was subjected to a 13% polyacrylamide gel electrophoresis (see: Laemmli, Nature, 227: 680-685,1970) which was followed by western blotting employing antibodies against VEGF and EPO (Santa Cruz, U. S. A.) (see: Figures 3a and 3b). Figure 3a and 3b are photographs of western blot of VEGF protein and EPO protein. As shown in Figures 3a and 3b, in cells without quercetin treatment, expression level of VEGF and EPO genes stayed same with time, while the expression of VEGF and EPO genes of quercetin-treated cells was found to be inhibited with time.

Example 6: Inhibitory effect of quercetin on angiogenesis To find out whether quercetin also inhibits angiogenesis in vivo, chick embryo chorioallantoic membrane (CAM) assay was performed: that is, using 3 day old chicken fertilized eggs, a part of egg shell was

cracked gently and the crack was sealed with cellophane tape. After incubation for 2 days under a condition of over 90% humidity and 37 to 38°C, ethanol (control group) or quercetin was injected at a concentration of 1,5, and 10, ug per each fertilized egg onto the CAM which was followed by 48 hour incubation to assay inhibition of angiogenesis of fertilized eggs (see: Figure 4). Figure 4 is a graph showing the inhibition of angiogenesis in fertilized eggs depending on quercetin concentration. As shown in Figure 4, inhibition of angiogenesis reached up to 70 to 80% when cells were treated with 5 to 10pg of quercetin.

Example 7: Inhibitory effect of quercetin on HIF-1 activity To find out which upper regulatory gene is involved in the inhibition of VEGF and EPO gene expression in hypoxic tumor cells, the rate of transcription of HIF-1 gene, which is known to control VEGF and EPO gene expression, was measured employing EMSA method: that is, RIF cells with or without quercetin treatment were exposed to a hypoxic condition for 0,0.5 and 1.5 hours, washed with phosphate buffer solution 3 times, resuspended in lml of buffer solution (l. 5mM MgCl2, lOmM HEPES, pH 7.9), incubated on ice for 15 minutes, and then centrifuged at 3000 x g for 15 minutes at 4°C to obtain nuclei pellet respectively. The nuclei pellet was resuspended in 200p1 of buffer solution (l. 5mM MgCl2,0.2mM DTT, 1mM PMSF, lpg/ml Aprotinin, lmM Leupeptin, 100mM KC1,10% (v/v) glycerol, lOmM HEPES, pH 7.9), incubated on ice for 15 minutes and then centrifuged at 12000 xg for 15 minutes at 4°C to obtain supernatant.

For test group, to 19p1 of binding buffer solution (l. 5mM MgCl2, 0.2mM DTT, 1mM PMSF, lpg/ml Aprotinin, 1mM Leupeptin, 100mM KC1,10% (v/v) glycerol, 0.5pg/pl poly (dI-dC), lOmM HEPES, pH 7.9), 8pg of the said

supernatant protein was added and then 1p1 (0. 05pmol/ul) of 32P-labelled probe 1 : 5'-agcttgccctacgtgctgtctcag-3' (SEQ ID NO : 5) and probe 2: 5'-acgggatgcacgacagagtcttaa-3' (SEQ ID N0 : 6) which have a binding site to HIF-1 DNA was added.

For control group, 5pmol of unlabelled said probes 1 and 2 were mixed with nucleus extract at room temperature, and then 32P-labelled probes 1 and 2 were added. Samples of control and test groups obtained above were subjected to a 4% polyacrylamide gel electrophoresis and then mRNA bound to 32P-labelled probes 1 and 2 were detected by sensitizing X-ray film (see: Figure 5). Figure 5 is a photograph of X-ray film representing HIF-1 mRNA : lane c is control group and lane free is 32P-labelled probes only to confirm labelling efficiency of probes. As shown in Figure 5, the cells without quercetin treatment did not show any change in HIF-1 gene activity, while HIF-1 gene activity of the quercetin-treated cells decreased.

Example 8: Inhibitory effect of quercetin on PKC 5 The relationship between reduction of HIF-1 gene transcriptional activity by quercetin treatment and PKC 5 gene was examined. RIF cells with or without quercetin treatment were exposed to a hypoxic condition for 0,0.5 and 1.5 hours and washed with a phosphate buffer solution 3 times. 5 x 106 cells were resuspended in 100p1 of buffer solution (10% SDS (w/v), 5% ß-mercaptoethanol (v/v), 10% glycerol (v/v), 25mM Tris-Cl, pH 6.8). After disrupting cell membrane using a sonicator, the mixture was centrifuged at 100,000 xg for 30 minutes at 4°C to obtain cytoplasmic fraction in supernatant. The said cytoplasmic fraction was mixed with 70u1 of buffer solution containing Triton X-100 (10% SDS (w/v), 5% mercaptoethanol (v/v), 10% glycerol (v/v), 1% triton X-100, 25mM Tris-Cl, pH 6.8), incubated at 4°C for 30 minutes and centrifuged at 100,000 xg for 30 minutes at 4°C to obtain particulate fraction in supernatant. Equal amounts of the

cytoplasmic fraction and the particulate fraction were subjected to electrophoresis and expression of PKC 5 was examined using PKC 5 antibody (see: Figures 6a and 6b).

Figures 6a and 6b are photographs of western blot representing expression of PKC 5 in the cytoplasmic fraction and PKC 5 in the particulate fraction, where c respresents positive control. As shown in Figures 6a and 6b, in cells without quercetin treatment, the level of PKC 5 expression was maintained both in the cytoplasmic fraction and in the particulate fraction, while in cells with quercetin treatment, the level of PKC 5 expression in the cytoplasmic fraction was maintained but the level of PKC 5 expression in the particulate fraction was decreased.

Thus, it has been verified that the inhibition of VEGF and EPO gene expression was caused by decrease in the expression of PKC 5 gene.

As clearly illustrated and demonstrated above, the present invention provides a method for inhibiting the expression of angiogenesis-associated factors, VEGF and EPO genes by treating hypoxic tumor cells with quercetin.

In accordance with the present invention, since quercetin is able to inhibit the expression of genes for angiogenesis-associated factors, VEGF and EPO in solid tumor cells, it would be practically applied for the elevation of therapeutic efficiency against hypoxic tumor cells and the inhibition of metastasis of cancer cells.