N T I O X I D A N T S A N D G E N E R E G U L A T I O N ! The Effects of Vitamins C and E on Estrogen Receptors J E O N G H O N A M RESEARCH S H O W S T H A T E S T R O G E N B I N D I N G TO ITS RECEPTOR PLAYS A ROLE I N BREAST CANCER D E V E L O P M E N T A N D T H A T A N T I O X I D A N T S POSSIBLY M I T I G A T E T H I S EFFECT. PAST RESEARCH E X A M I N E D W H E T H E R V A R I O U S T R E A T M E N T S LEAD TO ACCEL- ERATED CELL D I V I S I O N , BUT C H A R A C T E R I Z A T I O N A N D C O M P A R I S O N OF T H E EFFECTS OF D I F F E R E N T T R E A T M E N T S O N G E N E LEVEL E X P R E S S I O N OF T H E RECEPTOR WAS N O T A C C O M P L I S H E D . I N I T I A L L Y , T H E EFFECT OF A N T I O X I D A N T S O N T H E E S T R O G E N RECEP- TOR E X P R E S S I O N WAS I N V E S T I G A T E D , R E V E A L I N G T H E PRESENCE OF V I T A M I N S C A N D E. S U B S E Q U E N T LY, T H E I N D I V I D U A L EFFECTS OF V I T A M I N S C A N D E O N N I T R I C O X I D E RELEASE (A POSSIBLE CANCER R E D U C T I O N A G E N T ) S T I M U L A T E D BY E S T R O G E N A C T I N G O N T H E SURFACE E S T R O G E N RECEPTOR OF BREAST CANCER CELLS WAS O B S E R V E D . G E N E R A L LY, V I T A M I N E WAS M O S T EFFECTIVE FOR I M P R O V I N G N I T R I C OX IDE RELEASE. I N T R O D U C T I O N Breast cancer is one of the most common cancers among women worldwide. Statistical data have shown that among American women, nearly one out of three cancers diag- nosed is a breast cancer and in the year 2001 , approxi- mately 192,200 cases of breast cancer were diagnosed among women in the United States. Through many years of breast cancer research, it has been documented that es- trogen plays a critical role in the development of breast can- cer.1 Estrogens are steroid molecules and sex hormones that stimulate the development of female characteristics and sexual reproduction. The most common forms of human estrogens are ly-beta estradiol and estrone that are pro- duced and secreted by the ovaries. Two of the female or- gans that play a central role in sexual reproduction, the breast and the uterus, are the main target organs of the es- trogen molecule. Cells of the breast and the uterus have es- trogen receptors (alpha and beta) that have specific sites to which only estrogen can bind. Estrogen molecules are only effective when they bind to the receptors. When estrogen molecules pass through the membrane of the cell and bind to the estrogen receptors, the shape of the receptor changes and estrogen receptor complex attaches to the estrogen re- sponse elements (EREs) i n DNA, causing certain genes to become active. The active genes produce molecules of mes- senger RNA that influence cell activity i n a variety of ways by synthesizing specific proteins. 1 1 This study examines the interaction of estrogen and antioxidants on the expres- sion of estrogen receptor genes. Cancer is caused by mutations in growth regulatory genes. Mutations may be caused by transcription errors in DNA before the process of cell division. 1 1 1 As these cells prolifer- ate, the transcription errors are then carried on to the new generation of cells and may lead to uncontrollable prolifer- ation. Besides its role in developing female sexual characteristics, estrogen also impacts on the health of the immune system, and the body's response to the stress and the changing en- vironment.^ One of the main effects of estrogen is induc- ing stimulation for cell proliferation. v Cell proliferation is estrogen's natural role but it increases a woman's chance of developing breast cancer or uterine cancer. Previous stud- ies have demonstrated the effect of ij-beta estradiol on cell proliferation. I n several studies, rj-beta estradiol enhanced the rate of cell proliferation and also decreased the level of apoptosis of breast cancer cells by inducing the bcl-2, anti- apoptosis gene . V 1 Estrogen cannot distinguish between mutated cells and healthy cells. Proliferation stimulated by estrogen can result in the proliferation of mutant cells, thereby causing cancer.V11 To prevent estrogen from pro- moting cell proliferation, researchers have been developing anti-estrogen substances such as tamoxifen, and raloxifene that can block the estrogen receptor.™ Currently, two types of estrogen receptors (ER) have been identified, alpha and beta. They both bind to DNA but it has been shown that in MCF-7 adenocarcinoma human breast cancer cells, ER alpha is the prevalent form and ER beta is hardly detectable. Estrogen a^ha-receptor is also mainly involved with the development of a breast cancer.1X Most research on breast cancer regarding antioxidants has focused on the effect of melatonin (pineal indoleamine), a type of an antioxidant, on the proliferation of MCF-7 breast cancer cells. Various studies have demonstrated that mela- tonin has an anti-proliferative effect on estrogen responsive MCF-7 breast cancer cells and also that the administration of melatonin reduces the incidence and growth rate of chemically induced mammary tumors. x According to the several studies, not only does melatonin inhibit the MCF-7 cell growth but it also increases cell doubling time and de- lays the entry of MCF-7 cells into mitosis. x l Furthermore, it has been shown that melatonin decreases ER bonding ac- tivity and ER mRNA expression/ 1 1 However, when mela- tonin and estradiol were treated together, the anti-prolifer- ative effect of melatonin was reversed, demonstrating that the effect of melatonin can be counteracted by cell cycle ac- celeration stimulated by estradiol/ 1 1 1 E L E M E N T S S P R I N G 06 In addition, the effects of other types of antioxidants such as carotenoids, and retinoic acid on MCF-7 cell growth also have been widely studied. These antioxidants have been shown to have an inhibitory effect on the ER positive MCF- 7 cell growth but not on ER negative MDA-MB-231 breast cancer cells. x l v On the other hand, it has been reported that vitamin E, selenium, and palm oil tocotrienols have been shown to be effective anti-proliferative agents in both ER negative and positive cells breast cancer cells. x v Unlike pre- vious studies where researchers observed the effect of an- tioxidants on the proliferation of the cells, i n the first part of this study, the effect of the antioxidants ascorbic acid (vita- m i n C) and alpha tocopheral (vitamin E) on the gene ex- pression of estrogen receptor wi l l be observed. Another part of this study is to investigate whether ascorbic acid and alpha tocopherol can affect the nitric oxide release from the surface receptor of ER positive MCF-7 cells. Several studies have found the existence of surface estro- gen receptors in human peripheral monocytes and the gan- glionic nervous system of Mytilus edulis, the common mussel. These investigations also demonstrated that 17- beta estradiol stimulates nitric oxide (NO) release by bind- ing to the cell surface estrogen receptor.X V 1 Nitric oxide is a free radical that scavenges other free radicals and controls the activation state of various tissues such as immune cells, thereby helping to maintain appropriate levels of cellular activity. NO has also been shown to play a host defense against tumors, viruses and bacteria. I n addition, it has been shown that free radicals promote cancer by damaging DNA. Since NO release is one of estrogen receptors' criti- cal functions, investigating the effect of antioxidants on NO release by surface estrogen receptors on MCF-7 cells is e s " sential. However, it has been found out that while NO plays an important role in numerous physiological and patho- physiological conditions, excessive concentrations of NO may lead to a tissue damage and organ dysfunction.™ 1 I n the second phase of this experiment, cells are pre-treated with ascorbic acid and alpha tocopherol, and then the NO release triggered by ij-beta estradiol is measured by using NO-specific amperometric probe. M A T E R I A L S A N D M E T H O D S G E N E E X P R E S S I O N CELL C U L T U R I N G A flask with MCF-7 cells (American Type Culture Collection, ATCC), RPMI1640 10 percent Media (GIBCO, Invitrogen, Carlsbad, CA) and Tripsin (SIGMA, St Louis, MO) were prepared. From the original flask with MCF-7 cells, the media was removed, and then washed with 2 m l of trypsin. Three m l of trypsin was added to the flask contain- ing MCF-7 cells, and the flask was tilted back and forth so the trypsin would cover the whole surface with MCF-7 cells. The flask was then sat still for 5 to 15 minutes unti l all the cells detach from the flask surface. When all the cells were detached, 7 m l of media was added to neutralize the Tripsin. Remaining cells were rinsed with phosphate buffered saline (PBS) by using a pipet. Everything i n the flask was transferred to a 15ml tube and then centrifuged for 3 min- utes at 1000 rpm. After the tube was centrifuged, the liq- uid in the tube was decanted. The cell pellet in the tube was then resuspended by adding 1 0 m l of RPMI media. The cell pellet and the media were pipetted up and down unti l cell pellet was completely broken. 1 X 106 cells was added to each well of two six wells plates in 2 m l RPMI media and then incubated in a 37 oC incubator. P R E P A R I N G S O L U T I O N S Ascorbic acid (vitamin C) stock solution with i M concentra- tion was prepared by adding o . i8g of Ascorbic acid (SIGMA, St Louis, MO) to i m l of PBS (Phosphate Buffer Saline) (GIBCO, Invitrogen, Carlsbad, CA), and then 10II was added to 9 9 0 U I of PBS, to make i o m M solution. Alpha tocopherol solution (vitamin E) (SIGMA, St Louis MO) was prepared next. i M stock solution was made by adding o.43g to i m l of ethanol. 50 I I from theiM stock so- lution was added to 9 5 0 I I of ethanol for concentration of 5 o m M . Again 1011 of the solution was added to 9 o I I of P B S for concentration of 5 m M . A N T I O X I D A N T S A N D GENE R E G U L A T I O N : T H E EFFECTS OF V I T A M I N S C A N D E O N E S T R O N G E N RECEPTORS Estrogen solution was also prepared. 10-3 M stock solution was prepared by adding o.oo3g of 17-, estradiol (SIGMA, St Louis MO) to 1 0 m l of ethanol. r o l l of the solution was then added to 9 9 0 I I PBS for concentration of 10-5 M. C E L L T R E A T M E N T Two six wells plates were treated in the following order. First two wells were untreated as controls. Next two wells were treated with Ascorbic acid. 2 0 I I of i o m M Ascorbic acid solution was added to these wells for final concentra- tion of 1 0 0 I M for each well. Same procedure was repeated for next two wells but 17-beta estradiol solution was added with Ascorbic acid. 2 o i l of the 10-5 M 17-beta estradiol so- lution was added the wells for final concentration of 10-7 M along with 2 0 I I of i o m M Ascorbic acid solution. After treating first plate, second plate was treated with com- binations with alpha tocopherol solution. First two wells were treated with alpha tocopherol solution. 2 0 I I of 5 m M alpha tocopherol solution was added to the well for a final concentration was 5 0 I M for each well. Next two wells were treated with combination of alpha tocopherol and 17-beta estradiol solution. Again 2 0 I I of 5 m M alpha tocopherol so- lution was added to the two wells and then 2 0 I I of the 10-5 M 17-beta estradiol solution was added. For last two wells, Ascorbic acid and alpha tocopherol solutions were added together. 2 o i l of i o m M Ascorbic acid solution was added, and then 2 o i l of 5 m M alpha tocopherol solution was added. These plates were incubated for 24 hours at 39 degrees Celcius. R N A I S O L A T I O N U S I N G R N E A S Y ( R ) P R O T E C T M I N I K I T ( 2 5 0 ) ( Q I A G E N ) After 24 hr. incubation, each type of cells was collected into 15ml Falcon tubes using sterile cell scraper. Tubes were then centrifuged for five minutes at 300-x g. After cen- trifuge was done, supernatant from each tube was dis- carded, and then 6 0 0 II of Buffer RLT was added to each tube to disrupt the cells. When Buffer RLT was added to the tube, it was pipetted up and down several times for homog- enization. After that, 6 0 0 I I of 70 percent ethanol was added to the homogenized lysate of each tube and mixed well by pipetting. 7 0 0 I I of each sample was added to RNeasy m i n i spin column sitting in a 2-ml collection tube (supplied), and centrifuged for 15 seconds at 10 ,000 rpm. Volume of the each sample exceeded 7 0 0 I I , so flow- through of the collection tube was discarded and then rest of the aliquots were added to the same RNeasy column and the each column was centrifuged again. After the cen- trifuge, flow-through was discarded. 7 0 0 I I of Buffer RWi was pipetted onto each of RNeasy column, and columns were centrifuged for 15 seconds at 10 ,000 for washing. After that flow-through and collection tubes were dis- carded. RNeasy column were transferred into a new 2-ml collection tube (supplied). 5 0 0 I I of Buffer RPE were pipet- ted onto the each column, and the columns were cen- trifuged for two minutes at maximum speed to dry the RNeasy membrane. After the centrifuge was done, RNeasy spin column were placed in a new two-ml collection tube, which were not supplied and old collection tube with fil- trate was discarded. RNeasy spin column with new collec- tion tubes were centrifuged at full speed for 1 minute. After this step was completed, each RNeasy column was trans- ferred into a new 1.5-ml collection tube (supplied), and 3 0 I I of RNase-free water was directly added to the each of RNeasy column. Columns were centrifuged for one minute at io ,ooorpm as for final step in collecting mRNA. mRNA from each sample was collected into the tube and the columns were discarded. mRNA sample tubes were immediately placed on ice. R T ( R E V E R S E T R A N S C R I P T I O N ) Optical Density was first founded for each sample of mRNA, for the calculation for making the concentrations of RNA samples to be equal. RNA samples were diluted in RNase-free water in a new 2 0 0 I I tubes. These samples were then denatured at 95 degrees Celcius for one minute. After the denaturation step, to the each tube, following reagents (All from Invitrogen™, Carlsbad, CA) were added: 811 of dNTP's, 4 I I of 5X 1st Strand Buffer, 2 I I of 0.1 M DTT, i l l of Random Primer, i l l Rnase Inhibitor (SIGMA, St Louis, MO). After reagents were added, i l l of enzyme, E L E M E N T S : : S P R I N G 0 6 Super Script™ I I RNase H-Reverse Transcriptase (Invitrogen™, Carlsbad, CA) was added to the each sample. Samples were ran in Thermal Cycler (Gene Amp PCR System 9 7 0 0 , Applied Biosystem) for 6 0 minutes a t 4 0 ° C , and then 10 minutes at 65°C. After RT was done samples were placed in ice. PCR (Polymerase Chain Reaction) To the 2 0 0 I I tubes prepared for the cDNA (RT products), following components (Invitrogen™, Carlsbad, CA) were added for PCR: 5II of 10X PCR buffer, 1.5II of Magnesium Chloride, 2 I I of dNTP's, i l l of Estrogen - -receptor forward primer, i l l reverse primer, 2 9 I I of Rnase free water, i o i l of cDNA. In addition, primers specific for the ,-actin gene was also used as an internal control for the PCR reaction. Sequence of Forward and Reverse Estrogen Alpha Primer: Forward Primer: 5'-ATCCTGATGATTGGTCTCGTCT-3' Reverse Primer: 5'-GGATATGGTCCTTCTCTTCCAGA-3' Sequence of Forward and Reverse Estrogen Beta Primer: Forward Primer: 5'-GCTCATCTTTGCTCCAGATCTTG-3' Reverse Primer: 5'-CAATCACCCAAACCAAAGCATC-3' The samples were denatured at 95°C for 2 minute in a ther- mal cycler (Gene Amp PCR System 9 7 0 0 , Applied Biosystem), and then added 0.5II of Taq DNA polymerase to each tube. The PCR analysis was performed for 35 cycles at 95°C for 30 sec, 55°C for 30 sec, 72°C for 1 min , then as a final primer extension step, 72°C for 10 minutes. The PCR product was analyzed by gel electrophoresis. E L E C T R O P H O R E S I S ig of Agarose was added to a flask then 6 0 m l of iX TBE Buffer solution were added. The flask was then m i - crowaved for about 2 minutes unti l the solution started boil. After the solution cooled down, 5II of Ethidium Bromide was added and mixed well. The gel was then cast onto a horizontal gel electrophoresis apparatus. 45I I of each PCR product was mix with 9 I I of DNA loading buffer, and 3oi l from the mixture was loaded onto the gel. After electrophoresis, the gel was visualized and analyzed by a Gel Documentation system (UVP) containing an ultravio- let transilluminator. NITRIC OXIDE DETERMINATION CELL C U L T U R I N G Same step was performed as for gene expression except that nitric oxide determination was done to the cells in 9 6 wells plates, so 2 0 0 I I from mixture of cells and media were pipetted to the each well. This step was done to 15 wells. CELL T R EAT M ENT First five wells were left as control. To next five wells, 2 I I of Ascorbic acid (Vitamin C) solution with concentration of i o m M was added for the final concentration of i o o l l for each well. To last five wells, 2 I I of Alpha tocopherol (Vitamin E) solution with concentration of 5 m M was added to each well for the final concentration of 5 0 I M . Pre-treated cells were incubated for 24 hours at 39 degrees Celcius. N I T R I C O X I D E D E T E R M I N A T I O N For Nitric Oxide detection, Apollo 4 0 0 0 Free Radical Analyzer manufactured by World Precision Instrument (Sarasota, FL) was used. NO release was detected with an NO-selective microprobe manufactured also by World Precision Instruments (Sarasota, FL). The redox current was detected by a current-voltage converter circuit and con- tinuously recorded. The dimensions of the probe (30 I M di- ameter, 0 . 5 m m length) permitted the use of a micromanip- ulator (Zeiss-Eppendorff). The sensor was placed approximately 100 I M above the respective tissue surface. Calibration of the electrochemical sensor was performed by use of different concentrations ( i o i l , 2 0 I I , 4 0 I I , and 80I I ) of a nitrosothiol donor S-nitroso-N-acetyl-DL-penicil- lamine (SNAP). Each well was placed under the probe and the graph was observed by the computer interfaced DUO- 18 software (World Precision Instruments, Sarasota, FL). When the graph line stabilized, i o i l of 17-, estradiol solu- tion with concentration of 10-5 M was added to the well to stimulate NO release. Amount of NO release was graphed on the monitor and it was measured. RESEARCH PAPER T I T L E FIGURE 1 A FIGURE 1 B GENE EXPRESSION FOR ESTROGEN ALPHA RECEPTORS I N M C F - 7 CELLS Figure i A : The band in the left lane represents the amplified cDNA of ER-alpha genes in MCF-7 cells that were treated with vj-beta estra- diol. The band on the right lane is that of control group of untreated cells. The brightness of the band on the right lane (control group) is slightly brighter than brightness of the band on the left lane (17-beta) estradiol, suggesting that vj-beta, estradiol slightly down-regu- lated the gene expression. Figure iB : Expression levels for beta-actin primer remained constant for two samples in Figure iA, which verified equivalent sample load- ing. Figure 2A: The band on the left represents the amplified cDNA of HR-alpha genes in a group of MCF-7 cells that were treated only with ascorbic acid, and the band on the right lane represents amplified cDNA(ER-alpha) of the group treated in a combination of vj-beta estra- diol and ascorbic acid. The brightness of the band on the right lane (a group treated with the combination) is shown to be brighter than the brightness of the band in the left lane (a group treated with ascorbic acid), which means that more cDNA was amplified in the group treated with the combination. Also when the two bands in Figure 2A are compared to the control band in Figure iA, the control group of Figure 1 is brighter than the left lane of Figure 2 (ascorbic acid) but dimmer than the right lane of Figure 2 A (17-beta estradiol and ascor- bic acid). This indicates that ascorbic acid alone resulted in a down-regulation (less cDNA were amplified) of ER-alpha gene expression and the combination of ascorbic acid and iy-beta estradiol resulted in an up-regulation. Figure 2B: Expression levels for beta-actin primer remained constant for two samples in Figure 2A, which verified equivalent sample loading. G E N E E X P R E S S I O N PCR products were analyzed on a two percent Agarose gel and a gel documentation system containing a UV transillu- minator (UVP) (Figure 1-4). Brightness of the bands from the results of beta-actin gene expressions (Figure iB, 2B, 5B) verified equivalent sample loading. Figures iA and 2A below show bands that represent the amplified amount of cDNA for ER-alpha genes. The brighter the band is, the more amplified cDNA is present. Since the cDNA was gen- erated by a Reverse Transcription of mRNA, the brighter the band, the greater level of the expression of the ER-alpha gene that produced the mRNA. The right lane i n Figure iA shows the bands of amplified cDNA of a control group of untreated MCF-7 cells (right lane), and the experimental group treated with ly-beta estradiol (left lane). A slight dif- ference in the brightness of the two bands can be noticed. It appears that the control group band is brighter than the lj-beta estradiol treated group, which means that the ER- alpha gene had diminished expression i n the group treated with estradiol. In other words rj-bcta estradiol appears to have down-regulated the gene. Figure 2A shows the band of the group treated with ascorbic acid (left lane), and the band of the group treated with the combination of ascorbic acid and iy-beta estradiol (right lane). When the left lane in Figure 2A (ascorbic acid treatment) is compared to the right lane of Figure iA (control), the control again appears brighter, suggesting that ascorbic acid also down-regulates the expression of the ER-alpha gene. The right lane (ascor- bic acid and iy-beta estradiol treated) is brighter than both the left lane (ascorbic acid treatment) of Figure 2A and the right lane (control) of Figure iA, suggesting that when ascorbic acid is i n a combination with estrogen, it can up- regulate the ER-alpha gene. I n the RT-PCR for estrogen-beta receptors, it was difficult to compare the expression levels because the bands (shown i n Figures 3, and 4) were so faint. A possible explanation for such a low expression level might be because in MCF-7 cells, the alpha-receptor is the prevalent form and the beta- receptor is hardly detectable. x v m It appears that the anti-proliferative effect of ascorbic acid on MCF-7 c e u s reported in the literature x l x is related to the down-regulation of estrogen receptor gene expression in MCF-7 c e u s treated with ascorbic acid. There was also a slight down-regulation in the gene expression level in the group treated with rj-beta estradiol and it seems that this down regulation was caused by a negative feedback due to the addition of iy-beta estradiol. Previous work found that when estrogen is added to the melatonin, an antioxidant, the anti-proliferative effect of melatonin was reversed. Similarly in this research, it ap- pears that the down-regulating effect on the ER alpha-re- V I T A M I N E Nitric Oxide Determination Average amount of nitric oxide released for the con- trol group was 0.4 nM. Two groups treated with vi- tamin C and E released more NO than the control group but overall vitamin E caused more increase in NO release than vitamin C A N T I O X I D A N T S A N D GENE R E G U L A T I O N : T H E EFFECTS OF V I T A M I N S C A N D E O N E S T R O N G E N RECEPTORS FIGURE 4 FIGURE 5B FIGURE 5B GENE EXPRESSION FOR ESTROGEN BETA RECEPTORS I N M C F - 7 CELLS Figures 3 and 4: Possibly due to the fact that estrogen alpha-receptors are the prevalent form in MCF-7 cells and that beta-receptors are hardly detectable, a minimal amount of cDNA for beta receptors were amplified. This caused the brightness of the bands visualized on the gel to be very faint. No conclusions could be drawn. GENE EXPRESSION FOR ESTROGEN ALPHA RECEPTORS I N M C F - 7 CELLS Figure 5A: From left: control group (untreated), alpha tocopheral (vitamin E) treated group, and the group treated in a combination of 17- beta estradiol and alpha tocopheral. No significant difference in the brightness of the bands of control group and vitamin E treated group could be found, but when third band (vitamin E and ly-beta estradiol) is compared to other two bands, the third band is less bright. The combination of alpha tocopheral and estrogen appears to have down-regulated ER-alpha gene expression level. Figure 5B: Expression levels for beta-actin primer remained constant for all three samples, which verified equivalent sample loading. FIGURE Nitric Oxide Determination A direct comparison of the contol group and vitamir E group. !_ 7. S 6- ceptor gene caused by ascorbic acid was counteracted by the up-regulating addition of vy-beta estradiol. Figure 5A, shows by contrast that i n the alpha tocopheral (vitamin E) treatment, alpha tocopheral alone (middle lane) did not cause much change in the gene expression level of estrogen a^fca-receptors when compared to the control group (first lane from left). However, the band from the combination of alpha tocopheral and ly-beta estradiol (third lane from left) is dimmer and therefore the combina- tion of alpha tocopheral and xy-beta estradiol caused down- regulation in the gene expression level of ER-alpha. It ap- pears that alpha tocopheral and ascorbic acid have opposite effects when combined with the addition of iy-beta estra- diol. N I T R I C O X I D E D E T E R M I N A T I O N A total of 15 trials were performed for nitric oxide determi- nation. Figure 6 shows the bar graph of the average amounts of NO release for three groups of MCF-7 cells with a different treatment for each group. Overall, compared to the control group, both vitamin C and E increased the level of nitric oxide release. However, it ap- pears that vitamin E was more effective in boosting NO re- lease. Figure 7 shows that this is true i n a direct compari- son of the control and vitamine E. As mentioned in the in- troduction, an excessive amount of NO may have negative effects, but the amount of NO increased by vitamin E was not in a reach to be considered as an excessive level. Since NO is a scavenger for other free radicals that may promote a cancer, it appears that vitamin E may has positive effects in l imit ing cancer development by increasing NO release. F U T H E R RESEARCH More studies regarding antioxidants and estrogen recep- tors wi l l have to be done. I n the future, Real Time PCR may be conducted, which is the type of PCR that gives the exact quantity of amplified cDNAs. By getting an exact quantity through conducting Real Time PCR, it would be possible to get clearer data. More trials can be conducted in addition. Also in the future, a variety of other antioxidants other than vitamin C and E wi l l be used for similar types of experi- ments. C O N C L U S I O N Although further studies wi l l have to be done, this study demonstrated the overall effect of ascorbic acid (vitamin C), alpha tocopheral (vitamin E) and their combination with 17- beta estradiol on ER-alpha gene expression. This study A N T I O X I D A N T S A N D GENE R E G U L A T I O N : T H E EFFECTS OF V I T A M I N S C A N D E O N E S T R O N G E N RECEPTORS demonstrated that ascorbic acid might have positive effects in l imiting the spread of breast cancer by decreasing the es- trogen receptor level. However, this study also revealed that the combination of estrogen and ascorbic acid could cause up-regulation of ER-alpha gene expression, which may be a possible explanation for the phenomena of greater prolifer- ation that occurred i n the previous studies. It can be sus- pected that the combination of estrogen and ascorbic acid can make cells more vulnerable and may accelerate the MCF-7 c e u growth due to the increased level of estrogen re- ceptors. I n contrast to ascorbic acid (vitamin C), alpha tocopheral (vitamin E) caused a down-regulation of ER-alpha gene ex- pression level when the cells were treated in combination with ij-beta estradiol. Acting alone, alpha tocopheral showed no effect on regulation of this gene. This study also demonstrated the effect of antioxidants on the release of nitric oxide on the surface estrogen receptor of MCF-7 cells. This study showed that the both antioxi- dants increased NO level but vitamin E was more effective than vitamin C, suggesting that vitamin E may have posi- tive effects in preventing cancer. Vitamin C and Vitamin E appears to be working by different mechanisms since vita- m i n E cause a significant release of NO, whereas vitamin C caused a significant decrease of ER-alpha gene expression i n the MCF-7 human breast cancer cells. Overall, the data from this study wi l l help further understand how antioxi- dants work in the prevention of cancers and other type of diseases. E N D N O T E S i . Refernce 1 i i . Refernce 2 i i i . Refernce 2 iv. Refernce 2 v. Refernce 3 v i . Refernces 4-8 vi i . Refernce 2 v i i i . Refernces 9-10 ix. Refernces 11-12 x. Refernces 8-18 xi . Refernces 19-21 x i i . Refernce 22 x i i i . Refernce 23 xiv. Refernces 23-27 xv. Refernces 28-31 xvi. Refernces 32-33 xvii . Refernce 34 xvii i . Refernces 11-12 xix. Refernce 35 R E F E R E N C E S 1. Cheryl Cardinez, MSPH, Vilma Cokkinides, PhD, Ted Gansler, M D , Robert Greenlee, PhD, M P H , Apr i l Harris, Mary Beth H i l l Harmon, MSPH, Taylor Murray, Debbie Saslow, PhD, K i m Andrew Sawyer, Joann Schellenbach, Robert Smith, PhD, Susan Summers, Dawn Willis , PhD, M P H , Harriet Zoller: Breast Cancer Facts and Figures 2001-2002 American Cancer Society, Atlanta Georgia 2. Lewis J. Kleinsmith Ph.D, Donna Kerrigan M.S., Jeanne Kelly Understanding Estrogen Receptors, Tamoxifen, and Raloxifene: Science Behind News, National Cancer Institute: http://press2.nci.nih.g0v/sciencebehind/estrogen/estro- genoo.htm 3. Peter Collins & Carolyn Webb "Estrogen hits the surface" Nature Medicine Volume 5 1999 Nature America Inc. 4. Hafner F, Holler E, von Angerer E. Effect of growth factors on estrogen receptor mediated gene expression. J Steroid Biochem Mol Biol. 1996 Jul;58(4):385-93. 5. Weichselbaum RR, Hellman S, Piro AJ, Nove JJ, Little JB. Proliferation kinetics of a human breast cancer line i n vitro fol- lowing treatment w i t h iybeta-estradiol and i-beta-D-arabinofura- nosylcytosine. Cancer Res. 1978 Aug;38(8):2339-42. 6. Schor NF, Tyurina YY, Tyurin VA, Kagan VE. Differential membrane antioxidant effects of immediate and long-term estra- diol treatment of MCF-7 breast cancer cells. Biochem Biophys Res Commun. 1999 Jul 5;26o(2):4io- 7. Tesarik J, Garrigosa L, Mendoza C. Estradiol modulates breast cancer cell apoptosis: a novel nongenomic steroid action relevant E L E M E N T S S P R I N G O 6 http://press2.nci.nih.g0v/sciencebehind/estrogen/estro- to carcinogenesis. Steroids. 1999 Jan-Feb;64(i-2):22-7. 8. Perillo B, Sasso A, Abbondanza C, Palumbo G. tybeta-estra- diol inhibits apoptosis i n MCF-7 cells, inducing bcl-2 expression via two estrogen-responsive elements present i n the coding se- quence. Mol Cell Biol. 2000 Apr ;2o(8):2890-90i. 9. Prichard RS, H i l l A D , Dijkstra B, McDermott EW, O'Higgins NJ. : The prevention of breast cancer. Br J Surg. 2003 Jul;9o(7)772-83. 10. Wakeling AE, Newboult E, Peters SW: Effects of anioestro- gens on the proliferation of MCF-7 human breast cancer cells J Mol Endocrinol. 1989 May;2(3):225-34 11. Pace, P., J. Taylor, S. Suntharalingam, R. C. Coombes, and S. A l i . 1997. Human estrogen receptor binds i n a manner similar to and dimerises wi th estrogen receptor . J. Biol. Chem. 272:25832-25838 12. Shanmugam, M . , N . L. Krett, E. T. Maizels, R. E. Cutler, Jr., C. A. Peters, L. M . Smith, M . L. O'Brien, O. K. Park-Sarge, S. T. Rosen, and M . Hunzicker-Dunn. 1999. Regulation of protein k i - nase C delta by estrogen i n the MCF-7 human breast cancer cell line. Mol. Cell. Endocrinol. 148:109-118 13. Karasek M , Pawlikowski M : Antiproliferative effects of mela- tonin and CGP52608 Biol Signals Recept. 1999 Jan-Apr;8(i- 2)75-8 14. Cos S, Mediavilla M D , Fernandez R, Gonzalez-Lamuno D, Sanchez-Barcelo EJ: Does melatonin induce apoptosis i n MCF-7 human breast cancer cells i n vitro? J Pineal Res. 2002 Mar;32(2):90-6. 15. Blask DE, H i l l SM: Effects of melatonin on cancer: studies on MCF-7 human breast cancer cells i n culture. J Neural Transm Suppl. 1986;21:433-49 16. Cos S, Blask DE. Melatonin modulates growth factor activity i n MCF-7 human breast cancer cells: J Pineal Res. 1994 Aug;i7(i):25-32 17. Cos S, Sanchez-Barcelo EJ: Melatonin, experimental basis for a possible application i n breast cancer prevention and treatment. Histol Histopathol. 2000 Apr;i5(2):637"47 18. H i l l SM, Spriggs LL, Simon MA, Muraoka H , Blask DE. The growth inhibitory action of melatonin on human breast cancer cells is l inked to the estrogen response system. Cancer Lett. 1992 Jul io;6493):249-56. 19. Crespo D, Fernandez-Viadero C, Verduga R, Ovejero V, Cos S: Interaction between melatonin and estradiol on morphological and morphometric features of MCF-7 human breast cancer cells. J Pineal Res. 1994 May;i6(4):2i5-22 20. Cos S, Sanchez-Barcelo EJ: Melatonin inhibi t ion of MCF-7 human breast-cancer cells growth: influence of cell proliferation rate: Cancer Lett. 1995 Jul i3;93(2):207-i2 21. Cos S, Recio J, Sanchez-Barcelo EJ: Modulation of the length of the cell cycle time of MCF-7 human breast cancer cells by melatonin: Life Sci. i996;58(9):8n-6 22. Tina M . Molis, Louaine L. Spriggs, Steven M . H i l l : Modulation of estrogen receptor mRNA expression by melatonin i n MCF-7 human breast cancer cells. Molecular Endocrinology 8:1681-1690,1994 23. Ng JH, Nesaretnam K, Reimann K, Lai LC. Effect of retinoic acid and palm oil carotenoids on oestrone sulphatase and oestra- diol-i7beta hydroxysteroid dehydrogenase activities i n MCF-7 and MDA-MB-231 breast cancer cell lines. Int J Cancer. 2000 Octi ;88(i):i35-8 24. Prakash P, Russell RM, Krinsky N I . I n vitro inhibi t ion of pro- liferation of estrogen-dependent and estrogen-independent human breast cancer cells treated wi t h carotenoids or retinoids. J Nutr. 2001 May;i3i(5):i574-8o. 25. Nesaretnam K, Jin L i m E, Reimann K, Lai LC. Effect of a carotene concentrate on the growth of human breast cancer cells A N T I O X I D A N T S A N D GENE R E G U L A T I O N : T H E EFFECTS OF V I T A M I N S C A N D E O N E S T R O N G E N RECEPTORS and pS2 gene expression. Toxicology. 2000 Oct 26;i5i(i-3):ii7- 26. 26. Li Z, Wang Y, Mo B. [The effects of carotenoids on the prolif- eration of human breast cancer cell and gene expression of bcl-2] Zhonghua Yu Fang Yi Xue Za Z h i . 2002 }ul;36(4):254-7. 27. Lai LC. Role of steroid hormones and growth factors i n breast cancer: Clin Chem Lab Med. 2002 Oct;40 (io):96c)-74 28. Watrach A M , Milner JA, Watrach MA, Poirier KA. Inhibit ion of human breast cancer cells by selenium. Cancer Lett. 1984 Nov;25(i):4i-7. 29. Guthrie N , Gapor A, Chambers AF, Carroll KK. Inhibi t ion of proliferation of estrogen receptor-negative MDA-MB-435 and - positive MCF-7 human breast cancer cells by palm oil to- cotrienols and tamoxifen, alone and i n combination. J Nutr. 1997 Mar;i27(3):544S-548S. 30. Malafa MP, Neitzel LT. Vitamin E succinate promotes breast cancer tumor dormancy. J Surg Res. 2000 Sep;93(i):i63-70. 31. Heisler T, Towfigh S, Simon N, McFadden DW. Peptide YY and vitamin E inhibit hormone-sensitive and -insensitive breast cancer cells. J Surg Res. 2000 Jun i ; 9 i ( i ) : 9 - i 4 . 32. Stefano GB, Cadet P, Mantione K, Cho JJ, Jones D, Z h u W. Estrogen signaling at the cell surface coupled to nitric oxide re- lease i n Mytilus edulis nervous system. Endocrinology. 2003 Apr ;i44(4):i234-40 33. Stefano GB, Prevot V, Beauvillain JC, Fimiani C, Welters I , Cadet P, Breton C, Pestel J, Salzet M , Bilfinger TV. Estradiol cou- pling to human monocyte nitric oxide release is dependent on in- tracellular calcium transients: evidence for an estrogen surface receptor. J I m m u n o l . 1999 Oct i;i63(7):3758-63- 34. Lirk P, Hoffmann G, Rieder J. Inducible nitric oxide syn- thase-time for reappraisal. Curr Drug Targets In f lamm Allergy. 2002 Mar ; i ( i) :89- io8. 35. Noto V, Taper HS, Jiang Y H , Janssens J, Bonte J, De Loecker W. Effects of sodium ascorbate (vitamin C) and 2-methyl-i,4- naphthoquinone (vitamin K3) treatment on human tumor cell growth i n vitro. I . Synergism of combined vitamin C and K3 ac- t ion. Cancer. 1989 Mar i ;63(5):90i-6. E L E M E N T S S P R I N G 06