Effects of Two Isolated Compounds from Red Sorghum Bran on p53 and bcl 2 Gene Expression in Human Breast cancer Cell Line (MCF-7)

Aims: The aim of our research was to isolate anthocyanin from red sorghum bran and to study its effects on p 53 and bcl 2 gene expressions in Human Breast cancer cell line (MCF-7). Main methods: Two compounds were isolated from red sorghum bran by using Sephadex LH-20 column chromatography. The antiproliferative activity of isolated compounds were analysed by MTT assay and the expression studies were performed by RT-PCR analysis. Key ndings: Our research focusing on apigenin-5-β-D-glucopyranoside and luteolin-5-β-D-glucopyranoside another compound as a potention anticancer agent have demonstrated that the compounds isolated from red sorghum bran inhibited breast cancer cell lines by up regulation of P 53 gene expression and down regulation of Bcl 2 gene expression. Signicance: In conclusion, the two compounds isolated from red sorghum bran, were effective in up regulation of P 53 and down regulation of Bcl 2 gene expression.


Introduction
Consumption of fruits and vegetables that are adundant in avonoids has been associated with reduced incidence of many form of cancer (1,2). These agents are universally present in fruits, vegetables, grains, nuts, tea and fruits. Anthocyanins occur ubiquitously in the plant kingdom and confer the bright red, blue and purple colors to fruits and vegatables. Pure anthocyanins and anthocyanin -rich extracts from fruits and vegatables have exhibited anti-proliferative activity towards multiple cancer cell types in vitro. One of these promising properties is the perturbation of the cell -cycle progression. Research in our laboratory has been focused on apigenin-5-β-D-glucopyranoside (3). Apigenin-5-β-D-glucopyranoside is a avones that is widely distributed in many fruits, vegetables and grains. The mechanism(s) for the selective effect of the anthocyanins on the growth of cancer cell against normal cells is/are not known. Although anthocyanins have attracted much attention for their possible bene ts, little is known regarding their anti -cancer action.
In the present study, the antiproliferative activity of puri ed anthocyanin compounds isolated from red sorghum bran and its effects on p53 and bcl-2 gene expressions in Human Breast cancer cell line (MCF-7) was determined.

Sample extraction
The bran of red sorghum (200g) was extracted by incubation with 1% Hydrochloric acid in methanol. Overnight at room temperature, followed by a ltration through whatman lter paper no.4. Methanol was removed by a rotary evaporation under 35 C and the pigmented fraction extracts were stored for a further study.

Isolation of anthocyanin
The concentrated ltrates were then applied onto an amperlite XAD -7 resin column (Sigma Aldrich, Missouri, USA) (1.5 x 40 cm), and washed with distilled water, followed by an elution with 1% Tri uoroacetic acid in methanol (4,5). The elution was evaporated again after being collected by a fraction collector. Isolated anthocyanins were analysed on a UV-Visible spectrophotometer (Genesys 5 Technical trade links pvt.ltd). The fractions with the highest absorbance at 480nm were pooled and evaporated to remove methanol and dried under vaccum at 35 C.
2.4 Puri cation of Anthocyanin 5 ml of concentrated isolated methanolic pigments were fractioned by Sephadex LH-20 column (100 cm x 2.6 cm, sigma, USA) using water(0.1% TFA)-Methanol(40:60, v/v) as eluent (4,5). The ow rate was 0.3 mL/min. Fractions were collected by a fraction collector. The fractions were evaporated on a rotary evaporator to remove methanol to facilitate the removal of water remaining in the sample. The evaporation temperature was less than 38 C. The homogeneity of individual fractions was checked by HPLC analysis. The lyophilized sample was used for expression studies.

High -Performance Liquid Chromatography
The anthocyanin samples was diluted accordingly with methanol and HPLC analysis was carried out using a liquid chromatography system (Shimadzu, Kyoto, Japan) with a Photodidode array detector. The Detection was carried at 480 nm. The ow rate was maintained at 1.0ml/min. A Reverse Phase C 18 column having a particle size of 5.0 μm was used for HPLC analysis of anthocyanins. The sample injection was 20 μl. A gradient mobile phase was used for elution : elution A was 10% Formic acid in water and B was Acetonitrile/water/formic acid ( 5:4:1). Apigenin-5-β-D-glucopyranoside was used as a standard.

preparation of Test Solutions
For cytotoxicity studies, each weighed test drugs were separately dissolved in distilled DMSO and volume was made up with DMEM supplemented with 2% inactivated FBS to obtain a stock solution of 1 mg/ml concentration and sterilized by ltration. Serial two fold dilutions were prepared from this for carrying out cytotoxic studies.

Determination of cell viability by MTT Assay
Principle: The ability of the cells to survive a toxic insult has been the basis of most cytotoxicity assays. This assay is based on the assumption that dead cells or their products do not reduce tetrazolium. The assay depends both on the number of cells present and on the mitochondrial activity per cell. The principle involved is the cleavage of tetrazolium salt 3-(4, 5 dimethyl thiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) into a blue coloured product (formazan) by mitochondrial enzyme succinate dehydrogenase. The number of cells was found to be proportional to the extent of formazan production by the cells used Francis and Rita, (1986).
Procedure:The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 x 10 5 cells/ml using DMEM containing 10% FBS. To each well of the 96 well microtitre plate, 0.1 ml of the diluted cell suspension (approximately 10,000 cells) was added. After 24 h, when a partial monolayer was formed, the supernatant was icked off, washed the monolayer once with medium and 100 ml of different test concentrations of test drugs were added on to the partial monolayer in microtitre plates. The plates were then incubated at 37 o C for 3 days in 5% CO 2 atmosphere, and microscopic examination was carried out and observations were noted every 24 h interval. After 72 h, the drug solutions in the wells were discarded and 50 ml of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 h at 37 o C in 5% CO 2 atmosphere. The supernatant was removed and 100 ml of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (CTC 50 ) values is generated from the dose-response curves for each cell line.
Page 5/10 1 X 10 6 MCF-7 cells were plated per well of 6 well tissue culture plate and incubated at 37 o C/5% CO 2 overnight. These cells were treated with 20 mg/ ml of the test sample dissolved in DMEM containing 10% FBS. As a negative control DMEM containing 10% FBS was used and as a positive control cells were treated with 5µg/ml Doxorubicin hydrochloride. Plates were incubated for further 48 hours. Total RNA was isolated from these cells and mRNA expression levels of p53 and BCl-2 were carried out using semi quantitative reverse transcriptase polymerase chain reaction (RT-PCR).

1.Isolation and Puri cation
In red sorghum bran, many components such as sugar, acid and salts occur except for anthocyanins. To better isolate anthocyanins from the red sorghum bran, the crude extract generally was passed through Amberlite XAD -7 resin column exhibited a high spectral characteristics. The isolated anthocyanins were further puri ed through sephadex LH 20 column and two compounds were isolated from red sorghum bran anthocyanin. The isolated two compounds luteolin-5-β-D-glucopyranoside and apigenin-5-β-Dglucopyranoside-5-β-D-glucopyranoside was taken for invitro cytotoxicity assay and expression studies in MCF -7 cell line.

Hplc Analysis
The puri ed anthocyanins were checked for homogeneity with the standard apigenin-5-β-Dglucopyranoside. The second fraction matches with the standard apigenin-5-β-D-glucopyranoside with the same retention time. (Figs. 1 & 2). The rst fraction doesn't match with the standard. (Fig. 3) Thus the data indicate that two compounds was able to isolate from red sorghum bran by the combination of Amberlite XAD − 7 and Sephadex LH -20 column chromatography.

2.invitro Cytotoxity Assay
There was no signi cant cytotoxicity observed even at 20 mg/ml concentration by MTT assay. This could be due to the presence of color to the sample which interferes with the reading at 570 nm. 3.expression Of P Gene In Mcf-7 Cell Line P 53 protein is essential for check point control that arrests Human cells with damaged DNA in G1 phase of cell cycle. P 53 also leads to induction of proteins that promotes apoptosis.
In this study of P 53 gene expression, the MCF-7 cells were treated with 20mg/ml of apigenin-5-β-Dglucopyranoside and luteolin-5-β-D-glucopyranoside isolated from red sorghum bran. Our results indicated that the expression of P 53 increased after one hr incubation, and were maintained at a high level over 48 hr of treatment with 20 mg/ml of apigenin-5-β-D-glucopyranoside and luteolin-5-β-Dglucopyranoside isolated from red sorghum bran.
Expression of P 53 gene was investigated by RT-PCR in untreated and treated MCF-7 cells. Quantitation of each band was performed by densitometry analysis software and the results expressed as the ratio of P 53 /β -actin in percent to the medium (control). Doxorudicin hydrochloride (DOX) was used positive control. As shown in Fig. 4 and table 1 in comparision to the medium control, the P 53 gene expression is increased in the presence of apigenin-5-β-D-glucopyranoside and luteolin-5-β-D-glucopyranoside.

4.expression Of Bcl Gene In Mcf-7 Cell Line
Bcl 2 protein function is to suppress apoptosis or promotes cell survival. In our study Bcl 2 gene expression, MCF -7 cells were treated with 20 mg/ml.
In this study of Bcl 2 gene expression, the MCF-7 cells were treated with 20mg/ml of apigenin-5-β-Dglucopyranoside and luteolin-5-β-D-glucopyranoside isolated from red sorghum bran. Our results indicated that the expression of Bcl 2 markedly increased after one hr incubation, and were maintained at a high level over 48 hr of treatment with 20 mg/ml of apigenin-5-β-D-glucopyranoside and luteolin-5-β-Dglucopyranoside isolated from red sorghum bran.
The expression of p53 and bcl-2 gene in MCF-7 cells was rst determined by RT-PCR after incubation with luteolin-5-β-D-glucopyranoside and apigenin-5-β-D-glucopyranoside. An increase in p53 gene expression was observed after incubation with luteolin-5-β-D-glucopyranoside and apigenin-5-β-D-glucopyranoside when compared with the control. In the case of effect of bcl-2 gene expression on MCF-7 cells reduced bcl-2 gene expression was observed after incubation with luteolin-5-β-D-glucopyranoside and apigenin-5β-D-glucopyranoside. The relative gene expression level; REL was normalized and compared to the expression of β-actin (Fig. 5).
The mechanisms by which apigenin-5-β-D-glucopyranoside protected against skin carcinogensis in animal models seem to be related to the induction of cell cycle arrest at the G2/M and /or Go/G1 phase (11,12). Recently we demonstrated that apigenin-5-β-D-glucopyranoside inhibited the growth of three Human colon carcinoma cell lines in association with a reversible G2/M cell -cycle arrest (13). That arrest was associated with decreased activity of P 34 cdc2 kinase and reduced accumulation of P 34 cdc2 and cyclin B1 proteins (14).

Conclusion
Flavonoids are a major class of non nutritive components of plant -derived diets, and they are widely distributed in plant -based foods and have been suggested to play a role in cancer prevention. Current studies in our laboratory focusing on apigenin-5-β-D-glucopyranoside and luteolin-5-β-D-glucopyranoside as a potention anticancer agent have demonstrated that the compounds isolated from red sorghum bran inhibited breast cancer cell lines by upregulation of P 53 gene expression and down regulation of Bcl 2 gene expression. Among the two compounds isolated from red sorghum bran, the apigenin-5-β-Dglucopyranoside was more effective in upregualtion of P 53 and down regulation of Bcl 2 gene expression. Tables Table 1 Showing the quantitative analysis of RT PCR