OVER EXPRESSION OF CYCLOOXYGENASE 2 DETECTED IN MCF-7 BREAST CANCER CELL AND COMPARED WITH LUNG CARCINOMA CELLLINE (A549)

In advanced studies Cyclooxygenase-2 (COX-2) mRNA was determined mostly by in situ hybridization or Northern Blot analysis-methods not suitable for absolute quantification of mRNA copy numbers. Here we reported that, over expression of COX-2 mRNA was observed and calibrated through highly sensitive externally standardized real-time RT-PCR with Light Cycler instrument. The total RNA was isolated from two breast cancer cell lines (MCF-7, ZR-75-1), one lung cancer cell line (A549) and one normal breast cell line (HBL-100). The presence of COX-2 mRNA copy numbers was determined in all cell lines thourgh Reverse Transcription PCR by cDNA conversion method. In this study, we observed breast cancer cell lines and lung cancer cell lines were showing high expression levels of COX-2 mRNA at the same time there was the lowest expression was detected in normal breast cells. This low levels of COX 2 expression due to the tumorogenic action of a normal cells. Thus we evaluated COX-2 expression at different levels in breast and lung carcinoma cell lines. Results of our study provide insight view to the involvement of different carcinoma cells in pathogenesis with respect to COX-2 mRNA expression. This Light Cycler technology is currently considered to be  the most precise method for nucleic acid quantification and  which showed to be a powerful tool for further expression studies on cancer gene  pathogenesis.


INTRODUCTION
Cyclooxygenase (COX) is the critical enzyme involved in modulating inflammatory response through the synthesis of prostaglandins 1 . The inducible isoform of the enzyme, COX-2, is over expressed in several malignant and premalignant lesions 1 . Several epidemiological studies also be linked induction as well as over-expression of the cyclooxygenase-2 (COX-2) gene to cancer of the colon 2 esophagus, lung, prostate, skin, bladder and breast 3 . The

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www.ijbr.ssjournals.com cyclooxygenase (COX) genes are involved in the conversion of glycerophosphoric acid into arachidonic acid through COX pathway and the enzyme exists in two isoforms like, COX-1 and COX-2. COX-1 is an enzyme appears to be a responsible for mediating the production of prostaglandins (PGE 2 ). The isoenzyme COX-2 is primarily associated with inflammatory activity and tumorogenic process 3, 4 . Cytokines and growth factors increases the expression of COX-2, mainly at inflammatory sites, producing prostaglandins and resulting in inflammation, pain and fever 5 . Oncogenes and tumour promoters were also reported in associated with the inflammatory response 4 . Inhibition of the expression of the cyclooxygenase-2 (COX-2) enzyme has been associated with prevention or reversal of cancer development in several organs. 5 Higher COX-2 expression correlates with a poor clinical outcome in various cancers 5, 6 and selective inhibition of COX-2 suppresses tumour growth 7. An increased levels of COX-2 expression has been detected in adenocarcinomas (ADC) and squamous cell carcinomas (SQCC) 5, 7 in comparison with normal lung tissue; however, higher levels were observed in SQCC. COX-2 inhibitors are currently being tested in ongoing phase II studies for the chemoprevention of tumor suppression and limiting COX 2 expression at cellular level, based on preclinical studies in mice 12 . This study is mainly focused to investigate cyclooxygenase-2 (COX-2) mRNA expression in breast, lung when compared with normal cell line like HBL-100. For this study we used Real Time PCR method and we have isolated mRNA from the cancer cells. By making cDNA conversion with the use of reverse transcriptase through reverse transcription PCR method. These cDNA copies were amplified by using specific COX 2 primers, SYBR Green used as detector for the expression amplification of these reactions. SYBR-Green binding that is a quick, reliable, easily optimized and compares well with the other assays. This demonstrates its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy 25,26 . To investigate expression of Cyclooxygenase-2 (COX-2) in cancer cell lines, RT-PCR and western blot analysis were used. Western blot is one of the most specific methods for expression of COX 2 in all cancer cell lines. It is suggested that COX-2 is expressed in all cancer cell lines, which provides a basis for the chemoprevention of cancer 26 . Here, we reported, a new approach for rapid semi-quantitative multiplex RT-PCR method to study and COX-2 genes relative to a housekeeping gene (β-actin) as a means of analyzing gene expression. With this method, it is possible to rapidly analyze the effect of different expression patterns on Cyclooxygenase genes in multiple cell lines. In this study, multiplex RT-PCR was used to analyze expression of Cyclooxygenase 2 gene expressions in cells derived from breast adenocarcinoma, lung carcinoma and in normal breast cell lines as control for this study. The results were analyzed using the Applied Biosystems software developed by manufacturers. It will be more supportive and highly sensitive

CELL CULTURE MAINTENANCE
Two breast cancer cell lines, MCF-7 and ZR-75-1,One lung cancer cell line, A549 and normal breast cell line HBL-100 were purchased from NCCS, Pune, India MCF-7 and A549 were maintained in Dulbecco's Modified Eagles medium supplemented with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L Na bicarbonate , 0.1 mM nonessential amino acids, and 1.0 mM of Na pyruate. ZR-75-1 cell was maintained in RPMI 1640 Medium with 2mM Lglutamine.1.5 g/L Na bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM Na pyruate, and HBL-100 cell was maintained in McCoys 5A Medium, (HiMedia, Inc) with 2mM L-Glutamine adjusted to contain 1.5 g/L Glucose, 10mM HEPES and 1.0 mM Na pyruate and supplemented with 0.2 Units/ml calf insulin respectively with 10% fetal bovine serum (GIBCO,USA). Penicillin and Streptomycin (100IU/100µg) was adjusted 1 ml per liter. Cells were maintained at 37 0 C with 5% CO 2 atmosphere.

CELL VIABILITY
Cell viability was assessed by trypan blue dye exclusion test as reported by Chakraborty et al. (2004). The number of stained and unstained will be counted using haemocytometer. Cell viability was analyzed through 0.4 % Trypan blue dye exclusion, which was always greater than 95%. All cells were showed a very good number of viable cells Figure 1.

RNA EXTRACTION
Cells with logarithmic growth phase were seeded in the 25×cm 2 flask. Approximately 5x 10 6 cells were used for total RNA extraction. RNA was extracted with commercially available RNA preparation kit BIO BASIC INC Canada, as per manufacturer's protocol and extracted RNA was stored in -80°C for over night 26 .

REVERSE TRANSCRIPTION PCR
Extracted total RNA was reversetranscribed 29 to cDNA using dNTP's 2 µl, random primers. Brifely 1000ng of RNA, 200 units of Reverse Transcriptase (25 Units/µl)1µl (Promega; USA), RT buffer 2 µl, template was about 10 µl added and remaining part is nuclease free water mixed with making final volume of 20 µl reactions at 25°c for 10 min, 42°c for 45 min and 95°c for 3 min with only one repeated steps. Measurement of nucleic acid concentrations were done at OD 260 nm on spectrophotometer (BioPhotometer Eppendorf; Germany).

IJBR2[5][2011]320-333
www.ijbr.ssjournals.com Real Time PCR (RT-PCR) was performed using specific primers for both COX 2 and β-actin with SYBR Green (Applied Biosystems) dye as a target detector molecule. Briefly 12.5 µl, Reverse transcriptase (25 Units/µl) 1.0 µl and forward and reverse primers for COX-2 and β-actin 1.0 µl and remaining part was nuclease free water. 5µl of quantified template RNA added to make the final reaction volume to 25µl. The primer sequence for COX-2 and β actin is given in table 1. immersed was about over night. After that overnight incubated membrane gels were detected by UV Trans illuminator. The Exposed blot was kept to film for 15 sec -5 min.

DETECTION OF COX 2 AND β ACTIN GENE
The reverse transcripted cDNA samples were amplified by using specific primers for the evalution of COX 2 and β actin genes, it was showed the visualized bands of both the genes in all celllines based on their concentration of COX 2 and β actin cDNA levels. This indicated to us to proceed Real time PCR study Fig 2 and 3.
The cDNA concentrations were measured by using spectrophotometer (BioPhotometer Eppendorf; Germany).

QUANTIFICATION OF COX 2 AND β ACTIN
COX 2 mRNA expression was significantly higher in breast cancer cell line (MCF 7), followed by A549, ZR-75-1 and finally normal cell lines (HBL-100) whereas expression of β-actin was normal in all the cancer cell lines followed by MCF 7, A549, ZR-75-1 and HBL 100. MCF 7 amplification was started at the twenty second cycle whereas remaining cancer cell line required slightly higher than MCF7.But Normal cell line amplification started at the twenty sixth cycle. (Table 2). Table 2. Copy numbers, cyclic amplification and Ct value of both COX 2 and β actin genes in the cancer and normal cell lines.

REAL TIME PCR EXPRESSION
The isolated RNA was reverse transcribed to cDNA and it was subjected for RT PCR evalution of expression  (Fig 8).
The amplification pattern of all cDNA profiles were analysed by using dissociation stages for their amplification curves and their proper amplification. This indicates that proper dissociation gives the better amplification of all cDNA.
The amplification plots of COX-2 and βactin in cancer and normal cell lines are shown in Figure 8.For this expression correlation strategies we used student t-Test for analyse complete expression profiles of cDNA in all cancer cell lines.

DISCUSSION
In this study over expression of COX 2 was proved by means of RT PCR, western blot analysis and also the presence of cDNA copies of all cancer cell lines (Fig. 3 Compared to traditional methods of studying gene expression such as Northern blot analysis and Ribonuclease Protection Assay, the multiplex RT-PCR is a rapid method and expression of more than one gene can be analyzed in relation to a reference housekeeping gene from a small quantity of mRNA 17,18 . Compared to conventional RT-PCR, multiplex RT-PCR has an advantage of sensitivity. Comparing two genes amplified in different reactions, to the same housekeeping gene is less sensitive than comparing both of the genes to a housekeeping gene in a single reaction. In an optimized multiplex reaction, any of the PCR factors that become limiting factor will affect the amplification of each gene equally 18. The greatest advantage has been the elimination of the limitations imposed by traditional methods, such as gel electrophoresis. These include low sensitivity, non-detection of shoulder peaks, long analysis time with postanalysis visualization and low automation 19 . Also fewer steps to the final data eliminate the loss of sensitivity generated at each step manually or due to the instruments. According to our suggestions whereas the traditional method of slab gel electrophoresis can take up to 5 h to complete, the RT PCR based method can analyze and semiquantitate more samples at the earliest 15 . Considerable savings in reagents, sample size and time, coupled with reduction in size and number of the separation and detection apparatuses needed, has made this technique extremely cost-and time-effective. The molecular mechanisms controlling the expression of COX-2 are not completely understood but are important because this is a crucial component of inflammation and a rate-limiting step in colon carcinogenesis 20 . In this report we demonstrate that a major regulatory point of COX-2 gene expression occurs at the post-transcriptional level Minnie et al showed that cytoplasmic proteins specifically bind to the COX-2 and postulated that this interaction regulates the stability of COX-2 mRNA. Thus, the levels of COX-2 protein are determined by post-transcriptional regulation as well as by transcriptional mechanisms. This level of complexity is consistent with the requirement for tight control of the enzymatic action of COX-2, which has pathogenic effects if its expression is unregulated.