Relation Between Ornithine Decarboxylase And Proliferation And Invasion Of Breast Cancer

The polyamines include spermidine, spermine and putrescine; they are positively charged aliphatic amines. In mammalian cells, polyamines are synthesized from ornithine to putrescine, catalyzed by ornithine decarboxylase (ODC). Ornithines were decarboxylazed by ODC. Then the first adenosylmethionine is added to putrescine to produce spermidine, this step is catalyzed by spermidine synthetase, and the second adenosylmethionine is added to spermidine to produce spermine, catalyzed by spermine synthetase. Adenosylmethionine was provided by Sadenosylmethionine and decarboxylated by S-adenosylmethionine decarboxylase (SAMDC). So, the ODC is the first rate limiting enzyme of polyamine biosynthesis.There are high Polyamine concentration expressions in breast cancer tissue. Leveque J et al found in breast cancer tissue, polyamine's concentrations were significantly higher compared with the polyamine concentrations in controls, and he also found that polyamines concentrations have a close relationship with the tumours aggressiveness, histological grade and Ki-67 proliferative index. Norman JC, et al tested Concentrations of polyamines, DNA and oestrogen receptor (ER) states in 23 malignant and 2 benign breast tumors. He also found that Polyamine and DNA concentrations were significantly higher in ER-poor than in ER-rich tumours.Breast cancer is the most common malignancy among females, statistics show that in the United States alone, about 200,000 new cases are diagnosed every year and about 50,000 women die annually from the disease. Overall, about 15% of all women will be diagnosed with breast cancer during their lifetime. In China, although the incidence rate of breast cancer is relatively lower, about 36.2 per 100,000 population in 1993-1997. But it is increasing very fast, at 3% per year. and the mortality rates increased by 38.91% than before.In order to increase the diagnosis level and improve treatment, great efforts have been done on different kinds of breast tumour markers, polyamines and ODC maybe committed as an independent tumor marker of breast cancer. According reports, ODC can be a target for the inhibition of tumor's proliferation and invasion. DFMO, (alpha-difluoromethylornithine)was conceived as an enzyme-activated irreversible inhibitor of ODC, it can decrease the invasiveness of metastasis human breast cancer cell lines. The mechanism by which DFMO acts to reduce invasiveness may be DFMO treatment decreases the expression of the metalloprotease meprin alpha, and MMP-7 mRNA. But early clinical cancer therapeutic trials with DFMO were disappointing; because it caused a series side effects at high doses, such as: diarrhea, abdominal pain, moderate anemia and temporal loss of hearing. So, it is very essential to find a new way to inhibit ODC activity so as to block tumor growth and/or metastases. Previous Studies show that antisense RNA of ODC can significantly inhibit the growth and invasive ability of prostate cancer cells and colorectal cancer, but there is no report about using this technique on breast cancer cells. So, we took breast cancer as a target, study the effect of antisense RNA of ODC on breast cancer cells so as to provide a help for further gene therapy to breast cancer.In order to approach the possibility of antisense gene therapy targeting ODC ,we infected the antisense RNA recombined adenovirus vector of the third extron in ODC gene(rAd-ODC / Ex3as) to breast cancer cell line MDA-MB-231. Expressions of ODC in cells were determined by western blot. MTT assay and soft agar assay were used to analysis the effect on cell growth. Cell cycle was evaluated by FCM and cellular invasion by cell migration assay. A nude mouse xenograft model was used to examine tumorigenicity and the effect on the formed breast tumors. Part 1 Expression of ODC gene in breast cancer tissues and cell linesMethods:Using reverse transcription polymerase chain reaction (RT-PCR) and western blot assay we examined the mRNA and protein expression levels of the ODC gene in the lesions and the normal tissues.To find the relations between these expression levels and breast cancer, and its clinical stages, we tested the ODC protein levels in 4 breast cancer cell lines and normal breast epithelial cells lines by western blot.Next, we tested its localization and cell cycle expression by immunocytochemistry and cell cycle synchronization.Results:The expression of ODC gene in lesions of breast cancer was significantly higher than that in contiguous normal tissues (p<0.05). And the ODC protein expression in 4 breast cancer cell lines was significantly higher than that in normal breast epithelial cells lines. ODC was present in the cytoplasm, in resting cells, ODC staining was moderate and predominantly in the perinuclear region, whereas mitotic cells were strongly and homogeneously stained. The expression of ODC gene in MDA-MB-231 cells exhibited a time-dependent increase beginning at 6h after stimulation and then decrease at 9-12h, followed a second increase at 15-18h, decreased at 21-24h. The cyclin D1 also peaked at around 6h and and fell gradually.Conclusion: The more expression of ODC gene in human breast cancer than that in normal breast cells is to say that ODC plays an important role in mammary carcinogenesis.Part 2 Inhibition effect of breast cancer cells with rAd-ODC / Ex3as in vitroMethods:MDA-MB-231 was infected with rAd-ODC / Ex3as, MTT and soft agar assay was used to measure the infection rate and observe the growth inhibition ,western blot were used to observe the inhibition of ODC and cyclin D1 in infected tumor cells. Flow cytometry was used to analyze cell cycle progression.the MDA-MB-231 breast cancer cell line was assessed by cell migration assay. MDA-MB-231 cells were transfected with rAd-0DC/Ex3as or rAd-GFP viruses, or incubated with PBS as acontrol. The three polyamines (putrescine, spermidine and spermine) in the cell lysates were analyzed by HPLC.Results:Results showed that rAd-ODC / Ex3as could significantly inhibit the growth of MDA-MB-231 cells without cell toxicity. Western blot showed that 48.2% of ODC and 55% of cyclin D1protein expression was inhibited in MDA-MB-231 cells. Cell flow cytometry analysis showed that rAd-ODC / Ex3as could lead to the infected cell arrest at G₁ phase,but no effect to apoptosis.cell migration assay showed relatively low invasion. Transfection of rAd-ODC/Ex3as resulted in reduction of the three polyamines, compared with rAd-GFP or PBS. However, only the level of putrescine was significantly lower in rAd-ODC/Ex3as transfected cells than rAd-GFP or PBS treated cells.Conclusion:rAd-ODC / Ex3as could interfere with ODC expression,induceG₁ arrest ,suppress the proliferation and invasive ability of breast cancer cells.Part 3 Inhibition of effect of breast cancer cells with rAd-ODC / Ex3as in vivo Methods:To examine the effect of the antisense ODC on the formed breast cancer, the mouse MDA-MB-231 breast tumors were stablished by injecting 5×10⁶ cells (in medium) into nude female mice.When tumors had reached 5-7mm in diameter, the mice were treated with the recombined adenovirus every fifth day. Tumor size was measured once every fifth days.Results:Expression of antisense ODC inhibited the growth of MDA-MB-231 breast cancer cells in vivo, as evidenced by the reduction in tumor incidence and tumor size, when compared with those of rAd-GFP treated or no virus-treated tumors. Antisense ODC can also inhibit the growth of the breast tumors.ConclusionODC plays an important role in breast tumorgenicity; on the other hand, antisense ODC has inhibitory effect on the formed breast tumors in vivo.SummaryExpression of ODC gene in breast cancer tissues and cell lines were significantly high than that in contiguous normal tissues and normal breast epithelial cell lines. After treatment with rAd-ODC / Ex3as, expressions of ODC and cyclin Dl gene in breast cancer cell line MDA-MB-231 were reduced 48.2% and 50%, cell growth was substantially inhibited and cell cycle was arrested at G₁ phase. Cell migration assay showed relatively low invasion. Marked suppression of tumor formation and tumor growth was observed in the xenograft model.This study suggests that rAd-ODC / Ex3as has the antitumor effect on the human breast cancer cells .