Inhibition Of Herceptin On Cervical Cancer Cells And Its Synergistic Mechanism Of Action With Carboplatin

Cervical cancer is one of the most common malignant tumours. The morbility and mortality are in the first place of malignant tumours in female. Owing to the cytology screening, the morbility and mortality have decreased obviously. But the proportion of cervical adenocarcinoma, including adenosquamous carcinoma has been increasing for the past few years. Cervical adenocarcinoma is insensitive to radiotherapy, and its effect on conventional clinical chemotherapy is small. So it is important to find a new rational treatment to cervical adenocarcinoma.HER-2/neu is also called c-erbB-2 oncogene, the coding protein is called P185 c-erbB-2, which is a kind of transmembrance receptor glucoprotein. It participats cellular growth and differentiation modulation. Ras oncogene is coding P21ras protein, which is able to combine GTP or GDP with high specificity. It is the switch of cellular growth and differentiation modulation. When HER-2/neu is combined with ligand, it can activate Ras. Therefore, Ras/Raf/MAPK pathway is activated. Then it will induce genetic transcription and cell proliferation. Herceptin, which is a recombinant humanized monoclonal antibody (mAb) directed against the extracellular domain (ECD) of the HER-2 protein, is a target medicine aiming directly at tyrosine protein kinase. It can block downstream signaling pathways, which will impact epithelial cells growth and inhibit transcription and proliferation.In this study, we aim to evaluate the inhibitory effect of Herceptin on HeLa, SiHa cells, and to investigate the synergistic mechanism of action with carboplatin. At the same time, by detecting the changes of HER-2/neu and downstream Ras oncogene, we could infer the inhibitory mechanism of Herceptin on cervical cancer cells.And then to evaluate the validity of Herceptin on the treatment to cervical adenocarcinoma. AIM:1. To detect the expression of HER-2/neu and Ras oncogenes in cervical adenocarcinoma and squamous carcinoma tissues and cell lines,and to evaluate the correlation between clinicopathological indexes and HER-2/neu as well as Ras in cervix cancer, so as to investigate the role of HER-2/neu and Ras in genesis and development of uterine cervix cancer;2. To observe the inhibitory differences in adenocarcinoma HeLa cell and squamous carcinoma SiHa cell after treated with Herceptin alone or with carboplatin together, in order to study the synergistic mechanism of action with carboplatin;3. To investigate the inhibition mechanism of Herceptin on cervical cancer cells. METHODS:1. The SP immunohistochemical method was used to detect the expressions of P185c-erbB-2 and P21ras in 20 cases of normal cervix tissue, 40 cases of adenocarcinoma and 45 cases of squamous carcinoma, as well as in HeLa and SiHa cells;2. The inhibition of Herceptin on HeLa, SiHa cells and synergistic effect with carboplatin was studied by means of MTT;3. The morphological changes of cervix cells were detected by light microscope and transmission electron microscope(TEM);4. Flow cytometry (FCM) was used to detect the cell apoptosis and cell cycle;5. The fluorescence expression of Ras oncogene was detected by FITC immunofluorescin histochemistry after treated with Herceptin alone;6. The differences of mRNA expression of HER-2/neu and Ras were determined by RT-PCR;7. The protein expressions of HER-2/neu and Ras were studied by Western Blot.RESULTS:1. P185c-erbB-2 and P21ras were not observed in normal cervixes. Compared with normal cervix tissue, P185c-erbB-2 and P21ras positive expressions in adenocarcinoma and squamous carcinoma were different significantly (P<0.0125), and the expression between adenocarcinoma and squamous carcinoma had obvious difference (P<0.0125). P185c-erbB-2 and P21ras expressions are related to the cell differentiation level (P<0.0125). In addition, there was an obvious correlation between P185c-erbB-2 and P21ras in cervix cancer(P<0.01); P185c-erbB-2 and P21ras were most positive expressions in adenocarcinoma HeLa cells, and weakly positive or negative in squamous carcinoma SiHa cells;2. HeLa cells represented advanced stage of apoptosis after treated with Herceptin, but SiHa cells were most in early stage; Herceptin inhibited cervical cancer cells proliferation significantly, and synergistic effect with carboplatin was determined (P<0.05, P<0.01). It could induce cell apoptosis, especially with carboplatin together. In addition, Herceptin promoted G1 phase of cell cycle arrest. Interestingly, G2 phase was further arrested and S phase cell proportion was decreased when carboplatin was added; Compared with squamous carcinoma SiHa cells, changes of apoptosis and cell cycle were obvious in adenocarcinoma HeLa cells;3. The fluorescence expression of Ras oncogene was reduced after treated with Herceptin alone; The mRNA and protein expressions of HER-2/neu and Ras were decreased after treated with Herceptin alone or with carboplatin together (P<0.05). It was still significant in HeLa cell. CONCLUSION:1. P185c-erbB-2 and P21ras proteins were overexpressed in cervix cancer, especially in cervical adenocarcinoma;2. Herceptin promoted G1 phase of cell cycle arrest and decreased S phase cell proportion, it can induce cell apoptosis, and its synergistic mechanism of action with carboplatin is obvious;3. Herceptin inhibits proliferation of cervical cancer cells by restraining Ras/MAPK pathway; HeLa cells are more sensitive to Herceptin. Therefore, it is possible for Herceptin to be a target therapy in treatment of cervical adenocarcinoma.