Synergistic Effects Of Herceptin And 9-cis RA On HER2/neu-positive Breast Cancer And The Underlying Molecular Mechanism

The HER2/neu proto-oncogene (also known as c-ErbB2, neu, and HER2) encodes a 185-kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity that resembles the receptor for epidermal growth factor. Overexpression of HER2/neu was found to be a poor prognostic factor for survival from breast cancer. HER2/neu appears to play an important role in the biologic behavior of breast cancers and holds potential as a target for oncogene-directed therapies.Herceptin(Trastuzumab), a humanized monoclonal antibody to HER2/neu, directed against the extracellular domain of the tyrosine kinase receptor HER2/neu. The mechanisms of the action of this antibody involve disruption of DNA repair and induction of antibody-dependent cellular cytotoxicity. It is clear that targeted therapies are going to play a greater role in improving survival and quality of life in advanced breast cancer, with Herceptin serving as a successful model that is a relatively nontoxic agent associated with survival benefits. Studies indicate that Herceptin combined with chemotherapy such as paclitaxel, docetaxel and doxorubicin is superior to chemotherapy alone as first-line treatment of patients with HER2/neu-positive metastatic breast cancer in terms of overall survival, response rate, response duration, time to progression, and time to treatment failure with little additional toxicity.Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The family of compounds known as the retinoids include 9-cis Retinoic acid (9-cis RA), all-trans RA, 13-cis RA and so on. 9-cis RA and all-trans RA (ATRA) are naturally occurring hormones. The nuclear receptors that mediate the effects of retinoids are the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). ATRA binds RAR with high affinity but does not bind to RXR, whereas 9-cis RA, an isomer of ATRA, is a ligand that binds and transactivates both RARs and RXRs. Since their introduction 15 years ago, retinoids have been increasingly used for therapy and/or chemoprevention of epithelial skin cancer and cutaneous T cell lymphoma. A important study demonstrated the RXR-slective retinoids, LGD1069 could effectively surpress ER-negative tumor development in virus-ErbB2 transgenic mice with minimal toxicity. It suggest that RXR-agonists are promising agnets for treatment and prvention of ErbB2-overexpressing breast cancer.Based on the above analysis, the synergistic effect of Herceptin and 9-cis RA on the proliferation and apoptosis of tumor cells and its possible molecular mechanisms were studied. After treatment of Herceptin in combination with 9-cis RA on tumor cells, the cellular proliferation capacity was observed with MTT assay, the cell cycle distribution was measured with flow cytometry, and the apoptosis was analyzed with TUNEL test. At the same time, we inoculated the MCF-7 breast cancer cells with exogenous HER2/neu gene (MCF-7^HER2 cells) into athymic nude mice to observe the synergistic inhibitory effects of Herceptin and 9-cis RA in vivo. Finally, we explored the possible molecular mechanisms underlining those mentioned above by detecting the expression of ErbB2, pErbB2, CDK2, Cyclin E, COX-2, p27 and the RXRαwith RT-PCR and Western blot.The results and conclusions are as followed:1. After treatment with 5μg/ml Herceptin and 1μmol/L 9-cis RA for 24～72h, the proliferation capacity of MDA-MB-453 cells was significantly inhibited and the cell cycle progression was arrested at the G0/G1 phase as compared with control group. These findings suggested that 9-cis RA was able to effectively increase the sensitivity of MDA-MB-453 cells to Herceptin in vitro.2. The apoptotic rate of MDA-MB-453 cells was remarkably increased after 72h treatment with 5μg/ml Herceptin in combination with 1μmol/L 9-cis RA as compared with control group indicating that Herceptin and 9-cis RA could exert synergistic inhibitory effects on MDA-MB-453 cells by inducing apoptosis.3. The xenograft model transplanted with MCF-7^HER2cells was well established. Having being compared with control group, Herceptin group and 9-cis RA group, the tumor size of nude mice treated with Herceptin in combination with 9-cis RA for 20 days was remarkably decreased as well as the Ki-67 expression. Indicating that Herceptin and 9-cis RA could synergistically inhibited the cell cycle progression of HER2/neu-positive breast cancer cells in vivo.4. After treatment with 5μg/ml Herceptin and 1μmol/L 9-cis RA for 24h, the expression of ErbB2, pErbB2 and COX-2 were down-regulated, but the RXRαexpression was not changed. Indicating that Herceptin combined with 9-cis RA was able to synergistically inhibited the proliferation capacity of HER2/neu-positive breast cancer cells by down-regulate the expressions of relevant factors ErbB2 and COX-2.5. After treatment with 5μg/ml Herceptin and 1μmol/L 9-cis RA for 24h, The expression of CDK2, Cyclin E and the binding ability of Cyclin E/CDK2 complex of MDA-MB- 453 cells was significantly down-regulated, and the expression of p27 protein level was significantly increased. Indicating that Herceptin and 9-cis RA could synergistically inhibited the cell cycle progression of HER2/neu-positive breast cancer cells by regulate the expression and activity of relevant factors.Taken together, our study clearly suggests that 9-cis RA could synergistically cooperates with Herceptin to inhibit the HER2/neu overexpressing breast cancer both in vitro and in vivo.