The Preparation Of Rhein Lysinate And The Mechanism Of Its Anti-tumor Activity

Malignancy is the second cause of death, and in some areas it has been the first one. The mortality of malignancy increases yearly in the world. The important way for human to overcome malignancy is to explore new anti-tumor drugs. With the development of immunology and molecular biology, a number of receptors, genes and signal transduction pathways involved in tumor generation had been reported. Based on this discovery, targeted therapy of tumor has been made a breakthrough. The development of targeted drugs is being one of the most attractive fields in tumor research and therapy. The drugs targeting protein tyrosine kinase had already been the focus point. A tyrosine kinase is an enzyme that can transfer a phosphate group from ATP to a tyrosine residue in a protein, including receptor PTKs, non-receptor PTKs, IR and Janus. In the abnormal condition, PTKs are constructively activated, consequently blocking the regulation of cell differentiation, growth and apoptosis, and inducing tumor progression.More than 50% of the known receptor PTKs have been found to be either mutated or overexpressed in association with human malignancies and 30% of them have been implicated in human solid toumors. EGFR family, an important group of PTKs, includes four members: EGFR (ErbB-1), HER-2 (ErbB-2), HER-3 (ErbB-3) and HER-4 (ErbB-4). There are one million and five hundred thousand new cases of breast cancer around the world every year, 27%—30% of them with EGFR overexpression and 20%—25% of them with HER-2 overexpression. Therapeutic use of protein tyrosine kinase inhibitors (TKIs) and monoclonal antibody targeting EGFR or HER-2 have some limitations. The efficacy of an anti-tumor drug that targets only a single point is less than 30%. The anti-tumor activity can be improved by the combination of chemotherapeutic agents; however, resistance occours soon through the mutual compensation of receptor PTKs. So, dual receptor tyrosine kinase inhibitors targeting both EGFR and HER-2 may increase the response rate. Emodin, a tyrosine kinase inhibitor, suppresses HER-2 atuo-phosphorylation and trans-phosphorylation and blocks tyrosine kinase activity of ErbB-2. Emodin can inhibit the growth of HER-2 -overexpressing tumors in mice and also sensitize these tumors to taxol. In this article, we studied if rhein analog showed dual inhibition to EGFR and HER-2.Rhein, emodin and aloe-emodin are the main anthraquinone derivatives of rhubarb. It was reported that the anthraquinone derivatives of rhubarb could inhibit tumor cell proliferation, induce tumor cell apoptosis, block blood vessel formation and even reverse multi-drug resistance. However, all of them have poor water solubility and poor bioavailability. Their therapeutic applications are limited in cathartic. The most significant difference between rhein and other anthraquinone derivatives is that rhein has a free carboxyl group. Therefore structural modification can be directed to the free carboxyl group.Partâ… Preparation of rhein analog with high solubility in waterPurpose: To prepare rhein derivatives easily dissolving in water.Methods: One is the preparation of lysyl-rhein. The carboxyl group of rhein reacts with the amino group of lysine in special condition to form lysyl-rhein, which improves its solubility in water depending on the free amino group and the free carboxyl group of lysine. Another is the preparation of rhein lysinate (RHL). The carboxyl group of rhein reacts with the amino group of lysine in room temperature to form RHL, which easily dissolves in water. By MTT assay, we observed the anti-tumor activities of these new products in vitro.Results: We obtained two new products by the first method. One was yellow and did not dissolve in water. Another was purple and easy to dissolve. These two compounds were proved to be rheinyl lysine methyl ester, by infrared spectrum analysis. The anti-tumor efficacy of these two compounds was far less than that of rhein and the yield of them was very low, only 6% and 9% respectively. No further study was conducted. By second mthod we obtained a purple compound with good solubility. It was proved to be RHL by violet spectrophotometry, HPLC, and infrared spectrum analysis. By MTT assay, RHL had the same anti-tumor activity as rhein (in DMSO solution).Conclusions: Although rheinyl lysine methyl ester could partly improve the solubility, the anti-tumor activity was far less than rhein. No further study was conducted. RHL not only easily dissolved in water but also showed the same anti-tumor activity as rhein. That warrants further development of RHL.Partâ…¡The anti-tumor activity of rhein lysinate and its molecular mechanism in tumor treatment in vitroPurpose: To observe the anti-tumor activity of RHL and investigate its molecular mechanism.Methods: By MTT assay investigating tumor cell proliferation; by Western blot investigating the phosphorylation of protein and the expression level of proteins in PTKs signaling pathway; by immunocytochemistry observing the expression level of EGFR and HER-2; by RT-PCR observing HER-2 change in transcriptional level; by FACS observing cell apoptosis and cell cycle arrest.Results: RHL inhibited the proliferation of different breast cancer cells (MCF-7, SK-Br-3 and MDA-MB-231), ovarian cancer cells (SK-OV-3) and HUVEC. The IC₅₀ was 95μmol/L, 80μmol/L, 110μmol/L, 85μmol/L and 200μmol/L, respectively. It had the same anti-prliferation with rhein. In MCF-7 cells with EGFR overexpression and without HER-2 overexpression, RHL inhibited the phosphorylation of EGFR, the phosphorylation of downstream Raf/MEK/ERK signaling pathway and showed synergistic effect with chemotherapeutic drugs such as taxol. In SK-Br-3 cells with both EGFR and HER-2 overexpression, RHL inhibited the phosphorylation of EGFR and HER-2 and induced cell apoptosis through p53 and p21 pathway.Conclusions: RHL was a new dual target protein tyrosine kinase inhibitor aiming directly at EGFR and HER-2. RHL not only inhibited tumor cell proliferation but also induced tumor cell apoptosis.Partâ…¢The anti-tumor activity of rhein lysinate and its synergism with taxol in tumor treatment in vivoPurpose: To observe anti-tumor activity of RHL and explore its synergistic effect with taxol. Methods: Establishing the model of mouse H22 liver cancer and human MCF-7 breast cancer xenografts in athymic mice, administering RHL by intragastric administration and intraperitoneal injection, observing the tolerance in mice and the therapeutic effect of RHL, and observing the phosphorylation of EGFR in tumor tissue by immunohistochemistry method.Results: RHL inhibited tumor growth. The efficacy of RHL administered by intraperitoneal injection was better than by intragastric administration. RHL in combination with taxol showed the synergistic effect in inhibiting tumor growth in mouse H22 liver cancer and MCF-7 breast cancer xenografts in athymic mice. The result of immunohistochemistry demonstrated that RHL partly abrogated the increase of the phosphorylation of EGFR and ERK induced by taxol.Conclusions: RHL inhibited tumor growth of mouse liver cancer H22 and human breast cancer MCF-7 xenografts in athymic mice. The efficacy of RHL administered by intraperitoneal injection is better than that by intragastric administration. It had a synergistic anti-tumor activity with taxol.In summary, we obtained a new compound - RHL. That is easily dissolved in water. RHL inhibited the proliferation of breast cancer and ovarian cancer cells and had the same potency with rhein. Our results indicated that RHL, as a new dual tyrosine kinase inhibitor, was capable of repressing the tyrosine kinase activity of EGFR and HER-2, accordingly inhibiting the proliferation of EGFR overexpressing MCF-7 breast cancer cells and inducing cell apoptosis of EGFR and HER-2 overexpressing SK-Br-3 breast cancer cells. Moreover, it sensitized tumor cells to chemotherapeutic drugs such as taxol. These results may have important therapeutic implications in cancer chemotherapy.