Identification Of New Anti-Melanoma Natural Compounds From Chinese Medicinal Herbs By High Throughput Screening Technology

Melanoma is the sixth most common cancer and the malignancy with the highest rise in incidence and the most dangerous form of skin cancer. Melanoma is also a major health problem and its rates are increasing in the worldwide. The incidence rate for invasive melanoma is highest in whites, who are almost 30 times more likely to develop melanoma. Men aged 65 or older are more than twice as likely to develop melanoma as women in the same age group (The Lifetime risk for Melanoma is 1 in 35 women and 1 in 25 men) and number of cases is about 100,000 per year. According to the International Agency for Research on Cancer, the countries with the highest incidence of cutaneous melanoma are Australia and New Zealand. The incidence of melanoma is increasing, and the therapeutic options for malignant disease are limited, resulting in an increase in the death rate.The chemotherapy of melanoma is to prevent the metastasis and kill the cancer cells which is including Alkylating Agents (such Dacarbazine (DTIC), response rate is 15% to 20%), Platinum Drugs (such cisplatin, response of 15% as a single treatment) and Microtubule-toxin Agents (such paclitaxel, response rate of 16% as single treatment) and still the side effects are main problem of chemotherapy agents. The current treatment for metastatic melanoma is still poor in overall response and survival rate. Although some bio-chemotherapy trials have shown good outcomes, no agent or regimen has improved median response duration and overall survival in subsequent studies. New treatment options, as single or in combination, with the use of natural phyto-compounds or herbal medicines may provide new agents in the treatment of malignant melanoma.The natural products are the most consistently successful source of drug leads and provide a greater structural diversity than standard combinatorial chemistry and offer major opportunities for finding novel low molecular weight lead structures that are active against a wide range of assay targets. Traditional Chinese Medicine (TCM) has been practiced for thousands of years with a long history of 2000 to 3000 years and has contributed in the worldwide to the identification of active ingredients for a particular medical usage for several TCM composed of single or multiple herbal components. In the coming years, the stage is set for a more systematic, rigorous analysis and testing of therapeutic agents used in TCM that may eventually lead to the development of useful therapeutic agents for the entire world. TCM natural products have the potential for use in cancer therapy, for example; artesunate, homoharringtonine, arsenic trioxide and cantharidin, the controlled clinical studies have shown that homoharringtonine and arsenic trioxide can exert profound activity against leukaemia. Increased knowledge of the molecular mechanisms of TCM-derived drugs and recent developments in their applications demonstrate that the combination of TCM with modern cutting-edge technologies provides an attractive strategy for the development of novel and improved cancer therapeutics.Furthermore, high throughput screening is a new development of strategy to speed up the identification, isolation and characterization process and to discover small natural molecules that can be delivered to use in cancer treatment with less side effects on the human health in the near future. In the present study, we used high throughput screening approaches to investigate the melanoma properties of a compounds fraction library from 500 Chinese herbs and to identify new small natural molecules with anticancer properties against melanoma and other cancer cells.We have developed a novel HTS-based strategy to systematically identify active natural compounds against any drug target from Chinese medicinal herbs. An herbal compound fraction library was constructed from 500 herbs most frequently used in Traditional Chinese Medicine (TCM) that are likely to contain therapeutic ingredients for a broad spectrum of human diseases including virus infection. For construction of the TCM fraction library, crude herbal extracts were first prepared by 95% ethanol extraction on Soxhlet reflux apparatus followed by automated fractionation of the extracts using preparative HPLC. Eighty fractions were collected from each herb by 0-90% methanol gradient. The number of single compounds in each fraction was ranging from 1 to 17 (averaged 10.4). We have built up a natural compounds library with 40,000 fractions from 500 herbs that is suitable for high throughput screening. Single active compounds from positive fractions are identified by further fractionation, activity analysis and structure determination. For high throughput screening, a working TCM fraction library was prepared in 96-well format by dissolving each fraction in DMSO to make a 20 mM solution suppose average compounds molecular weight is 500 dolton. A final concentration of 100μM was used to screen for anti-melanoma activity. Among 240 Chinese herbs containing 19,200 fractions screened against human melanoma cancer cell line A375, we identified 154 positive fractions from 67 herbs. We tested the toxicity of positive fractions on freshly isolated mouse splenocytes to exclude fractions toxic to normal cells. The results indicated 110 fractions with remarkable toxicity and 44 fractions with no significant effect on mouse splenocytes. We choose a positive fraction with anti-melanoma activity and least toxicity on splenocytes from a Chinese herb called Artemisia Argyi to isolate positive single compounds that are responsible for the anti-melanoma activity. Organic extraction followed by preparative HPLC fractionation was used to isolate anti-melanoma single compounds from herbal plant Artemisia Argyi. We have successfully isolated two compounds with anti-proliferative effect on A375 melanoma cells. The chemical structures of the two compounds were determined as an O-methylated flavone; Eupatilin (5, 7-dihydroxy-3’, 4’, 6-trimethoxyflavone) and jaceosidin (4, 5, 7-trihydroxy-3’, 6-dimethoxyflavone) by using MS, 1H NMR and 13C NMR.The anti-proliferative effect of eupatilin in human melanoma A375 cells was performed by MTT assay. The EC50 value is about 150μM. Eupatilin specifically induced morphological changes in A375 cells and was less toxic to the normal mouse splenocytes. The inhibitory effects of A375 cells were associated with the DNA damage, apoptosis, and cell cycle arrest at G2/M phase in a dose-dependent manner. The apoptotic effects of eupatilin were further verified by using Annexin V-FITC/propidium iodide staining in flow cytometry. It was observed that the apoptosis rates were 13.05% and 29.2% in A375 cells treated with 150 and 300μM eupatilin for 24 h, respectively, compared to the control cells at 2.04%. Furthermore we indicated apoptosis effect by using Hoechst staining (easily detect the fragmentation of DNA in cell nucleus). Flow cytometry was performed to determine whether eupatilin induced cell cycle arrest. The percentage of A375 cells in G2/M phase was increased from 8.82% in untreated cells to 21.70%, and 29.86% in the cells treated for 24h with 150 and 300μM eupatilin, respectively.These results suggest that our high throughput screening strategy using herbal fraction library is promising in identifying small natural molecules with anti-melanoma activity. The two identified flavones compounds with confirmed anti-melanoma activity perhaps worth further mechanistic and therapeutic studies in human melanoma. Isolation and functional characterization of single compounds from other positive fraction of different herbs may identify new natural compounds with new mechanisms and ideal therapeutic properties against human melanoma.