Pharmaceutical Analysis Of Three Highly Volatile Organosulfur Compounds In Garlic And Their Inhibition Mechanism Of Proliferation Of Human Lung Cancer H460Cells

Garlic is the bulbs of Liliaceae allium sativum. Thousand years of epidemiological studies and clinical uses have shown that garlic possesses serious pharmacological effects such as tumor reduction, anti-atherosclerosis, blood lipids and sugar modulation, antifungal, antimicrobial, and stimulating immune system. Studies have indicated that the beneficial effects of garlic are closed related to organosulfur compounds in garlic. The organosulfur compounds are mainly allyl sulfides such as diallyl disulfide (DADS) and diallyl sulfide (DAS). These organosulfur compounds have been found to inhibit the growth of various tumors, but the mechanism of the effects and structure-efficacy relationship are not clearly understood.Our previous studies have found that the major highly volatile organosulfur compounds detected in a variety of garlic preparations at room temperature include diallyl sulfide (DAS), methyl allyl disulfide (AMDS) and diallyl disulfide (DADS) with AMDS as the most abundant. In this work, we used the GC-MS method to determine the purity and impurity profiles of synthetic DADS, AMDS and DAS in order to further explore the feasibility of these three garlic organosulfur compounds as new drug candidates. The stability study of these three organosulfur compounds was conducted under the conditions of25℃,4℃and-20℃. The second part of this project was to study the anti-proliferation effects of these three garlic organosulfur compounds on human lung cancer H460cells. The mechanistic study of apoptosis and evaluation of structure-activity relationship of these three organosulfur compounds included the characterization of inhibition rates, cell death, mitochondria membrane potentials, cell cycle, and other biological properties.The pharmaceutical analysis of three synthetic organosulfur compounds DADS, AMDS and DAS by using GC-MS indicated that the purity of DADS, AMDS and DAS was determined to98.8%,96.5%and100.0%, respectively. The structural identification of impurities in the DADS, AMDS and DAS lots showed that the major impurity in the DADS lot was DAS (1.2%) while the main impurities of the AMDS lot were found to be DADS (2.2%) and1-propenyl methyl disulfide (1.3%). The stability assay of DADS, AMDS and DAS lots demonstated that DADS, AMDS and DAS were unstable under25℃conditions, particularly AMDS whose contents was dropped to50%within three months. However, they were stable at-20℃with the content decrease of less than2%. Therefore, these three organosulfur compounds should be stored at-20℃conditions.In recent years, the study of signal transduction pathways of apoptosis or cell cycle arrest of tumors induced by chemotherapy drugs have become hot topics in modern biological research of anti-tumor drugs. In this work, we evaluated the mechanism of apoptosis and cell arrest of lung cancer H460cells induced by DADS, AMDS and DAS in vitro. The SRB assay results show that when H460cells were treated with50-600μM DADS, AMDS and DAS for24,48,72h, the maximum inhibition ratios of DADS and AMDS at600μM for72h were74.8%and65.3%, respectively, while DAS within600μM had no effects on H460cells apoptosis. Therefore, the inhibtion rates of DADS, AMDS and DAS on H460cells were in the order of DADS> AMDS> DAS. The microscopic analysis indicated that DADS and AMDS could induce the nuclear chromatin and condensation of H460cells and the number of round cells increased significantly and the cells became larger with the time. The DAPI staining analysis showed that the phenomena of chromatin condensation and nuclear condensation (typical apoptotic bodies) were observed after H460cells were treated with400μM DADS or AMDS while DAS even at600μM could not induce any H460cells apoptosis. FITC-Annexin V/PI double staining assay found that the apoptotic ratio of H460cells was increased with increased concentrations of DADS or AMDS, showing a significant dose-dependent relationship while no effect was observed for DAS up to600μM. The Elisa assay showed that300μM and600μM DADS or AMDS could significantly increase the expression of caspase3, caspase8and caspase9with a dose-dependent manner while600μM DAS did not show any effects. The flow cytometry analysis indicated that DADS and AMDS decreased the mitochondria membrane potentials and elevated levels of intracellular reactive oxygen species while600μM DAS could not have significant effects. Western blotting assay results show that DADS and AMDS significantly reduced the expression of Bcl-2and Bcl-XL proteins, and increased the expressions of Bax cytochrome c and FADD in the death receptor pathway. RT-PCR assay showed that DADS and AMDS also decreased the level of Bcl-2expressions at the mRNA level, and increased expression levels of Bax, caspase3and FADD, indicating DADS and AMDS induced apoptosis of H460cells via mitochondrial and death receptor pathways. The cell cycle analysis with PI staining indicated that H460cells were arrested in the G2/M phase by DADS or AMDS while DAS could not. Western blotting assay indicated that DADS and AMDS significantly upregulated the expressions of p53and p21proteins and reduced PCNA, Cyclin B1, Cyclin A2protein expressions in a dose-dependent manner. RT-PCR analysis demonstrated that DADS and AMDS increased the mRNA level of Cyclin B1. The results of the wound scratching analysis show that DADS and AMDS significantly increased the width of scratches comparing with a control after the treatment for24h while600μM DAS had no effect. Western blotting assay also showed that DADS and AMDS increased the level of E-cadherin adhesion protein.In conclusion, the preliminary pharmaceutical analysis of three garlic organosulfur compounds diallyl sulfide (DAS),allyl methyl disulfide (AMDS), diallyl disulfide (DADS) indicated that the purity of these compounds has met or nearly met as the purity requirements for pharmaceutical substances. However, their stability must be further improved through special formulation techniques. The structure-activity evaluation of these three organic sulfur compounds on lung cancer H460cells demonstrated that DADS and AMDS could effectively inhibit H460cell proliferation while DAS showed a very limited anti-tumor effect. DADS and AMDS could block lung cancer H460cells in G2/M phase, induce cell cycle arrest, up-regulate p53and p21protein expressions, and reduce expressions of PCNA, cyclin B1and cyclin A2proteins. DADS and AMDS could also decrease the migration ability of lung cancer cells H460by increasing the E-cadherin protein expression level. These studies will certainly contribute to the deeper understanding of the anticancer mechanism of these garlic organosulfur compounds and provide a solid starting point for the use of these compounds in the clinical treatment of non-small cell lung cancer.