Effects of diallyl trisulfide on induction of apoptotic death in murine leukemia WEHI‐3 cells in vitro and alterations of the immune responses in normal and leukemic mice in vivo

Diallyl trisulfide (DATS), a chemopreventive dietary constituent and extracted from garlic, has been shown to against cultured many types of human cancer cell liens but the fate of apoptosis in murine leukemia cells in vitro and immune responses in leukemic mice remain elusive. Herein, we clarified the actions of DATS on growth inhibition of murine leukemia WEHI‐3 cells in vitro and used WEHI‐3 cells to generate leukemic mice in vivo, following to investigate the effects of DATS in animal model. In in vitro study, DATS induced apoptosis of WEHI‐3 cells through the G0/G1 phase arrest and induction of caspase‐3 activation. In in vivo study DATS decreased the weight of spleen of leukemia mice but did not affect the spleen weight of normal mice. DATS promoted the immune responses such as promotions of the macrophage phagocytosis and NK cell activities in WEHI‐3 leukemic and normal mice. However, DATS only promotes NK cell activities in normal mice. DATS increases the surface markers of CD11b and Mac‐3 in leukemia mice but only promoted CD3 in normal mice. In conclusion, the present study indicates that DATS induces cell death through induction of apoptosis in mice leukemia WHEI‐3 cells. DATS also promotes immune responses in leukemia and normal mice in vivo. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1343–1353, 2015.     

Sodium 2-propenyl thiosulfate derived from garlic induces phase II detoxification enzymes in rat hepatoma H4IIE cells

There is evidence that onions and garlic protect against cancer in humans. It has been suggested that this effect is partly due to the organosulfur compounds in Allium vegetables and that these substances act through induction of phase II detoxification enzymes. Here, we hypothesized that alk(en)yl thiosulfates, sodium n-propyl thiosulfate (NPTS), and sodium 2-propenyl thiosulfate (2PTS), which were identified in onions and garlic, respectively, may induce phase II enzymes. Therefore, rat hepatoma cells (H4IIE) were cultured with 1 to 100 μmol/L of NPTS or 2PTS for 48 hours at 37°C; and the activities and messenger RNA (mRNA) expression levels of phase II enzymes in H4IIE cells were investigated. The effects of diallyl trisulfide and tert-butylhydroquinone, known as phase II inducers, were also examined as positive controls and compared with the responses of NPTS and 2PTS. Quinone reductase (QR) activity and mRNA expression levels of QR and epoxide hydrolase 1 were significantly increased by 2PTS (P < .05-.005). In particular, QR activity was increased at a relatively low concentration of 2PTS (10 μmol/L). However, glutathione S-transferase activity and mRNA expression levels of glutathione S-transferase A5 and uridine diphosphate glucuronosyl transferase 1A1 were not changed by 2PTS. In contrast, NPTS did not affect the activities and mRNA expression levels of these phase II enzymes. These results show that 2PTS can induce phase II enzymes, and its inductive effect is comparable or superior to that of diallyl trisulfide and tert-butylhydroquinone.     

大蒜素微球体外释放研究

目的:研究聚乳酸大蒜素微球释放规律.方法:采用动态透析技术研究聚乳酸大蒜素微球的体外释药性能,并用高效液相色谱法测定大蒜素含量,以累计释药百分率进行不同模型的拟合.结果:聚乳酸大蒜素微球的释放曲线符合双相动力学方程.结论:聚乳酸大蒜素微球具有良好的缓释作用.     

Diallyl trisulfide inhibits cell migration and invasion of human melanoma a375 cells via inhibiting integrin/facal adhesion kinase pathway

Melanoma is the leading cause of death from skin disease due to its propensity for metastasis. Studies have shown that integrin‐mediated focal adhesion kinase (FAK) signal pathway is implicated in cell proliferation, survival and metastasis of tumor cells. Our previous results indicated that diallyl trisulfide (DATS) provided its antimelanoma activity via inducing cell cycle arrest and apoptosis. The aim of this study was to explore DATS mediated antimetastatic effect and the corresponding mechanism in human melanoma A375 cells. We found that DATS exhibited an inhibitory effect on the abilities of migration and invasion in A375 cells under noncytotoxic concentrations analyzed by wound healing assays and Matrigel invasion chamber system. DATS attenuated invasion of A375 cells with characteristic of decreased activities and protein expressions of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9. Moreover, DATS exerted an inhibitory effect on cell adhesion of A375 cells, which is in correlation with the change in integrin signaling pathway. Results of Western blotting showed that DATS decreased the levels of several integrin subunits, including α4, α5, αv, β1, β3 and β4. Subsequently, DATS induced a strong decrease in total FAK, phosphorylated FAK Tyr‐397,‐576, ?577, and disorganized F‐actin stress fibers, resulting in a nonmigratory phenotype. These results suggest that the antimetastatic potential of DATS for human melanoma cells might be due to the disruption of integrin/FAK signaling pathway.     

DATS通过NF-κB抑制LPS诱导小鼠肺泡巨噬细胞促炎细胞因子表达

目的探讨二烯丙基三硫(DATS)抑制脂多糖(LPS)诱导小鼠肺泡巨噬细胞肿瘤坏死因子-α(TNF-α)及白介素-1β表达的信号转导机制。方法体外培养MH-S细胞,用DATS和(或)LPS进行干预。反转录PCR检测细胞中TNF-α、IL-1β mRNA表达,电泳迁移率改变分析(EMSA)检测细胞核因子-κB(NF-κB)活性,Western blot检测细胞磷酸化(p-IκB)及非磷酸化IκB的表达。结果LPS刺激MH-S细胞可导致TNF-α、IL-1β mRNA、p-IκB表达增加及NF-κB活性升高。用DATS(0.1、0.5、2.5、5.0mg.L-1)预处理细胞30min后再给予LPS刺激,可使TNF-α、IL-1β mRNA表达降低,并呈剂量依赖性;升高的NF-κB活性及p-IκB表达均显不同程度的抑制。单独DATS对TNF-α、IL-1β mRNA表达及NF-κB活性无影响。结论DATS可通过抑制IκB磷酸化及NF-κB活化,进而下调LPS诱导小鼠肺泡巨噬细胞TNF-α、IL-1β mRNA表达。     

大蒜素自微乳的制备与质量评价

目的:制备大蒜素(DATS)自微乳制剂,并评价其质量.方法:采用伪三元相图法,以聚氧乙烯氢化蓖麻油RH-40为表面活性剂,无水乙醇为助表面活性剂,油酸为油相,绘制该系统的相图,在此基础上制备DATS自微乳;建立HPLC法测定DATS自微乳中DATS的含量;对自微乳的外观性状、相对密度、黏度、pH值、电导率、形态、粒径及粒度分布、Zeta电位、含量、配伍和稳定性等进行研究.结果:DATS自微乳为无色澄明液体,稳定性良好,相对密度为0.840 g·mL-1,黏度为5.447 mPa·s,遇水形成O/W型微乳,稀释250倍后电镜下观察成圆球形,平均粒径26.4 nm,Zeta电位0.398 mV,pH值5.62,电导率为8.37μs·cm-1,3批制剂的含量分别为31.77,31.45,32.15 mg·mL-1.DATS自微乳与葡萄糖注射液、氯化钠注射液等配伍稳定.结论:DATS自微乳制备工艺简单,性质稳定,质量易控.     

A cell-based system to identify and characterize the molecular mechanism of drug-metabolizing enzyme (DME) modulators

Many naturally occurred or synthetic compounds can modulate the body's drug-metabolizing enzymes to enhance carcinogen detoxification, and some have demonstrated remarkable cancer prevention effects. Understanding the molecular mechanism behind each candidate agent is critically important in designing rational cancer chemoprevention strategies. In this work, we have employed a set of molecular mechanism-based assays and characterized eight classes of known drug-metabolizing enzyme (DME) modulators in a cellular system. Examination of mRNA and protein levels of representative phase I and phase II enzymes validated the results obtained in our cell-based system. Our data confirmed that the antioxidant ethoxyquin (EQ) and the isothiolcyanate sulfurophane (SFP) exclusively activate the antioxidant response element (ARE), and thus represent monofunctional inducers. We were also able to reclassify some compounds, and to use the system to identify structure-activity relationships among structurally related but different compounds. Finally, this cell-based system permitted us to identify a potential novel mechanism for cross-talk between the ARE and the xenobiotic response element (XRE)-mediated pathways.     

DATS通过p38MAPK通路抑制LPS诱导的小鼠MH-S细胞TNF-α及IL-1β表达

目的探讨p38MAPK在二烯丙基三硫(DATS)抑制脂多糖(LPS)诱导小鼠肺泡巨噬细胞促炎细胞因子表达中的作用。方法体外培养MH-S细胞,用DATS和(或)LPS进行干预,Western blot检测细胞p38及磷酸化p38(p-p38)的表达;用LPS和(或)SB203580孵育细胞,反转录PCR检测细胞中TNF-α、IL-1βmRNA表达,Western blot检测细胞磷酸化(p-IκB)及非磷酸化IκB的表达。结果 LPS刺激MH-S细胞可导致p-p38表达增加,呈时间依赖性;用DATS(0.1、0.5、2.5、5.0 mg.L-1)预处理细胞30 min后再给予LPS刺激,p-p38表达呈剂量依赖性下降;单独DATS对p-p38表达无明显影响。p38特异性抑制剂SB203580可剂量依赖性地抑制LPS诱导的p-IκB蛋白、TNF-α及IL-1βmR-NA表达。结论 DATS可通过抑制p38MAPK通路抑制IκB磷酸化及NF-κB活化,进而下调LPS诱导小鼠肺泡巨噬细胞TNF-α、IL-1βmRNA表达。     

Diallyl trisulfide-induced apoptosis of bladder cancer cells is caspase-dependent and regulated by PI3K/Akt and JNK pathways

Diallyl trisulfide (DATS) is one of the major organosulfur components of garlic (Allium sativum L.), which inhibits the proliferation of various cancer cells, but the exact mechanisms of this action in human bladder cancer cells still remain largely unresolved. In this study, we investigated how DATS induces apoptosis in T24 human bladder cancer cells in vitro. Treatment of T24 cells with DATS resulted in potent anti-proliferative activity. Additionally, some typical apoptotic characteristics, such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells, were observed. With respect to the mechanism underlying the induction of apoptosis, DATS reduced the expression of anti-apoptotic Bcl-2 and Bcl-xL, and inhibitor of apoptosis protein family proteins, but the expression of pro-apoptotic Bax and death receptor-related proteins was increased compared with the controls. DATS also activated caspase-8 and -9, the respective initiator caspases of the extrinsic and the intrinsic apoptotic pathways. The increase in mitochondrial membrane depolarization was correlated with activation of effector caspase-3 and cleavage of poly-ADP-ribose polymerase, a vital substrate of activated caspase-3. Blockage of caspase activation through treatment with a pan-caspase inhibitor consistently inhibited apoptosis and abrogated growth inhibition in DATS-treated T24 cells. The study further investigated the roles of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) pathways with respect to the apoptotic effect of DATS, and showed that DATS deactivates Akt. Additionally, DATS activates extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK), but not p38 MAPK, in T24 cells. Unlike ERK, JNK inhibitors reversed DATS-induced apoptosis and growth inhibition; however, inhibition of PI3K/Akt notably enhanced the apoptotic action of DATS. The results suggest that the pro-apoptotic activity of DATS is probably regulated by a caspase-dependent cascade through the activation of both intrinsic and extrinsic signaling pathways, which is mediated through the blocking of PI3K/Akt and the activation of the JNK pathway.