| BackgroundsGarlic(Allium sativum)has long been used both for flavoring and for the potential benefits of preventing and curing ailments in many cultures.Epidemiologic evidence showed that the raw garlic consumption was associated with reduced risk of various cancers.Over the past more than half a century,studies of antitumor activity of garlic have become a hot spot.Part of experimental studies have documented garlic and its organosulfur components are effective in inhibiting various cancers,especially in the most concentrated study of prostate cancer,followed by breast cancer,colon cancer,lung cancer,gastric cancer.According to incomplete statistics,the anti-tumor study of the main organosulfur compounds in garlic,such as diallyl sulfide(DAS),diallyl disulfide(DADS),diallyl trisulfide(DATS),S-allylcysteine(SAC)and S-allymercaptocysteine(SAMC),have been reported.So far,the marketed garlic products are usually used as health care products,such as garlic oil,aged garlic extracts,common garlic capsules and tablets.Among them,the efficacy of garlic oil and aged garlic extracts was severely limited by its mixture of complex components and low content of active ingredients.While a small amount of garlic drugs on the market were also used as anti-bacterial and anti-fungal drugs.These drugs were sold less and not used in a wider range of clinical applications such as anti-tumor.The reasons for this phenomenon were the lack of more in-depth and normative researches.Comprehensive analysis of existing literature showed that there are many unreasonable studies on the anti-tumor effects of garlic.First,the selection of the target compounds was too random.For example,the anticancer effects of DAS,DADS,DATS,AMDS,SAC,SAMC,black garlic extracts and garlic powder had been reported.But few articles investigated the structure-activity relationship or took the solubility,stability as well as metabolism into account.Secondly,the anti-tumor effect and its underlying mechanisms of some garlic compounds were evaluated just on cell level.Due to the lack of in vivo processes,such as drug absorption,distribution,metabolism and excretion,the cellular model was considered too ideal to be recognized.In particular,the cellular model cannot evaluate the role of garlic in the immune regulation.Some studies used the animal model for further investigation,but the establishment of xenograft model was not standardized.For example,some researchers started the treatment process as soon as cells were injected into the animals or when the size of tumor hadn’t reached the quantification;some other researchers directly injected drugs into the tumor,contrary to the intention of in vivo studies.Finally,most of the studies investigated the anticancer effect of single compound in vitro and in vivo.The combination therapy is rare.Besides,none of them considered the process of absorption,metabolism,and distribution after the organosulfur compounds were taken into the body.Therefore,most of the early anti-tumor activity studies were not systematic and non-standard,leading to the lower application value.The target compound of garlic organic sulfur components and the tumor treatment model were determined by the comprehensive consideration of the activity,stability,toxicity and epidemiology of compounds.Studies on the structure-activity relationship of antitumor drugs in early stage showed that allyl sulfide was a functional group in garlic organic sulfur compounds.The anti-cancer activity increased with the increase of the number of sulfur elements,but its stability decreased and the toxicity increased.After the comprehensive consideration,the diallyl trisulfide(DATS)single component was selected as the compound Object.The choice of anti-tumor application model was based on epidemiological statistics,large data statistics showed that daily consumption of garlic had a positive effect in reducing the incidence of gastric cancer and lung cancer.The in vivo and in vitro experimental of this subject was designed to evaluate the pharmacological and antitumor activity of DATS with high activity in garlic.The main research content consists of the following three pairts:(1)the study of in vitro and in vivo drug metabolism.(2)the preclinical evaluation of pharmacodynamics in anti-gastric and anti-lung cancer,(3)the study of DATS combined with cisplatin to reduce the side effects of cisplatin.Our study would compensate for the inadequate experimental design flaws in previous researches and provide some useful evidence for the development and utilization of garlic.Methods and ResultsFirst,we did an in-depth study and comprehensive evaluation to choose the research objective.The structure-activity relationship study indicates that Allyl group and Sulfur play a key role in the anti-cancer activity of garlic compounds.Numerous researches about DAS,DADS,DATS,and four Sulfur compounds also show that with the increasing of Sulfur,the anti-cancer activity has significantly enhanced along with increased toxicity.Above all,DATS alone was chosen as the research objective.We choose the gastric cancer model and lung cancer model from the enormous cancer models to evaluate the anticancer activity.First,given that the incidence of gastric and lung cancer shows an upward trend and the mortality remains high,it’s urgent to find more effective therapeutic method.Secondly,epidemiological studies indicated that a garlic-rich diet decreases risk of some cancers such as gastric,stomach,and lung cancer.Furthermore,from the point of pharmacokinetic,most Sulfur compounds linger in the stomach and are digested later and its volatility makes it easier to be excreted to the outside of the body through lung instead of other organs.So the stomach and lung are more likely to be exposed to drug.In conclusion,the main content of this study included three sections:(1)Metabolism and transformation of garlic organic sulfur compounds DATS invitro and in vivo:In the study of drug metabolism,the GC-MS analysis showed that only two metabolites were Methyl allylsulfoxide(AMSO)and methylallyl sulfone(AMS02)in plasma,and the substrate peak of DATS was not detected by single dose and multi-dose administration of DATS,indicating that DATS had a faster metabolism in the blood.We chose the blood as a model of in vitro metabolism.The results showed that DATS was metabolized into two major metabolites DADS and allyl mercaptan(AM)in blood cells by HPLC analysis.In order to explain the inconsistency of metabolic results in vitro and in vivo,we conducted a more in-depth study of DATS metabolic transformation.Comprehensive literatures suggested that AMS may be its intermediate product.According to the principle of methylation reaction,AM was methylated in blood cells to form AMS,and then AMSO and AMS02 were produced by oxidative reaction of AMS in liver microsomes.While previous studies have reported that DADS could be transformed into AM.Therefore,we get a transformation pathway of DATS→DADS→AM→AMS→AMSO→AMS02.In dynamics study,DATS has a rapid metabolism process in vivo,while its metabolites AMSO and AMS02 have a long retention in rat plasma.(2)To evaluate the anticancer effect of DATS on gastric cancer and lung cancer.The antitumor activity of DATS was assessed on gastric cancer SGC-7901 and BGC-823 cells and lung cancer NCI-H460 cells.The results showed that DATS had a significant inhibitory effect on these tumor cells.The mechanism of DATS was achieved by regulating cyclins protein to arrest cell cycle progress,activating caspase cascade,regulating bcl-2 family protein and MAPK pathway to induce cell apoptosis.In the in vivo antitumor study,we established the xenograft model firstly.When tumor volume reached 100-200 mm,mice were randomly divided into groups.The evaluation indicators include tumor volume,relative tumor volume,and relzative rate of tumor growth.We observed that DATS administration in mice effectively inhibited the growth of xenografts,while the body weight was not declined obviously.However.the relative tumor growth rate T/C(%)value of DATS on each tumor was calculated.which did not meet the requirement of less than 40%.Although DATS had certain anti-gastric cancer and lung cancer effect,might need to be used in conjunction with other anti-tumor drugs.Besides,DDP group(5 mg/kg)caused a notable loss of body weight in mice,which indicated a severe injure to the body.In view of the chemo-protection role of garlic compounds,we designed DDP and DATS combined treatment.Results showed that DATS had enhanced the antitumor effect of DDP.More importantly,DATS significantly alleviated the loss of body weight caused by DDP-treated alone group.(3)To investigate the protective effect of DATS and its active metabolites on cisplatin-induced nephrotoxicity.Cisplatin has a variety of side effects in clinical practice,in which nephrotoxicity was a very common clinical adverse reaction.Therefore,we focused on the protective effect of DATS by establishing DDP-induced BAL b/c mouse kidney injury model.The results showed that DATS pretreatment effectively reduced the renal dysfunction caused by DDP,such as the regulation of inflammatory factor expression,modification of the renal tubular injury,reduction of inflammation cell infiltration,and so on.To further investigate the mechanism,we examined the cytotoxic effects of cisplatin and possible protective effects of DATS on proximal tubular HK-2 cells by using SRB assay.The results showed that DATS didn’t have the protective effect in vitro test.Then we detected the blood and kidney tissues homogenate by GC-MS and found a certain amount of AMS02 in blood and kidney.So we examined the protective effect of AMS02 on cisplatin-induced cytotoxicity.The effect of AMS02 on cell viability,apoptosis and related caspase proteins was examined on HK-2 cells.AMS02 was confirmed to significantly attenuate cisplatin-induced renal damage.Moreover,AMS02 effectively reduced cisplatin-induced ROS in kidney cells.DDP activated the phosphorylation of ERK,JNK,p38,increased pro-inflammatory cytokine levels(COX2,NFκB)and inhibited the expression of anti-inflammatory cytokine,IκBα After the combination of AMS02,the situation had improved significantly.In terms of role of MAPK-specific inhibitors,JNK and ERK inhibitors significantly increased cell viability and reduced cell apoptosis.Thus,JNK and ERK may be a key factor in the role of AMS02.ConclusionsThe biological transformation pathway of DATS was evaluated by various kinds of in vitro and in vivo models and the main metabolites were confirmed.These results in vitro and in vivo anti-tumor activity of DATS showed that its anti-gastric cancer and lung cancer efficacy did not reach the regulatory requirements of T/C(%)value of less than 40%.Based on the anti-tumor characteristic of DATS,we creatively proposed a combination therapy for cisplatin and DATS administration and demonstrated the positive effect of DATS on improving the efficacy of cisplatin.After evaluating the antitumor activity of DATS in vitro and in vivo,the treatment regimen of cisplatin was put forward according to its anti-tumor characteristics,and the positive effect of DATS on improving the curative effect of cisplatin was verified.Due to the common injury in kidney,we further investigated whether DATS has the protective effect against cisplatin-induced nephrotoxicity.We focused on the active metabolites AMS02 at the first time and explored the possible mechanism underlying this nephroprotective effect.This study provided theoretical and experimental reference data for further metabolic and pharmacodynamics of garlic-based organic sulfur compounds. |
