Anti-cancer And Anti-warburg Effects Of Rosmarinic Acid In Nude Mice Xenograft Model Bearing MKN-45 Gastric Cancer Cells

Gastric cancer is one of the most common cancers worldwide and has a high mortality rates. In china, the high incidence of advanced gastric cancer is attributed to delayed diagnosis and lack of effective therapies for metastasis. Although more extensive use of neoadjuvant chemotherapy, operation,chemotherapy together with Intraperitoneal Hyperthermic Perfusion Chemotherapy have been applied to clinical, overall survival for patients with advanced stage disease remains poor. The reason is associated with invasion and metastasis.The tumor microenvironment is involved in the process of gastric cancer. The intermediates of tumor cells play an important role in the formation of microenvironment. Most cancer cells exhibit increased glycolysis and use this metabolic pathway for generation of ATP as a main source of their energy supply. This phenomenon is known as the Warburg effect and is considered as one of the most fundamental metabolic alterations during malignant transformation. It has become widely accepted that the glycolytic intermediates provide essential anabolic support for cell proliferation and tumor growth. The Warburg metabolic phenotype is a widespread cancer-associated trait. Indeed, enhanced glucose uptake by cancer cells has become the basis for positron emission tomography (PET) with 18-fluorodeoxyglucose (FDG), which preferentially accumulates in tumor cells as a result of their rapid uptake of glucose. Because of the prevalence of this phenotype, PET has been an effective clinical imaging technique to detect most cancers and monitor therapeutic responses. With the help of gas chromatography-mass spectrometry, a quantitative measurement of organic acids in tissues from gastric cancer patients indicates increased glucose metabolism in gastric cancer, the levels of glycolytic products, including pyruvic acid and lactic acid, as well as the levels of ketone bodies, including 3-hydroxybutyric acid, were also significantly increased in cancer tissues compared to normal tissues. Application of an unrestricted ketogenic diet enriched with omega-3 fatty acids delayed tumour growth in a mouse xenograft model, and citrate could Induces apoptotic cell death, which indicated a promising way to treat gastric carcinoma. But further studies of the effective inhibitors that have anti-warburg effect are needed.The research of anti-tumor Chinese medicine has made great progress and has been internationally recognized recent years, and medicinal plant resources are rich in our country, most of them had shown good prospects. Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. It is commonly found in species of the Boraginaceae and the subfamily Nepetoideae of the Lamiaceae. However, it is also found in species of other higher plant families and in some fern and hornwort species. Rosmarinic acid has a number of interesting biological activities, e.g. antiviral, antibacterial, anti-inflammatory and antioxidant. The presence of rosmarinic acid in medicinal plants, herbs and spices has beneficial and health promoting effects. In plants, rosmarinic acid is supposed to act as a preformed constitutively accumulated defence compound. It has been reported that rosemary can suppress the development of tumors in several organs including the colon, breast, liver, as well as melanoma and leukemia cells. RA also has an anti-Warburg effect in gastric carcinoma in vitro. However, there is no report about whether RA has an anti-Warburg effect in gastric carcinoma in vivo, and the mechanism for the anti-Warburg effect is also ambiguous. This study aimed to study them.Objective:This study aimed to identify the anticancer and anti-warburg effect effects of rosmarinic acid (RA) in vivo, the effects of RA on tumor growth and proliferation in gstric cancer xenograft in nude mice. The underlying mechanisms involing in the expression of hexokinase-2(HK-2) and lactate dehydrogenase-A(LDHA) were also investigated in vivo after RA treatment.Methods:For subcutaneous implantation, 1×109 MKN45 cells suspended in 100 μL of phosphate-buffered saline were injected subcutaneously into the flank of BALB/c-nu mice. When the tumor cubage reached(74.08±17.89)mm3 after mouse xenograft models were successfully established,24 BALB/C (nu/nu) nude mice were randomly divided into 4 groups(n=6 each). And an intraperitoneal injection of rosmarinic acid (RA) was administered at 1 mg/kg (low-dose),2 mg/kg (middle-dose) and 4 mg/kg (high-dose) groups, twice per day respectively for 18 days while the mice in the control group were injected with saline. All the nude mice were sacrificed 19 days later, The growth curves of xenografts in nude mice were drawn and weight inhibition rates measured. An enzyme-linked immunosorbent assay was used to detect proinflammatory cytokines interleukin-6 (IL-6). The glucose and lactate concentration in samples was determined by glucose assay kit and lactate assay colorimetric kit. The mRNA transcription of IL-6,STAT3,HIF-1a,HK-2,LDHA were measured by Real Time-PCR. The expression levels of signal transducer and activators transcription 3α (STAT-3α) and phosphorylated of STAT-3α (pSTAT-3α), hypoxia-inducible factor lalpha (HIF-1α) were measured by Western blotting assay, the expression of glycolytic enzymes as hexokinases 2 (HK-2) and lactate dehydrogenase A (LDHA) were also detected by Western blotting. All experiments involving animals were approved by The Ethics Committee of the Southern Medical University. The animal experiment was conducted in accordance with national laws on animal welfare in the People’s Republic of China.Results:When solid tumors in BALB/c-nu mice were treated with RA, Tumor volume in nude mice treated with RA was smaller than that in control group, When tumor-bearing mice were treated with 2 mg/kg of RA for 12 days, tumor volume was less than the control group, the difference was statistically significant (F=137.34, P<0.001). when compared with the control group during the whole treatment period, there was no loss of body weight in the mice trated with 1 mg/kg and 2 mg/kg of RA, but the mice losed weight rather dramatically in the group of 4 mg/kg. Tumor weights of RA groups were lighter than those of the control(F=51.592, P<0.05).The rate of tumor inhibition in RA groups were 10.76%、16.03%、24.29%, respectively.The effect of RA on glucose consumption was decreased, meanwhile a graded decrease of lactate generation with increasing doses of RA was found in tumor tissues homogenate.Result of ELISA showed that treatment with RA resulted in decreased IL-6 secretion, the cytokine linked with chronic inflammation and cancer development.The expression mRNA of IL-6,STAT3,HIF-1a,HK-2,LDHA were detected by real-time PCR, compared with the control group, the mRNA levels of IL-6,STAT3,HIF-1α,HK-2,LDHA were lower in RA groups;The expression of STAT-3α, pSTAT-3α, HIF-1α, HK-2 and LDHA was measured by western blotting assay, according to the results, RA could suppress the expression of STAT-3α, pSTAT-3α, HIF-1α, HK-2 and LDHA compared with control group.Conclusions:RA can effectively inhibit the in vivo growth of gastric tumor. The mechanism may be correlated with the down-regulated expressions of IL-6, surppress the activation of STAT-3α, decreased expression of HIF-1α, HK-2, and LDHA. These results demonstrated that rosmarinic acid had an anti-Warburg effect in vivo. Therefore, RA might be a potential therapeutic agent to suppress the Warburg effect in gastric carcinoma.