Carnosic Acid Reverses The Multidrug Resistance Of Leukemia Cells In Vivo

Carnosic acid (CA) is one of the main active compounds of rosemary (Rosmarinus), has a variety of biological activity of anti-oxidant, anti-inflammatory and anti-tumors. CA anti-inflammatory and anti-tumor activity is obtained from animal in vivo and cell lines in vitro.There is no animal toxicity testing data reported of CA.Purpose:Increasing interest in carnosic acid (CA) is due to its pharmacological properties. The aim of this study was to evaluate the acute and 30-day oral toxicity of CA.Methods:The acute oral toxicity study in Kunming mice design followed the OECD-guidelines 423, and a 30-day chronic oral toxicity study in Wistar rats based on the enhanced OECD test guideline 407 were performed.The acute oral toxicity study of Kunming mice were divided into six groups, Group 1 (control, Omg/kg) received olive oil, and groups 2 to 6 received CA at the dosage of 3500,4500,5500,7500 and 8500 mg/kg body weight, respectively. All the test substances in the study were prepared in distilled olive oil and administered oral gavage. Histopathological examination was performed for all living mice on day 15.Wistar rats, at 7 weeks of age, were weighted and randomly assigned to four groups. CA was suspended in the olive oil and was administered to rats once daily by oral gavage at doses of 0 (control),150,300, and 600 mg/kg/day for at least 28 days. Blood samples were obtained from the abdominal aorta immediately prior to necropsy for hematology and clinical chemistry.All animals were subjected to complete necropsy at different time points, and the major organs and tissues were also collected and subjected to gross and microscopic examination.Results:In the acute oral mice toxicity study, the LD50 of CA to mouse was 7100 mg/kg (95% CI,6060-8940 mg/kg) body weight. The histopathological changes were only observed in the heart and liver of all mice treated with CA. Slight hydrops, cytoplasmic degeneration, and a single cell point necrosis were observed in the liver, and myocardial fibrosis and inflammatory cell infiltration were found in the heart for all survival mice treated with a single-dose CAIn 30-day oral toxicity study, no deaths were observed during the time. The mean values of the increased body weight in the three experimental groups were lower than that in the control group. However, when compared to control values the terminal body weights and body weight gains were not statistically reduced throughout the 4 weeks’ study。Treatment-related clinical chemistry changes found the mean of serum TP and AST in the high-dose and the intermediate-dose groups showed significant decline and significantly increased respectively when compared with the control group. For other tested terms there were not significant differences among the four groups.The histopathological changes could only be observed in the heart, liver and kidneys of the rats treated with high-dose CA. In the heart the myocardial fibrosis and inflammatory cell infiltration could occasionally be observed. In the liver a single cell point necrosis and inflammatory cell infiltration were detected in a few regional or hepatic sinus. In the kidneys, we found a few epithelial cells with cloudy swelling and empty. There were not significant difference of histopathological changes between low-dose CA group and control group.Conclusion:The LD50 of CA was 7100 mg/kg body weight for mice, high-dose CA (600 mg/kg per day) could result in the injury of the liver and myocardial muscle, especially for male rats and the toxicity of 30-day oral administration of CA in rats, which suggests that CA has a relatively low oral toxicity profile but its prolonged use, based on the enhanced OECD test guideline 407 at least 90-day oral toxicity study are needed to examine the sub chronic toxicity.The treatment outcome of acute myeloid leukemia (AML) has achieved great improvement, however it still faces up with a tremendous challenge. The drug resistance is also an obstacle in the treatment of acute leukemia. The resistance may be mediated by the multidrug resistance (MDR-1/P-gp) gene. Many compounds have been reported to be capable of modulating the MDR phenotype, however, few can be clinically applied due to their unacceptable side effects or toxicity at the doses required to be effective.Carnosic acid (CA), has been studied for the antitumor or antineoplastic behavior on different types of cancer cell lines/rat or mouse models. In the article the the antileukemic activity of CA combining with adriamycin was investigated in vivo by a K562/A02/SCID mouse model of human AML.Purpose:A NOD/SCID AML mouse model, which was set up by inoculation with K562/A02 cells, was used to study whether leukemia cells growth in vivo can be inhibited by CA combining with adriamycin.Methods:All NOD/SCID mice were given cyclophosphamide 2mg/mouse for three days. Using an adjusted K562/A02 cell concentration of 5x107/ml, each mouse was inoculated with 1x107/ml of the cells through their tail vein.After inoculation of K562/A02 cells in NOD/SCID mice for two weeks, six animals were killed to determinate whether the K562/A02/SCID leukemia model was successful established. White blood cells were separated from the blood. Expressions of human mdr and bcr/abl mRNA were performed using RT-PCR. The visible leukemia cells were determined from peripheral blood, bone marrow smears.The experimental group was fed with 1%(v/v) CA powder. After 2 weeks of feeding, a solution of 1 mg/kg adriamycin was injected by intraperitoneal injection, with an interval time of two days, and a total of 3 injections for the animals in the two groups. After feeding for 4 weeks, tail vein blood was collected, the number of the leukemia cells was calculated in the blood smears, and expressions of human mdr and bcr/abl mRNA were determined by RT-PCR. The percentages of apoptotic cells were determined for the two groups by flow cytometry analysis.Results:Two weeks after inoculation of K562/A02 cells in the NOD/SCID mice, a large number of infiltrated K562/A02 cells were found in the peripheral blood, bone marrow smears, and slices of neck lymph nodes and liver of mice. Expressions of human mdr1 and bcr/abl mRNA in the experimental animals were nearly at the same levels as in the positive control.Much more leukemia cells were in the peripheral blood and bone marrow smears. In peripheral blood smears the percentages of visible leukemia cells in 200 white cell counts were 20.0% and 32.5% for the CA-treated and the control groups respectively, and the difference was statistically significant. The percentages of apoptotic cells of mice treated with single adriamycin (11.2±1.80) and the mice treated with CA combining with adriamycin (15.5±1.56) have a significant difference (P<0.05)Expressions levels of mdr1 and bcr-abl mRNA and protein in the experimental animals were significantly lower than that of the control animals. In the slices of lung, liver, kidney and bone marrow, the pathological changes have no significant difference, but micro-vessels show much more leukemia cells in the control animals than those in the CA-treated animals.The median of 95% CI survival time is 19 (10.0-44.2) and 33 (29.4-36.6) days for the control group and the CA-treated group, respectively. The difference is statistically significant (P<0.05)Conclusion:In the present work we confirmed that CA combining with adriamycin can significantly extend the survival time in the K562/AO2 leukemia animal model. CA is a promising adjuvant chemical drug candidate for clinical AML therapy.