Enhancement Of Antitumor Activity And Attenuation Of Cardiotoxicity Of Anthracyclines By Schisandrin B In Vitro And In Vivo

Cancer is a major threat to human's life. Chemotharapy is one of the main approaches to cancer therapy. However, cancer chemotherapy has been significantly hindered by the cancer multidrug resistance and chemo-agent-associated side effects. Multidrug resistance (MDR) refers to cross resistance of cancer cells toward a diverse varieties of structurally and functionally unrelated anticancer drugs. Over expression of drug transport proteins such as P-glycoprotein(P-gp), multidrug resistance-associated protein 1(MRP1) , accounts for most of the clinical MDR cancers. These drug transport proteins can unilaterally extrude the intracellular drugs out of cells, and keep the intracellular drug at sublethal levels, by which cancer cells acquire MDR. In addition to MDR, the side effects of anticancer drugs have significantly interfered chemotherapy. Notably, anthracycline antibiotics, although indispensable in cancer therapy, has demonstrated severe cardiotoxicities, which could be even exuberated when combined with other anticancer drugs such as taxol, Trastuzumab, and Cox-2 inhibitors.One of the keys to increase the effectiveness of chemotherapy is to overcome cancer MDR and simultaneously attenuate the anthracycline-associated cardiotoxicities. Although there are several MDR reversal agents which have entered clinical trial, none isavailable clinically. Only one drug, dexrazoxane (ICRF-187 ) , has been approved by US FDA, for the use against doxorubicin- associated cardiotoxicities. However, this drug has been concerned on the aspects of its interference of the efficacies of anthracyclines, the potential carcinogencity toward bone marrow cells, and among others. Therefore, agents that can reverse cancer drug resistance and simultaneously mitigate anthracycline-associated cardiotoxicity are badly needed clinically.Many compounds have demonstrated activities to reverse cancer MDR in vitro. There also have been some reports on agents which could mitigate anthracycline-associated cardiac toxicities. However, these agents showed only single function, either reversing cancer MDR or mitigating doxorubicin-associated cardiotoxicity. There is no report about an agent which has dual functions. It could be expected that an agent with dual function would have broader clinical application than a mono-funcitonal agent.In our previous studies, we found that naturally-occurring dibenzocyclooctadiene lignan, is a novel class of P-gp inhibitors. Because the structural and functional similarity between P-gp and MRP1, here, we propose that this class of compounds may have the capacity to reverse MRP1 mediated cancer drug resistance. We also assume that this class of compound can increase the apoptosis in cancer cells induced by anticancer drug. Lastly, we hypothesized that this class of compound can attenuate anthracycline-associated cardiotoxicities, because dibenzocyclooctadiene lignan is able to increase cellular GSH redox cycling, which can neutralize the peroxides generated from doxorubicin.Therefore, if dibenzocyclooctadiene lignan could have dual function against cancer MDR and anthracycline-associated cardiotoxicities, this class of agents would have potentials for broad clinical application.In this study, Firstly ,we investigated the reversal of MRP1-mediated multidrug resistance in human leukemia cell lines HL60/ ADR and HL60/ MRP by dibenzocyclooctadiene lignans. Secondly, we measured the effect of Sch B on DOX-induced apoptosis in SMMC7721, a human hepatic carcinoma cell line; lastly, we evaluated the role of Sch B in the mice or rat models in comparison with that of Dox with the following specific aims: 1. to evaluate the effect of SchB on the antitumour activity of DOX in S180 mice; 2. to detedrmine the cardioprotective effect and other roles of Sch B in S180 mice; 3. to discuss the cardioprotective effects and mechanisms of Sch B in the acute heart injurymodel induced by DOX in ICR mice and in S-D rat. Based on the above studies, we hope to interpret mechanisms of dibenzocyclooctadiene lignans enhanced-antitumour activity and reduced-cardiotoxicity of anthracycline.Part Ⅰ The study of dibenzocyclooctadiene lignans-inhibited multidrug resistance-associated protein 1 in vitro.The activities of 5 dibenzocyclooctadiene lignans (schisandrin A, schisandrin B, schisantherin A, schisandrol A, and schisandrol B) to reverse MRP1-mediated drug resistance were tested using HL60/ADR and HL60/MRP, the human promyelocytic leukemia cell lines with the overexpression of MRP1 but not P-gp. The growth inhibition assay of schisandrin A, schisandrin B, schisantherin A, schisandrol A, and schisandrol B combining with vincristine, daunorubicin, and VP-16 in HL60/ADR and HL60/MRP detected by FCM ;The DNR and CFDA accumulation assay in the presence or absence of schisandrin A, schisandrin B, schisantherin A, schisandrol A, or schisandrol B or probenecid also using FCM. The results were described as below:1. The 5 lignans could effectively reverse drug resistance of the 2 cell lines toward vincristine, daunorubicin, and VP-16. The reversal folds of drug resistance are from 2 to 70.2. Dibenzocyclooctadiene lignans differentially restore intracellular drug accumulation in HL60/ADR and HL60/MRP.Part Ⅱ The study of Schisandrin B-enhanced doxorubicin-induced apoptosis of cancer cells in vitroThe toxicity of SchB was evaluated by incubation of SMMC7721 cells with Sch B, and followed by a MTT assay to calculate the IC50 and by a flow cytometry (FCM) asay for apoptosis; SchB enhanced apoptosis induced by doxorubicin was determined by incubation of SMMC7721 cells with doxorubicin in the presence or absence of Sch B. The general caspase inhibitor, zVAD-FMK., was added to incubation to check if Sch B-enhanced apoptosis was associated with caspase activation. The apoptotic cells were determined by propidium iodide(PI) staining and analyzed by FCM; The effect of Sch B on DOX uptake and efflux was determined by incubation of SMMC7721 with doxorubicin in the presenceor absence of Sch B, and followed by measuring intracellular doxorubicin by a FACs Caliber; MDR1 and MRP1 mRNA and protein were determined by quantitative real-time RT-PCR and FACS; Loss of mitochondrial membrane potential was assessed by FCM, using Rhodanminl23; The expression of caspase-3,-8,-9 and PARP were checked with Western Blot. The results were described as below:1. In SMMC7721 cells,. the early and later apoptotic cells constituted for 5.1±2.8% and 15.5±4.3%, respectively, when the cells were treated with DOX alone, whereas they constituted for 12.2± 1.1% and 27.0± 6.7%, respectively, when the cells were treated with DOX and Sch B combined. Since Sch B alone did not induce apoptosis, it is obvious that Sch B is able to augment the potency of DOX.2. In rat cardiomyocytes, the apoptotic rates induced by DOX alone or combined with Sch B constituted 7.8 ±2.1% and 8.82 ± 1.03%,respectively. Similar results were observed when human fibroblasts were treated with DOX in the presence or absence of Sch B. Showed that Sch B does not enhance DOX-induced apoptosis of normal cells.3. The expression of MDR1 mRNA in SMMC7721 cells treated with DOX for different time intervals were determined using quantitative real-time RT-PCR. Although MDR1mRNA was detected after exposure to DOX, it only constituted less than 0.1% of the positive control. We determined P-gp in those cells by FACS analysis. The results demonstrated that expression of P-gp is negligible in the cells treated with DOX. Therefore, Sch B enhanced DOX-induced apoptosis is unlikely via its action on P-gp. We next checked the expression of MRP1 mRNA in SMMC7721 cells treated with DOX for different times using RT-PCR . MRP1 mRNA was not detected ,proving that the Sch B enhanced DOX potency is not a consequence of its inhibition of MRP1.SMMC7721 cells were exposed to 5 mM DOX in the presence or absence of Sch B (50 mM), and intracellular DOX was determined by FCM at the indicated times. The intracellular DOX in the presence or absence of Sch B was comparable without a significant difference. Likewise, there was no significant difference of DOX efflux between the two groups. These data indicated that Sch B enhanced DOX-induced apoptosis was not a result of its action on any DOX-efflux pumps.4. zVAD-FMK, a general caspase inhibitor, was applied to SMMC7721 cells which weretreated with a mixture of DOX and Sch B. The results showed that zVAD-FMK not only blocked the apoptosis induced by DOX alone, but also abolished the difference in the apoptotic rate between the two groups, indicating Sch B enhanced apoptosis is caspase dependent.5. In SMMC7721 cells, DOX combined with Sch B treatment caused a pronounced reduction of procaspase-3, as compared with DOX alone, indicating the enhanced cleavage of the pro-caspase-3 to its active form Correspondingly, the cleavage of PARP was the most pronounced by the DOX-Sch B combined treatment, agreeable with the amount change of pro-caspase-3. Sch B alone activated neither pro-caspase-9 nor pro-capase-8 in SMMC7721 cells. The activation of caspase-8 was comparable between DOX alone and combined treatment. On the other hand, Sch B and DOX combined treatment caused a cleavage of pro-caspase-9 to its active forms, significantly more than DOX alone. These results indicated that Sch B enhanced DOX-induced apoptosis most likely via mitochondrial pathway. After exposure to 1 mM DOX in the presence of 50 mM Sch B for 48 h, cells exhibited much lower Rhl23 staining (270.08 ±3.93) than control's 408.46± 5.22 (P < 0.01), although DOX alone reduced Rhl23 staining mildly (360.55± 3.65). There were no significant differences between Sch B alone and control.. These results proved that combined treatment caused the loss of the mitochondrial membrane potential, it agreed to the former.6. SMMC7721 cells were treated with DOX, mitoxantrone or vincristine in the presence or absence of Sch B. The results demonstrated that Sch B indiscriminately enhanced the potency of the three drugs, indicating that apoptotic enhancement by Sch B was irrelevant to the latter's capacity to generate ROS.Part Ⅲ The study of Schisandrin B enhanced antitumor effect and lessened toxicity of DOX in S180 miceFor the cell viability assay, S180 cells were incubated with Sch B and/or DOX for 2 days, followed by a MTT assay. S180 cells were injected into the bilateral flanks of male ICR mice, then they were randomly assigned to 5 groups: ①SI 80 control,②Sch B group,③Dox group,④Dox+Sch B1 group, ⑤Dox+Sch B2 group. DOX(2mg/kg) was administered ip to mice after intragastrically dosed with Sch B(50mg/kg or 100mg/kg) or 0.9% NaCl, every 2 days for 14 days. The tumor volume and weight of mouse were measured every 2 days. At the end of the experiment (24 hours following the last Dox injection), mice were bled from the reterorbital sinus and then killed by cervical dislocation. Serum was separated and stored at -80°C until analysis. Subsequently, the tumor, liver, themus, kidney, heart, spleen were removed and weighed. Serum CK,LDH,SOD,AST and MDA were measured. The results were as below:1. In vitro, DOX alone showed concentration-dependent cytotoxic effects in S180 cells. Sch B combination with DOX resulted in a significant potentiation of DOX cytotoxicity compared to DOX alone. Sch B alone up to 200 μM had no significant cytotoxicity on S180 cells.2. Mice given DOX alone significantly inhibited tumor weight and volume cmpared with control mice(P<0.05). Intraperitoneal doxorubicin plus intragastrically Sch B inhibited the tumor growth more effectively than Dox alone. There was a significant difference in tumor weight and volume between the group receiving doxorubicin and those received doxorubicin plus Sch B(100 mg/kg) (P<0.05). Sch B alone had no inhibitory effect on tumor growth. Therefore, Sch B increased the antitumor activity of doxorubicin .3. A remarkable reduction in body weight was observed in the mice receiving doxorubicin compared with control mice. Administration of Sch B before DOX significantly prevented the doxorubicin-induced reduction in body weight(P<0.05).4. Mice given DOX alone resulted in a reduction in weight of liver, heart, kidney, spleen and thymus compared with control mice(P<0.05). Mice given Sch B before DOX prevented the reduced weight of the organs. There was a significant difference in themus weight and kidneys between the group receiving doxorubicin alone and those receiving doxorubicin plus Sch B(100 mg/kg) (P<0.05). These results indicate that the toxicity and other side effect of of doxorubicin could be at least partially prevented by the Sch B.5. In Mice given DOX alone, serum LDH, CK, AST activities and MDA content were significantly increased compared with the S180 control while SOD activity were significantly decreased (P<0.05). In mice given Sch B(100 mg/kg) combination withDOX showed a significant tendency to reduce serum CK, LDH, AST, and MDA and improving serum SOD activity compared to DOX group(P<0.05). To GOT and MDA , Sch B(50 mg/kg) combination with DOX group also had a significant difference compared with DOX alone group (P<0.05). Sch B alone showed no significant effect on the enzyme levels. The results demonstrated that intragastrically pretreatment of the animals with schisandrin B prior to DOX administration attenuated the Dox-induced cardiac toxicity.Part Ⅳ The protective role of schisandrin B against doxorubicin-induced acute cardiotoxicity in mice216 ICR mice were randomly assigned to 6 groups: ①S180 control,②Sch B group, ③Dox group,④Dox+Sch B1 group, ⑤Dox+Sch B2 group,⑥Dox+Sch B3 group. Groups ①② and ③ were treated intragastrically with saline, and groups ④⑤⑥ were treated intragastrically with Sch B (100mg/kg, 50mg/kg or 25mg/kg) for 3 days, Then DOX(25mg/kg) was administered ip to mice. One part of mice were bled and then killed by cervical dislocation. Serum was separated and stored at -80℃ until analysis. Serum CK,CK-MB,LDH and GOT were measured. The heart was excised quickly and myocardial homogenates were prepared for MMP analysis. Five days after the Dox injection, the second part of mice were killed and left ventricle was excised and myocardial homogenates were prepared for the detection of MDA, GST, GR, GSH-PX, CAT, SOD, GSH/GSSG. Left ventricles were also processed for ultrathin section preparation for ultrastructural morphological examination by electron microscopy. The remaining mice were used for survival study. The results were as below:1. In Mice given DOX alone, serum LDH, CK, AST activities, and MDA levels were significantly increased compared with the saline-treated control (P<0.05). DOX+Sch B groups showed a significant tendency to reduce serum CK, CK-MB, LDH and GOT activity compared to DOX group. All the enzymes in Dox+Sch B1 (100 mg/kg ) group had a significant difference compared with DOX groups(P<0.05). CK and CK-MB activity in Dox+Sch B2 (50 mg/kg ) group and Dox+Sch B3(25 mg/kg ) groups also had a significant difference compared with DOX groups (P<0.05). LDH activity in Dox+SchB2 (50 mg/kg) group had a significant difference compared with DOX group(P<0.05). Sch B group showed no significant effect on the enzyme levels.2. Using metalloproteinase zymography analysis, A significantly increase of MMP-2 activity was observed in the left ventricle of mice receiving doxorubicin compared with control mice. All the Dox+Sch B groups showed a significant tendency to reduce MMP-2 activity compared to DOX alone treatment. Dox+Sch B1 (100 mg/kg )group had the best effect.3. The activities of GST, GR, GSH-PX, SOD, and GSH were lower and the levels of MDA and GSSG were higher in DOX group than in the saline group(P<0.05). The activities of CAT were also lower in DOX groups than in the saline group with no significant difference. The levels of MDA in all the 3 Dox+Sch B groups were significantly lower than that in DOX group (P<0.05). The activities of GST and SOD in Dox+Sch B1 (100 mg/kg ) group and Dox+Sch B2(50 mg/kg ) group were significantly higher than that in DOX group(P<0.05). The activities of GR and GSH-PX, Sch B (100 mg/kg ) significantly attenuated the Dox-induced reduction of GR and GSH-PX activities (P<0.05). The activities of CAT in all the 3 Dox+Sch B groups were also higher than in the DOX group but with no statistical significance. The activities of all the enzyme in Sch B groups had no significant difference from those in saline groups. The ratio of GSH/GSSG in all the 3 Dox+Sch B groups had a significant increase compared with DOX groups(P<0.05). The levels of GSH in Dox+Sch B1 (100 mg/kg ) groups were higher compared with DOX groups(P<0.05). The levels of GSSG in all the 3 Dox+Sch B groups were lowerer than in the DOX group, but had no significant difference. The results indicated that Sch B pretreatment could protect the myocardium against cardiotoxicity of DOX by enhancing the heart tissue glutathione status and alleviating the injury induced by lipid peroxidation.4. Administration of DOX in mice led to myocardial ultrastructural damage such as disarrayed myofibril, cytoplasmic vacuolization, mitochondrial swelling with lost cristae. Pretreatment with Sch B suppressed the damage, particularly alleviated the degree of cytoplasmic vacuolization, mitochondrial swelling, and cristae disappearance.5. No deaths were observed in animals receiveng saline or Sch B treatment alone. The survival rate of DOX groups was 16.7%, whereas Sch B at 100, 50, or 25mg/kg combinedwith DOX groups were 50%, 41.7%, and 33.4%, respectively, and the difference in DOX-induced mortality in the absence and the presence of Sch B was significant(P<0.05). Sch B pretreatment increased the survival rate of DOX-treatment groups.Part Ⅴ The protective role of schisandrin B against doxorubicin-induced acute cardiotoxicity in rats42 S-D rats were randomly assigned to 3 groups: ①control,②Dox group,③Dox+Sch B group. Groups ① and ② were treated intragastrically with saline, and group ③ was treated intragastrically with Sch B at 100mg.kg^-1.d^-1 for 3days. On day 4, DOX at 25mg/kg was administered ip to every group rats. Five days after the Dox injection, The Myocardial Contractile Function of the rats was recorded, observed and analyzed using the MedLab-U/4c biological signal collecting system. Results: Compared with control group, the LVSP, LVEDP and ±dp / dt of DOX group decreased significantly(P<0.05), while the LVSP, LVEDP and ±dp / dt of Sch B+DOX group increased significantly compared with those of DOX group(P<0.05), indicating that Sch B pretreatment could improve the cardiac contraction and diastolic functions in DOX groups.Conclusion:1. Dibenzocyclooctadiene lignan is a novel class of MRP1 inhibitors.2. Sch B, a member of dibenzocyclooctadiene lignans, significantly enhanced DOX-induced apoptosis of hepatic cancer cell SMMC7721. This enhancement was irrelevant to the action of Sch B on P-glycoprotein or on the production of ROS, but associated with the activation of caspase-9 rather than caspase-8. On the other hand, at the same experimental conditions, Sch B did not enhance the DOX-induced apoptosis of primary rat cardiomyocytes and primary human fibroblasts.3. In S180-bearing mice, Sch B significantly potentiated the anticancer activities of DOX while attenuated its cardiotoxicities.4. Using acute cardiotoxcity animal (mice and rat) models, we proved that SchB significantly prevented Dox-induced cardiac damage, including survival rate, myocardical function, changes of serum cardical enzymes, and levels of peroxides. The mechanism was associated with the potency of SchB that promotes the cellular antioxidant status including GSH redox cycling, SOD, which neutralized the peroxidative species generated from Dox.5. Sch B, a member of dibenzocyclooctadiene, is an only agent reported or found so far that has dual functions against cancer multidrug resistance and anthracycline-induced cardiotoxicities.