Intervention Of Huayu Pills On Growth And Metastasis Of 4T1 Breast Cancer And Its Anti-tumor Mechanism Based On Platelet Pathway

Background:Breast cancer is the most common cancer for women and the second cancer-related deaths among females in the world.The major cause of the mortality is metastasis of the cancer cells.Recent research found that the blood hypercoagulation emerged in many cancer patients,which increased the risk of metastasis and postoperative recurrence.Reviewing the tumor microenvironment,platelets play important roles in creating the blood hypercoagulation and promoting hematogenous tumor metastasis.Present anti-platelet agents such as heparin and asprine have been shown therapeutic efficacy on tumor metastasis by depleting the platelets in the whole body.However,this kind of therapeutic strategy has clinical limitations as it is associated with some complications,such as gastrointestinal bleedings and hemorrhagic strokes,especially in patients who are already thrombocytopenic due to chemotherapy or radiotherapy.Thus,safer but effective anti-platelet agents should be investigated.Huayuwan(HYW)is a prescription and a Chinese herbal mixes,which was demonstrated that it is useful to treat the cancer patients.Curcumin,which is extracted from the plant curcuma Tonga,is a natural compound with no significant toxicity to humans and has been used for treatment of variety of diseases.Accumulating evidence suggests that curcumin is a highly pleiotropic molecule,having diverse biological activities.Although extensive research has shown that curcumin possesses anti-tumor effects,but its anti-metastasis potency and the underlying mechanisms are scarcely reported.Hence,in this study,we tried to explore the function of curcumin on regulating the systemic circulation coagulation in 4T1 breast tumor-bearing mice model by testing blood routine,coagulationand thrombelastograph.Metherd:(1)Cell culture and 4T1 tumor-bearing mice model4T1-luc cells(1×104)were implanted into the mammary fat pad of normal female BALB/c mice.From day12,100mg/kg Curcumin(Cur-L),300mg/kg Curcumin(Cur-H),23.4g/kg HYW and 46.8g/kg HYW were administered(p.o.)everyday for 33 days.(2)Bioluminescent imagingMice were then placed onto the warmed stage inside the light-tight camera box with continuous exposure to 1-3%isoflurane.The low levels of light emitted from the bioluminescent tumors or cells were detected by the IVISTM camera system,integrated,digitized,and displayed.(3)Metastatic score Formalin-fixed lungs were submitted for histopathology and the hematoxylin-eosin-stained microscopic sections were scored for the presence of pathological lesions.The whole lungs were imaged by a high speed digital slide scanners Pannoramic P250(3DHISTECH,Hungary).(4)Blood samples and routine blood tests.For laboratory measurements,100 ?L mouse whole blood was collected into tubes containing EDTA(BD Vacutainer,UK),an in vitro anticoagulant,prior to sacrifice.Routine blood tests were immediately performed using a Sysmex XT-2000i automated hematology analyzer(Sysmex Corp.,Hyogo,Japan)for the platelets(PLT)counts.(5)Coagulation tests of Activated partial tromboplastin time(APTT),prothrombin time(PT)and Fibrinogen(Fbg)The plasma samples were tested by a coagulation method for APTT,PT,and Fbg as previously described,respectively.In addition,blood was diluted 10 times by DI water in the test of Fbg.The results were expressed in s(PT,APTT),and g/1(Fbg level).(6)Thrombelastograph AssayThe TEG Hemostasis Analyzer(Haemonetics Corporation,USA)measures clot formation,clot strength,and clot degradation in whole blood by adding kaolin and calcium chloride(final concentration,10.5mMol/L),to enable a quantitative analysis of platelet function.(7)Flow Cytomery Analysis of P-Selectin Expression in Activated PlateletsPlatelet CD41(PE)expression was determined by flow cytometry to quantitate the total number of platelets and the proportion of activated platelets was determined using phycoerythrin-conjugated anti-P-selectin(CD62P,FITC).(8)Flow Cytomery Analysis of Annexin-V Expression in Apoptotic PlateletsCD41 to quantitate the total number of platelets,and the proportion of apoptotic platelets was determined using Annexin-V f.Samples were analyzed using a fluorescence-activated cell sorter.(9)Western BlotDetection of integrin(31 protein and integrin ?3 protein levels was performed by Western blot assays.Membranes were imaged using a LiCor Odyssey scanner.Boxes were manually placed around each band of interest,which returned near-infrared fluorescent values of raw intensity with intra-lane background subtracted using Odyssey 3.0 analytical software(LiCor,USA).(10)Statistical Analysis Data are presented as mean ± SEM.Statistical analysis was performed using GraphPad Prism(Graphpad Software,La Jolla,CA,USA).P Values<0.05 were considered statistically significant.Result:(1)HYW and Curcumin inhibited 4T1 tumor growth and lung metastasis in mice BALB/c female mice were inoculated with 4T1-luc cells.When the tumor volume reached 80 mm3(12 days' after implantation),saline(control),low dose curcumin(Cur-L,100mg/kg)and high dose curcumin(Cur-H,300mg/kg),low dose HYW(HYW-L,2.34g/kg)and high dose HYW(HYW-H,4.68g/kg)were respectively administered by daily oral feeding,and tumor volume was measured at the same time.After 3 weeks' treatment,the total luc signal at the tumor site in both Cur-L and Cur-H,HYW-L and HYW-H was lower than the control group,which was consistent with the tumor weightand tumor growth curves.These results exhibited that the treatment with HYW or curcumin significantly attenuated tumor growth at the dose-dependent manner,which was consistent with previous studies.Although the curcumin cannot totally inhibit the tumor growth,the metastasis was significantly decreased after the curcumin treatment,especially the Cur-H group.According to the the lung hematoxylin-eosin(H&E)stained slices,both the area and the number of lung metastasis were significantly decreased in the curcumin groups and HYW groups,and Cur-H group had the best efficacy.In addition,the lung weight provides an indirect evidence for the anti-metastasis efficiency of curcumin.(2)Suppression of High Blood Coagulation from HYW and Curcumin Distribution of PLT in tumor-bearing mice.PLT count is the measure of platelet cells/?l blood.Blood testing revealed that the average PLT count was limited in normal mice and increased in tumor-bearing mice.In addition,PLT were not decreased significantly following HYW/curcumin treatment compared with the control group.Results of fibrinogen(Fbg)level in tumor-bearing mice.Fbg level were expressed in g/1 in coagulation tests.Fbg determination results indicated tumor-associated high blood coagulation in mice,characterized as increased Fbg values compared with normal miceI.In addition,HYW/curcumin treatment was demonstrated no significant decrease of the Fbg levels compared with the control group.Results of PT and APTT in tumor-bearing mice.Both PT and APTT determination were expressed in second(s).A shorter PT or APTT indicates a hyper coagulable state.In 4T1 tumor-bearing mice,values of PT and APTT,were significantly different from those in normal mice.They were all significantly decreased following tumor cell implantation compared with the normal group.Meanwhile,we also obseflochceheofin HYW/curcumin on PT and APTT.HYW/Curcumin treatment was demonstrated to significantly increase the values of PT compared with the control group.In addition,APTT was increased following curcumin treatment,although the difference was not significan.Then,we tried to investigate whether curcumin could hav effent en integrated fuction of blood coagulation using TEG hemostasis analyzer.The statistics result reveals that compared to normal mice,tumor-bearing ones showed about a shorter R time,indicating a hypercoagulability.While R time was increased under the treatment of curcumin compared with group control.Results of MA are presented,pairwise comparison of the amplitudes show significant difference between normal and control groups.By presenting a higher MA value,hypercoagulability was observed in tumor-bearing mice.According to MA,treatment with different concentrations of HYW/curcumin could decrease the High Blood Coagulation in a dose-dependent manner.(3)HYW/Curcumin Inhibits Platelet Activation and supplements Platelet Apoptosis in 4T1 tumor-bearing miceWe first examined the effects of curcumin on platelet activation in tumor-burdened mice.Representative images of P-selectin levels platelets with curcumin treatment drawn from flow cytomety analyses are given in figure 5A.P-selectin(CD62P)is a widely used cell surface marker of activated platelets.By flow cytometry analysis,we found that HYW-H and Cur-H could inhibit P-selectin expression to a great extent in platelets.By contrast,platelet apoptosis is conducive to inhibiting hematogenous metastasis.In order to determine whether the Platelet activation inhibition by HYW/curcumin was associated with apoptosis,we further examined the potential stimulative effects of HYW/curcumin on platelet apoptosis in vivo.Treatment of tumor-burdened mice with different concentrations of HYW/curcumin resulted in an increase of the proportion of apoptotic platelets in a dose-dependent manner.(4)HYW and Curcumin Down-regulates the Expression of ?3 and ?1 Integrins in 4T1 TumorTumor cells and platelets both express integrins,such as integrin ?3 and ?1,which were shown to support tumor cell interaction with platelets allowing adhesion to the vasculature and metastasis.We next sought to address these specific proteins affected by curcumin in 4T1 tumor tissues in the mouse model.The expression of the integrin ?1 and ?3protein levels related to celladhesion molecule were decreasedafterbeingtreated with HYW/curcumin.Conclution:These results indicated that the HYW and curcumin greatly inhibited 4T1 tumor growth and lung metastasis in mice.Moreover,our present study further confirms that among breast cancer-bearing mice,widespread cancer-associated thromboses were observed,and HYW/curcumin has the ability to normalize the blood coagulation function.And then,results show that curcumin inhibits activation of platelets and stimulate platelet apoptosis might act as a potential inhibitor of platelet-related metastasis progresses.