Experimental Studies On Anti-tumor Effects Of Sorafenib Combined With Heparin/Low Molecular Weight Heparin

BackgroundEvery year,millions of hepatocellular carcinoma(HCC) patients are dying in China,which contains 50%of the world HCC cases.But few drugs were effective for this disease.Multikinase inhibitor Sorafenib,which blocks RAF/MEK/ERK cascade and VEGFR2,PDGFR,FLT3,Ret,c-Kit,was showing its inhibitor role of HCC proliferation.The same thing was already confirmed both in vitro and in vivo.Heparin and low molecular weight heparin were found having antitumor effects these years.They exhibit anti-proliferation,anti-invasion and anti-angiogenesis effects through their ability of bonding ECM proteins,including FGF,HGF,P and L selectin and so on.Tumor microenvironment is important in cancer malignant processes.Tumor cells connect with their surroundings by a great amount of active/inhibitive factors. New vessels from the microenvironment feed tumor cells,and the surrounding blood cells,such as platelet may help tumor cells metastasis to beyond places.A combined drug therapy containing of proliferation-target reagent and also microenvironment-target reagent may exhibit more effective than single target therapy.We chose sorafenib and heparin/dalteparin(a kind of low molecular weight heparin) as the examination drugs and high potential invasion HCC cells HCCLM6 as the object cells,to verify our assumption.Materials and MethodsCompounds and cell lines.Sorafenib was dissolved in 100%DMSO and diluted with H-DMEM to the desired concentration.Heparin and dalteparin were diluted with H-DMEM to the desired concentration.HCCLM6 are human HCC tumor cells with high potency of invasion.Cell proliferation detection MTT assay.In vitro cytotoxicity to HCCLM6 was determined using the MTT assay.Cells were cultured in 96-well plates in the presence of different concentrations of sorafenib,heparin/dalteparin and combined reagents.Hours later,MTT was added and the plates were incubated for an additional 4 hours.The MTT is reduced to purple-blue formazan crystals by viable cells. Formazan crystals were dissolved by DMSO and the optical density(OD) was measured at 490 nm on a enzyme mark instrument.Flow cytometry cell cycle analysis.HCCLM6 Cells were plated in six-well plates at 1×10~6 per well.After compounds treatment,cells were harvested and stained with propidium iodide(PI),The stained cells were analyzed by flow cytometry.Apoptosis detection with Annexin V-FITC and PI.Compounds treatment cells were harvested and stained with Annexin V-FITC and PI.And then apoptotic cells population was analyzed immediately by flow cytometryWestern blot detect.1×10~6 compound treatment cells were lysed.The protein was loaded on gels electrophoresis,and transferred onto PVDF membranes,incubated with ERK/PERK antibody and then second antibody conjugated with horseradish peroxidase.Cell Adhesion Assay.The 96-well plates were coated with Matrigel.Cells were cultured with prepared drugs in it for 1 h and gently washed.The cells that remained attached to the bottom of the plate were stained with hematoxylin-eosin and counted under a microscope.Cell Invasion Assay.A Transwell system was added with Matrigel to the upper chamber and dried.The cells were added to the upper chamber of the Transwell insert. The lower chamber was filled with 500μ1 H-DMEM containing FBS.After 12 h,the cells that penetrated to the lower surface of the filter were stained with Giemsa and counted.Chick Embryo Chorioallantoic Membrane(CAM) Assay.8 days hatched fertilized eggs were applied with prepared drugs or only H-DMEM as control to the CAM.After 72 h,the vessel numbers(VN) of CAM were counted.Statistical analysis.Data were analyzed with SPSS 11.5.Comparisons between groups was calculated by One way ANOVA,P-Values were considered to be significant at＜0.05. ResultsGrowth inhibitory effects of sorafenib and heparin/dalteparin on HCCLM6. Cells were observed under a microscope.The cells of the control group were big and compact,but the heparin/dalteparin treated cells were smaller and the intervals were wider than the untreated group.Sorafenib treated cells were pycnotic and dead as concentration elevated.Cell proliferation were detected with MTT assay,Sorafenib dose-dependently inhibited the proliferation of HCCLM6(P＜0.05),and heparin/dalteparin showed additive antineoplastic effects to sorafenib(P=0.000). Dalteparin performed more synergy to sorafenib than heparin at the same concentration(P＜0.05).Cell cycle analysis.The S phase distribution of each groups were significantly different(F=3.508,P=0.035).Cell cycle analysis by flow cytometry showed a increase of S phase alter sorafenib treatment for 24hours(P＜0.05),heparin/dalteparin did not show synergy to sorafenib.Induction of apoptosis of drugs.The early apoptosis percentage of each groups were significantly different(F=13.916,P=0.000).Drug treated groups had higher early apoptosis percentage than the control(P＜0.05).The similar results were obtained when compare early apoptosis between combining-drug groups and single-drug groups(P＜0.05).The late apoptosis percentage of each groups were significantly different(F=373.217,P=0.000).The additive effects of single drug on late apoptosis were only observed between sorafenib and the control group(P =0.000),but not between the combining-drug groups and sorafenib group.The heparin and dalteparin did not express different anyway.Western blot of ERK1/2 and p-ERK1/2.The differences between groups of ERK1/2 were faint(F=1.302,P=0.326).The p-ERK1/2 expression of groups were different(F=47.011,P=0.000 ),it decreased when added single drugs or combining drugs(P＜0.05).Cell adhesion analysis.The cell adhesion percentage of each groups were significantly different(F=149.871,P=0.000).Compared with the control group,the adhesion percentage decreased in heparin/dalteparingroups(P=0.000) but not in sorafenib group(P=0.097).Sorafenib did not increase the anti-adhesion effects of heparin/dalteparin significantly(P=0.582/P=0.934 ).Cell invasion analysis.The cell invasion percentage of each groups were significantly different(F=78.773,P=0.000).Compared with control group,sorafenib, heparin,dalteparin,inhibited the invasion ability of HCCLM6(P=0.000 ).Sorafenib increased the inhibition effects of heparin/dalteparin(P=0.004/P=0.000).CAM assay.In morphology,the sorafenib decreased the VN and also induced vessels deformity,whereas heparin/dalteparin mainly decreased small vessel number and slightly increased the big ones.The VN of each groups were significantly different(F=206.659,P=0.000).The VN decreases were observed in treated groups than control group(P=0.000),and in combining-drug groups than single-drug groups(P＜0.05).There is no significant difference of effects on CAM between heparin and dalteparin(P= 0.447 ),(heparin+sorafenib) and(dalteparin+sorafenib) (P=0.633).DiscussionThe tumor microenvironment is focused on the role of the microenvironment in which a tumor originates and the critical role it plays in tumor initiation and progression.The interaction between the tumors and their surrounding cellular environment,collectively known as the stroma,co-evolve during the tumor initiation and progression,strongly effects the establishment and treatment of cancer.Evidence is emerging that critical stromal elements of the tumor are attractive targets for cancer prevention,because they primarily influence tumor cells in the early stages of cancer progression.Drugs aiming these elements could be sorted as two groups,one is aiming at the acceptors on tumor cells,such as sorafenib,gefitinib,and the other is aiming at the factors in microenvironment stroma,such as bevacizumab,cetuximab, heparin and so on.Sorafenib(BAY 43-9006,Nexavar) is a multikinase inhibitor with activity against Raf kinase and several receptor tyrosine kinases,including vascular endothelial growth factor receptor 2(VEGFR2),platelet-derived growth factor receptor(PDGFR),FLT3,Ret and c-Kit.Heparin/low-molecular-weight heparins(LMWHs) have been validated in both the prevention and treatment of thromboembolic disease in cancer patients and the improvement of patients' survival time in retrospective and prospective trials.This is believed to relate to factors such as direct anti-neoplastic effects,the modulation of the immune system,the suppression of the effect of coagulation proteases in the tumor microenvironment and anti-angiogenic effects.Tumor initiation and progression involve a multifactor network.Combining independent function mechanism drugs may block multitargets to obtain more extensive result.We selected sorafenib and heparin/dalteparin,targeting the tumor proliferation and environment proteins separately,to verify our presumption. Sorafenib plus heparin/dalteparin more effectively inhibit the HCCLM6 cell proliferation and angiogenesis than single drugs,and also the anti-adhesion and anti-invasion effects.There is no significant difference of effects between heparin and dalteparin.The results advise us to use these drugs together in clinic,which may bring more benefits to cancer patients.