Decipering Therapeutic Efficacy And Underlying Molecular Mechanism In Virto Of Apatinib In Mantle Cell Lymphoma

BackgroundMantle cell lymphoma(MCL)is an uncommon subtype of malignant B cell lymphoma,and accounts for 3-6%of non-Hodgkin lymphoma with a higher incidence in elder patients.The typical morphological features are that monoclonal lymphoid tissue diffuse or nodular grow in the mantle zone.MCL is accompanied with immunophenotypes like CD5 CD20 positive,CD23 negative.One of the most frequent genetic abnormalities is t(11:14),(q 13:q32),which results in the cell cycle protein Cyclin D1 overexpressing in nuclear.MCL contains duel features with the fast progess of aggressive lymphoma and difficult to cure of indolent lymphoma,even the 5-year survival rate less than 30%after a conventional chemotherapy.In the recent years,for the clinical efficacy of MCL patients,there exists two main problems:one is for the treatment of MCL,there is no a standard therapy,while the traditional rituxan(rituximab)CHOP(cyclophosphamide,doxorubicin,vincristine,prednisolone)is not the best induced regimen and most of the older patients can’t tolerate a high dose chemotherapy such as hyper-CVAD/MA and the subsequent autologous hematopoietic stem cell transplantation;Another is that even if the patient get completely remission after the initial induction,patients are easy to relapse since the short remission period.Once relapsed,the remission rate decreases extremely,and the prognosis is poor.Targeted drugs,characterized by low toxicity and high selectivity,have always been popular in cancer research.In recent years,with the development of biology research,more and more targeted drugs have been applied to clinic,which has increased the possibility of curing MCL.Therefore,it is crucial to actively develop new targeted drugs and explore effective combination of new drugs and chemotherapy to improve the survival of MCL patients.Apatinib is a self-developed vascular endothelial growth factor receptor-2(VEGFR-2)inhibitor,which target to ATP binding site and block vascular endothelial factor(VEGF)and its combination.Apatinib is generated by inhibiting tumor angiogenesis and downstream signal transduction pathways to inhibit tumor growth and nutrition,and ultimately achieve the goal of killing the tumor.As a safe and effective of a new generation oral small molecule TKIs,Apatinib mainly applied to standardized treatment failure in patients with advanced gastric cancer and had literature reported about its antitumor effect among other solid tumor such as lung cancer,liver cancer,breast cancer.Receptor tyrosine kinases(RTKs)is a categories of important enzyme-linked receptors family in cell surface and comprises of vascular endothelial growth factor receptor(VEGFR),epidermal growth factor receptor(EGFR),insulin and insulin-like growth factor 1 receptor(IGF-1)and so on.Ras,one of G protein family,mediated RTKs-Ras-MAPK signaling is one of the important transduction pathways downstream of RTKs.When the ligand(such as growth factors)combined with RTKs subsequent causing RTKs dimers,activating of RTKs via growth factor receptor-bound protein 2(Grb2)and son of sevenless(SOS).The activation of the Ras proteins then cause the phosphorylation of downstream mitogen activated protein kinases(MAPKs)cascade reaction and conduct the extracellular signals into the nucleus,involved in regulating cell proliferation,differentiation,promote cell survival and other physiological processes.Objectivethe chemotherapy works a poor efficacy for the Mantle Cell Lymphoma(MCL) and there is an urgent need for the new targeting agents.Apatinib,exhibits anti-cancer activities among various tumors.In this study,we investigate the effects and mechanism about apatinib,a novel VEGFR2 inhibitor,on the proliferation and apoptosis toward mantle cell lymphoma cell lines(Mino and Z138).Furthermore,we will explore the relationship between apatinib and the Ras-MAPK signaling pathway.Method1.cell lines:MCL cell lines,Mino and Z138.2.after 48h treatment with various concentration(2.5、5、10、20、40μmol/L)of apatinib among Mino and Z138 cells,using CCK-8 kit to detect the proliferation ratio and using graph prism5 to count the IC50 values.2.after 48h treatment with various concentration(10、20、30、40μmol/L)of apatinib in Mino and Z138 cells,using the Annexin-V-APC/PI double staining kit in flow cytometric to compare the percentage of apoptotic cells.3.Western blotting to detect the protein levels of the Ras-MAPK signaling pathway(Ras、c-Raf、pMEKl/2 and pERKl/2)among the treatment(20、40μmol/L of Apatinib for 48h)or non-treatment..4.Using the statistical software SPSS 20.0 to process the statistics.For the small samples,non-parametric test Kruskal-Wallis H test was used to compare the difference of mean.An value of P<0.05 was accepted as an indication of statistical significance.Results represent the mean ± SD of at least three independent experiments.All tests are bilateral test.Results1.The CCK-8 experiment results shows that treated with various concentration apatinib for 48h can inhibit the MCL cells’(Mino and Z138)proliferation through a dose-dependent maner.In the Mino cells,the proliferation inhibition rate is(5.72±2.43)%、(8.19±2.2)%、(17.70±4.01)%、(44.58±7.59)%、(85.12±2.67)%respectively and the results has statistical significance(check by Kruskal-Wallis H test,X2=22.016,P=0.001).the IC50 are 20.63±0.18.In Z138 cells,the inhibition rate is(7.19±1.71)%、(10.60±1.43)%、(20.24±5.93)%、(53.46±4.91)%、(87.07 ± 1.41)%and the difference is statistical significance compared with control group(X2=16.460,P=0.006).The IC50 is(18.15±0.18)μM.2.Apoptosis assay results showed that treatment with different concentrations(10、20、30、40 μmol/L)of apatinib triggered obvious dose-dependently apoptosis against Mino and Z138.The apoptosis rates are(8.10±0.98)%、(29.46±7.18)%、(35.06±4.17)%、(50.01±2.14)%and(10.58±2.74)%、(16.51±0.81)%、(23.07±3.14)%、(34.48 ± 6.40)%,and the compare with control group((4.03±0.99)%,(5.45±1.82)%)is significant respectively(the statistical value is X2=13.033,P=0.011 and X2=13.500,P=0.009).3.Western blotting results demonstrated that decreased the Ras-MAPK signaling pathway(Ras,c-Raf,pMEKl/2,pErkl/2)in Mino cells when treated with apatinib(20,40uM)for 48h.After the analysis of relative grayscale of the protiens,the expression of Ras,c-Raf,pMEK1/2,pERK1/2 protein in the apatinib group(20,40μmol/L)has the obvious difference compared with the control group(Oμmol/L).Conclusions1.Apatinib can potentially inhibit the proliferation and lead to an apoptosis efficacy in Mino and Z138 cells.2.Apatinib selectively binds to and inhibits VEGFR2,which relative to the VEGF/VEGFR2 signaling pathway,and then inhibited the activation of Ras-MAPK pathway in the downstream,thereby playing a killing effect on MCL cells.