Study Of Bufadienolides And Their Inhibition Effects On Both Proliferation And Metastasis Of Tumor Cells

Background and aimsCinobufacini and other related toad preparations as more promising,safe and efiective medicine widely used in a variety of advanced cancer in clinical with remarkable effect.Modern pharmacological research showed that,bufadienolides achieved anti-tumor effect through a variety of ways with fewer side effects.Chemical researches have shown that the main active constituents of cinobufacini injection were bufadienolides,which is a class of small molecules between 300-500. However, in view of its traditional formulations and use for the treatment of gastrointestinal cancer in clinical,it is regrettable that few scholars pay attention to the effect on brain cancer.In addition,metastasis is a critical factor of death,once transferred；it will be out of control no matter gastrointestinal cancer or brain cancer.Seeing that the strong pharmacological activities and less side effect of cinobufacini combine the special of the major anti-tumor active ingredients,we suspected that it could be applied to the treatment of brain cancer with a bright future. In this study,bufadienolides have been chosen to clairify the chemical components and their pharmacological activities on human pancreatic cancer cell SW1990 and human glioblastoma cell U-87.MethodsBufadienolides are the main anti-tumor active constituents both in cinobufacini and toad venom.To identify the active components in bufo melanostictus Schneider and clarify the difference between fresh and dried Venenum Bufonis,a UPLC-MS/MS method has been established,laying the foundation of the development of its chemical compositions.Through XTT assay,DNA fragmentation assay,LDH assay and Hoechst 33342 & PI staining assay,the pharmacological activity on inhibition of proliferation were evaluated.The mechanism of gamabufotalin was studied deeply.The mechanisms of regulating cancer cell proliferation were checked through the test of Akt,ERK,P38 MAPK and other key factors by western blot.At the same time,wound healing assay and transwell assay were used to observe the impact of gamabufotalin on cell migration.The effect of drug on RNA expression levels of MMp family was tested by Real-time PCR assay.Besides, the preliminary evaluation of the drug toxicity on immune system was studied by XTT and flow cytometry.Results:1. Analysis of chemical composition and selection of effective anti-tumor compound1.1 In this study, the chemical compositions of the fresh and dried toad venom of Bufo Melanostictus were investigated by UHPLC-high-resolution-mass spectrometry in the positive ion mode. Preliminarily,39 major constituents were identified via the precise molecular weight and the fragmentation ions of compounds in the fresh toad venom of Bufo Melanostictus. Further, a significant difference of the chemical profile between the fresh and dried toad venom were existed, namely, the species and contents of bufadienolides in the fresh toad venom were less by comparison with the dried toad venom.1.2 On the basis of the chemical analysis in Bufo Melanostictus,10 single bufadienolides were selected to further investigate their pharmacological activity of pancreatic cancer cell line SW1990 and human glioblastoma cell line U-87. Firstly, the XTT result showed that gamabufotalin, arenobufagin and bufadienolides had better anti-tumor activity with IC50 less than 40 ng/mL in both two kinds of cancer cells while all of them exhibited weak cytotoxicity effect on normal human Peripheral Blood Mononuclear Cell (PBMCs). Especially, the IC50 of gamabufotalin in PBMCs was 7 times of that in SW1990 cells and 4 times in U-87 cells, respectively.2 Gamabufotalin impact proliferation on U-87 and SW1990 cells2.1 DNA fragmentation assay shows after administration of Gamabufotalin in U-87 and SW1990 cell lines 24 hours, neither DNA ladder nor DNA smear was observed. However, DNA smear was observed in U-87 cell lines after 48 hours administrated with gamabufotalin more than 200 ng/mL.2.2 LDH assay showed that an obvious release of LDH occurred after dosing more than 40 ng/mL of gamabufotalin in both U-87 and SW1990 cell lines, While 1.6 ng/mL of arenobufagin in U-87 cells and 40 ng/mL in SW1990 cells were observed.2.3 Hoechst 33342 & PI staining showed that with increasing doses, the proportion of positive cells of PI increased.2.4 Western blot assay showed that a significant increase of p-P38 after dosing 8 ng/mL of gamabufotalin and an obvious decrease of p-Akt and p-ERK at the dose of 100 and 200 ng/mL, which exceeded its IC50 in U-87 cells. However, to SW1990 cells, no significant change of p-P38 and p-Akt was observed, except that the level of p-ERK depressed in more than 40 ng/mL of gamabufotalin.3. Gamabufotalin impact migration on U-87 and SW1990 cells3.1 Wound healing assay revealed that the healing of two kinds of cancer cells was apparently inhibited after dosing 4 ng/mL of gamabufotalin.3.2 Transwell migration assay displayed that there is a significant inhibition on the migrated cell number of pancreatic cancer at the dose of 4 ng/mL. Further, gamabufotalin could also inhibit the migrated number of glioblastoma cells at the dose of 8 ng/mL, which had stronger ability of transferring.3.3 Real-time PCR results showed that the RNA expression of MMP-9 decreased significantly with the increasing of drug concentration although the content of MMP-2 and MMP-7 barely varied. Simultaneously, an obvious rising trend of TIMP-1 was observed with no significant difference.3.4 Zymography assay demonstrated that MMP-2 in the supernatant was suppressed by 16 ng/mL of gamabufotalin.4. Bufadienolides effects on human peripheral blood mononuclear cells4.1 XTT assay shows selective anti-tumor functions on both cancer cells dealing with gamabufotalin.4.2 The proportion of treg cells in normal human PBMCs was reduced when dealing with gamabufotalin.Conclusions1. There is a significant difference between the fresh anddried toad venom, the species and contents of bufadienolides in the fresh toad venom were less than the dried toad venom.2. The 10 kinds of bufadienolides had different effects on pancreatic cancer cell line SW1990 and human glioblastoma cells U-87.3. Gamabufotalin could obviously inhibit the proliferation of two kinds of cancer cells (SW1990 and U-87), which is likely to cause the cell death. The mechanism research suggested that gamabufotalin might cause the cell death via inhibiting the level of phosphorylation of Akt and ERK1/2 as well as improving the phosphorylation of P38 MAPK. The migration related tests showed that gamabufotalin had excellent anti-metastasis effects on both SW1990 and U-87 cells. Together with the PCR results, it can be speculated that the impact of gamabufotalin on the influence of MMP-9, which was a member in the MMPs family, may be one of the key factors influencing the cell migration.4. Gamabufotalin didn’t suppress the autoimmune system.