The Effects Of Docetaxel Combined With 4-AP In Human Breast Cancer MCF-7 Cell

Docetaxel is an antineoplastic agent which acts by promoting the assembly of tubulin into stable microtubules and inhibits their disassembly which leads to a marked decrease of free tubulin in order to affect the cell caryomitosis.At present,Docetaxel has been used in the all kinds of cancer such as breast cancer;stomach cancer,lung cancer and esophagus cancer.At the same time,the kalium ion channel on the cellular membrane plays very important role in the phase of G₁ in the cell caryomitosis.There is evidence that the kalium ion channel blocker can inhibit the proliferation of cell,but the specific mechanism is unclear.The previous study had showed doctaxel not only can inhibit their disassembly which leads to a marked decrease of free tubulin and genetic expression in order to affect the cancer cells,but also can block the kalium ion channel in order to affect the cell caryomitosis.The study is to approach the synergistic action in which doctaxel combined with 4-AP affect the proliferation,cycle and apoptosis in the human breast cancer.Then this can provide the theory to the drugs combination in the clinic.Methods1.To investigate the effects of DOC,4-AP and the combination of both drugs on cell proliferation of MCF-7 by MTT assays.Inoculate and incubate the logarithm growth cells,add 5μg·μL^-1MTT 10μl every slot after giving drugs 24,48 and 72 h, cultivate 4 h,add DMSO 100μl,agitate 60 rpm 10 min,measure the OD₅₇₀with ELx800,and calculate the inhibition ratio.Inhibition Ratio(%)=(control OD- experiment OD)/control OD×100%.2.The cell cycles are measured by flow cytometry after the cells were stained by PI.Inoculate and incubate the logarithm growth cells,collect cells after giving drugs 24 h,centrifuge 1000 rpm 5 min,add cool PBS 10³Î¼l,repeat the steps above three times, add 75%cool Alc 10³Î¼l under 4℃.The second day,centrifuge 1500 rpm 10 min,add cool PBS 10³Î¼l,centrifuge 1500 rpm 5 min,add 0.1μg·μL^-1PI and 0.1μg·μL^-1Rnase A 500μl respectively.Detect cell cycles after cultivating 40 min under 37℃.3.The apoptosis cells are measured by flow cytometry after the cells were stained by both Annexin V-FITC and PI.Inoculate and incubate the logarithm growth cells, collect cells after giving drugs 24 h,centrifuge 1000 rpm 10 min,add cool PBS 10³Î¼l, repeat the steps above twice,add Binding Buffer 200μl,add Annexin V-FITC 10μl and PI 5μl,react 15 min away from light under room temperature,add Binding Buffer 300μl,detect cell apoptosis within 1 h.Results1.DOC and 4-AP can inhibit the proliferation of human breast cancer cell MCF-7 respectively.4-AP of 5×10³Î¼mol·L^-1have additive effects on inhibiting the proliferation of MCF-7,and the effects present the dose-time dependence(P＜0.05).2.MCF-7 cells can be blocked at G₂/M period by DOC,the cell ratio can be increased from 8.83%±0.44%of the control group to 53.58%±1.44%of the 5μmol·L^-1DOC group,(P＜0.01).They can be blocked at G₀/G₁ period by 4-AP.The cell ratio can be increased from 63.89%±0.98%of the control group to 76.56%±1.34% of the 5×10³Î¼mol·L^-14-AP group,(P＜0.05).But,the G₂/M period which be blocked by 4-AP has decreased from 8.83%±0.44%of the control group to 0.42%±0.17%of the 5×10³Î¼mol·L^-14-AP group,(P＜0.05).The combination of docetaxel and 4-AP could decrease the rate of the percentage of cell in G₂/M and increase the rate of the percentage of cell cycle in G₀/G₁.3.The effects of improving MCF-7 apoptosis are not obvious when given DOC of 5μmol·L^-1for 24 h.But when DOC are combined with 4-AP of 5×10³Î¼mol·L^-1,both the apoptosis and the cell death are very transparent.The apoptosis cells are increased from 4.60%±0.91%of the control group to 12.20%±0.82%of the combination group,(P＜0.05).And the cellular necrosis could increase very obviously from 6.97%±0.75%to 58.42%±0.31%,(P＜0.05).ConclusionThis study observed that the effects of docetaxel and 4-AP on the cell proliferation cycle and apoptosis,the study results showed that doctaxel and 4-AP could inhibit the MCF-7 cell's proliferation in the way of preventing the cell cycle in the G₂/M phase and G₀/G₁ phase respectively,and 4-AP could induce the cell apoptosis in order to inhibit the cell proliferation.