The Study On Synergistic Antitumor Mechanism For Zoledronic Acid And Paclitaxel In Vitro

Backgroud:Malignancy is the most common disease which is seriously endanger the people in good health.Lung cancer and breast cancer have a high incidence rate and easily metastase to bone.At present,bisphosphonates are well established in the management of bone disease in clinical setting,especialy the third generation bisphosphonates,such as zoledronic acid.According to the recent studies,zoledronic acid not only could inhibit bone metastasis,but also has direct or indirect antitumor effects.Zoledronic acid can prevents tumor cell invasion and adhesion,inhibits tumor cell proliferation and vitality,induces tumor cell apoptosis and inhibits angiogenesis;in addition, combining with cytotoxic chemotherapy may provide further antitumor synergies, which people are very interested in.According to Neville-Webbe's report~[3],sequential application of doxorubicin followed by zoledronic acid has been shown to induce significantly more tumor cell apoptosis than either agent alone.Among the most chemotherapy agents,paclitaxel as a microtubule stabilizing agent is used universally in the first line regimens for the therapy of malignancy.Paclitaxel could prevent microtubule dissociation and make it stable,thus inhibit the normal integration of microtubule net and prevent the cells in the stage of G2/M.Both paclitaxel and zoledronic acid are part of the treatment of lung cancer and breast cancer patients. Weather there are synergistic effects between zoledronic acid and paclitaxel and weather the synergistic interaction is drug sequencing dependent is investigated all over the world.Objective:To investigate if there is any difference in the inhibition between sequential and simultaneous treatment using zoledronic acid and paclitaxel for breast cancer cell line MCF-7 and lung cancer cell line A-549.To analyse the relationships among these datas and find out if there is any interaction between zoledronic acid and paclitaxel.To establish weather the drug interaction is sequencing dependent and supply the experimental basis for the clinical chemotherapy. Methods:The breast cancer cell line MCF-7 and lung cancer cell line A-549 were devided into 15 groups which were treated with increasing concentrations of the zoledronic acid or paclitaxel.The effects of zoledronic acid or paclitaxel were determined by assessing inhibiton rates of different groups.According to the experimental concentrations used before and the clinical achievable concentrations,we could find out the appropriate concentrations of zoledronic acid and paclitaxel.We combined zoledronic acid with paclitaxel and treated both cell lines in sequential or simultaneous.At the same time,we set up a contrast group which didn't accept any treatment.In the experiment,we assessed inhibition rate,apoptosis rate and the changes of cell cycle by MTT and flow cytometer.Results:1.The results of MTT showed us that zoledronic acid and paclitaxel could inhibit the growth of breast cancer cell line MCF-7 and lung cancer cell line A-549 alone; zoledronic acid and paclitaxel caused a dose-dependent inhibition in cell growth; combining clinically achievable concentrations and drug concentrations other people used in experiments,we chosed the 10μM and 100μM as the proper concentrations of zoledronic acid in this experiment;the proper concentration of paclitaxel is 2nM.2.The results of MTT also showed us that zoledronic acid and paclitaxel could inhibit cell growth together,which is greater than either zoledronic acid alone or paclitaxel alone,and the levels of synergistic inhibiton are dependent on drug sequencing.Maximum inhibition was induced by the sequence order:paclitaxel then zoledronic acid＞zoledronic acid and paclitaxel together＞zoledronic acid then paclitaxel,P＜0.05.3.The Flow cytometry showed that giving paclitaxel before zoledronic acid could induce maximum apoptosis,then zoledronic acid and paclitaxel together,and the third is giving zoledronic acid before paclitaxel.The synergistic effects are greater than either drug alone,P＜0.05;4.In order to investigate whether the treatments outlined above affected specific stages of the cell cycle,we determined the distribution of cells in the various phases of the cell cycle by flow cytometry.Paclitaxel induces suppression of microtubule dynamics,which leads to an arrest of the cells in G2/M phase.Zoledronic acid induced a concentration-dependent increase of cells in S phase,with a corresponding decrease in G2/M phase,and an increase in subGl shoulder.For cell cycle analysis of drug sequencing,the difference among three sequencing groups is whether zoledronic acid and paclitaxel have effects on S phase or G2/M phase.The sequence that induced the most apoptosis(paclitaxel then zoledronic acid) was the only sequence inducing an accumulation of cells in S phase then inhibiting the cells division.The effect was significantly great than control alone,P＜0.01.Conclusions: Both zoledronic acid and paclitaxel could inhibit the growth of the tumor cells,and the inhibitions are dose dependent.There are synergistic effects for the combined treatment with zoledronic acid and paclitaxel.The levels of apoptosis are dependent on drug sequencing,with the greatest effect achieved when paclitaxel is administered prior to zoledronic acid.Also,when paclitaxel is administered prior to zoledronic acid,it could induce an accumulation of cells in S phase then inhibit the cells division.