Reversal Of Doxorubicin Resistance By Vardenafil In MCF-7/ADR Cells

Objective: Multidrug resistance(MDR)is an important cause of cancer chemotherapy failure.A major mechanism of MDR is the over expression of the binding cassette-ABC transporter of adenosine ATP.The ABC transporter can reduce the intracellular drug by promoting the drug to move out of cancer cells and then result in MDR.Research about tumor MDR reversal agents is expected to find ways to overcome drug resistance,reduce cancer recurrence,and then improve the quality of life of patients.Most of the MDR reversal agents developed in recent years were P-gp inhibitors.Those inhibitors inhibited p-gp function by inhibiting the transport activity of P-gp or competing the substrate binding site of P-gp with the chemotherapeutic drug.However,most studies about drug resistance reversal strategies were in vitro stage.The reversal agents have been reported to be ineffective in the treatment of tumors because of their low specificity and insufficient side effects in clinical practice.Therefore,it is a long-term process to develop low-toxic MDR reversal agents.Vardenafil is a new generation of phosphodiesterase 5(PDE5)inhibitor with good oral absorption and high security.This study is mainly to detect doxorubicin resistance reversal and the mechanism of vardenafil in human breast cancer MCF-7/ADR cells.Methods:1 MTT assay was used to detect the effect of vardenafil,sildenafil and tadalafil combined with doxorubicin on the proliferation of MCF-7/ADR cells.2 Hoechst 33342 staining was used to detect the efftct of vardenafil combined with doxorubicin on the apoptosis of MFC-7/ADR cells.3 The laser accumulation confocal microscopy(LSCM)was used to detect the content and distribution of doxorubicin in the MCF-7/ADR cells.4 The content of doxorubicin in different parts of MCF-7/ADR cells was detected by high performance liquid chromatography-mass spectrometry(HPLC-MS).5 Western blot assay was used to detect the effect of vardenafil combination with doxorubicin on the expression of p-gp,Bax,Bcl-2 and cleaved-caspase 3 proteins in MCF-7/ADR cells.6 Establishing BALB/c Nude mice model with breast cancer MCF-7/ADR,and detecting the doxorubicin resistance reversal by vardenafil in vivo.The tumor volume was measured every day,and the protein expression was detected.Results:1 The inhibitory effects of vardenafil,sildenafil,tadalafil combined with doxorubicin on MCF-7/ADR cellsThe results showed that vardenafil,sildenafil,tadalafil alone or doxorubicin alone did not inhibit the growth of MCF-7/ADR cells.Vardenafil,sildenafil or tadalafil in combination with doxorubicin significantly inhibited the growth of MCF-7/ADR cells.The inhibition rate was increased with the increasing concentrations of vardenafil,sildenafil or tadalafil.We compared the reversal effect of vardenafil,sildenafil and tadalafil on MCF-7/ADR cells,The reversal effect of vardenafil is the best among three drugs.2 The effect of vardenafil in combination with doxorubicin on the apoptosis of MCF-7/ADR cellsThe cell nuclei in normal control group were round with evenly dispersed blue,and did not show the feature of apoptosis.There were no difference among control group,doxorubicin alone and vardenafil alone group in nuclei morphology and the density of fluorescence.The nuclei morphology appeared typical apoptosis feature in vardenafil combined with doxorubicin group.The cell nuclei were concentrated,and most of cell nuclei were like half-moon in vardenafil combined with doxorubicin group.3 The effect of vardenafil on the content and distribution of doxorubicin in MCF-7/ADR cells by laser confocal microscopyThe results showed that red fluorescence was observed in doxorubicin group and combination group 6 h after drugs administration,indicating that doxorubicin entered the cell and was not completely pumped out.There was no red fluorescence in control group and vardenafil group.The intensity of red fluorescence(doxorubicin)in the vardenafil and doxorubicin combination group was significantly higher than that in doxorubicin alone group,suggesting that vardenafil could improve the accumulation of doxorubicin in the cells.These results indicated that doxorubicin was located both in nucleus and mitochondria.4 The effect of vardenafil on the content and distribution of doxorubicin in MCF-7/ADR cells by HPLC-MSThe results of HPLC-MS showed that vardenafil increased the content of doxorubicin in cell nuclei,cytoplasm and mitochondria.The content of doxorubicin in cell nuclei was significantly higher than that in cytoplasm and mitochondria.In the nuclei,the accumulation of doxorubicin in vardenafil and doxorubicin combination group was higher than that in doxorubicin alone group at three time points.The content of doxorubicin in vardenafil and doxorubicin combination group at 12 h were higher than that at 6 h or 2 h.The content of doxorubicin in the cytoplasm and mitochondria reached a peak at 6 h.5 The effect of vardenafil combination with doxorubicin on the expression of p-gp,Bcl-2,Bax and cleaved-caspase 3 proteins in MCF-7/ADR cellsThe Bax/Bcl-2 ratio was increased in vardenafil in combination with doxorubicin group compared with that in control group.There was no diffenrence for Bax/Bcl-2 ratio among control group,doxorubicin alone group and vardenafil alone group.The expression of cleaved-caspase3 appeared in combination group.There was no difference for p-gp expression among four groups.6 The effect of vardenafil combination with doxorubicin on the growth of tumor in mice with MCF-7/ADR tumorThe mice with MCF-7/ADR tumor were successfully established.The weights of mice in doxorubicin alone group and combination group were significantly decreased after 35 days of continuous administration compared with the control group.There was no difference between vardenafil group and control group in the weights of mice.There was no significant difference in the tumor growth rate between the doxorubicin alone group,control group and the vardenafil group,and the tumor growth rate in the combination group was significantly decreased.Conclusions:1 Vardenafil,sildenafil,tadalafil alone or doxorubicin alone do not inhibit the growth of MCF-7/ADR cells.Vardenafil,sildenafil,or tadalafil combined with doxorubicin significantly inhibit the growth of MCF-7/ADR cells.Vardenafil is the most effective drug among the three drugs.2 Vardenafil increases the content of doxorubicin in cell nucleus,cytoplasm and mitochondria.The content of doxorubicin in cell nucleus was significantly higher than that in cytoplasm,and mitochondria.3 The tumor volume in vardenafil alone and vardenafil combination group was significantly decreased compared with that in control group,which indicates that vardenafil can increase the sensitivity of tumor cells to doxorubicin.4 Vardenafil in combination with doxorubicin induces the apoptosis of MCF-7/ADR cells,increases the ratio of Bax/Bcl-2 and the expression of cleaved-caspase 3,and have no effect on the expression of p-gp.