Lenalidomide Improves The Antitumor Effects Of Chemotherapeutic Drugs In Triple-negative Breast Cancer

BackgroundTriple-negative breast cancer(TNBC)accounts for 15 to 20%of breast cancer.TNBC often occurs in younger women and has poor histological differentiations.It is known to be the most aggressive subgroup.Due to the negative expression of estrogen receptor,progesterone receptor and human epithelial growth factor receptor 2,TNBC doesn't benefit from endocrine therapy and molecular target therapy.Therefore,chemotherapy plays a key role in the treatment of TNBC.The drugs for first-line chemotherapy include anthracycline,paclitaxel,cisplatin and 5-Fluorouracil.Although the TNBC patients have received the intensive chemotherapy,the prognosis is poor.Besides conventional chemotherapy,immunomodulatory drugs may be applied to improve the antitumor effect of chemotherapeutic drugs.Lenalidomide is known as the second generation of immunomodulatory drugs.In addition to immunomodulatory properties,lenalidomide demonstrates antiapoptotic and antiangiogenic effects.Lenalidomide has achieved good clinical results in multiple myeloma and myelodysplastic syndromes.Clinical trials have demonstrated that lenalidomide has antitumor effect on several solid tumors including colorectal,prostate,pancreatic and renal cell cancer.In addition.lenalidomide can enhance the antitumor effect of chemotherapeutic drugs on prostate and colorectal cancer.However,the research of lenalidomide in breast cancer is only limited to cell level.Study showed that lenalidomide combined with vitamin D induced TNBC cell apoptosis.There is no study on the effect of combination of lenalidomide and chemotherapeutic drugs on MDA-MB-231 cells proliferation and apoptosis.Breast tumorigenesis is accompied with large amounts of angiogenesis.Antiangiogenic therapy can partially inhibit the growth of breast cancer metastatic focus.Therefore,antigngiogenic therapy has some value on the breast cancer treatment.Tumor angiogenesis involves a series of complex steps.Endothelial cells proliferation and migration are critical in tumor angiogenesis.It is a therapeutic strategy for antiangiogenic therapy to inhibit endothelial cells proliferation and migration.In addition to induction of MDA-MB-231 cells apoptosis,lenalodmide can inhibit angiogenesis by preventing endothelial cells migration.Chemotherapeutic drugs in high dose and long interval administration mode mainly kill tumor cells and have a little effect on tumor angiogenesis.But chemotherapeutic drugs in low dose and short interval administration mode may inhibit angiogenesis by preventing endothelial cells migration.Not all chemotherapeutic drugs are applied for inhibiting angiogenesis.Only when the dose of drugs to inhibit vascular endothelial cells is lower than that of drugs to tumor cells are chemotherapeutic drugs suitable for inhibiting angiogenesis.The effect of ariamycin,paclitaxel,cisplatin and 5-Fluorouracil on inhibiting angiogenesis in TNBC is in dispute.Lenalidomide and chemotherapeutic drugs had a direct effect on MDA-MB-231 cells and endothelial cells.But the mechanins are different.Therefore,it may be possible for the combination of lenalidomide and chemotherapeutic drugs to treat TNBC.The combination of lenalidomide and chemotherapeutic drugs may be the new mode in TNBC treatment.At present,there is no report on the effect of combination of lenalidomide and chemotherapeutic drugs on MDA-MB-231 proliferation,apoptosis and HUVEC proliferation and migration.Neither is there report on the effect of combination in TNBC nude mice.ObjectsThe aim of the present study was to investigate the antitumor effects and mechanisms of combination of lenalidomide with chemotherapeutic drugs on tumor cells,endothelial cells and nude mice at the cellular and animal levels.Human TNBC cells MDA-MB-231 and human umvilical vein vessel endothelial cells HUVEC were studied at tumor cellular and endothelial cellular level.The effect of lenalidomide,ariamycin,paclitaxel,cisplatin and 5-Fluorouracil(5-FU)and combination on proliferation and apoptosis of MDA-MB-231 cells and proliferation and migration of HUVEC cells were investigated.The difference between the effects of chemotherapeutic drugs and combination of chemotherapeutic drugs with lenalidomide on MDA-MB-231 proliferation was compared.The difference between the sensitivity of MDA-MB-231 cells and HUVEC cells to chemotherapeutic drugs was detected.Based on these results,we will indentify one of four chemotherapeutic drugs which have synergistic effect with lenalidomide but also inhibit antiangiogenic effects.To determine pERK,Bcl-2 and Caspase-3 expression in MDA-MB-231 cells and pERK and pAkt expression in HUVEC cells after treated with lenalidomide,chemotherapeutic drugs and combination.At the same time,the subcutaneous transplanted tumor model of TNBC line MDA-MB-231 cells in nude mice will be established.The inhibitory effect of combination of lenalidomide with chemotherapy drug on TNBC in animal experiment was investigated.The effects of drugs on tumor growth,apoptosis and microvessel density(MVD)were investigated.The aim of the study was to explore the mechamisms of lenalidomide improvement of chemotherapeutic drugs antitumor efficacy on TNBC.Methods1.MTT was used to detect the effect of drugs on proliferation of MDA-MB-231 cells.MDA-MB-231 cells were treated with different concentrations of lenalidomiade,chemotherapeutic drugs(lenalidomide,ariamycin,paclitaxel,cisplatin and 5-FU)and combination for 24 h.48 h,and 72 h,respectively.The inhibitory effect of drugs on proliferation of MDA-MB-231 cells was investigated.Based on MTT results,the chemotherapeutic drugs which had synergetic effect with lenalidomide will be screened.2.Flow cytometry was used to determine the effect of drugs on apoptosis of MDA-MB-231 cells.MDA-MB-231 cells were treated with lenalidomide,screened chemotherapeutic drugs and combination for 72 h.Then,the effect of drugs on apoptosis of MDA-MB-231 cells was investigated.3.Western Blot was used to detect protein expression.MDA-MB-231 cells were treated with lenalidomide,screened chemotherapeutic drugs and combination for 72 h.The change of pERK,Bcl-2 and Caspase-3 in MDA-MB-231 cells was investigated.4.MTT was used to deternmine the effect of drugs on proliferation of HUVEC cells.HUVEC cells were treated with different concentrations of lenalidomide and screened chemotherapeutic drugs for 24 h,48 h and 72 h,respectively.The inhibitory effect of drugs on proliferation was investigated.5.Second screening of chemotherapeutic drugs.To screen more sensitive drug to HUVEC than to MDA-MB-231 based on the proliferation curve of MDA-MB-231 and HUVEC cells from the first screened chemotherapeutic drugs.6.MTT was used to determine the effect of drugs on proliferation of HUVEC cells.HUVEC cells were treated with combination of lenalidomide with second screened chemotherapeutic drug for 72 h.The inhibitory effect of drugs on proliferation of HUVEC was investigated.7.Scratch healing test was used to investigate the effect of drugs on migration of HUVEC cells.HUVEC cells were treated with lenalidomide,second screened chemotherapeutic drug and combination in 1%EGM2 medium for 24 h.The effect of drugs on HUVEC cells migration was investigated.8.Western Blot was used to determine protein expression.HUVEC cells were treated with lenalidomide,second screened chemotherapeutic drug and combination for 72 h and the change of pAkt protein was investigated.9.The subcutaneous transplanted tumor model of TNBC MDA-MB-231 cells in nude mice was established.Thirty transplanted mice were randomly divided into six groups.There were five mice in each group.PBS group(PBS,intraperitoneal injection with 10 mg/kg,once a day for fourteen days);LEN group(LEN,oral gavage with 25 mg/d,once a day for fourteen days);5 mg/kg 5-FU group(5-FU,intraperitoneal injection with 5 mg/kg,once a day for fourteen days);25 mg/kg 5-FU group(5-FU,intraperitoneal injection with 25 mg/kg,once a day for five days);LEN+5 mg/kg 5-FU group(LEN,oral gavage with 25 mg/d,once a day for fourteen days;5-FU,intraperitoneal injection with 5 mg/kg,once a day for fourteen days);LEN+25 mg/kg 5-FU group(LEN,oral gavage with 25 mg/d LEN,once a day for fourteen days;5-FU,intraperitoneal injection with 25 mg/kg,once a day for five days);Nude mice were executed after 28 days of drug administration.The tumor volume was measured and tumor growth curve was drawn.Ki67 expression,microvessel density were detected by immunohistochemistry.Tumor apoptosis was measured by TUNEL staining.The change of pERK,Bcl-2 and Caspase-3 was investigated by Western Blot.Results1.The inhibitory effect of lenalidomide,chemotherapeutic drugs and combination on MDA-MB-231 cells proliferationMTT results showed that lenalidomide didn't have inhibitory effect on MDA-MB-231 cell proliferation.All of the four chemotherapeutic drugs had inhibitory effect on MDA-MB-231 cells proliferation in a dose-and time-dependent manner.Besides,lenalidomide enhanced the inhibitory effect of cisplatin and 5-FU on MDA-MB-231 cells proliferation.Lenalidomide had no synergetic effect of adriamycin and paclitaxel on MDA-MB-231 cells proliferation.2.The effect of lenalidomide,chemotherapeutic drugs and combination on MDA-MB-231 cells apoptosisFlow cytometry results showed that the apoptotic rate of MDA-MB-231 cells was significantly increased after treated with lenalidomide and cisplatin,respectively.The apoptotic rate was significantly higher after treated with combination of lenalidomide with cisplatin compared with treated with cisplatin.Lenalidomide enhanced the effect of cisplatin on MDA-MB-231 cell apoptosis.Flow cytometry results showed that the apoptotic rate of MDA-MB-231 cells was significantly increased after treated with 5-FU.The apoptotic rate was singinficantly increased after treated with combination of lenalidomide with 5-FU compared with treated with 5-FU.3.The effect of drugs on pERK,Bcl-2 and Caspase-3 expression in MDA-MB-231 cellsWestern Blot results showed that lenalidomide alone decreased the level of phosphorylated extracellular signal-regulated kinase(pERK)in MDA-MB-231 cells,which is known to regulate cell proliferation,whereas pERK level was increased after treated with cisplatin alone.Their combination significantly reduced the pERK level in MDA-MB-231 cells compared with cisplatin alone.Both lenalidomide and cisplatin decreased anti-apoptotic protein Bcl-2 expression and increased pro-apoptotic protein Caspase-3 expression in MDA-MB-231 cells.Besides,the combination significantly downregulated Bcl-2 expression and upregulated Caspase-3 expression compared with single treatment.Lenalidomide downregulated pERK expression.5-FU upregulated pERK expression in MDA-MB-231 cell.Combination reduced pERK expression compared with 5-FU alone.Lenalidomide and 5-FU alone decreased Bcl-2 and increased Caspase-3 expression.Combination significantly downregulated Bcl-2 expression and upregulated Caspase-3 expression compared to lenalidomide and 5-FU alone.4.The inhibitory effect of lenalidomide,chemotherapeutic drugs and combination on HUVEC proliferationMTT results showed that lenalidomide didn't have inhibitory on HUVEC cell proliferation.Both cisplatin and 5-FU had inhibitory effect on HUVEC cell proliferation in a dose-and time-dependent manner.5.The sensitivity of MDA-MB-231 and HUVEC to cisplatin and 5-FUThe same concentration of cisplatin had higher inhibitory effect on MDA-MB-231 cells than on HUVEC cells.It indicated that MDA-MB-231 cells were more sensitive to cisplatin than HUVEC cells.But that was a different thing.The same concentration of 5-FU had higher inhibitory effect on HUVEC cells than on MDA-MB-231 cells.It indicated that 5-FU preferentially inhibited HUVEC cells proliferation relative to MDA-MB-231 cells.These findings indicated that it was suitable for 5-FU as an antiangiogenic drug.6.The effect of combination of lenalidomide with 5-FU on HUVEC proliferationLenalidomide significantly enhanced the inhibitory effect of 5-FU on HUVEC proliferation compared with 5-FU alone.7.The effect of combination of lenalidomide with 5-FU on HUVEC migrationLenalidomide and 5-FU inhibited HUVEC cells migration,respectively.Besides,lenalidomide significantly enhanced the inhibitory effect of 5-FU on HUVEC cells migration compared with 5-FU alone.8.The effect of lenalidomide,5-FU and combination on pAkt expression in HUVEC cellsWestern Blot results showed that lenalidomide and 5-FU downregulated pAkt expression,respectively.Lenalidomide combined with 5-FU significantly decreased pAkt expression compared with 5-FU alone.9.The inhibitory effect of lenalidomide,5-FU and combination on the subcutaneous transplanted tumor model of TNBC.Thirty cases of nude mice models were successfully constructed.Significant increasement of apoptotic cells and decreasement of microvessel density(MVD)was detected and no significant change of Ki67 expression was found in LEN group compared with PBS group.Apoptotic cells was increased MVD was significantly decreased in low-dose(5 mg/kg)5-FU.But no significant change of Ki67 expression was investigated in low-dose(5 mg/kg)5-FU.Significant decreasement of Ki67 expression and MVD and significant increasement of apoptotic cells were investigated in high-dose(25 mg/kg)5-FU group compared with PBS group.Significant decreasement of Ki67 expression and MVD and significant increasement of apoptotic cells were detected in combination of lenalidmomide with 5-FU group compared with 5-FU group.Western Blot results showed that pERK and Bcl-2 level was slightly decreased and that Caspase-3 level was slightly increased in LEN group as compared to PBS group.Bcl-2 level was slightly decreased and pERK and Caspase-3 level were slightly increased in low-dose(5 mg/kg)5-FU group.Bcl-2 level was significantly decreased and pERK and Caspase-3 level was significantly increased in high-dose 5-FU(25 mg/kg)group as compared to PBS group.Significant decreasement of pERK and Bcl-2 and significant increasement of Caspase-3 were detected in combination of lenalidmomide with 5-FU group compared with 5-FU group(Fig.12).Conclusion1.Lenalidomide can inhibit TNBC growth by inducing tumor cell apoptosis,inhibting human endothelial cell migration.2.Among ariamycin,paclitaxel,cisplatin and 5-Fluorouracil,lenalidomide can enhance the inhibitory effect of cisplatin and 5-FU on MDA-MB-231 proliferation.Lenalidomide can also enhance the inhibitory effect of 5-FU on HUVEC migration.3.Lenalidomide can improve the antitumor effect of 5-FU on TNBC growth by decreasing the level of pERK and Bcl-2,increasing the level of Caspase-3 and inhibiting angiogenesis.