Effect And Mechanisms Of Excessive Human Serum Albumin Weakening Anticancer Effect Of Cisplatin And Etoposide

ObjectiveHuman serum albumin(HSA)is usually abused due to incorrect medication information health literacy both in developed and less-developed rural area in our country.In most circumstance,HSA is often mistaken for tonic and used for cancer patients.However,the possible effect of excessiveHSA on chemotherapy is unclear.In this research we hope:1.Investigation of cases(1)Application of HSA in patients with lung cancer were investigated to clarify the extent of the abuse of HSA in major hospitals.2.To detected the change of pharmacodynamics of chemotherapy drugs and concrete mechanisms in cell-level of chemotherapy drugs cisplatin(DDP)and etoposide(VP-16)alone and in combination with excessive human serum albumin(eHSA):(1)to test the cell toxicity in cell-level of chemotherapy drugs cisplatin(DDP)and etoposide(VP-16)alone and in combination with eHSA in human lung cancer cell line A549;(2)to study the effect of eHSA on cell apoptosis,cell colony formation,and cell migration by flow cytometry,cell cloning assay,and cell migration assay;(3)to detect the intracellular drug concentrations of chemotherapy drugs alone and in combination with eHSA with LC-MS/MS;(4)to explore the mechanisms on the molecular level of ERCC1,TOP2 A protein and m RNA expression.3.To explore the efficacy of chemotherapy drugs DDP and VP-16 alone and in combination with eHSA in animal level :(1)to measured serum album in control group,DDP/VP-16 alone or in combination with eHSA;(2)to study the effect of excessive human serum albumin on body weight;(3)to measured lung tumor weight and volume changes and the inhibition rate in DDP and VP-16 alone group or in combination with eHSA group;(4)to explore the mechanisms on the molecular level of ERCC1,TOP2 A protein and m RNA expression.Methods1.Investigation of case(1)Choose all cases of patients from January 2016 to April 2016 of cancer center,oncology,surgical oncology,respiratory medicine in two three-level general hospitals.Inclusion criteria: patients with lung cancer which used DDP and VP-16 alone or combination.The survey included gender,age,tumor stage,serum albumin level,chemotherapy drugs and the use of human serum albumin.2.Cellular test(1)To test the cell toxicity of cisplatin and etoposide alone and in combination with excessive human serum albumin in human lung cancer cell line A549 by CCK-8 assay.To dilute cisplatin was diluted to 0,1,5,10,15,20?mol/L concentration,etoposide was diluted to 0,2.5,5,10,20?mol/L,human serum albumin to 10,20?mol/L.Cell proliferation inhibition rate was conducted after 72 h treatment.Combination concentration of cisplatin and etoposide was chosen at 10?mol/L.Experiments are divided into separate groups: control group,VP-16 alone,DDP alone,HSA alone group,VP-16 and DDP+10?mol/L HSA combination group(VP-16/DDP+HSA),VP-16 and DDP+ 20?mol/L HSA(VP-16/DDP+HSA).(2)Cell apoptosis effect of eHSA combination was assayed by flow cytometry after 24 h and 48 h treatment.(3)Cell migration assay was used to determine the changes of tumor cell migration rate after 48 h treatment using transwell chamber.The migration lasted for 24 h.(4)LC–MS/MS was used to determine the intracellular drug concentration.(5)Real-time quantitative PCR and western blot was used to study the change of drug-sensitive gene mutations--ERCC1 and TOP2 A.After 72 h drug treatment,total protein was extracted for western blot assay to detect the expression of ERCC1,TOP2 A.Total RNA was extracted for real-time quantitative PCR experiment.One-way ANOVA statistical method was used based on homogeneity of variance.T test was used to compare the difference between groups.(6)A549 Cells(150000 cells per well)were seeded into 6-well plates for 24 h,ERCC1/TOP2 A were respectively interfered with OPTI-MEM 500 ?L +si RNA-mate 1000 pmol +corresponding si RNA 1000 pmol.Then A549 cells and RNAi A549 cells treated with DDP,VP-16,HSA alone or DDP+HSA,VP-16+HSA combination for 72 h and collected for Western blotting and q RT-PCR.A549 cells(5000 cells per well)were seeded into 96-well plates for 24 h,ERCC1 and TOP2 A were respectively interfered with OPTI-MEM 50?L+si RNA-mate100pmol+corresponding si RNA 80 pmol.Eight hours later,A549 cells and RNAi A549 cells treated with DDP,VP-16,HSA alone or DDP+HSA,VP-16+HSA combination for 72 h and collected for CCK-8 test.(1)LLC lung cancer cells(1×107 cells)suspended in 0.2m L PBS was subcutaneously inoculated in the right flank of C57BL/6 mice.The model was established at day 10 after inoculation.Normal mice were set as control without any treatment.All mice inoculated with tumor cells were given 1,2,4 g/kg/d of HSA i.v.via tail vein for 3 days respectively,and then were randomized into 12 groups as follows: Group A: NS + NS,Group B: HSA(1 g/kg/d)+ NS,Group C: HSA(2 g/kg/d)+ NS,Group D: HSA(4 g/kg/d)+ NS,Group E: NS + DDP,Group F: HSA(1 g/kg/d)+ DDP,Group G: HSA(2 g/kg/d)+ DDP,Group H: HSA(4 g/kg/d)+ DDP,Group I: NS + VP-16,Group J: HSA(1 g/kg/d)+ VP-16,Group K: HSA(2 g/kg/d)+ VP-16,Group L: HSA(4 g/kg/d)+ VP-16.On day 4 the mice were given 2,10 mg/kg/d of DDP/VP-16 i.p.for 18 days,respectively.(2)After HSA administration 3 days later,the blood albumin level was detected before the chemotherapy initiation(on day 4)and at the end of treatment(on day 21).Blood albumin level was detected by using Beckman Coulter(AU5800).(3)During the treatment,tumor length(L)and width(W)was calibrated using caliper twice a week and tumor volume was calculated as the formula: V=L×W 2 /2;(4)After treatment,tumors were isolated and weighed.The inhibition rate was calculated as ratio of(1-tumor weight of experiment group/control group)× 100%;(5)After being treated with indicated drugs,tumor tissue(groups A,C,E,G,I,K)were sliced and sonicated while cells were collected in lysis buffer on ice.The protein was quantified with BCA protein assay kit.The expression of ERCC1 and TOP2A were detected using western blot.Results1.Investigation of cases(1)Through the survey we found that in 100 cases of patients,Serum 3.Animal experiment albumin levels lower than 35g/L,12 patients were intravenous injection of HSA;Higher than 35g/L about 88 patients,24 patients of them intravenous injection of HSA,unnecessary use of HSA as high as 27.27%.2.Cell experiment(1)CCK-8 results showed that excess HSA reduced the inhibition effe ct of cisplatin and etoposide on A549 cells.The inhibition rate of the proli feration of VP-16/DDP+HSA(10?mol/L)is 62.49%±1.50%/67.51%±2.58%.T he inhibition rate of proliferation of VP-16/DDP+HSA(20?mol/L)is 62.10%±1.54%/64.99%±3.29%.The combination group percent rate was significant ly lower than that of the single drug group VP-16/DDP(65.20%±0.85%/73.04%±2.31%)(P<0.05).(2)Cell apoptosis was detected by flow cytometry(FCM).HSA alone could not induce apoptosis(1.98%±0.59%,1.81%±0.16%),VP-16 and DD P could obviously induce apoptosis.Compared with DDP and VP-16 alone,excessiveHSA(VP-16-HSA and DDP-HSA)significantly reduced cell apo ptosis,and this effect was dose-dependent.After cells were treated 24 h o r 48 h VP-16 alone,cell apoptosis rate is 19.70%±0.99%/34.79%±1.92%,th e combination group of drug VP-16+HSA(10?mol/L)apoptosis rate is 13.21%± 0.56%/29.19%±1.04%,the apoptosis rate in combination group VP-16+HSA(20?mol/L)is9.89%±0.68%/22.28%±1.40%(P < 0.05).After DDP treatm ent 24 h or 48 h alone,cell apoptosis rate was31.50%± 0.95%/40.15%±0.69%,the combination group of drug DDP+HSA(10?mol/L)apoptosis rate is22.70%±2.21%/35.22%±0.40%,the apoptosis rate in combination group D DP+HSA(20?mol/L)is 17.89%± 0.53%/33.28%±1.89%(P < 0.05).(3)Compared to DDP/VP-16 alone,excessiveHSA increased cancer cell migration and colony formation rate,and reduced the concentration of DDP/VP-16 in cells.Western blot and real time fluorescent quantitative PCR assay showed that HSA enhanced the expression of drug sensitive gene mutations ERCC1 and TOP2 A.(4)The results showed that eHSA significantly weakened the inhibiting effect of DDP/VP-16 in A549 cells.However,in RNAi A549 cells,eHSA no longer weakened but enhanced the inhibiting effect of DDP alone,meanwhile eHSA no longer altered the effect of VP-16 alone.Similarly,eHSA supplement significantly enhanced the m RNA expression of ERCC1 and TOP2 A compared with the monotherapy group in A549 cells.In ERCC1 and TOP2 A RNA interfered A549 cells,m RNA expression of ERCC1 and TOP2 A were apparently reduced.eHSA supplement enhanced the protein expression of ERCC1 and TOP2 A compared with the monotherapy group in A549 cells.In ERCC1 and TOP2 A RNA interfered A549 cells,protein expression of ERCC1 and TOP2 A were significantly decreased.These results suggested that ERCC1 and TOP2 A might be involved in cell viability in A549 cells.3.Animal experiment(1)The results displayed that blood albumin levels were positively correlated with HSA supplement in mice before the chemotherapy initiation [HSA(H)>HSA(M)>HSA(L)>NS].However,at the end of treatment their levels in 12 groups were almost the same,suggesting that supplemented HSA was catabolized completely with the time;(2)At the end of treatment,the body weight of mice in Groups A~D increased gradually,meanwhile decreased gradually in Groups E~H and Groups I~L.However,the body weight of mice in Group E was slightly less than that Groups F,G,and H.Similarly,the body weight of mice in Group I was slightly less than that in Groups J,K,and L,although no statistically difference existed among these groups(P > 0.05);(3)Furthermore,we observed that the tumor weight in Groups E and I decreased more than that in Groups F,G,H and Groups J,K,L,respectively(P < 0.05).The tumor growth inhibition rates of DDP treatment Groups E,F,G,H were 44.35,36.89,30.92,29.42%,respectively.The tumor growth inhibition rates of VP-16 treatment Groups I,J,K,L is 32.41,28.78,21.54,19.83%,respectively.Similarly,the tumor volume in Groups E and I decreased more than that in Groups F,G,H and Groups J,K,L,respectively(P < 0.05);(4)Histopathological results displayed that the tumor tissue was soft with smooth or festering surface in Groups A,B,C,and D.In Groups E~L,the tumor texture was very hard with a slightly uneven surface,more bleeding as compared with that in Groups A~D;(5)We found that eHSA supplement significantly enhanced protein expression of ERCC1 and TOP2 A compared with DDP/VP-16 monotherapy group.ConclusionsIn summary,our research about eHSA combination with DDP/VP-16 in vivo and in vitro showed negative effect to anticancer drugs,which suggested that eHSA weaken the anticancer effect of DDP/VP-16 via up-regulating ERCC1/TOP2 A expression,respectively.But further molecular mechanism studies are warranted to investigate whether eHSA is not conducive to lung cancer chemotherapy.