| Methotrexate,the first choice for treating choriocarcinoma whose casefatality was very high before, has a severe problem of acquired drug-resistant.Reversing MDR by drug is an effective method to solve drug-resistant oftumor, in which revering in gene level is the important point. The focus of ourstudy is to reverse drug-resistant and regain the sensitivity to Methotrexate. Ch.Medicine ,with strong reversing cavity, is dominant in reversing multidrugresistance (MDR). Some Ch. Medicine can improve the therapy of tumorwhich can't be achieved by Western medicine. Survivin and Bcl-2 areapoptosis inhibiting genes, which can lead to drug-resistant of tumor withexcessive express. Base on melocular mechanism of MDR, our study observedcytomorphology, the rate of cell adhesion,detection of β-HCG,check proteinexpression through immunocytochemistry by S-P method, and detection ofmRNA expression of genes survivin and Bcl-2 by RT-PCR test to observeanti-tumor effect of shikonin,research the co-effect to inhibit MDR cellJAR/MTX of shikonin and MTX and detect the relationship between thiseffect and express of genes survivin and Bcl-2, then to explore the reversingdrug-resistant mechanism of choriocarcinoma JAR/MTX cell by shikonin invitro。Objective:To explore the effect on inhibiting drug-resistant cell JAR/MTX ofchoriocarcinoma by shikonin, the mechanism of MTX to occur drug-resistantand the effect and mechanism of shikonin to reverse drug-resistant ofchoriocarcinoma.Methods:Culture cells in vitro and divide them into 5 groups:①control group,with saline. ②MTX group ,with MTX2ug/ml. ③shikonin A group, withshikonin 0.2ug/ml. ④shikonin B group,with shikonin 2ug/ml. ⑤MTX andshikonin A group, with MTX 2ug/ml and shikonin 0.2ug/ml. Observe thegrowth condition in 24,48,72 hours of each group and calculate rate of celladhesion;checkβ-HCG value in 24,48 hours after adding drugs;Fix cells andperform H-E stain to observe the change of cytomorphology. Check proteinexpress of genes survivin and Bcl-2 by S-P immunocytochemistry method.Collect cells to check mRNA expression of genes survinin and Bcl-2 byRT-PCR method.Results:1. The inhibitive effect of shikonin to cell growth and the character ofmorphologyAfter been dealed with drugs, cells in controlled group and MTX groupsgrew well. Rate of cell adhesion in shikonin B group had significant differencewith controlled group (p<0.01) and shikonin A group(p<0.05), the differencewere greater in 48 and 72 hours.HE stain showed cells in controlled group andshikonin A group had big nuclear, dark stained and multinuclear, whilepyknosis, karyorrhexis, karyolysis and eosinophilic body can be seen inshikonin groups. The outline of chromatin and some necrosis fragments can beseen in apoptotic body, which showed cell apoptosis occur. So shikonin hasstrong effect on inhibiting JAR/MTX cell growth and leading to cell apoptosisand this effect is time dependent and dose dependent.2. Effect on reversing drug-resistant of JAR/MTX cells to MTX by shikoninand characters of cytomorphology.Our study showed JAR/MTX cells have severe drug-resistant to MTX.There was no significant different between JAR/MTX group and controlledgroup after 50% lethal dose(LD50%) for JAR cells were given (p>0.05).shikonin A group had inhibitive effect compared with controlled group. Butwhen two groups together, cell apoptosis and necrosis occur after only 24hours, and rate of cell adhesion was 22% after 48 hours,6%. after 72hours.Cells dropped, floated and broken ,having significant difference withcontrolled group(p<0.01) and statistic difference with shikonin A group andMTX group(p<0.05). It showed while inhibiting the growth and inducingapoptosis of JAR/MTX, shikonin can reverse the drug-resistant of JAR/MTXto MTX.3. Shikonin inhibit secretion of β-HCG by JAR/MTX.Shikonin can inhibit the secretion of β-HCG by JAR/MTX and thiseffect is time and dose dependent. Secretion of β-HCG in MTX+shivinon Agroup has statistic drop compared with MTX group and shikonin A group(p<0.05) and significant drop with controlled group(p<0.01).4. The molecular biologic mechanism of shikonin to inhibit the growth ofJAR/MTX cellAccording to S-P immuno-histo-chemistry test and RT-PCR test, proteinand mRNA of Bcl-2 and survivin expressed in drug-resistant JAR/MTX cellsin choriocarcinoma in controlled group, while the expression decreaseddominant in shikonin B group with the same trend of mRNA and protein. Inshikonin A group, the expression decreased a little.The inhibitive effect ofshikonin to drug-resistant cell JAR/MTX is dose dependent.5. Mechanism of drug-resistant occurring in JAR/MTX cells to MTXThere were expressions of the protein and mRNA of genes survivin andBcl-2 in JAR/MTX cells in controlled group.After MTX was added, express ofprotein and mRNA of gene survivin increase significantly compared withcontrolled group;protein express of gene Bcl-2 had no change,but mRNA ofgene Bcl-2 increase significantly compared with controlled group.It showeddrug-resistant occurred. MDR of JAR/MTX is relative with excessiveexpression of survivin and Bcl-2 which is actived by MTX in chemistrytherapy.6. The relationship between reversing drug-resistant effect of JAR/MTX cell toMTX by shikonin and expression of survivin and Bcl-2.Our study showed drug-resistant occurred in JAR/MTX cells to 50%lethal dose of MTX for primary culture cells and there was no significantdifference between the MTX group and controlled group.Drug has mildtoxicity to cells while shikonin in shikonin A group.But strong inhibitingkilling effect occurred after put MTX and shikonin A together,with statistaticdifference compared with MTX group and shikonin A group(p<0.05) andsignificant difference compared with controlled group(p<0.01). According toimmuonhistochemistry test and RT-PCR test, compared with controlled group,expression of survivin and Bcl-2 decreased a little in shikonin A group,expression of survivin increased in MTX group.while expression of survivin,Bcl-2 decreased when the two drugs were used together (p<0.01),which can beconcluded that the mechanism of reversing drug-resistant and regainsensitivity of JAR/MTX cells to MTX by shikonin is to decrease expression ofinhibiting apoptosis gene survinin and Bcl-2 and induce cell apoptosis.Conclusions:1. shikonin has strong effect on inhibiting the growth of JAR/MTX and theeffect has dominant time dependent an dose dependent.2. Shikonin can reverse drug-resistant of JAR/MTX to MTX while inhibitinggrowth and inducing apoptosis of JAR/MTX cells.3. Shikonin decrease the secretion of β-HCG in JAR/MTX and this effect istime dependent and dose dependent. On one hand, shikonin induced cellapoptosis and necrosis,so number of cells decreased and secretion of β-HCG decreased. On the other hand, shikonin inhibit secretion of β-HCGby JAR/MTX cells. This two effects influenced each other.4. The inhibiting effect on JAR/MTX cells by shikonin is relative todecreased expressions of survivin and Bcl-2.5. MDR of cell JAR/MTX to MTX has relationship with survivin and Bcl-2.6. The mechanism of reversing drug-resistant and regain sensitivity ofJAR/MTX cells to MTX by shikonin is to decrease expression ofinhibiting apoptosis gene survinin and Bcl-2 and induce cell apoptosis.To sum up, shikonin can inhibit growth of JAR/MTX, induce cellapoptosis and necrosis and reverse drug resistant of JAR/MTX to MTX. Themechanism is to inhibit the activity of survivin and Bcl-2.Expression ofsurvivin and Bcl-2 can be used to evaluate the drug-resistant condition oftumor cells.Ch. Medicine ,with strong reversing cavity, is dominant inreversing multidrug resistance (MDR). Some Ch. Medicine can improve thetherapy of tumor which can't be achieved by Western medicine. Becauseshikonin has multiple effects on human body. it has the possibility to become anew method to cure drug-resistant choriocarcinoma.
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