| Objective:There are three pathways for indirubin, an anti-tumor component of Chinese traditional medicine, to inhibit the growth of cancer cells. The three pathways are competitively inhibit CDKs with the kinase's ATP-binding pocket, directly inhibits c-Src kinase and block constitutive Stat3 signaling, and inhibition of nucleoside transporters respectively. In view of indirubin can be regulated through the binding with the receptor of its ligand AhR, we deduce that indirubin can inhibit the growth of human cancer cells related to CDK or Src or kinase. The conclusions have been verified on many kinds of human cancer cells using clonal cell culturing method and MTT method. The cancer cells include leukemia cell line(K562), gastric cancer cell line, lymphoma cell line, uterine cervix cancer cell line, promyelocyte leukemia cell line(HL-60), carcinoma of gallbladder cell line, hepatocarcinoma cell line, and hilar cholangiocarcinoma cell line. In the meantime, we have also observed the effect of indirubin on the growth of haemopoietic progenitor cell through the invto culture of haemopoietic progenitor cell.Methods:The effects of indirubin on the growth of the following cancer cells have been observed using semisolid cell colony culture method, which include leukemia cell line(K562), gastric cancer cell line, lymphoma cell line, uterine cervix cancer cell line, promyelocyte leukemia cell line(HL-60), carcinoma of gallbladder cell line, hepatocarcinoma cell line, and hilar cholangiocarcinoma cell line respectively. The indirubin was resolved using dimethyl sulfoxide(DMSO), and three groups with the concentration 1mg/ml , 100μg/ml and 10μg/ml are obtained. Then, after the mixture of drug and cell lines is put in the 37℃water for 30 minutes and centrifuge for 20 minutes, the drug was washed out. The cell lines was rebuilt into cell suspension. Next, the cells in cell suspension are counted, and cell suspension is put into the culture box with 37℃saturated humidity 5%CO2 for six days, and the number of cell colony are observed.Finally, the experimental animals (mouse, rabbit) were given the indirubin from the mouth. The dosages for mouse are 4mg/kg,8mg/kg,16mg/kg respectively. The blood from mouse eyes is extracted and the serum containing the sero-drug is separated after 24 hours. The dosage for rabbit is 133mg/kg. The blood plasma of albino rabbit is extracted given medicine in 2h, 4h, 6h, 8h, and 24h. The number of cell clones of uterine cervix cancer , gastric cancer ,leukemia(K562), carcinoma of gallbladder , hilar cholangiocarcinoma is observed after 6-day culture. The MTT method was used to investigate the effects of indirubin on the cell growth of K562,carcinoma of gallbladder ,hilar cholangiocarcinoma. In the meantime, semisolid cell colony culture method was used to observe the effects of indirubin and sero-drug/ blood plasma of drug on granulocyte colony–forming cell,erythroid progenitor cell,megakaryocytic progenitor cell,mix haemopoietic progenitor cell.Results:The inhibition of indirubin on cell growth has been widely observed in many kinds of cancer cells.1. The inhibition results of indirubin on growth of cancer sells in vitro culture: indirubin with dosage from 0.5 to100μg/ml has significant inhibition effects on uterine cervix cancer cell, hepatocarcinoma cell, lymphoma cell, hilar cholangiocarcinoma cell, leukemia cell(K562) and promyelocyte leukemia cell(HL-60),but has not significant effect on gastric cancer cell.2. The inhibition effects of sero-drug of mus with different concentration on the growth of cancer cells: sero-drug of mus has effects in inhibiting the growth of uterine cervix cancer and gastric cancer.3.The inhibition effects of the blood plasma of albino rabbit with the same dosage of indirubin on the growth of cancer cells: The blood plasma of albino rabbit with indirubin has significant effects in inhibiting the growth of gastric cancer cell after given medicine in 2 hours, and the effects can last 96 hours after given medicine. For cancel cells such as K562, carcinoma of gallbladder and hilar cholangiocarcinoma,The inhibition effects of the blood plasma of albino rabbit with indirubin take place after given medicine in 4 to 6 hours, and gradually decrease after given medicine in 96 hours.4 The inhibition effects of mus sero-drug with different dosage on the growth of K562 cells: mouse serum after given medicine in 24 hours has inhibition effects on the growth of K562 cells in different dosage from 0.05ml to 0.4ml, but effect on the K562 cells in 0.05ml dosage is weaker than that in other dosages from 0.1-0.4ml.5.The results from MTT method: The rabbit serum with indirubin has inhibition effects on growth of K562, carcinoma of gallbladder, hilar cholangiocarcinoma. Each cell line has different sensitivity to the serum with indirubin. The growth of K562 cell line is inhibited after given medicine in 2 hours to 24 hours. The growth of carcinoma of gallbladder cell line is inhibited after given medicine in 8 hours, and the maximum inhibition is reached in 24 hours. The growth of hilar cholangiocarcinoma cell line is inhibited after given medicine in 2 hours, and subsequently the inhibition gradually decrease.6.The inhibition effects of indirubin on all myeloid haemopoietic progenitor cell:There is no inhibition effects of mouse serum with indirubin on the growth of haemopoietic progenitor cell, which includes CFU-GM, CFU-E,BFU-E, CFU-Meg,and CFU-Mix. For CFU-GM,CFU-E and BFU-E, there is a light effect to stimulate the growth of cell lines, but the effect is unrelated to the concentration of indirubin in serum. In addition, there also exists stimulation effects of rabbit serum with indirubin on the growth of granulocyte colony-forming cell. Moreover, the intro results verified the above conclusion too. |
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