| ObjectiveCancer is a very severe threat to human lives. Although there are many anti-tumor drugs for cancer patients, the effect seems poor with the severe side-effects and drug resistance. Nowadays, it is necessary for us to find some new anti-tumor drugs.There are many treating experiences to cancer in Chinese medicine. Numerous researches proved that Chinese Medicine had an effect of anti-tumor with not only compound but also single herb or active ingredients. Researchers have made some Chinese medicines to treat cancer, but the effects are limited. Therefore, the field of anti-tumor Chinese medicine is in the ascendant to the whole world.Modern researches proved that there are some active ingredients with directed cytotoxic effect to cancer cells, such as curcumin, Elemene, Matrine, Garlicin, taxol, tanshinone, etc. There is a close relationship between Gap Junction Intercellular Communication(GJIC) and genesis and development of neoplasm, which has become a hot spot. But the reports of Chinese medicine and GJIC are still few, such as tanshinone IIA, etc.This study was to choose active ingredients with the inhibited or cytotoxic effect to five cancer cell lines, and choose the up-regulated active ingredients to GJIC. So we can find out some new data to Chinese medicine.Methods1 Measurement of growth time and density on five cancer cell line In this experiment, we measured the absorbent of five cancer cell line of SGC-7901,Hep-G2,H22,HL-60 and B16, with different densities and time. The reduction rate of Alamar Blue would be used to identify the most perfect factors to cell growth.2 Recover the active ingredients to tumor cell lines 2. 1 The active ingredientsAstragalosideⅣ, Ginsenoside Rh2, Ginsenoside Rg3, Icariin, xuduan, Ferulic Acid, Echinacoside, trigonelline, cordycepin, Ganoderma lucidum, ganoderic acid and Shenfu Injection.2. 2 The inhibited or cytotoxic experimentHavest SGC-7901, B16, Hep-G2 and culture overnight for 24h, and then plant in 96-well plate with 4000 per well. Havest HL-60 and H22, and plant in 96-well plate with 4000 per well. Add AstragalosideⅣ, Ginsenoside Rh2, Ginsenoside Rg3, Icariin, xuduan, Ferulic Acid, Echinacoside, trigonelline and cordycepin respectively with an eventual concentration of 0μM, 5μM, 10μM, 20μM, 40μM, 80μM, Ganoderma lucidum and ganoderic acid with Omg/L, 5mg/L, 10mg/L, 20mg/L, 40mg/L, 80mg/L, and Shenfu Injection with OmL/L, 5mL/L, 10mL/L, 20mL/L, 40mL/L, 80mL/L, for five wells. Culture in 37℃, 5％CO2 with 180μl complete medium, then add 20μl Alamar Blue per well, and measure the absorbent of 570hm and 630hm for 0h,24h,48h and 72h. The survival rate was determined, with the curve of growth and IC50 value.2.3 Flow cytometry analysisWe planted HL-60 in an 6-well plate with 2.5×106 per well, with control group, ECH 20μM group, ECH 40μM group, cordycepin 20μM, cordycepin 40μM, Shenfu injection 25mL/L and Shenfu injection 50mL/L. After cultured with these drugs, we harvested the cells and measure with the flow cytometry.2.4 Agar electrophoresisHavest H22 cells with treatment of Shenfu injection(25mL/L and 50mL/L), wash with PBS, and attract DNA to electrophoresis with 1.5％agar, 30V for 2 hours.Then observe with UV and take the picture.3 The influence of GJIC and Cx43 with active ingredients We developed 15 groups of control group, apigenin, AGA, AstragalosideⅣ, Ginsenoside Rh2, Ginsenoside Rg3, Icariin, xuduan, Ferulic Acid, Echinacoside, trigonelline, cordycepin, Ganoderma lucidum, ganoderic acid and Shenfu injection. After treated 36 hours with these ingredients, the effect on the GJIC of Hep-G2 were detected by SL/DT. The expression of Cx43 in cells were determined by FITC indirect immuno-fluorescent assay.Result1 The reduction rate of Alamar Blue showed 4000 cells per well with total 200μl have a linear relationship with time for 24 to 72 hours. 2 Inhibited or cytotoxic experiment showed①The severe cytotoxic effects were observed under microscope on ECH treated Hep-G2, cordycepin treated HL-60, and Shenfu injection treated HL-60 and H22.②Alamar Blue assay showed ECH had an inhibit effect to Hep-G2 in 80μM, IC50 is 467.8μM(368.3μg/mL), cordycepin to HL-60, IC50 is 155μM(46.5μg/mL). After treated with Shenfu injection, the survival rates of H22 were 100±4.93％,104.31±3.19％,97.31±4.53％,66.44±2.94％,18.01±3.93％,2.54±1.35％, respectly, with IC50 value of 26.51mL/L. Other ingredients showed no cytotoxic effects. HL-60 cells treated with Shenfu Injection were detect by MTT assay. The survival rates of HL-60 were 87.5±8.02％, 56.45±5.03％and 38.38±7.48％, respectly, with IC50 value of 68mL/L。③The apoptotic rate with HL-60 treated by cordycepin is 1.36％, Shenfu injection is 4.20％. The sub-G1 peak was observed in HL-60 treated with Shenfu Injection. The S phase was stuck in HL-60 treated cordycepin.④DNA ladder was observed on H22 treated by Shenfu injection.3 The effect of active ingredients to GJIC and Cx43①SL/DT showed there were an up-regulation of GJIC on Hep-G2 treated with Apigenin, ECH, cordycepin and Shenfu Injection.②The result of Cx43 detection is control group with 1.4±0.55 scores, ECH with 1.6±0.55, cordycepin with 0.6±0.45, ICA with 3.4±0.55, trigonelline with 2.0±0.71, Shenfu injection with 3.4±0.89. There were a up-regulation to ECH, trigonelline, G-Rh2, ICA and Shenfu injection.Conclusion①There are cytotoxic effects on H22 and HL-60 with treated Shenfu injection. A. We could observe the changes under microscope. B. MTT and Alamar Blue assay showed the survival rates decreased. C. The IC50 of HL-60 was 26.51mL/L, and H22 was 68mL/L. D. The flow cytometry and agar electrophoresis had demonstrated that apoptotic cells were existed.②There are inhibited effects on HL-60 with treated cordycepin. A. We could observe no changes under microscope. B. Alamar Blue assay showed the survival rates decreased. C. The IC50 of HL-60 was 155μM(46.5μg/mL). D. The flow cytometry had demonstrated that the S phase was stagnation.③There are low effects on Hep-G2 treated with ECH. The IC50 was 467.8μM (368.3μg/mL).④The other nine active ingredients showed no inhibited and cytotoxic effects of the five cell lines.⑤ECH, trigonelline and Shenfu injection showed a up-regulation effect to GJIC.⑥Shenfu injection, trigonelline, ICA and G-Rh2 showed a up-regulation to Cx43.
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