| Objectives In China, primary hepatocellular carcinoma (PHC) is themalignancy with high incidence and the second fatal cancer, and in developedcountry of Europe and America the incidence of PHC is increasing. However, noeffective measures control its occurrence and development so far. Because Chineseherbal medicines have special advantages of multi-target, multi-effect, few sideeffects, improving immune response, hardly drug-resistance, it plays an importantrole in cancer treatment. It has been a research hot spot that using modern medicaltechnology to investigate anti-tumor effects of Chinese herbal medicine, extracteffective components, and develop anticancer drugs with Chinese characteristicsand independent knowledge property-right. Recent evidences reveal thatAgrimonia Pilosa Ledeb. (APL), a Chinese medicine herb, can inhibit tumorgrowth in vivo and in vitro. In the present study, the anti-cancer effects of APLwater extracts on cultured hepatocellular cancer line SMMC-7721 cells wereobserved in vitro and the underlined mechanism was investigated, which wasexpected to provide experimental proofs for developing a new anti-PHC drug.Methods Dried APL plus water boiled down to prepare water extracts withdried APL concentration of 1.25g/ml. The extracts were sterilely filtered and storedin refrigerator, and diluted with culture medium into different concentrationsaccording to experimental design when using. SMMC-7721 cells were revitalizedin vitro, maintained in RPMI1640 medium with 10% fetal calf serum. Cells inlogarithm growth phase were digested into suspended cells, and incubated into96-well plates. The medium containing different concentrations of APL extractwere used for further culture when cells grew well. The effects of APL onSMMC-7721 cells were observed by the MTT assay at three time points (24, 48,72h) after culturing with the medicine-containing medium, and inhibitory rate (IR)and IC50 were calculated. This experiment was divided into A, B, C, D groupaccording to APL water extracts concentration of 40, 20, 10, 5mg/ml, respectively,and blank control group (medium without APL), 5-Fu control group (medium with10μg/ml 5-Fu). A series of tests of SMMC-7721 cell apoptosis induced by APLwater extracts were performed in group A and B which presented obviousapoptotic effects: 1cell-climbing slide were prepared, and cell morphologicchanges after HE stain were observed under a light microscope; 2 apoptotic ratiowere measured by Flow Cytometer after Annexin V/PI double stain; 3theexpressions of Bcl-2 and P53 protein on the cells were examined byimmunohistochemical staining method. Results 1 The APL water extracts could inhibit the proliferation ofSMMC-7721 cells in vitro, and with features of depending on time-dose. Theinhibition effects were observed at 24h in 40mg/ml concentration group, and IRswere 23.32%, 51.71%, 71.55% at three time points, respectively, and with higherIRs than 5-Fu control group. The inhibition effects were observed at 48h in20mg/ml concentration group, and IRs were 23.94%, 27.51% at 48, 72h timepoints, respectively. Slight inhibition effects were observed at 72h in 10mg/mlconcentration group, and IR was 13.47%. No inhibition effect was observed in5mg/ml concentration group. The IC50 of APL on SMMC-7721 cells at 24, 48, 72htime points were 76.38, 38.66, 33.21mg/ml, respectively. 2 The HE stainmorphologic changes of SMMC-7721 cells after APL treatment were compatiblewith apoptosis under light microscope. It can be observed that cells changed intosmaller and rounder, karyopyknosis, crescent formation, and cells spilt to severalpieces containing condensed nuclear after the highest concentration APL treatment.3The apoptotic ratio of SMMC-7721 cell treated by APL were raised detected bythe Flow Cytometer, and with trend of time-dose dependent .The cell apoptoticratio were 33.41%, 42.73% in 40mg/ml concentration group at 48, 72h,respectively, and 19.47%, 23.02% in 20mg/ml concentration group at 48, 72h,respectively, and with significant differences between groups (P<0.05~0.01). 4Immunohistochemical staining tests revealed that the expressions of Bcl-2 proteinon SMMC-7721 cells were down-regulated, and P53 protein up-regulated afterAPL treatment, and with time-dose dependent. In 40mg/ml concentration group,the positive cell ratio of Bcl-2 was from (47.87±4.70)% at 48h down to(26.48±5.69)% at 72h, and the positive cell ratio of p53 was from (48.67±4.35)%at 48h up to (83.71±3.17)% at 72h. In 20mg/ml concentration group, the positivecell ratio of Bcl-2 was from (71.85±4.07)% at 48h down to (58.53±5.27)% at 72h,and the positive cell ratio of p53 was from (22.87±2.32)% at 48h up to(50.61±3.98)% at 72h. There were significant differences between groups and timepoints (P<0.01). Conclusions 1. The Agrimonia Pilosa Ledeb. water extracts could inhibit the proliferationof SMMC-7721 cells in vitro and with characteristics of time-dose dependent. TheIC50 of APL for SMMC-7721 is 33.21mg/ml at 72h. 2. The Agrimonia Pilosa Ledeb. water extracts could induce the apoptosis ofSMMC-7721 cell in vitro, the apoptotic ratio with trend of time-dose dependent. 3. The Agrimonia Pilosa Ledeb. water extracts could regulate down theexpression of Bcl-2 protein and up the expressions of P53 protein, and both withcharacteristics of time-dose dependent. 4. The apoptosis of SMMC-7721 induced by Agrimonia Pilosa Ledeb. waterextracts may be the basis of proliferation inhibition, and its underling mechanismsis down-regulate the expression of Bcl-2 protein and up-regulate the expression ofP53 protein. |
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