Study On Anti - Tumor Growth And Metastasis Of Pulsatilla Saponins And Its Mechanism

Malignant cancer is a serious threat to human life. It brings a huge economic burden and pain to the patient. Breast cancer is a common malignant tumor in women. Surgery, radiotherapy and chemotherapy are the basic therapies for cancer patient. The treatment effect of chemotherapy drugs is poor for the advancerd and metastatic patient, accompanied by adverse reactions and drug resistance. Therefore, it is very urgent to develop anti-tumor new drugs with low side effect. Until now, researchers pay more attention to the Pulsatilla chinensis compound or the extract mixture on anti tumor. The Pulsatilla chinensis saponin monomer on anti tumor is rarely reported. The role of saponins on tumor invasion and metastasis has not been reported and the mechanisms of how saponins exert the anti-tumor functions are still poorly characterized. Here, we screen the anti-tumor effect of the five saponin monomers which are extracted from Pulsatilla chinensis. We obtained the strong anti-tumor effect of R13 and saponin D. The anti-tumor effect of R13 is stronger than saponin D. We confirmed the effect on anti tumor of R13 and saponin D and their effect on metastasis. We further research the mechanism of R13 and saponin D on anti tumor and inhibiting metastasis.Object The five saponin monomers were screened on anti-tumor effect. They were saponin D、R13、saponin A、saponin B and β-Hederin. We determined the stronger anti-tumor effect of R13 and saponin D. We further research the mechanism of R13 and saponin D on anti-tumor through cell cycle arrest, apoptosis, energy metabolism and autophagy. We for the first time research the anti tumor metastasis and their mechanism of R13 and saponin D. Antitumor effect and inhibition of metastasis were verified in animal model. We aim to find the effective components and the best one on anti tumor in order to lay a foundation of new drug development and clinical application of saponin monomers.MethodsPart one Screening of antitumor active ingredient effect of Pulsatilla saponinsThe anti-tumor effect of the five Pulsatilla saponins were studied in five cell lines which are human breast cancer ZR75-1 and MCF-7 cells, human liver cancer HepG2 cell、human cervical carcinoma Hela cell, human colon cancer HCT-116 cell. The IC50 values were determined according to the inhibition rates of six different concentrations based on the statistics method of the compounds. The IC50 was fitted by software Origin 9.0.Part two Study on the antitumor effect and mechanism of Pulsatilla saponins1. The anti tumor efficacy experiment was performed in the five cell lines. Then OD value was tested by CCK-8 kit. The cell growth curve was measured.2.’Cell soft agar colony formation and cell colony formation were excuted in ZR75-1 and MCF-7 cells. The cells were treated with R13 and soponin D. Then Two weeks later, colony number was counted.3. Cells treated with R13 and saponin D were respectively stained with PI or Annexin V-FITC/PI. Then the effect of cell cycle and apotosis of R13 and saponin D were measured by Flow cytometry. And the expression of cycle maker proteins cyclin D1, cyclin A, cyclin E, P21, P27, and apotosis related protein caspase-3 and PARP was detected by western blotting.4. Cells were treated with R13 and saponin D for 24h-48h. Then glucose uptake, ATP, PFK, pyruvate and lactate were detected according to the manufacturer’s protocol5. The GFP-LC3 plasmid was transfected into breast cancer cells by Lipofectmine 2000 reagent. Then R13 and saponin D were respectively added into the cells. The cells were observed with a fluorescence microscope. Meanwhile, the expression of LC31I was analyzed by western blotting.Part three Study on the effect and mechanism of saponin monomer against tumor metastasis1. Wound healing experiment was performed in five cell lines, in order to screen the effect of the five Pulsatilla saponins on metastasis.2. Cell invasion was performed with matrigel invasion chambers. The cells treated with R13 and saponin D were observed under microscope and invasion number was counted.3. Cells were treated with R13 and saponin D. Then the expression of EMT related proteins E-cadherin、N-cadherin and Vimentin was detected by western blotting.Part four Animal study on the effect of R13 on anti tumor and metastasis1. The inhibitary effect of R13 on cancer cell growth were performed in HepG2 cell tumor bearing nude mice model and 4T1 cell tumor bearing nude mice model. Tumor size was measured at indicated times using calipers. The expression of cycle related protein, apotosis related protein, EMT related protein and autophagy marker LC3Ⅱ were analyzed by western blotting.2. The inhibitary effect of R13 on cancer cell metastasis was performed by using 4T1-LUC cell tumor metastasis nude mice model. Then the tumor formation in lung tissue was observed by IVIS200 imaging system. And the cancer cell distribution in lung tissue was observed by HE staining under microscope.Result1. We found that the IC50 value of R13 between 0.54-0.86 × 10-5mol/L was the minimum among the five saponins, while the the IC50 value of saponin D between 1.09-1.93× 10-5mol/L was larger than that of R13. So, we selected R13 and saponin D for further anti-tumor study.2. We mapped cell growth curves of the five saponin monomers in five cancer cell lines. The data presented with statistical significance showed that R13 at high and low dosage significantly inhibited cell growth in the five cancer cell lines in a dose-dependent manner (p<0.05 or p<0.01). The inhibitary effect of R13 on cancer cell was consistent with the positive control cisplatin, and the effect of R13 at high dosage was equivalent to cisplatin. Saponin D manifested a lower inhibition effect in a dose-dependent manner(p<0.05 or p<0.01), compared to R13 and cisplatin. The inhibitory effect of R13 and saponin D on cancer cells was more sensitive than that of normal cells.3. The rusults of cell soft agar colony formation and cell colony formation displayed that the cells intervined with R13 growed slowerly, compared to the negative control. And the colony number in R13 was less than the negative control (p<0.01), and was consistent with cisplatin.4. Compared with the control cells, treatment with R13 and saponin D in ZR75-1 and MCF-7 cells significantly increased the proportion of cells in G0/G1 phase, which associated with decreased proportion of cells in S-phase. And the result showed that R13 significantly inhibited the expression othe G1/S-phase markers cyclin D1, cyclin A and cyclin E but increased that of P21 and P27 in ZR75-1 and MCF-7 cells. These results were similar with positive control cisplatin. The regulatory effect of R13 on cell cycle and G1 phase-related protein was stronger than that of saponin D5. The resluts showed that the percentage of apoptotic cells treated with R13 and saponin D were higher than the mock-treated in ZR75-1 and MCF-7. The effect of R13 was comparable to cisplatin. Consistent with the results of saponin D and R13 modulation of apoptosis, saponin D and R13 revealed increased cleaved (active) caspase-3 and cleaved PARP level. The effect of R13 on apoptosis was stronger saponin D.6. The data displayed that R13 could decrease the content of glucose uptake, ATP, pyruvate and lactate and the level of PFK. But saponin D could decrease glucose uptake lactate and the level of PFK, while had no significant change on ATP and pyruvate.7. Both R13 and saponin D could promote the occurrence of autophagy in ZR75-1 and MCF-7 cells. Cells treated with R13 and saponin D were observed with a fluorescence microscope. And autophagosome was found. The expression level of LCII was increased in R13 and saponin D groups.8. Wound healing assay and Cell invasion assays showed together that R13 and saponin D inhibit BC cell migration and reduced the number of invaded cells, with morphologic changes from an elongated fibroblastoid phenotype to a polarized epithelial phenotype. R13 and saponin D increased the expression of the epithelial marker E-cadherin and decreased that of N-cadherin and Vimentin, two mesenchymal markers.9. Compared with the saline group, mice administered with R13 significantly reduced the size of BC or Liver tumor volume. The intensity of photonic radiance in the lungs of the R13 group was much lower than that in the saline group in 4T1-LUC tumor metastasis mice model. Histologic analysis on the lungs confirmed the metastasis foci. The number of tumor foci found in the lungs in the R13 group was much less than that in the saline group. The expression level of related proteins were consistent with that of cells in vitro.ConclusionR13 has the strongest anti-tumor effect and the ability to inhibit metastasis among the five saponin monomers in five cancer cell lines, which is more sensitve to cancer cells than nomal cells. R13 inhibits cell growth by suppressing G1/S phase transition, inducing cell apoptosis, regulating cell energy metabolism and promoting cell autophagy. R13 suppresses BC cell metastasis with decreased EMT. Importantly, R13 exhibits the strongest effect on both anti-proliferation and anti-metastasis abilities among all of the reported Pulsatilla saponin monomers. Pulsatilla chinensis, as a natural traditional Chinese medicine, R13 is easy to access. Thus, R13 would be used at both non-metastatic and metastatic stages of breast cancer.