Antitumor Activity Of Volatile Oil Extracted From Saussurea Lappa On Hepatocellular Carcinoma

?Background & Objectives? Saussurea Lappa,the root of Aucklandia Lappa Decne from compositae,contains a variety of ingredients like volatile oil,sterols,alkaloids,organic acids and so on.Based on the screening strategy of activity,we found that the volatile oil from Saussurea Lappa(VOSL)is the main active anticancer ingredient.Costunolide(Cos)and Dehydrocostus lactone(Dehy),the key anticancer molecules,account for about 75% in VOSL.Many studies have shown that Cos and Dehy have a good pharmacological efficacy in anti-inflammatory,anti-tumor,anti-virus,inhibiting the contraction of blood vessels,immunoregulation and so on.We found that Cos and Dehy have synergistic antitumor effect in vivo.VOSL and Cos-Dehy showed stronger inhibition effect on MCF-7 tumor-bearing nude mice than Cos or Dehy alone.Moreover,VOSL not only restrains the growth of MCF-7 xenograft tumors,but also maintains body weight and vitality of tumor-bearing nude mice,which has a great significance to “tumor-bearing survival” for mice.Application of Traditional Chinese Medicine(TCM)to cancer therapy has a long history.So far it has not become the primary method of cancer treatment.One of the most important reasons is that the chemical compounds of TCM are very complicated,which lead to the dimness of active ingredients and pharmacological mechanisms,and the difficulty of quality control and modern industrial production.Therefore,it is difficult to be admitted by modern medicine system,especially in western countries.The goal of TCM modernization is to keep the characteristics of “multiple components,more target points,system treatment”,and strive to illuminate the active ingredients and pharmacological mechanisms,and achieve controllable quality.To some extent,as to the complexity of chemical composition in Saussurea lappa,it could be regard as a “black box”.While the chemical composition of VOSL is much more clear(Cos and Dehy),it could be regard as a "grey box".Furthermore,Cos and Dehy could be compared to a “white box” for their clear and definite chemical structure and pharmacological mechanism.Therefore,in this study,VOSL was used to treat HCC in vivo and in vitro to evaluate its anticancer effects and explore its mechanisms,with the progressive design from “white box”,“grey box” to “black box” step by step.The results would provide the foundation for development of new anticancer drugs,which acted as the “multiple ingredients,more targets,low toxicity”.Our research work on clarifying the anti-cancer mechanisms of VOSL would make contribution for our country's TCM modernization strategy.?Methods? 1.Screening sensitive cell lines: Using MTT method,HCC cell lines which are more sensitive to VOSL and its effective active ingredients were screened to conduct the subsequent experiments.From SMMC-7721,Hep3 B,Hep G2,Huh-7,HCC-LM3,L02 and WRL-68 cell lines,SMMC-7721 and Hep-3B cell lines were demonstrated to be more sensitive to the tested drugs.2.Cell cycle analysis and clone formation assay: SMMC-7721 and Hep3 B cells were treated with Cos,Dehy,C+2D and VOSL for 48 hours,respectively.Cell cycle analysis and clone formation assay were carried out to evaluate the effects of different ingredients.3.Apoptosis analysis: SMMC-7721 and Hep3 B cells were treated with Cos,Dehy,C+2D or VOSL for 48 hours,and then the apoptosis was detected by Hochest and Flow cytometry analysis.4.Migration and invasion assays: Cell lines were treated with Cos,Dehy,C+2D or VOSL for 48 hours,and cell migration and invasion were detected by Scarification test and Transwell assay.5.Cell protein expression: Using Western blot,the expression levels of relevant proteins which are associated wirh regulation of cell apoptosis and invasion in SMMC-7721 and Hep3 B cells were detected after treated with different concentrations of VOSL,Cos,Dehy and C+2D.6.Animal experiment: SMMC-7721 cells were used to establish the HCC xenograft models.After treated with Cos,Dehy,C+2D,VOSL and 5-Fu,the tumor growth and the general situation of mice were observed,and the tumors were harvested and weighed.Formaldehyde-fixed,paraffin-embedded tissue sections were prepared for examining the expression of relevant protein by immunohistochemistry.?Results? 1.Effects of VOSL on cell biology behaviors of HCC cell lines a.The effects of VOSL and its active ingredients on the proliferation of five HCC cell lines and two normal liver cell lines showed that SMMC-7721 and Hep3 B cells were more sensitive than Hep G2,Huh-7,HCC-LM3,L02 and WRL-68 cells to these compounds.(The half maximal inhibitory concentrations of VOSL on SMMC-7721 and Hep3 B cells were 6.202±0.337,5.405±0.210 ?g/ml).Simultaneously,the inhibitory rates of VOSL on normal liver cells were lower,which revealed the relative weak toxicity on normal cells.b.VOSL,C+2D,Cos,and Dehy significantly inhibited the clone formation activity of SMMC-7721 and Hep3 B cells in a dose-dependent manner.c.VOSL,C+2D,Cos,and Dehy inhibited cell cycle progression in SMMC-7721 cells,with an increase of S phase and a depletion of G0/G1 and G2/M phases,and Cos inhibited Hep3 B cell cycle progression with the similar results.However,VOSL,C+2D and Dehy induced Hep3 B cell cycle arrest with an increase of S phase and G2/M phase and a depletion of G0/G1 phase.d.The results from Hoechst analysis demonstrated that the cell nuclei in the drug-treated groups experienced a stronger blue fluorescence than those in the control group,which meant that VOSL,C+2D,Cos,and Dehy can induce HCC cell apoptosis accompanied with karyopyknosis.e.VOSL,C+2D,Cos,and Dehy treatments led to the dramatic increase of apoptotic rates in both SMMC-7721 and Hep3 B cells,which higher than the control groups(3.46±0.69,8.66±1.74)%.And the apoptotic rates were all above 50% when gave the concentration of IC50.f.Scarification test results showed that VOSL,C+2D,Cos,and Dehy at low concentrations significantly reduced the migration capabilities of HCC cells in a dose-dependent manner when compared with the untreated group.g.Transwell test indicated that VOSL,C+2D,Cos,and Dehy reduced the migration capabilities of HCC cells in a dose-dependent manner when compared with the untreated group.2.Effects of VOSL on protein expression associated with cell apoptosis and invasion.a.VOSL,C+2D and Dehy treatments up-regulated the expression of cleaved-Caspase3,and down-regulated the expression of Caspase3 in SMMC-7721 and Hep3 B cells.However,the expression of Caspase3 and cleaved-Caspase3 in the Cos-treated group had no obvious changes compared with the control group,which meant that the apoptotic induction mechanism of Cos on HCC cells may be different from VOSL,C+2D and Dehy.b.The expression of MMP-9 was significantly reduced both in SMMC-7721 and Hep3 B cells after drug treatments.c.The activation of MEK/P38 cascade in SMMC-7721 and Hep3 B cells was significantly inhibited by VOSL,C+2D,Cos,and Dehy in a dose-dependent manner,but they had no effect on ERK activation.VOSL,C+2D,Cos,and Dehy also caused a dramatically decrease of p-Akt levels in a dosage-dependent manner in SMMC-7721 and Hep3 B cells,however,the p-Akt level had no obvious changes in the Cos-treated group.d.The expression levels of p-EGFR in HCC cells were significantly suppressed after VOSL,C+2D,Cos,or and Dehy treatments.3.Curative effect of VOSL on SMMC-7721 nude mouse transplantation tumor.a.The nude mice were treated with Cos,Dehy,C+2D,VOSL and 5-Fu for 24 days at the dose of 15mg/kg.The results showed that the xenograft tumor growth of the treatment groups were lower than the NT group(the inhibitory rates were 23.15%,30.23%,37.51%,55.71% and 42.22%,respectively),and VOSL revealed better anti-tumor activity than the other treatment groups.b.The result of TUNEL assay showed that,compared with the negative control,the number of apoptotic cells was all increased in the VOSL,C+2D,Cos,and Dehy-treated groups,and the VOSL group exerted the greatest effect on inducing apoptosis in HCC xenografts.c.The expression levels of Ki67,p-P38,p-Akt and p-EGFR to the VOSL,C+2D,Cos,and Dehy treatments-treated in the xenografts derived from SMMC-7721 cells were significantly reduced compared with the control group.Therefore,we concluded that VOSL may suppress HCC progression by inhibiting the EGFR-mediated signaling.?Conclusion? The effects of VOSL on signal pathways and protein expression in HCC were explored to investigate the possible mechanisms of the drug.From the experiments,VOSL,Cos,Dehy and C+2D inhibited HCC cell proliferation,induced cell apoptosis and decreased the migration and invasion capabilities in vitro.VOSL suppressed the phosphorylation of EGFR,thereafter inhibited its downstream cascades MEK/P38 and PI3-K/Akt signalings,which are essential in the invasion and apotosis of HCC.The antitumor activity of VOSL was verified by the experiments of the HCC xenograft tumor.VOSL can delay the tumor growth rate and decrease tumor volume in mouse model.In addition,we speculated that there should exist other anti-HCC compounds besides Cos and Dehy within VOSL because VOSL exhibited better anti-HCC effect than C+2D in vivo.To date,the other anti-HCC compounds besides Cos and Dehy in VOSL have not yet been figured out,which is our topic of further research.