Study On Anti-growth Activity And Its Mechanism In Human Gynecological Tumor Cell Lines By Compounds From Inula Britanica L.

Objective: There are about 100 kinds of Inula plants all over the world, distributed in Europe, Asia and Africa, mainly in the Mediterranean region. More than 20 kinds of Inula plants exist in our country. Inula britanica is the perennial herb, which has been used for the therapy of gastroenteritis, bronchitis and tuberculosis diarrhea as a traditional Chinese herbal medicine. It is reported currently that Inula britanica has the major chemical composition of flavonoids, sesquiterpene lactones and terpenoids. It is proved that majority of pharmacological activity is sesquiterpene lactones compounds with the effects of anti-inflammatory, anti-liver injury, anti-tumor and cytotoxicity, also having effect on diabetes. In the present study, human cervical cancer cell line(HeLa), human endometrial adenocarcinoma cell line(HEC-1), human ovarian clear-cell carcinoma cell line(SHIN-3), human ovarian clear-cell carcinoma cell line(HOC-21) and human ovarian cystadenocarcinoma cell line(HAC-2) were used. The aim of the experiment is to study the effects of Isoalantolactone, 1-O-Acetylbritannilactone and Britannilactone from Inula britanica on inhibiting proliferation of 5 cultured gynecological tumor cells in vitro and further to explore the mechanism of Isoalantolactone on inhibiting proliferation of HeLa.Methods:1 MTT assay1.1 Preparation for tumor cell lineTo modulate HeLa tumor cell lines with the concentration of 2×105 / mL by RPMI1640 complete medium.1.2 Anti-tumor experiments of compounds in vitroHela Cells were harvested at mid-exponential phase and then planted in 96 well plates for 50μL each. Cells were treated for 48 hours with Phosphate buffer as blank control, 0.05% DMSO as solvent control, cisplatin as positive control and 3 compounds of different concentration (1μmol/L, 10μmol/L and 100μmol/L) for 50μL. Each group was equipped with 3 duplicate wells. Each well was added 5 mg/mL MTT 10μL before 4 hours to the end of cultivation. Then absorbed culture supernatant carefully, added reaction stop solution 150μL, and oscillate 10 min to dissolve the crystallization completely. Finally the hole absorbance (OD) values is measured by Enzyme-linked immunosorbent assay with determination of wavelengthλ= 570 nm and the reference wavelengthλ= 620 nm. So the survival rate of tumor cells could be calculated.1.3 The broad-spectrum anti-tumor cell proliferation experiment of active compoundsTo select the active monomer compound and investigate the effects of active compound on cell growth inhibition rates of HEC-1, SHIN-3, HOC-21 and HAC-2 cell lines.2 Analysis of apoptosis by Flow cytometry and AnnexinⅤ-FITC kitCells were harvested at mid-exponential phase. Cells were treated for 48 hours with Phosphate buffer as blank control and Isoalantolactone of the concentration of 12.5μmol/L. Washed cells twice with 4℃precooling PBS, and re-suspended cells with binding buffer 250μL to mediate cells concentration of 1×106/mL. Took cell suspension 100μL in 5 mL streaming tube, added AnnexinⅤ/ FITC 5μL and 20μg/mL propidium iodide 10μL, mixing, and added diluted binding buffer 400μL in the reaction tube after incubation at room temperature away from light 15 minutes. Then analyzed data by flow cytometry in time.Results:1 The results of MTT assayThe compounds from the Inula britanica had significant inhibition on the growth of cultured gynecological tumor cell in vitro. There were different inhibitory effects on the proliferation of human gynecological tumor cells among Isoalantolactone, 1-O-Acetylbritannilactone and Britannilactone, which were HeLa, HEC-1, SHIN-3, HOC-21 and HAC-2. Isoalantolactone had remarkable effect on inhibiting proliferation of HeLa, HEC-1, HOC-21 and HAC-2. After administration of Isoalantolactone with different concentrations of 1μmol/L, 10μmol/L and 100μmol/L for 48 hours, the survival rate of HeLa cell were 91.81%, 66.67% and 18.65%, the survival rate of HEC-1 were 92.96%, 68.67% and 33.96%, the survival rate of HOC-21 were 83.57%, 73.37% and 20.72%, and the survival rate of HAC-2 were 85.62%, 49.75% and 20.53%, respectively. Administration of 1-100μmol/L Isoalantolactone can significantly inhibit proliferation of HeLa, HEC-1, HOC-21 and HAC-2 cells in a dose-dependent manner. The cell survival rates in Isoalantolactone group are all much higher than that in PBS control group with the concentration of 100μmol/L (P<0.01), with the IC50 of 17.67μmol/L, 32.74μmol/L, 19.65μmol/L and 11.49μmol/L, respectively.2 The detection of apoptosis by flow cytometry and Annexin-Ⅴ/PI double stainingAfter administration of Isoalantolactone with the concentration of 12.5μmol/L for 48 hours, Hela cells were stained with AnnexinⅤand propidium iodide. The flow cytometry analysis showed that the apoptotic rate in Isoalantolactone group (40.75±5.16)% was higher than that in PBS group (3.47±3.82)% (P<0.05). The data from FCM further established that Isoalantolactone might inhibit proliferation and induce apoptosis in Hela in vitro.Conclusions: The compounds from the Inula britanica have the effects of inhibiting proliferation of cultured gynecological tumor cell, and Isoalantolactone might induce the apoptosis of human Hela cells in vitro. Cancer has the relationship to the imbalance between cell proliferation and apoptosis. Apoptosis is closely related to tumor occurrence, development and degradation. To induce apoptosis of tumor cell is becoming a new target of tumor therapy in recent years. There are efficient and specific inhibitory effect on the proliferation of human cervical cancer cell line in vitro, endometrial cancer cell line and ovarian cancer cell line among the compounds from the Inula britanica. With deeper understanding of the pathogenesis of cervical cancer and endometrial cancer and better acknowledging of resistance mechanism of ovarian cancer, gynecological tumor treatment steps to a new level. The compounds from the Inula britanica provide a new therapeutic strategy for cervical cancer, endometrial cancer and ovarian cancer with drug resistance, which is the hot topic of the prevention and treatment on cancer. The compounds are expected to become a widely applied anti-cancer drug.