Effects of SP600125 on Proliferation and Invasion of Human Cervical Cancer HeLa Cells / 肿瘤防治研究

Objective To investigate the effect of SP600125 on the proliferation, cell cycle, apoptosis and invasion of human cervical cancer HeLa cells. Methods CCK-8 method was used to detect the proliferation of HeLa cells treated with different concentrations of SP600125 at different time points. The 20 μmol/L of SP600125 was determined for subsequent experiments. Cell proliferation ability was detected using plate clone formation assay; nuclear morphology was observed by DAPI staining; cell cycle and apoptosis were measured by flow cytometry; cell migration and invasion were detected by cell scratch and Transwell methods; the mRNA and protein levels of p53, Mad2L1 and CDC20 were measured by qRT-PCR and Western blot after SP600125 treatment at different time points. Results Compared with control group (0.1%DMSO), cells proliferative activity were reduced by 10, 20, 30, 40 and 50 μmol/L SP600125 treatment for 24h. Compared with control group, the rate of apoptosis was significantly increased in SP600125 treatment groups, and the cell proportion in G2/M phase increased (P < 0.001), while the cell proportion in G0/G1 phases cells was reduced after SP600125 treatment for 24h and 48h (P < 0.001), and the clonal number, migration and invasion ability of HeLa cells also decreased significantly (P < 0.001). qRT-PCR and Western blot results showed a significant decrease in Mad2L1 mRNA and protein expression (P < 0.05) and a significant increase in p53 and CDC20 mRNA and protein expression (P < 0.01). Conclusion SP600125 can induce cell cycle arrest of cervical cancer HeLa cells in G2/M phase by upregulating p53 and CDC20 and downregulating Mad2L1 expression, and promote cell apoptosis to inhibit cell proliferation, migration and invasion.

Enhanced Effect of miR-148a Targeting STAT3 on Chemosensitivity of Cervical Cancer HeLa Cells to Cisplatin / 肿瘤防治研究

Objective To investigate the effect of miR-148a on the chemosensitivity of cervical cancer HeLa cells to cisplatin and its related mechanism. Methods Cervical cancer HeLa cells were cultured in vitro and the concentration gradient of cisplatin was set to detect IC20 value. Control group, mimic control group, miR-148a mimic group, inhibitor control group and miR-148a inhibitor group were set up. qRT-PCR was used to detect the expression of miR-148a and STAT3 mRNA after transfection. After 4 μmol/L cisplatin treatment, the proliferation of HeLa cells was detected by MTT assay; the apoptosis and cell cycle distribution were detected by flow cytometry; Western blot was used to detect the protein expression of p-STAT3/STAT3, CyclinD1, Bcl-2, Bax and Cleaved caspase-3. Results The IC20 of cisplatin on HeLa cells was about 4 μmol/L. Compared with the mimic control group, the level of miR-148a in the miR-148a mimic group was significantly increased, and the level of STAT3 mRNA was significantly decreased (P < 0.05). Compared with the inhibitor control group, the level of miR-148a in HeLa cells in miR-148a inhibitor group was significantly decreased, and the level of STAT3 mRNA was significantly increased (P < 0.05). On the basis of cisplatin treatment, compared with the mimic control group, the apoptosis rate, G0/G1 phase cell ratio, the protein levels of Bax and Cleaved caspase-3 were significantly increased in miR-148a mimic group, while OD value, the proportions of cells in S and G2/M phase, the protein levels of p-STAT3/STAT3, CyclinD1, Bcl-2 were significantly decreased (P < 0.05); compared with the inhibitor control group, the above indicators of HeLa cells in miR-148a inhibitor group changed significantly in the opposite direction (P < 0.05). Conclusion MiR-148a could enhance the chemosensitivity of cervical cancer HeLa cells to cisplatin by targetedly inhibiting STAT3.

Fermentation conditions and antitumor products of an ocean-derived fungus Alternaria sp. 114-1G: a preliminary investigation / 国际药学研究杂志

Objective: To investigate and optimize the fermentation conditions of an ocean-derived fungus, Alternaria sp. 114- 1G, and isolate metabolites in the fermentation products to explore antitumor compounds in the products via the structure elucidation and in vitro antitumor activity assay. Another aim of this study is to accomplish a fundamental work for further research on new compounds from Alternaria sp. 114- 1G. Methods: The in vitro antitumor activity was assayed for the crude extracts and isolated compounds by the CCK-8 method using a human cervical cancer HeLa cell line. The fermentation conditions were investigated and opti- mized based on the biomass of the fermentation and the in vitro antitumor activity of crude extracts of the fermentation. For the separation and isolation of metabolites, the column chromatography was performed on silica gel and Sephadex LH-20 columns, and semi-preparative high performance liquid chromatography(semi-HPLC)was conducted on a COSMOSIL C18-MS-Ⅱ column(10 mm×250 mm). The obtained compounds were identified according to the MS, 1H NMR and13C NMR data. Results: The relatively favored fermentation conditions were as follows: mannitol 25 g/L, maltose 15 g/L, glucose 10 g/L, monosodium glutamate 10 g/L, soybean peptone 5 g/L, yeast extract 3 g/L; 60% sea water in whole medium, initial pH 7.5; and fermentation at 10℃ for 15 days under a 150 r/min shaking speed on a rotary shaker. Four compounds 1-4 were isolated from the fermentation products of 114-1G strain and identified as cyclo (Gla-Tyr)(1), cyclo(Ala-Ile)(2), thymidine DNA nucleotide(3)and pachybasin(4). Among them, 4 showed a relatively stronger inhibitory activity on HeLa cells, with the 57.8% of inhibition rate at 100 μg/ml. Some other compounds were also isolated, and a phenolic one had been shown to be a new compound by a literature survey according to the planar structure deduced. Further studies on their structures were in progress. Conclusion: Fermentation conditions for Alternaira sp. 114-1G were investigated preliminarily, and four compounds 1-4 have been isolated from the fermentation products of the 114-1G strain. Among them, pachybasin(4)showed a relatively higher inhibitory effect on human cancer HeLa cells. Alternaira sp. 114-1G could produce new metabolites and the present study has provided a reliable groundwork for further research on new compounds from Alternaira sp. 114-1G.

Separation and Purification of Alhagi sparsifolia n -butanol Extract Monomeric Compounds and Study on Their Effects on Human Cervical Cancer HeLa Cells / 中国药房

OBJECTIVE： To separate and purify Alhagi sparsifolia n-butanol extract monomeric compounds， and to investigate its effects on the proliferation and metastasis of human cervical cancer HeLa cells. METHODS： The n-butanol extract was separated and purified by silica gel column， Sephadex LH-20 gel column and prep-HPLC. The structures of compounds were analyzed and identified according to physicochemical properties and spectrum （mass spectrum， hydrogen spectrum， carbon spectrum） data. Using human cervical cancer HeLa cells as objects， 5-FU as positive control， MTT assay was used to detect the inhibitory rate of HeLa cells pretreated with different doses of compounds （6.25， 12.5， 25， 50， 100， 200 μg/mL）； IC50 was calculated to screen active monomers. Scratch test was used to investigate the effects of above active monomers （all 50 μg/mL） on the migration ability of HeLa cells. Kim’s formula was used to evaluate the effects of 5-FU separately combined with above active monomers [（3.125+6.25），（6.25+12.5），（12.5+25），（25+50）μg/mL]. RESULTS： Six compounds were isolated from the n-butanol extract part of A. sparsifolia and identified as butin （Ⅰ）， 3′，4′，7-trihydroxyisoflavone （Ⅱ）， p-methoxyphenylacetic acid （Ⅲ）， 4-hydroxyacetophenone （Ⅳ）， aurantiamide acetate （Ⅴ）， protocatechualdehydea （Ⅵ）. Compared with blank control group， 5-FU and each compound （5-FU：6.25-200 μg/mL， compound Ⅰ： 12.5-200 μg/mL； compound Ⅱ： 25， 50， 200  μg/mL； compound Ⅲ： 6.25， 100， 200 μg/mL； compound Ⅳ： 50， 100， 200 μg/mL； compound Ⅴ： 12.5， 25， 200 μg/mL； compound Ⅵ： 6.25-200 μg/mL） could significantly increase the cell inhibition rate. IC50 of compound Ⅰ， Ⅴ， Ⅵ were decreased significantly （P＜0.05 or P＜0.01）， and those of compound Ⅰ and Ⅵ were lower relatively. The migration distance of cells in 5-FU and compound Ⅰ and Ⅵgroups were decreased significantly， compared with blank control group （P＜0.05 or P＜0.01）. 5-FU separately combined with compound Ⅰ and Ⅵ showed additive and enhanced inhibitory effects on the proliferation of HeLa cells （synergistic index＞0.9）. CONCLUSIONS： Compounds Ⅰ-Ⅵ are isolated from Alhagi for the first time. Butin and protocatechualdehydea are active monomers of its n-butanol extract part. Above two monomers can inhibit the proliferation and migration of human cervical cancer Hela cells， with strong inhibitory effect in vitro， and stronger inhibitory effect combined with 5-FU than any compound alone.

Analysis of Pharmacological Effect of Total Tannins from Spatholobi Caulis on Cervical Cancer HeLa Cells by 3D Microfluidic Chip / 中国实验方剂学杂志

Objective:To study on the chemical constituents of total tannins from Spatholobi Caulis,and to analyze the pharmacodynamics and mechanism of total tannins from Spatholobi Caulis on cervical cancer HeLa cells. Method:UPLC-Q-TOF/MS was used to qualitatively analyze the composition of total tannins from Spatholobi Caulis.The appropriate concentration and time of administration were screened by 3 dimensional(3D) microfluidic chip.Flow cytometry was used to determine the effect of total tannins from Spatholobi Caulis on the cell cycle and apoptosis of cervical cancer HeLa cells and analyzed by FlowJo v10.0.7 and ModFit LT 3.2 software.Enzyme-linked immunosorbent assay(ELISA) was used to determine the changes of vascular endothelial growth factor(VEGF)-A and Caspase-3 factors in cervical cancer HeLa cells supernatant treated with total tannins from Spatholobi Caulis. Result:Total of 15 components in total tannins from Spatholobi Caulis were identified or inferred.The low,medium and high dosages of total tannins from Spatholobi Caulis were 0.5,1.0, 2.0 g·L-1 and the best time of administration was 36 h.The proportions of early and late apoptosis of cervical cancer HeLa cells increased significantly in the apoptosis analysis after being treated by total tannins from Spatholobi Caulis.The DNA synthesis early phase(G0/G1 phase) of cervical cancer HeLa cells significantly increased,and the DNA synthesis phase(S phase) and the DNA synthesis late phase(G2/M phase) reduced.After being treated with total tannins from Spatholobi Caulis,the expression of VEGF-A in cervical cancer HeLa cells supernatant was significantly decreased and the expression of Caspase-3 was significantly increased. Conclusion:Spatholobi Caulis is rich in tannins,which can significantly inhibit the proliferation of cervical cancer HeLa cells and promote its apoptosis.This paper can provide the basis for further research of total tannins from Spatholobi Caulis.