The Antitumor Efficacy Screening Of Extracts From Stellera Chamaejasmel In Both Vitro And Vivo And The Mechanism

Objective: To screen the best antitumor components of Stellera chamaejasme L.and their sensitive cell lines. Moreover，the antitumor mechanism of it by blocking thecell cycle was also been studied.Methods:（1）16different components of ethanol extracts from Stellerachamaejasme L.（ESC）：HH，H1^H8，JH and J1^J8， were got by gradient columnchromatography eluted with ethanol in different concentrations. Then the human cancercell lines such as hepatic carcinoma BEL-7402，SK-HEP-1，and lung cancer A549，NCI-H157were processed with all the components in the concentration of100μg/mL todo the first screening. SRB assay were used to detect the inhibition rate of all the celllines mentioned above in48h in order to select the most inhibitive components and themost sensitive cell lines to proceed to pharmacodynamics authentication. Eachcomponent in the concentration of6.25¹00μg/mL was used to treat the sensitive celllines and the inhibition rates to each cell line for24h，48h and72h were detected withSRB. Also，the IC₅₀of each component was calculated and their main chemicalcomposition was analyzed by UPLC-MS.（2）40%to100%ethanol extract in the above experiments were combined and beendried by using rotary evaporation and freeze-drying to get the steam powder andfreeze-dried powder. Then each kind in the concentration of1.5625²00μg/mL wasused to treat A549，NCI-H157，NCI-H460，BEL-7402and SK-HEP-1cell lines，andthe inhibition rates to each cell line of24h，48h and72h were detected by SRB assay.Also，the IC₅₀of each component was calculated to select the sensitive cell lines tofurther study and compare the anti-tumor activity of two kinds of powder. Then，themore advanced powder were selected to study its effect on the H22tumor-bearing miceand compare the anti-tumor effect between intragastric administration andintraperitoneal injection. After the mice transplanted tumor model was set up，they wereweighed and administer0.2mL/10g gavage or intraperitoneal injection once per day for10consecutivedays. The mice were weighed and coat color，mental state，activities and diet were observed closely everydayduringtheadministration.24hafterthelastadministration，the spleen，thymus and tumor were weighed to calculate the index ofspleen，thymus and tumor inhibition rate. Changes in white blood cell count and plateletcount in the blood of mice were analyzed with whole blood analyzer. The tumor tissuespecimen were reserved to acquire proper tissue slice. Moreover， the morphology oftumor cells were observed by light microscopy.（3） The appearance changes of sensitive cell lines was observed under opticalmicroscope after intervention with ESC for24h，and after immunofluorescence staining,use confocal laser microscope to observe the cytoskeleton changes. With propidiumiodide（PI） staining，the change after drug intervention of cell cycle were detected byflow cytometry. Also，the ESC including25，50，100μg/mL affect the sensitive celllinesafter6hand36hrespectively，the cells were collected to observe their effects onnucleic acid transcription and protein expression level of cyclinE1.Base on the results，RT-PCR was used to observe the mRNA level of NF-κB pathway-related cytokineDR5，TNF-α，TNFR，IL-1βto preliminarily explore the possible mechanism and potentialtargets of antitumor effect of ESC.Results:（1）The inhibition effect to the proliferation of the different cancer cellshave great difference among16components， and the lung cancer cells are moresensitive to them than the hepatocarcinoma cells. Besides，the inhibition rate of J5，J6and H8are higher than the other components and their effect have a certain time andconcentration dependence. At72h，the inhibition rate of each component ranges from（60.573.83）%to（96.660.51）%for lung cancer cells，and IC₅₀from（9.61±0.79）μg/mL to （55.76±2.31）μg/mL. J5，J6and H8are the biflavonoids.（2） There is no significant difference between freeze-dried powder androtary-evaporated powder of the inhibition rate on every kinds of cell lines. The highestinhibition rate is （80.39±4.01）%to NCI-H157and the least IC₅₀is （49.00±0.85）μg/mL toSK-HEP-1. Administering the different concentrations rotary-evaporated powder of theethanol extraction of Stellera chamaejasme L by intragastric administration can significantly inhibit the tumor growth of H22tumor-bearing mice，inhibition rates were29.68%，44.00%，59.02%，positive drug84.28%and intraperitoneal injection25.98%.Compared with the solvent group， gavage group had no significant effect on the spleenindex of tumor-bearing mice. However，gavage high-dose group can reduce the thymusindex of tumor-bearing mice （P<0.05）. And intraperitoneal injection group can reducethe thymus index and spleen index of tumor-bearing mice （P<0.05）. Gavage high-dosegroup and positive drug capecitabine tablets can significantly reduce the number ofwhite blood cells and platelets in the blood of tumor-bearing mice （P<0.05）. Comparedwith the intraperitoneal injection solvent group，the intraperitoneal injection group cansignificantly reduce the number of platelets in the blood of H22tumor-bearing mice（P<0.05）， while a smaller impact on leukocytes， but the count of white blood cell andplatelet in each mouse still within the normal range. Results of Light microscopyshowed that: the tumor cells in the intragastric solvent control group were atypia，invasive， arranged irregularly. The atypia， apoptosis and necrosis of tumor cellsinpositive drug and in the high-dose group is significantly lessen than that in controlgroup. Moreover，atypia of the tumor cells in the middle dose group also significantlyreduced （P<0.05） and necrosis， apoptosis had the trend to reduce （P<0.1）. Thesechange show a certain anti-tumor effect. The atypia， apoptosis and necrosis of tumorcellsin the small-dose group also reduced than that in control group， but the differencehad no significance. tumor cells of the low-dose group Shaped， apoptosis andnecrosis lesions compared with the control group has been reduced， but no significantdifference. Tumor cell atypia in intraperitoneal injection of solvent and gavage solventcontrol group was not significantly different. Compared with control group， tumor cellatypia， apoptosis and necrosis in intraperitoneal injection group had no significantdifference.（3） Flow cytometry results showed thatSK-HEP-1cells were processed by25，50,100ug/mL rotary-evaporated powder of ESC，and the major cell cycle arrest is in theS phase. compared with the negative control group， the cell of S phase had a significant increase （P<0.05）. RT-PCR and Western Blot results showed thatSK-HEP-1cells were processed by25μg/mL，50μg/mL100ug/mL rotary-evaporatedpowder of ESC for36H，andcyclinE1mRNA level and protein expression levels in eachgroup were lowered. Compared with the control group，medium and high dose groupswere significantly different（P<0.05）.Further RT-PCR experiments suggests that DR5，IL-1β mRNA levels in each dose group increased. Compared with the control group，medium and high dose groups were significantly different（P<0.05）. TNF-αmRNAexpression levels in each group were raised in a concentration-dependent manner，butno significant difference compared with the control group. After36h inventation，IL-1βmRNA levels were increasd and continue to increase in each group，and TNFRmRNA level rised in a concentration-dependent manner，but compared with the controlgroup， the difference had no significance.Conclusion:（1） The biflavonoids in ESC have exerted an satisfactory inhibitoryeffect on the lung cancer cell proliferation.（2） The rotary-evaporated powder of the ethanol extraction of Stellerachamaejasme L showed a strong inhibitory effect. different routes of administrationincluding intraperitoneal injection intragastric administration are able to inhibit tumorgrowth inH22tumor-bearing mice. Moreover，they do not have obvious side effects.（3） ESC can change the cell appearance and mitosis by affecting cell skeleton，inhibit SK-HEP-1cells proliferation by blocking the S phase，which may concernwithincreasedDR5，TNF-α，TNFR，IL-1βlevels and activated NF-κB signal pathway，thereby reducingcyclinE1expression levels.