Study On Anti-growth Activity And Its Mechanism In Human Lung Cells By Sesquiterpene Lactone Compounds From Achillea Millefolium L.

Achillea millefolium Linnaeas (which also is commonly referred to as‘yarrow’), is a perennial herb compositae. According to legend, our ancestorFu Hsi used it as a symbol in divination. The genus Achillea comprises over100species, mainly distributed in Northeast, Inner Mongolia and Sinkiang inChina. It survives in the partial shade and cold areas and has a lowrequirement for soil. Achillea millefolium Linnaeas tastes spicy and bitter andhas a property of coolness. It is widely used in folk medicine for thepreparation of herbal teas with antiphlogistic and spasmolytic activities. It hasbeen playing an important part for a long time as a drug in traditional andmodern medical practices for the treatment of fever, cough, bronchitis, asthma,skin inflammation, and liver ailments, and as food flavorings andpreservatives.The present experiment investigated the effects of21sesquiterpenelactone compounds extracted from the flower head of Achillea millefoliumLinnaeas on the proliferation of the human lung tumor cells for the first time.We screened out four compounds with obvious inhibitory activity and themechanism of anti-proliferation of A549cells was investigated in order toprovide experimental basis and theoretical foundation for developing thehigh-efficiency and low-toxin drugs.Part one The inhibitory effects of21sesquiterpene lactone compoundson proliferation of human lung tumor cellsObjective: To observe the inhibitory effects of21sesquiterpene lactonecompounds--(1) Millifolide C,(2) isololiolide,(3)6-epiloliolide,(4) artemina,(5) achillinin B,(6) achillinin C,(7)1a,3a-dihydroxy-7b,11aH-germacra-4Z,10-(14)-dien-12,6a-olide,(8)1-hydroxy-2R,3R-isopyliden-hex-5-en-4-one, (9) iso-secotanapartholide,(10) arteludooicinolide A,(11)3-acetyl-iso-seco-tanapartholide,(12)3-methoxy-anapartholide,(13) secotanapartholide A,(14)secotana-partholide B,(15)5-epi-secotanapartholide,(16)4-dehydroxy-1,10-epoxy-Achillinin A,(17)4-dehydroxy-Achillinin A,(18) Achillinin A,(19)2,3-dihydroxy-Achillinin A,(20) Millifolide A,(21) Millifolide B onproliferation of five human lung tumor cells; and to screen out the compoundswith significantly inhibitory effects on proliferation of tumor cells.Methods: The proliferation of Human lung cancer cells which treatedwith21sesquiterpene lactone compounds was measured by monotetrazolium(MTT) colorimetric assay. The concentration effect curve of experimentalcompounds on the tumor cells was fit by Hill mathematical model to calculatethe IC50of medicine.Results:(1)10μmol/L cisplatin inhibited the proliferation of humansmall-cell lung cancer cells (QG-90and PC-6) strongly. The inhibition rates ofcell proliferation were82.33%and75.29%. Compounds1,16,20,21(at finalconcentration of10μmol/L) presented the proliferation inhibition of QG-90and PC-6cell lines stronger than the effect of cisplatin;(2)10μmol/L cisplatinacted on human nonsmall-cell lung cancer cells (A549, RERF-LC-kj andQG-56), the inhibition rates of cell proliferation were71.02%、44.66%and39.54%.21sesquiterpene lactone compounds acted on human nonsmall-celllung cancer cells (A549, RERF-LC-kj and QG-56), compounds1,16,20,21displayed inhibitory activities of the proliferation of A549, RERF-LC-kj andQG-56cells as the same or better effect than cisplatin;(3) Compounds2~15and17,18,19demonstrated weak anti-proliferative activity of five humanlung tumor cells;(4) through logarithm regression equation of proliferativerate of tumor cells on the concentration of compounds1,16,20,21, we got theIC50values of the four compounds on the five human lung cancer cells. All theIC50values were below10mol/L except those of compounds20,21on A549cells; the IC50values of compounds1and16on QG-90cells were0.12and0.59mol/L; the IC50value of compound1on QG-56cells was0.91mol/L;the IC50value of compound21on PC-6cells were0.19mol/L. Conclusions: Compounds1,16,20and21from Achillea millefoliumLinnaeas presented strong anticancer activity on human lung cancer cells; theantitumor effect on lung cancer cells of guaianolide isomers sesquiterpenelactone compounds was affected by the structure of ternary oxygen ring, andthe more ternary oxygen rings, the stronger effect.Part two The mechanisms of inhibitory effects of sesquiterpene lactonecompound Millifolide C on proliferation of human lung can-cer cellsObjective: A lot of experiments in vivo and in vitro have confirmed thatmany anticancer drugs can induce different types of sensitive tumor cellsapoptosis. Apoptosis could also be induced by radiotherapy, thermotherapyand biological agent in clinic. Traditional Chinese medicinal herbs containedmany anticancer compounds which could induce apoptosis. The first part ofour study had confirmed that four sesquiterpene lactone compounds inhibitedthe proliferation of human lung cancer cells significantly. In this part, weexplored the antitumor mechanism of the four sesquiterpene lactonecompounds, and identified whether sesquiterpene lactone compoundMillifolide C display its antilung cancer effects through the activities ofinducing human lung cancer cells (A549) apoptosis.Methods:(1) We detected the reporter genes by luciferase reporter geneassay. The reporter genes were pG13-Luc, p21-Luc, Bax-Luc, hNoxa-Luc,SRE-Luc, IgK-Luc, NFAT-Luc and CRE-Luc.(2) We examined cell apoptosisby FCM. A549cells were incubated24h by Millifolide C at final concentra-tion of10μmol/L.(3) We examined the expressions of p53, Bax andCaspase-3protein by Western blot.(4) We observed the reversal effects ofCaspase-inhibitor on Millifolide C’s inhibitory activities on proliferation ofA549cells.Results:(1) The expressions of reporter gene in A549cells which weretreated with compounds1,16,20and21(at final concentration of10mol/L):The results showed that Millifolide C can induce the expressions of reportergenes pG13-Luc, p21-Luc, Bax-Luc, hNoxa-Luc which were associated with the p53-dependent signaling pathways of A549cells significantly. But therewere no effects to the expressions of reporter gene SRE-Luc, IgK-Luc,NFAT-Luc and CRE-Luc, which were associated with other signalingpathways of A549cells. And the other three sesquiterpene lactone compoundsshowed no activities to the expressions of the reporter genes.(2) Effects ofMillifolide C on apoptosis of A549cells: After treated for24h with MillifolideC at final concentration of10μmol/L, A549cells’ early apoptosis rate[(25.0±3.7)%], had significant difference compared with that [(5.8±0.3)%]of blank group,(P<0.05). Millifolide C increased apoptosis rate of A549cells.(3) Millifolide C could increase the expressions of p53, Bax and Caspase-3ofA549cells significantly.(4) The reversal effects of Caspase-inhibitor onMillifolide C’s inhibition on proliferation of A549cells: After treated with0.1,1and10μmol/L cisplatin and Millifolide C, the cell survival rates of A549cells were95.10%、73.66%、28.27%and70.82%、29.87%、3.64%; Aftertreated with20μmol/L Caspase-inhibitor and cisplatin or Millifolide C, theA549cells’ survival rates were100.00%、80.90%、56.34%and84.79%、49.67%、17.05%.Conclusions: The apotosis of human lung cancer cells (A549cells) couldbe induced by sesquiterpene lactone compound Millifolide C significantly; theexpression of p53was increasely stimulated in A549cells with Millifolide C,and the expression of Bax and Caspase-3increased as followed. It wasprobably one of the mechanisms of A549cell apotosis induced bysesquiterpene lactone compound Millifolide C. And Caspase-inhibitor had thereversal effects of Millifolide C’s inhibitory activities on proliferation of A549cells.