The Effects Of Chinese Traditional Medicine X On Proliferation And Cell Cycle Of Lung Cancer Cell

PrefaceHuman' s health and life were greatly threatened by cancer. The incidence of and the death rate of lung cancer is primary in all malignant tumors in china, and it increases at the speed of 0. 5% annually. The therapy of Chinese medical science and Chinese traditional medicine may alleviate the symptoms and pains of patients, improve the qualities of survival, prolong the lives, cut down the death rate, which is very important. The Chinese scholars have been studying how the Chinese traditional medicine induces the tumor apoptosis in recent years. Up to these days, they have found many extracts of traditional Chinese drugs could induce tumor apoptosis in vitro experiment. It can reveal the mechanism how the Chinese traditional medicine treat tumors and accelerate further development investigation of anti - tumors. Our lab studied the Chinese traditional medicine and naturally occurring drugs with abundant experiences and the major active components have been extracted from Chinese traditional medicine which is called artemisiae capillaris. The Chinese traditional medicine X is one of the important drugs which our lab is studying and its possible mechanisms of anti -tumor effect will be discussed in this experiment.MaterialA549 cell strain: human adenocarcinoma of lung.Reagent;Chinese traditional medicine X ,NBCS(new -born calf serum) , Rnase A PI,RPMI - 1640 medium,Trypsin,MTT kit.Instruments;FCM, fluorescence microscope,super clean bench,steam steri-lizer^ centrifuge ^ analytical balance, C02 incubator.Methods1. MTT experimentThe cells were collected and made them the suspended single cell in culture fluids, then adjusted the concentration of the cells to 5 x 104cells/ml. Added the culture fluid of 90jxl in every well of 96 wells tissue culture plate, so that the amount of cells was about 4000 ~ 5000 in every well. Then added the drugs which the concentrations are different and 0. 9% sodium chloride injection to the culture plate respectively with precision microliter pipette, each group had three wells, 10|xl/well. The culture plate was put in CO2 incubator. Added MTT of IOjjlI to different wells after the cells were cultured in 72 hours ^ 48 hours ^24 hours separately, then continued to culture in 4 hours. Added the IOOjjlI fluid which dissolved the formanzan to each well, above mentioned overnight, and examined the absorbency A in 570nm until all the formanzan were dissolved under the optical microscope.2. Examine the percent of A549 apoptosis and analyzed cell cycleThe cells were collected and made them the suspended single cell in culture fluid, then adjusted the concentration of the cell to 1 ~2 x 106cells/ml. Added the culture fluid of 1. 8ml to the every well of 12 wells tissue culture plate, then added the drugs which the concentrations are different and 0. 9% sodium chloride injection to the culture plate respectively with micropipette, each group had three wells, 200 |xl/well. The culture plate was put in CO2 incubator. After the cells were cultured in 72 hours N48 hours ^24 hours, their suspended fluids were collected to the different 5ml centrifuge tubes and marked. To discard the clear supernatant liquids, after the cell suspended fluids were centrifuged with 1000rpm,5min. The cold alcohol of 70% of lml was added to each tube, in order to fix cells, then in A°C refrigerator overnight. The propidium iodide staining solution of 1 ml was added to every centrifuge tube, after the cell suspended fluids were centrifuged and the clear supernatant liquids were discarded. Then dyed the cells for 30min away from lights and used the FCM to examine them.Statistical analysisAll results were showed by means ± SD. All of data were statistic by SPSS. The significant differences were evaluated according to one - way ANOVA. A value of P <0. 05 was considered to indicate a significant difference.Results1. Chinese traditional medicine X had inhibitory effect on lung cancer cell The results of two experiments which were used the same methods are nearly consistent;this indicates that the experiments have repeatability. The results reveal that the Chinese traditional medicine X have obvious inhibitory effect on the A549 from the beginning of 24 h, the inhibitory effects increased gradually with the time prolonged;and the inhibitory effects increased gradually with the dose being increased. The inhibition rates of the group that the dose was 250jxg/ ml were 88.78% and 86.40% in two experiments.2. The percent of A549 apoptosis and cell cycleThe results of two experiments which were used the same methods are nearly consistent;this indicates that the experiments have repeatability. The results revealed that the percentage of S phase and G2/M phase cells decreased obviously with the doses being increased gradually;but the percentage of Go/ G, phase cells increased gradually with the doses increased gradually. The effect of time was not obvious. The results indicated that the Chinese traditional medicine X could inhibit multiplication cycle of A549 and block the cell growth in the Go / G, phase, the cells that entered the active S phase and G2/M phase diminished.DiscussionApoptosis is an active death process that only a few of cells generated and it is a programmed cell death that genes mediated. Occurrences of tumors are notonly the cell proliferation and differentiation but also in the mechanism of apop-tosis. The dynamic balance between cell proliferation and apoptosis is a most elemental biological procedure. In this procedure, multicellular organisms preserve their structural stability, balance of internal environment, growth and development. Cell proliferation and apoptosis make all tissues and organs in a normal condition from bis -aspects. Once the balance is broken, human will have diseases. The disequilibrium between cell proliferation and apoptosis may make tumors occur, the degree of disorders may determine whether tumors generate or not and developing speed. Therefore in recent years, we have discovered many drugs which inhibited cell proliferation and promoted tumor apoptosis, demolished the dynamic balance between proliferation and apoptosis, and could make the tumor cells dead. That the traditional Chinese medicine induces the tumor apoptosis is a newly emerging question for study or discussion. A lot of scholars have accepted its significance. The studies about the traditional Chinese drug and apoptosis have got a few satisfying outcomes and are continuing. At present, the studies have transformed from apoptosis to molecular levels and gene levels, it is very important to indicate the mechanisms of traditional Chinese drug and sieve the new drugs of anti - tumors. In this experiment, the results revealed that the Chinese traditional medicine X had obvious inhibitory effects on the A549 from the beginning of 24h, the inhibitory effects increased gradually with the time prolonged;and the inhibitory effects increased gradually with the dose being increased. The inhibition rates of the group that the dose was 250|ig/ml were 88.78% and 86.40% in two experiments. We used FCM to examine the percent of A549 apoptosis and cell cycle in experiment. The results revealed that the percentage of S phase and G2/M phase cells decreased obviously with the doses being increased gradually;but the percentage of Go/ G, phase cells increased gradually with the doses being increased gradually. The effect of time was not obvious. The percent of A549 apoptosis of the group that the dose was 250jxg/ml were 16.08% and 15.44% in two experiments;the topmost percent of Go/ G, phase of the group that the dose was 250jjLg/ml were 79. 21% and 74. 50% , but the percent of Go/ Gl phase of the control group were 49. 16% and 50. 53% , the result was P <0. 01 by comparison, there was a significant differ-. 8 ?jnce;the percent of S phase of the group that the dose was 250|xg/ml were 15. 70% and 20. 37% , but the percent of S phase of the control group were 43. 14% and 39. 68% , the result was P <0. 01 by comparison, there was a significant difference;the percent of G2/M phase of the group that the dose was 25O|xg/ml were 5. 09% and 5.13%, but the percent of G2/M phase of the control group were 9.79% and 7. 69% , the result was P <0.05 by comparison, the difference had statistical significance. The results above that the traditional Chinese drug effect on A549 was got after in 72 hours. It was clear that the drug effected on DNA synthesis period and the phase of mitosis, in which proliferations were more active, in other words the drug affected on S phase and G2/M phase. The percentage of S phase and G2/M phase decreased obviously but the percentage of Go/ G, phase increased obviously with the concentration of the drugs being increased, so the cells were blocked in Go/ G, phase. The traditional Chinese drug made the tumor cells reduce DNA synthesis and inhibited the growth of tumor cells. So the traditional Chinese drug had anti - tumor functions. Its pharmaceutical potentials were worth for further investigating.ConclusionChinese traditional medicine X had inhibitory effect on lung cancer cells in vitro. Through suppressing DNA synthesis, it blocked the cell in the Go/ G, phase, therefore suppressed cell's proliferation. Its pharmaceutical potentials were worth for further investigating.