Research About The Mechanism Of Cantharidin(?) Inhibition To The Proliferation Of Lung Cancer A549 Cell

PrefaceCantharidin is the effective composition of the telinifly. As the traditional Chinese medicine of our country, Cantharidin having anti - tumor function, regulating the immunity, rising white blood cells and protecting functions of hepato-cytes, etc. , but its anticancer mechanism still needs to be expounded.Cell cycles performance are regulated by many relevant genes, the core of this regulating mechanism is the cyclin - dependent kinase, regard CDKs as the core, cyclins have the positive regulation function to them, while CDK inhibitors have the negative functions. The regulation and control network formed by CDKs/cyclins/CKIs can regulate cell cycle to run meticulously. Nearly all the oncogenes, anti - oncogenes' biological effects are all sum up in cell cycle' s mechanism finally. A lot of oncogenes participate in the cell cycle' s regulation directly, or itself is the important components of cell cycle regulating complex. The variation of these genes causes the cell cycle out of control. It is most common to lose the regulation with the checkpoint, which usually caused by some changes in the cyclin/CDK complex. The cell cycle molecule can act as the anti - tumor therapeutic target, through regulating the cell cycle, it can prevent tumor growing, and promote the cell to be apoptosis or death, thus achieve the therapeutic purpose. Now, cyclins, CDKS, CKIS have already become the most important kind of research objects in the anti - tumor therapy.Apoptosis is cell dies as a series of waterfall activated initiative death course after being activated by all kinds of death signal. It not only exists in the normal organism, but also exists extensively in the tumor organism. In recent years, the further investigation of apoptosis and its mechanism verifies that emer-gence, development and drug resistance of the tumor are all having close relation to apoptosis. The reduction of cell's apoptosis can cause the emergence of the tumor, and promote the malignant transformation of the tumor cell and gradual progress through escaping the apoptosis. Meanwhile, peculiar induction to tumor cell's apoptosis has become the target of tumor therapeutic study, and also become the sign of appraising the result of treatment of tumor, it offers the reliable basis in order to study the method to treat the tumor effectively too. Apoptosis is controlled by a lot of genes in cells similarly as cell division and cell differentiation, these genes control the apoptosis of the cell through different signals.The signal conducts play an important role in the cell's control, the change of tumor cell's molecule level may take place in different levels, from growth factor to receptor, the signal molecules in the cell to the cell nucleus'transcription factor, etc. Changes of different component parts in the signal pathway will all cause cell proliferation to be out of control, then produces the carcinomatous change. Studies of signal conduction mechanism of tumor cells, blocking tumor cells autocrine or paracrine signal conduction pathway selectively, destroy of its automatic control growth regulation mechanism, are becoming the extremely attractive research focus. The cytoplasmic kinases are mostly Serine/Threonine protein kinases. The most closed relations to the cell growth, it is the mitogen -activated protein kinase pathway that was also studied most in the international front investigation in recent years ( mitogen - activated protein kinase pathway, MAPK pathway). MAPK participates in many kinds of life course of cell extensively , including growth, differentiation, cell cycle regulation, the tumor occurrence , apoptosis, etc. Extra cellular signal regulated kinase is the main and classical pathway of MAPK unit (extra cellular signal regulated kinase, ERK) , it mainly transmits the mitogenous signals, participates in regulating the cell cycle and promoting cell proliferation and differentiation.In this study, we oberserved Cantharidin s inhibition to lung cacer A549 cell through three respects: cell cycle 's regulation, apoptosis induction and MAPK signal transduction pathway. Meantime, we checked the expression or ac-tivitie changes of some proteins, such as; cyclinB1, P34cdc2, phos - P34cdc2, P21, survivin, Bcl-2, Bax, ERK1/ERK2, phos - ERK1/phos - ERK2 in orderto discuss the mechanism of cantharidin's inhibition mechanism in molecules level.Method1. Cell cultureUse the 10% RPMI 1640 medium of 56℃ inactivated foetus catties serum, cultured in the 37℃ of saturation degree of wetness, 5% of the CO2 incubator, the cell grew as single storey sticks to the wall, fetches growth period of logarithm cells for the experiment.2. MTT technique drug sensitive testDigested and collected logarithm growth period cells and adjusted to the u-nicellular suspensive liquid with the concentration for 5 × 10~4 ml, inoculated it in the 96 pore plate, added cantharidin dispensed to the end concentration (mg/ L) 204.8,102.4,51.2,25.6,12.8,6.4,3.2,1.6,0.8,0.4,0.2,0.1,0.05,0. 025 after 24 hours, every density has 6 replies holes, continue training 24h, 48h, 72h respectively. Lost the superstratum, every hole added MTT 50μl, make the end concentration with 0. 42mg/ml, mixes evenly, cultured in the 37℃ 5% CO2 incubator for 4h, lost the superstratum, dissolved the blue purple particle with DMSO. Using Ascent enzyme mark meter detecting the value of light density of every hole (OD value) that the appearance measures wavelength 570nm, the experiment is repeated for 2 times. Adopted SPSS statistical analysis software, and used straight line regression method to calculate the IC50 value.3. The cell culture taking synchronization with cell cycleUsing the starvation law of serum to make A549 cell in the G0 phase in same step. The exponential growth period cells are diluted to 1 × 10 /ml in the culture flask, then add RPMI1640 media, and cultured in 37℃ CO2 incubator after 48 hours, the cells arrest in G0 phase at this moment. Then lose the anserum RPMI1640 media, and added media of RPMI1640 containing 10% foetus cattle serum,did experiments.4. FACScan measures cell cycleThe cells were digested by 0. 25% trypsin, collected after blowing afloat,rinsed in PBS centrifugation 2 times, 1000rpm, 10 minutes, then lost the superstratum, added 70% (vol/vol) cold ethanol, placed in the . -20℃ , get rid of the ethanol , rinse twice by PBS, added the PBS 1ml including RNaseA to the cells, placed in the incubator with 37℃ for 30 minutes. Then, the cell dyes for 30 minutes in 1ml 50 μg/ml propidium iodide in 4℃ without light. Then use the FACScan to carry on the cell DNA content and cell cycle analysis, the software used is CELLQEST.5. The morphologic detection of cell apoptosis5. 1 Observation of the living cells in the culture flask with the Oylmpus IX - 70 type invert microscope.5. 2 Observation of the ultrastructure: The cell passes 2. 5% Glutaral, 1% acid's double fixation, gradient ethanol and acetone dehydratation, Epon812 resin embeded ultramicrotome ( AO type) section, sodium acetate and citromalic acid lead dyed doubly, observed and took a picture under JEM -1200EX and H-600 transmission electron microscope.6. AnnexinV - FITC mark method to detect the apoptosis quantitivelyTo digest the cells with trypsin(0.25% ) , collected after the cells blowing afloat , then rinsed with PBS and centrifugated for one time, 1000rpm, 10 minutes. Get rid of the superstratum after centrifugation, added the conjunctive buffer, suspensed cells again, adjusted the concentration of cells to be 5 × 10~5/ ml. Added 5 μl AnnexinV - FITC into 195 μl suspensive cells, mixed at room temperature and incubated for 10 minutes. Then added 200 μl conjunctive buffer, rinsed and centrifugated the cells, 1000rpm, 10 minutes. 190 μl conjunctive buffer was added after Superstratum was abandoned to resuspense the cells. Then 10 μl propidium iodide of 20 μg/ml was added. The expression of phosphatidyl serine protein of cell membrane and the contention of DNA of dead cells were detected with FACScan.7. DNA abstraction of cells and agarose gel electrophoresisAfter digested with trypsin(0.25% ), the cells were collected after blowing afloat, rinsed with PBS for 2 times, 1000rpm, 10 minutes. The cells was resus-pend with 500 μl nucleus splitting fluid, and incubated over night at 37℃. Next day, superstratum shifted to EP after centrifugation, 12000rpm, 15 minutes.Extracted with 0. 5ml chloroform isoamyl alcohol, reversed the mixture for several times, we could see the white floccose precipitate. Put into liquid nitrogen for 10 minutes, centrifugated 12000rpm, 10 minutes, then deposit the DNA, abandoned the superstratum, then draw remnant liquid to vacuum. 100μl TE buffer and 5μl RNaseA were added, then incubated in water bath at 37V. for 30 minutes. Added DNA sample 10μl and 6 × loading buffer 2 μl, 1.2% agarose e-lectrophoresis (voltage 150V,40min). Dyed by bromoethidine, then we used automatic electrophoresis gel image analysis meter to detect it and take a photograph.8. Western blottingTreated cells were rinsed with PBS, centrifuged and added cracked buffer of 700μl. After crushed with ultrasonic and centrifuged, 12000rpm, 1 hour, 4℃ , superstratum was collected. The deposit was crushed with ultrasonic, centrifuged, 12000rpm, 1 hour, 4℃., then collected superstratum. After sucking the superstratum, we use the phenol regent method to detect the proteins to be stipulated by laws. The sample added to its buffer to boil for 5 minutes. Used 10% SDS - PAGE to have an electrophoresis with BIORAD electrophoresis board, the condition of double board is; 150V, 40mA. Colloxylin equilibrated in the met - stamp liquid for 10 minutes, then after the 50 V met - stamp, we rinsed it 5 minutes with TBS, and with the TBS to stay, blocked, all these are following the rule that gel is in negative pole while membrane is in the other pole. Incubated with the primary antibody, rinsed with TTBS 2 times after blocking, each time for 5 minutes, then transfer into cyclinB1 , P34cdc2 , phos -P34cdc2, P21, survivin, Bcl - 2, Bax, ERK1/ERK2, phos - ERK1/phos - ERK2o-vernight. Incubated with the second antibody. Incubated with the secondary antibody. Transferred into the secondary anti - body of Sheep anti - Mouse IgG -HRP (1:5000), Sheep anti - Rabbit IgG - HRP ( 1:5000) , Horse anti -Sheep IgG - HRP ( 1:5000) and incubated for 1 hour. Detection was carried out using ECL chemiluminescence system. Mixed A and B liquid according to the concentration of 0. 125ml/cm2. Expose the x - ray film in a dark room, then develop 5 min and fix for 5 min.Result1. MTT - medicine sensitivity testThe inhibition of cantharidin to A549 was depended on time and concentration. The value of IC50 for 24,48,72 hours is3.2mg/L,2,5 mg/L,1.4 mg/L2. Cell cycle detectionAbandoned RPMI1640 without serum, we obtained the cells synchronized at G0 through serum hungry, and cultured the cells with RPMI1640 containing 10% FBS. We examined the cells by flow cytometry every three hours. After 2 hours'convalescence, cells reached phase of G2 after 17 hours, and arrived the end of G2 after 22 hours. The whole cell cycle was about 23 hours.3. Cell cycle blockCantharidin were added into each of 3 bottles of experimental groups of cells synchronized at G0 by serum hungry and convalesced for 2h, the final concentration of which is 3. 2mg/L. Three bottles of cells after 18 hours convalesced were obtained, added cantharidin to make the final concentration 3. 2mg/ L. A bottle of cells was collected every 3 hours, measured by flow cytometry. Collected 6 periods of cells that mentioned above and got them measured by flow cytometry. Cantharidin acted on G1 inducing G1 blocked slightly; acted on G2 phase causing G2 block, the cell staying at G2 no longer transiting G2/M, and leading to G2/M block. We could see the subpeak of apoptosis at any period.4. Morphology of apoptosis cells4. 1 We observed the cells with inverse microscopes, founded some cells became round, small, and some others' surface sprouted. The sprouts may be close to the membrane, inside or outside, formed cell an irregular shape. Apoptosis bodies were come from which the buds failed off, shaped round or ellipse. Inside them concentrated chromatin contour could be seen, some of these cells failed off floating in the medium, while others swelled and then ruptured.4. 2 Observed cells by transmission electric microscope, we could see clearly the structures of nucleolus, mitochondria and endoplast of contrast A549 cells, and the nucleolus taking the majority of cell. After cantharidin added toA549 cell, nucleus crimpled, cracked without karyon membrane wraped up, chromosome gathered to cell membrane, and the cell transformed to apoptosis bodies.5. Annexin V -FITC detected quantificationally apoptosis cellsCells were collected which were dealed with 1.4 mg/L cantharidin at 24h, 48h,72h and normal controls, then marked Annexin V - FITC and analysed by flow cytometry to examine the quantity of apoptosis cells. We found that the proportion of A549 apoptosis cells were lower after cantharidin was added for 24h and 48 h, however the proportion at 72 h was higher.6. DNA agarose gel electrophoresisWe collected the cells treated with cantharidin about 72 h, 120h and normal controls, then DNA agarose gel electrophoresis was executed. There was a " ladder strip" appeared which was the characteristic of apoptosis at 72h because DNA of apoptosis cells was degraded to regular segment. Moreover, at 120h, a majority of cells were necrosis and DNA of these cells degraded unregularly, then a continuous "membrane strip" appeared. Howerer, because the molecular of DNA of normal cells was large and moved short distance, so they remained near at the adding hole.7. Western BlottingCells obtained from synchronous convalescence for 18h, were added cantharidin, which the final concentration was 3. 2mg/L. Three hours later, we collected the cells respectively which were 18h , 21h and treated group. After the cells were treated with cantharidin for 3h, we found there were no changes within expression of P34cdc2, phos - P34 cdc2, whereas cyclinB1 and survivin were decreased, P21 was increased, and ERK1/ERK2, phos - ERK1/phos - ERK2 decreased. The cells which were treated for 24h, 48h, 72h and a group of normal cells were collected respectively to be examined the expression of apoptosis relevant proteins Bcl -2, Bax, survivin. Then we found Bax was increased gradually with time extending, but the expression of Bcl - 2 and survivin were declined.