The Study Of Parthenolide And Cisplatin On The Proliferation And Apoptosis Of SW620Colon Cancer Cells

BackgroundColon cancer is one of the common malignant tumors in human body. It is the secondary frequent gastroenteric tumor with a high morbidity in China, Europe and America. Each year, more than one million new colon cancer patients are diagnosed in the world. The morbidity of colon cancer in China with4.2%is higher than the international standard of2%. Nowadays, the combination of operation and chemotherapy is the main treatment of colon cancer. Although the5years survival rate of colon cancer of early stage exceed70～90%, the curative effect is still not satisfying to advanced colon cancer. Only palliative surgery or chemotherapy could be performed to these patients because of the metastasis and extensive invasion of cancer at the time of the diagnosis, even though the comprehensive treatment of operation and chemotherapy is still suitable to the middle and advanced stage of colon cancer patients. However, the clinical effect of this combinative therapy is not satisfying because of the anti-drug sensitivity from cancer cells or serious adverse reactions from chemotherapy. Generally, the anti-drug mechanisms of tumor cells is very complicated, which includes three pathways:cytokinetics, biochemistry and pharmacological drug resistance. If synergy effect exised in two different anti-tumor drugs, it is believed that the low generate and high deactivation or even side effect of active metabolite will be possibly avoided while the treating effect will be saved even the dosage is reduced. It should be great clinical significant if the clinical therapeutic efficacy be executed with the increase of drug sensitivity and the decrease of dosage and chemotherapy correlated adverse reactions.Prior studies found that the parithenolide, one of sesquiterpene lactone obtained from feverfew, has a high antineoplasmic activity. It can inhibit the growth of many kinds of tumor cells, such as liver cancer cells, cholangiocarcinoma cells, multipl myeloma cells, and induces to death in vitro. Moreover, it also can enhance the drug sensitivities to cancer cells, such as the sensitivities of cisplatin to liver cancer cells. Recently, few papers were found about parthenolide to colon cancer, especially associated with other drugs. Can it affect the drug sensitivities to colon cancer cells and the proliferation and apoptosis of colon cancer cells or not? Whether it has a synergy with other anti-tumor drugs and decrease the dosage of chemotherapeutics or even reduce the chemotherapy correlated adverse reactions? Therefore, the researches about it should be having great clinical significance because of the lack of Prior studies. On the other hand, Cisplatin is widely used to treat human entity tumor, including colon cancer, especially to the ascites caused by advanced disease. Even with wide anticancer spectrum and powerful effect, cisplatin also be partly limited in clinical application because of serious toxic reaction and easy to drug resistance. With the object of SW620, the purpose of this study is to investigate the effects of parthenolide and cisplatin on the proliferation and apoptosis of colon cancer cells and of the potential synergy effect. It should be have some guiding roles to find the ways of enhancing the drug’s sensitivity and decreasing the dosage of chemotherapeutics and chemotherapy correlated adverse reactions.1Objective To observe the effects of parthenolide and cisplatin on the proliferation and apoptosis of colon cancer cells by using Flow cytometry and MTS assay. To preliminarily investigate the possible mechanisms and provide the evidence for clinical treatment with the expression of apoptosis-related genes and patterns under the treatments of parthenolide and (or) cisplatin were analyzed by using quantitative real-time PCR (Q-PCR) and Western blot.2Methods2.1Experimental designAccording to the medication, this experiment was divided into the following groups:A). Groups of cell proliferation with MTS assay including:◆Solvent control group (DMSO group)◆PTL groups(5μmol/L;10μmol/L;15μmol/L;20μmol/L),◆DDP groups (2.5μmol/L;5μmol/L;7.5μmol/L;10μmol/L)◆PTL+DDP groups (DDP5μmol/L+PTL10μmol/L; DDP5μmol/L+PTL20μmol/L; DDP10μmol/L+PTL10μmol/L; DDP10μmol/L+PTL20μmol/L)B). The experiment of Flow cytometry, Q-PCR and Western blot were all divided into the following groups:◆Solvent control group(DMSO group)◆PTL group (10μmol/L)◆DDP group (5μmol/L)◆PTL+DDP group (10μmol/L+5μmol/L)2.2Methods2.2.1Cell culture and medicationDistribute the cell suspension into the culture capsule and put the RPMI-1640 nutrient medium with10%FBS. Take the culture plate into the incubator with CO2at the presence of37℃. The state of cell medication as following:2.2.2Detecting the cell proliferation by MTS assaya) Take cells from each group for the following experiment.b) To catter and count cells after digestion, and adjusting the cells density to1×105/ml. and then, cells were divided into96-well plates (each well contains1×104cells).c) Conventional cell culture, drug treatments be in progress after cell adherence, and then collecting the different time point cells to MTS assay.d) Collecting cells from each time point (Oh,24h,48h,72h) for MTS assay with1:10ratio (lOul MTS to100ul culture fluid).e) To read the absorbance at490nm on the microplate reader after4hours incubation.2.2.3Detecting cell apoptosis by FCMa) To collect sampl for cell apoptosis dectection after48h drug treatment. b) Transferring each group’s nutrient medium to15ml metuliform tube and lays it on the ice.c) Rinsing the cells in the culture plate with2ml PBS and remove PBS.d) Putting0.5m10.25%Pancreatin with no EDTA and incubating cells until it starts to separate from culture plate under microscope observation.e) Lightly and continuously tap the culture plate and make cells totally separate from it.f) Lighly resuspension cells to nutrient medium or1×combination buffer and make it’s density about to1×106cells/ml。g) To transfer0.5ml cells suspension (5×105cells) to a new clean centrifuge tube from culture plate.h) Putting1.25ul Annexin V-FITC.i)15minute Reaction away from light under room temperature(18-24℃).j) To centrifugate cells5minute with800rpm under room temperature, and then remove supernatantk) Lightly repeated to suspense cells with0.5ml prechill buffer.1) putting10μl Propidium Iodide。m) Preserving sample on the ice away from light. n) FCM detection.2.2.4Fluorescence quantitative PCR(q-PCR) detectionSW620cells were inoculated into wells of6-well plates and treated with drugs for24h and then collected. Putting1ml Trizol and scatter cells to misce bene and making cells fully clearage. After5min standing, putting200ul chloroform and precipitate protein. Centrifugating cells and removing supernatant and putting isometric isopropanol to precipitate RNA. The Precipitate was rinsed twice with75%ethanol and then to be dissolved by15～60μL DEPC.1μL RNA samples were volumetrically diluted to50times and to the detect the OD value with BioPhotometer plus Eppendorf s nucleic acid protein analyzer. It will be indicated that the RNA samples were pure or without protein pollution if the ratio of OD260/OD280higher than1.8. RNA was reverse transcripted in the RNase-free PCR Tubes and then obtained cDNA was used to the PCR experiment. Primers used in PCR experiment were shown in tablel. The PCR amplification conditions were as follows:Initial denaturation at95℃for5min was followed by30cycles of at95℃for15sec, at50℃for15sec and at72℃for32sec, then the final cycle had an extended incubation at72℃for5minutes.2.2.5Western Blot detection SW620cells were inoculated into6-well plates. Collecting cells sample after48h drug treatment and then extracting total cellular protein with RIPA lysate. The protein density was determined by BCA protein assay.3Oμg of total proteins in each well were electrophoresed on SDS-polyacrylamide separating gels (8%-12%) Proteins were transferred to polyvinyl difluoride (PVDF) membrane in transfer buffer and then the membrane was incubated with blocking solution (5%skim milk) for1h. It was incubated with primary antibodys which were diluted with PBS (Caspase-3,l:1500; Caspase-9,1:1000; RARP,1:1000; Bcl-2,1:1000) at4℃overnight. The PVDF membrane was washed with TBST3times,5minute for each time, at the next day. And then incubating it with suitable secondary antibody for one hour under room temperature. Washing PVDF membrane with TBST in3times,5minute for each time, and to expose it in darkroom.3Results3.1The result of MTS detection demonstrated that parthenolide and cisplatin were both able to inhibit the proliferation of SW620cells respectively, which had statistical significance compared with DMSO group (P<0.05). And compared with cisplatin, parthenolide with5μmol/L shown stronger inhibition on proliferation of SW620cells with48h drug-treating. Both of parthenolide and cisplatin showed a concentration-dependent inhibition to sw620cells proliferation. The combined effect of these two drugs were stronger than single agent. Jin Zhengjun method analyze that q value of the drug combination group (10μmol/L parthenolide+5μmol/L cisplatin) exceed1.15, which implies that there is a synergistic effect in this two drugs. The q values of other groups were from0.85to1.15, which implies only an additive effect in these two drugs.3.2FCM detection showed that the apoptosis rates of sw620cells in the parthenolide group and the cisplatin group are higher than that of DMSO group and apoptosis rate of parthenolide+cisplatin group was higher than that in single agent group. The early apoptosis rates of the DMSO group, parthenolide group, cisplatin group and combined group were1.25%、5.69%、3.65and8.36%respectively, while no significant difference among any groups (5.21%、6.58%、6.03%and7.86%)3.3The Q-PCR detection indicated that parthenolide and cisplatin can both significantly down-regulate the expression of Bcl-2and up-regulate the expression of caspase3, caspase9, Bax and PARP. Compare with single agent groups, the regulation to these apoptosis-related genes was more obvious in combined group.3.4The detection of Western blot demonstrated that the expression of Bcl-2protein in sw620cells treated with parthenolide decreased significantly, while that of Bax protein was ascendant. And the protein expression of caspase3、caspase9and PARP in sw620cells were also up-regulated. There were similar results in cisplatin groups. Moreover, the change of above-mentioned protein expression level was greater when parthenolide and cisplatin combined treat with SW620cells.4Conclusions4.1Parithenolide and Cisplatin both have the inhibitory effect with concentration dependent to the colon cancer cell SW620. And they had additive effect but no synergy function to inhibit the proliferation and induce the apoptosis of colon cancer cells SW620when they are used together.4.2The effect of Parithenolide and Cisplatin to induce the apoptosis of SW620possibly caused by adjusting the expression of the apoptosis-related genes, such as Bcl-2, Bax, Caspase3, Caspase9and PARP. It’s possible correlated to the pathway of mitochondria with influencing the balance of Bcl-2/Bax and stimulating the cascade reaction of caspase proteins.