小白菊内酯及顺铂对人结肠癌SW620细胞增殖、凋亡影响的研究
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摘要
研究背景
结肠癌是人体最常见的恶性肿瘤之一,占胃肠道肿瘤的第2位。在欧美及中国都有很高的发病率,全球结肠癌每年新发病例超过100万例。中国结肠癌发病率由上世纪70年代初的12/10万增长到目前的56/10万,升速约为每年4.2%,远超2%的国际水平。目前,针对该病的治疗主要是采用手术、化疗为主的多种治疗手段结合的综合治疗。对早期结肠癌的治疗效果相对较好,Ⅰ～Ⅱ期结肠癌患者5年生存率可以达到70～90%以上。但对于中晚期结肠癌患者而言,效果并不理想。因肿瘤侵犯、多处转移等原因导致较多患者就诊时即失去根治性手术治疗的机会而仅能进行姑息性手术、化疗等姑息性治疗。当然,手术+化疗为主的综合治疗仍是中晚期结肠癌患者较为合适的选择。但因有较多结肠癌患者长期化疗后可能出现耐药,对化疗药物敏感性较低而影响疗效,甚至部分还可因出现较严重的化疗副反应而无法进行化疗,从而影响整体治疗效果。我们知道,肿瘤耐药机制复杂,一般由以下三方面原因导致,即细胞动力学所致的耐药、生化原因所致的耐药和药理学原因所致的耐药。临床上如果两药能起协同作用,则可在不影响疗效前提下减少相互药物用量,进而有可能减少各自的毒副作用,克服由活性代谢物的低生成或高失活所引起的耐药。临床上如何克服肿瘤耐药、提高癌细胞对化疗药物的敏感性、减少临床化疗药物的用量,进而减少化疗相关副反应,顺利完成化疗过程对提高治疗效果具有重要的临床意义。
此前有研究表明,从菊科植物中提取的一种倍半萜内酯化合物小白菊内酯具有较强的抗肿瘤活性,在体外可抑制多种肿瘤细胞株的生长增殖,并能诱导细胞凋亡,比如肝癌、胆管癌及多发性骨髓瘤等。另外,小白菊内酯还可以增强癌细胞对化疗药的敏感性,如增强肝癌细胞对顺铂的敏感性。但目前该药对结肠癌的作用方面的研究较少,它对结肠癌细胞的增殖与凋亡有何影响?是否可与其它化疗药物起协同作用、减少化疗药物的用量、进而在临床上增强疗效、减少化疗毒副反应?顺铂是在人体实体肿瘤治疗中使用较为广泛的抗肿瘤药,结肠癌治疗中亦有广泛应用,尤其在晚期结肠癌腹腔转移产生腹水的治疗中有较好效果。虽然顺铂抗癌谱广、作用强,但因其毒性反应大、易产生耐药性,导致顺铂在临床应用上受到一定的限制。因此寻找能够增敏顺铂的治疗药物,减轻顺铂的毒副反应具有重要的临床意义。本研究中以人的结肠癌细胞SW620作为研究对象,采用小白菊内酯及顺铂作用于SW620后,观察它们对结肠癌细胞凋亡及增殖有何影响,二者联用是否有协同、增效功能。这对临床上能否提高结肠癌细胞对化疗药物的敏感性,减少化疗药物的用量,进而减少化疗相关副反应等具有一定的指导作用。
1目的
结肠癌细胞SW620经过小白菊内酯及顺铂作用后,通过MTS法及流式细胞仪检测,观察小白菊内酯及顺铂对人结肠癌细胞SW620细胞增殖及凋亡方面的影响,了解小白菊内酯与顺铂二者联合使用是否可产生协同效应?是否可以增强结肠癌细胞对顺铂的敏感性?同时通过荧光定量PCR和western blot方法检测凋亡相关基因、蛋白的表达,初步探讨其可能的影响机制,为小白菊内酯用于临床治疗结肠癌,特别是与化疗药物联合应用治疗结肠癌提供理论依据。
2方法
2.1实验分组
按细胞培养后药物处理情况,实验分组情况如下：
A). MTS法检测细胞增殖实验分组为：
◆溶剂对照组(DMSO组)
◆小白菊内酯(PTL)各浓度组(5pmol/LL.10μol/L.15μμmol/L、20μmol/L)
◆顺铂(DDP)各浓度组(2.5μmol/L.5μmol/L.7.5μmol/L、101/L)
◆小白菊内酯和顺铂联合组(DDP5μmol/L+PTL10μmol/L、DDP5μmol/L+PTL20μmol/L、DDP10μmol/L+PTL10μmol/L.DDP10μmol/L+PTL20μmol/L)
B).流式细胞仪(FCM)检测细胞凋亡及PCR、Western Blot检测凋亡相关基因、蛋白实验分组均为：
◆溶剂对照组(DMSO组)
◆小白菊内酯组(10μmol/L)
◆顺铂组(5μmol/L)
◆小白菊内酯+顺铂组(10μmol/L+5μmol/L)
注：从A部分实验MTS检测两药联合各浓度组中选出抑制率最高的一组作为B部分实验各组用药浓度的选择依据。
2.2实验方法
2.2.1细胞培养及药物处理
SW620细胞接种于含10%胎牛血清的RPMI-1640培养基中,置37℃5%CO2培养箱中培养,常规胰酶消化传代。各组细胞药物处理如下：
2.2.2MTS法检测细胞增殖
将对数期生长的SW620细胞消化后,计数,调整细胞浓度为1×105个/ml,分到96孔板,每孔100ul,即每孔细胞为1×104个,常规培养,待细胞贴壁后,进行药物处理。药物作用时间是24h、48h、72h。收集各个时间点的细胞(0h,24h,48h,72h)加入MTS,比例为1/10。即100ul培养液加入10ul检测液。孵育4小时后,酶标仪读板,MTS检测读取OD490数据。采用金正均法判定两药物是否具有协同作用。也称概率相加法,具体的公式：q=EAB/(EA+EB-EA×EB),公式中EAB为联合处理组的抑制率EA、EB分别为A药和B药单独处理的抑制率,若q值在0.85-1.15间为两药合用单纯相加,若q>1.15为有协同作用,若q<0.85表示两药合用有拮抗作用。
2.2.3流式细胞仪检测细胞的凋亡
将SW620细胞接种于6孔板,药物处理48h后收样进行流式细胞凋亡检测。胰酶消化细胞后,用预冷的PBS清洗细胞,将细胞轻轻重悬于预冷的1×结合缓冲液中使得其密度大约为1×108细胞/ml。加入1.25μl Annexin V-FITC。室温(18-24℃)避光反应15分钟。室温800r/min离心5分钟,去除上清。将细胞用0.5ml预冷的l×结合缓冲液轻轻重悬。加入10μl Propidium Iodide。将样本放置在冰上避光保存。用流式细胞仪检测分析。
2.2.4荧光定量PCR检测
将SW620细胞接种于6孔板,处理24h后收集细胞样品,加入lml Trizol溶液,吹打混匀,使细胞充分裂解,静置5min；加入200μ L氯仿,沉淀蛋白；离心去上清,加入等体积的异丙醇沉淀RNA,用75%乙醇洗涤两次,加入15～60μL DEPC水溶解沉淀。取1μL RNA样品50倍稀释,在BioPhotometer plus艾本德核酸蛋白测定仪上测定OD值,OD260/OD280的比值大于1.8,说明制备的RNA较纯,无蛋白质污染。在RNase free的PCR管中进行RNA的逆转录,得到的cDNA用于PCR实验。各因子检测引物如表1所示。扩增条件是：95℃预变性5min,95℃15sec,50℃15sec,72℃32sec,30个循环,72℃后延伸5min。
2.2.5Western Blot检测
将SW620细胞接种于6孔板,处理48h后收集细胞样品,RIPA裂解液裂解细胞提取细胞总蛋白。BCA法测定蛋白总浓度,每孔上样量为30μg,8-12.的分离胶分离总蛋白,转膜至PVDF膜,5的脱脂奶粉室温封闭1h,PBS稀释一抗(Bax,1:500；Caspase-3,1:1500；Caspase-9,1:1000；RARP,1:1000: Bcl-2,1:1000)4℃孵育过夜,次日,TBST洗膜3次,每次5min；合适二抗室温孵育1h;TBST洗膜3次,每次5min；于暗室中进行曝光。
3结果
3.1MTS检测结果：将小白菊内酯和顺铂稀释成不同的浓度梯度,分别处理SW620细胞并进行MTS检测,单因素方差分析的结果显示各组间比较有统计学差异,小白菊内酯及顺铂均对SW620细胞的增殖有抑制作用。多重比较显示各试验组均与溶剂对照组有统计学差异。不同试验时间和不同试剂类型均会影响SW620细胞的增殖抑制率,对于同一试剂,不同浓度对抑制率亦有影响。对试剂类型和试验时间进行多重比较,结果显示试验类型三组间均有显著差异,小白菊内酯和顺铂(PTL+DDP)组对SW620细胞的增殖抑制率最高,其次是小白菊内酯(PTL)组,顺铂(DDP)组最低。相同浓度下小白菊内酯和顺铂对SW620细胞的增殖抑制率亦显示小白菊内酯组比顺铂组高,二者对SW620细胞的增殖抑制作用均表现出浓度依赖性。联合作用时对SW620细胞的增殖抑制作用较单药作用时增强。金正均法检测显示,小白菊内酯与顺铂联合组各组q值均介于0.85-1.15之间,说明两药联用时对SW620细胞的增殖的抑制作用可相加,亦无拮抗作用。但两药在抑制SW620细胞的增殖方面没有协同效应。
3.2流式细胞仪检测显示小白菊内酯和顺铂单用组的SW620细胞凋亡率显著高于溶剂对照组,二者联用组细胞凋亡率高于单药组。溶剂对照组、小白菊内酯组、顺铂组和小白菊内酯与顺铂联用组早期凋亡的比例分别为1.25%、5.69%、3.65和8.36%,晚期凋亡的比例却变化不大,分别为5.21%、6.58%、6.03%和7.86%。
3.3荧光定量PCR检测检测凋亡相关基因caspase3、caspase9、PARP、Bcl-2及Bax的mRNA水平的表达。结果提示：小白菊内酯和顺铂的单独作用可以明显下调Bcl-2的表达水平,二者联合作用时效果更明显。顺铂处理SW620细胞后,caspase3、caspase9、Bax及PARP的mRNA水平的表达量出现了上调；小白菊内酯处理后,caspase3、caspase9、Bax及PARP的mRNA水平的表达量亦出现了上调；联合作用后,四个凋亡相关基因的mRNA水平的表达较单用组上调更明显。
3.4用western blot方法检测凋亡相关蛋白的表达,结果显示小白菊内酯作用后SW620细胞中的Bcl-2蛋白的表达水平明显降低,而Bax蛋白的表达水平则明显增高；小白菊内酯处理后,SW620细胞中caspase3、caspase9及PARP的蛋白水平的表达量也出现了上调。使用顺铂处理的细胞,检测蛋白的表达趋势与小白菊内酯相似。二者联合时,上述变化更加明显。
4结论
4.1小白菊内酯及顺铂对SW620细胞的增殖均有抑制作用,并均表现出浓度依赖性。两药联用时对SW620细胞的增殖的抑制作用可相加,无拮抗作用。但两药在抑制SW620细胞的增殖方面没有协同效应。
4.2小白菊内酯及顺铂均能促进SW620细胞凋亡,二者联用时效果更明显。二者对SW620细胞凋亡的影响可能是通过下调Bcl-2及上调Bax、Caspase3、 Caspase9和PARP等凋亡相关基因的表达而发挥作用的。且可能与线粒体途径密切相关,即通过影响Bcl-2/Bax的表达平衡,从而使线粒体功能紊乱,进而激发Caspase级联反应,促使细胞凋亡。
Background
Colon 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.2Methods
2.1Experimental design
According 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)
2.2Methods
2.2.1Cell culture and medication
Distribute 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 assay
a) 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 FCM
a) 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 supernatant
k) 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) detection
SW620cells 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.
3Results
3.1The 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.
4Conclusions
4.1Parithenolide 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.
结肠癌是人体最常见的恶性肿瘤之一,占胃肠道肿瘤的第2位。在欧美及中国都有很高的发病率,全球结肠癌每年新发病例超过100万例。中国结肠癌发病率由上世纪70年代初的12/10万增长到目前的56/10万,升速约为每年4.2%,远超2%的国际水平。目前,针对该病的治疗主要是采用手术、化疗为主的多种治疗手段结合的综合治疗。对早期结肠癌的治疗效果相对较好,Ⅰ～Ⅱ期结肠癌患者5年生存率可以达到70～90%以上。但对于中晚期结肠癌患者而言,效果并不理想。因肿瘤侵犯、多处转移等原因导致较多患者就诊时即失去根治性手术治疗的机会而仅能进行姑息性手术、化疗等姑息性治疗。当然,手术+化疗为主的综合治疗仍是中晚期结肠癌患者较为合适的选择。但因有较多结肠癌患者长期化疗后可能出现耐药,对化疗药物敏感性较低而影响疗效,甚至部分还可因出现较严重的化疗副反应而无法进行化疗,从而影响整体治疗效果。我们知道,肿瘤耐药机制复杂,一般由以下三方面原因导致,即细胞动力学所致的耐药、生化原因所致的耐药和药理学原因所致的耐药。临床上如果两药能起协同作用,则可在不影响疗效前提下减少相互药物用量,进而有可能减少各自的毒副作用,克服由活性代谢物的低生成或高失活所引起的耐药。临床上如何克服肿瘤耐药、提高癌细胞对化疗药物的敏感性、减少临床化疗药物的用量,进而减少化疗相关副反应,顺利完成化疗过程对提高治疗效果具有重要的临床意义。
此前有研究表明,从菊科植物中提取的一种倍半萜内酯化合物小白菊内酯具有较强的抗肿瘤活性,在体外可抑制多种肿瘤细胞株的生长增殖,并能诱导细胞凋亡,比如肝癌、胆管癌及多发性骨髓瘤等。另外,小白菊内酯还可以增强癌细胞对化疗药的敏感性,如增强肝癌细胞对顺铂的敏感性。但目前该药对结肠癌的作用方面的研究较少,它对结肠癌细胞的增殖与凋亡有何影响?是否可与其它化疗药物起协同作用、减少化疗药物的用量、进而在临床上增强疗效、减少化疗毒副反应?顺铂是在人体实体肿瘤治疗中使用较为广泛的抗肿瘤药,结肠癌治疗中亦有广泛应用,尤其在晚期结肠癌腹腔转移产生腹水的治疗中有较好效果。虽然顺铂抗癌谱广、作用强,但因其毒性反应大、易产生耐药性,导致顺铂在临床应用上受到一定的限制。因此寻找能够增敏顺铂的治疗药物,减轻顺铂的毒副反应具有重要的临床意义。本研究中以人的结肠癌细胞SW620作为研究对象,采用小白菊内酯及顺铂作用于SW620后,观察它们对结肠癌细胞凋亡及增殖有何影响,二者联用是否有协同、增效功能。这对临床上能否提高结肠癌细胞对化疗药物的敏感性,减少化疗药物的用量,进而减少化疗相关副反应等具有一定的指导作用。
1目的
结肠癌细胞SW620经过小白菊内酯及顺铂作用后,通过MTS法及流式细胞仪检测,观察小白菊内酯及顺铂对人结肠癌细胞SW620细胞增殖及凋亡方面的影响,了解小白菊内酯与顺铂二者联合使用是否可产生协同效应?是否可以增强结肠癌细胞对顺铂的敏感性?同时通过荧光定量PCR和western blot方法检测凋亡相关基因、蛋白的表达,初步探讨其可能的影响机制,为小白菊内酯用于临床治疗结肠癌,特别是与化疗药物联合应用治疗结肠癌提供理论依据。
2方法
2.1实验分组
按细胞培养后药物处理情况,实验分组情况如下：
A). MTS法检测细胞增殖实验分组为：
◆溶剂对照组(DMSO组)
◆小白菊内酯(PTL)各浓度组(5pmol/LL.10μol/L.15μμmol/L、20μmol/L)
◆顺铂(DDP)各浓度组(2.5μmol/L.5μmol/L.7.5μmol/L、101/L)
◆小白菊内酯和顺铂联合组(DDP5μmol/L+PTL10μmol/L、DDP5μmol/L+PTL20μmol/L、DDP10μmol/L+PTL10μmol/L.DDP10μmol/L+PTL20μmol/L)
B).流式细胞仪(FCM)检测细胞凋亡及PCR、Western Blot检测凋亡相关基因、蛋白实验分组均为：
◆溶剂对照组(DMSO组)
◆小白菊内酯组(10μmol/L)
◆顺铂组(5μmol/L)
◆小白菊内酯+顺铂组(10μmol/L+5μmol/L)
注：从A部分实验MTS检测两药联合各浓度组中选出抑制率最高的一组作为B部分实验各组用药浓度的选择依据。
2.2实验方法
2.2.1细胞培养及药物处理
SW620细胞接种于含10%胎牛血清的RPMI-1640培养基中,置37℃5%CO2培养箱中培养,常规胰酶消化传代。各组细胞药物处理如下：
2.2.2MTS法检测细胞增殖
将对数期生长的SW620细胞消化后,计数,调整细胞浓度为1×105个/ml,分到96孔板,每孔100ul,即每孔细胞为1×104个,常规培养,待细胞贴壁后,进行药物处理。药物作用时间是24h、48h、72h。收集各个时间点的细胞(0h,24h,48h,72h)加入MTS,比例为1/10。即100ul培养液加入10ul检测液。孵育4小时后,酶标仪读板,MTS检测读取OD490数据。采用金正均法判定两药物是否具有协同作用。也称概率相加法,具体的公式：q=EAB/(EA+EB-EA×EB),公式中EAB为联合处理组的抑制率EA、EB分别为A药和B药单独处理的抑制率,若q值在0.85-1.15间为两药合用单纯相加,若q>1.15为有协同作用,若q<0.85表示两药合用有拮抗作用。
2.2.3流式细胞仪检测细胞的凋亡
将SW620细胞接种于6孔板,药物处理48h后收样进行流式细胞凋亡检测。胰酶消化细胞后,用预冷的PBS清洗细胞,将细胞轻轻重悬于预冷的1×结合缓冲液中使得其密度大约为1×108细胞/ml。加入1.25μl Annexin V-FITC。室温(18-24℃)避光反应15分钟。室温800r/min离心5分钟,去除上清。将细胞用0.5ml预冷的l×结合缓冲液轻轻重悬。加入10μl Propidium Iodide。将样本放置在冰上避光保存。用流式细胞仪检测分析。
2.2.4荧光定量PCR检测
将SW620细胞接种于6孔板,处理24h后收集细胞样品,加入lml Trizol溶液,吹打混匀,使细胞充分裂解,静置5min；加入200μ L氯仿,沉淀蛋白；离心去上清,加入等体积的异丙醇沉淀RNA,用75%乙醇洗涤两次,加入15～60μL DEPC水溶解沉淀。取1μL RNA样品50倍稀释,在BioPhotometer plus艾本德核酸蛋白测定仪上测定OD值,OD260/OD280的比值大于1.8,说明制备的RNA较纯,无蛋白质污染。在RNase free的PCR管中进行RNA的逆转录,得到的cDNA用于PCR实验。各因子检测引物如表1所示。扩增条件是：95℃预变性5min,95℃15sec,50℃15sec,72℃32sec,30个循环,72℃后延伸5min。
2.2.5Western Blot检测
将SW620细胞接种于6孔板,处理48h后收集细胞样品,RIPA裂解液裂解细胞提取细胞总蛋白。BCA法测定蛋白总浓度,每孔上样量为30μg,8-12.的分离胶分离总蛋白,转膜至PVDF膜,5的脱脂奶粉室温封闭1h,PBS稀释一抗(Bax,1:500；Caspase-3,1:1500；Caspase-9,1:1000；RARP,1:1000: Bcl-2,1:1000)4℃孵育过夜,次日,TBST洗膜3次,每次5min；合适二抗室温孵育1h;TBST洗膜3次,每次5min；于暗室中进行曝光。
3结果
3.1MTS检测结果：将小白菊内酯和顺铂稀释成不同的浓度梯度,分别处理SW620细胞并进行MTS检测,单因素方差分析的结果显示各组间比较有统计学差异,小白菊内酯及顺铂均对SW620细胞的增殖有抑制作用。多重比较显示各试验组均与溶剂对照组有统计学差异。不同试验时间和不同试剂类型均会影响SW620细胞的增殖抑制率,对于同一试剂,不同浓度对抑制率亦有影响。对试剂类型和试验时间进行多重比较,结果显示试验类型三组间均有显著差异,小白菊内酯和顺铂(PTL+DDP)组对SW620细胞的增殖抑制率最高,其次是小白菊内酯(PTL)组,顺铂(DDP)组最低。相同浓度下小白菊内酯和顺铂对SW620细胞的增殖抑制率亦显示小白菊内酯组比顺铂组高,二者对SW620细胞的增殖抑制作用均表现出浓度依赖性。联合作用时对SW620细胞的增殖抑制作用较单药作用时增强。金正均法检测显示,小白菊内酯与顺铂联合组各组q值均介于0.85-1.15之间,说明两药联用时对SW620细胞的增殖的抑制作用可相加,亦无拮抗作用。但两药在抑制SW620细胞的增殖方面没有协同效应。
3.2流式细胞仪检测显示小白菊内酯和顺铂单用组的SW620细胞凋亡率显著高于溶剂对照组,二者联用组细胞凋亡率高于单药组。溶剂对照组、小白菊内酯组、顺铂组和小白菊内酯与顺铂联用组早期凋亡的比例分别为1.25%、5.69%、3.65和8.36%,晚期凋亡的比例却变化不大,分别为5.21%、6.58%、6.03%和7.86%。
3.3荧光定量PCR检测检测凋亡相关基因caspase3、caspase9、PARP、Bcl-2及Bax的mRNA水平的表达。结果提示：小白菊内酯和顺铂的单独作用可以明显下调Bcl-2的表达水平,二者联合作用时效果更明显。顺铂处理SW620细胞后,caspase3、caspase9、Bax及PARP的mRNA水平的表达量出现了上调；小白菊内酯处理后,caspase3、caspase9、Bax及PARP的mRNA水平的表达量亦出现了上调；联合作用后,四个凋亡相关基因的mRNA水平的表达较单用组上调更明显。
3.4用western blot方法检测凋亡相关蛋白的表达,结果显示小白菊内酯作用后SW620细胞中的Bcl-2蛋白的表达水平明显降低,而Bax蛋白的表达水平则明显增高；小白菊内酯处理后,SW620细胞中caspase3、caspase9及PARP的蛋白水平的表达量也出现了上调。使用顺铂处理的细胞,检测蛋白的表达趋势与小白菊内酯相似。二者联合时,上述变化更加明显。
4结论
4.1小白菊内酯及顺铂对SW620细胞的增殖均有抑制作用,并均表现出浓度依赖性。两药联用时对SW620细胞的增殖的抑制作用可相加,无拮抗作用。但两药在抑制SW620细胞的增殖方面没有协同效应。
4.2小白菊内酯及顺铂均能促进SW620细胞凋亡,二者联用时效果更明显。二者对SW620细胞凋亡的影响可能是通过下调Bcl-2及上调Bax、Caspase3、 Caspase9和PARP等凋亡相关基因的表达而发挥作用的。且可能与线粒体途径密切相关,即通过影响Bcl-2/Bax的表达平衡,从而使线粒体功能紊乱,进而激发Caspase级联反应,促使细胞凋亡。
Background
Colon 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.2Methods
2.1Experimental design
According 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)
2.2Methods
2.2.1Cell culture and medication
Distribute 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 assay
a) 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 FCM
a) 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 supernatant
k) 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) detection
SW620cells 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.
3Results
3.1The 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.
4Conclusions
4.1Parithenolide 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.
引文
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[16]Sun Y, St Clair, Fang F, et al. The radiosensitization effect of parthenolide in prostate cancer cells is mediated by nuclear factor-kappaB inhibition and enhanced by the presence of PTEN[J]. Mol Cancer Ther,2007,6(9):2477-2786.
[17]Park JH, Liu L, Kim IH, et al. Identification of the genes involved in enhanced fenretinide-induced apoptosis by parthenolide in human heptoma cells[J]. Cancer Res,2005,65(7):2804-2814.
[18]Sweeney CJ, Mehrotra S, Sadaria MR, et al. The sesquiterpene lactone parthenolide in combination with docetaxel reduces metastasis and improves survival in a xenograft model of breath cancer [J]. Mol Cancer Ther, 2005,4(6):1004-1012.
[19]Yip-Schneider MT, Nakshatri H, Sweeney CJ, et al. Parthenolide and sulindac cooperate to mediate growth suppreassion and inhibition the nuclear factor-kappa B pathway in pancreatic carcinoma cells[J]. Mol Cancer Ther,2005,4(4):587-594.
[20]黄东胜,刘军伟,陈建峰,等.小白菊内酯对胰腺癌细胞BxPC-3增殖和凋亡的影及机制[J].中国病理生理杂志,2008,24(4)：661-665.
[21]Kwok BH, Koh B, Ndubuisi MI, et al. The anti-inflamm atory natural product parthenolide from te medicinal herb Feverfew directly binds to and inhibits/kappa B kinase(J]. Chem Biol,2001; 8(8):759-66.
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[13]SONG Xiao-Kai, TU Peng-Fei, WU Li-Jun, et al. A New Sesquiterpene Lactone from Ts00ngiodendron odorum Chun [J]. J Asia Natural Products Res, 2002,3(4):285-291.
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[15]Nakabayashi H, Shimizu K. Involvement of Akt/NF-KB pathway in antitumor effects of parthenolide on glioblastoma cells in vitro and in vivo[J]. BMC Cancer.2012,5 (12):453.
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[17]Sheehan M, Wong HR, Hake PW, et al. Parthenolide, an inhibitor of the nuclear factor-kappa B pathway, ameliorates cardiovascular derangement and outcome in endotoxic shock in rodents[J]. MolPharmacol,2002,61(5):953-63.
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[20]Shanmugam R, Jayaprakasan V, Gokem-Polar Y, et al. Restoring chemotherapy and refractory prostate cancer model[J]. Prostate,2006,66(14):1498-1511.
[21]Mendoca MS, Chin-Sinex H, Gomez-Millan J, et al. Parthenolide sensitizescells to X-ray-induced cell killing through inhibition of NF-kappaB and split-dose repair[J]. Radiat Res,2007,168(6):689-697.
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[24]Sweeney CJ, Mehrotra S, Sadaria MR, et al. The sesquiterpene lactone parthenolide in combination with docetaxel reduces metastasis and improves survival in a xenograft model of breath cancer [J]. Mol Cancer Ther, 2005,4(6):1004-1012.
[25]Yip-Schneider MT, Nakshatri H, Sweeney CJ, et al. Parthenolide and sulindac cooperate to mediate growth suppreassion and inhibition the nuclear factor-kappa B pathway in pancreatic carcinoma cells [J]. Mol Cancer Ther,2005,4(4):587-594.
[26]黄东胜,刘军伟,陈建峰,等.小白菊内酯对胰腺癌细胞BxPC-3增殖和凋亡的影及机制[J].中国病理生理杂志,2008,24(4)：661-665.
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[29]Wong HR, Menendez IY. Sesquiterpene lactones inhibit induciblenitric oxide synthase gene expression in cultured rat aortic smooth musclec ells[J]. Biochem Biophys Res Commun.1999,262(2):375:80.
[30]Kreuger MR, Grootjans S, Biavatti MW, et al. Sesquiterpene lactones as drugs with multiple targets in cancer treatment:focus on parthenolide[J]. Anticancer Drugs.2012,23(9):883-96.
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