DATS逆转人类骨肉瘤多药耐药株耐药及对类肿瘤干细胞作用的初步研究
摘要
研究背景:骨肉瘤是骨外科常见于青少年患者的恶性肿瘤,虽然近年来在外科手术以及新辅助化疗上取得了很大进展,但死亡率仍很高。骨肉瘤易复发转移及患者对化疗药物的原发或继发耐药是导致治疗失败的主要原因。尽管临床已开发出多种针对肿瘤耐药的药物,但临床疗效仍不满意,肿瘤干细胞假说为探寻骨肉瘤的发生转移及耐药机制,寻找有效治疗药物指明了方向。
肿瘤干细胞(Tumor stem cells)的发现为揭开肿瘤的发生、发展及侵袭转移耐药机制,寻找肿瘤预防治疗的方法等展示出广阔的前景。早在上世纪中叶,科学家们从恶性畸胎瘤与胎儿组织的相似性,特别是癌组织与胚胎组织相似的多向潜能分化性,认识到二者可能密切相关。近年来的研究表明,在白血病以及部分实体瘤中(如乳腺癌、脑瘤等),只有一小部分肿瘤细胞具有无限增殖能力,并可形成新的肿瘤灶,这种细胞被认为是肿瘤干细胞。当前所指的肿瘤细胞的异质性不仅包括肿瘤内细胞的异形性,对化疗放疗反应的差异以及侵袭和转移能力的不同,还包括肿瘤中存在有肿瘤干细胞和非肿瘤干细胞。多数细胞没有无限增殖和成瘤能力,只有肿瘤干细胞可自我更新产生新的肿瘤干细胞和无致瘤能力的肿瘤细胞。
肿瘤干细胞假说的提出是人类认识肿瘤发生机制的一大进步,它在理论上丰富了肿瘤发生的理论,在临床实践中提示人们杀伤肿瘤细胞(虽然它们也不断地增殖)并不能治愈肿瘤,原因是肿瘤干细胞表达ABC转运蛋白,参与多药耐药,并表达与干细胞共同的维持未分化的关键调控基因,如Sox2,Nanog,Oct4等,能够保持并具有很强的自我更新能力和抗凋亡能力,使得它们可逃逸当前的标准治疗方法,成为复发转移的“源泉”,因此,肿瘤干细胞被认为是肿瘤治疗研究的新靶点,成为根治恶性肿瘤的希望所在。
寻找肿瘤干细胞的表面分子标记,一直是研究的热点。1997年,Bonnet等研究发现,人急性粒细胞白血病(acute myeloid leukemia,AML)中的CD34+CD38-亚群细胞,虽然仅占总数的0.2%,但是唯一可在免疫缺陷鼠具有成瘤性的细胞;2003年,Singh SK等发现脑肿瘤细胞在无血清培养中仅占总细胞数少数的CD133+群细胞可形成肿瘤细胞球,而CD133-群细胞不能形成,在第二年他们又发现CD133+群细胞移植到NOD/SCID鼠脑内,可诱发脑肿瘤,但CD133-群细胞却不能。2006年后,研究发现越来越多的肿瘤干细胞与CD133+群细胞有关,Suetsugua从肝癌细胞株Huh-7中,Ricci-Vitiani L和O'Brien CA分别在结肠癌组织中均分离出CD133+细胞,并证明该群细胞为肿瘤干细胞。目前,骨肉瘤肿瘤干细胞尚无公认的表面标记。
当前的许多研究认为,肿瘤干细胞是造成肿瘤耐药的最根本原因,肿瘤细胞对化疗药物的耐受性是肿瘤治疗的主要障碍。现有治疗肿瘤的方法主要是针对肿瘤组织内的大多数细胞,而不是肿瘤干细胞。肿瘤干细胞与正常干细胞具有许多相似的特征,如大多处于细胞周期的G0期,具有自我更新和分化能力,表达特定的干细胞表面标记,共用相同的干细胞调控通路等。研究证明,肿瘤干细胞致瘤性强,并多表达特异性的ATP结合盒转运蛋白(ATP-binding cassettetransporter,ABC transporter),如ABCB1、ABCG2等介导的膜泵耐药分子,使其对化疗天然耐药。ABCB1、ABCG2是从肿瘤组织中分离出来的最基本的肿瘤多药耐药基因,ABCB1编码P-gp,ABCG2编码BCRP;这些转运蛋白通过利用ATP分解产生的能量主动将细胞内的药物泵出,从而保护自身免受细胞毒性药物的损伤,对化疗药物不敏感。肿瘤干细胞高表达这类分子,可以将化疗药物泵出细胞,降低细胞内的药物浓度,导致肿瘤化疗后少数肿瘤干细胞仍然存活,目前认为这是肿瘤耐药、复发转移的重要机制。
为了寻找逆转肿瘤多药耐药的方法,科学家们实验了许多药物,诸如:钙离子通道阻滞剂,免疫抑制剂以及激素类药物等等药物,取得了一定的效果,但是,这类药物在产生逆转效应剂量上,都具有一些毒副作用,限制了临床应用。因此,许多研究者开始转向一些天然药物。大蒜中的成分DAS,就已经被证明在低毒性的剂量上,有能力逆转P-gp介导的K562白血病耐药株的多药耐药性。近年来的流行病学研究已经证实,大蒜与人体许多种癌症的发病率下降有密切的关系,这应归功于从粉碎的新鲜大蒜中提取的有机硫化合物(organosulfurcompounds,OSCs),主要包括DAS(diallyl sulfide,二烯丙基一硫化物)、DADS(diallyl disulfide,二烯丙基二硫化物)、DATS(diallyl trisulfide,二烯丙基三硫化物,又称为大蒜素)。我们曾经对DATS对于骨肉瘤细胞系Saos-2作用进行研究,发现DATS对Saos-2细胞具有明显的抑制作用。而对于DATS是否具有和DAS类似的逆转P-gp介导的多药耐药性的作用,目前还未见有报道。
本研究对人类骨肉瘤及Saos-2细胞系中的肿瘤干细胞标记物CD133表达进行研究,并用免疫磁珠细胞分选(MACS)方法分离Saos-2细胞系中的CD133阳性(CD133+)群细胞,对其分化、细胞周期、致瘤性以及与多药耐药相关和干细胞调控相关的MDR1、Sox2基因表达进行研究,以研究该群细胞的干细胞特性。使用逐步递增氨甲喋呤(MTX)浓度方法诱导建立骨肉瘤多药耐药系Saos-2/R,评估大蒜素(DATS)逆转骨肉瘤耐药的能力,并研究DATS逆转耐药后,培养基中MTX对Saos-2/R中肿瘤干细胞标记物CD133表达百分率的影响。
目的:检测肿瘤干细胞的表面分子标记CD133在骨肉瘤组织细胞中的表达,分析骨肉瘤细胞系Saos-2中CD133+群细胞的干细胞特性。以Saos-2为母本,诱导建立骨肉瘤耐药细胞系,研究大蒜素(DATS)逆转骨肉瘤耐药的潜力,并研究逆转耐药前后,对骨肉瘤肿瘤干细胞群的影响。
方法:免疫组化研究CD133的表达:取自本院病理科的骨肉瘤常规石蜡包埋组织蜡块标本55例,其中骨旁骨肉瘤4例,纤维母细胞型骨肉瘤13例,软骨母细胞型骨肉瘤12例,骨母细胞型骨肉瘤26例,切成3μm的组织切片备用。取Saos-2细胞系对数增长期细胞,传代于放置有消毒后盖玻片的培养皿中,培养24h制备细胞爬片,使用70%乙醇固定20分钟备用。CD133表达流式细胞仪分析:分析对数生长期的骨肉瘤细胞系Saos-2细胞的CD133表达百分率。分离去除死细胞后,使细胞与CD133-PE抗体结合,上机检测CD133表达百分比率。CD133+Saos-2细胞分化研究:检测CD133阳性细胞加入完全培养基前后的细胞周期及CD133百分率变化;收集细胞后,按照操作手册,MACS去除死细胞,然后同样用MACS方法分离CD133+群细胞,流式细胞分析该群细胞在加入全培养基前的第0天,以及加入全培养基后第3及10天的CD133阳性细胞群的百分率的变化,并以Saos-2细胞做对照,分析第0天及第10天的细胞周期变化。CD133+/-群细胞MDR1,Sox2基因表达Real-time PCR分析:使用Real-time PCR分析CD133+群细胞涉及多药耐药及干细胞分化调控的关键基因MDR1、Sox2的表达情况;收集细胞,按照操作步骤去除死细胞,分选出CD133+/-群细胞,按照步骤行RNA抽提及逆转录,并行Real-time PCR分析。CD133+/-群细胞平板克隆形成实验:细胞平板克隆形成实验检测CD133+/-群的细胞克隆形成能力,分析其致瘤性差别;收集细胞后,按照操作手册,MACS去除死细胞,然后MACS方法分离CD133+/-群细胞,将细胞悬液做倍数稀释,按每皿150、100、200个细胞梯度密度,分别接种于含10ml预温37℃培养液的培养皿中,晃动培养皿使细胞分散均匀,将平皿移入培养箱中,在37℃,5%CO2及饱和湿度环境下,静止培养3周,将平皿放置在显微镜下,肉眼直接计数克隆数,按照公式:克隆形成率(%)=克隆数/接种细胞数×100,计算克隆形成率。
使用氨甲喋呤(MTX)浓度递增法诱导培养Saos-2细胞系,使Saos-2逐渐产生耐药性,命名为Saos-2/R,使用RT-PCR及Western blotting方法研究其P-gp(MDR1)的表达情况;使用MTT法验证Saos-2/R对多种化疗药物的药物敏感性变化,确定其是否产生多药耐药;DATS逆转耐药研究:应用含梯度浓度的DATS的完全培养基孵育Saos-2/R 48h,RT-PCR及Western blotting方法检测Saos-2/R的P-gp(MDR1)表达变化,分析能够下调P-gp(MDR1)表达的最低DATS逆转浓度;MTT药敏实验检测DATS作用后,Saos-2/R对不同化疗药物敏感性的变化。DATS逆转耐药后对CD133+群细胞的影响:使用含有最低逆转浓度的DATS及梯度浓度MTX的完全培养基孵育Saos-2/R细胞48h,并与不含DATS,但含有同样梯度浓度MTX完全培养基孵育的Saos-2/R细胞做对照,流式细胞仪分析梯度浓度的化疗药物MTX对Saos-2/R中的CD133+群细胞所占比例的影响。
结果:在人体骨肉瘤组织中普遍存在CD133+群细胞,约占5-14%,而在对人类骨肉瘤细胞系Saos-2的研究中发现,CD133+群Saos-2细胞在Saos-2细胞中约占5%左右,在使用MACS分离提纯后,对比CD133+群细胞在加入完全培养基前后的细胞周期及CD133表达百分率变化,发现CD133+群百分率在第0,3及10天出现迅速的时间依赖性下降,并且第10天时的细胞周期的G0/G1,G2及S期细胞比例与对照组Saos-2细胞的细胞周期相似,而与第0天细胞周期相比,第10天的细胞周期G0/G1期细胞明显减少,G2及S期细胞比例明显增加。我们还发现CD133+群比CD133-群细胞具有更高的细胞克隆形成率,并且CD133+群细胞与CD133-群细胞相比过表达MDR1及Sox2基因,而MDR1了是肿瘤干细胞耐药性的一个重要原因,Sox2基因则是干细胞调节网络的核心因子之一。
RT-PCR及Westem blotting结果显示,Saos-2/R相对于Saos-2细胞过表达P-gp(MDR1),MTT药敏实验显示Saos-2/R细胞对多种化疗药物耐药,具有多药耐药性,DAYS逆转耐药实验显示,随着DAYS梯度浓度增高,Saos-2/R的P-gp(MDR1)表达出现浓度依赖性下调,10μM DATS即可有效下调P-gp(MDR1)表达,MTT实验显示,10μM DATS存在下,Saos-2/R对多种化疗药物的敏感性增加,多药耐药性被逆转。使用CD133作为肿瘤干细胞标志物,研究了耐药株Saos-2/R在DAYS逆转耐药后,CD133+群细胞比例的变化。我们假设CD133+群的细胞百分比在DATS逆转耐药后,会无法耐受化疗药物的毒性而出现下降。我们检测对比了仅有梯度浓度MTX孵育的和同时有DAYS存在的Saos-2/R细胞中的CD133百分比率,我们发现,在没有DATS存在下,CD133+群百分比率在梯度浓度的MTX作用下是稳定的,但是在10μM DAYS存在下,这一百分比率随着MTX梯度浓度的提高而出现浓度依赖性下降。
结论:骨肉瘤Saos-2细胞系中的CD133+群细胞具有少量、处于静止期、分化增殖潜能特点,并表达干细胞关键调控基因及耐药相关基因等肿瘤干细胞的特征,我们称其为“类肿瘤干细胞”。DAYS可有效下调P-gp(MDR1)的表达并逆转耐药,是有应用潜力的逆转骨肉瘤P-gp介导多药耐药的逆转剂。经过DATS逆转耐药后的Saos-2/R中的类肿瘤干细胞---CD133+群细胞---无法耐受MTX的作用,而出现浓度依赖性百分率降低,我们推断DATS逆转耐药同时,可能具有逆转类肿瘤干细胞耐药的作用,使得类肿瘤干细胞对MTX的耐受性下降而出现细胞比例下降。
Background: Osteosarcoma is the most common primary bone malignancy in children and adolescents. Despite advances in surgery and multi-agent chemotherapy, long-term survival rates have stagnated at approximately 65%. The perplexing problem preventing successful chemotherapy is recurrence, metastasis and multidrug resistance of osteosarcoma. Although several drugs have been developed to treat osteosarcoma, the clinical effects are not so good. However, the hypothesis of tumor stem cells might point out the direction for future cancer research.
The hypothesis of tumor stem cells have very important prospect in the study of mechanism tumorigenicity, metastasis and drug resistance. In fact, scientists had found the correlation of stem cell and cancer cells through observeing the similarity between cancer tissue and embryonic tissue. Recent reserches have shown that there are only a small fraction of tumor cells having unlimited proliferative capacity in leukemia and part of solid tumors cells. The small fraction of tumor cells, which are able to form new tumor foci, are referred to as tumor stem cells. The current concept of heterogeneity of tumor cells also includes the differences of tumor stem cells and non-tumor stem cells in addition to differences in response to chemotherapy and in the capacity of invasion and metastasis outside. Most tumor cells do not have unlimited capacity of proliferation except for tumor stem cells, which can generate a new self-renewing tumor stem cells and non-tumorigenic capacity of tumor cells.
It is a big step forward in the process of exploring the mechanism of human tumor oncogenesis, etastasis and drug resistance mechanisms that tumor stem cell hypothesis. It has enriched the mechanism of human tumor oncogenesis in theory. In clinical practice, it remind us that the reason of our traditional failure chemotherapy is the tumor stem cell. The tumor stem cell over-express the protein of ABC transporters, which result in tumor stem cells escaping the current standard method of treatment, and to be the "source" of recurrence and metastasis. Therefore, the tumor stem cells have been considered a new target for the treatment of the tumor reserch, which has become a "radical" malignant hope.
It is the research hotspot to seek for the tumor stem cells marker. In 1997, Bonnet reported that CD34+ CD38- cells of acute myeloid leukemia cells have the stem cell characteristics. Even though they accounted for only 0.2 percent of the total number of cells, but they are only oncogenous cells group in the SCID rat. In 2003, Singh SK found that CD133 + cells group, which are only a small number of the total cell number of the brain tumor cells, can form tumor cell balls in serum-free culture and CD133- cells group didn't. In the next year, they found that the CD133 + cells group of the brain tumor cells, which were transplanted into NOD / SCID mouse's brain, can cause brain tumors. But the CD133- cells group didn't. After 2006, more and more studies showed that CD133+ group cells is closely related to tumor stem cells. Suetsugua, Ricci-Vitiani L and O'Brien CA showed that they isolated CD133 + cells group from the hepatoma cell line Huh-7 and colon cancer tissues, respectively. And they proved that the cells group were tumor stem cells. At present, there is no recognized surface tumor stem cells markers of human osteosarcoma.
Lot of the current studies suggest that tumor stem cells are the most fundamental reason that result in chemotherapeutic drugs resistant of tumor cells, which are the main obstacles to the treatment of malignant. The existing method of treatment of tumor is mainly directed against the majority of cells, rather than tumor stem cells. Tumor and normal stem cells have many similar characteristics, which are in the GO cell cycle phase and expression of specific ATP-binding cassette (ATP-binding cassette, ABC), such as ABCB1, ABCG2 and so on. These membrane pump-mediated resistance elements resulted in their natural resistance to chemotherapy. ABCB1, ABCG2 from tumor tissues are tumor multidrug resistance gene. ABCB1 encode P-gp, and ABCG2 encode BCRP. These transporter protein, which use the energy generated for ATP decomposition, will pump the intracellular drugs out and protect tumor stem cells from cytotoxic drug damage. Because tumor stem cells over expressed ATP-binding cassette, chemotherapeutic drugs can be pumped out of cells and intracellular drug concentrations be reduced. After tumor chemotherapy, there are still a small number of tumor stem cells survive. We believed that this is an important mechanism of the tumor drug resistant, recurrence and metastasis.
In order to look for the way of reversal multidrug resistance of tumor, scientists have experimented with many drugs, such as calcium channel blockers, immunosuppressive agents, as well as hormone drugs and so on. They have achieved some results. However, some side effects of these drugs have limited the clinical application. Therefore, many researchers began to study some natural medicines. The ingredients of garlic, DAS, has been proved to have the ability to reverse P-gp-mediated leukemia K562 multidrug resistance at the low toxicity doses. In recent years, epidemiological studies have confirmed that garlic has a close relationship with the many types of human cancer incidence rates drop. This should be attributed to crush fresh garlic from the extracted organic sulfur compounds (organosulfur compounds, OSCs), mainly including the DAS (diallyl sulfide, diallyl sulfide 1), DADS (diallyl disulfide, diallyl disulfide ) and DATS (diallyl trisulfide, diallyl trisulfide, also known as allicin). We ever studied the role of DATS on osteosarcoma cell line Saos-2. We found that DATS is able to inhibite osteosarcoma cells. However, as for the question of the DAS and DATS have a similar reversal of P-gp-mediated multidrug resistance role, there are not related studies yet.
In this study, we studied the tumor stem cell markers CD133 expression on human osteosarcoma cells. And we sort out the CD133-positive (CD133 +) group of cells from Saos-2 osteosarcoma cell lines using magnetic separation methods. We studied these cells differentiation, cell cycle, tumorigenicity and MDR1, Sox2 gene expression, which are related to multidrug resistance-associated and stem cell regulation and control. Additionally, we set up multi-drug resistance in osteosarcoma line Saos-2/R using methotrexate (MTX)-induced concentration ways. The ability of DATS reversing drug-resistant osteosarcoma cells were assessed. Finally, the imact of MTX on CD133 marker percentage were studied after DATS reversing drug-resistant of Saos-2/R.
Objective: To analyze the authentication osteosarcoma tumor stem cell surface molecular markers. To set up drug-resistant osteosarcoma cell lines and research DATS's potential of reversing drug resistance in osteosarcoma cell.To study the imact of MTX on CD133 marker percentage before and after DATS reversing drug resistance of osteosarcoma tumor cells.
Methods: Immunohistochemical study of CD133 expression: 55 cases osteosarcoma conventional paraffin-embedded tissues specimens come from Pathology Department Qilu Hospital Shandong University, including 4 cases parosteal osteosarcoma, 13 cases fibroblast-type osteosarcoma , 12 cases chondroblastic osteosarcoma and 26 cases Osteoblastoma osteosarcoma. They were cut into 3μm standby. Check logarithmic growth phase Saos-2 cells subcultured in Petri dish with the coverslip, which has been sterilized. When the cells have adhered to coverslip after being cultured for 24h, we use 70% ethanol to fix the cells for 20 minutes standby. CD133 expression by flow cytometry analysis: To analyze percentage of CD133 expression in the logarithmic phase of osteosarcoma cell line Saos-2 cells. After removing dead cells using Dead Die cell separation kit, cells were incubated with monoclonal anti-CD 133 conjugated to PE and detected the percentage of CD133 expression using a FACScan. CD133-positive Saos-2 cell differentiation: To detect the changes of CD133 positive percentage and the cell cycle before and after CD133-positive Saos-2 cell incubated in complete medium and. To remove dead cells using MACS MicroBeads separation, then sort out CD133 + cells from Saos-2 cells and analyze changes of CD133 positive percentage at 0~(th), 3~(th) and 10~(th) day after incubating in complete culture medium. And to analyze changes of the cell cycle at 0~(th) and 10~(th) day after incubating in complete culture medium. MDR1, Sox2 gene expression of CD133 + / - cells, by Real-time PCR analysis: Using Real-time PCR to analyze MDR1 and Sox2 expression of CD133+ cells, which are related to multidrug resistance and stem cell differentiation regulation. To remove dead cells using MACS MicroBeads separation, then sort out CD133 +/- cells from Saos-2 cells. According to operation Manual, RNA extraction and reverse transcription-line were done and then MDR1 and Sox2 expression were analyzed by Real-time PCR. CD133 + / - cells colony forming test: To analyze the differences of CD133+/- cells tumorigenicity using flat cell cloning test. Using MACS to remove dead cells and sort out CD133+/-cells from Saos-2 cells according to operating manuals. To dilute make cell suspension according to 50,100 and 200 cells per dish density gradient. They were inoculated in the Petri dish with 10ml pre-heating at 37℃culture medium. To shake up the Petri dish so that cells dispersed sloshing uniform. And then the Petri dish were plate into the incubator at 37℃, 5% CO2 and saturated humidity environment. After the static cultivate for three weeks, the Petri dish were placed on the plate under the microscope and the number of clones were counted directly by the naked eye. Then, Cell s colony forming efficiency was calculated according to the formula: cells colony forming efficiency (%) = Cloning number / inoculation cell number×100. A cell line resistant to MTX cytotoxicity (named for the Saos-2 / R) was established in our laboratory by progressive adaptation of MTX-sensitive Saos-2 parental cells to increasing concentrations of MTX. To study the P-gp (MDR1) expression of Saos-2/R using RT-PCR and Western blotting. To detect changes in drug sensitivity on a variety of chemotherapy drugs and to determine whether they have multi-drug resistance with MTT test. DATS reverse drug resistance research: Saos-2 / R cells were incubated for 48h with the complete medium containing the gradient concentration of DATS of incubated with 48h. To detect the changes of P-gp (MDR1) expression using RT-PCR and Western blotting method to analyze the lowest DATS reversing concentration. Assay of MTX reducing the tumor stem-like cells after DATS reversing drug resistance of Saos-2/R: Saos-2 / R cells were incubated with complete medium containing DATS of lowest reversing concentration, and the MTX gradient concentration for 48h. And the Saos-2 / R incubated in complet medium without DATS to be the control cells. They also contain the same MTX concentration gradient. To analyze the changes of CD133+ cells percentage using flow cytometry analysis.
Results: CD133 + cells were is commonly found in human osteosarcoma tissue accounting for about 5-14%, while they were also found in the human osteosarcoma cell line Saos-2 accounting for around 5%. CD133 + cells were put in complete medium after being sorted out from Saos-2 cells using MACS. And then, the percentage of CD133 + cells were detected by FCM. We found the percentage declined rapidly with the extension of time to cultivate. At the same time, the cell cycle at 10~(th) day is similar to nomal Saos-2 cell cycle, and there is significant difference compared with that at 0~(th) day. We also found that CD133 + cells have higher rates of colony-forming cells than that of CD133- cells. And CD133 + cells over-expressed MDR1 and Sox2 genes compared with CD133- cells. As mentioned earlier, it is an important reason of tumor stem cells MDR that MDR1 over-expression. And Sox2 gene is one of important core member of stem cell gene regulatory network. In the light of the above, we believe that CD133 + cells group of Saos-2 cells have some of key features of tumor stem cells. We call them tumor stem-like cells. We use CD133 as a marker to study the percentage of changes of CD133+ Saos-2/R cells after the Saos-2 / R drug-resistant being reversed by DATS. We assume that groups of CD133 + cells percentage will decline after drug resistance being reversed by DATS for not tolerating the toxicity of chemotherapy drugs. We compared the CD133 percentage changes of Saos-2/R incubated in complete medium with MTX concentration gradient with/without 10uM DATS. We found that the presence of CD133 + cells is stable in the group absence of DATS, while the percentage declined gradiently in the presence of 10μM DATS.
Conclusion: CD133 + cells were is commonly found in human osteosarcoma tissue and human osteosarcoma cell line(Saos-2) with a small amount. The CD133 + group of Saos-2 cells are at a quiescent cell cycle period and over-express key regulatory genes of nomal stem cells and drug resistance-related genes. And they have highly tumorigenic characteristics. These results showed CD133 + group of Saos-2 cells have the characteristics of tumor stem cells. We called tumor stem-like cells. The drug-resistance cells line Saos-2/R, which we set up using MTX inducing, have MDR characteristics. DATS can effectively reduce P-gp (MDR1) expression of Saos-2/R and reverse its drug resistance. After the Saos-2 / R drug-resistant being reversed, CD133 + cells percentage of Saos-2/R reduce for not being tolerated the toxicity of MTX. DATS may reverse the role of drug resistance of tumor stem-like cells while reversing drug resistance of Saos-2/R.
肿瘤干细胞(Tumor stem cells)的发现为揭开肿瘤的发生、发展及侵袭转移耐药机制,寻找肿瘤预防治疗的方法等展示出广阔的前景。早在上世纪中叶,科学家们从恶性畸胎瘤与胎儿组织的相似性,特别是癌组织与胚胎组织相似的多向潜能分化性,认识到二者可能密切相关。近年来的研究表明,在白血病以及部分实体瘤中(如乳腺癌、脑瘤等),只有一小部分肿瘤细胞具有无限增殖能力,并可形成新的肿瘤灶,这种细胞被认为是肿瘤干细胞。当前所指的肿瘤细胞的异质性不仅包括肿瘤内细胞的异形性,对化疗放疗反应的差异以及侵袭和转移能力的不同,还包括肿瘤中存在有肿瘤干细胞和非肿瘤干细胞。多数细胞没有无限增殖和成瘤能力,只有肿瘤干细胞可自我更新产生新的肿瘤干细胞和无致瘤能力的肿瘤细胞。
肿瘤干细胞假说的提出是人类认识肿瘤发生机制的一大进步,它在理论上丰富了肿瘤发生的理论,在临床实践中提示人们杀伤肿瘤细胞(虽然它们也不断地增殖)并不能治愈肿瘤,原因是肿瘤干细胞表达ABC转运蛋白,参与多药耐药,并表达与干细胞共同的维持未分化的关键调控基因,如Sox2,Nanog,Oct4等,能够保持并具有很强的自我更新能力和抗凋亡能力,使得它们可逃逸当前的标准治疗方法,成为复发转移的“源泉”,因此,肿瘤干细胞被认为是肿瘤治疗研究的新靶点,成为根治恶性肿瘤的希望所在。
寻找肿瘤干细胞的表面分子标记,一直是研究的热点。1997年,Bonnet等研究发现,人急性粒细胞白血病(acute myeloid leukemia,AML)中的CD34+CD38-亚群细胞,虽然仅占总数的0.2%,但是唯一可在免疫缺陷鼠具有成瘤性的细胞;2003年,Singh SK等发现脑肿瘤细胞在无血清培养中仅占总细胞数少数的CD133+群细胞可形成肿瘤细胞球,而CD133-群细胞不能形成,在第二年他们又发现CD133+群细胞移植到NOD/SCID鼠脑内,可诱发脑肿瘤,但CD133-群细胞却不能。2006年后,研究发现越来越多的肿瘤干细胞与CD133+群细胞有关,Suetsugua从肝癌细胞株Huh-7中,Ricci-Vitiani L和O'Brien CA分别在结肠癌组织中均分离出CD133+细胞,并证明该群细胞为肿瘤干细胞。目前,骨肉瘤肿瘤干细胞尚无公认的表面标记。
当前的许多研究认为,肿瘤干细胞是造成肿瘤耐药的最根本原因,肿瘤细胞对化疗药物的耐受性是肿瘤治疗的主要障碍。现有治疗肿瘤的方法主要是针对肿瘤组织内的大多数细胞,而不是肿瘤干细胞。肿瘤干细胞与正常干细胞具有许多相似的特征,如大多处于细胞周期的G0期,具有自我更新和分化能力,表达特定的干细胞表面标记,共用相同的干细胞调控通路等。研究证明,肿瘤干细胞致瘤性强,并多表达特异性的ATP结合盒转运蛋白(ATP-binding cassettetransporter,ABC transporter),如ABCB1、ABCG2等介导的膜泵耐药分子,使其对化疗天然耐药。ABCB1、ABCG2是从肿瘤组织中分离出来的最基本的肿瘤多药耐药基因,ABCB1编码P-gp,ABCG2编码BCRP;这些转运蛋白通过利用ATP分解产生的能量主动将细胞内的药物泵出,从而保护自身免受细胞毒性药物的损伤,对化疗药物不敏感。肿瘤干细胞高表达这类分子,可以将化疗药物泵出细胞,降低细胞内的药物浓度,导致肿瘤化疗后少数肿瘤干细胞仍然存活,目前认为这是肿瘤耐药、复发转移的重要机制。
为了寻找逆转肿瘤多药耐药的方法,科学家们实验了许多药物,诸如:钙离子通道阻滞剂,免疫抑制剂以及激素类药物等等药物,取得了一定的效果,但是,这类药物在产生逆转效应剂量上,都具有一些毒副作用,限制了临床应用。因此,许多研究者开始转向一些天然药物。大蒜中的成分DAS,就已经被证明在低毒性的剂量上,有能力逆转P-gp介导的K562白血病耐药株的多药耐药性。近年来的流行病学研究已经证实,大蒜与人体许多种癌症的发病率下降有密切的关系,这应归功于从粉碎的新鲜大蒜中提取的有机硫化合物(organosulfurcompounds,OSCs),主要包括DAS(diallyl sulfide,二烯丙基一硫化物)、DADS(diallyl disulfide,二烯丙基二硫化物)、DATS(diallyl trisulfide,二烯丙基三硫化物,又称为大蒜素)。我们曾经对DATS对于骨肉瘤细胞系Saos-2作用进行研究,发现DATS对Saos-2细胞具有明显的抑制作用。而对于DATS是否具有和DAS类似的逆转P-gp介导的多药耐药性的作用,目前还未见有报道。
本研究对人类骨肉瘤及Saos-2细胞系中的肿瘤干细胞标记物CD133表达进行研究,并用免疫磁珠细胞分选(MACS)方法分离Saos-2细胞系中的CD133阳性(CD133+)群细胞,对其分化、细胞周期、致瘤性以及与多药耐药相关和干细胞调控相关的MDR1、Sox2基因表达进行研究,以研究该群细胞的干细胞特性。使用逐步递增氨甲喋呤(MTX)浓度方法诱导建立骨肉瘤多药耐药系Saos-2/R,评估大蒜素(DATS)逆转骨肉瘤耐药的能力,并研究DATS逆转耐药后,培养基中MTX对Saos-2/R中肿瘤干细胞标记物CD133表达百分率的影响。
目的:检测肿瘤干细胞的表面分子标记CD133在骨肉瘤组织细胞中的表达,分析骨肉瘤细胞系Saos-2中CD133+群细胞的干细胞特性。以Saos-2为母本,诱导建立骨肉瘤耐药细胞系,研究大蒜素(DATS)逆转骨肉瘤耐药的潜力,并研究逆转耐药前后,对骨肉瘤肿瘤干细胞群的影响。
方法:免疫组化研究CD133的表达:取自本院病理科的骨肉瘤常规石蜡包埋组织蜡块标本55例,其中骨旁骨肉瘤4例,纤维母细胞型骨肉瘤13例,软骨母细胞型骨肉瘤12例,骨母细胞型骨肉瘤26例,切成3μm的组织切片备用。取Saos-2细胞系对数增长期细胞,传代于放置有消毒后盖玻片的培养皿中,培养24h制备细胞爬片,使用70%乙醇固定20分钟备用。CD133表达流式细胞仪分析:分析对数生长期的骨肉瘤细胞系Saos-2细胞的CD133表达百分率。分离去除死细胞后,使细胞与CD133-PE抗体结合,上机检测CD133表达百分比率。CD133+Saos-2细胞分化研究:检测CD133阳性细胞加入完全培养基前后的细胞周期及CD133百分率变化;收集细胞后,按照操作手册,MACS去除死细胞,然后同样用MACS方法分离CD133+群细胞,流式细胞分析该群细胞在加入全培养基前的第0天,以及加入全培养基后第3及10天的CD133阳性细胞群的百分率的变化,并以Saos-2细胞做对照,分析第0天及第10天的细胞周期变化。CD133+/-群细胞MDR1,Sox2基因表达Real-time PCR分析:使用Real-time PCR分析CD133+群细胞涉及多药耐药及干细胞分化调控的关键基因MDR1、Sox2的表达情况;收集细胞,按照操作步骤去除死细胞,分选出CD133+/-群细胞,按照步骤行RNA抽提及逆转录,并行Real-time PCR分析。CD133+/-群细胞平板克隆形成实验:细胞平板克隆形成实验检测CD133+/-群的细胞克隆形成能力,分析其致瘤性差别;收集细胞后,按照操作手册,MACS去除死细胞,然后MACS方法分离CD133+/-群细胞,将细胞悬液做倍数稀释,按每皿150、100、200个细胞梯度密度,分别接种于含10ml预温37℃培养液的培养皿中,晃动培养皿使细胞分散均匀,将平皿移入培养箱中,在37℃,5%CO2及饱和湿度环境下,静止培养3周,将平皿放置在显微镜下,肉眼直接计数克隆数,按照公式:克隆形成率(%)=克隆数/接种细胞数×100,计算克隆形成率。
使用氨甲喋呤(MTX)浓度递增法诱导培养Saos-2细胞系,使Saos-2逐渐产生耐药性,命名为Saos-2/R,使用RT-PCR及Western blotting方法研究其P-gp(MDR1)的表达情况;使用MTT法验证Saos-2/R对多种化疗药物的药物敏感性变化,确定其是否产生多药耐药;DATS逆转耐药研究:应用含梯度浓度的DATS的完全培养基孵育Saos-2/R 48h,RT-PCR及Western blotting方法检测Saos-2/R的P-gp(MDR1)表达变化,分析能够下调P-gp(MDR1)表达的最低DATS逆转浓度;MTT药敏实验检测DATS作用后,Saos-2/R对不同化疗药物敏感性的变化。DATS逆转耐药后对CD133+群细胞的影响:使用含有最低逆转浓度的DATS及梯度浓度MTX的完全培养基孵育Saos-2/R细胞48h,并与不含DATS,但含有同样梯度浓度MTX完全培养基孵育的Saos-2/R细胞做对照,流式细胞仪分析梯度浓度的化疗药物MTX对Saos-2/R中的CD133+群细胞所占比例的影响。
结果:在人体骨肉瘤组织中普遍存在CD133+群细胞,约占5-14%,而在对人类骨肉瘤细胞系Saos-2的研究中发现,CD133+群Saos-2细胞在Saos-2细胞中约占5%左右,在使用MACS分离提纯后,对比CD133+群细胞在加入完全培养基前后的细胞周期及CD133表达百分率变化,发现CD133+群百分率在第0,3及10天出现迅速的时间依赖性下降,并且第10天时的细胞周期的G0/G1,G2及S期细胞比例与对照组Saos-2细胞的细胞周期相似,而与第0天细胞周期相比,第10天的细胞周期G0/G1期细胞明显减少,G2及S期细胞比例明显增加。我们还发现CD133+群比CD133-群细胞具有更高的细胞克隆形成率,并且CD133+群细胞与CD133-群细胞相比过表达MDR1及Sox2基因,而MDR1了是肿瘤干细胞耐药性的一个重要原因,Sox2基因则是干细胞调节网络的核心因子之一。
RT-PCR及Westem blotting结果显示,Saos-2/R相对于Saos-2细胞过表达P-gp(MDR1),MTT药敏实验显示Saos-2/R细胞对多种化疗药物耐药,具有多药耐药性,DAYS逆转耐药实验显示,随着DAYS梯度浓度增高,Saos-2/R的P-gp(MDR1)表达出现浓度依赖性下调,10μM DATS即可有效下调P-gp(MDR1)表达,MTT实验显示,10μM DATS存在下,Saos-2/R对多种化疗药物的敏感性增加,多药耐药性被逆转。使用CD133作为肿瘤干细胞标志物,研究了耐药株Saos-2/R在DAYS逆转耐药后,CD133+群细胞比例的变化。我们假设CD133+群的细胞百分比在DATS逆转耐药后,会无法耐受化疗药物的毒性而出现下降。我们检测对比了仅有梯度浓度MTX孵育的和同时有DAYS存在的Saos-2/R细胞中的CD133百分比率,我们发现,在没有DATS存在下,CD133+群百分比率在梯度浓度的MTX作用下是稳定的,但是在10μM DAYS存在下,这一百分比率随着MTX梯度浓度的提高而出现浓度依赖性下降。
结论:骨肉瘤Saos-2细胞系中的CD133+群细胞具有少量、处于静止期、分化增殖潜能特点,并表达干细胞关键调控基因及耐药相关基因等肿瘤干细胞的特征,我们称其为“类肿瘤干细胞”。DAYS可有效下调P-gp(MDR1)的表达并逆转耐药,是有应用潜力的逆转骨肉瘤P-gp介导多药耐药的逆转剂。经过DATS逆转耐药后的Saos-2/R中的类肿瘤干细胞---CD133+群细胞---无法耐受MTX的作用,而出现浓度依赖性百分率降低,我们推断DATS逆转耐药同时,可能具有逆转类肿瘤干细胞耐药的作用,使得类肿瘤干细胞对MTX的耐受性下降而出现细胞比例下降。
Background: Osteosarcoma is the most common primary bone malignancy in children and adolescents. Despite advances in surgery and multi-agent chemotherapy, long-term survival rates have stagnated at approximately 65%. The perplexing problem preventing successful chemotherapy is recurrence, metastasis and multidrug resistance of osteosarcoma. Although several drugs have been developed to treat osteosarcoma, the clinical effects are not so good. However, the hypothesis of tumor stem cells might point out the direction for future cancer research.
The hypothesis of tumor stem cells have very important prospect in the study of mechanism tumorigenicity, metastasis and drug resistance. In fact, scientists had found the correlation of stem cell and cancer cells through observeing the similarity between cancer tissue and embryonic tissue. Recent reserches have shown that there are only a small fraction of tumor cells having unlimited proliferative capacity in leukemia and part of solid tumors cells. The small fraction of tumor cells, which are able to form new tumor foci, are referred to as tumor stem cells. The current concept of heterogeneity of tumor cells also includes the differences of tumor stem cells and non-tumor stem cells in addition to differences in response to chemotherapy and in the capacity of invasion and metastasis outside. Most tumor cells do not have unlimited capacity of proliferation except for tumor stem cells, which can generate a new self-renewing tumor stem cells and non-tumorigenic capacity of tumor cells.
It is a big step forward in the process of exploring the mechanism of human tumor oncogenesis, etastasis and drug resistance mechanisms that tumor stem cell hypothesis. It has enriched the mechanism of human tumor oncogenesis in theory. In clinical practice, it remind us that the reason of our traditional failure chemotherapy is the tumor stem cell. The tumor stem cell over-express the protein of ABC transporters, which result in tumor stem cells escaping the current standard method of treatment, and to be the "source" of recurrence and metastasis. Therefore, the tumor stem cells have been considered a new target for the treatment of the tumor reserch, which has become a "radical" malignant hope.
It is the research hotspot to seek for the tumor stem cells marker. In 1997, Bonnet reported that CD34+ CD38- cells of acute myeloid leukemia cells have the stem cell characteristics. Even though they accounted for only 0.2 percent of the total number of cells, but they are only oncogenous cells group in the SCID rat. In 2003, Singh SK found that CD133 + cells group, which are only a small number of the total cell number of the brain tumor cells, can form tumor cell balls in serum-free culture and CD133- cells group didn't. In the next year, they found that the CD133 + cells group of the brain tumor cells, which were transplanted into NOD / SCID mouse's brain, can cause brain tumors. But the CD133- cells group didn't. After 2006, more and more studies showed that CD133+ group cells is closely related to tumor stem cells. Suetsugua, Ricci-Vitiani L and O'Brien CA showed that they isolated CD133 + cells group from the hepatoma cell line Huh-7 and colon cancer tissues, respectively. And they proved that the cells group were tumor stem cells. At present, there is no recognized surface tumor stem cells markers of human osteosarcoma.
Lot of the current studies suggest that tumor stem cells are the most fundamental reason that result in chemotherapeutic drugs resistant of tumor cells, which are the main obstacles to the treatment of malignant. The existing method of treatment of tumor is mainly directed against the majority of cells, rather than tumor stem cells. Tumor and normal stem cells have many similar characteristics, which are in the GO cell cycle phase and expression of specific ATP-binding cassette (ATP-binding cassette, ABC), such as ABCB1, ABCG2 and so on. These membrane pump-mediated resistance elements resulted in their natural resistance to chemotherapy. ABCB1, ABCG2 from tumor tissues are tumor multidrug resistance gene. ABCB1 encode P-gp, and ABCG2 encode BCRP. These transporter protein, which use the energy generated for ATP decomposition, will pump the intracellular drugs out and protect tumor stem cells from cytotoxic drug damage. Because tumor stem cells over expressed ATP-binding cassette, chemotherapeutic drugs can be pumped out of cells and intracellular drug concentrations be reduced. After tumor chemotherapy, there are still a small number of tumor stem cells survive. We believed that this is an important mechanism of the tumor drug resistant, recurrence and metastasis.
In order to look for the way of reversal multidrug resistance of tumor, scientists have experimented with many drugs, such as calcium channel blockers, immunosuppressive agents, as well as hormone drugs and so on. They have achieved some results. However, some side effects of these drugs have limited the clinical application. Therefore, many researchers began to study some natural medicines. The ingredients of garlic, DAS, has been proved to have the ability to reverse P-gp-mediated leukemia K562 multidrug resistance at the low toxicity doses. In recent years, epidemiological studies have confirmed that garlic has a close relationship with the many types of human cancer incidence rates drop. This should be attributed to crush fresh garlic from the extracted organic sulfur compounds (organosulfur compounds, OSCs), mainly including the DAS (diallyl sulfide, diallyl sulfide 1), DADS (diallyl disulfide, diallyl disulfide ) and DATS (diallyl trisulfide, diallyl trisulfide, also known as allicin). We ever studied the role of DATS on osteosarcoma cell line Saos-2. We found that DATS is able to inhibite osteosarcoma cells. However, as for the question of the DAS and DATS have a similar reversal of P-gp-mediated multidrug resistance role, there are not related studies yet.
In this study, we studied the tumor stem cell markers CD133 expression on human osteosarcoma cells. And we sort out the CD133-positive (CD133 +) group of cells from Saos-2 osteosarcoma cell lines using magnetic separation methods. We studied these cells differentiation, cell cycle, tumorigenicity and MDR1, Sox2 gene expression, which are related to multidrug resistance-associated and stem cell regulation and control. Additionally, we set up multi-drug resistance in osteosarcoma line Saos-2/R using methotrexate (MTX)-induced concentration ways. The ability of DATS reversing drug-resistant osteosarcoma cells were assessed. Finally, the imact of MTX on CD133 marker percentage were studied after DATS reversing drug-resistant of Saos-2/R.
Objective: To analyze the authentication osteosarcoma tumor stem cell surface molecular markers. To set up drug-resistant osteosarcoma cell lines and research DATS's potential of reversing drug resistance in osteosarcoma cell.To study the imact of MTX on CD133 marker percentage before and after DATS reversing drug resistance of osteosarcoma tumor cells.
Methods: Immunohistochemical study of CD133 expression: 55 cases osteosarcoma conventional paraffin-embedded tissues specimens come from Pathology Department Qilu Hospital Shandong University, including 4 cases parosteal osteosarcoma, 13 cases fibroblast-type osteosarcoma , 12 cases chondroblastic osteosarcoma and 26 cases Osteoblastoma osteosarcoma. They were cut into 3μm standby. Check logarithmic growth phase Saos-2 cells subcultured in Petri dish with the coverslip, which has been sterilized. When the cells have adhered to coverslip after being cultured for 24h, we use 70% ethanol to fix the cells for 20 minutes standby. CD133 expression by flow cytometry analysis: To analyze percentage of CD133 expression in the logarithmic phase of osteosarcoma cell line Saos-2 cells. After removing dead cells using Dead Die cell separation kit, cells were incubated with monoclonal anti-CD 133 conjugated to PE and detected the percentage of CD133 expression using a FACScan. CD133-positive Saos-2 cell differentiation: To detect the changes of CD133 positive percentage and the cell cycle before and after CD133-positive Saos-2 cell incubated in complete medium and. To remove dead cells using MACS MicroBeads separation, then sort out CD133 + cells from Saos-2 cells and analyze changes of CD133 positive percentage at 0~(th), 3~(th) and 10~(th) day after incubating in complete culture medium. And to analyze changes of the cell cycle at 0~(th) and 10~(th) day after incubating in complete culture medium. MDR1, Sox2 gene expression of CD133 + / - cells, by Real-time PCR analysis: Using Real-time PCR to analyze MDR1 and Sox2 expression of CD133+ cells, which are related to multidrug resistance and stem cell differentiation regulation. To remove dead cells using MACS MicroBeads separation, then sort out CD133 +/- cells from Saos-2 cells. According to operation Manual, RNA extraction and reverse transcription-line were done and then MDR1 and Sox2 expression were analyzed by Real-time PCR. CD133 + / - cells colony forming test: To analyze the differences of CD133+/- cells tumorigenicity using flat cell cloning test. Using MACS to remove dead cells and sort out CD133+/-cells from Saos-2 cells according to operating manuals. To dilute make cell suspension according to 50,100 and 200 cells per dish density gradient. They were inoculated in the Petri dish with 10ml pre-heating at 37℃culture medium. To shake up the Petri dish so that cells dispersed sloshing uniform. And then the Petri dish were plate into the incubator at 37℃, 5% CO2 and saturated humidity environment. After the static cultivate for three weeks, the Petri dish were placed on the plate under the microscope and the number of clones were counted directly by the naked eye. Then, Cell s colony forming efficiency was calculated according to the formula: cells colony forming efficiency (%) = Cloning number / inoculation cell number×100. A cell line resistant to MTX cytotoxicity (named for the Saos-2 / R) was established in our laboratory by progressive adaptation of MTX-sensitive Saos-2 parental cells to increasing concentrations of MTX. To study the P-gp (MDR1) expression of Saos-2/R using RT-PCR and Western blotting. To detect changes in drug sensitivity on a variety of chemotherapy drugs and to determine whether they have multi-drug resistance with MTT test. DATS reverse drug resistance research: Saos-2 / R cells were incubated for 48h with the complete medium containing the gradient concentration of DATS of incubated with 48h. To detect the changes of P-gp (MDR1) expression using RT-PCR and Western blotting method to analyze the lowest DATS reversing concentration. Assay of MTX reducing the tumor stem-like cells after DATS reversing drug resistance of Saos-2/R: Saos-2 / R cells were incubated with complete medium containing DATS of lowest reversing concentration, and the MTX gradient concentration for 48h. And the Saos-2 / R incubated in complet medium without DATS to be the control cells. They also contain the same MTX concentration gradient. To analyze the changes of CD133+ cells percentage using flow cytometry analysis.
Results: CD133 + cells were is commonly found in human osteosarcoma tissue accounting for about 5-14%, while they were also found in the human osteosarcoma cell line Saos-2 accounting for around 5%. CD133 + cells were put in complete medium after being sorted out from Saos-2 cells using MACS. And then, the percentage of CD133 + cells were detected by FCM. We found the percentage declined rapidly with the extension of time to cultivate. At the same time, the cell cycle at 10~(th) day is similar to nomal Saos-2 cell cycle, and there is significant difference compared with that at 0~(th) day. We also found that CD133 + cells have higher rates of colony-forming cells than that of CD133- cells. And CD133 + cells over-expressed MDR1 and Sox2 genes compared with CD133- cells. As mentioned earlier, it is an important reason of tumor stem cells MDR that MDR1 over-expression. And Sox2 gene is one of important core member of stem cell gene regulatory network. In the light of the above, we believe that CD133 + cells group of Saos-2 cells have some of key features of tumor stem cells. We call them tumor stem-like cells. We use CD133 as a marker to study the percentage of changes of CD133+ Saos-2/R cells after the Saos-2 / R drug-resistant being reversed by DATS. We assume that groups of CD133 + cells percentage will decline after drug resistance being reversed by DATS for not tolerating the toxicity of chemotherapy drugs. We compared the CD133 percentage changes of Saos-2/R incubated in complete medium with MTX concentration gradient with/without 10uM DATS. We found that the presence of CD133 + cells is stable in the group absence of DATS, while the percentage declined gradiently in the presence of 10μM DATS.
Conclusion: CD133 + cells were is commonly found in human osteosarcoma tissue and human osteosarcoma cell line(Saos-2) with a small amount. The CD133 + group of Saos-2 cells are at a quiescent cell cycle period and over-express key regulatory genes of nomal stem cells and drug resistance-related genes. And they have highly tumorigenic characteristics. These results showed CD133 + group of Saos-2 cells have the characteristics of tumor stem cells. We called tumor stem-like cells. The drug-resistance cells line Saos-2/R, which we set up using MTX inducing, have MDR characteristics. DATS can effectively reduce P-gp (MDR1) expression of Saos-2/R and reverse its drug resistance. After the Saos-2 / R drug-resistant being reversed, CD133 + cells percentage of Saos-2/R reduce for not being tolerated the toxicity of MTX. DATS may reverse the role of drug resistance of tumor stem-like cells while reversing drug resistance of Saos-2/R.
引文
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