Using ABCG2-molecule-expressing side population cells to identify cancer stem-like cells in a human ovarian cell line

CSCs (cancer stem cells) are a small subset of cells within a tumour that possesses the characteristics of stem cells and are considered to be responsible for resistance to chemoradiation. Identification of CSCs through stem cell characteristics might have relevant clinical implications. In this study, SP (side population ) cells were sorted from a human ovarian cancer cell line by FACS to determine whether cancer stem cell-like SP cells were present. A very small fraction of SP cells (2.6%) was detected in A2780 cells. SP cells possessed the following characteristics: highly proliferative activity, marked ability for self-renewal in soft agar and culture medium, high expression of ABCG2, drug resistance to vinblastine in vitro, and strong tumourigenic potential in Balb/c nude mice. It is concluded that there exists in the A2780 cell line a small number of SP cells with high expression of ABCG2. The cells have the characteristics of cancer stem-like cells, and identification and cloning of such human SP cells can help in improving therapeutic approaches to ovarian cancer in patients.     

5-Fluorouracil Chemotherapy of Gastric Cancer Generates Residual Cells with Properties of Cancer Stem Cells

Background: 5-Fluorouracil (5Fu) chemotherapy is the first treatment of choice for advanced gastric cancer (GC), but its effectiveness is limited by drug resistance. Emerging evidence suggests that the existence of cancer stem cells (CSCs) contributes to chemoresistance. The aim of the present study was to determine whether 5Fu chemotherapy generates residual cells with CSC-like properties in GC. Methods: Human GC cell lines, SGC7901 and AGS, were exposed to increasing 5Fu concentrations. The residual cells were assessed for both chemosensitivity and CSC-like properties. B lymphoma Mo-MLV insertion region 1 (BMI1), a putative CSC protein, was analyzed by immunohistochemical staining and subjected to pairwise comparison in GC tissues treated with or without neoadjuvant 5Fu-based chemotherapy. The correlation between BMI1 expression and recurrence-free survival in GC patients who received 5Fu-based neoadjuvant chemotherapy was then examined. Results: The residual cells exhibited 5Fu chemoresistance. These 5Fu-resistant cells displayed some CSC features, such as a high percentage of quiescent cells, increased self-renewal ability and tumorigenicity. The 5Fu-resistant cells were also enriched with cells expressing cluster of differentiation (CD)133⁺, CD326⁺ and CD44⁺CD24^-. Moreover, the BMI1 gene was overexpressed in 5Fu-resistant cells, and BMI1 knockdown effectively reversed chemoresistance. The BMI1 protein was highly expressed consistently in the remaining GC tissues after 5Fu-based neoadjuvant chemotherapy, and BMI1 levels were correlated positively with recurrence-free survival in GC patients who received 5Fu-based neoadjuvant chemotherapy. Conclusions: Our data provided molecular evidence illustrating that 5Fu chemotherapy in GC resulted in acquisition of CSC-like properties. Moreover, enhanced BMI1 expression contributed to 5Fu resistance and may serve as a potential therapeutic target to reverse chemoresistance in GC patients.     

解毒消癞饮逆转氟尿嘧啶诱导的肝癌干细胞耐药的机制研究

目的探讨解毒消瘾饮乙酸乙酯提取物（EE—JXY）能否降低肝癌细胞株Huh7对小剂量氟尿嘧啶（5-FU）化疗耐药的影响,并从肿瘤干细胞的角度探讨可能的机制。方法EE-JXY联合小剂量5-FU体外干预人肝癌细胞株Huh7。MTT法检测细胞存活率;流式细胞仪分析Huh7肝癌细胞株中侧群（sidepopulation,SP）细胞的比例;PCR检测肝癌干细胞相关基因ATP结合盒转运蛋白G2（ABCG2）和八聚体结合转录因子4（Oct4）mRNA的表达。采用单因素方差分析对数据进行统计。结果d,N量5-FU对Huh7细胞的增殖有抑制作用[24h存活率（84．54±2．88）％,96h存活率（58．36±3．52）％],但SP细胞的比例也从24h的4．72％升高至96h的12．07％,同时诱导了ABCG2和Oct4mRNA表达的上调。EE．JXY能够增强小剂量5-FU对Huh7细胞增殖的抑制能力[24h存活率从（84．54±2．88）％下降到（31．23±2．42）％,96h存活率从（58．36±3．52）％下降到（25．37±0．99）％],SP含量从24h的4．72％下降至1．69％,96h的12．07％下降至9．97％,同时也抑制了ABCG2和Oct4 mRNA的表达。结论肝癌Huh7细胞对小剂量5．FU存在化疗抵抗。EE—JXY能通过降低肝癌干细胞相关基因ABCG2 mRNA和Oct4 mRNA水平及SP的比例,增强Huh7细胞对小剂量5-FU的化疗敏感性,这可能是解毒消瘤饮降低肿瘤复发和转移的机制之一。     

ABCG2: A potential marker of stem cells and novel target in stem cell and cancer therapy

ABCG2 is a member of the ATP binding cassette (ABC) transporters, which can pump a wide variety of endogenous and exogenous compounds out of cells. Widely expressed in stem cells, ABCG2 is also found to confer the side population phenotype and is recognized as a universal marker of stem cells. Although the precise physiological role of ABCG2 in stem cells is still unclear, existing data strongly suggest that ABCG2 plays an important role in promoting stem cell proliferation and the maintenance of the stem cell phenotype. In addition, ABCG2 is also found to be expressed in a number of cancer cells and appears to be a marker of cancer stem cells. Moreover, ABCG2 expression in tumors may contribute to their formation and progression. Thus, ABCG2 has potential applications in stem cell and tumor therapy.     

Low Molecular Weight Heparin Ablates Lung Cancer Cisplatin-Resistance by Inducing Proteasome-Mediated ABCG2 Protein Degradation

Cancer side population (SP) cells, which are often referred to as cancer stem cells, are thought to be responsible for lung cancer chemotherapy resistance, and currently no drug can specifically target these cells. We hypothesize low-molecular-weight heparin (LMWH) may affect the biological properties of SP cells and could be used to clinically target these cells. To test this, SP cells were isolated from cisplatin (DDP)-resistant lung adenocarcinoma A549/DDP cells by flow cytometric sorting. Compared to non-SP cells, SP cells formed increased numbers of colonies in vitro, and had a 1000-fold increase in tumorigenicity in vivo. Proliferation and apoptosis assays demonstrated LMWH had no significant effect on lung SP cell proliferation or apoptosis. However, LMWH reduced lung SP cell colony formation ability and protein expression of the multidrug transporter, ABCG2, by FACS and western blot analyses without affecting its mRNA levels by RT-PCR. Consistently, immunohistochemistry stainings of ABCG2 in LMWH-treated tumor tissues were significantly reduced compared with those in controls. Further, we found proteasomal inhibitor MG132, but not lysosomal inhibitors leupeptin and pepstatin A, could restore ABCG2 protein levels in LMWH-treated SP cells. These suggest LMWH ablates lung SP cell chemoresistance by proteasome-mediated reduction of ABCG2 protein levels without affecting its mRNA levels. We also determined LMWH combined with cisplatin could overcome cisplatin-resistance and induced lung SP cells apoptosis both in vitro and in vivo. This study provides an experimental basis for using a combination of LMWH, which targets lung SP cells, with chemotherapy to improve lung cancer survival.     

Curcumin Improves the Tumoricidal Effect of Mitomycin C by Suppressing ABCG2 Expression in Stem Cell-Like Breast Cancer Cells

Cancer cells with stem cell–like properties contribute to the development of resistance to chemotherapy and eventually to tumor relapses. The current study investigated the potential of curcumin to reduce breast cancer stem cell (BCSC) population for sensitizing breast cancer cells to mitomycin C (MMC) both in vitro and in vivo. Curcumin improved the sensitivity of paclitaxel, cisplatin, and doxorubicin in breast cancer cell lines MCF-7 and MDA-MB-231, as shown by the more than 2-fold decrease in the half-maximal inhibitory concentration of these chemotherapeutic agents. In addition, curcumin sensitized the BCSCs of MCF-7 and MDA-MB-231 to MMC by 5- and 15-fold, respectively. The BCSCs could not grow to the fifth generation in the presence of curcumin and MMC. MMC or curcumin alone only marginally reduced the BCSC population in the mammospheres; however, together, they reduced the BCSC population in CD44⁺CD24^−/low cells by more than 75% (29.34% to 6.86%). Curcumin sensitized BCSCs through a reduction in the expression of ATP-binding cassette (ABC) transporters ABCG2 and ABCC1. We demonstrated that fumitremorgin C, a selective ABCG2 inhibitor, reduced BCSC survival to a similar degree as curcumin did. Curcumin sensitized breast cancer cells to chemotherapeutic drugs by reducing the BCSC population mainly through a reduction in the expression of ABCG2.     

酪氨酸磷酸酶PRL-3增加胃癌细胞BGC823的SP细胞比例并提高其对化疗药物的耐受性

蛋白酪氨酸磷酸酶PRL-3是近年发现的蛋白酪氨酸磷酸酶家族成员,能促进肿瘤细胞的侵袭、转移及上皮细胞间质转型,提示PRL-3可能在肿瘤发生发展及诱导肿瘤干细胞生成中发挥重要作用.由于侧群(SP)细胞具有许多干细胞的性质,SP细胞分选是目前筛选和分离获得干细胞或前体细胞常用的有效方法.为探讨PRL-3在诱导干细胞生成中的潜在作用,本文在建立过表达PRL-3的人胃癌细胞BGC823的基础上,通过SP分选和CCK-8的方法分析PRL-3对BGC823细胞中SP细胞的比例以及对化疗药物耐受性的影响.结果提示,高表达PRL-3提高BGC823中SP细胞的比例（2.5% vs 9.4%）,同时增加BGC823对化疗药物紫杉醇和顺铂的耐受性（相对于对照细胞,其耐药指数分别为1.75和1.29）.由于SP细胞的产生和细胞耐药性的提高与ABC家族基因表达水平上调密切相关,通过定量 RT-PCR和Western印迹检测发现,PRL-3能上调ABCB1和ABCG2的表达.上述研究结果表明,PRL-3有可能通过上调ABCB1和ABCG2的表达,增加胃癌细胞BGC823的SP细胞比例并增加其对化疗药物的耐受性.     

Dexamethasone down-regulates ABCG2 expression levels in breast cancer cells

The breast cancer resistance protein ABCG2 effluxes a variety of drugs and is believed to play an important role in multidrug resistance to chemotherapy. We show here for the first time that dexamethasone (DEX) and progesterone (PROG) are able to strongly inhibit ABCG2 expression in progesterone receptor (PR)-positive MCF7 and PR-negative MDA-MB-231 breast cells. In contrast, in the latter cells stably-transfected with progesterone receptor isoforms A and B, ABCG2 expression was strongly up-regulated by DEX and PROG. In addition, two other ligands of Pregnane X Receptor (PXR) and/or Glucocorticoid Receptor (GR) were also able to down-regulate ABCG2 expression in PXR- and GR-positive MCF7 cells. ABCG2 expression regulation by DEX likely resulted from the activation of PR-, PXR-, and/or GR-signaling pathways. ABCG2 expression inhibition by DEX was associated with increased sensitivity to mitoxantrone, a known ABCG2 substrate. The findings suggest that DEX may be useful in improving drug efficacy under certain conditions.     

Blockade of Hedgehog Signaling Synergistically Increases Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Cell Lines

Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs.     

Hedgehog/GLI-mediated transcriptional inhibitors from Zizyphus cambodiana

The aberrant hedgehog (Hh)/GLI signaling pathway causes the formation and progression of a variety of tumors. By screening tropical plant extracts by using our screening system, Zizyphus cambodiana was found to include Hh/GLI signaling inhibitors. Bioassay-guided fractionation of this plant extract led to the isolation of three active pentacyclic triterpenes, colubrinic acid (1), betulinic acid (2) and alphitolic acid (3), as potent inhibitors. The inhibition of GLI-related protein expression with 1 or 2 was observed in HaCaT cells with exogenous GLI1, or human pancreatic cancer cells (PANC1), which express Hh/GLI components aberrantly. The expressions of GLI-related proteins PTCH and BCL2 were clearly inhibited by 1 or 2. We also examined the cytotoxicity of these active compounds against PANC1, human prostate cancer cells (DU145) and mouse embryo fibroblast cells (C3H10T1/2). The cytotoxicity against cancer cells (PANC1 and DU145) by 1 or 2 would be caused by inhibition of the expression of the anti-apoptosis protein BCL2. These pentacyclic triterpene inhibitors showed an important relationship between Hh/GLI signaling inhibition, the decrease of BCL2, and cytotoxicity against cancer cells.     

Prostate cancer cell lines under hypoxia exhibit greater stem-like properties

Hypoxia is an important environmental change in many cancers. Hypoxic niches can be occupied by cancer stem/progenitor-like cells that are associated with tumor progression and resistance to radiotherapy and chemotherapy. However, it has not yet been fully elucidated how hypoxia influences the stem-like properties of prostate cancer cells. In this report, we investigated the effects of hypoxia on human prostate cancer cell lines, PC-3 and DU145. In comparison to normoxia (20% O(2)), 7% O(2) induced higher expressions of HIF-1α and HIF-2α, which were associated with upregulation of Oct3/4 and Nanog; 1% O(2) induced even greater levels of these factors. The upregulated NANOG mRNA expression in hypoxia was confirmed to be predominantly retrogene NANOGP8. Similar growth rates were observed for cells cultivated under hypoxic and normoxic conditions for 48 hours; however, the colony formation assay revealed that 48 hours of hypoxic pretreatment resulted in the formation of more colonies. Treatment with 1% O(2) also extended the G(0)/G(1) stage, resulting in more side population cells, and induced CD44 and ABCG2 expressions. Hypoxia also increased the number of cells positive for ABCG2 expression, which were predominantly found to be CD44(bright) cells. Correspondingly, the sorted CD44(bright) cells expressed higher levels of ABCG2, Oct3/4, and Nanog than CD44(dim) cells, and hypoxic pretreatment significantly increased the expressions of these factors. CD44(bright) cells under normoxia formed significantly more colonies and spheres compared with the CD44(dim) cells, and hypoxic pretreatment even increased this effect. Our data indicate that prostate cancer cells under hypoxia possess greater stem-like properties.     

Generation of an ABCG2 transgenic line - A tool to study ABCG2 expression in mice

The ATP-binding cassette (ABC) transporter 2 (ABCG2) is expressed by stem cells in many organs and in stem cells of solid tumors. These cells are isolated based on the side population (SP) phenotype, a Hoechst 3342 dye efflux property believed to be conferred by ABCG2. Because of the limitations of this approach we generated transgenic mice that express Nuclear GFP (GFPn) coupled to the Puromycin-resistance gene, under the control of ABCG2 promoter/enhancer sequences. We show that ABCG2 is expressed in neural progenitors of the developing forebrain and spinal cord and in embryonic and adult endothelial cells of the brain. Using the neurosphere assay, we isolated tripotent ABCG2-expressing neural stem cells from embryonic mouse brain. This transgenic line is a powerful tool for studying the expression of ABCG2 in many tissues and for performing functional studies in different experimental settings.     

Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996

The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substrate Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer.