Renyi Qin

CD44+CD133+ population exhibits cancer stem cell-like characteristics in human gallbladder carcinoma

Cancer stem cells (CSCs)/tumor-initiating cells have been defined as a subset of tumor cells responsible for initiating and sustaining tumor development. Emerging evidence strongly supports the existence of CSCs in various solid tumors, but they have not yet been identified in human gallbladder carcinomas (GBC). In this study, we identified CSCs in primary GBC and in the cell line GBC-SD using the cell surface markers CD44 and CD133. The percentages of CD44+CD133+ cells were 1.76-3.05% in primary tumors and 40.29% in GBC-SD cells. These cells showed stem cell properties, including self-renewal, differentiation potential, and high tumorigenicity. In vitro culture experiments revealed that CD44+CD133+ GBC cells possessed a higher spheroid-colony forming ability in serum-free media than other subpopulations. When injected into nonobese diabetic/severe combined immunodeficient mice, these cells formed new tumors and generated CD44+CD133+, CD44-, and CD133- progeny. CD44+CD133+ cells also showed a high degree of chemoresistance, possibly due to upregulation of the breast cancer resistance protein (ABCG2) and the transcription factor Gli1 in these highly tumorigenic cells. These results suggest that the CD44+CD133+ population exhibited CSC-like characteristics and may thus provide a novel approach to the diagnosis and treatment of GBC.

Isolation and characterization of tumorigenic extrahepatic cholangiocarcinoma cells with stem cell-like properties

Recent studies suggest that the ability to form and grow tumors specifically resides in a small cell population called cancer stem cells (CSCs). These studies were conducted mainly on various human cancers; however, isolation and characterization of stem cells from cholangiocarcinoma have not been attempted. The molecular markers CD24, CD44, CD34, and epithelial cell adhesion molecule (EpCAM) are widely used, individually or in combination, to characterize some types of CSCs. In this study, we used these markers to identify a subpopulation of cells in extrahepatic cholangiocarcinoma (ECC) with cancer stem/progenitor cell‐like properties. We found that CD24+CD44+EpCAMhigh cells (0.39–2.27%) were present in human ECC tissues. The expression of a CD24+CD44+EpCAMhigh subpopulation was consistent with primary cancers and could be duplicated during serial in vivo passaging in NOD/SCID mice. CD24+CD44+EpCAMhigh cells isolated from 3 cholangiocarcinoma xenografts showed high tumorigenic potential compared with CD24−CD44−EpCAMlow/− cells. These tumorigenic ECC cells exhibited the stem cell properties of self‐renewal and ability to produce heterogeneous progeny. We report the identification of a CSC population in ECC characterized by CD24, CD44 and EpCAM phenotypes. Our findings could provide new insight into the tumorigenesis of cholangiocarcinoma and offer a potential target for anti‐cancer therapy.

Systematic review and meta-analysis comparing three techniques for pancreatic remnant closure following distal pancreatectomy

Established closure techniques for the pancreatic remnant after distal pancreatectomy include stapler, suture and anastomotic closure. However, controversy remains regarding the ideal technique; therefore, the aim of this study was to compare closure techniques and risk of postoperative pancreatic fistula (POPF).

Synergistic antitumor activity of withaferin A combined with oxaliplatin triggers reactive oxygen species-mediated inactivation of the PI3K/AKT pathway in human pancreatic cancer cells

Application of oxaliplatin for the treatment of pancreatic cancer (PC) is restricted owing to its toxic side effects and drug resistance. We investigated how withaferin A (WA), a bioactive component isolated from the medicinal plant Withania somnifera, acts synergistically with oxaliplatin on human PC in vitro and in vivo. We found that WA enhanced oxaliplatin-induced growth suppression and apoptosis in PC cells dramatically through a mechanism involving mitochondrial dysfunction and inactivation of the PI3K/AKT pathway. Combination treatment resulted in significant accumulation of intracellular reactive oxygen species (ROS). Pretreatment of cells with the ROS scavenger N-acetylcysteine completely blocked the apoptosis induced by combination treatment, and recovered expression of AKT inactivation, which revealed the important role of ROS in apoptosis and AKT regulation. In vivo, combination therapy showed the strongest anti-tumor effects compared with single agents, without obvious additional toxicity. These results support the notion that combination treatment with oxaliplatin and WA could facilitate development of an effective strategy for PC treatment.

CD133 + gallbladder carcinoma cells exhibit self-renewal ability and tumorigenicity

AIM To identify cancer stem cells (CSCs) in human gallbladder carcinomas (GBCs). METHODS Primary GBC cells were cultured under serum-free conditions to produce floating spheres. The stem-cell properties of the sphere-forming cells, including self-renewal, differentiation potential, chemoresistance and tumorigenicity, were determined in vitro or in vivo. Cell surface expression of CD133 was investigated in primary tumors and in spheroid cells using flow cytometry. The sphere-colony-formation ability and tumorigenicity of CD133(+) cells were assayed. RESULTS In vitro culture experiments revealed that floating spheroids were generated from primary GBC cells, and these sphere-forming cells could generate new progeny spheroids in serum-free media. Spheroid cells were differentiated under serum-containing conditions with downregulation of the stem cell markers Oct-4, Nanog, and nestin (P < 0.05). The differentiated cells showed lower spheroid-colony-formation ability than the original spheroid cells (P < 0.05). Spheroid cells were more resistant to chemotherapeutic reagents than the congenetic adherent cells (P < 0.05). Flow cytometry showed enriched CD133(+) population in sphere-forming cells (P < 0.05). CD133(+) cells possessed high colony-formation ability than the CD133(-) population (P < 0.01). CD133(+) cells injected into nude mice revealed higher tumorigenicity than their antigen-negative counterparts (P < 0.05). CONCLUSION CD133 may be a cell surface marker for CSCs in GBC.

Balanced Tiam1-rac1 and RhoA drives proliferation and invasion of pancreatic cancer cells.

Tiam1 is a rac1-specific guanine nucleotide exchange factor, and Tiam1-rac1 is involved in a number of cellular processes. Rac1 and RhoA act as molecular switches that cycle between GTP- and GDP-bound states to balance the activities of rac1 and RhoA. The downregulation of rac1 activity leads to upregulation of RhoA activity, which promotes invasion and migration of pancreatic cancers cells. At present, however, the role of Tiam1-rac1 and RhoA in pancreatic cancers is not fully understood. We found that Tiam1 was upregulated in pancreatic cancers and was significantly expressed in tumors without lymph node involvement or distant metastasis compared with cancers where there was involvement. Although Tiam1-rac1 signaling promoted pancreatic cancer cell proliferation and tumor growth via the Wnt signaling pathway in vitro and in vivo, inhibiting Tiam1-rac1 signaling did not prolong the overall survival time in vivo. This provided evidence that there was a balance between rac1 and RhoA activities in pancreatic cancers. Furthermore, only the combined inhibition of Tiam1-rac1 and RhoA had a beneficial effect on the growth of pancreatic cancers in vivo. Taken together, these results suggest that the progression of pancreatic tumors is partially controlled by the balance between Tiam1-rac1 and RhoA. Mol Cancer Res; 11(3); 230–9. ©2012 AACR.

CD133 and ALDH may be the molecular markers of cholangiocarcinoma stem cells.

Dear Editor, We read the comments of Angela Gradilone et al. on our article ‘‘Isolation and characterization of tumorigenic extrahepatic cholangiocarcinoma cells with stem cell–like properties.’’ The authors concluded that CD133 and ALDH1 are two other molecular markers of putative cancer stem cells (CSCs) in extrahepatic cholangiocarcinoma (CC). This is an interesting point and very beneficial for further identification of CC stem cells. Actually, this is a big question about how to confirm putative molecular markers for CSCs. Usually, there are two main ways to identify the markers for new type CSCs. First, some normal stem/progenitor cells markers could be used to identify the CSCs, such as leukemia stem cells (CD34þCD38 ), brain CSCs (CD133) and breast CSCs (CD24 CD44þESAþ). Second, some molecular markers that have been confirmed on some CSCs can be used to identify new type CSCs. For example, CD133 first is used to identify brain CSCs, now is the marker for CSCs in liver, colon, pancreas, leukemia and endometrial tumors. CD24/CD44/ESA first are used to identify breast CSCs, individually or in combination, are markers for CSCs in pancreas, prostate, ovarian and colorectal cancers. CD133 is very important and extensively used in characterization CSCs. Just as Angela Gradilone et al. mentioned, hepatocellular carcinoma and CC share the same origin. One would expect that CC CSCs express CD133, which is found highly expressed on hepatic progenitor cells. However, whether CD133þ could be added to CD44, CD24 and ESA to further restrict the phenotypic definition of CC stem cells and isolate an even more tumorigenic subset remains to be determined. A recent work has been demonstrated that several different stem/progenitor cell–like subpopulations can exist in leukemia and some solid cancers. Wright et al. showed Brca1 breast tumors contain distinct CD44þ/CD24 and CD133þ cells with CSC characteristics. Hwang-Verslues et al. demonstrated multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers. Either CD133 is a further restrict the phenotype or it is a distinct stem/progenitor cell molecular maker, which is really meaningful to detect CD133 expression and identify the CSCslike properties in these subset cells of CC. In a series of recent studies, ALDH1 has been described to play a crucial role in identification of putative CSCs and/or potential cancer-initiating cells in solid cancers (e.g., breast, ovarian, colon and head and neck squamous cancer). However, the correlation between the CD44/CD24 phenotype and ALDH1 expression is controversial. Some literatures showed that ALDH1 could be added to CD44 and CD24 to further restrict the phenotypic definition of CSCs and isolate an even more tumorigenic subset. Ginestier et al. investigated the overlap between the ALDEFLUOR-positive population and CD44þ/CD24 /lin in breast cancer. They showed that these two phenotypes, ALDEFLUOR and CD44þ/CD24 /lin , identified a small overlapping cell fraction. The cell population bearing both CSC phenotypes had higher tumorigenic capacity. Tanei et al. showed that ALDH1þ seems to be a more significantly predictive marker than CD44þ/CD24 for the identification of breast CSCs in terms of resistance to chemotherapy. Chen et al. also verified this point in head and neck squamous cancer. However, some other reports showed the different data. Charafe-Jauffret et al. demonstrated there was no correlation between the CD44þ/CD24 phenotype and ALDH1 expression by immunohistochemistry using double staining on paraffin sections. Dylla et al. showed that tumorigenicity is strictly conferred by ESAþCD44þALDHþ cells in the UM-C4 colorectal tumor line. In contrast, tumors also can arise from ESAþCD44þALDH cells taken from the UM-C6, OMP-C5 and OMP-C8 tumor lines, an ALDHþ subset of TG ESAþCD44þ cells does exist in all xenogeneic colorectal tumor lines investigated to date. A subpopulation of CD44 cells with high ALDH activity also exists in all tumor lines, but these cells fail to generate tumors on transplantation. In conclusion, CD133 and ALDH1 are two very important molecular markers for putative CSCs. The further work to detect their expressions and the CSC-like properties of these subpopulations in CC would remarkably improve the CC stem cells research. Moreover, to clarify the correlation among CD133, ALDH and CD24/CD44/EpCAM and to identify cells bearing multiple CSC phenotypes may improve the sensitivity and specificity of the test, further elucidating the role of single stemness markers in CC just as Angela Gradilone et al. mentioned. Yours sincerely, Min Wang Juan Xiao

Circulating myeloid-derived suppressor cells in patients with pancreatic cancer

BACKGROUND Myeloid-derived suppressor cells (MDSCs) are heterogeneous cell types that suppress T-cell responses in cancer patients and animal models, some MDSC subpopulations are increased in patients with pancreatic cancer. The present study was to investigate a specific subset of MDSCs in patients with pancreatic cancer and the mechanism of MDSCs increase in these patients. METHODS Myeloid cells from whole blood were collected from 37 patients with pancreatic cancer, 17 with cholangiocarcinoma, and 47 healthy controls. Four pancreatic cancer cell lines were co-cultured with normal peripheral blood mononuclear cells (PBMCs) to test the effect of tumor cells on the conversion of PBMCs to MDSCs. Levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and arginase activity in the plasma of cancer patients were analyzed by enzyme-linked immunosorbent assay. RESULTS CD14+/CD11b+/HLA-DR- MDSCs were increased in patients with pancreatic or bile duct cancer compared with those in healthy controls, and this increase was correlated with clinical cancer stage. Pancreatic cancer cell lines induced PBMCs to MDSCs in a dose-dependent manner. GM-CSF and arginase activity levels were significantly increased in the serum of patients with pancreatic cancer. CONCLUSIONS MDSCs were tumor related: tumor cells induced PBMCs to MDSCs in a dose-dependent manner and circulating CD14+/CD11b+/HLA-DR- MDSCs in pancreatic cancer patients were positively correlated with tumor burden. MDSCs might be useful markers for pancreatic cancer detection and progression.

Simultaneous inhibition of the ubiquitin-proteasome system and autophagy enhances apoptosis induced by ER stress aggravators in human pancreatic cancer cells

ABSTRACT In contrast to normal tissue, cancer cells display profound alterations in protein synthesis and degradation. Therefore, proteins that regulate endoplasmic reticulum (ER) homeostasis are being increasingly recognized as potential therapeutic targets. The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. However, interactions between autophagy, the proteasome, and ER stress pathways in cancer remain largely undefined. This study demonstrated that withaferin-A (WA), the biologically active withanolide extracted from Withania somnifera, significantly increased autophagosomes, but blocked the degradation of autophagic cargo by inhibiting SNARE-mediated fusion of autophagosomes and lysosomes in human pancreatic cancer (PC) cells. WA specifically induced proteasome inhibition and promoted the accumulation of ubiquitinated proteins, which resulted in ER stress-mediated apoptosis. Meanwhile, the impaired autophagy at early stage induced by WA was likely activated in response to ER stress. Importantly, combining WA with a series of ER stress aggravators enhanced apoptosis synergistically. WA was well tolerated in mice, and displayed synergism with ER stress aggravators to inhibit tumor growth in PC xenografts. Taken together, these findings indicate that simultaneous suppression of 2 key intracellular protein degradation systems rendered PC cells vulnerable to ER stress, which may represent an avenue for new therapeutic combinations for this disease.

Proteome analysis of human pancreatic ductal adenocarcinoma tissue using two-dimensional gel electrophoresis and tandem mass spectrometry for identification of disease-related proteins.

A comparative proteomic approach has been used to identify and analyze proteins related to pancreatic cancer. Proteomes of eight pairs of clinical pancreatic ductal adenocarcinoma (PDAC) tissue samples and samples of normal adjacent tissue were obtained by two-dimensional gel electrophoresis (2DE). Comprehensive analysis of proteins was focused on total protein spots for which there were statistical differences between the two groups. Proteins were identified by peptide mass fingerprinting with tandem mass spectrometry (MS–MS). Western blotting and immunohistochemistry (IHC) were also performed to verify the expression of some candidate proteins. Thirty protein spots were identified, including proteases, antioxidant proteins, signal-transduction proteins, calcium-binding proteins, structural proteins, chaperones, and others. Western blotting and IHC confirmed up-regulated expression of two candidate proteins, nucleotide diphosphatase kinase (NDPK) and annexin II, in tumorous tissues. These results suggest that combination of 2DE with MS is an effective strategy for discovery of differently expressed proteins in PDAC which may be molecular markers for diagnosis or therapeutic targets.