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Sterol regulatory element-binding protein 1 (SREBP1) is dysregulated in a variety of types of human cancer. However, the functional roles of SREBP1 in esophageal squamous cell carcinoma (ESCC) remain poorly understood. The present study investigated the function of SREBP1 in cell proliferation and motility. Microarray datasets in Oncomine, reverse transcription-quantitative PCR and western blot analysis revealed that SREBP1 was overexpressed in ESCC tumors when compared with normal tissues. In addition, SREBP1 overexpression was significantly associated with tumor differentiation, lymphatic metastasis and Ki67 expression. Results suggested that silencing SREBP1 inhibited the proliferation, migration and invasion of ESCC cells, whereas overexpression of SREBP1 had opposite effects on proliferation and metastasis. In addition, loss of SREBP1 significantly increased E-cadherin and decreased N-cadherin, Vimentin, Snail, matrix metalloproteinase 9 and vascular endothelial growth factor C expression levels, which were restored via SREBP1-overexpression. Mechanistically, loss of SREBP1 suppressed T-cell factor 1/lymphoid enhancer factor 1 (TCF1/LEF1) activity and downregulated TCF1/LEF1 target proteins, including CD44 and cyclin D1. Moreover, knockdown of SREBP1 downregulated the expression levels of stearoyl-CoA desaturase 1 (SCD1), phosphorylated glycogen synthase kinase-3β and nuclear β-catenin. Furthermore, the inhibitors of SREBP1 and/or SCD1 and small interfering RNA-SCD1 efficiently inhibited the activation of the Wnt/β-catenin pathway driven by constitutively active SREBP1. Finally, in vivo results indicated that SREBP1-knockdown suppressed the proliferation and metastasis of ESCC. Taken together, these findings demonstrated that SREBP1 exerts oncogenic effects in ESCC by promoting proliferation and inducing epithelial-mesenchymal transition via the SCD1-induced activation of the Wnt/β-catenin signaling pathway.

Manganese superoxide dismutase (MnSOD) promotes invasive and migratory activities by upregulating Forkhead box protein M1 (FoxM1) expression. The present study investigated whether modulation of MnSOD and FoxM1 expression was responsible for the antitumor effects of genistein on cancer stem-like cells (CSLCs) derived from non-small cell lung cancer cells (NSCLCs). Spheroids prepared from H460 or A549 cells were defined as lung cancer stem-like cells (LCSLCs) and were treated with genistein. The Cell Counting Kit-8 assay was performed to assess human lung fibroblast IMR-90 cell proliferation, as well as NSCLC H460 and A549 cell proliferation following treatment with genistein. MnSOD, FoxM1, cluster of differentiation (CD)133, CD44, BMI1 proto-oncogene, polycomb ring finger (Bmi1) and Nanog homeobox (Nanog) protein expression levels were examined via western blotting. The sphere formation assay was conducted to evaluate LCSLC self-renewal potential, and LSCLC migratory and invasive activities were analyzed using the wound healing and Transwell invasion assays, respectively. Knockdown and overexpression of MnSOD and FOXM1 via short hairpin-RNA or cDNA transfection were performed. The results indicated that genistein (80 and 100 µM) suppressed H460 and A549 cell viability compared with IMR-90 cells. Sub-cytotoxic concentrations of genistein (20 and 40 µM) inhibited sphere formation activity and decreased the protein expression levels of CD133, CD44, Bmi1 and Nanog in LCSLCs compared with the control group. Genistein also suppressed the migratory and invasive activities of LCSLCs compared with the control group. MnSOD and FoxM1 overexpression antagonized the effects of genistein (40 µM), whereas MnSOD and FoxM1 knockdown enhanced the inhibitory effects of genistein (20 µM) on CSLC characteristics of LCSLCs. Overall, the results suggested that genistein suppressed lung cancer cell CSLC characteristics by modulating MnSOD and FoxM1 expression levels.

Pancreatic cancer is a malignant neoplasm with high invasiveness and poor prognosis. In a previous study, a highly invasive pancreatic cancer cell line was established and found to feature enhanced interleukin-32 (IL-32) expression. However, whether IL-32 promotes the invasiveness by enhancing or suppressing the expression of IL-32 through regulating downstream molecules was unclear. To investigate the effect of IL-32, cells were established with high levels of expression or downregulated IL-32; their invasive ability was measured using a real-time measurement system and the expression of some candidate downstream molecules involved in invasion was evaluated in the two cell types. The morphological changes in both cell types and the localization of IL-32 expression in pancreatic cancer tissues were studied using immunohistochemistry. Among the several splice variants of IL-32, cells transfected with the ε isoform had increased invasiveness, whereas the IL-32-suppressed cells had reduced invasiveness. Several downstream molecules, whose expression was changed in the two cell types, were monitored. Notably, changes of E-cadherin, CLDN1, CD44, CTGF and Wnt were documented. The morphologies of the two cell types differed from the original cell line. Immunohistochemically, the expression of IL-32 was observed only in tumor cells and not in normal pancreatic cells. In conclusion, IL-32 was found to promote the invasiveness of pancreatic cancer cells by regulating downstream molecules.

Serine/arginine-rich splicing factor 3 (SRSF3; also known as SRp20), an important member of the family of SRSFs, is abnormally expressed in tumors, resulting in aberrant splicing of hub genes, such as CD44, HER2, MDM4, Rac family small GTPase 1 and tumor protein p53. Under normal conditions, the splicing and expression of SRSF3 are strictly regulated. However, the splicing, expression and phosphorylation of SRSF3 are abnormal in tumors. SRSF3 plays important roles in the occurrence and development of tumors, including the promotion of tumorigenesis, cellular proliferation, the cell cycle and metastasis, as well as inhibition of cell senescence, apoptosis and autophagy. SRSF3-knockdown significantly inhibits the proliferation and metastatic characteristics of tumor cells. Therefore, SRSF3 may be suggested as a novel anti-tumor target. The other biological functions of SRSF3 and its regulatory mechanisms are also summarized in the current review.

The high rate of mortality associated with ovarian cancer (OC) is due in part to the development of resistance to chemotherapy, which allows the resistant tumour cells to invade and metastasise. Clarifying the mechanistic basis for drug resistance may reveal novel avenues for treatment. The present study investigated the mechanism of paclitaxel (PTX) resistance in human epithelial OC by evaluating the expression of stem cell-associated cell surface markers endoglin (CD105), CD44 antigen and vascular cell adhesion molecule 1 (CD106), in association with the malignant potential of the human OC OVCAR3 cell line and its PTX-resistant derivative OC3/TAX300. The expression of CD105, CD44 and CD106 was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and flow cytometry, and cell invasion was evaluated using a Transwell invasion assay. CD105, CD44 and CD106 levels were increased in OC3/TAX300 cells compared with the OVCAR3 cells, as determined by flow cytometry (P

The long non-coding RNA (lncRNA) imatinib-upregulated (IUR) has been recently reported as a tumor suppressor in leukemia. Preliminary microarray data revealed a downregulation of IUR in pancreatic adenocarcinoma (PAAD) and a positive correlation with microRNA-34a (miR-34a) expression. The present study aimed to investigate the role of IUR in PAAD. This study included samples from 58 patients with PAAD and the PAAD cell lines Capan-2 and HPAC. Reverse transcription quantitative PCR was performed to determine gene expression levels. Cell transfections were carried out to assess gene interactions between IUR, miR-34a and CD44. Transwell assays were performed to explore the effects of transfections on cell invasive and migratory abilities. The results demonstrated that IUR was downregulated in PAAD tissue compared with adjacent non-tumor tissue samples and that low expression levels of IUR correlated with poor survival in patients with PAAD. In PAAD tissue samples, the expression of IUR positively correlated with miR-34a expression but negatively correlated with CD44 expression, which is a target of miR-34a. In PAAD cells, overexpression of IUR resulted in miR-34a upregulation and CD44 downregulation. miR-34a overexpression did not affect the expression of IUR but downregulated CD44. In PAAD cells, overexpression of IUR and miR-34a led to decreased invasive and migratory abilities. However, CD44 overexpression played an opposite role and attenuated the effects of IUR and miR-34a overexpression. In conclusion, the results from this study demonstrated that IUR may upregulate miR-34a expression in order to inhibit PAAD cell migration and invasion by downregulating CD44.

Cancer stem cells (CSCs) and epithelial mesenchymal transition (EMT) markers are considered useful indicators associated with metastasis and prognosis of colorectal cancers (CRCs). However, only a few studies have focused on the expression of these useful markers in metastases. Metastasectomy is widely used in advanced CRCs, and thus the postoperative prognostic factors are worth investigating. The present study investigated the consistency and differences of target proteins between primary and metastatic lesions of colorectal cancer, and discussed the prognostic indicators following resection of colorectal liver metastases. Clinical data of 56 patients with liver metastases from colorectal cancer were collected and the expression levels of target proteins (Ki-67, CD133, CD44, Snail, E-cadherin and β-catenin) were detected in primary tumor and matched liver metastases via immunohistochemistry analysis. Paired comparison between both tissue types was performed. The prognostic values of the target proteins for resectable colorectal cancer liver metastases were assessed. No significant differences were observed between the primary tissues and metastatic tissues. The consistency rates of these protein expression levels ranged from 51.8-78.6%. The maximum diameter of the liver metastases was

Substantial evidence suggests that cancer stem cells (CSCs) are the main cause of the initiation, progression and recurrence of tumors. Benzidine has been identified as a risk factor for bladder cancer. The aim of the present study was to investigate the effects of benzidine on bladder CSCs (BCSCs) and the possible mechanism underlying its action. The bladder cancer cell lines UM-UC-3 and EJ were maintained in serum-free medium and cells forming three-dimensional spheres were characterized as BCSCs. The sphere-forming cells were exposed to different concentrations of benzidine and vismodegib, and western blotting was performed to evaluate the expression of markers associated with CSCs and the Sonic hedgehog (SHH) signaling pathway. Flow cytometry was used to detect the distribution of cells in different phases of the cell cycle, and immunofluorescence staining was used to detect the protein expression of CD44. The results revealed that the levels of BCSC markers, namely CD133, CD44, aldehyde dehydrogenase 1-A1, Nanog and octamer-binding transcription factor-4, in the cell spheres were markedly elevated compared with those in cells cultured in serum-supplemented medium. Furthermore, benzidine increased the expression of BCSC markers and promoted the sphere-forming ability of the cells. In addition, it was observed that benzidine activated the SHH pathway, while inhibition of the Shh pathway using vismodegib diminished the promoting effects of benzidine on BCSCs. The findings of the present study indicate that benzidine promoted the stemness of BCSCs via activation of the SHH pathway, which may support further exploration of the molecular basis of the association between benzidine exposure and bladder oncogenesis.

Gastric cancer is a common tumor of the digestive system, which can occur in any part of the stomach. Kallikrein 6 (KLK6) is a trypsin-like serine protease and has been found to be involved in extracellular matrix remodeling, tumor invasion and nervous system plasticity. Our previous study reported that KLK6 suppressed HGC-27 gastric cancer cell growth by inhibiting epithelial-mesenchymal transition; however, the mechanism of action underlying the effect of KLK6 still remains unclear. The aim of the present study was to investigate the effect and the underlying mechanism of KLK6 on stem cell-like properties and metabolism in gastric carcinoma cells. The HGC-27 cell line was transfected with KLK6 overexpression (OV-KLK6) and interference (short hairpin-KLK6) vectors, then the transfection efficiency was confirmed using western blot analysis and reverse transcription-quantitative PCR. The percentage of CD133(+) and CD44(+) cells was detected using flow cytometry, while the protein expression levels of the stem-associated genes, Nanog, Oct-4, SOX2 and Notch1, the metabolic markers, hexokinase (HK)1, HK2, GLUT1, and the proteins within the PI3K signaling pathway, phosphorylated (p)-PI3K, p-AKT and p-mTOR, were determined using western blot analysis. Biochemical kits were used to measure ATP production, lactic acid content and glucose uptake. A tumorigenicity assay was performed with nude mice to detect gastric tumor volume, and the protein expression level of Oct-4, Nanog, HK1, HK2 and GLUT1, and the mRNA expression level of KLK6 was also determined in gastric tumor tissues of mice. Compared with that in the control group, KLK6 protein and mRNA expression levels were significantly decreased in the four sh-RNA groups (P

Cancer stem cells (CSCs) exhibit self-replication, self-differentiation, drug resistance and immune evasion activities. In recent years CSCs have become increasingly important for the treatment of malignant tumors. CSCs express specific markers, including cluster of differentiation (CD)44, CD44 variant 9 (CD44v9), ATP-binding cassette sub-family G member 2 (ABCG2), CD24, B lymphoma Mo-MLV insertion region 1 homolog (BMI-1) and aldehyde dehydrogenase 1 (ALDH1). However, the prognostic value of their expression in patients with oral squamous cell carcinoma (OSCC) are not well known. The present study evaluated these markers in stage I and II patients with OSCC and examined the association between T classification, histological differentiation, classification of invasion mode, lymph node metastasis and disease-free survival rate. Tissue specimens were obtained from 70 patients with stage I or II OSCC following either surgery or biopsy. Immunohistochemistry was performed and positive staining was defiend as 10% positive cells. CD44 and CD44v9 expressions were strongly detected in all OSCC tissues compared with normal epithelial cells. A total of 22 (31.4%) cases expressed ABCG2 and there was a significant association between ABCG2 expression and invasion. A total of 41 cases (59.0%) expressed CD24 and there was a significant association between CD24 expression and invasion. A total of 33 cases (47.1%) expressed BMI-1 and there was a significant association between BMI-1 expression and the disease-free survival rate. A total of 18 cases (25.7%) expressed ALDH1. Although there was no association between ALDH1 expression and T classification, there were significant associations between ALDH1 expression and histological differentiation, invasion mode, metastasis and the disease-free survival rate. Multivariate analysis revealed that ALDH1 expression was the only prognostic factor for disease-free survival rate. The results of the present study suggest that the positivity of ALDH1 detected in patients with OSCC correlates with the number of cells undergoing epithelial mesenchymal transition and metastasis. These findings indicated that the expression of ALDH1 may be an effective prognostic marker indicating the survival of patients with stage I and II OSCC.

Gastric cancer is one of the most common types of cancer in the world. Patients with gastric cancer often respond poorly to conventional chemotherapies, therefore more comprehensive therapy is required. Melanoma differentiation-associated gene-7 (MDA-7), also termed interleukin-24, is a potent tumor suppressor gene. Numerous studies have demonstrated that MDA-7 suppresses the growth and induces the apoptosis of cancer cells. In the present study, the MDA-7 gene was transfected into human gastric cancer AGS cells using adenovirus. Transwell and wound healing assays were performed to evaluate AGS cell invasion and migration, respectively. Western blotting was used to detect the expression of epithelial (E)-cadherin, cluster of differentiation (CD)44 and matrix metalloproteinase (MMP)-2 and MMP-9 proteins. A recombinant virus package was successfully constructed, and it was verified using western blotting that exogenous MDA-7 was highly expressed in the AGS cells. MDA-7 overexpression inhibited invasion and migration, decreased CD44, MMP-2 and MMP-9 expression, and increased epithelial (E-)cadherin expression in the AGS cells. Results of the present study revealed that MDA-7 inhibits gastric cancer invasion and metastasis by inhibiting CD44, MMP-2 and MMP-9 expression and by promoting E-cadherin expression.

Epithelial-mesenchymal transition (EMT) is a notable mechanism underlying cancer cell metastasis. Transforming growth factor β1 (TGF-β1) has been used to induce EMT; however, there is a lack of information regarding the role of TGF-β1 in mesenchymal-epithelial transition (MET). In the present study, EMT was induced in A549 lung cancer cells using TGF-β1 (TGF-β1-treated group) and MET was induced sequentially from the TGF-β1-treated group by removing the TGF-β1 (MET/return group). Untreated A549 lung cancer cells were used as a control. Characteristic features, including cancer stem cell markers [cluster of differentiation (CD)24, CD44 and CD133], cell proliferation and migration and diverse intracellular mechanisms, were observed in all groups. Using western blot analysis, the TGF-β1-treated group demonstrated increased vimentin and reduced E-cadherin expression, whereas the MET/return group demonstrated the opposite trend. Among cancer stem cell markers, the population of CD24low cells was reduced in the TGF-β1-treated group. Furthermore, the G2/M phase cell cycle population, cisplatin-sensitivity, and cell proliferation and migration ability were increased in the TGF-β1-treated group. These features were unaltered in the MET/return group when compared to the TGF-β1-treated group. Immunoblotting revealed an increase in the levels of SMAD3, phosphorylated SMAD3, phosphorylated extracellular signal-regulated kinase and caspase-3, and a decrease in active caspase-3 levels in the TGF-β1-treated group. Increased caspase-3 and reduced active caspase-3 levels were observed in the MET/return group, similar to those in the TGF-β1-treated group; however, levels of other signalling proteins were unchanged compared with the control group. EMT induced by TGF-β1 was not preserved; however, stemness-associated properties (CD24 expression, caspase-3 expression, cell proliferation and cisplatin-resistance) were sustained following removal of TGF-β1.

Emerging evidence has suggested that pancreatic adenocarcinoma is sustained by pancreatic cancer stem cells. The present study aimed to investigate the expression patterns of the pancreatic cancer stem cell surface markers cluster of differentiation CD44 and CD24 in a pancreatic adenocarcinoma cell line, and to investigate the possible mechanisms for their radiation resistance. Flow cytometry was used to analyze the expression patterns of CD44 and CD24 in the pancreatic adenocarcinoma PANC-1 cell line. In addition, a multi-target click model was used to fit cell survival curves and determine the sensitizer enhancement ratio. The apoptosis and cycle distribution of the four cell subsets was determined using flow cytometry, and the level of reactive oxygen species (ROS) was determined using the 2',7'-dichlorofluorescin diacetate probe. The present results identified that the ratios of CD44+ and CD24+ in the sorted PANC-1 cell line were 92.0 and 4.7%, respectively. Prior to radiation, no statistically significant differences were observed among the four groups. Following treatment with 6 MV of X-rays, the rate of apoptosis was decreased in the CD44+CD24+ group compared with other subsets. The percentage of G0/G1 cells was highest in the CD44+CD24+ group compared with the three other groups, which exhibited increased radiosensitivity. In addition, the level of ROS in the CD44+CD24+ group was reduced compared with the other groups. In summary, the results of the present study indicated that CD44+CD24+ exhibited stem cell properties. The lower level of ROS and apoptosis in CD44+CD24+ cells may contribute to their resistance to radiation in pancreatic adenocarcinoma.

Tissues surrounding hepatocellular carcinomas (HCCs) lack glucose. Hepatocyte selection medium (HSM) is deficient in glucose and is supplemented with galactose. HCC cells were cultured in HSM to investigate the stem cell markers α-fetoprotein (AFP) and cluster of differentiation 44 (CD44). HCC cells (HLF and PLC/PRF/5 cells) were cultured in HSM. Viable cell numbers were determined on days 0 and 7 following culture in HSM. RNA was isolated and subjected to reverse transcription-quantitative PCR (RT-qPCR) to analyze the mRNA expression levels of AFP and CD44. Immunostaining was performed to analyze the protein levels of AFP and CD44. The number of viable cells was significantly decreased on day 7 following culture in HSM. The expression levels of AFP and CD44 increased on day 7 as assessed using RT-qPCR. Immunostaining confirmed the results of RT-qPCR analysis. The number of viable HCC cells was decreased in HSM, whereas the expression levels of AFP and CD44 increased. Therefore, HSM is potentially useful for the enrichment of HCC cells with cancer stem cell characteristics.

Salinomycin (SAL), a polyether ionophore antibiotic, has been demonstrated to selectively kill cancer stem cells (CSCs) in various types of human tumor. The aim of the present study was to investigate the effects of SAL on canine mammary CSCs. CSCs in canine mammary carcinoma cell lines (CMT7364 and CIPp) were identified using a sphere formation assay and flow cytometry. The chemoresistance, invasive potential and expression of stem cell-associated proteins of these spheres was then analyzed. This demonstrated that the spheres exhibited characteristics of CSCs, including a cluster of differentiation (CD)44+/CD24-/low phenotype, upregulation of Wnt/β-catenin signaling pathway-associated proteins and chemoresistance. The viability of the spheres was decreased in a concentration- and time-dependent manner following treatment with SAL, and the spheres did not exhibit increased resistance to SAL compared with their parental cells. In addition, exposure to SAL inhibited sphere-formation and invasive potential in canine mammary CSCs in a dose-dependent manner. Furthermore, SAL decreased the CD44+/CD24-/low population and downregulated the expression of Wnt/β-catenin signaling-associated proteins (β-catenin, Cyclin D1 and octamer-binding transcription factor 4) in the spheres. In conclusion, the present study demonstrated that SAL is an effective inhibitor of canine mammary CSCs in vitro, indicating that SAL is a promising chemotherapeutic agent for the treatment of canine mammary carcinoma.

Cancer stem cell (CSC) properties have been recently proposed to explain tumor carcinogenesis and multidrug resistance in several human cancers, including non-small cell lung cancer (NSCLC). The present study examined the protein expression of three CSC-associated markers, namely ATP binding cassette subfamily B member 1 (ABCB1), aldehyde dehydrogenase 1 family member A1 (ALDH1A1) and cluster of differentiation (CD) 44, by immunohistochemistry in 194 NSCLC patients who underwent complete resection of NSCLC tumors. The association between the expression of these proteins and patient prognosis was evaluated to clarify the prognostic significance of CSC-associated markers in NSCLC patients. Positive staining for ABCB1 demonstrated a trend toward worse survival compared with negative staining in stage I–III NSCLC. Negative staining for ALDH1 or CD44 exhibited a trend toward worse survival compared with positive staining in stage I–III NSCLC. It was observed that patients with stage I lung adenocarcinoma (ADC) showing positivity for ABCB1 expression had significantly poorer survival than those with negative ABCB1 staining (P=0.03). Furthermore, stage I ADC patients with wild-type epidermal growth factor receptor (EGFR) who exhibited positive staining for ABCB1 had significantly shorter disease-free survival (DFS) compared with patients with negative staining for ABCB1 (P

Aromatase inhibitors (AIs), such as letrozole, are considered as first-line treatment for estrogen receptor-positive breast cancer in postmenopausal women. Despite the successful use of letrozole, resistance to therapy, tumor relapse and metastasis remain principal causes of patient mortality. Although there is no therapy currently available for AI-resistant breast cancer, previous reports have demonstrated that AI resistance is associated with hormone independence, increased growth factor signaling, enhanced cellular motility and epithelial to mesenchymal transition (EMT). This suggests a convergence of EMT and cancer stem cells (CSCs) in endocrine resistance. The present study evaluated the contribution of mammospheres in letrozole-resistant breast cancer by characterizing mammospheres and their potential impact on cellular motility. Ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with either letrozole-resistant MCF-7 cells (LTLT-Ca) or letrozole-sensitive MCF-7 cells (AC-1). Subsequently, intratumoral CSC marker expression was assessed by immunohistochemistry. The results indicated that LTLT-Ca tumors were CD44(+)/CD24(+), while AC-1 tumors presented low CD44/CD24 expression. Since mammosphere formation depends on CSCs, both cell lines were cultured either adherently (2D) or as mammospheres (3D) to assess the CD44/CD24 protein expression profile. When 3D culturing both cell lines, higher expression levels of CD44 and CD24 were observed when compared with their adherent counterparts, with the most robust change observed in the LTLT-Ca cell line. To quantitate the breast cancer stem cell activity, mammosphere formation assays were performed, and the LTLT-Ca cells formed mammospheres at a 3.4-fold higher index compared with AC-1 cells. Additionally, targeted gene expression arrays were conducted to compare the LTLT-Ca 3D and 2D cells, revealing that LTLT-Ca 3D cells displayed decreased expression levels of genes involved in cell adhesion and tumor suppression (e. g., E-cadherin, caveolin 1 and β-catenin). To validate this finding, wound healing assays were performed, and LTLT-Ca mammospheres exhibited a 70% wound closure, whereas AC-1 mammospheres exhibited a 39% wound closure. Collectively, the present findings demonstrated a strong association between AI-resistant mammospheres and an increased propensity for migration, which may be indicative of a poor prognosis.

Our previous study found that highly metastatic breast cancer cells, such as MDA-MB-231 cells, release higher levels of ATP and exhibit greater P2Y2 receptor (P2Y2R) activity than lowly metastatic breast cancer cells, and that P2Y2R activation mediated by ATP plays a significant role in tumor progression and metastasis. In addition, we reported that radiotherapy-resistant (RT-R) breast cancer cells promote invasion and tumor growth through the activation of P2Y2R by ATP released from RT-R-breast cancer cells than breast cancer cells. Moreover, increased numbers of cancer stem cells (CSCs) were observed among the RT-R-breast cancer cell population. Therefore, in this study, we investigated the expression level of five CSC markers (CD24, CD44, Oct3/4, Notch-4 and ALDH1A1) as well as P2Y2R in the tumor tissues of patients with breast cancer and determined which CSC marker correlates with P2Y2R in breast cancer. According to the immunohistochemical analysis, CD44, Oct3/4 and Notch-4 but not ALDH1A1 were significantly expressed in the tumor tissues (n=180) compared with the normal epithelial tissues (n=20) of patients with breast cancer. It was demonstrated that P2Y2R expression was increased in tumor tissues of patients with breast cancer compared with normal epithelial tissue. Notably, it was identified that P2Y2R expression has a significant correlation with only the CSC marker Notch-4 in patients with breast cancer. The results of this study suggested for the first time to the best of our knowledge that Notch-4 has a notable correlation with P2Y2R, which has important roles in tumor progression and metastasis.

Increasing evidence supports the concept that cancer stem cells (CSCs) are responsible for cancer progression and metastasis, therapy resistance and relapse. In addition to conventional therapies for colon cancer, the development of immunotherapies targeting cancer stem cells appears to be a promising strategy to suppress tumor recurrence and metastasis. In the present study, dendritic cells (DCs) were pulsed with whole-tumor cell lysates or total RNA of CD44+ colon cancer stem cells (CCSCs) isolated from mouse colon adenocarcinoma CT-26 cell cultures and investigated for their antitumor immunity against CCSCs in vivo and in vitro. In a model of colon adenocarcinoma using BALB/c mice, a sequential reduction in tumor volume and weight was associated with an extended survival in tumor-bearing mice vaccinated with DCs pulsed with RNA or CCSC lysate. In addition, a lactate dehydrogenase assay indicated that cytotoxic T-cells derived from the treated mice exhibited strong cytotoxic activity. Additionally, an enzyme-linked immunosorbent assay revealed that the cytotoxic T-cells of the treated mice released higher levels of interferon-γ against CCSCs compared with those of the control group. In all experiments, the antitumor efficacy of the lysate-pulsed DC-treated and RNA-pulsed DC-treated groups were significantly higher compared with that of the DC-treated and control groups. The results of the present study indicated the potential use of DCs pulsed with cancer stem cell lysates as a potent therapeutic antigen to target CSCs in colon cancer. Additionally, the results provided a rationale for using lysate-pulsed DCs in vivo to eliminate residual tumor deposits in post-operative patients.

Epithelial cell adhesion molecule (EpCAM) is a marker for circulating tumor cells (CTCs) in various types of cancer, while cluster of differentiation 44 (CD44) is a marker for gastric cancer (GC) stem cells. To evaluate the clinical significance of CD44+ CTCs in patients with GC in the present study, the number of EpCAM+CD44+ and EpCAM+CD44− cells were detected in the peripheral blood of 26 GC patients and 12 healthy volunteers using flow cytometry. The number (mean ± standard deviation) of EpCAM+CD44+ cells in the GC patients and healthy volunteers was 69.9±52.0 and 0.91±2.10, respectively (P=0.0001), while that of EpCAM+CD44− cells was 59.1±88.0 and 9.83±9.91, respectively (P=0.0313). The sensitivity and specificity of EpCAM+CD44+ cell detection for the identification of GC patients were 92.3 and 100%, respectively. By contrast, the values of EpCAM+CD44− cell detection were 76.9 and 83.3%, respectively. The number of EpCAM+CD44+ cells in the GC patients was correlated with the disease stage (P=0.0423), the depth of the tumor (P=0.0314) and venous invasion (P=0.0184) in the resected tumor specimens, while the number of EpCAM+CD44− cells did not correlate with any clinicopathological factors. The number of EpCAM+CD44+ cells significantly decreased following surgical resection of the tumor or induction of systemic chemotherapy. Additionally, atypical cells with a high nuclear to cytoplasmic ratio were morphologically detected in the sorted EpCAM+CD44+ cells. These results suggested that CD44+ CTCs, but not CD44− CTCs, reflect the malignant status of the primary tumor in patients with GC, providing a candidate biomarker for diagnosis and treatment response.

The wound healing process after surgery alters the area surrounding the original tumor and around the scar, and the modified microenvironment is more favorable for tumor recurrence. Intraoperative radiotherapy (IORT) is one of the more novel strategies in breast cancer (BC) treatment. Irradiation during surgery has effects on the tumor microenvironment, abrogating the proliferative cascade induced by surgical wound healing. The aim of the present study was to determine the effect of surgical wound fluids from IOERT treatment (RT-WF) compared with wound fluids from conservative-breast surgery only (WF) on the cancer stem cell phenotype in a panel of BC cell lines. Post-operative wound fluids were derived from patients with BC who underwent a tumor resection (quadrantectomy) plus intraoperative electron radiotherapy using a single dose of ≤10 Gy on the tumor bed and surrounding tissues, or from those who underwent a tumor resection without IOERT. Cell lines were incubated with 10% wound fluids, and after 4 days, the cluster of differentiation (CD)44+/CD24−/low phenotype and aldehyde dehydrogenase 1 (ALDH1) activity were determined by flow cytometry. The two types of fluid each affected the CD44+/CD24−/low phenotype. The results varied markedly between each cell line, even for the same histological subtypes. RT-WF decreased the CD44+/CD24−/low populations in the basal-like BT-549 and MDA-MB-468 cell lines, whereas in the luminal type MCF7 cell line, the two fluids inhibited these populations. The HER-OE subtypes harbored a minimal CD44+/CD24−/low population, but the growth of SK-BR-3 was stimulated by the two post-operative fluids. WF exhibited a stronger effect on ALDH1 activity compared with RT-WF. The stimulatory effect was dependent on the histological subtype of the cell line and the strongest dependence was observed in luminal subtypes characterized by low dehydrogenase activity in the control group. The present results enable a better understanding of the mechanism of recurrence and metastases following BC surgery. With respect to histological phenotype, its effect on tumor progression, either local or systemic, strongly suggests the requirement for further research and clinical validation.

Cluster of differentiation (CD)44 has been implicated in cancer metastasis to bone. Clinical and experimental studies have suggested that the standard isoform of CD44 (CD44s) and the variant isoform of CD44 (CD44v) enhance metastasis. The present study examined the differential roles of CD44s and CD44v, particularly CD44v8-10, in the development of bone metastases. For this purpose, MDA-MB-231 human breast cancer cells and A549 human lung cancer cells were stably transduced with epithelial splicing regulatory protein 1 (ESRP1), which regulates the alternative splicing of several genes, including CD44. The introduction of ESRP1 induced a splicing switch from CD44s to CD44v, particularly to CD44v8-10, while the total amount of CD44 was rarely affected. However, ESRP1 did not significantly affect cell proliferation, migration, invasion or tumor sphere formation in vitro. Furthermore, ESRP1 did not cause significant differences in the development of bone metastases in a mouse model. As an alternative approach, cancer cells transduced with the CD44v8-10 gene were also established. The overexpression of CD44v8-10 in MCF-7 human breast cancer cells, which rarely express any isoform of CD44, promoted cell migration and sphere formation, whereas the overexpression of CD44v8-10 in MDA-MB-231 cells, which endogenously express high levels of CD44s, did not exert these effects. The results of the present study collectively suggest that the ability of CD44v8-10 to promote tumor aggressiveness and bone metastases is similar to that of CD44s. CD44v8-10 and CD44s may represent potential therapeutic targets for the treatment of bone metastases.

The aim of the present study was to analyze the stem cell marker, Nanog gene, for the diagnosis and prognosis of lung cancer cases, and to study its application in the diagnosis of lung cancer. In total, 100 patients diagnosed with lung cancer between April, 2013 and May, 2015 were included in the present study. The patients were randomly divided into group A (lung cancer) and group B (squamous cell lung carcinoma). RT-PCR was used to detect the cancer and adjacent tissues, and Nanog gene expression was detected in groups A and B in cells. The results showed that, analysis of Nanog gene expression in the two groups of patients varied to different degrees. There was no significant difference between the two groups with regard to age, gender, disease stage and lymph node metastasis. Nanog gene expression in patients with carcinoma were significantly higher than that in the adjacent tissues (p

Significant efforts have been made to gain a better understanding of the heterogeneity of triple-negative breast cancers from the histological to the molecular and genomic levels. In this study, we attempted to bring forward gene expression subtypes of triple-negative breast cancer (TBNC) to the clinic, by translating gene stratification to clinically accessible immunohistochemical (IHC) classification. Using IHC analysis, we categorized 154 TBNC cases into three main subclasses. Differences in the frequencies of basic characteristics and clinicopathological parameters between the subtypes were examined using Chi-square tests. We defined three main groups among the 154 triple-negative cases. The basal-like (BL) group expressed cytokeratin (CK) 5/6 and/or CK14 (83 cases), the AR+ group demonstrated positivity for androgen receptor (18 cases), and the final group exhibited a CD44+CD24-/low phenotype (39 cases). There were three overlapping cases between the BL subgroup and the CD44+CD24-/low phenotype subgroup, and 11 unclassified cases. In this new IHC classification, three subcategories exhibited a statistical difference with regard to age, tumor size, histological grade, tumor necrosis, Ki67 labeling index, relapse-free survival, breast cancer-specific survival and response to chemotherapy. According to our definition, the BL group and CD44+CD24-/low phenotype could be observed in tumors that were not triple-negative, and BL tumors that were triple-negative demonstrated almost undistinguishable clinicopathological characteristics compared with BL tumors that were not triple-negative. The same observation was made with CD44+CD24-/low tumors that were triple-negative vs. CD44+CD24-/low tumors that were not. The AR+ group demonstrated undistinguishable clinicopathological characteristics compared with the luminal subtype. We successfully distinguished three subtypes exhibiting diverse clinicopathological and prognostic characteristics with the minimum use of IHC markers.

Recent evidence indicates that tumor-initiating cells (TICs), also called cancer stem cells (CSCs), are responsible for tumor initiation and progression, therefore representing an important cell population that may be used as a target for the development of future anticancer therapies. In the present study, Cryptotanshinone (CT), a traditional Chinese herbal medicine, was demonstrated to regulate the behaviors of LNCaP prostate cells and prostate LNCaP TICs. The results demonstrate that treatment with CT alters cellular proliferation, cell cycle status, migration, viability, colony formation and notably, sphere formation and down-regulation of stemness genes (Nanog, OCT4, SOX2, β-catenin, CXCR4) in TICs. The present study demonstrates that CT targets the LNCaP CD44+CD24- population that is representative of prostate TICs and also affects total LNCaP cells as well via down-regulation of stemness genes. The strong effect with which CT has on prostate TICs suggests that CT may potentially function as a novel natural anticancer agent that specifically targets TICs.

Dipyridamole, a traditional anti-platelet drug, has been reported to inhibit the proliferation of cancer cells. The present study aimed to investigate the possibility of dipyridamole as an adjuvant of chemotherapy by enhancing the cytotoxicity of an anti-cancer drug. The cytotoxicity of colorectal cancer cells (HCT-8), CD133+/CD44+ stem-like subpopulation of HCT-8 cells and lymphoma cells (U937) to dipyridamole and/or doxorubicin was evaluated using MTT proliferation and colony forming assays. The expression levels of phosphorylated cAMP-regulatory element-binding protein (pCREB) and poly(ADP-ribose) polymerase-1 (PARP-1) in cells were analyzed via western blotting and immunofluores- cence. The present study reported controversial data regarding the anti-cancer effect of dipyridamole. Dipyridamole increased, rather than inhibited, the proliferation of HCT-8 and U937 cells in a dose-dependent manner. Furthermore, it was found that dipyridamole significantly increased the expression levels of pCREB and PARP-1. However, the combined usage of dipyridamole significantly enhanced the cytotoxicity of doxorubicin to HCT-8 cells at particular doses. Based on the current findings, dipyridamole likely induces the phosphoryla- tion of CREB to promote the proliferation of cancer cells, but may enhance the cytotoxicity of anti-cancer drugs at particular doses., Oncology Letters, 21(4), art.no.251; 2021

The aim of the present study was to investigate the expression profiles and prognostic values of CD133 and CD44 in a cohort of patients with pancreatic neuroendocrine tumours (PNETs). PNET data from patients who underwent radical resection at the Guangdong Provincial People's Hospital were retrospectively analysed. Immunohistochemistry was performed on PNET samples, and CD133 and CD44 expression was examined. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. A total of 71 cases were included in the study. The mean age of the patients was 45.2 years, and the mean tumour size was 3.3 cm. CD44 expression was positively associated with poor tumour differentiation (P=0.007), high Ki-67 index (P=0.001), added mitotic count (P=0.003), high histological grade (P=0.001) and advanced stage (P=0.025). Similarly, CD133 expression was positively associated with high Ki-67 index (P=0.014) and added mitotic count (P=0.012). However, CD133 expression was not associated with tumour differentiation (P=0.118), histological grade (P=0.126) and stage (P=0.203). Survival analysis revealed that both CD44 and CD133 were prognostic factors for overall survival (OS) and/or disease-free survival (DFS), and that increased co-expression of CD44 and CD133 indicated poor OS and DFS rates in patients with PNET. In patients with no expression or low expression of either CD44 or CD133, a DFS rate of 100% was observed, indicating a low recurrence risk. The present findings suggested that high CD44 and CD133 expression was associated with a poor prognosis in patients with PNET. CD44 and CD133 may be used as prognostic indicators of OS and/or DFS in patients with PNETs.

Prostate cancer is the fifth most common cause of cancer-associated mortality in males worldwide. The survival of prostate cancer-initiating cells (CICs) is an important factor behind the metastasis and recurrence of prostate cancer. The cluster of differentiation (CD) 44 antigen is considered an important marker for prostate CICs. Salinomycin is a potent therapeutic drug against CICs. The present study demonstrated that salinomycin exerts potent activity against CD44(+) prostate CICs. To further enhance this anticancer effect, salinomycin-encapsulated lipid-poly(lactic-co-glycolic acid) nanoparticles linked with CD44 antibodies (SM-LPN-CD44) were generated. The anticancer effect of the nanoparticles was investigated in a series of assays, including a cytotoxicity assay, flow cytometry and anticancer assay in prostate cancer-bearing mice in vivo. The results revealed that SM-LPN-CD44 could efficiently and specifically promote the delivery of salinomycin to CD44(+) prostate CICs, and there by achieve greater inhibition of the cells compared with that achieved by salinomycin and non-targeted nanoparticles. To the best of our knowledge, this is the first study to report improved therapeutic effects against prostate CICs achieved by the enhancement of targeted drug delivery via nanoparticles conjugated with CD44 antibodies. Therefore, SM-LPN-CD44 nanoparticle-based therapy represents a novel approach to eliminate prostate CICs and is a promising potential treatment strategy for prostate cancer.

Docetaxel treatment is a standard chemotherapy strategy for castration-resistant prostate cancer (CRPC), and patients with CRPC eventually develop resistance to treatment. However, little is understood regarding the underlying mechanism of resistance. The present study aimed to identify the underlying crucial genes and regulatory networks associated with docetaxel resistance in prostate cancer using bioinformatics analyses. For this purpose, one expression profile dataset (GSE33455), which included two docetaxel-sensitive and two docetaxel-resistant cell lines, was downloaded from the Gene Expression Omnibus database, and analyses of differential gene expression and function enrichment were performed. A protein-protein interaction (PPI) network was constructed, and the associated hub genes were investigated using the Search Tool for the Retrieval of Interacting Genes/Proteins and Cytoscape software. A total of 756 differentially expression genes (DEGs) were identified, including 509 downregulated and 247 upregulated genes. Enrichment analysis revealed that the DEGs were associated with the interferon-γ-mediated signaling pathway, protein binding, bicellular tight junctions and cancer pathways. Two modules were screened from the PPI network, and the corresponding genes were identified to be largely enriched in the interferon-γ-mediated signaling pathway and the negative regulators of the DExD/H-Box helicase 58/interferon induced with helicase C domain 1 signaling pathway, and enriched in cell-cell adhesion and the Rap1 signaling pathway. Among the ten hub genes, epidermal growth factor receptor, spleen tyrosine kinase (SYK), intracellular adhesion molecule 1 (ICAM1), interleukin (IL)6, CXC motif chemokine ligand 8 (CXCL8), cyclin dependent kinase 1 and CD44 molecule (CD44) were significantly differentially expressed in prostate cancer tissues compared with healthy tissues based on The Cancer Genome Atlas data. The Gene Expression Profiling Interactive Analysis database revealed that ICAM1 was positively associated with IL6 and CXCL8, and epidermal growth factor receptor was positively associated with CD44 and SYK. Additionally, ten hub genes, which were identified to be associated with the drug resistance of docetaxel in prostatic carcinoma in the present study, were predominantly associated with tumor progression and metastasis. Reverse transcription-quantitative PCR analysis performed on docetaxel-sensitive and docetaxel-resistant prostate cancer cell lines demonstrated that certain hub genes, including CDK1, 2'-5'-oligoadenylate synthetase 3, CXCL8 and CDH1, were highly expressed in the docetaxel-resistant cell lines, which confirmed the bioinformatics results. In conclusion, the present study identified a number of important genes that are associated with the molecular mechanism of docetaxel resistance by integrated bioinformatical analysis, and these genes and regulatory networks may assist with identifying potential gene therapy targets for CRPC. Further functional analyses are required to validate the current findings.

The present study focused on the roles of members of the Hedgehog (Hh) signaling pathway in the maintenance of malignant biological characteristics, such as tumorigenesis, similar to that of pancreatic tumor cells. Cluster of differentiation (CD)44+CD24+/CD44-CD24- cells were isolated from three different pancreatic cancer cell lines by flow cytometry. Among the three pancreatic cancer cell lines, the SW1990 cell line exhibited the highest percentage of CD44+CD24+ cells, which accounted for 39.9% of the total. The expression of members of the Hh signaling pathway in CD44+CD24+/CD44-CD24- cells was detected using reverse transcription-polymerase chain reaction and western blot analysis. The results demonstrated that members of the Hh signaling pathway were differentially expressed in CD44+CD24+ cells compared with CD44-CD24-, normal pancreatic duct cells and unsorted SW1990 cells. In addition, lentiviral expression vectors expressing Smoothened (Smo) small interfering RNA (siRNA) were constructed. Following transfection with the lentiviral expression vectors, Smo expression was markedly reduced in CD44+CD24+ cells. The present study represents a preliminary investigation into the biological characteristics of CD44+CD24+ pancreatic cancer cells.

Breast cancer stem-like cells (BCSLCs) with a CD44+/CD24-/low phenotype initiate the invasion and metastasis of breast cancer. The expression of New York oesophageal squamous cell carcinoma 1 (NY-ESO-1), one of the most immunogenic cancer-testicular antigens, is largely restricted to cancer and germ cells/placental trophoblasts, with little to no expression in normal adult somatic cells. Currently, few studies have reported the expression or function of NY-ESO-1 in BCSLCs. In the present study, immunohistochemistry indicated enhanced expression levels of NY-ESO-1/CD44 (P

Periostin (POSTN) secreted by intrahepatic cholangiocarcinoma stem cells (ICSCs) serves important roles in promoting tumor progression. The present study aimed to investigate POSTN-recruited tumor-associated macrophages (TAMs) in intrahepatic cholangiocarcinoma (ICC). A total of 50 cases were used to investigate the distribution of ICSCs and TAMs in ICC. HCCC-9810 cells were sorted by cluster of differentiation (CD)44, the expression of POSTN of CD44+ (cancer stem cells) and CD44− cells (non-cancer stem cells), and medium were evaluated by western blot analysis. HCCC-9810 cells and THP-1 macrophages were used to detect the effects of POSTN on recruiting TAMs in vitro. The present study revealed that CD44+ cells in ICC tissues and the HCCC-9810 cell line were associated with high POSTN secretion levels. Furthermore, POSTN was associated with TAM density in primary ICC tissues. Additionally, POSTN increased the migration of TAMs derived from THP-1 cells. These findings suggested that POSTN secreted by ICSCs may serve important functions in TAM recruitment, and it may be a potential curative strategy to target the tumor microenvironment in ICC.

Previous studies have demonstrated that long noncoding RNAs (lncRNAs) exhibit critical regulatory roles in cancer biology. However, few lncRNAs have been well characterized in bladder cancer. In the previous study, we demonstrated that gastric adenocarcinoma associated, positive CD44 regulator, long intergenic noncoding RNA (GAPLINC) was significantly upregulated in bladder cancer tissues compared with normal tissues in The Cancer Genome Atlas (TCGA) cohort (P=0.039) and a validated cohort of 80 patients with bladder cancer (P=0.021). Statistical analysis revealed that GAPLINC expression level was associated with tumor stage in the validated cohort (P=0.017). Kaplan-Meier analysis demonstrated that patients in the high GAPLINC expression group had a worse overall survival (P=0.0386), indicating that GAPLINC may be a sensitive prognostic biomarker for patients with bladder cancer. Furthermore, knockdown of GAPLINC inhibited cell proliferation and colony formation, promoted cells cycle arrest at G1 phase and suppressed cells migration and invasion. The findings of the present study suggest that GAPLINC exhibits an oncogenic role in bladder cancer and may be a potential prognostic biomarker and therapeutic target.

The resistance of breast cancer to radiotherapy remains a major obstacle to successful cancer management. Radiotherapy may result in DNA damage and activate breast cancer stem cells. DNA damage may lead to activation of the checkpoint kinase (CHK) signaling pathway, of which debromohymenialdisine (DBH) is a specific inhibitor. Radiotherapy also increases the expression of phosphorylated CHK1/2 (pCHK1/2) in the breast cancer cell line, MCF-7, in vitro in a dose-dependent manner. DBH is a relatively stable effective inhibitor that significantly reduces pCHK1/2 expression and MCF-7 proliferation. Low-dose radiotherapy combined with DBH resulted in a higher MCF-7 inhibition rate compared with high-dose radiation alone. This result indicates that the inhibition of the CHK1/2 signal pathway may significantly reduce DNA damage within radiated cells. Radiotherapy may also regulate the proportion of CD44+/CD24− MCF-7 cancer stem cells in a dose- and time-dependent manner. However, the stem cell proportion of MCF-7 cells was significantly reduced by treatment with DBH. The inhibition is relatively stable and time dependent. Significant reductions were observed after 3 days of culture (P

It has been proposed that the Notch signaling pathway may serve a pivotal role in cellular differentiation, proliferation and apoptosis. However, the function of Notch signaling in gastric cancer stem cells (GCSCs) is largely unknown. The present study aimed to delineate the role of the Notch1 pathway in GCSCs and during epithelial-mesenchymal transition (EMT). Flow cytometry was used to isolate CD44+ cells from the human gastric cancer cell line, MKN45. CD44+ cells displayed the characteristics of CSCs and exhibited higher Notch1 expression compared with CD44- cells. To investigate the role of the Notch1 pathway in GCSCs, CD44+ cells were treated with the γ-secretase inhibitor DAPT. DAPT treatment inhibited the expression of the Notch1 downstream target Hes1 and EMT markers, suppressed the properties of CSCs and impaired the invasion and proliferation capabilities of CD44+ cells. In addition, intraperitoneal treatment with DAPT effectively inhibited the growth of CD44+ cell xenograft tumors. The present study indicated that CD44+ GCSCs possess the characteristics of CSCs and that the Notch1 pathway serves a critical role in the maintenance of CSCs and EMT.