Protein Expression Levels Of CD44 Research Articles

Baicalin Antagonizes Prostate Cancer Stemness via Inhibiting Notch1/NF-κB Signaling Pathway.

To investigate the molecular mechanisms underlying the effect of baicalin on prostate cancer (PCa) progression both in vivo and in vitro. The in situ PCa stem cells (PCSCs)-injected xenograft tumor models were established in BALB/c nude mice. Tumor volume and weight were respectively checked after baicalin (100 mg/kg) treatment. Hematoxylin-eosin (HE) staining was used to observe the growth arrest and cell necrosis. mRNA expression levels of acetaldehyde dehydrogenase 1 (ALDH1), CD44, CD133 and Notch1 were determined by reverse transcription-polymerase chain reaction. Protein expression levels of ALDH1, CD44, CD133, Notch1, nuclear factor κB (NF-κB) P65 and NF-κB p-P65 were detected by Western blot. Expression and subcellular location of ALDH1, CD44, CD133, Notch1 and NF-κB p65 were detected by immunofluorescence analysis. In vitro, cell cycle distribution and cell apoptosis of PC3 PCSCs was assessed by flow cytometry after baicalin (125 µmol/L) treatment. The migration and invasion abilities of PCSCs were assessed using Transwell assays. Transmission electron microscopy scanning was utilized to observe the structure and autophagosome formation of baicalin-treated PCSCs. In addition, PCSCs were infected with lentiviruses expressing human Notch1. Compared with the control group, the tumor volume and weight were notably reduced in mice treated with 100 mg/kg baicalin (P<0.05 or P<0.01). Histopathological analysis showed that baicalin treatment significantly inhibited cell proliferation and promoted cell apoptosis. Furthermore, baicalin treatment reduced mRNA and protein expression levels of CD44, CD133, ALDH1, and Notch1 as well as the protein expression of NF-κB p-P65 in the xenograft tumor (P<0.01). In vitro, the cell proliferation of PCSCs was significantly attenuated after treatment with 125 µmol/L baicalin for 72 h (P<0.01). The cell migration and invasion rates were decreased following treatment with baicalin for 48 and 72 h (P<0.01). Baicalin notably induced cell apoptosis and seriously damaged the structure of PCSCs. The mRNA and protein expressions of CD133, CD44, ALDH1 and Notch1 in PCSCs were significantly downregulated following baicalin treatment (P<0.01). Importantly, the inhibitory effects of baicalin on PCa progression and stemness were reversed by Notch1 overexpression (P<0.05 or P<0.01). Mechanistically, baicalin exhibited a potential therapeutic effect on PCa via inhibiting the Notch1/NF-κB signaling pathway and its mediated cancer stemness.

CD44 regulates biological behavior and Ras signaling pathway in nasopharyngeal carcinoma stem cells

Objective: To study the biological behavior of nasopharyngeal carcinoma stem cells and to explore the activation of Ras signaling pathway regulated by CD44. Methods: CNE2-SC and 5-8F-SC were nasopharyngeal carcinoma stem cells and obtained by serum-free suspension culture. Cell counting kit-8 (CCK-8) assay, colony formation assay, Transwell migration assay, cell adhesion array were used to investigate the growth, proliferation, migration and adhesion of nasopharyngeal carcinoma stem cells. Western blot test was used to detect the expressions of Ras signaling pathway related proteins and siRNA-mediated interference was used to determine the activation of Ras signaling pathway regulated by CD44. Results: The growth rates of CNE2-SC and 5-8F-SC cells were significantly lower than those of nasopharyngeal carcinoma cells at 24, 48 and 72 hours after inoculation (P<0.05). After 14 days of implantation, the colony formation rates of CNE2-SC (44.5±1.9)% and 5-8F-SC (47.4±1.8)% were higher than those of CNE2 (34.9±1.5)% and 5-8F (37.2±1.7)%, respectively(P<0.01). The migration cell number of CNE2-SC was (87.6±7.8), 3.97 times higher than that of CNE2 (P<0.01). The migration cell number of 5-8F-SC was (67.2±5.7), 3.07 times higher than 5-8F (P<0.01). The adhesion rates of CNE2-SC and CNE2 cells were (42.1±7.6)% and (8.9±2.0)%, respectively at 3 hours after inoculation and were (82.4±5.0)% and (12.1±2.2)% at 6 hours after inoculation, respectively. The adhesion rate of CNE2-SC cells was higher than that of CNE2 cells (all P<0.01). The adhesion rates of 5-8F-SC and 5-8F cells were (53.6±6.1)% and (7.3±1.5)% at 3 hours after inoculation, and (90.7±3.6)% and (11.0±1.2)% at 6 hours after inoculation, respectively. The adhesion rate of 5-8F-SC cells was higher than that of 5-8F cells (P<0.01). The expression levels of CD44, Ras and N-cadherin were significantly higher, while phosphatase and tensin homolog deleted on chromosome 10 (PTEN), E-cadherin in nasopharyngeal carcinoma stem cells were lower than those of the nasopharyngeal carcinoma cells. Furthermore, the levels of phosphorylated mitogen extracellular kinase1/2 (p-MEK1/2) and phosphorylated extracellular signal-regulated protein kinase1/2 (p-ERK1/2)were significantly increased in nasopharyngeal carcinoma stem cells (P<0.01). Correlation analysis showed that the protein expression levels of CD44 was highly positively correlated with RAS in nasopharyngeal carcinoma stem cells(r=0.985, P=0.002; r=0.962, P=0.038). Deletion of CD44 in CNE2-SC decreased the expression levels of HER-2, Ras and p-ERK1/2, p-Akt and phosphorylated protein kinase C-δ(p-PKCδ) (P<0.01). Conclusions: Despite compare to the nasopharyngeal carcinoma cell, nasopharyngeal carcinoma stem cells grows at a relatively slow rate, the capacities of clone formation, migration, adhesion are promoted. This may be related to the CD44-regulated abnormal activation of Ras signaling pathway.

Study on the effect of curcumin on the chemoresistance of colon carcinoma cells and its mechanism

Objective To investigate the effect of curcumin (Cur) onresistance toirinotecan (CPT-11) inhuman colon carcinoma cells and explore the mechanism involved. Methods Drug-resistanthuman colon carcinoma cell line (LoVo/CPT-11) was established by exposure to gradually increased concentrationof CPT-11, cell counting kit-8 (CCK-8) assay was applied to detect the growth inhibition rate and figure up the resistance index. Western blotting was used to examine the change of cancer stem cell (CSC) markers CD44 and CD133. The effect of Cur on resistance to CPT-11 was detected after treatment with the mixture of Cur (5, 10, 20 μmol/L) and CPT-11. Additionally, drug-resistant cells were separatedinto four groups: the control group, the Cur group, the CPT-11 group and the mixture group. The protein expression levels of CSC markers and the proportion of CD133+ cellswere detectedinthe four groups. Results The resistance index of LoVo/CPT-11 cell was 6.21 and itsprotein expression levels of CD44 and CD133 weresignificantly higher than LoVo cell (1.63±0.42 vs. 0.59±0.07, 0.75±0.13 vs. 0.24±0.02; P=0.023, 0.019). The 50% inhibition dose (IC50)of CPT-11 in LoVo/CPT-11 cellstreated with Cur (5, 10, 20 μmol/L) and CPT-11 was (144.48±3.22), (100.72±2.08), (146.87±2.73) μmol/L respectively, there wassignificant decrease compared with the control group [(268.72±2.43) μmol/L, P=0.031, 0.020, 0.036]. The protein expression levels of CD44 and CD133 in the Cur group and the mixture group were significantly decreased compared with the CPT-11 group (2.11±0.45, 1.79±0.55 vs. 3.04±0.64; P=0.041, 0.034; 1.34±0.13, 0.52±0.04 vs. 1.60±0.15; P=0.044, 0.020), and the proportion of CD133+ cells wasalso significantly lower than the CPT-11 group[(2.91±0.29)%, (1.11±0.14)% vs. (3.53±0.37)%; P=0.028, 0.016]. Conclusion CSC was related to the acquired chemoresistance of human colon carcinoma cells, Cur could reduce chemoresistance in human colon carcinoma cells by inhibiting the characterization of CSC. Key words: Curcumin; Irinotecan; Colon carcinoma; Chemoresistance; Cancer stem cells

NIBP impacts on the expression of E-cadherin, CD44 and vimentin in colon cancer via the NF-κB pathway.

NIBP, a novel nuclear factor-κB (NF-κB)-inducing kinase (NIK) and IκB kinase β (IKKβ) binding protein, directly interacts with NIK and IKKβ, and acts as the 'bridge' of the NF‑κB classical and alternative signaling pathways. However, its influence on epithelial‑mesenchymal transition markers in colon cancer remains to be fully elucidated. The aim of the present study was to investigate the roles of NIBP impacting on the expression of E‑cadherin, CD44 and vimentin. In the present study, the associations between NIBP and E‑cadherin, CD44 and vimentin in clinical samples were analyzed by making pairwise comparisons between normal colon tissue, non‑metastatic colon cancer tissue and metastatic colon cancer tissue. In invitro experiments, after changing the expression of NIBP in cells, the protein expression levels of CD44, vimentin, E‑cadherin were analyzed by western blot analysis. The results revealed that the protein expression levels of NIBP, CD44 and vimentin were markedly increased, and E‑cadherin was markedly decreased, in metastaticcolon cancer tissue compared with normal colon tissue and non‑metastatic colon cancer tissue. Upregulation of NIBP expression decreased the levels of E‑cadherin, whereas the downregulation of NIBP increased E‑cadherin levels, while no significant differences were observed in the levels of CD44 and vimentin. In addition, cells that were treated with the NF‑κB inhibitor, pyrrolidine dithiocarbamate (PDTC), also tended to exhibit increased levels of CD44 and vimentin expression in the NIBP upregulated expression group (29‑NIBP group) compared with the mock group, whereas the expression levels of E‑cadherin, CD44 and vimentin were similar in the NIBP downregulated expression group (116‑NIBPmir group) and the HCT116 blank control group (116‑mock group) on treatment of the cells with tumor necrosis factor‑α. These findings indicated that NIBP, E‑cadherin, CD44 and vimentin are possibly associated with metastasis in colon cancer. When the NF‑κB pathway is not subjected to any interventions, NIBP may predominantly regulate the NF‑κB classical pathway, rather than the alternative pathway. When the classical pathway was completely inhibited, NIBP was able to activate the NF‑κB alternative pathway. NIBP is therefore necessary for the interaction between the NF‑κB classical and alternative pathways. In conclusion, NIBP impacts on the expression levels of E‑cadherin, CD44 and vimentin via the NF‑κB classical and alternative pathways. Therapeutic regimens for patients with colorectal cancer may comprise NIBP inhibitors in the future.

Comprehensive proteome quantification reveals NgBR as a new regulator for epithelial-mesenchymal transition of breast tumor cells.

Our previous publication showed that NgBR is highly expressed in human breast invasive ductal carcinoma. However, the roles of NgBR and NgBR-mediated signaling pathway in breast tumor cells are still unclear. Here, we not only demonstrated that the quantitative proteomics analysis is a powerful tool to investigate the global biological function of NgBR, but also revealed that NgBR is involved in the transition of breast epithelial cells to mesenchymal stem cells, which is one of the major mechanisms involved in breast cancer metastasis. These findings provide new insights for understanding the roles of NgBR in regulating breast epithelial cell transform during the pathogenesis of breast cancer.

Abstract 3078: CD44 mRNA stability is regulated by RNA binding protein HuR in ovarian cancer cells

Abstract CD44, a major hyaluronic acid (HA) receptor, is involved in cell migration, metastasis and tumor progression. While in normal ovarian epithelium CD44 is not expressed, ovarian carcinoma cells express multiple CD44 isoforms as the tumor progresses and eventually CD44 expression is abrogated in advanced ovarian cancer metastatic sites. We as well as others have observed that the levels of CD44 mRNA transcript not always correspond to the CD44 protein expression levels. This observation suggests that CD44 is post-transcriptionally regulated. To test this hypothesis we first analyzed the CD44 3’ untranslated region (3’ UTR) sequence and found numerous AU-rich elements (ARE) in this region. Some of these sequences are known HuR binding site consensus sequences. HuR is an RNA binding protein that is known to stabilize transcripts. HuR is expressed in the nucleus and is transported to the cytoplasm where it functions as an mRNA stabilizer. We performed mRNA half-life experiments that showed a two-fold reduction in the stability of CD44 transcript in siHuR transfected ovarian cancer cells. Consistent with this result, treatment of these cells with inhibitors of DNA methylation and histone deacetylation (i.e., 5-azacytidine and Trichostatin A, respectively) known to diminish the presence of HuR in the cytoplasm, also decreased the stability of CD44 mRNA. Western blot analysis showed the expected reduction in cytoplasmic HuR as well as down-regulation in CD44 protein expression. A direct interaction of HuR with the CD44 3’ UTR was detected by biotin pulldown assays. These results indicate that HuR posttranscriptionally stabilizes the expression of CD44 in the cytoplasm. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3078. doi:10.1158/1538-7445.AM2011-3078