CD44v6 Gene Research Articles

Anticancer Effects of Valproic Acid via Regulation of Epigenetic Mechanisms in Non-small-cell Lung Cancer A549 Cell Line.

Epigenetic mechanisms are the most important factors contributing to both the development and metastasis of cancer cells. We aimed to scrutinize the role of epigenetic alternations of genes involved in cancer metastasis, including CD44v6 (metastasis indicator) and Nm23-H1 (a novel tumor suppressor), in the A549 lung cancer cell line. The A549 cells were cultured in the DMEM medium. Valproic acid (VPA) was used as a histone deacetylase inhibitor. Caspase-3 activity was assessed by adding DEVD-pNA substrate to the cell lysate. Gene expression was determined by real-time PCR. Finally, protein expression was assessed by western blot. The results showed that VA significantly decreased the expression of the CD44v6 gene and its protein level. This was further accompanied by lower expressions of MMP-2 and MMP-9 genes. On the other hand, the expression of Nm23-H1 and its protein were significantly increased in the cells accompanied by higher activity of caspase-3 (P ˂ 0.05). Our results showed that epigenetic regulation of CD44v6, Nm23-H1, MMP-2, and MMP-9 might be involved in the pathogenesis and metastasis of lung cancer. Therefore, the use of histone deacetylase inhibitors can be effective in the suppression of metastases and the treatment of these tumors.

Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice.

CD44v6, a splice variant of the cell surface glycoprotein CD44, acts as a co-receptor for c-Met and is upregulated in tumors with high metastatic potential.Methods: We screened a phage-displayed peptide library for peptides that selectively bind to CD44v6-overexpressing cells and exploited them to block CD44v6 and deliver a pro-apoptotic peptide to tumors for cancer therapy.Results: CNLNTIDTC (NLN) and CNEWQLKSC (NEW) peptides bound preferentially to CD44v6-high cells than to CD44v6-low cells. The binding affinities of NLN and NEW to CD44v6 protein were 253 ± 79 and 85 ± 18 nM, respectively. Peptide binding to CD44v6-high cells was inhibited by the knockdown of CD44v6 gene expression and competition with an anti-CD44v6 antibody. A pull-down assay with biotin-labeled peptides enriched CD44v6 from cell lysates. NLN and NEW induced CD44v6 internalization and inhibited hepatocyte growth factor-induced c-Met internalization, c-Met and Erk phosphorylation, and cell migration and invasion. In mice harboring tumors, intravenously administered NLN and NEW homed to the tumors and inhibited metastasis to the lungs. When combined with crizotinib, a c-Met inhibitor, treatment with each peptide inhibited metastatic growth more efficiently than each peptide or crizotinib alone. In addition, KLAKLAKKLAKLAK pro-apoptotic peptide guided by NLN (NLN-KLA) or NEW (NEW-KLA) killed tumor cells and inhibited tumor growth and metastasis. No significant systemic side effects were observed after treatments.Conclusions: These results suggest that NLN and NEW are promising metastasis-inhibiting peptide therapeutics and targeting moieties for CD44v6-expressing metastases.

Alterations of Hyaluronan Metabolism in Acute Coronary Syndrome: Implications for Plaque Erosion

BackgroundSuperficial erosion currently causes at least one-third of acute coronary syndromes (ACS), and its incidence is increasing. Yet, the underlying mechanisms in humans are still largely unknown. ObjectivesThe authors sought to assess the role of hyaluronan (HA) metabolism in ACS. MethodsPeripheral blood mononuclear cells were collected from ACS (n = 66), stable angina (SA) (n = 55), and control (CTRL) patients (n = 45). The authors evaluated: 1) gene expression of hyaluronidase 2 (HYAL2) (enzyme degrading high-molecular-weight HA to its proinflammatory 20-kDa isoform) and of CD44v1, CD44v4, and CD44v6 splicing variants of HA receptor; and 2) HYAL2 and CD44 protein expression. Moreover, they compared HYAL2 and CD44 gene expression in ACS patients with plaque erosion (intact fibrous cap and thrombus) and in ACS patients with plaque rupture, identified by optical coherence tomography analysis. ResultsGene expression of HYAL2, CD44v1, and CD44v6 were significantly higher in ACS as compared with SA (p = 0.003, p < 0.001, and p = 0.033, respectively) and CTRL subjects (p < 0.001, p < 0.001, and p = 0.009, respectively). HYAL2 protein expression was significantly higher in ACS than in SA (p = 0.017) and CTRL (p = 0.032), whereas no differences were found in CD44 protein expression. HYAL2 and CD44v6 gene expression was significantly higher in patients with plaque erosion than in those with plaque rupture (p = 0.015 and p = 0.029, respectively). ConclusionsHYAL2 and CD44v6 splicing variants seem to play an important role in ACS, in particular when associated with plaque erosion. After further validation, HYAL2 might represent a potentially useful biomarker for the noninvasive identification of this mechanism of coronary instability.

Detection and Clinical Significance of CD44v6 and Integrin-β1 in Pancreatic Cancer Patients using a Triplex Real-Time RT-PCR Assay

The cell adhesion molecules CD44v6 and integrin-β1 are associated with the progression and metastasis of cancer. A novel triplex real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to quantify CD44v6 and integrin-β1 gene expression in peripheral blood mononuclear cells from 30 pancreatic cancer (PC) patients and 12 healthy individuals. The standard curve of the triplex qRT-PCR was constructed by optimizing the reaction condition and the amplification efficiency was 102.5, 101.1, and 100.6 % for CD44v6, integrin-β1 and endogenous gene (β-actin) amplification. Nonspecific bands were not observed from the triplex qRT-PCR amplification and the detection limit of this assay was 100 copies. Expression levels of CD44v6 and integrin-β1 gene were significantly lower in healthy individuals than PC patients (P<0.05). CD44v6 and integrin-β1 gene expression were not associated with the sex, age, and tumor position in PC (P>0.05). CD44v6 gene expression was significantly associated with clinical stage, liver metastasis, and tumor size (P<0.05). Integrin-β1 gene expression was significantly associated with clinical stage and liver metastasis (P<0.05). This triplex qRT-PCR assay may provide a useful tool for diagnosis, prognosis, and therapeutic evaluation in PC.

The establishment of the duplex real‐time RT‐PCR assay for the detection of CD44v6 in pancreatic cancer patients and clinical application

Cell adhesion molecule CD44v6 has been found to be associated with the progression and metastasis of numerous cancers. In this study, a novel duplex real-time quantitative reverse-transcription PCR (qRT-PCR) assay was developed to quantitatively detect the CD44v6 gene expression in pancreatic cancer patients. The primers and probes of CD44v6 and β-actin genes were designed and standard curve of the duplex qRT-PCR was constructed by optimizing the reaction conditions. The specificity and reproducibility of this assay were satisfactory and the detection limit was 100 copies, which was 10 times more sensitive than the conventional RT-PCR assay. This assay was also used to detect the expression levels of CD44v6 messenger RNA in peripheral blood mononuclear cell in 37 pancreatic cancer patients and 12 healthy people. The results showed that 37 clinical samples were tested positive by the duplex qRT-PCR compared with only 30 by the conventional RT-PCR. The levels of CD44v6 expression showed significant correlation with sex, tumor size, tumor differentiation, clinical stage, lymph node, and liver metastasis (P < 0.05). Compared with the control group, CD44v6 levels in patients prior and 10 days post cryosurgery were significantly increased (P < 0.05) but had no significant change in those 1 month post cryosurgery (P > 0.05). The duplex qRT-PCR assay may provide a useful tool for the evaluation of prognosis and curative effect of pancreatic cancer.

Relationship between CD44 expression or glycosylation and hepatocellular carcinoma metastasis

To explore the relationship of CD44 expression or glycosylation and hepatocellular carcinoma (HCC) metastasis. IHC, Quantum dots detection, RT-PCR, Western blot, Cellular immune fluorescence and MS-PCR were used to identify CD44 expression in HCC samples and a series of human HCC cell lines with different metastatic potentials. Lectin array was used to reveal the relationship of CD44v6 glycosylation and human HCC metastasis. Immunohistochemistry analysis showed that CD44v6 was mainly distributed on the cell membrane, while CD44S immunoreactivity was prominently in the cytoplasm, CD44v3 and CD44v4/5 were in cytoplasm on membrane. Among CD44S and those CD44 variants, only the expression of CD44v6 was higher in metastasis HCC samples as compared to that in non-metastasis group (x²=8.828, P less than 0.05). This result was also re-confirmed by the result of Quantum dots (t = 2.392, P < 0.05) and serum detection (t = 2.56, P < 0.05). We found completely methylation of CD44v6 gene in Hep3B and incomplete methylation in MHCC97H and MHCC97L cell lines with metastatic potentials. The lectin affinity assay indicated that lectin MAL, SNA and WGA showed more affinity to MHCC97H and MHCC97Lcell lines than that of the non-metastatic Hep3B cell lines. CD44v6 over-expression presents a positive correlation with HCC metastatic potential, which may be associated with DNA methylation level in promoter sequence. The increasing sialic acid modified glycan of CD44v6 might be related to HCC metastatic ability.

Expression ofCD44v6gene in normal human peripheral blood

To investigate if CD44v6 could be used as a molecular marker of cancer progression and metastasis through the detection of CD44v6 gene expression in normal human peripheral blood. RNA was extracted from the peripheral blood mononuclear cells of 50 healthy donors, the expression of CD44v6 was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). CD44v6 mRNA was detected in 58% of healthy volunteers under the proper controls. Our results suggest that the measurement of CD44v6 expression in peripheral blood by RT-PCR is not suitable for detection of circulating tumor cells.

Expression and clinical significance of nm23-H1 and CD44v6 in non-small cell lung cancer

To study the role of nm23-H1 and CD44v6 gene in non-small cell lung cancer (NSCLC). Expressions of nm23-H1 and CD44v6 genes were analyzed in 147 cases of NSCLC by SP immunohistochemistry method. The overall positive rate of nm23-H1 staining was 62.6% (92/147). Significant differences in the positive rate of nm23-H1 were found between well and poor differentiated groups [68.6% (83/121) [WTBX]vs 34.6% (9/26)], and between adenocarcinoma and squamous cell carcinoma , and between adenocarcinoma and squamous cell carcinoma [78.8% (52/66) [WTBX]vs 50.8% (32/63) ] ( (P < 0.05), but not between stage I+II and stage III+IV [ 65.3% (62/95) [WTBX]vs 57.7% (30/52) ], and between N0 and N1-3 , and between N0 and N1 3 [64.7% (55/85) [WTBX]vs 59.7% (37/62) ]( ( P > 0.05). The positive rate of nm23-H1 of patients who survived for more than 3 years was 71.4% (55/77), which was significantly higher than 52.9% (37/70) of patients who survived for less than 3 years (Chi-square=5.4, P < 0.05). For CD44v6, the overall positive rate was 63.9% (94/147). Significant differences in the positive rate of CD44v6 were found between well and poor differentiated groups [68.6% (83/121) [WTBX]vs 42.3% (11/26)], and between adenocarcinoma and squamous cell carcinoma , and between adenocarcinoma and squamous cell carcinoma [53.0% (35/66) [WTBX]vs 77.8% (49/63) ] ( (P < 0.05), but not between stage I+II and stage III+IV [ 66.3% (63/95) [WTBX]vs 59.6% (31/52) ], and between N0 and N1-3 , and between N0 and N1 3 [63.5% (54/85) [WTBX]vs 64.5% (40/62) ]((P > 0.05). The positive rate of CD44v6 of patients who survived for more than 3 years was much lower than that of patients who survived for less than 3 years (43/74 vs 51/70, P < 0.01 ). The 3-year survival rate in patients with nm23-H1(+)CD4v6(-) was significantly higher than those with nm23-H1(-)CV44v6(+) (22/32 vs 11/34, P < 0.01). There are some relationships among expressions of nm23-H1 and CD44v6 in NSCLC and cell differentiation, histological classification and prognosis, and no relation in stage and lymph node metastasis.

Pitfall in chemotherapy for ovarian cancer.

Concerning the biological properties of recurrent ovarian cancer, other than drug resistance, we revealed that the expressions of mutant p53 and CD44v6 genes were significantly greater in recurrent ovarian cancer than in those of its primary counterpart. These findings suggest that chemotherapeutic agents may modify some biological characteristics of cancer by altering gene expressions. The biological behavior concerning the metastatic potential of a recurrent disease must be elucidated in order to develop an optional treatment regimen against recurrent tumors. Therefore, we established in-vivo cisplatin-resistant cell lines by repeated administration, in order to find a more suitable model for reflecting the biological aggressiveness of clinically recurrent ovarian cancer following chemotherapy. Chemotherapeutic agents have given a substantial advantage to cancer patients. It must be borne in mind that the cancer cells surviving following chemotherapy possibly present different biological properties from primary cancer cells, and that these properties might be developed by the chemotherapeutic agents.