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The Association of CXC Receptor 4 Mediated Signaling Pathway with Oxaliplatin-Resistant Human Colorectal Cancer Cells

View Article: PubMed Central - PubMed

ABSTRACT

The stromal cell–derived factor-1 (SDF-1)/CXC receptor 4 (CXCR4) axis plays an important role in tumor angiogenesis and invasiveness in colorectal cancer (CRC) progression. In addition, metastatic CRC remains one of the most difficult human malignancies to treat because of its chemoresistant behavior. However, the mechanism by which correlation occurs between CXCR4 and the clinical response of CRC to chemotherapy remains unknown. We generated chemoresistant cells with increasing doses of oxaliplatin (OXA) and 5-Fluorouracil (5FU) to develop resistance at a clinical dose. We found that the putative markers did not change in the parental cells, but HCT-116/OxR and HCT-116/5-FUR were more aggressive and had higher tumor growth (demonstrated by wound healing, chemotaxis assay, and a nude mice xenograft model) with the use of oxaliplatin. Apoptosis induced by oxaliplatin treatment was significantly decreased in HCT-116/OxR compared to the parental cells. Moreover, HCT-116/OxR cells displayed increased levels of p-gp, p-Akt p-ERK, p-IKBβ, CXCR4, and Bcl-2, but they also significantly inhibited the apoptotic pathways when compared to the parental strain. We evaluated the molecular mechanism governing the signaling pathway associated with anti-apoptosis activity and the aggressive status of chemoresistant cells. Experiments involving specific inhibitors demonstrated that the activation of the pathways associated with CXCR4, ERK1/2 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/Akt is critical to the functioning of the HCT-116/OxR and HCT-116/5-FUR characteristics of chemosensitivity. These findings elucidate the mechanism of CXCR4/PI3K/Akt downstream signaling and provide strategies to inhibit CXCR4 mediated signaling pathway in order to overcome CRC’s resistance to chemotherapy.

No MeSH data available.


Expression of markers CD133, CD44, SOX-2 and OCT-4 in parental cells of HCT-116 and chemoresistant cell lines of HCT-116/OxR and HCT-116/5FUR.(A) Western blotting showed that CD133, CD44, SOX-2 and OCT-4 proteins were enriched expressed in the chemoresistant cells compared with parental cell. Protein levels were quantified by densitometric analysis with the control being set at 100%. Data presented in western blot are derived from a representative study, and comparisons of protein expression are calculated from three independent experiments. *P < 0.05, when compared with control HCT-116 group. (B) Immunofluorescence analysis showed expression of markers CD133, CD44 and E-cadherin in parental cell line of HCT-116 and chemoresistant cell line of HCT-116/OxR and HCT-116/5-FUR.
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pone.0159927.g001: Expression of markers CD133, CD44, SOX-2 and OCT-4 in parental cells of HCT-116 and chemoresistant cell lines of HCT-116/OxR and HCT-116/5FUR.(A) Western blotting showed that CD133, CD44, SOX-2 and OCT-4 proteins were enriched expressed in the chemoresistant cells compared with parental cell. Protein levels were quantified by densitometric analysis with the control being set at 100%. Data presented in western blot are derived from a representative study, and comparisons of protein expression are calculated from three independent experiments. *P < 0.05, when compared with control HCT-116 group. (B) Immunofluorescence analysis showed expression of markers CD133, CD44 and E-cadherin in parental cell line of HCT-116 and chemoresistant cell line of HCT-116/OxR and HCT-116/5-FUR.

Mentions: To establish stable colon cancer cell lines continuously resistant to OXA or 5FU, HCT-116 cells were exposed to increasing concentrations of OXA or 5FU [19]. Chemoresistant cells displayed elongated and dyscohesive features. These cells are referred to as having the characteristics of tumor-initiating cells, tumor-promoting cells, or cancer stem cells (CSC). The expression profiles of parental HCT-116 human CRC cells and cells resistant to OXA or 5FU (HCT-116/OxR or HCT-116/5FUR) were evaluated by western blot and immunofluorescence stain. HCT-116/OxR and HCT-116/5FUR cells expressed significantly more CD133 and CD44, putative CSC markers as well as SOX2 and OCT4 [27] than did parental HCT-116 cells (Fig 1). Transcription factors for maintaining the survival of cancer stem cell–like cells and anti-apoptotic activity were found in colorectal cancers.


The Association of CXC Receptor 4 Mediated Signaling Pathway with Oxaliplatin-Resistant Human Colorectal Cancer Cells
Expression of markers CD133, CD44, SOX-2 and OCT-4 in parental cells of HCT-116 and chemoresistant cell lines of HCT-116/OxR and HCT-116/5FUR.(A) Western blotting showed that CD133, CD44, SOX-2 and OCT-4 proteins were enriched expressed in the chemoresistant cells compared with parental cell. Protein levels were quantified by densitometric analysis with the control being set at 100%. Data presented in western blot are derived from a representative study, and comparisons of protein expression are calculated from three independent experiments. *P < 0.05, when compared with control HCT-116 group. (B) Immunofluorescence analysis showed expression of markers CD133, CD44 and E-cadherin in parental cell line of HCT-116 and chemoresistant cell line of HCT-116/OxR and HCT-116/5-FUR.
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pone.0159927.g001: Expression of markers CD133, CD44, SOX-2 and OCT-4 in parental cells of HCT-116 and chemoresistant cell lines of HCT-116/OxR and HCT-116/5FUR.(A) Western blotting showed that CD133, CD44, SOX-2 and OCT-4 proteins were enriched expressed in the chemoresistant cells compared with parental cell. Protein levels were quantified by densitometric analysis with the control being set at 100%. Data presented in western blot are derived from a representative study, and comparisons of protein expression are calculated from three independent experiments. *P < 0.05, when compared with control HCT-116 group. (B) Immunofluorescence analysis showed expression of markers CD133, CD44 and E-cadherin in parental cell line of HCT-116 and chemoresistant cell line of HCT-116/OxR and HCT-116/5-FUR.
Mentions: To establish stable colon cancer cell lines continuously resistant to OXA or 5FU, HCT-116 cells were exposed to increasing concentrations of OXA or 5FU [19]. Chemoresistant cells displayed elongated and dyscohesive features. These cells are referred to as having the characteristics of tumor-initiating cells, tumor-promoting cells, or cancer stem cells (CSC). The expression profiles of parental HCT-116 human CRC cells and cells resistant to OXA or 5FU (HCT-116/OxR or HCT-116/5FUR) were evaluated by western blot and immunofluorescence stain. HCT-116/OxR and HCT-116/5FUR cells expressed significantly more CD133 and CD44, putative CSC markers as well as SOX2 and OCT4 [27] than did parental HCT-116 cells (Fig 1). Transcription factors for maintaining the survival of cancer stem cell–like cells and anti-apoptotic activity were found in colorectal cancers.

View Article: PubMed Central - PubMed

ABSTRACT

The stromal cell&ndash;derived factor-1 (SDF-1)/CXC receptor 4 (CXCR4) axis plays an important role in tumor angiogenesis and invasiveness in colorectal cancer (CRC) progression. In addition, metastatic CRC remains one of the most difficult human malignancies to treat because of its chemoresistant behavior. However, the mechanism by which correlation occurs between CXCR4 and the clinical response of CRC to chemotherapy remains unknown. We generated chemoresistant cells with increasing doses of oxaliplatin (OXA) and 5-Fluorouracil (5FU) to develop resistance at a clinical dose. We found that the putative markers did not change in the parental cells, but HCT-116/OxR and HCT-116/5-FUR were more aggressive and had higher tumor growth (demonstrated by wound healing, chemotaxis assay, and a nude mice xenograft model) with the use of oxaliplatin. Apoptosis induced by oxaliplatin treatment was significantly decreased in HCT-116/OxR compared to the parental cells. Moreover, HCT-116/OxR cells displayed increased levels of p-gp, p-Akt p-ERK, p-IKB&beta;, CXCR4, and Bcl-2, but they also significantly inhibited the apoptotic pathways when compared to the parental strain. We evaluated the molecular mechanism governing the signaling pathway associated with anti-apoptosis activity and the aggressive status of chemoresistant cells. Experiments involving specific inhibitors demonstrated that the activation of the pathways associated with CXCR4, ERK1/2 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/Akt is critical to the functioning of the HCT-116/OxR and HCT-116/5-FUR characteristics of chemosensitivity. These findings elucidate the mechanism of CXCR4/PI3K/Akt downstream signaling and provide strategies to inhibit CXCR4 mediated signaling pathway in order to overcome CRC&rsquo;s resistance to chemotherapy.

No MeSH data available.