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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.


Related in: MedlinePlus

Representative chemosensitivity showed wound healing assay and assessment of cell death on HCT-116/OxR and HCT-116/5-FUR cells depending on the concentration of OxA and 5-FU.(A) HCT-116, HCT-116/OxR and HCT-116/5-FUR cells were incubated with OxA and 5-FU for 6, 12 and 24 h, and the migration using the scratch-wound assay was visualized as described in Methods. The percentage of surface area filled by the parental HCT-116 CRC cell line was subsequently quantified by densitometric analyses relative to that of the control, which was set at 100% as shown in the graph. The variable *, #, & symbols indicate means that are significantly different when compared to the control group of different times incubation with P < 0.05, respectively. (B) Determination of the effects of OxA and 5-FU treatment on cell migration and invasiveness cells. Invasion through a layer of Matrigel was determined by a Boyden Chamber method as described in Methods. The lower and upper chemotaxis cells were separated by a polycarbonate membrane. Microscopy images detected cells that migrated into the inner membrane. Magnification: × 200. The cell migration was quantified by counting the number of cells that migrated into the inner membrane. Control cells remained untreated. The invasiveness was quantified and is presented in the graph. (C) Treatment for 24 h, the cells were stained with FITC-conjugated Annexin-V and PI for flow cytometry analysis as described in Materials and Methods. P < 0.01, compared with the control group. *p < 0.05, cell death assays, untreated group versus OxA treated HCT-116 group. #P < 0.05, untreated group versus OxA treated HCT-116/OxR group.
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pone.0159927.g002: Representative chemosensitivity showed wound healing assay and assessment of cell death on HCT-116/OxR and HCT-116/5-FUR cells depending on the concentration of OxA and 5-FU.(A) HCT-116, HCT-116/OxR and HCT-116/5-FUR cells were incubated with OxA and 5-FU for 6, 12 and 24 h, and the migration using the scratch-wound assay was visualized as described in Methods. The percentage of surface area filled by the parental HCT-116 CRC cell line was subsequently quantified by densitometric analyses relative to that of the control, which was set at 100% as shown in the graph. The variable *, #, & symbols indicate means that are significantly different when compared to the control group of different times incubation with P < 0.05, respectively. (B) Determination of the effects of OxA and 5-FU treatment on cell migration and invasiveness cells. Invasion through a layer of Matrigel was determined by a Boyden Chamber method as described in Methods. The lower and upper chemotaxis cells were separated by a polycarbonate membrane. Microscopy images detected cells that migrated into the inner membrane. Magnification: × 200. The cell migration was quantified by counting the number of cells that migrated into the inner membrane. Control cells remained untreated. The invasiveness was quantified and is presented in the graph. (C) Treatment for 24 h, the cells were stained with FITC-conjugated Annexin-V and PI for flow cytometry analysis as described in Materials and Methods. P < 0.01, compared with the control group. *p < 0.05, cell death assays, untreated group versus OxA treated HCT-116 group. #P < 0.05, untreated group versus OxA treated HCT-116/OxR group.

Mentions: Using the scratch-wound assay, a lasting rapid movement was observed in resistant cells. A resultant movement of an HCT-116 cell migration front was clearly evident at 24 h, where a highly confluent (90%–100%) monolayer region gradually moved into the cell-free scratch region. In the presence of OXA and 5FU, at a clinically relevant plasma concentration, migration in HCT-116/OxR and HCT-116/5-FUR were highly increased after 12 h and 24 h of incubation, whereas OXA and 5-FU led to a virtually complete inhibition of parental HCT-116 cell proliferation and sensitivity to drugs (Fig 2A). The Boyden chamber assay was used to evaluate the in vitro migration and invasion effect of treatment with antineoplastic drugs associated with the aggressive status of HCT-116, HCT-116/OxR and HCT-116/5-FUR cells. The observations revealed that OXA and 5-FU resulted in a remarkable inhibition of parental HCT-116 cell migration as compared to no treatment alone, but HCT-116/OxR and HCT-116/5-FUR cells showed highly evident movement and invasive properties (Fig 2B). To quantify the anti-apoptosis activity of OXA upon resistant cells, we subjected HCT-116 and HCT-116/OxR cells to a dose range of OXA for 24 h and performed viability assays. The half-maximal inhibitory concentration (IC50) of an OXA treatment upon proliferation was sensitively 2.0 μg/ml in HCT-116 cells. HCT-116/OxR cells were resistant to OXA as expected with 95% of viable cells. Similarly, as expected, oxaliplatin-resistant cells were resistant to oxaliplatin as determined by annexin V-FITC/PI dye (Fig 2C). In addition, to determine the in vivo effects of OXA on tumor-growth resistance in HCT-116/OxR cells, we exposed human parental HCT-116 and HCT-116/OxR cells to indicated OXA doses for 18 days and examined them by assay of xenografted tumor in nude mice, as described previously. The results of previous toxicity-assessment experiments suggested that the in vivo dosage of OXA should not exceed 5 mg/kg/day; therefore, daily intraperitoneal injections of OXA were used in these experiments. Fig 3 shows the time course of xenograft growth with and without OXA treatment. Evaluation of the xenograft HCT-116 tumor volume showed that a significant inhibition of tumor growth by OXA was caused in a time-dependent manner. However, the HCT-116/OxR xenograft volumes were reduced 2.5 fold more than parental HCT-116, which was the control group (P < 0.05). At the end of this experiment, the xenograft tumors were removed. Evaluation of a proliferative mitosis by H&E staining of the tumor sections showed that OXA led to an increase in the number of proliferating HCT-116/OxR cells in tumors compared with those untreated in the control group. Fig 4 shows the representative photo images from the tumor sections.


The Association of CXC Receptor 4 Mediated Signaling Pathway with Oxaliplatin-Resistant Human Colorectal Cancer Cells
Representative chemosensitivity showed wound healing assay and assessment of cell death on HCT-116/OxR and HCT-116/5-FUR cells depending on the concentration of OxA and 5-FU.(A) HCT-116, HCT-116/OxR and HCT-116/5-FUR cells were incubated with OxA and 5-FU for 6, 12 and 24 h, and the migration using the scratch-wound assay was visualized as described in Methods. The percentage of surface area filled by the parental HCT-116 CRC cell line was subsequently quantified by densitometric analyses relative to that of the control, which was set at 100% as shown in the graph. The variable *, #, & symbols indicate means that are significantly different when compared to the control group of different times incubation with P < 0.05, respectively. (B) Determination of the effects of OxA and 5-FU treatment on cell migration and invasiveness cells. Invasion through a layer of Matrigel was determined by a Boyden Chamber method as described in Methods. The lower and upper chemotaxis cells were separated by a polycarbonate membrane. Microscopy images detected cells that migrated into the inner membrane. Magnification: × 200. The cell migration was quantified by counting the number of cells that migrated into the inner membrane. Control cells remained untreated. The invasiveness was quantified and is presented in the graph. (C) Treatment for 24 h, the cells were stained with FITC-conjugated Annexin-V and PI for flow cytometry analysis as described in Materials and Methods. P < 0.01, compared with the control group. *p < 0.05, cell death assays, untreated group versus OxA treated HCT-116 group. #P < 0.05, untreated group versus OxA treated HCT-116/OxR group.
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pone.0159927.g002: Representative chemosensitivity showed wound healing assay and assessment of cell death on HCT-116/OxR and HCT-116/5-FUR cells depending on the concentration of OxA and 5-FU.(A) HCT-116, HCT-116/OxR and HCT-116/5-FUR cells were incubated with OxA and 5-FU for 6, 12 and 24 h, and the migration using the scratch-wound assay was visualized as described in Methods. The percentage of surface area filled by the parental HCT-116 CRC cell line was subsequently quantified by densitometric analyses relative to that of the control, which was set at 100% as shown in the graph. The variable *, #, & symbols indicate means that are significantly different when compared to the control group of different times incubation with P < 0.05, respectively. (B) Determination of the effects of OxA and 5-FU treatment on cell migration and invasiveness cells. Invasion through a layer of Matrigel was determined by a Boyden Chamber method as described in Methods. The lower and upper chemotaxis cells were separated by a polycarbonate membrane. Microscopy images detected cells that migrated into the inner membrane. Magnification: × 200. The cell migration was quantified by counting the number of cells that migrated into the inner membrane. Control cells remained untreated. The invasiveness was quantified and is presented in the graph. (C) Treatment for 24 h, the cells were stained with FITC-conjugated Annexin-V and PI for flow cytometry analysis as described in Materials and Methods. P < 0.01, compared with the control group. *p < 0.05, cell death assays, untreated group versus OxA treated HCT-116 group. #P < 0.05, untreated group versus OxA treated HCT-116/OxR group.
Mentions: Using the scratch-wound assay, a lasting rapid movement was observed in resistant cells. A resultant movement of an HCT-116 cell migration front was clearly evident at 24 h, where a highly confluent (90%–100%) monolayer region gradually moved into the cell-free scratch region. In the presence of OXA and 5FU, at a clinically relevant plasma concentration, migration in HCT-116/OxR and HCT-116/5-FUR were highly increased after 12 h and 24 h of incubation, whereas OXA and 5-FU led to a virtually complete inhibition of parental HCT-116 cell proliferation and sensitivity to drugs (Fig 2A). The Boyden chamber assay was used to evaluate the in vitro migration and invasion effect of treatment with antineoplastic drugs associated with the aggressive status of HCT-116, HCT-116/OxR and HCT-116/5-FUR cells. The observations revealed that OXA and 5-FU resulted in a remarkable inhibition of parental HCT-116 cell migration as compared to no treatment alone, but HCT-116/OxR and HCT-116/5-FUR cells showed highly evident movement and invasive properties (Fig 2B). To quantify the anti-apoptosis activity of OXA upon resistant cells, we subjected HCT-116 and HCT-116/OxR cells to a dose range of OXA for 24 h and performed viability assays. The half-maximal inhibitory concentration (IC50) of an OXA treatment upon proliferation was sensitively 2.0 μg/ml in HCT-116 cells. HCT-116/OxR cells were resistant to OXA as expected with 95% of viable cells. Similarly, as expected, oxaliplatin-resistant cells were resistant to oxaliplatin as determined by annexin V-FITC/PI dye (Fig 2C). In addition, to determine the in vivo effects of OXA on tumor-growth resistance in HCT-116/OxR cells, we exposed human parental HCT-116 and HCT-116/OxR cells to indicated OXA doses for 18 days and examined them by assay of xenografted tumor in nude mice, as described previously. The results of previous toxicity-assessment experiments suggested that the in vivo dosage of OXA should not exceed 5 mg/kg/day; therefore, daily intraperitoneal injections of OXA were used in these experiments. Fig 3 shows the time course of xenograft growth with and without OXA treatment. Evaluation of the xenograft HCT-116 tumor volume showed that a significant inhibition of tumor growth by OXA was caused in a time-dependent manner. However, the HCT-116/OxR xenograft volumes were reduced 2.5 fold more than parental HCT-116, which was the control group (P < 0.05). At the end of this experiment, the xenograft tumors were removed. Evaluation of a proliferative mitosis by H&E staining of the tumor sections showed that OXA led to an increase in the number of proliferating HCT-116/OxR cells in tumors compared with those untreated in the control group. Fig 4 shows the representative photo images from the tumor sections.

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.


Related in: MedlinePlus