A MCP1 fusokine with CCR2-specific tumoricidal activity

<p>Abstract</p> <p>Background</p> <p>The CCL2 chemokine is involved in promoting cancer angiogenesis, proliferation and metastasis by malignancies that express CCR2 receptor. Thus the CCL2/CCR2 axis is an attractive molecular target for anticancer drug development.</p> <p>Methods</p> <p>We have generated a novel fusion protein using GMCSF and an N-terminal truncated version of MCP1/CCL2 (6-76) [hereafter GMME1] and investigated its utility as a CCR2-specific tumoricidal agent.</p> <p>Results</p> <p>We found that distinct to full length CCL2 or its N-truncated derivative (CCL2 5-76), GMME1 bound to CCR2 on mouse lymphoma EG7, human multiple myeloma cell line U266, or murine and human medulloblastoma cell lines, and led to their death by apoptosis. We demonstrated that GMME1 specifically blocked CCR2-associated STAT3 phosphorylation and up-regulated pro-apoptotic BAX. Furthermore, GMME1 significantly inhibited EG7 tumor growth in C57BL/6 mice, and induced apoptosis of primary myeloma cells from patients.</p> <p>Conclusion</p> <p>Our data demonstrate that GMME1 is a fusokine with a potent, CCR2 receptor-mediated pro-apoptotic effect on tumor cells and could be exploited as a novel biological therapy for CCR2⁺malignancies including lymphoid and central nervous system malignancies.</p

Alterations in glutathione levels and apoptotic regulators are associated with acquisition of arsenic trioxide resistance in multiple myeloma.

Arsenic trioxide (ATO) has been tested in relapsed/refractory multiple myeloma with limited success. In order to better understand drug mechanism and resistance pathways in myeloma we generated an ATO-resistant cell line, 8226/S-ATOR05, with an IC50 that is 2-3-fold higher than control cell lines and significantly higher than clinically achievable concentrations. Interestingly we found two parallel pathways governing resistance to ATO in 8226/S-ATOR05, and the relevance of these pathways appears to be linked to the concentration of ATO used. We found changes in the expression of Bcl-2 family proteins Bfl-1 and Noxa as well as an increase in cellular glutathione (GSH) levels. At low, clinically achievable concentrations, resistance was primarily associated with an increase in expression of the anti-apoptotic protein Bfl-1 and a decrease in expression of the pro-apoptotic protein Noxa. However, as the concentration of ATO increased, elevated levels of intracellular GSH in 8226/S-ATOR05 became the primary mechanism of ATO resistance. Removal of arsenic selection resulted in a loss of the resistance phenotype, with cells becoming sensitive to high concentrations of ATO within 7 days following drug removal, indicating changes associated with high level resistance (elevated GSH) are dependent upon the presence of arsenic. Conversely, not until 50 days without arsenic did cells once again become sensitive to clinically relevant doses of ATO, coinciding with a decrease in the expression of Bfl-1. In addition we found cross-resistance to melphalan and doxorubicin in 8226/S-ATOR05, suggesting ATO-resistance pathways may also be involved in resistance to other chemotherapeutic agents used in the treatment of multiple myeloma

ATO and Melphalan resistance pathways overlap while Doxorubicin resistance pathway(s) may be related.

<p>8226/S, 8226/S-CR, 8226/S-ATOR05 cells, as well as 8226/S-ATOR05 cells cultured in the absence of ATO for 7, 14, 21, 35, and 50 days were treated with the indicated concentrations of ATO, Mel, or Dox for 24 h and apoptosis was determined by flow cytometry and graphed as percent of control Annexin V positive cells versus drug concentration. The data are presented as the mean ± SD of at least 3 independent experiments.</p

ABC genes relative expression.

<p>Genes associated to drug resistance are in bold. Genes that were not expressed are not listed.</p

8226/S-ATOR05 cell line is resistant to ATO and resistance is not due to changes in uptake.

<p>(<b>A</b>) 8226/S, 8226/S-CR, 8226/S-ATOR05 cell lines were treated with the indicated concentrations of ATO for 24 h and apoptosis was assessed by flow cytometry using Annexin V-FITC and PI staining and graphed as percent of control Annexin V positive cells versus drug concentration. (<b>B</b>) 8226/S, 8226/S-CR, 8226/S-ATOR05 cells were treated with 2 µM ATO for 24 h and then collected for intracellular arsenic determination. The data are presented as the mean ± SD of at least 3 independent experiments. Student’s <i>t</i>-test was used to compare differences between 8226/S and 8226-CR and 8226/S-ATOR05 cells, with 95% confidence intervals. *, P<0.05; **, P<0.01; ***, P<0.001.</p

The antioxidant response is unchanged in 8226/S-ATOR05 cells while the expression of Bcl-2 family members is altered.

<p>8226/S, 8226/S-CR, 8226/S-ATOR05 cells were treated with 2 µM ATO for 0, 6, or 24 h, protein expression was determined by Western blot and membranes were probed with antibodies against proteins involved in the (<b>A</b>) antioxidant response or (<b>B</b>) the Bcl-2 family. (<b>C</b>) A heatmap of Bcl-2 genes expressed in 8226/S cells. The scale represents the change relative to the median expression for each gene. (<b>D</b>) Protein expression of Noxa and Bfl-1 was determined by Western blot in 8226/S, 8226/S-CR, 8226/S-ATOR05 control cells. Actin expression demonstrates protein loading. Quantitation of changes in Noxa and Puma are provided. The data are normalized to actin and presented as fold change relative to untreated parental cells.</p

The ATO resistance phenotype is lost upon removal of arsenic.

<p>(<b>A</b>) 8226/S, 8226/S-CR, 8226/S-ATOR05 cells, as well as 8226/S-ATOR05 cells cultured in the absence of ATO for 7, 14, 21, 35, and 50 days were treated with the indicated concentrations of ATO for 24 h and apoptosis was determined by flow cytometry and graphed as percent of control Annexin V positive cells versus drug concentration. Statistical analysis compared to 8226/S are provided in the lower panel. (<b>B</b>) 8226/S, 8226/S-CR, 8226/S-ATOR05 control cells, as well as control cells from the indicated time points were harvested and the intracellular GSH concentration was determined as described in the Materials and Methods. (<b>C</b>) Real-time PCR was performed on 8226/S, 8226/S-CR, 8226/S-ATOR05 cells, as well as 8226/S-ATOR05 cells cultured in the absence of ATO for 7, 14, 21, 35, and 50 days to determine the expression patterns of Bfl-1. Data is graphed using the Delta CT, which represents the number of cycles needed to reach threshold, normalized against GAPDH. High delta Ct values represent low expression while low delta Ct values indicate high expression. The data in (B) and (C) are presented as the mean ± SD of at least 3 independent experiments. Student’s <i>t</i>-test was used to compare differences between 8226/S-ATOR05 and all other cell lines, with 95% confidence intervals. *, P<0.05; **, P<0.01; ***, P<0.001, No difference (ND).</p

GSH inversely correlates with response to high concentrations of ATO while Bfl-1 expression inversely correlates with the response to clinically relevant ATO concentrations.

<p>GSH levels (<b>A</b>) or Bfl-1 expression (<b>B</b>) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052662#pone-0052662-g004" target="_blank">Figure 4</a> was correlated with apoptosis data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052662#pone-0052662-g004" target="_blank">Figure 4</a>. Pearson coefficient data are presented as a determinant of the strength of the correlation. (<b>C</b>) Cells were treated with the indicated concentrations of ATO in the presence or absence of BSO (1 µM) and apoptosis determined at 24 and 48 h. The data are presented as the mean ± SD of at least 3 independent experiments. The lower panel provides the statistical analyses of the data (student’s t test).</p

ATO resistant cells are also resistant to melphalan and doxorubicin, but remain sensitive to darinaparsin, bortezomib and ABT-737.

<p>8226/S, 8226/S-CR, and 8226/S-ATOR05 cells were treated with the indicated concentrations of ATO, DAR, Mel, Bz, ABT-737 or Dox for 24 h and apoptosis was assessed by flow cytometry and graphed as percent of control Annexin V positive cells vs. drug concentration. The data are presented as the mean ± SD of at least 3 independent experiments.</p

The intracellular GSH level is elevated in 8226/S-ATOR05 cells and is important for arsenic resistance.

<p>(<b>A</b>) 8226/S, 8226/S-CR, 8226/S-ATOR05 control cells were harvested and the intracellular GSH concentration was determined using the Glutathione Assay kit from Calbiochem. (<b>B</b>) 8226/S, 8226/S-CR, 8226/S-ATOR05 cells were treated with 2 µM ATO alone or in combination with either 100 µM BSO or 10 mM NAC for 24 h and apoptosis was determined using flow cytometry. Data are graphed as percent of control Annexin V positive cells. Mean ± SD of at least 3 independent experiments. **, P<0.01.</p