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. 2015 Mar 9;16(3):5386-99.
doi: 10.3390/ijms16035386.

Different apoptotic pathways activated by oxaliplatin in primary astrocytes vs. colo-rectal cancer cells

Matteo Zanardelli  1 , Laura Micheli  2 , Raffaella Nicolai  3 , Paola Failli  4 , Carla Ghelardini  5 , Lorenzo Di Cesare Mannelli  6
Affiliations

Different apoptotic pathways activated by oxaliplatin in primary astrocytes vs. colo-rectal cancer cells

Matteo Zanardelli et al. Int J Mol Sci. .

Abstract

Oxaliplatin-based chemotherapy improves the outcomes of metastatic colorectal cancer patients. Its most significant and dose-limiting side effect is the development of a neuropathic syndrome. The mechanism of the neurotoxicity is unclear. The limited knowledge about differences existing between neurotoxic and antitumor effects hinders the discovery of effective and safe adjuvant therapies. In vitro, we suggested cell-specific activation apoptotic pathways in normal nervous cells (astrocytes) vs. colon-cancer cells (HT-29). In the present research we compared the apoptotic signals evoked by oxaliplatin in astrocytes and HT-29 analyzing the intrinsic and extrinsic apoptotic pathways. In astrocytes, oxaliplatin induced a mitochondrial derangement measured as cytosolic release of cytochrome C, increase in superoxide anion levels and decreased expression of the antiapoptotic protein Bcl-2. Caspase-8, a main initiator of the extrinsic process remained unaltered. On the contrary, in HT-29 oxaliplatin increased caspase-8 activity and Bid expression, thus activating the extrinsic apoptosis, while the Bcl-2 increased expression blocked the mitochondrial damage. Data suggest the preferred activation of the intrinsic apoptosis as oxaliplatin damage signaling in normal nervous cells. The extrinsic pathway prevails in tumor cells indicating a possible strategy for planning new molecules to treat oxaliplatin-dependent neurotoxicity without negatively influence chemotherapy.

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Figures

Figure 1
Figure 1
Cytosolic release of cytochrome C. Astrocytes (5 × 104 cells/slide) and HT-29 (5 × 104 cells/slide) were exposed to 100 μM oxaliplatin for 8 h. Specimens were stained with anti-cytochrome C and a secondary antibody conjugated with Alexa Fluor 488 (green) and DAPI (blue) for nucleus visualization. (A1–A3) control astrocytes; (B1–B3) oxaliplatin-treated astrocytes; (C1–C3) control HT-29; (D1–D3) oxaliplatin-treated HT-29. Calibration: 20 µm. Bars represent the mean ± S.E.M of cells displaying a diffuse cytosolic distribution of cytochrome C as percentage of total analyzed cells. Cells were counted using the “cell counter” plugin of ImageJ 1.33, free-share image analysis software (ImageJ, NIH, Bethesda, MD, USA). At least three fields (40X 0.75NA objective) per slide and two slides for each condition were analyzed, repeating the experiment three times. ** p < 0.01 vs. control.
Figure 2
Figure 2
O2.− concentrations. Astrocytes (5 × 105 cells/well) and HT-29 (3 × 105 cells/well) were exposed to 100 μM oxaliplatin for 4 h. O2.− concentration was evaluated by cytochrome C assay. The nonspecific absorbance was measured in the presence of superoxide dismutase (SOD; 300 mU/mL) and subtracted from the total value. Values are expressed as µM/mg protein/4 h. Bars represent the mean ± S.E.M. of three experiments. * p < 0.05 vs. control and # p < 0.05 vs. astrocytes control.
Figure 3
Figure 3
Bcl-2 protein expression. Astrocytes (106 cells/flask) and HT-29 (8 × 105 cells/flask) were incubated with 100 μM oxaliplatin for 8 h and then cell lysates were analyzed by Western blot. Densitometric analysis (top) and representative immunoblot (bottom) are shown. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was used as loading control and GAPDH normalization was performed for each sample. Values are expressed as integrated density making the ratio between Bcl-2 and GAPDH specific band intensities. Bars represent the mean ± S.E.M. of three experiments. * p < 0.05 vs. control, ** p < 0.01 vs. control and # p < 0.05 vs. astrocytes control.
Figure 4
Figure 4
Death receptor 5 (DR5) protein expression. Astrocytes (106 cells/flask) and HT-29 (8 × 105 cells/flask) were incubated with 100 μM oxaliplatin for 8 h and then Western blot analysis were performed. Densitometric analysis (top) and representative immunoblot (bottom) are shown. GAPDH was used as loading control and GAPDH normalization was performed for each sample. Values are expressed as integrated density making the ratio between DR5 and GAPDH specific band intensities. Bars represent the mean ± S.E.M. of three experiments.
Figure 5
Figure 5
Caspase-8 activity. Astrocytes (5 × 105 cells/well), HT-29 (3 × 105 cells/well) and PC12 (3 × 105 cells/well) were treated with 100 μM oxaliplatin for 8 h. Caspase-8 activity was expressed as fluorescent arbitrary unit/mg protein. Bars represent the mean ± S.E.M. of three experiments. * p < 0.05 vs. control.
Figure 6
Figure 6
Bid protein expression. HT-29 (8 × 105 cells/flask) were incubated with 100 μM oxaliplatin for 8 h and then cell lysates were analyzed by Western blot. Densitometric analysis (top) and representative immunoblot (bottom) are shown. GAPDH was used as loading control and GAPDH normalization was performed for each sample. Values are expressed as integrated density making the ratio between Bid and GAPDH specific band intensities. Bars represent the mean ± S.E.M. of three experiments. * p < 0.05 vs. control.
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References

    1. André T., Boni C., Mounedji-Boudiaf L., Navarro M., Tabernero J., Hickish T., Topham C., Zaninelli M., Clingan P., Bridgewater J., et al. Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. N. Engl. J. Med. 2004;350:2343–2351. doi: 10.1056/NEJMoa032709. - DOI - PubMed
    1. Hoff P.M., Saad E.D., Costa F., Coutinho A.K., Caponero R., Prolla G., Gansl R.C. Literature review and practical aspects on the management of oxaliplatin-associated toxicity. Clin. Colorectal Cancer. 2012;11:93–100. doi: 10.1016/j.clcc.2011.10.004. - DOI - PubMed
    1. Kidani Y., Noji M., Tashiro T. Antitumor activity of platinum (II) complexes of 1,2-diamino-cyclohexane isomers. Gann. 1980;71:637–643. - PubMed
    1. Tashiro T., Kawada Y., Sakurai Y., Kidani Y. Antitumor activity of a new platinum complex, oxalato (trans-l-1,2-diaminocyclohexane) platinum (II): New experimental data. Biomed. Pharmacother. 1989;43:251–260. doi: 10.1016/0753-3322(89)90004-8. - DOI - PubMed
    1. De Gramont A., Figureer A., Seymour M., Homerin M., Hmissi A., Cassidy J., Boni C., Cortes-Funes H., Cervantes A., Freyer G., et al. Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer. J. Clin. Oncol. 2000;18:2938–2947. - PubMed

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