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Sensitization of U937 leukemia cells to doxorubicin by the MG132 proteasome inhibitor induces an increase in apoptosis by suppressing NF-kappa B and mitochondrial membrane potential loss.

Ortiz-Lazareno PC, Bravo-Cuellar A, Lerma-Díaz JM, Jave-Suárez LF, Aguilar-Lemarroy A, Domínguez-Rodríguez JR, González-Ramella O, De Célis R, Gómez-Lomelí P, Hernández-Flores G - Cancer Cell Int. (2014)

Bottom Line: In this work, we studied the sensitizing effect of the MG132 proteasome inhibitor on the antitumor activity of DOX.Therefore, MG132 decreases senescence, p65 phosphorylation, and the DOX-induced Bcl-2 antiapoptotic protein.MG132 sensitizes U937 leukemia cells to DOX-induced apoptosis, increasing its anti-leukemic effectiveness.

View Article: PubMed Central - HTML - PubMed

Affiliation: División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO), Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco, México. gina.geodic1967@gmail.com.

ABSTRACT

Background: The resistance of cancerous cells to chemotherapy remains the main limitation for cancer treatment at present. Doxorubicin (DOX) is a potent antitumor drug that activates the ubiquitin-proteasome system, but unfortunately it also activates the Nuclear factor kappa B (NF-кB) pathway leading to the promotion of tumor cell survival. MG132 is a drug that inhibits I kappa B degradation by the proteasome-avoiding activation of NF-кB. In this work, we studied the sensitizing effect of the MG132 proteasome inhibitor on the antitumor activity of DOX.

Methods: U937 human leukemia cells were treated with MG132, DOX, or both drugs. We evaluated proliferation, viability, apoptosis, caspase-3, -8, and -9 activity and cleavage, cytochrome c release, mitochondrial membrane potential, the Bcl-2 and Bcl-XL antiapoptotic proteins, senescence, p65 phosphorylation, and pro- and antiapoptotic genes.

Results: The greatest apoptosis percentage in U937 cells was obtained with a combination of MG132 + DOX. Likewise, employing both drugs, we observed a decrease in tumor cell proliferation and important caspase-3 activation, as well as mitochondrial membrane potential loss. Therefore, MG132 decreases senescence, p65 phosphorylation, and the DOX-induced Bcl-2 antiapoptotic protein. The MG132 + DOX treatment induced upregulation of proapoptotic genes BAX, DIABLO, NOXA, DR4, and FAS. It also induced downregulation of the antiapoptotic genes BCL-XL and SURVIVIN.

Conclusion: MG132 sensitizes U937 leukemia cells to DOX-induced apoptosis, increasing its anti-leukemic effectiveness.

No MeSH data available.


Related in: MedlinePlus

Western blot analysis of caspases −3, -8, and −9 and cytochromecin U937 cells treated with MG132, Doxorubicin (DOX) or MG132 + DOX. U937 cells were cultured and treated with MG132 (1 μM), DOX 1 μM, or with MG132 + DOX. After 24 h, the cells were harvested and lysed. Equivalent amounts of individual lysates were placed onto 10% SDS gradient polyacrylamide gels for electrophoresis and then were electrotransferred onto Immobilon-P PVDF membranes. A representative study is shown. Relative density was calculated using IMAGEJ ver.1.46r software.
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Figure 3: Western blot analysis of caspases −3, -8, and −9 and cytochromecin U937 cells treated with MG132, Doxorubicin (DOX) or MG132 + DOX. U937 cells were cultured and treated with MG132 (1 μM), DOX 1 μM, or with MG132 + DOX. After 24 h, the cells were harvested and lysed. Equivalent amounts of individual lysates were placed onto 10% SDS gradient polyacrylamide gels for electrophoresis and then were electrotransferred onto Immobilon-P PVDF membranes. A representative study is shown. Relative density was calculated using IMAGEJ ver.1.46r software.

Mentions: To confirm our results, we determined cleavage in caspases and in cytochrome c release. The results in Figure 3 allow us to observe that cell treatment with the MG132 proteasome inhibitor increased the cleavage of caspase-9 (3.7-fold), caspase-8 (2.4-fold), and of caspase-3 (15.9-fold), and also the release of cytochrome c (5.3-fold) compared with the UCG. Similarly, DOX treatment increased the cleavage of caspase-9 by 6.2-fold, of caspase-8 by 1.9-fold, and of caspase-3 by 34.7-fold, and cytochrome c release by 4.4-fold compared with that of the UCG. It is noteworthy that when we used MG132 + DOX, we observed considerably more cleavage of caspase-3 (48.5-fold) compared with MG132 or DOX alone and also when compared with the UCG. Additionally, when we used both drugs simultaneously, we observed an increase in cytochrome c release (4.5-fold) and the cleavage of caspase-8 (2-fold) and of caspase-9 (5.1-fold) in comparison with the UCG. These results show the sensitization of leukemia cells to chemotherapy induced by the MG132 proteasome inhibitor and demonstrates that caspases possess significant participation.


Sensitization of U937 leukemia cells to doxorubicin by the MG132 proteasome inhibitor induces an increase in apoptosis by suppressing NF-kappa B and mitochondrial membrane potential loss.

Ortiz-Lazareno PC, Bravo-Cuellar A, Lerma-Díaz JM, Jave-Suárez LF, Aguilar-Lemarroy A, Domínguez-Rodríguez JR, González-Ramella O, De Célis R, Gómez-Lomelí P, Hernández-Flores G - Cancer Cell Int. (2014)

Western blot analysis of caspases −3, -8, and −9 and cytochromecin U937 cells treated with MG132, Doxorubicin (DOX) or MG132 + DOX. U937 cells were cultured and treated with MG132 (1 μM), DOX 1 μM, or with MG132 + DOX. After 24 h, the cells were harvested and lysed. Equivalent amounts of individual lysates were placed onto 10% SDS gradient polyacrylamide gels for electrophoresis and then were electrotransferred onto Immobilon-P PVDF membranes. A representative study is shown. Relative density was calculated using IMAGEJ ver.1.46r software.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927225&req=5

Figure 3: Western blot analysis of caspases −3, -8, and −9 and cytochromecin U937 cells treated with MG132, Doxorubicin (DOX) or MG132 + DOX. U937 cells were cultured and treated with MG132 (1 μM), DOX 1 μM, or with MG132 + DOX. After 24 h, the cells were harvested and lysed. Equivalent amounts of individual lysates were placed onto 10% SDS gradient polyacrylamide gels for electrophoresis and then were electrotransferred onto Immobilon-P PVDF membranes. A representative study is shown. Relative density was calculated using IMAGEJ ver.1.46r software.
Mentions: To confirm our results, we determined cleavage in caspases and in cytochrome c release. The results in Figure 3 allow us to observe that cell treatment with the MG132 proteasome inhibitor increased the cleavage of caspase-9 (3.7-fold), caspase-8 (2.4-fold), and of caspase-3 (15.9-fold), and also the release of cytochrome c (5.3-fold) compared with the UCG. Similarly, DOX treatment increased the cleavage of caspase-9 by 6.2-fold, of caspase-8 by 1.9-fold, and of caspase-3 by 34.7-fold, and cytochrome c release by 4.4-fold compared with that of the UCG. It is noteworthy that when we used MG132 + DOX, we observed considerably more cleavage of caspase-3 (48.5-fold) compared with MG132 or DOX alone and also when compared with the UCG. Additionally, when we used both drugs simultaneously, we observed an increase in cytochrome c release (4.5-fold) and the cleavage of caspase-8 (2-fold) and of caspase-9 (5.1-fold) in comparison with the UCG. These results show the sensitization of leukemia cells to chemotherapy induced by the MG132 proteasome inhibitor and demonstrates that caspases possess significant participation.

Bottom Line: In this work, we studied the sensitizing effect of the MG132 proteasome inhibitor on the antitumor activity of DOX.Therefore, MG132 decreases senescence, p65 phosphorylation, and the DOX-induced Bcl-2 antiapoptotic protein.MG132 sensitizes U937 leukemia cells to DOX-induced apoptosis, increasing its anti-leukemic effectiveness.

View Article: PubMed Central - HTML - PubMed

Affiliation: División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO), Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco, México. gina.geodic1967@gmail.com.

ABSTRACT

Background: The resistance of cancerous cells to chemotherapy remains the main limitation for cancer treatment at present. Doxorubicin (DOX) is a potent antitumor drug that activates the ubiquitin-proteasome system, but unfortunately it also activates the Nuclear factor kappa B (NF-кB) pathway leading to the promotion of tumor cell survival. MG132 is a drug that inhibits I kappa B degradation by the proteasome-avoiding activation of NF-кB. In this work, we studied the sensitizing effect of the MG132 proteasome inhibitor on the antitumor activity of DOX.

Methods: U937 human leukemia cells were treated with MG132, DOX, or both drugs. We evaluated proliferation, viability, apoptosis, caspase-3, -8, and -9 activity and cleavage, cytochrome c release, mitochondrial membrane potential, the Bcl-2 and Bcl-XL antiapoptotic proteins, senescence, p65 phosphorylation, and pro- and antiapoptotic genes.

Results: The greatest apoptosis percentage in U937 cells was obtained with a combination of MG132 + DOX. Likewise, employing both drugs, we observed a decrease in tumor cell proliferation and important caspase-3 activation, as well as mitochondrial membrane potential loss. Therefore, MG132 decreases senescence, p65 phosphorylation, and the DOX-induced Bcl-2 antiapoptotic protein. The MG132 + DOX treatment induced upregulation of proapoptotic genes BAX, DIABLO, NOXA, DR4, and FAS. It also induced downregulation of the antiapoptotic genes BCL-XL and SURVIVIN.

Conclusion: MG132 sensitizes U937 leukemia cells to DOX-induced apoptosis, increasing its anti-leukemic effectiveness.

No MeSH data available.


Related in: MedlinePlus