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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

Assessment of senescence in U397 human leukemic cells treated with MG132 (1 μM), Doxorubicin (DOX) 1 μM, or MG132 + DOX. U937 cells (1 × 106) were incubated alone or with different treatments for 24 h at 37°C in a humid atmosphere containing 5% CO2 and 95% air in RPMI-S medium. After incubation, the cells were washed in the same medium, and senescence was determined by measuring SA-β-gal utilizing flow cytometry. For each sample, at least 20,000 events were acquired in a FACSAria-I cell sorter and the data were analyzed with FACSDiva software. Results are shown as % and represent the mean ± the Standard deviation (SD) of three independent experiments performed in triplicate. Statistical analysis, the Mann–Whitney U test. *p <0.05 MG132 + DOX vs DOX; ♦p <0.05 DOX vs all groups. UCG = Untreated control group.
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Figure 5: Assessment of senescence in U397 human leukemic cells treated with MG132 (1 μM), Doxorubicin (DOX) 1 μM, or MG132 + DOX. U937 cells (1 × 106) were incubated alone or with different treatments for 24 h at 37°C in a humid atmosphere containing 5% CO2 and 95% air in RPMI-S medium. After incubation, the cells were washed in the same medium, and senescence was determined by measuring SA-β-gal utilizing flow cytometry. For each sample, at least 20,000 events were acquired in a FACSAria-I cell sorter and the data were analyzed with FACSDiva software. Results are shown as % and represent the mean ± the Standard deviation (SD) of three independent experiments performed in triplicate. Statistical analysis, the Mann–Whitney U test. *p <0.05 MG132 + DOX vs DOX; ♦p <0.05 DOX vs all groups. UCG = Untreated control group.

Mentions: It is well known that cells in senescence are SA-β-gal-positive; in Figure 5, we can observe that U937 leukemia-cell cultures, either without treatment or treated with MG132 alone, were practically non SA-β-gal-positive (6.2 ± 3.8% and 6.8 ± 3.2%, respectively); in contrast, DOX-treated cells exhibited a higher percentage of SA-β-gal-positive cells (45.8 ± 4.5%, respectively). However, when U937 cells were treated with MG132 + DOX, we observed a very important reduction in senescence (19.8 ± 3.8%; p <0.05) in comparison with cells treated only with DOX.


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)

Assessment of senescence in U397 human leukemic cells treated with MG132 (1 μM), Doxorubicin (DOX) 1 μM, or MG132 + DOX. U937 cells (1 × 106) were incubated alone or with different treatments for 24 h at 37°C in a humid atmosphere containing 5% CO2 and 95% air in RPMI-S medium. After incubation, the cells were washed in the same medium, and senescence was determined by measuring SA-β-gal utilizing flow cytometry. For each sample, at least 20,000 events were acquired in a FACSAria-I cell sorter and the data were analyzed with FACSDiva software. Results are shown as % and represent the mean ± the Standard deviation (SD) of three independent experiments performed in triplicate. Statistical analysis, the Mann–Whitney U test. *p <0.05 MG132 + DOX vs DOX; ♦p <0.05 DOX vs all groups. UCG = Untreated control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Assessment of senescence in U397 human leukemic cells treated with MG132 (1 μM), Doxorubicin (DOX) 1 μM, or MG132 + DOX. U937 cells (1 × 106) were incubated alone or with different treatments for 24 h at 37°C in a humid atmosphere containing 5% CO2 and 95% air in RPMI-S medium. After incubation, the cells were washed in the same medium, and senescence was determined by measuring SA-β-gal utilizing flow cytometry. For each sample, at least 20,000 events were acquired in a FACSAria-I cell sorter and the data were analyzed with FACSDiva software. Results are shown as % and represent the mean ± the Standard deviation (SD) of three independent experiments performed in triplicate. Statistical analysis, the Mann–Whitney U test. *p <0.05 MG132 + DOX vs DOX; ♦p <0.05 DOX vs all groups. UCG = Untreated control group.
Mentions: It is well known that cells in senescence are SA-β-gal-positive; in Figure 5, we can observe that U937 leukemia-cell cultures, either without treatment or treated with MG132 alone, were practically non SA-β-gal-positive (6.2 ± 3.8% and 6.8 ± 3.2%, respectively); in contrast, DOX-treated cells exhibited a higher percentage of SA-β-gal-positive cells (45.8 ± 4.5%, respectively). However, when U937 cells were treated with MG132 + DOX, we observed a very important reduction in senescence (19.8 ± 3.8%; p <0.05) in comparison with cells treated only with DOX.

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