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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

Apoptosis and caspase activity in U937 cells treated with Doxorubicin (DOX), MG132, or MG132 + DOX. U937 cells were incubated alone or in combination with MG132 (1 μM), DOX 1 μM, or MG132 + DOX for 24 h at 37°C in a humid atmosphere containing 5% CO2 in RPMI-S culture medium. After incubation, the cells were washed and apoptosis was assessed by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC) (a). For each sample, at least 20,000 events were acquired in a FACSAria-I cell sorter and the data were analyzed with FACSDiva software. The activity of caspase-3 (b), -8 (c), and −9 (d) was evaluated utilizing a caspase colorimetric staining kit. The results represent the mean ± the Standard deviation (SD) of three independent experiments performed in triplicate. Statistical analysis was performed by means of the Mann–Whitney U test. ●p <0.05 MG132 + DOX vs MG132, DOX, or the Untreated control group (UCG); *p <0.05 all groups vs the UCG.
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Figure 2: Apoptosis and caspase activity in U937 cells treated with Doxorubicin (DOX), MG132, or MG132 + DOX. U937 cells were incubated alone or in combination with MG132 (1 μM), DOX 1 μM, or MG132 + DOX for 24 h at 37°C in a humid atmosphere containing 5% CO2 in RPMI-S culture medium. After incubation, the cells were washed and apoptosis was assessed by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC) (a). For each sample, at least 20,000 events were acquired in a FACSAria-I cell sorter and the data were analyzed with FACSDiva software. The activity of caspase-3 (b), -8 (c), and −9 (d) was evaluated utilizing a caspase colorimetric staining kit. The results represent the mean ± the Standard deviation (SD) of three independent experiments performed in triplicate. Statistical analysis was performed by means of the Mann–Whitney U test. ●p <0.05 MG132 + DOX vs MG132, DOX, or the Untreated control group (UCG); *p <0.05 all groups vs the UCG.

Mentions: At 24 h post-treatment, apoptosis was evaluated in U937 cells treated with the different schedules. In Figure 2a, we can observe that the UCG showed a lower percentage of apoptosis (7.8 ± 1.6%) compared with that of the groups treated exclusively with MG132 or with DOX (45.4 ± 5.3% and 55.1 ± 5.4% of apoptosis, respectively; p <0.05). Interestingly, cell cultures exposed to MG132 + DOX exhibited superior values of apoptosis in comparison with the cells treated only with one drug, with a percentage of apoptosis of 75.1 ± 0.5% (p <0.05 when comparing MG132 + DOX vs. MG132- or DOX-treated cells), with an average increase for the three groups representing ∆% = 1017 in relation to the UCG. In order to confirm caspase participation, we evaluated caspase activity; in Figure 2b, we are able to observe that MG132 proteasome inhibitor and DOX-induced caspase-3 activity (p <0.05) in comparison with the UCG. Therefore, the MG132 + DOX group demonstrated most important induction of caspase-3 activity (p <0.05) in comparison with that of the other groups. In Figure 2c, we observe that the MG132, DOX, and MG132 + DOX groups induced caspase-8 activity in comparison with the UCG (p <0.05), the highest induction noted in the groups treated with MG132 or MG132 + DOX. In Figure 2d, we can also observe that the highest activity of caspase-9 was observed in DOX- or MG132 + DOX-treated groups (p <0.05).


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)

Apoptosis and caspase activity in U937 cells treated with Doxorubicin (DOX), MG132, or MG132 + DOX. U937 cells were incubated alone or in combination with MG132 (1 μM), DOX 1 μM, or MG132 + DOX for 24 h at 37°C in a humid atmosphere containing 5% CO2 in RPMI-S culture medium. After incubation, the cells were washed and apoptosis was assessed by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC) (a). For each sample, at least 20,000 events were acquired in a FACSAria-I cell sorter and the data were analyzed with FACSDiva software. The activity of caspase-3 (b), -8 (c), and −9 (d) was evaluated utilizing a caspase colorimetric staining kit. The results represent the mean ± the Standard deviation (SD) of three independent experiments performed in triplicate. Statistical analysis was performed by means of the Mann–Whitney U test. ●p <0.05 MG132 + DOX vs MG132, DOX, or the Untreated control group (UCG); *p <0.05 all groups vs the UCG.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Apoptosis and caspase activity in U937 cells treated with Doxorubicin (DOX), MG132, or MG132 + DOX. U937 cells were incubated alone or in combination with MG132 (1 μM), DOX 1 μM, or MG132 + DOX for 24 h at 37°C in a humid atmosphere containing 5% CO2 in RPMI-S culture medium. After incubation, the cells were washed and apoptosis was assessed by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC) (a). For each sample, at least 20,000 events were acquired in a FACSAria-I cell sorter and the data were analyzed with FACSDiva software. The activity of caspase-3 (b), -8 (c), and −9 (d) was evaluated utilizing a caspase colorimetric staining kit. The results represent the mean ± the Standard deviation (SD) of three independent experiments performed in triplicate. Statistical analysis was performed by means of the Mann–Whitney U test. ●p <0.05 MG132 + DOX vs MG132, DOX, or the Untreated control group (UCG); *p <0.05 all groups vs the UCG.
Mentions: At 24 h post-treatment, apoptosis was evaluated in U937 cells treated with the different schedules. In Figure 2a, we can observe that the UCG showed a lower percentage of apoptosis (7.8 ± 1.6%) compared with that of the groups treated exclusively with MG132 or with DOX (45.4 ± 5.3% and 55.1 ± 5.4% of apoptosis, respectively; p <0.05). Interestingly, cell cultures exposed to MG132 + DOX exhibited superior values of apoptosis in comparison with the cells treated only with one drug, with a percentage of apoptosis of 75.1 ± 0.5% (p <0.05 when comparing MG132 + DOX vs. MG132- or DOX-treated cells), with an average increase for the three groups representing ∆% = 1017 in relation to the UCG. In order to confirm caspase participation, we evaluated caspase activity; in Figure 2b, we are able to observe that MG132 proteasome inhibitor and DOX-induced caspase-3 activity (p <0.05) in comparison with the UCG. Therefore, the MG132 + DOX group demonstrated most important induction of caspase-3 activity (p <0.05) in comparison with that of the other groups. In Figure 2c, we observe that the MG132, DOX, and MG132 + DOX groups induced caspase-8 activity in comparison with the UCG (p <0.05), the highest induction noted in the groups treated with MG132 or MG132 + DOX. In Figure 2d, we can also observe that the highest activity of caspase-9 was observed in DOX- or MG132 + DOX-treated groups (p <0.05).

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