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N-(2-hydroxyphenyl)acetamide (NA-2) and Temozolomide synergistically induce apoptosis in human glioblastoma cell line U87.

Hanif F, Perveen K, Jawed H, Ahmed A, Malhi SM, Jamall S, Simjee SU - Cancer Cell Int. (2014)

Bottom Line: MTT and TUNEL assays were used to detect the growth inhibitory effect and apoptotic activity of NA-2 alone and in combination with TMZ.The combine administration of NA-2 (0.33 mM) and temozolomide (0.1 mM) significantly enhanced the cell growth inhibition and apoptosis.Furthermore RT-PCR and imunocytochemistry data revealed that cooperative apoptosis induction was associated with increased ratio of Bax to Bcl-2 and active Caspase-3 expression.

View Article: PubMed Central - PubMed

Affiliation: Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270 Pakistan.

ABSTRACT

Background: Despite the modern therapies available for treating glioblastoma multiforme (GBM), it is still a deadly disease. The development of new therapeutic strategies for the management of gliomas is therefore crucial. The present study is designed to analyze the therapeutic potentials of synthetic compound N-(2-hydroxyphenyl)acetamide (NA-2) in the treatment of GBM as a single agent or in combination with Temozolomide (TMZ) on glioblastoma cells.

Methods: MTT and TUNEL assays were used to detect the growth inhibitory effect and apoptotic activity of NA-2 alone and in combination with TMZ. Synergy was assessed using combination Index method. The expression of apoptosis related markers Bax, Bcl-2 and caspase-3 were assessed by RT-PCR, whereas, the active caspase-3 protein expression was determined using imunocytochemistry.

Results: Both NA-2 and TMZ inhibited the growth of U87 in a dose dependent manner. The combine administration of NA-2 (0.33 mM) and temozolomide (0.1 mM) significantly enhanced the cell growth inhibition and apoptosis. Furthermore RT-PCR and imunocytochemistry data revealed that cooperative apoptosis induction was associated with increased ratio of Bax to Bcl-2 and active Caspase-3 expression.

Conclusion: Our findings support that NA-2 possesses strong apoptotic activity and the combined administration of NA-2 and TMZ may be therapeutically exploited for the management of GBM.

No MeSH data available.


Related in: MedlinePlus

Effect of NA-2, TMZ and their combination on Bcl-2, Bax and Caspase-3 expression. Representative bands for BCL-2, BAX and Caspase-3 detected by RT PCR in vehicle control and treated cells. The housekeeping gene β-actin was amplified as an internal control. Graphic illustration of Bcl-2, Bax, Bax/ Bcl-2 ratio and Caspase-3 gene after normalizing with their β-actin expression also shown. Combination treatment with NA-2 and TMZ induce marked changes in the expression of all three mentioned genes as compared to their individual treatment. Each bar represents mean ± S.E.M of three independent experiments. Significant difference between vehicle control and treated cells was indicated by *P <0.05, **P <0.01, ***P <0.001. The significant difference between the individual drugs NA-2 and TMZ and their combination treatment is indicated by $$P <0.01, $$$P <0.001 and ###P <0.001 respectively.
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Fig4: Effect of NA-2, TMZ and their combination on Bcl-2, Bax and Caspase-3 expression. Representative bands for BCL-2, BAX and Caspase-3 detected by RT PCR in vehicle control and treated cells. The housekeeping gene β-actin was amplified as an internal control. Graphic illustration of Bcl-2, Bax, Bax/ Bcl-2 ratio and Caspase-3 gene after normalizing with their β-actin expression also shown. Combination treatment with NA-2 and TMZ induce marked changes in the expression of all three mentioned genes as compared to their individual treatment. Each bar represents mean ± S.E.M of three independent experiments. Significant difference between vehicle control and treated cells was indicated by *P <0.05, **P <0.01, ***P <0.001. The significant difference between the individual drugs NA-2 and TMZ and their combination treatment is indicated by $$P <0.01, $$$P <0.001 and ###P <0.001 respectively.

Mentions: Our results clearly shows that treatment with NA-2 alone leads to significant decrease in Bcl-2 expression as compare to control While no significant difference was observed in case of TMZ treatment (Figure 4). Moreover, further decrease in Bcl-2 was observed in the group receiving combination treatment of TMZ with NA-2. On the contrary, combination treatment showed more significant increase in Bax expression as compared to the control and individual drug treatment and these changes in Bcl-2 and Bax gene expression leads to dramatic increase in Bax to Bcl-2 ratio in cells exposed to combined treatment of TMZ and NA-2 than their individual treatment. Likewise, profound increase in caspase-3 expression was observed in cells treated with TMZ and NA-2 in combination.Figure 4


N-(2-hydroxyphenyl)acetamide (NA-2) and Temozolomide synergistically induce apoptosis in human glioblastoma cell line U87.

Hanif F, Perveen K, Jawed H, Ahmed A, Malhi SM, Jamall S, Simjee SU - Cancer Cell Int. (2014)

Effect of NA-2, TMZ and their combination on Bcl-2, Bax and Caspase-3 expression. Representative bands for BCL-2, BAX and Caspase-3 detected by RT PCR in vehicle control and treated cells. The housekeeping gene β-actin was amplified as an internal control. Graphic illustration of Bcl-2, Bax, Bax/ Bcl-2 ratio and Caspase-3 gene after normalizing with their β-actin expression also shown. Combination treatment with NA-2 and TMZ induce marked changes in the expression of all three mentioned genes as compared to their individual treatment. Each bar represents mean ± S.E.M of three independent experiments. Significant difference between vehicle control and treated cells was indicated by *P <0.05, **P <0.01, ***P <0.001. The significant difference between the individual drugs NA-2 and TMZ and their combination treatment is indicated by $$P <0.01, $$$P <0.001 and ###P <0.001 respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4319240&req=5

Fig4: Effect of NA-2, TMZ and their combination on Bcl-2, Bax and Caspase-3 expression. Representative bands for BCL-2, BAX and Caspase-3 detected by RT PCR in vehicle control and treated cells. The housekeeping gene β-actin was amplified as an internal control. Graphic illustration of Bcl-2, Bax, Bax/ Bcl-2 ratio and Caspase-3 gene after normalizing with their β-actin expression also shown. Combination treatment with NA-2 and TMZ induce marked changes in the expression of all three mentioned genes as compared to their individual treatment. Each bar represents mean ± S.E.M of three independent experiments. Significant difference between vehicle control and treated cells was indicated by *P <0.05, **P <0.01, ***P <0.001. The significant difference between the individual drugs NA-2 and TMZ and their combination treatment is indicated by $$P <0.01, $$$P <0.001 and ###P <0.001 respectively.
Mentions: Our results clearly shows that treatment with NA-2 alone leads to significant decrease in Bcl-2 expression as compare to control While no significant difference was observed in case of TMZ treatment (Figure 4). Moreover, further decrease in Bcl-2 was observed in the group receiving combination treatment of TMZ with NA-2. On the contrary, combination treatment showed more significant increase in Bax expression as compared to the control and individual drug treatment and these changes in Bcl-2 and Bax gene expression leads to dramatic increase in Bax to Bcl-2 ratio in cells exposed to combined treatment of TMZ and NA-2 than their individual treatment. Likewise, profound increase in caspase-3 expression was observed in cells treated with TMZ and NA-2 in combination.Figure 4

Bottom Line: MTT and TUNEL assays were used to detect the growth inhibitory effect and apoptotic activity of NA-2 alone and in combination with TMZ.The combine administration of NA-2 (0.33 mM) and temozolomide (0.1 mM) significantly enhanced the cell growth inhibition and apoptosis.Furthermore RT-PCR and imunocytochemistry data revealed that cooperative apoptosis induction was associated with increased ratio of Bax to Bcl-2 and active Caspase-3 expression.

View Article: PubMed Central - PubMed

Affiliation: Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270 Pakistan.

ABSTRACT

Background: Despite the modern therapies available for treating glioblastoma multiforme (GBM), it is still a deadly disease. The development of new therapeutic strategies for the management of gliomas is therefore crucial. The present study is designed to analyze the therapeutic potentials of synthetic compound N-(2-hydroxyphenyl)acetamide (NA-2) in the treatment of GBM as a single agent or in combination with Temozolomide (TMZ) on glioblastoma cells.

Methods: MTT and TUNEL assays were used to detect the growth inhibitory effect and apoptotic activity of NA-2 alone and in combination with TMZ. Synergy was assessed using combination Index method. The expression of apoptosis related markers Bax, Bcl-2 and caspase-3 were assessed by RT-PCR, whereas, the active caspase-3 protein expression was determined using imunocytochemistry.

Results: Both NA-2 and TMZ inhibited the growth of U87 in a dose dependent manner. The combine administration of NA-2 (0.33 mM) and temozolomide (0.1 mM) significantly enhanced the cell growth inhibition and apoptosis. Furthermore RT-PCR and imunocytochemistry data revealed that cooperative apoptosis induction was associated with increased ratio of Bax to Bcl-2 and active Caspase-3 expression.

Conclusion: Our findings support that NA-2 possesses strong apoptotic activity and the combined administration of NA-2 and TMZ may be therapeutically exploited for the management of GBM.

No MeSH data available.


Related in: MedlinePlus