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Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level.

Munoz JL, Rodriguez-Cruz V, Ramkissoon SH, Ligon KL, Greco SJ, Rameshwar P - Oncotarget (2015)

Bottom Line: We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance.TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein.Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells.

View Article: PubMed Central - PubMed

Affiliation: New Jersey Medical School, Rutgers, Newark, NJ, USA.

ABSTRACT
Glioblastoma Multiforme (GBM), the most common and lethal adult primary tumor of the brain, showed a link between Sonic Hedgehog (SHH) pathway in the resistance to temozolomide (TMZ). PTCH1, the SHH receptor, can tonically represses signaling by endocytosis. We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance. TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein. Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells. Computational studies, real-time PCR, reporter gene studies, western blots, target protector oligos and ectopic expression identified miR-9 as the target of PTCH1 in resistant GBM cells with concomitant activation of SHH signaling. MiR-9 mediated increases in the drug efflux transporters, MDR1 and ABCG2. MiR-9 was increased in the tissues from GBM patients and in an early passage GBM cell line from a patient with recurrent GBM but not from a naïve patient. Pharmacological inhibition of SHH signaling sensitized the GBM cells to TMZ. Taken together, miR-9 targets PTCH1 in GBM cells by a SHH-independent method in GBM cells for TMZ resistance. The identified pathways could lead to new strategies to target GBM with combinations of drugs.

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Related in: MedlinePlus

Combined treatment to inhibit SHH and P-gp enhanced cell death with TMZ treatment(A) U87 and T98G were treated with 3 nM, 3 μM GDC-0449 and/or 200 μM TMZ. After 72 h, cell viability was accessed by Cell Titer Blue. The percent cell viability is presented as mean±SD, n = 4. (B) Whole cell extracts from U87 and T98G, treated with 3 μM GDC-0449 and/or 200 μM TMZ. After 72 h, the cells were studied for caspase 3. (C) The band densities for active caspase 3 in ‘B’ were normalized with the bands for β-actin.
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Figure 5: Combined treatment to inhibit SHH and P-gp enhanced cell death with TMZ treatment(A) U87 and T98G were treated with 3 nM, 3 μM GDC-0449 and/or 200 μM TMZ. After 72 h, cell viability was accessed by Cell Titer Blue. The percent cell viability is presented as mean±SD, n = 4. (B) Whole cell extracts from U87 and T98G, treated with 3 μM GDC-0449 and/or 200 μM TMZ. After 72 h, the cells were studied for caspase 3. (C) The band densities for active caspase 3 in ‘B’ were normalized with the bands for β-actin.

Mentions: U87 and T98G cells were treated with 3 nM or 3 μM GDC-0449 (Vis) and/or 200 μM TMZ for 72 h. Cell viability showed significant decrease in viable cells as compared to TMZ or Vis alone (Figure 5A). We performed similar treatment as for Figure 5A, except with 3 μM GDC-0449 (Vis) to blunt both SMO and P-gp. Whole cell extracts were studied for active caspase 3 by western blot. The results showed 30% increase in cleaved caspase 3 (active) when the cells were treated with Vis and TMZ GDC-0449 (Vis) (Figures 5B, 5C).


Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level.

Munoz JL, Rodriguez-Cruz V, Ramkissoon SH, Ligon KL, Greco SJ, Rameshwar P - Oncotarget (2015)

Combined treatment to inhibit SHH and P-gp enhanced cell death with TMZ treatment(A) U87 and T98G were treated with 3 nM, 3 μM GDC-0449 and/or 200 μM TMZ. After 72 h, cell viability was accessed by Cell Titer Blue. The percent cell viability is presented as mean±SD, n = 4. (B) Whole cell extracts from U87 and T98G, treated with 3 μM GDC-0449 and/or 200 μM TMZ. After 72 h, the cells were studied for caspase 3. (C) The band densities for active caspase 3 in ‘B’ were normalized with the bands for β-actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359226&req=5

Figure 5: Combined treatment to inhibit SHH and P-gp enhanced cell death with TMZ treatment(A) U87 and T98G were treated with 3 nM, 3 μM GDC-0449 and/or 200 μM TMZ. After 72 h, cell viability was accessed by Cell Titer Blue. The percent cell viability is presented as mean±SD, n = 4. (B) Whole cell extracts from U87 and T98G, treated with 3 μM GDC-0449 and/or 200 μM TMZ. After 72 h, the cells were studied for caspase 3. (C) The band densities for active caspase 3 in ‘B’ were normalized with the bands for β-actin.
Mentions: U87 and T98G cells were treated with 3 nM or 3 μM GDC-0449 (Vis) and/or 200 μM TMZ for 72 h. Cell viability showed significant decrease in viable cells as compared to TMZ or Vis alone (Figure 5A). We performed similar treatment as for Figure 5A, except with 3 μM GDC-0449 (Vis) to blunt both SMO and P-gp. Whole cell extracts were studied for active caspase 3 by western blot. The results showed 30% increase in cleaved caspase 3 (active) when the cells were treated with Vis and TMZ GDC-0449 (Vis) (Figures 5B, 5C).

Bottom Line: We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance.TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein.Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells.

View Article: PubMed Central - PubMed

Affiliation: New Jersey Medical School, Rutgers, Newark, NJ, USA.

ABSTRACT
Glioblastoma Multiforme (GBM), the most common and lethal adult primary tumor of the brain, showed a link between Sonic Hedgehog (SHH) pathway in the resistance to temozolomide (TMZ). PTCH1, the SHH receptor, can tonically represses signaling by endocytosis. We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance. TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein. Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells. Computational studies, real-time PCR, reporter gene studies, western blots, target protector oligos and ectopic expression identified miR-9 as the target of PTCH1 in resistant GBM cells with concomitant activation of SHH signaling. MiR-9 mediated increases in the drug efflux transporters, MDR1 and ABCG2. MiR-9 was increased in the tissues from GBM patients and in an early passage GBM cell line from a patient with recurrent GBM but not from a naïve patient. Pharmacological inhibition of SHH signaling sensitized the GBM cells to TMZ. Taken together, miR-9 targets PTCH1 in GBM cells by a SHH-independent method in GBM cells for TMZ resistance. The identified pathways could lead to new strategies to target GBM with combinations of drugs.

Show MeSH
Related in: MedlinePlus