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Combination of 9-aminoacridine with Campath-1H provides effective therapy for a murine model of adult T-cell leukemia.

Ju W, Zhang M, Petrus M, Maeda M, Pise-Masison CA, Waldmann TA - Retrovirology (2014)

Bottom Line: Alone, 9AA did not cause significant drops in surrogate tumor markers, soluble IL-2Rα or β2-micorglobulin (β2μ) levels with only a slight increase in survival of MET-1-bearing mice.Consistent with reduced tumor cell burden, combination treatment significantly increased survival of MET-1-bearing mice compared to mice treated with either drug alone.Consistent with increased tumor suppressor activity, we found increased PARP-1 cleavage in 9AA and combination treated cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, NIH, 10 Center Drive, Building 10, Room 4 N115, Bethesda, MD 20892-1374, USA. masisonc@mail.nih.gov.

ABSTRACT

Background: Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy of CD4+CD25+ lymphocytes caused by human T-cell lymphotropic virus type 1. While much progress has been made in understanding the mechanisms of cellular dysregulation, the prognosis for aggressive ATL still remains poor. Therefore, new therapeutic approaches need to be developed.

Results: Previously, we demonstrated that the viral protein Tax inactivates p53 in HTLV-1-infected T-cells. Here we show that 9-aminoacridine (9AA) through p53 reactivation and NF-κB inhibition has selective toxicity for infected leukemic cells independent of their p53 status. We further demonstrate that 9AA activates caspase-3/7 resulting in PARP cleavage. Next we investigated the efficacy of 9AA in the MET-1 ATL model. Alone, 9AA did not cause significant drops in surrogate tumor markers, soluble IL-2Rα or β2-micorglobulin (β2μ) levels with only a slight increase in survival of MET-1-bearing mice. However, in combination with Campath-1H, 9AA treatment resulted in low soluble IL-2Rα and β2μ levels at 2 and 4 weeks. Consistent with reduced tumor cell burden, combination treatment significantly increased survival of MET-1-bearing mice compared to mice treated with either drug alone. Splenic cells isolated from 9AA or combination treated mice showed increased p53 protein levels and transcriptional activity. Consistent with increased tumor suppressor activity, we found increased PARP-1 cleavage in 9AA and combination treated cells.

Conclusion: Our results indicate that targeting reactivation of p53 and inhibition of NF-κB with acridine-derivatives in combination with other chemotherapeutics could result in increased efficacy and selective killing of tumor cells.

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Related in: MedlinePlus

9AA increased expression of p53 and activity of caspase-3/7, downregulated the NF-κB pathway, and induced apoptosis in ATL cell lines. Jurkat, MT-1, ED40515 (-), and 43 Tb (-) cells were treated for 48 hours with and without 9AA at 5 μM. Data are representative of 3 independent experiments. (A) Western blot analysis of the expression levels of proteins in ATL cell lines before and after 9AA treatment. Cell lysates were immunoblotted with the indicated antibodies: PARP, p53, phospho-p65, p65, XIAP, and phosph-IKKα/β, phosphr-IκBα and IκBα. β-actin was used as an input control. (B) Detection of caspase-3/7 activity in ATL cell lines before and after 9AA treatment. Cells were measured for the activities of caspase-3/7. Fold change was calculated based on the activity of caspase-3/7 before treatment. Data are representative of 3 independent experiments and are presented as means ± SD. (C) Induction of p53 responsive genes in 9AA treated cells. Total cellular mRNA was extracted from cell lysates of 9AA (10 μM) or control cells 48 hours after treatment. Quantitative PCR was performed to measure the expression levels of hdm2 (left panel) and bax (right panel) genes. RNA levels of control treated cells were set at 1. Each sample was run in triplicate from two independent experiments. The MT1 cell line was run in triplicate from one experiment. Expression levels of p53 responsive genes were normalized to expression of gapdh for each cell line tested.
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Figure 3: 9AA increased expression of p53 and activity of caspase-3/7, downregulated the NF-κB pathway, and induced apoptosis in ATL cell lines. Jurkat, MT-1, ED40515 (-), and 43 Tb (-) cells were treated for 48 hours with and without 9AA at 5 μM. Data are representative of 3 independent experiments. (A) Western blot analysis of the expression levels of proteins in ATL cell lines before and after 9AA treatment. Cell lysates were immunoblotted with the indicated antibodies: PARP, p53, phospho-p65, p65, XIAP, and phosph-IKKα/β, phosphr-IκBα and IκBα. β-actin was used as an input control. (B) Detection of caspase-3/7 activity in ATL cell lines before and after 9AA treatment. Cells were measured for the activities of caspase-3/7. Fold change was calculated based on the activity of caspase-3/7 before treatment. Data are representative of 3 independent experiments and are presented as means ± SD. (C) Induction of p53 responsive genes in 9AA treated cells. Total cellular mRNA was extracted from cell lysates of 9AA (10 μM) or control cells 48 hours after treatment. Quantitative PCR was performed to measure the expression levels of hdm2 (left panel) and bax (right panel) genes. RNA levels of control treated cells were set at 1. Each sample was run in triplicate from two independent experiments. The MT1 cell line was run in triplicate from one experiment. Expression levels of p53 responsive genes were normalized to expression of gapdh for each cell line tested.

Mentions: Jurkat, MT-1, ED40515 (-), 43 Tb (-), ED40515 (+), and LM-Y1 cells were untreated or treated with 2 μM or 5 μM 9AA for 48 hours. After treatment, cells were stained with antibodies that detected active caspase-3 and cleaved PARP. A minor double positive population of active caspase-3/cleaved PARP was detected in all the cell lines in the absence of 9AA (Figure 2; top panels). A two- to 20-fold increase in caspase-3/cleaved PARP double positive cells was seen in the five ATL cell lines depending on the concentration of 9AA used (Figure 2). In contrast, the percentage of caspase-3/cleaved PARP double positive Jurkat cells did not change after 9AA treatment (Figure 2). Consistent with the increase in caspase-3/7 activity in 9AA treated MT-1, 43 Tb (-), and ED40515 (-) cells (Figure 3B), western blot analysis showed increased PARP cleavage (Figure 3A). These changes were not detected in 9AA treated Jurkat cells (Figure 3A and B). These results indicate that 9AA induces apoptosis in ATL cells.


Combination of 9-aminoacridine with Campath-1H provides effective therapy for a murine model of adult T-cell leukemia.

Ju W, Zhang M, Petrus M, Maeda M, Pise-Masison CA, Waldmann TA - Retrovirology (2014)

9AA increased expression of p53 and activity of caspase-3/7, downregulated the NF-κB pathway, and induced apoptosis in ATL cell lines. Jurkat, MT-1, ED40515 (-), and 43 Tb (-) cells were treated for 48 hours with and without 9AA at 5 μM. Data are representative of 3 independent experiments. (A) Western blot analysis of the expression levels of proteins in ATL cell lines before and after 9AA treatment. Cell lysates were immunoblotted with the indicated antibodies: PARP, p53, phospho-p65, p65, XIAP, and phosph-IKKα/β, phosphr-IκBα and IκBα. β-actin was used as an input control. (B) Detection of caspase-3/7 activity in ATL cell lines before and after 9AA treatment. Cells were measured for the activities of caspase-3/7. Fold change was calculated based on the activity of caspase-3/7 before treatment. Data are representative of 3 independent experiments and are presented as means ± SD. (C) Induction of p53 responsive genes in 9AA treated cells. Total cellular mRNA was extracted from cell lysates of 9AA (10 μM) or control cells 48 hours after treatment. Quantitative PCR was performed to measure the expression levels of hdm2 (left panel) and bax (right panel) genes. RNA levels of control treated cells were set at 1. Each sample was run in triplicate from two independent experiments. The MT1 cell line was run in triplicate from one experiment. Expression levels of p53 responsive genes were normalized to expression of gapdh for each cell line tested.
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Figure 3: 9AA increased expression of p53 and activity of caspase-3/7, downregulated the NF-κB pathway, and induced apoptosis in ATL cell lines. Jurkat, MT-1, ED40515 (-), and 43 Tb (-) cells were treated for 48 hours with and without 9AA at 5 μM. Data are representative of 3 independent experiments. (A) Western blot analysis of the expression levels of proteins in ATL cell lines before and after 9AA treatment. Cell lysates were immunoblotted with the indicated antibodies: PARP, p53, phospho-p65, p65, XIAP, and phosph-IKKα/β, phosphr-IκBα and IκBα. β-actin was used as an input control. (B) Detection of caspase-3/7 activity in ATL cell lines before and after 9AA treatment. Cells were measured for the activities of caspase-3/7. Fold change was calculated based on the activity of caspase-3/7 before treatment. Data are representative of 3 independent experiments and are presented as means ± SD. (C) Induction of p53 responsive genes in 9AA treated cells. Total cellular mRNA was extracted from cell lysates of 9AA (10 μM) or control cells 48 hours after treatment. Quantitative PCR was performed to measure the expression levels of hdm2 (left panel) and bax (right panel) genes. RNA levels of control treated cells were set at 1. Each sample was run in triplicate from two independent experiments. The MT1 cell line was run in triplicate from one experiment. Expression levels of p53 responsive genes were normalized to expression of gapdh for each cell line tested.
Mentions: Jurkat, MT-1, ED40515 (-), 43 Tb (-), ED40515 (+), and LM-Y1 cells were untreated or treated with 2 μM or 5 μM 9AA for 48 hours. After treatment, cells were stained with antibodies that detected active caspase-3 and cleaved PARP. A minor double positive population of active caspase-3/cleaved PARP was detected in all the cell lines in the absence of 9AA (Figure 2; top panels). A two- to 20-fold increase in caspase-3/cleaved PARP double positive cells was seen in the five ATL cell lines depending on the concentration of 9AA used (Figure 2). In contrast, the percentage of caspase-3/cleaved PARP double positive Jurkat cells did not change after 9AA treatment (Figure 2). Consistent with the increase in caspase-3/7 activity in 9AA treated MT-1, 43 Tb (-), and ED40515 (-) cells (Figure 3B), western blot analysis showed increased PARP cleavage (Figure 3A). These changes were not detected in 9AA treated Jurkat cells (Figure 3A and B). These results indicate that 9AA induces apoptosis in ATL cells.

Bottom Line: Alone, 9AA did not cause significant drops in surrogate tumor markers, soluble IL-2Rα or β2-micorglobulin (β2μ) levels with only a slight increase in survival of MET-1-bearing mice.Consistent with reduced tumor cell burden, combination treatment significantly increased survival of MET-1-bearing mice compared to mice treated with either drug alone.Consistent with increased tumor suppressor activity, we found increased PARP-1 cleavage in 9AA and combination treated cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, NIH, 10 Center Drive, Building 10, Room 4 N115, Bethesda, MD 20892-1374, USA. masisonc@mail.nih.gov.

ABSTRACT

Background: Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy of CD4+CD25+ lymphocytes caused by human T-cell lymphotropic virus type 1. While much progress has been made in understanding the mechanisms of cellular dysregulation, the prognosis for aggressive ATL still remains poor. Therefore, new therapeutic approaches need to be developed.

Results: Previously, we demonstrated that the viral protein Tax inactivates p53 in HTLV-1-infected T-cells. Here we show that 9-aminoacridine (9AA) through p53 reactivation and NF-κB inhibition has selective toxicity for infected leukemic cells independent of their p53 status. We further demonstrate that 9AA activates caspase-3/7 resulting in PARP cleavage. Next we investigated the efficacy of 9AA in the MET-1 ATL model. Alone, 9AA did not cause significant drops in surrogate tumor markers, soluble IL-2Rα or β2-micorglobulin (β2μ) levels with only a slight increase in survival of MET-1-bearing mice. However, in combination with Campath-1H, 9AA treatment resulted in low soluble IL-2Rα and β2μ levels at 2 and 4 weeks. Consistent with reduced tumor cell burden, combination treatment significantly increased survival of MET-1-bearing mice compared to mice treated with either drug alone. Splenic cells isolated from 9AA or combination treated mice showed increased p53 protein levels and transcriptional activity. Consistent with increased tumor suppressor activity, we found increased PARP-1 cleavage in 9AA and combination treated cells.

Conclusion: Our results indicate that targeting reactivation of p53 and inhibition of NF-κB with acridine-derivatives in combination with other chemotherapeutics could result in increased efficacy and selective killing of tumor cells.

Show MeSH
Related in: MedlinePlus