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Mapping of apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy.

Jangamreddy JR, Panigrahi S, Lotfi K, Yadav M, Maddika S, Tripathi AK, Sanyal S, Łos MJ - Oncotarget (2014)

Bottom Line: Majority of chronic myeloid leukemia patients experience an adequate therapeutic effect from imatinib however, 26-37% of patients discontinue imatinib therapy due to a suboptimal response or intolerance.The synthetic apoptin is able to inhibit cell proliferation in murine (32Dp210), human cell line (K562), and ex vivo in both imatinib-resistant and imatinib sensitive CML patient samples.The apoptin based single or combination therapy may be an additional option in CML treatment and eventually be feasible as curative therapy.

View Article: PubMed Central - PubMed

Affiliation: Dept. Clinical & Experimental Medicine, Integrative Regenerative Med. Center (IGEN), Linköping University, Sweden. Authors contributed equally.

ABSTRACT
Majority of chronic myeloid leukemia patients experience an adequate therapeutic effect from imatinib however, 26-37% of patients discontinue imatinib therapy due to a suboptimal response or intolerance. Here we investigated derivatives of apoptin, a chicken anemia viral protein with selective toxicity towards cancer cells, which can be directed towards inhibiting multiple hyperactive kinases including BCR-ABL1. Our earlier studies revealed that a proline-rich segment of apoptin interacts with the SH3 domain of fusion protein BCR-ABL1 (p210) and acts as a negative regulator of BCR-ABL1 kinase and its downstream targets. In this study we show for the first time, the therapeutic potential of apoptin-derived decapeptide for the treatment of CML by establishing the minimal region of apoptin interaction domain with BCR-ABL1. We further show that the apoptin decapeptide is able to inhibit BCR-ABL1 down stream target c-Myc with a comparable efficacy to full-length apoptin and Imatinib. The synthetic apoptin is able to inhibit cell proliferation in murine (32Dp210), human cell line (K562), and ex vivo in both imatinib-resistant and imatinib sensitive CML patient samples. The apoptin based single or combination therapy may be an additional option in CML treatment and eventually be feasible as curative therapy.

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Apoptin-derived proline-rich motif preferentially kills BCR-ABL1-positive cells(a) The effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl non-expressing 32DDSMZ cells (MTT assay). (b) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing 32Dp210 cells (MTT assay). (c) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing K562 cells (MTT assay). (d, e, f). Both Imatinib responsive and resistant patient samples are sensitive to apoptin derived decapeptide but not healthy donor samples. MTT assay results show a time dependent cell death by apoptin decapeptide at 16h, 36h and 48h. N=3. *p<0.05.
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Figure 4: Apoptin-derived proline-rich motif preferentially kills BCR-ABL1-positive cells(a) The effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl non-expressing 32DDSMZ cells (MTT assay). (b) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing 32Dp210 cells (MTT assay). (c) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing K562 cells (MTT assay). (d, e, f). Both Imatinib responsive and resistant patient samples are sensitive to apoptin derived decapeptide but not healthy donor samples. MTT assay results show a time dependent cell death by apoptin decapeptide at 16h, 36h and 48h. N=3. *p<0.05.

Mentions: To study the biological activity of the apoptin derived cell-penetrating synthetic peptide on murine 32Dp210 cell lines and human K562 cell lines expressing Bcr-Abl1p210, Tat-conjugated peptide (rkkrrqrrr-PKPPSKKRSC) was added at a concentration of 1μM to the growing cells in culture and cell survival was estimated by MTT cell survival assay at different time points over a period of 48 hours. The murine IL3-dependent primary hematopoietic murine cell line 32DDSMZ was used as the control cell line. In another set of parallel experiments a scrambled Tat-conjugated peptide sequence (rkkrrqrrr-PRRPSRSPKC) was used as treatment control. The results obtained from these three cell lines (32DDSMZ 32Dp210 and K562) treated with both test and control peptides are portrayed in figure 4a-c respectively. Cells grown without any treatment (control) were set to 100% proliferation and the cell survival was expressed as normalized average. As shown in figure 4a apoptin derived decapeptide treatment does not show any significant cellular toxicity among 32DDSMZ as compared to control and scrambled peptide treated cells. However, apoptin-derived decapeptide induced significant inhibition of cell proliferation and/or cell death among 32Dp210 as shown in figure 4b compared to control and scrambled peptide treated counterparts. These results further confirm the anti-proliferative effect of apoptin and apoptin derived peptides mediated through their SH3 domain interacting proline rich regions. Interestingly, similar peptide treatments on the BCR-ABL1p210 expressing K562 cells also have similar results (Fig. 4c).


Mapping of apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy.

Jangamreddy JR, Panigrahi S, Lotfi K, Yadav M, Maddika S, Tripathi AK, Sanyal S, Łos MJ - Oncotarget (2014)

Apoptin-derived proline-rich motif preferentially kills BCR-ABL1-positive cells(a) The effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl non-expressing 32DDSMZ cells (MTT assay). (b) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing 32Dp210 cells (MTT assay). (c) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing K562 cells (MTT assay). (d, e, f). Both Imatinib responsive and resistant patient samples are sensitive to apoptin derived decapeptide but not healthy donor samples. MTT assay results show a time dependent cell death by apoptin decapeptide at 16h, 36h and 48h. N=3. *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196195&req=5

Figure 4: Apoptin-derived proline-rich motif preferentially kills BCR-ABL1-positive cells(a) The effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl non-expressing 32DDSMZ cells (MTT assay). (b) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing 32Dp210 cells (MTT assay). (c) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing K562 cells (MTT assay). (d, e, f). Both Imatinib responsive and resistant patient samples are sensitive to apoptin derived decapeptide but not healthy donor samples. MTT assay results show a time dependent cell death by apoptin decapeptide at 16h, 36h and 48h. N=3. *p<0.05.
Mentions: To study the biological activity of the apoptin derived cell-penetrating synthetic peptide on murine 32Dp210 cell lines and human K562 cell lines expressing Bcr-Abl1p210, Tat-conjugated peptide (rkkrrqrrr-PKPPSKKRSC) was added at a concentration of 1μM to the growing cells in culture and cell survival was estimated by MTT cell survival assay at different time points over a period of 48 hours. The murine IL3-dependent primary hematopoietic murine cell line 32DDSMZ was used as the control cell line. In another set of parallel experiments a scrambled Tat-conjugated peptide sequence (rkkrrqrrr-PRRPSRSPKC) was used as treatment control. The results obtained from these three cell lines (32DDSMZ 32Dp210 and K562) treated with both test and control peptides are portrayed in figure 4a-c respectively. Cells grown without any treatment (control) were set to 100% proliferation and the cell survival was expressed as normalized average. As shown in figure 4a apoptin derived decapeptide treatment does not show any significant cellular toxicity among 32DDSMZ as compared to control and scrambled peptide treated cells. However, apoptin-derived decapeptide induced significant inhibition of cell proliferation and/or cell death among 32Dp210 as shown in figure 4b compared to control and scrambled peptide treated counterparts. These results further confirm the anti-proliferative effect of apoptin and apoptin derived peptides mediated through their SH3 domain interacting proline rich regions. Interestingly, similar peptide treatments on the BCR-ABL1p210 expressing K562 cells also have similar results (Fig. 4c).

Bottom Line: Majority of chronic myeloid leukemia patients experience an adequate therapeutic effect from imatinib however, 26-37% of patients discontinue imatinib therapy due to a suboptimal response or intolerance.The synthetic apoptin is able to inhibit cell proliferation in murine (32Dp210), human cell line (K562), and ex vivo in both imatinib-resistant and imatinib sensitive CML patient samples.The apoptin based single or combination therapy may be an additional option in CML treatment and eventually be feasible as curative therapy.

View Article: PubMed Central - PubMed

Affiliation: Dept. Clinical & Experimental Medicine, Integrative Regenerative Med. Center (IGEN), Linköping University, Sweden. Authors contributed equally.

ABSTRACT
Majority of chronic myeloid leukemia patients experience an adequate therapeutic effect from imatinib however, 26-37% of patients discontinue imatinib therapy due to a suboptimal response or intolerance. Here we investigated derivatives of apoptin, a chicken anemia viral protein with selective toxicity towards cancer cells, which can be directed towards inhibiting multiple hyperactive kinases including BCR-ABL1. Our earlier studies revealed that a proline-rich segment of apoptin interacts with the SH3 domain of fusion protein BCR-ABL1 (p210) and acts as a negative regulator of BCR-ABL1 kinase and its downstream targets. In this study we show for the first time, the therapeutic potential of apoptin-derived decapeptide for the treatment of CML by establishing the minimal region of apoptin interaction domain with BCR-ABL1. We further show that the apoptin decapeptide is able to inhibit BCR-ABL1 down stream target c-Myc with a comparable efficacy to full-length apoptin and Imatinib. The synthetic apoptin is able to inhibit cell proliferation in murine (32Dp210), human cell line (K562), and ex vivo in both imatinib-resistant and imatinib sensitive CML patient samples. The apoptin based single or combination therapy may be an additional option in CML treatment and eventually be feasible as curative therapy.

Show MeSH
Related in: MedlinePlus