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A novel derivative of doxorubicin, AD198, inhibits canine transitional cell carcinoma and osteosarcoma cells in vitro.

Rathore K, Cekanova M - Drug Des Devel Ther (2015)

Bottom Line: Because of the adverse effects of DOX, including hair loss, nausea, vomiting, liver dysfunction, and cardiotoxicity, novel derivatives of DOX have been synthesized and validated.AD198 had lower IC50 values as compared to DOX for all tested K9TCC and K9OSA cell lines.In addition, AD198 cleaved PKC-δ, which subsequently activated the p38 signaling pathway, resulting in the apoptosis of tested K9TCC and K9OSA cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Small Animal Clinical Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN, USA.

ABSTRACT
Doxorubicin (DOX) is one of the most commonly used chemotherapeutic treatments for a wide range of cancers. N-benzyladriamycin-14-valerate (AD198) is a lipophilic anthracycline that has been shown to target conventional and novel isoforms of protein kinase C (PKC) in cytoplasm of cells. Because of the adverse effects of DOX, including hair loss, nausea, vomiting, liver dysfunction, and cardiotoxicity, novel derivatives of DOX have been synthesized and validated. In this study, we evaluated the effects of DOX and its derivative, AD198, on cell viability of three canine transitional cell carcinoma (K9TCC) (K9TCC#1-Lillie, K9TCC#2-Dakota, K9TCC#4-Molly) and three canine osteosarcoma (K9OSA) (K9OSA#1-Zoe, K9OSA#2-Nashville, K9OSA#3-JJ) primary cancer cell lines. DOX and AD198 significantly inhibited cell proliferation in all tested K9TCC and K9OSA cell lines in a dose-dependent manner. AD198 inhibited cell viability of tested K9TCC and K9OSA cell lines more efficiently as compared to DOX at the same concentration using MTS (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium) assay. AD198 had lower IC50 values as compared to DOX for all tested K9TCC and K9OSA cell lines. In addition, AD198 increased apoptosis in all tested K9TCC and K9OSA cell lines. AD198 increased the caspase activity in tested K9TCC and K9OSA cell lines, which was confirmed by caspase-3/7 assay, and cleavage of poly (ADP-ribose) polymerase (PARP) was confirmed by Western blotting analysis. In addition, AD198 cleaved PKC-δ, which subsequently activated the p38 signaling pathway, resulting in the apoptosis of tested K9TCC and K9OSA cell lines. Inhibition of the p38 signaling pathway by SB203580 rescued DOX- and AD198-induced apoptosis in tested K9TCC and K9OSA cell lines. Our in vitro results suggest that AD198 might be considered as a new treatment option for K9TCC and K9OSA cell lines cancers in vivo.

No MeSH data available.


Related in: MedlinePlus

The inhibition of the p38 signaling pathway reduced AD198 (AD)- and DOX-induced apoptosis in tested K9TCC and K9OSA cell lines in vitro.Notes: (A) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. Caspase-3/7 activities were measured using the Caspase-Glo 3/7 luminescence assay. Relative caspase activities were normalized to control. The results are representative of three replicates in two independent experiments. Values represent mean ± standard error (n=6). Paired Student’s t-tests comparing treatment to control (*P<0.05, ***P<0.001) SB untreated to SB treatments ($$P<0.01, $$$P<0.001), and SB + DOX and SB + AD treatments (#P<0.05, ##P<0.01) were used. (B) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. The expressions of PARP (cleaved fragment) were evaluated by WB analysis. Actin was used as loading control. The results are representative of two independent experiments. (C) Densitometry evaluation of PARP protein bands from WB analysis was done using ImageJ software. Values represent the mean of measured densitometry of each protein’s band from three replicates from two independent experiments ± standard error (n=6). Paired Student’s t-tests were used to compare treatment to control groups (*P<0.05, ***P<0.001) and SB untreated to SB treatments groups ($$P<0.01, $$$P<0.001). (D) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. The expressions of p-ATF2 and p-CREB were evaluated by WB analysis. Actin was used as loading control. The results are representative of two independent experiments. (E) Densitometry evaluation of p-ATF2 and p-CREB protein bands from WB analysis was done using ImageJ software. Values represent the mean of measured densitometry of each protein’s band from three replicates from two independent experiments ± standard error (n=6). Paired Student’s t-tests comparing treatment to control (*P<0.05, **P<0.01, ***P<0.001), SB untreated to SB treatments ($P<0.05, $$P<0.01, $$$P<0.001), and SB + DOX and SB + AD treatments (#P<0.05, ##P<0.01) were used.Abbreviations: AD, AD198; ATF2, activating transcription factor 2; PARP(CF), PARP cleaved fragment; CREB, cyclic AMP response element binding protein; DOX, doxorubicin; K9OSA, canine osteosarcoma; K9TCC, canine transitional cell carcinoma; PARP, poly (ADP-ribose) polymerase; SB, SB203580; PARP(T), total PARP; WB, western blot.
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f5-dddt-9-5323: The inhibition of the p38 signaling pathway reduced AD198 (AD)- and DOX-induced apoptosis in tested K9TCC and K9OSA cell lines in vitro.Notes: (A) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. Caspase-3/7 activities were measured using the Caspase-Glo 3/7 luminescence assay. Relative caspase activities were normalized to control. The results are representative of three replicates in two independent experiments. Values represent mean ± standard error (n=6). Paired Student’s t-tests comparing treatment to control (*P<0.05, ***P<0.001) SB untreated to SB treatments ($$P<0.01, $$$P<0.001), and SB + DOX and SB + AD treatments (#P<0.05, ##P<0.01) were used. (B) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. The expressions of PARP (cleaved fragment) were evaluated by WB analysis. Actin was used as loading control. The results are representative of two independent experiments. (C) Densitometry evaluation of PARP protein bands from WB analysis was done using ImageJ software. Values represent the mean of measured densitometry of each protein’s band from three replicates from two independent experiments ± standard error (n=6). Paired Student’s t-tests were used to compare treatment to control groups (*P<0.05, ***P<0.001) and SB untreated to SB treatments groups ($$P<0.01, $$$P<0.001). (D) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. The expressions of p-ATF2 and p-CREB were evaluated by WB analysis. Actin was used as loading control. The results are representative of two independent experiments. (E) Densitometry evaluation of p-ATF2 and p-CREB protein bands from WB analysis was done using ImageJ software. Values represent the mean of measured densitometry of each protein’s band from three replicates from two independent experiments ± standard error (n=6). Paired Student’s t-tests comparing treatment to control (*P<0.05, **P<0.01, ***P<0.001), SB untreated to SB treatments ($P<0.05, $$P<0.01, $$$P<0.001), and SB + DOX and SB + AD treatments (#P<0.05, ##P<0.01) were used.Abbreviations: AD, AD198; ATF2, activating transcription factor 2; PARP(CF), PARP cleaved fragment; CREB, cyclic AMP response element binding protein; DOX, doxorubicin; K9OSA, canine osteosarcoma; K9TCC, canine transitional cell carcinoma; PARP, poly (ADP-ribose) polymerase; SB, SB203580; PARP(T), total PARP; WB, western blot.

Mentions: To confirm the role of the p38 signaling pathway in AD198- and DOX-induced apoptosis, we co-treated cells with a p38 inhibitor, SB203580. The inhibition of the p38 signaling pathway by SB203580 rescued DOX- and AD198-induced apoptosis in tested K9TCC#2-Dakota and K9OSA#1-Zoe cell lines, as shown in Figure 5. SB203580 rescued DOX- and AD198-induced caspase-3/7 activity (Figure 5A), inhibited the production of cleaved PARP (Figure 5B and C), and inhibited the phosphorylation of p-ATF2 and p-CREB (Figure 5D and E) in tested K9TCC and K9OSA cell lines.


A novel derivative of doxorubicin, AD198, inhibits canine transitional cell carcinoma and osteosarcoma cells in vitro.

Rathore K, Cekanova M - Drug Des Devel Ther (2015)

The inhibition of the p38 signaling pathway reduced AD198 (AD)- and DOX-induced apoptosis in tested K9TCC and K9OSA cell lines in vitro.Notes: (A) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. Caspase-3/7 activities were measured using the Caspase-Glo 3/7 luminescence assay. Relative caspase activities were normalized to control. The results are representative of three replicates in two independent experiments. Values represent mean ± standard error (n=6). Paired Student’s t-tests comparing treatment to control (*P<0.05, ***P<0.001) SB untreated to SB treatments ($$P<0.01, $$$P<0.001), and SB + DOX and SB + AD treatments (#P<0.05, ##P<0.01) were used. (B) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. The expressions of PARP (cleaved fragment) were evaluated by WB analysis. Actin was used as loading control. The results are representative of two independent experiments. (C) Densitometry evaluation of PARP protein bands from WB analysis was done using ImageJ software. Values represent the mean of measured densitometry of each protein’s band from three replicates from two independent experiments ± standard error (n=6). Paired Student’s t-tests were used to compare treatment to control groups (*P<0.05, ***P<0.001) and SB untreated to SB treatments groups ($$P<0.01, $$$P<0.001). (D) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. The expressions of p-ATF2 and p-CREB were evaluated by WB analysis. Actin was used as loading control. The results are representative of two independent experiments. (E) Densitometry evaluation of p-ATF2 and p-CREB protein bands from WB analysis was done using ImageJ software. Values represent the mean of measured densitometry of each protein’s band from three replicates from two independent experiments ± standard error (n=6). Paired Student’s t-tests comparing treatment to control (*P<0.05, **P<0.01, ***P<0.001), SB untreated to SB treatments ($P<0.05, $$P<0.01, $$$P<0.001), and SB + DOX and SB + AD treatments (#P<0.05, ##P<0.01) were used.Abbreviations: AD, AD198; ATF2, activating transcription factor 2; PARP(CF), PARP cleaved fragment; CREB, cyclic AMP response element binding protein; DOX, doxorubicin; K9OSA, canine osteosarcoma; K9TCC, canine transitional cell carcinoma; PARP, poly (ADP-ribose) polymerase; SB, SB203580; PARP(T), total PARP; WB, western blot.
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Related In: Results  -  Collection

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Show All Figures
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f5-dddt-9-5323: The inhibition of the p38 signaling pathway reduced AD198 (AD)- and DOX-induced apoptosis in tested K9TCC and K9OSA cell lines in vitro.Notes: (A) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. Caspase-3/7 activities were measured using the Caspase-Glo 3/7 luminescence assay. Relative caspase activities were normalized to control. The results are representative of three replicates in two independent experiments. Values represent mean ± standard error (n=6). Paired Student’s t-tests comparing treatment to control (*P<0.05, ***P<0.001) SB untreated to SB treatments ($$P<0.01, $$$P<0.001), and SB + DOX and SB + AD treatments (#P<0.05, ##P<0.01) were used. (B) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. The expressions of PARP (cleaved fragment) were evaluated by WB analysis. Actin was used as loading control. The results are representative of two independent experiments. (C) Densitometry evaluation of PARP protein bands from WB analysis was done using ImageJ software. Values represent the mean of measured densitometry of each protein’s band from three replicates from two independent experiments ± standard error (n=6). Paired Student’s t-tests were used to compare treatment to control groups (*P<0.05, ***P<0.001) and SB untreated to SB treatments groups ($$P<0.01, $$$P<0.001). (D) K9TCC#2-Dakota and K9OSA#1-Zoe cells were co-treated with p38 inhibitor SB (10 μM) in combination with 1 μM DOX and 1 μM AD for 24 hours. The expressions of p-ATF2 and p-CREB were evaluated by WB analysis. Actin was used as loading control. The results are representative of two independent experiments. (E) Densitometry evaluation of p-ATF2 and p-CREB protein bands from WB analysis was done using ImageJ software. Values represent the mean of measured densitometry of each protein’s band from three replicates from two independent experiments ± standard error (n=6). Paired Student’s t-tests comparing treatment to control (*P<0.05, **P<0.01, ***P<0.001), SB untreated to SB treatments ($P<0.05, $$P<0.01, $$$P<0.001), and SB + DOX and SB + AD treatments (#P<0.05, ##P<0.01) were used.Abbreviations: AD, AD198; ATF2, activating transcription factor 2; PARP(CF), PARP cleaved fragment; CREB, cyclic AMP response element binding protein; DOX, doxorubicin; K9OSA, canine osteosarcoma; K9TCC, canine transitional cell carcinoma; PARP, poly (ADP-ribose) polymerase; SB, SB203580; PARP(T), total PARP; WB, western blot.
Mentions: To confirm the role of the p38 signaling pathway in AD198- and DOX-induced apoptosis, we co-treated cells with a p38 inhibitor, SB203580. The inhibition of the p38 signaling pathway by SB203580 rescued DOX- and AD198-induced apoptosis in tested K9TCC#2-Dakota and K9OSA#1-Zoe cell lines, as shown in Figure 5. SB203580 rescued DOX- and AD198-induced caspase-3/7 activity (Figure 5A), inhibited the production of cleaved PARP (Figure 5B and C), and inhibited the phosphorylation of p-ATF2 and p-CREB (Figure 5D and E) in tested K9TCC and K9OSA cell lines.

Bottom Line: Because of the adverse effects of DOX, including hair loss, nausea, vomiting, liver dysfunction, and cardiotoxicity, novel derivatives of DOX have been synthesized and validated.AD198 had lower IC50 values as compared to DOX for all tested K9TCC and K9OSA cell lines.In addition, AD198 cleaved PKC-δ, which subsequently activated the p38 signaling pathway, resulting in the apoptosis of tested K9TCC and K9OSA cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of Small Animal Clinical Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN, USA.

ABSTRACT
Doxorubicin (DOX) is one of the most commonly used chemotherapeutic treatments for a wide range of cancers. N-benzyladriamycin-14-valerate (AD198) is a lipophilic anthracycline that has been shown to target conventional and novel isoforms of protein kinase C (PKC) in cytoplasm of cells. Because of the adverse effects of DOX, including hair loss, nausea, vomiting, liver dysfunction, and cardiotoxicity, novel derivatives of DOX have been synthesized and validated. In this study, we evaluated the effects of DOX and its derivative, AD198, on cell viability of three canine transitional cell carcinoma (K9TCC) (K9TCC#1-Lillie, K9TCC#2-Dakota, K9TCC#4-Molly) and three canine osteosarcoma (K9OSA) (K9OSA#1-Zoe, K9OSA#2-Nashville, K9OSA#3-JJ) primary cancer cell lines. DOX and AD198 significantly inhibited cell proliferation in all tested K9TCC and K9OSA cell lines in a dose-dependent manner. AD198 inhibited cell viability of tested K9TCC and K9OSA cell lines more efficiently as compared to DOX at the same concentration using MTS (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium) assay. AD198 had lower IC50 values as compared to DOX for all tested K9TCC and K9OSA cell lines. In addition, AD198 increased apoptosis in all tested K9TCC and K9OSA cell lines. AD198 increased the caspase activity in tested K9TCC and K9OSA cell lines, which was confirmed by caspase-3/7 assay, and cleavage of poly (ADP-ribose) polymerase (PARP) was confirmed by Western blotting analysis. In addition, AD198 cleaved PKC-δ, which subsequently activated the p38 signaling pathway, resulting in the apoptosis of tested K9TCC and K9OSA cell lines. Inhibition of the p38 signaling pathway by SB203580 rescued DOX- and AD198-induced apoptosis in tested K9TCC and K9OSA cell lines. Our in vitro results suggest that AD198 might be considered as a new treatment option for K9TCC and K9OSA cell lines cancers in vivo.

No MeSH data available.


Related in: MedlinePlus