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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

AD198 (AD)- and DOX-induced apoptosis and caspase activation in tested K9TCC and K9OSA cell lines in vitro.Notes: (A) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours and induced apoptosis was measured using the TACS Anexin V-FITC assay using a flow cytometer. Relative apoptotic activities were normalized to control groups. Values represent mean ± standard error (n=9) of three replicates of three independent experiments. Paired Student’s t-tests comparing treatment to control groups (*P<0.05, **P<0.01, ***P<0.001) and comparing among DOX and AD treatment groups (##P<0.01, ###P<0.001) were used. (B) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours and caspase activities were measured using the Caspase-Glo 3/7 luminescence assay. Relative caspase activities were normalized to control. Values represent mean ± standard error (n=6) of two replicates of three independent experiments. Paired Student’s t-tests comparing treatment to control (***P<0.001) and comparing among DOX and AD treatments (##P<0.01) were used. (C) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours. The expressions of PARP(CF) were evaluated by WB analysis. Actin was used as loading control. The results are representative of three independent experiments (n=3). (D) 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 two replicates of three independent experiments ± standard error (n=6). Paired Student’s t-tests were used to compare controls to DOX and AD treatments (*P<0.05, ***P<0.001) and to compare DOX to AD treatment (##P<0.01).Abbreviations: AD, AD198; PARP(CF), PARP-cleaved fragment; DOX, doxorubicin; K9OSA, canine osteosarcoma; K9TCC, canine transitional cell carcinoma; PARP, poly (ADP-ribose polymerase); PARP(T), total PARP; WB, Western blot.
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f3-dddt-9-5323: AD198 (AD)- and DOX-induced apoptosis and caspase activation in tested K9TCC and K9OSA cell lines in vitro.Notes: (A) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours and induced apoptosis was measured using the TACS Anexin V-FITC assay using a flow cytometer. Relative apoptotic activities were normalized to control groups. Values represent mean ± standard error (n=9) of three replicates of three independent experiments. Paired Student’s t-tests comparing treatment to control groups (*P<0.05, **P<0.01, ***P<0.001) and comparing among DOX and AD treatment groups (##P<0.01, ###P<0.001) were used. (B) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours and caspase activities were measured using the Caspase-Glo 3/7 luminescence assay. Relative caspase activities were normalized to control. Values represent mean ± standard error (n=6) of two replicates of three independent experiments. Paired Student’s t-tests comparing treatment to control (***P<0.001) and comparing among DOX and AD treatments (##P<0.01) were used. (C) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours. The expressions of PARP(CF) were evaluated by WB analysis. Actin was used as loading control. The results are representative of three independent experiments (n=3). (D) 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 two replicates of three independent experiments ± standard error (n=6). Paired Student’s t-tests were used to compare controls to DOX and AD treatments (*P<0.05, ***P<0.001) and to compare DOX to AD treatment (##P<0.01).Abbreviations: AD, AD198; PARP(CF), PARP-cleaved fragment; DOX, doxorubicin; K9OSA, canine osteosarcoma; K9TCC, canine transitional cell carcinoma; PARP, poly (ADP-ribose polymerase); PARP(T), total PARP; WB, Western blot.

Mentions: A representative cell line from each type of cancer was selected to further evaluate the molecular mechanisms of AD198 and DOX actions. We selected K9TCC#2-Dakota and K9TCC#1-Zoe cell lines to further evaluate the AD198-and DOX-induced apoptosis based on chemosensitivity tests, as shown in Figure 2 and Table 2. DOX and AD198 both significantly increased apoptosis in tested K9TCC#2-Dakota and K9OSA#1-Zoe cell lines; furthermore, AD198 was more effective in inducing apoptosis than DOX treatment in both tested cell lines, as shown in Figure 3A. DOX and AD198 increased the caspase-3/7 activities, as shown in Figure 3B. AD198 was more effective in activating caspase-3/7 enzymes as compared to DOX in K9TCC#2-Dakota and K9OSA#1-Zoe cells. In addition, increased cleavage of PARP by AD198 and DOX in both tested cell lines were confirmed (Figure 3C). Densitometry analysis of cleaved PARP protein showed an up to threefold increase in K9TCC#2-Dakota and an up to fourfold increase in K9OSA#1-Zoe when treated with AD198, as shown in Figure 3D. A statistically significant increase in PARP cleavage was observed after AD198 treatment as compared to DOX treatment 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)

AD198 (AD)- and DOX-induced apoptosis and caspase activation in tested K9TCC and K9OSA cell lines in vitro.Notes: (A) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours and induced apoptosis was measured using the TACS Anexin V-FITC assay using a flow cytometer. Relative apoptotic activities were normalized to control groups. Values represent mean ± standard error (n=9) of three replicates of three independent experiments. Paired Student’s t-tests comparing treatment to control groups (*P<0.05, **P<0.01, ***P<0.001) and comparing among DOX and AD treatment groups (##P<0.01, ###P<0.001) were used. (B) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours and caspase activities were measured using the Caspase-Glo 3/7 luminescence assay. Relative caspase activities were normalized to control. Values represent mean ± standard error (n=6) of two replicates of three independent experiments. Paired Student’s t-tests comparing treatment to control (***P<0.001) and comparing among DOX and AD treatments (##P<0.01) were used. (C) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours. The expressions of PARP(CF) were evaluated by WB analysis. Actin was used as loading control. The results are representative of three independent experiments (n=3). (D) 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 two replicates of three independent experiments ± standard error (n=6). Paired Student’s t-tests were used to compare controls to DOX and AD treatments (*P<0.05, ***P<0.001) and to compare DOX to AD treatment (##P<0.01).Abbreviations: AD, AD198; PARP(CF), PARP-cleaved fragment; DOX, doxorubicin; K9OSA, canine osteosarcoma; K9TCC, canine transitional cell carcinoma; PARP, poly (ADP-ribose polymerase); PARP(T), total PARP; WB, Western blot.
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f3-dddt-9-5323: AD198 (AD)- and DOX-induced apoptosis and caspase activation in tested K9TCC and K9OSA cell lines in vitro.Notes: (A) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours and induced apoptosis was measured using the TACS Anexin V-FITC assay using a flow cytometer. Relative apoptotic activities were normalized to control groups. Values represent mean ± standard error (n=9) of three replicates of three independent experiments. Paired Student’s t-tests comparing treatment to control groups (*P<0.05, **P<0.01, ***P<0.001) and comparing among DOX and AD treatment groups (##P<0.01, ###P<0.001) were used. (B) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours and caspase activities were measured using the Caspase-Glo 3/7 luminescence assay. Relative caspase activities were normalized to control. Values represent mean ± standard error (n=6) of two replicates of three independent experiments. Paired Student’s t-tests comparing treatment to control (***P<0.001) and comparing among DOX and AD treatments (##P<0.01) were used. (C) K9TCC#2-Dakota and K9OSA#1-Zoe cells were treated with 1 μM DOX and 1 μM AD for 24 hours. The expressions of PARP(CF) were evaluated by WB analysis. Actin was used as loading control. The results are representative of three independent experiments (n=3). (D) 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 two replicates of three independent experiments ± standard error (n=6). Paired Student’s t-tests were used to compare controls to DOX and AD treatments (*P<0.05, ***P<0.001) and to compare DOX to AD treatment (##P<0.01).Abbreviations: AD, AD198; PARP(CF), PARP-cleaved fragment; DOX, doxorubicin; K9OSA, canine osteosarcoma; K9TCC, canine transitional cell carcinoma; PARP, poly (ADP-ribose polymerase); PARP(T), total PARP; WB, Western blot.
Mentions: A representative cell line from each type of cancer was selected to further evaluate the molecular mechanisms of AD198 and DOX actions. We selected K9TCC#2-Dakota and K9TCC#1-Zoe cell lines to further evaluate the AD198-and DOX-induced apoptosis based on chemosensitivity tests, as shown in Figure 2 and Table 2. DOX and AD198 both significantly increased apoptosis in tested K9TCC#2-Dakota and K9OSA#1-Zoe cell lines; furthermore, AD198 was more effective in inducing apoptosis than DOX treatment in both tested cell lines, as shown in Figure 3A. DOX and AD198 increased the caspase-3/7 activities, as shown in Figure 3B. AD198 was more effective in activating caspase-3/7 enzymes as compared to DOX in K9TCC#2-Dakota and K9OSA#1-Zoe cells. In addition, increased cleavage of PARP by AD198 and DOX in both tested cell lines were confirmed (Figure 3C). Densitometry analysis of cleaved PARP protein showed an up to threefold increase in K9TCC#2-Dakota and an up to fourfold increase in K9OSA#1-Zoe when treated with AD198, as shown in Figure 3D. A statistically significant increase in PARP cleavage was observed after AD198 treatment as compared to DOX treatment 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