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A Synthetic Aptamer-Drug Adduct for Targeted Liver Cancer Therapy.

Trinh TL, Zhu G, Xiao X, Puszyk W, Sefah K, Wu Q, Tan W, Liu C - PLoS ONE (2015)

Bottom Line: AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure.In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox.We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure. AS1411 has shown promising utility as a treatment for cancers in Phase I and Phase II clinical trials without causing major side-effects. AS1411 inhibits tumor cell growth by binding to nucleolin which is aberrantly expressed on the cell membrane of many tumors. In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox. We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

No MeSH data available.


Related in: MedlinePlus

In vivo evaluation of AS1411-Dox adduct in Huh7 tumor xenograft mouse model.(a) Mice treated with free Dox or AS1411-Dox adduct had significant lower rates of tumor growth than those treated with AS1411 or control DNA-Dox adduct. (b, c) Side effects and cytotoxicity of each drug was illustrated by total body weight lost at the end of treatment (b) or activation of Caspase-3 in tumors, heart and kidney tissues (c). Mice treated with Dox lost 21.7% of their total body weight while with AS1411-Dox only 6.6% (P<0.001). Cleaved Caspase-3, which indicates the activation of Caspase-3 and induced apoptosis, was only identified in the heart and kidney of free Dox-treated mice, suggesting the specific cytotoxicity and reduced side effects of AS1411-Dox adduct compared to Dox.
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pone.0136673.g005: In vivo evaluation of AS1411-Dox adduct in Huh7 tumor xenograft mouse model.(a) Mice treated with free Dox or AS1411-Dox adduct had significant lower rates of tumor growth than those treated with AS1411 or control DNA-Dox adduct. (b, c) Side effects and cytotoxicity of each drug was illustrated by total body weight lost at the end of treatment (b) or activation of Caspase-3 in tumors, heart and kidney tissues (c). Mice treated with Dox lost 21.7% of their total body weight while with AS1411-Dox only 6.6% (P<0.001). Cleaved Caspase-3, which indicates the activation of Caspase-3 and induced apoptosis, was only identified in the heart and kidney of free Dox-treated mice, suggesting the specific cytotoxicity and reduced side effects of AS1411-Dox adduct compared to Dox.

Mentions: There are several well documented major side effects of doxorubicin treatment including; weight loss, cardio cytotoxicity and nephro cytotoxicity [32–38]. Doxorubicin is highly toxic and is administered intra-hepatically in normal circumstances [39]. To determine the efficacy of the AS1411-Dox adduct, a murine model of HCC was tested. The AS1411-Dox adduct was prepared as described and determined to have about 5.4 ± 0.1 copies of Dox per each strand of AS1411. Again we used a control DNA sequence which does not bind to target liver cancer cells, to prepare a control-Dox adduct. In brief, NOD Cg-Prkdc (scid) IL2 mice were each inoculated with 1 million Huh7 cells. Dorsal tumor nodules were allowed to grow to a volume of ~100 mm3 before treatment initiation. Tumor-bearing mice were randomly assigned into 4 groups (n = 5), and were treated by tail vein injection with either; (i) AS1411, (ii) free Dox, (iii) control-Dox adduct, or (iv) AS1411-Dox adduct, respectively. The Dox dosage was kept at 2 mg/kg, and the dosage of AS1411 in groups (i) and (iv) were equal. Tumor size and mouse weight were measured every other day, as indices of drug efficacy and non-specific cytotoxicity. The average tumor growth rates were calculated accordingly (Fig 5A). The mice treated with free Dox or AS1411-Dox adduct all had significantly lower rates of tumor growth than those treated with AS1411 alone or the control-Dox adduct. The control-Dox adduct also partially inhibited tumor progression, which is somewhat surprising. This is most likely due to the gradual release of Dox from the DNA, during an extended period of time in the circulation, as the control sequence does not target either tumor or normal cells. The antitumor efficacy of AS1411-Dox adduct was less than that of free doxorubicin, however the side effects of AS1411-Dox are greatly reduced compared to free Dox. At the end of treatment, the group of mice treated with free Dox lost 22.4% of total body weight on the average, while the group treated with the AS1411 Dox-adduct lost on average only 7.17% of total body weight (P<0.01) (Fig 5B). These side effects were further studied by examination of induced apoptosis in the heart and kidney tissue of mice treated in groups (i-iv). The induction of apoptosis in tumor tissue and heart and kidney tissue collected from mice was assessed by measuring the presence of cleaved Caspase 3 by western blot analysis. The data show that cleaved Caspase-3 was present in tumor samples collected from mice treated with the AS1411-Dox adduct, control-Dox adduct- and free Dox, indicating the cytotoxicity of Dox towards tumor tissue. There were no detectable levels of cleaved Caspase-3 in tumor samples treated with AS1411 only (Fig 5C). The data suggest that, under our experimental conditions, the aptamer alone did not induce apoptosis or cause cytotoxicity to either tumors or normal healthy tissue and that the effects of conjugating doxorubicin to AS1411 are beneficial. These data corroborate the previous observation that the AS1411 aptamer is not cytotoxic to Huh7 cells but a potent cell cycle inhibitor (Fig 2C). Unsurprisingly treatment with free Dox not only inhibited tumor growth, but also had the side effects of inducing apoptosis in heart and kidney tissues, providing evidence of typical Dox non-specific cytotoxicity, which is well documented. On the other hand, the AS1411-Dox adduct targeted tumor tissue with high specificity and induced tumor cell death, inhibiting tumor growth without inducing apoptosis in non-tumor tissues (Fig 5C). These results imply that the slight reduction in efficacy of AS1411-Dox could be compensated for by an increase in dosage as there were no major side effects detected for this adduct. The data provide evidence to suggest that the AS1411-Dox adduct has a similar tumor inhibition effect as that of free Dox, but with more targeted specificity to tumor and reduced non-specific systemic cytotoxicity to normal tissues. Conjugating Dox to AS1411 enables specific targeting of liver cells, and the majority of Dox is released in tumor cells, which provides a protective effect and prevents non-specific systemic cytotoxicity. The AS1411 aptamer has previously been shown to induce tumor cell apoptosis in glioma, leukemia, breast cancer and renal cell carcinoma [16,18,19,25,26,32]. Two recent studies have looked into the potential use of AS1411 in liver cancer cells. Yu and colleagues assessed the intracellular uptake of AS1411 conjugated to poly lactide co-glycolide (PLGA) nanoparticles [40]. The main findings were that AS1411 coated PLGA particles were preferentially absorbed by the HCC cell line, QGY-7703, when compared with an immortalized porcine hepatocyte cell line, HepLi [40,41]. The study showed that PLGA nanoparticles coated with AS1411 were absorbed by QGY-7703 cells by the binding of nucleolin and that nanoparticles entered the cell by endocytosis and macro pinocytosis [40]. This study provides additional supporting evidence to show that AS1411 targets liver cancer cells by the recognition of aberrantly expressed nucleolin on the surface of cancer cells. However, this study did not assess the properties of the unconjugated AS1411 aptamer and did not study any of the potential cytotoxic effects of AS1411 in vitro or in vivo. Another recent study by Zhang and colleagues utilized mesoporous nanoparticles conjugated with Dox and AS1411 and cytochrome c, this study assessed the efficacy of these novel conjugates in vitro and in vivo using Hep-G2 cells [42]. The study demonstrated that nanoparticles conjugated with Dox and AS1411 could reduce tumor growth in vivo while preventing associated weight loss in treated mice, thus also supporting AS1411 as a liver cancer specific drug. However, the study did not address concerns regarding the cardio cytotoxicity and renal cytotoxicity of nanoparticle conjugate with doxorubicin, which is an important aim in such a study. In addition, the study demonstrated the accumulation of nanoparticles in the liver, spleen and lung without assessing any putative side effects or potential toxicity, which may be caused by nanoparticle accumulation. This is an important issue, as silica based nanoparticles have been shown to induce an increase in the fraction of insoluble ubiquitinated proteins in the tissues that they accumulate in. Accumulation of these nanoparticles has been linked to accelerated ageing in c.elegans [43].


A Synthetic Aptamer-Drug Adduct for Targeted Liver Cancer Therapy.

Trinh TL, Zhu G, Xiao X, Puszyk W, Sefah K, Wu Q, Tan W, Liu C - PLoS ONE (2015)

In vivo evaluation of AS1411-Dox adduct in Huh7 tumor xenograft mouse model.(a) Mice treated with free Dox or AS1411-Dox adduct had significant lower rates of tumor growth than those treated with AS1411 or control DNA-Dox adduct. (b, c) Side effects and cytotoxicity of each drug was illustrated by total body weight lost at the end of treatment (b) or activation of Caspase-3 in tumors, heart and kidney tissues (c). Mice treated with Dox lost 21.7% of their total body weight while with AS1411-Dox only 6.6% (P<0.001). Cleaved Caspase-3, which indicates the activation of Caspase-3 and induced apoptosis, was only identified in the heart and kidney of free Dox-treated mice, suggesting the specific cytotoxicity and reduced side effects of AS1411-Dox adduct compared to Dox.
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pone.0136673.g005: In vivo evaluation of AS1411-Dox adduct in Huh7 tumor xenograft mouse model.(a) Mice treated with free Dox or AS1411-Dox adduct had significant lower rates of tumor growth than those treated with AS1411 or control DNA-Dox adduct. (b, c) Side effects and cytotoxicity of each drug was illustrated by total body weight lost at the end of treatment (b) or activation of Caspase-3 in tumors, heart and kidney tissues (c). Mice treated with Dox lost 21.7% of their total body weight while with AS1411-Dox only 6.6% (P<0.001). Cleaved Caspase-3, which indicates the activation of Caspase-3 and induced apoptosis, was only identified in the heart and kidney of free Dox-treated mice, suggesting the specific cytotoxicity and reduced side effects of AS1411-Dox adduct compared to Dox.
Mentions: There are several well documented major side effects of doxorubicin treatment including; weight loss, cardio cytotoxicity and nephro cytotoxicity [32–38]. Doxorubicin is highly toxic and is administered intra-hepatically in normal circumstances [39]. To determine the efficacy of the AS1411-Dox adduct, a murine model of HCC was tested. The AS1411-Dox adduct was prepared as described and determined to have about 5.4 ± 0.1 copies of Dox per each strand of AS1411. Again we used a control DNA sequence which does not bind to target liver cancer cells, to prepare a control-Dox adduct. In brief, NOD Cg-Prkdc (scid) IL2 mice were each inoculated with 1 million Huh7 cells. Dorsal tumor nodules were allowed to grow to a volume of ~100 mm3 before treatment initiation. Tumor-bearing mice were randomly assigned into 4 groups (n = 5), and were treated by tail vein injection with either; (i) AS1411, (ii) free Dox, (iii) control-Dox adduct, or (iv) AS1411-Dox adduct, respectively. The Dox dosage was kept at 2 mg/kg, and the dosage of AS1411 in groups (i) and (iv) were equal. Tumor size and mouse weight were measured every other day, as indices of drug efficacy and non-specific cytotoxicity. The average tumor growth rates were calculated accordingly (Fig 5A). The mice treated with free Dox or AS1411-Dox adduct all had significantly lower rates of tumor growth than those treated with AS1411 alone or the control-Dox adduct. The control-Dox adduct also partially inhibited tumor progression, which is somewhat surprising. This is most likely due to the gradual release of Dox from the DNA, during an extended period of time in the circulation, as the control sequence does not target either tumor or normal cells. The antitumor efficacy of AS1411-Dox adduct was less than that of free doxorubicin, however the side effects of AS1411-Dox are greatly reduced compared to free Dox. At the end of treatment, the group of mice treated with free Dox lost 22.4% of total body weight on the average, while the group treated with the AS1411 Dox-adduct lost on average only 7.17% of total body weight (P<0.01) (Fig 5B). These side effects were further studied by examination of induced apoptosis in the heart and kidney tissue of mice treated in groups (i-iv). The induction of apoptosis in tumor tissue and heart and kidney tissue collected from mice was assessed by measuring the presence of cleaved Caspase 3 by western blot analysis. The data show that cleaved Caspase-3 was present in tumor samples collected from mice treated with the AS1411-Dox adduct, control-Dox adduct- and free Dox, indicating the cytotoxicity of Dox towards tumor tissue. There were no detectable levels of cleaved Caspase-3 in tumor samples treated with AS1411 only (Fig 5C). The data suggest that, under our experimental conditions, the aptamer alone did not induce apoptosis or cause cytotoxicity to either tumors or normal healthy tissue and that the effects of conjugating doxorubicin to AS1411 are beneficial. These data corroborate the previous observation that the AS1411 aptamer is not cytotoxic to Huh7 cells but a potent cell cycle inhibitor (Fig 2C). Unsurprisingly treatment with free Dox not only inhibited tumor growth, but also had the side effects of inducing apoptosis in heart and kidney tissues, providing evidence of typical Dox non-specific cytotoxicity, which is well documented. On the other hand, the AS1411-Dox adduct targeted tumor tissue with high specificity and induced tumor cell death, inhibiting tumor growth without inducing apoptosis in non-tumor tissues (Fig 5C). These results imply that the slight reduction in efficacy of AS1411-Dox could be compensated for by an increase in dosage as there were no major side effects detected for this adduct. The data provide evidence to suggest that the AS1411-Dox adduct has a similar tumor inhibition effect as that of free Dox, but with more targeted specificity to tumor and reduced non-specific systemic cytotoxicity to normal tissues. Conjugating Dox to AS1411 enables specific targeting of liver cells, and the majority of Dox is released in tumor cells, which provides a protective effect and prevents non-specific systemic cytotoxicity. The AS1411 aptamer has previously been shown to induce tumor cell apoptosis in glioma, leukemia, breast cancer and renal cell carcinoma [16,18,19,25,26,32]. Two recent studies have looked into the potential use of AS1411 in liver cancer cells. Yu and colleagues assessed the intracellular uptake of AS1411 conjugated to poly lactide co-glycolide (PLGA) nanoparticles [40]. The main findings were that AS1411 coated PLGA particles were preferentially absorbed by the HCC cell line, QGY-7703, when compared with an immortalized porcine hepatocyte cell line, HepLi [40,41]. The study showed that PLGA nanoparticles coated with AS1411 were absorbed by QGY-7703 cells by the binding of nucleolin and that nanoparticles entered the cell by endocytosis and macro pinocytosis [40]. This study provides additional supporting evidence to show that AS1411 targets liver cancer cells by the recognition of aberrantly expressed nucleolin on the surface of cancer cells. However, this study did not assess the properties of the unconjugated AS1411 aptamer and did not study any of the potential cytotoxic effects of AS1411 in vitro or in vivo. Another recent study by Zhang and colleagues utilized mesoporous nanoparticles conjugated with Dox and AS1411 and cytochrome c, this study assessed the efficacy of these novel conjugates in vitro and in vivo using Hep-G2 cells [42]. The study demonstrated that nanoparticles conjugated with Dox and AS1411 could reduce tumor growth in vivo while preventing associated weight loss in treated mice, thus also supporting AS1411 as a liver cancer specific drug. However, the study did not address concerns regarding the cardio cytotoxicity and renal cytotoxicity of nanoparticle conjugate with doxorubicin, which is an important aim in such a study. In addition, the study demonstrated the accumulation of nanoparticles in the liver, spleen and lung without assessing any putative side effects or potential toxicity, which may be caused by nanoparticle accumulation. This is an important issue, as silica based nanoparticles have been shown to induce an increase in the fraction of insoluble ubiquitinated proteins in the tissues that they accumulate in. Accumulation of these nanoparticles has been linked to accelerated ageing in c.elegans [43].

Bottom Line: AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure.In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox.We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure. AS1411 has shown promising utility as a treatment for cancers in Phase I and Phase II clinical trials without causing major side-effects. AS1411 inhibits tumor cell growth by binding to nucleolin which is aberrantly expressed on the cell membrane of many tumors. In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox. We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

No MeSH data available.


Related in: MedlinePlus