The synthesis and tumor cell growth inhibition by doxazolidine carbamate prodrugs are reported. The carbamates were designed for selective hydrolysis by one or more human carboxylesterases to release doxazolidine (Doxaz), the formaldehyde-oxazolidine of doxorubicin that cross-links DNA to trigger cell death. Simple butyl and pentyl, but not ethyl, carbamate prodrugs inhibited the growth of cancer cells that overexpress carboxylesterase CES1 (hCE1) and CES2 (hiCE). Relative CES1 and CES2 expression levels were determined by reverse transcription of the respective mRNAs, followed by polymerase chain reaction amplification. More complex structures with a p-aminobenzyl alcohol (PABA) self-eliminating spacer showed better growth inhibition (IC50=50 nM for Hep G2 liver cancer cells) while exhibiting reduced toxicity toward rat cardiomyocytes, relative to the parent drug doxorubicin. Pentyl 4-(N-doxazolidinylcarbonyloxymethyl)phenylcarbamate, the lead compound for further investigation, appears to be activated in Hep G2 cells that express both CES1 and CES2.
The mechanism of doxorubicin is compared with that of doxazolidine, a doxorubicin−formaldehyde conjugate. The IC50 for growth inhibition of 67 human cancer cell lines, but not cardiomyocytes, is 32-fold lower with doxazolidine than with doxorubicin. Growth inhibition by doxazolidine correlates better with growth inhibition by DNA cross-linking agents than with growth inhibition by doxorubicin. Doxorubicin induces G2/M arrest in HCT-116 colon cancer cells and HL-60 leukemia cells through a well-documented topoisomerase II dependent mechanism. Doxazolidine fails to induce a G2/M arrest in HCT-116 cells but induces apoptosis 4-fold better than doxorubicin. The IC50 for doxazolidine growth inhibition of HL-60/MX2 cells, a topoisomerase II deficient derivative of HL-60 cells, is 1420-fold lower than the IC50 for doxorubicin, and doxazolidine induces apoptosis 15-fold better. Further, doxazolidine has little effect in a topoisomerase II activity assay. These data indicate that doxorubicin and doxazolidine induce apoptosis via different mechanisms and doxazolidine cytotoxicity is topoisomerase II independent.
Abstract We have reported the synthesis and biological evaluation of a prodrug to a doxorubicin active metabolite. Under physiologic conditions, release of the active metabolite, a conjugate of doxorubicin with formaldehyde, occurs with a half-life of 1 hour. To direct this prodrug to tumor, we designed two conjugates of the prodrug, doxsaliform, with the αvβ3-targeting peptides, CDCRGDCFC (RGD-4C) and cyclic-(N-Me-VRGDf) (Cilengitide). We now report the synthesis of these doxsaliform-peptide conjugates and their evaluation using MDA-MB-435 cancer cells. A hydroxylamine ether tether was used to attach 5″-formyldoxsaliform to RGD-4C in its acyclic form via an oxime functional group. The construct acyclic-RGD-4C-doxsaliform showed good binding affinity for αvβ3 in the vitronection cell adhesion assay (IC50 = 10 nmol/L) and good growth inhibition of MDA-MB-435 breast cancer cells (IC50 = 50 nmol/L). In its bicyclic forms, RGD-4C showed less affinity for αvβ3 and significantly less water solubility. Cyclic-(N-Me-VRGDf) was modified by substitution of d-4-aminophenylalanine for d-phenylalanine to provide a novel attachment point for doxsaliform. The conjugate, cyclic-(N-Me-VRGDf-NH)-doxsaliform, maintained a high affinity for αvβ3 (IC50 = 5 nmol/L) in the vitronectin cell adhesion assay relative to the peptide bearing only the tether (0.5 nmol/L). The IC50 for growth inhibition of MDA-MB-435 cells was 90 nmol/L. Flow cytometry and growth inhibition experiments suggest that the complete drug construct does not penetrate through the plasma membrane, but the active metabolite does on release from the targeting group. These drug conjugates could have significantly reduced side effects and are promising candidates for in vivo evaluation in tumor-bearing mice.
Osteosarcoma is a malignant bone tumor that afflicts over 10,000 dogs and 450 adolescents yearly. Most dogs and approximately 25% of children succumb to metastatic disease. We identified elevated insulin‐like growth factor II mRNA binding protein 1 (IGF2BP1) as a biomarker of poor prognosis in canine osteosarcoma. IGF2BP1 is an oncofetal protein that regulates mRNA subcellular localization, nuclear export, stability and translation. This empowers IGF2BP1 to control expression of oncogene targets that contribute to cellular proliferation and resistance to chemotherapy. IGF2BP1 expression correlates with poor outcome in a variety of human cancers. We over‐expressed IGF2BP1 in a canine osteosarcoma cell line and used shRNA knockdown constructs to decrease expression in a human osteosarcoma cell line. IGF2BP1 over‐expressing clones show a 1.67 fold increase in doxorubicin resistance compared to control and IGF2BP1 expression correlates to IC50 values (r2 = 0.89). IGF2BP1 knockdown increased sensitivity to doxorubicin by ≥ 10 fold compared to control and significant reductions in cellular migration, invasion, proliferation, and tumor growth in nude mice were also observed (p < 0.05). These results support the hypothesis that elevated expression of IGF2BP1 may contribute to tumor progression and inadequate therapeutic response. Microarray analysis of control and IGF2BP1 over‐expressing cells was used to detect global changes in gene expression and to identify potential targets for IGF2BP1. Differentially expressed genes were cross‐referenced to the RNA‐Binding Protein Immunoprecipitation database to identify direct mRNA targets bound by IGF2BP1 in osteosarcoma. We identified 162 genes that were differentially expressed (FC ≥2, FDR< 0.05). Pathway analysis identified enrichment for genes involved in regulation of cell adhesion, migration, and extracellular matrix. We identified 13 differentially expressed genes that directly bind IGF2BP1 and are involved in cell adhesion and protein kinase activity. We investigated mechanisms that contribute to increased IGF2BP1 expression in panels of human and canine osteosarcoma cell lines and tumors. Using qPCR analysis, we observed genomic amplification in 35% of canine tumors and cell lines and correlated amplification with IGF2BP1 transcript expression (p < 0.05, r2 = 0.8). Genomic amplification was not observed in human cell lines. Significant loss of 3’UTR regulatory sequences was observed in 20% of canine cell lines (p<0.05). IGF2BP1 promoter analysis indicated that most regulatory elements were located within 580 bp of the start site of translation in both species including transcriptional response elements for Myc and β‐catenin. Overall, our data suggests that increased IGF2BP1 expression contributes to the development and progression of human and canine osteosarcoma. Multiple mechanisms contribute to elevated IGF2BP1 expression including gene amplification and altered mRNA expression and stability. These results can be used to develop new treatment strategies that target elevated IGF2BP1. Support or Funding Information Morris Animal Foundation, CVMBS Research Council, Cancer League of Colorado This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
We have reported the synthesis and biological evaluation of a prodrug to a doxorubicin active metabolite. Under physiologic conditions, release of the active metabolite, a conjugate of doxorubicin with formaldehyde, occurs with a half-life of 1 hour. To direct this prodrug to tumor, we designed two conjugates of the prodrug, doxsaliform, with the alphavbeta3-targeting peptides, CDCRGDCFC (RGD-4C) and cyclic-(N-Me-VRGDf) (Cilengitide). We now report the synthesis of these doxsaliform-peptide conjugates and their evaluation using MDA-MB-435 cancer cells. A hydroxylamine ether tether was used to attach 5''-formyldoxsaliform to RGD-4C in its acyclic form via an oxime functional group. The construct acyclic-RGD-4C-doxsaliform showed good binding affinity for alphavbeta3 in the vitronection cell adhesion assay (IC50 = 10 nmol/L) and good growth inhibition of MDA-MB-435 breast cancer cells (IC50 = 50 nmol/L). In its bicyclic forms, RGD-4C showed less affinity for alphavbeta3 and significantly less water solubility. Cyclic-(N-Me-VRGDf) was modified by substitution of D-4-aminophenylalanine for D-phenylalanine to provide a novel attachment point for doxsaliform. The conjugate, cyclic-(N-Me-VRGDf-NH)-doxsaliform, maintained a high affinity for alphavbeta3 (IC50 = 5 nmol/L) in the vitronectin cell adhesion assay relative to the peptide bearing only the tether (0.5 nmol/L). The IC50 for growth inhibition of MDA-MB-435 cells was 90 nmol/L. Flow cytometry and growth inhibition experiments suggest that the complete drug construct does not penetrate through the plasma membrane, but the active metabolite does on release from the targeting group. These drug conjugates could have significantly reduced side effects and are promising candidates for in vivo evaluation in tumor-bearing mice.
The purpose of this study was to explore the role of transcription factor Ets1 in estrogen receptor α (ERα)-positive breast cancer progression. We expressed human Ets1 or empty vector in four human ERα-positive breast cancer cell lines and observed increased colony formation. Further examination of cellular responses in stable Ets1-expressing MCF7 clones displayed increased proliferation, migration, and invasion. Ets1-expressing MCF7 tumors grown in the mammary fat pads of nude mice exhibited increased rates of tumor growth (7.36±2.47 mm3/day) compared to control MCF7 tumors (2.52±1.70 mm3/day), but maintained their dependence on estradiol for tumor growth. Proliferation marker Ki-67 staining was not different between control and Ets1-expressing tumors, but Ets1-expressing tumors exhibited large necrotic centers and elevated apoptotic staining. Ets1 was shown to cooperate with ERα and the p160 nuclear receptor coactivator (NCOA/SRC) family to increase activation of a consensus estrogen response element luciferase reporter construct. Ets1-expressing MCF7 cells also exhibited elevated expression of the ERα target genes, progesterone receptor and trefoil factor 1. Using GST-pulldown assays, Ets1 formed stable complexes containing both ERα and p160 nuclear receptor coactivators. Taken together, these data suggest that the Ets1-dependent estradiol sensitization of breast cancer cells and tumors may be partially due to the ability of Ets1 to cooperate with ERα and nuclear receptor coactivators to stimulate transcriptional activity of estrogen-dependent genes.
Abstract Osteosarcoma (OSA) is the most common bone tumor in children arising in the metaphyseal regions of the appendicular skeleton and frequently metastasizing to the lungs. Standard treatment for OSA of surgical resection with neoadjuvant and adjuvant chemotherapy using multi-agent dose intensive therapy has resulted in survival rates of 75%, however for patients with metastasis at diagnosis, the survival rate is only 20%. Consequently, increased understanding of the mechanisms that contribute to metastasis and resistance to therapy is needed. Unfortunately, the availability of OSA samples for study is extremely limited. However, over 10,000 canine patients spontaneously develop OSA annually and canine tumors share common histological features, genetic mutations and gene expression profiles with human OSA. To identify factors that contribute to metastasis and chemotherapeutic resistance of OSA, we assessed the gene expression signatures of normal bone and groups of primary canine OSA tumors surgically resected from dogs with short and long disease free intervals (DFI) following standard treatment of amputation and therapy with doxorubicin or platinum-based drugs. Since the average DFI following treatment in canine osteosarcoma is approximately 200 days, these groups included tumors from dogs with DFI<100 days and DFI>300 days. We identified IGF2 mRNA binding protein 1 (IGF2BP1) as a gene with elevated expression in osteosarcomas from patients with a DFI<100 days compared to the DFI>300 group (7-fold, p=0.047) and normal bone (920-fold, p<0.001). IGF2BP1 is an oncofetal protein that binds multiple mRNA targets to regulate their nuclear transport, stability, translation and subcellular localization. In five human OSA cell lines we measured an average 14-fold increase in IGF2BP1 mRNA transcripts compared to normal human osteoblasts. More importantly, we measured elevated mRNA transcripts (five-fold, p=0.0368) and protein levels (seven-fold) in the MG63.2 cell line, a metastatic variant of the MG63 human OSA cell line, implicating IGF2BP1 in metastasis of osteosarcoma. IGF2BP1 knockdown in shRNA-expressing MG63.2 clones reduced cell invasion by 42% (p<0.05). In addition, IGF2BP1 knockdown in MG63.2 cells resulted in a three-fold (p<0.001) decrease in cellular proliferation compared to cells stably expressing a non-targeted shRNA construct. A similar experiment was performed in vivo using a subcutaneous model in nude mice (n=5/group) where the IGF2BP1 knockdown tumors from two independent shRNA constructs displayed significantly delayed tumor appearance and reduced tumor volume compared to control at days 9 (p<0.05), 12 and 14 (p<0.001) following tumor cell inoculation. Finally, IGF2BP1 knockdown resulted in a two-fold increase in doxorubicin sensitivity in MG63.2 cells (95% confidence interval). Overall, these data suggest IGF2BP1 drives OSA growth, metastasis and chemotherapeutic resistance. Citation Format: Brian T. Kalet, Liza E. O'Donoghue, Dawn L. Duval. IGF2 mRNA binding protein 1 drives growth, metastasis and chemoresistance in osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3008. doi:10.1158/1538-7445.AM2013-3008
The anthracycline antitumor drug, doxorubicin (DOX), has long been used as a broad spectrum chemotherapeutic. The literature now documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced activity in vitro and in vivo. We have recently reported the design, synthesis, and preliminary evaluation of a doxorubicin-formaldehyde conjugate targeted, via 4-hydroxytamoxifen, to the estrogen receptor (ER) and antiestrogen binding site (AEBS), which are commonly present in breast cancer cells. The lead targeted doxorubicin-formaldehyde conjugate, called DOX-TEG-TAM, was found to possess superior cell growth inhibition characteristics relative to clinical doxorubicin and an untargeted control conjugate, especially in ER-negative, multidrug resistant MCF-7/Adr cells. The enhanced activity in the absence of estrogen receptor raised the possibility that targeting was also mediated via AEBS. Fluorescence microscopy of an ER-negative, AEBS-positive cell line as a function of time showed initial DOX-TEG-TAM localization in cytosol, in contrast to initial DOX and untargeted doxorubicin-formaldehyde conjugate localization in the nucleus. DOX-TEG-TAM was taken up by four AEBS-positive cell lines to a greater extent than doxorubicin and an untargeted doxorubicin-formaldehyde conjugate. Of the four cell lines, three were ER negative. DOX-TEG-TAM uptake was inhibited in a dose-dependent manner by the presence of a competing AEBS ligand. DOX-TEG-TAM retains 60% of the affinity of 4-hydroxytamoxifen for AEBS. DOX-TEG-TAM was also taken up by the AEBS-negative, ER-positive cancer cell line Rtx-6; with these cells uptake was inhibited in a dose-dependent manner by the ER ligand, estradiol. The data support the hypothesis that uptake of 4-hydroxytamoxifen targeted doxorubicin-formaldehyde conjugate is mediated by both the antiestrogen binding site and estrogen receptor.
Abstract Osteosarcoma (OSA) is the most common bone tumor in children. Dose intensive therapies have resulted in survival rates of 75%, however for patients with metastasis at diagnosis, the survival rate is only 20%, indicating the need for improved therapeutic strategies for metastatic osteosarcoma. Unfortunately, the availability of human OSA samples for study is extremely limited; however, over 10,000 canine patients spontaneously develop OSA annually and canine tumors share common histological features, genetic mutations and gene expression profiles with human OSA. To identify factors that contribute to metastasis and chemotherapeutic resistance of OSA, we assessed the gene expression signatures of normal bone and groups of primary canine OSA tumors surgically resected from dogs with short (<100 days) and long (>300 day) disease free intervals (DFI) following standard treatment. Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) was identified as a negative prognostic indicator in this canine OSA study. IGF2BP1 is an oncofetal protein that binds multiple RNA targets to regulate their nuclear transport, stability, translation and subcellular localization. We have further investigated the role of IGF2BP1 in the development and metastatic progression of both human and canine osteosarcoma. IGF2BP1 transcripts were virtually undetectable on microarray analysis of normal bone and up-regulated greater-than 65-fold in both canine tumor groups relative to normal bone. RT-qPCR analysis validated these findings demonstrating a stair-step pattern of expression in which normal bone had virtually no IGF2BP1 expression, and IGF2BP1 expression was elevated 132-fold and 915-fold compared to normal bone in the long and short DFI tumor groups, respectively. Array CGH analysis revealed no genomic amplification of the IGF2BP1 locus, but 3′ UTR truncation may contribute to increased IGF2BP1 mRNA half-life. Similarly, human osteosarcoma cell lines, on average, expressed 14-fold higher levels of IGF2BP1 transcripts compared to human osteoblasts. Further, in the MG63.2 cell line, a metastatic variant of the MG63 human OSA cell line, we measured elevated mRNA transcripts (five-fold, p=0.0368) and protein levels (seven-fold), implicating IGF2BP1 in metastasis of osteosarcoma. IGF2BP1 knockdown in shRNA-expressing MG63.2 clones resulted in a three-fold (p<0.001) decrease in cellular proliferation compared to cells stably expressing a non-targeted shRNA construct. IGF2BP1 knockdown also reduced cell migration and invasion using Boyden chamber-based assays (p<0.05). MG63.2 cells from two independent IGF2BP1 knockdown shRNA constructs grown as subcutaneous tumors in nude mice (n=5/group) exhibited significantly delayed tumor appearance and reduced volume compared to scrambled controls (p<0.001). Finally, IGF2BP1 stable knockdown cell lines were up to 10-fold (p<0.05) more sensitive to doxorubicin compared to the control knockdown cell line. The sensitivity to doxorubicin correlated directly with IGF2BP1 knockdown (p = 0.0049, r2 = 0.9901). Overall, these data implicate IGF2BP1 gene expression in the development, metastatic progression, and chemoresistance of human and canine osteosarcoma. Further, these studies provide evidence that spontaneous canine OSA represents a valuable model for both target discovery and the development of novel therapeutic strategies for the treatment of metastatic OSA. Citation Format: Liza E. Pfaff, Brian T. Kalet, Deanna D. Dailey, Dawn L. Duval. Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) drives growth, metastasis and chemoresistance in human and canine osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A23.
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