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Protein expression profile of HT-29 human colon cancer cells after treatment with a cytotoxic daunorubicin-GnRH-III derivative bioconjugate.

ABSTRACT: Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau) was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III) derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac)-His-Asp-Trp-Lys(Da ?= Aoa)-Pro-Gly-NH2; Glp = pyroglutamic acid, Ac = acetyl; Aoa?=?aminooxyacetyl). This bioconjugate exerted in vitro cytostatic/cytotoxic effect on human breast, prostate and colon cancer cells, as well as significant in vivo tumor growth inhibitory effect on colon carcinoma bearing mice. In our previous studies, H-Lys(Dau = Aoa)-OH was identified as the smallest metabolite produced in the presence of rat liver lysosomal homogenate, which was able to bind to DNA in vitro. To get a deeper insight into the mechanism of action of the bioconjugate, changes in the protein expression profile of HT-29 human colon cancer cells after treatment with the bioconjugate or free daunorubicin were investigated by mass spectrometry-based proteomics. Our results indicate that several metabolism-related proteins, molecular chaperons and proteins involved in signaling are differently expressed after targeted chemotherapeutic treatment, leading to the conclusion that the bioconjugate exerts its cytotoxic action by interfering with multiple intracellular processes.

SUBMITTER: Schreier VN

PROVIDER: S-EPMC3981732 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Protein expression profile of HT-29 human colon cancer cells after treatment with a cytotoxic daunorubicin-GnRH-III derivative bioconjugate.

Schreier Verena Natalie VN Pethő Lilla L Orbán Erika E Marquardt Andreas A Petre Brindusa Alina BA Mező Gábor G Manea Marilena M

PloS one 20140409 4

Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau) was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III) derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac)-His-Asp-Trp-Lys(Da = Aoa)-Pro-Gly-NH2; Glp = pyroglutamic acid, Ac = acetyl; Aoa = aminoox ...[more]

PMID: 24718594

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Project description:As part of our research efforts in the area of titanium-based antitumor agents, we have investigated the cytotoxic activity of [Ti(4)(maltolato)(8)(mu-O)(4)], (Cp-R)(2)TiCl(2) and (Cp-R)CpTiCl(2) (R = CO(2)CH(3) and CO(2)CH(2)CH(3)), and three water-soluble titanocene-amino acid complexes-[Cp(2)Ti(aa)(2)]Cl(2) (aa = L: -cysteine, L: -methionine, and D: -penicillamine)-on the human colon adenocarcinoma cell line, HT-29. The capacity of [Ti(4)(maltolato)(8)(mu-O)(4)] to donate Ti(IV) to human apo-transferrin and its hydrolytic stability have been investigated and compared to the previously reported data on modified titanocenes with either hydrophilic ancillary ligands or the functionalized cyclopentadienyl ligands. Notably, the titanium-maltolato complex does not transfer Ti(VI) to human apo-transferrin at any time within the first seven days of its interaction, demonstrating the inert character of this species. Stability studies on these complexes have shown that titanocene complexes decompose at physiological pH while the [Ti(4)(maltolato)(8)(mu-O)(4)] complex is stable at this pH without any notable decomposition for a period of ten days. The antitumor activity of these complexes against colon cancer HT-29 cells was determined using an MTT cell viability assay at 72 and 96 h. The titanocene-amino acid and the (Cp-R)(2)TiCl(2)/(Cp-R)CpTiCl(2) (R = CO(2)CH(3)) complexes were not biologically active when human transferrin was absent; they also were inactive when human transferrin was present at dose-equivalent concentrations. (Cp-R)(2)TiCl(2) and (Cp-R)CpTiCl(2) (R = CO(2)CH(2)CH(3)) showed cytotoxic activity in HT-29 cells comparable to that which is displayed by titanocene dichloride. The titanium-maltolato complex had higher levels of cytotoxic activity than any other titanocene complex investigated. Transferrin may be important in protecting the titanium center from hydrolysis, but this may be achieved by selecting ligands that could result in hydrolytically stable, yet active, complexes.

Project description:5-fluorouracil (5-FU) and doxorubicin (DOX) are potent anti-tumour agents commonly used for colon and breast cancer therapy, respectively. However, their clinical application is limited by their side effects and the development of drug resistance. Honeybee venom is a complex mixture of substances that has been reported to be effective against different cancer cells. Its active compound is melittin, a positively charged amphipathic peptide that interacts with the phospholipids of the cell membrane, forming pores that enable the internalization of small molecules with cytotoxic activities,. and consequently, causing cell death. Some central nervous system (CNS) drugs have recently demonstrated great anti-cancer potential, both in vitro, in vivo and in clinical trials, being promising candidates for drug repurposing in oncology. The present work evaluated the anti-cancer efficacy of honeybee venom in combination with chemotherapeutic or CNS drugs in HT-29 colon and MCF-7 breast cancer cell lines. The chemical characterization of a Portuguese sample of honeybee venom was done by LC-DAD-ESI/MSn analysis. For single treatments, cells were incubated with increasing concentrations of bee venom. For combination treatments, increasing concentrations of bee venom were first combined with the half-maximal inhibitory concentration (IC50) of 5-FU and DOX, in HT-29 and MCF-7 cells, respectively. Cells were also treated with increasing concentrations of bee venom in combination with the IC50 value of four CNS drugs (fluphenazine, fluoxetine, sertraline and thioridazine). Cytotoxicity was evaluated by MTT and SRB assays. The combination index (CI) value was calculated using CompuSyn software, based on the Chou-Talalay method. Synergy scores of different reference models (HSA, Loewe, ZIP and Bliss) were also calculated using SynergyFinder. The results demonstrate that honeybee venom is active against HT-29 colon and MCF-7 breast cancer cells, having better anti-tumour activity in MCF-7 cells. It was found that bee venom combined with 5-FU and fluphenazine in HT-29 cells resulted in less cytotoxic effects compared to the co-treatment of fluoxetine, sertraline and thioridazine plus bee venom, which resulted in less than 15% of viable cells for the whole range of concentrations. The combination of MCF-7 cells with repurposed drugs plus honeybee venom resulted in better anti-cancer efficacies than with DOX, notably for lower concentrations. A combination of fluoxetine and thioridazine plus honeybee venom resulted in less than 40% of viable cells for all ranges of concentrations. These results support that the combination of honeybee venom with repurposed drugs and chemotherapeutic agents can help improve their anti-cancer activity, especially for lower concentrations, in both cell lines. Overall, the present study corroborates the enormous bioactive potential of honeybee venom for colon and breast cancer treatments, both alone and in combination with chemotherapy or repurposed drugs.

Project description:Resistance of tumor cells to chemotherapy, such as 5?fluorouracil (5?FU), is an obstacle for successful treatment of cancer. As a follow?up of a previous study we have investigated the effect of conditioned media (CM) from macrophages of M1 or M2 phenotypes on 5?FU cytotoxicity on the colon cancer cell lines HT?29 and CACO?2. HT?29 cells, but not CACO?2 cells, having been treated with a combination of M1 CM and 5?FU recovered their cell growth to a much larger extent compared to cells having been treated with 5?FU alone when further cultured for 7 days in fresh media. M1 CM treatment of HT?29, but not CACO?2 cells, induced cell cycle arrest in the G0/G1 and G2/M phases. 5?FU treatment induced accumulation of cells in S?phase in both HT?29 and CACO?2 cells. This accumulation of cells in S?phase was attenuated by combined M1 CM and 5?FU treatment in HT?29 cells, but not in CACO?2 cells. The mRNA expression of cell cycle regulatory proteins and 5?FU metabolic enzymes were analyzed in an attempt to find possible mechanisms for the M1 CM induced attenuation of 5?FU cytotoxicity in HT?29. Thymidylate synthetase (TS) and thymidine phosphorylase (TP) were found to be substantially downregulated and upregulated, respectively, in HT?29 cells treated with M1 CM, making them unlikely as mediators of reduced 5?FU cytotoxicity. Among cell cycle regulating proteins, p21 was induced in HT?29 cells, but not in CACO?2 cells, in response to M1 CM treatment. However, small interfering RNA (siRNA) knockdown of p21 had no effect on the M1 CM induced cell cycle arrest seen in HT?29 and neither did it change the growth recovery after combined treatment of HT?29 cells with M1 CM and 5?FU. In conclusion, treatment of HT?29 cells with M1 CM reduces the cytotoxic effect of 5?FU and this is mediated by a M1 CM induced cell cycle arrest in the G0/G1 and G2/M phases. So far, we lack an explanation why this action is absent in the CACO?2 cells. The current findings may be important for optimization of chemotherapy in colon cancer.

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Dataset Information

Protein expression profile of HT-29 human colon cancer cells after treatment with a cytotoxic daunorubicin-GnRH-III derivative bioconjugate.

Publications

Protein expression profile of HT-29 human colon cancer cells after treatment with a cytotoxic daunorubicin-GnRH-III derivative bioconjugate.

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