Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

With the recent advent of gene therapy, anti-apoptotic therapy has been receiving spotlight as a potential modality to inhibit the deterioration of pump function in the failing heart. We hypothesized that anti-apoptotic therapy using survivin gene delivery will 1) salvage H9c2 cells exposed to doxorubicin toxicity, and 2) ameliorate the progressive decline in left ventricular function in a rat model of doxorubicin-induced cardiomyopathy. The in vitro data suggested that survivin successfully prevented cell death under doxorubicin stress by both direct and indirect/paracrine mechanisms. Doxorubicin-treated animals developed progressive left ventricular dysfunction as evident by echocardiography and invasive pressure-volume loop analysis, which was prevented by ultrasound-mediated survivin plasmid delivery, but not empty plasmid delivery. Post-mortem analysis of myocardial tissue indicated a lowered apoptotic index in survivin-treated hearts, with evidence of decrease in interstitial fibrosis. In conclusion, survivin gene therapy was shown to ameliorate doxorubicin-induced cardiomyopathy, by decreasing apoptosis and preventing adverse remodeling.

Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

Cancer is a group of diseases in which normal cells are converted to cells capable of autonomous growth and invasion. In the chemotherapeutic control of cancer, drugs are usually given systemically so they reach toxic levels in healthy cells as well as cancer cells. This causes serious side effects. Another important problem with chemotherapy is resistance developed to cytotoxic drugs (multi drug resistance). Doxorubicin (Dox) occupies a central position in the treatment of breast cancer. However doxorubicin induced cardiac toxicity is associated with a high incidence of morbidity and mortality. Resistance of malignant tumors to Dox is another important cause of treatment failure in patients with cancer. One approach to overcome Dox-related toxicity is to use polymeric drug carriers, which direct the Dox away from heart tissue, and allow usage of lower dosages. In this present study two different anti-cancer drug delivery methods were evaluated. Dox was encapsulated in PLGA microparticles by single and double microemulsion solvent evaporation techniques. The highest entrapment of doxorubicin within PLGA microspheres obtained by optimization of process parameters. A sustained release of doxorubicin was obtained for 20 days. Several protein transduction domains are known to have the ability to pass through biological membranes. One such peptide is HIV-1 TAT. In this study TAT was evaluated for its ability to carry Dox into Dox resistant MCF-7 tumor cells. Dox peptide conjugate was more potent than free drug. The concentration of drug in resistant cancer cells was increased indicating a partial reversal of drug resistance.

Resistance to anticancer drugs is a serious obstacle to cancer chemotherapy. A common form of multidrug resistance (MDR) is caused by the overexpression of transmembrane transporter proteins P-glycoprotein and MRP1, encoded by MDR1 and MRP1 genes, respectively. These proteins lead to reduced intracellular drug concentration and decreased cytotoxicity by means of their ability to pump the drugs out of the cells. Breast cancer tumor resistance is mainly associated with overexpression of P-gp/MDR1. Although some chemical MDR modulators aim to overcome MDR by impairing the function of P-gp, they exhibit severe toxicities limiting their clinical relevance. Consequently, selective blocking of the expression of P-gp/MDR1 specific mRNA through RNA interference strategy may be an efficient tool to reverse MDR phenotype and increase the success of chemotherapy. Aim of this study was re-sensitizing doxorubicin resistant breast cancer cells to anticancer agent doxorubicin by selective downregulation of P-gp/MDR1 mRNA. The effect of the selected MDR1 siRNA and MRP1 expression after MDR1 silencing was determined by qPCR analysis. XTT cell proliferation assay was performed to v determine the effect of MDR1 silencing on doxorubicin sensitivity.Intracellular drug accumulation and localization was investigated by confocal laser scanning microscopy after treatment with MDR1 siRNA or other MDR modulators / verapamil or promethazine. The role of P-gp in migration characteristics of resistant cells was evaluated by wound healing assay. The results demonstrated that approximately 90% gene silencing occurred by the selected siRNA targeting MDR1 mRNA. However the level of MRP1 mRNA did not change after MDR1 downregulation. Introduction of siRNA resulted in about 70% re-sensitization to doxorubicin. Silencing of P-gp encoding MDR1 gene resulted in almost complete restoration of the intracellular doxorubicin accumulation and re-localization of the drug to the nuclei. Despite the considerably high concentration of the modulators, verapamil and promethazine were not as effective as siRNA for reversal of the drug efflux. According to wound healing assay, MDR1 silencing did not have any effect on migration characteristics of resistant cells, that is, P-gp expression does not seem to affect the motility of the cells. Selected siRNA duplex was shown to effectively inhibit MDR1 gene expression, restore doxorubicin accumulation and localization, and enhance chemo-sensitivity of resistant cells, which makes it a suitable future candidate for therapeutic applications.

One of the main problems of current cancer chemotherapy is the lack of selectivity of anti-cancer drugs to tumor cells which leads to systemic toxicity and adverse side effects. In order to overcome these limitations, researches on controlled drug delivery systems have gained much attention. Nanoscale based drug delivery systems provide tumor targeting. Among many types of nanocarriers, superparamagnetic nanoparticles with their biocompatible polymer coatings can be targeted to an intented site by an external magnetic field. Thus, the drug can be carried to the targeted site safely. The aim of this study is to prepare poly(dl-lactic-co-glycolic acid) (PLGA) coated magnetic nanoparticles and load anti-cancer drug, doxorubicin to them. For this purpose, magnetite (Fe3O4) iron oxide nanoparticles were synthesized as a magnetic core material (MNP) and then coated with oleic acid. Oleic acid coated MNP (OA-MNP) was encapsulated into PLGA. Effects of different OA-MNP/PLGA ratios on magnetite entrapment efficiency were investigated. Doxorubicin loaded magnetic polymeric nanoparticles (DOX-PLGA-MNP) were prepared. After the characterization of prepared nanoparticles, their cytotoxic effects on MCF-7 cell line were studied. PLGA coated magnetic nanoparticles (PLGA-MNP) had a proper size and superparamagnetic character. The highest magnetite entrapment efficiency of PLGA-MNP was estimated as 63 % at 1:8 ratio. Cytotoxicity studies of PLGA-MNP did not indicate any notable cell death between the concentration ranges of 2 and 250 &mu / g ml-1. It was observed that DOX-PLGA-MNP showed significant cytotoxicity on MCF-7 cells compared to PLGA-MNP. The results showed that prepared nanoparticles have desired size and superparamagnetic characteristics without serious toxic effects on cells. These nanoparticles may be suitable for targeted drug delivery applications. The findings obtained from drug studies may contribute to further work.

Insensitivity to chemotherapy is an ongoing issue in cancer treatment, one that appears to be highly dependent on patient-specific variations. It has been shown clinically that while a subset of patients will successfully respond to a particular chemotherapeutic regimen, there exists another subset of patients who when exposed to the same course of therapy will remain resistant to treatment or exhibit signs of relapse after treatment has been administered. This discrepancy raises interesting questions regarding the role that patient-specific variations play in controlling the efficacy of chemotherapy treatment regimens. Doxorubicin (Dox) is a common chemotherapeutic agent used in the treatment of a variety of solid tumors and leukemias and resistance to Dox treatment is a major issue in cancer chemotherapy, oftentimes leading to patient relapse. To gain a deeper understanding of the processes that influence Dox resistance, we must first understand the mechanisms that underlie and contribute to Dox's toxicity. To this end, the metabolic reactions that activate Dox have been implicated as major determinants of Dox cytoxicity and as possible factors that control Dox resistance in cancer cells. There are several lines of evidence that redox-dependent metabolism plays a large role in Dox toxicity. The Dox bioactivation network is comprised of a system of reduction/oxidation (redox) reactions that lead to the formation of toxic Dox metabolites and reactive oxygen species (ROS). Moreover, multi-drug resistant acute lymphoblastic leukemia cells derived from relapsed patients have elevated levels of the antioxidant glutathione and show insensitivity to Dox treatment. The redox dependence of Dox bioactivation, the understanding that Dox treatment generates ROS, and the evidence that Dox resistant cells exhibit increased antioxidant capacity, suggest the possibility that redox pathways modulate the efficacy of Dox treatment in cancer cells. The overall objectives of the proposed dissertation, therefore, were to investigate how the redox properties of the Dox bioactivation network influence Dox toxicity in acute lymphoblastic leukemia cells, and to provide evidence that cell-specific variations in the intracellular levels of these redox components influences the degree to which Dox treatment will induce cancer cell death. The significant findings of this study are that the redox reactions involved in Dox metabolism are dual-natured, containing a toxicity-generating module characterized by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent Dox reductive conversion, as well as an ROS signal-generating module characterized by NADPH- and oxygen-dependent Dox redox cycling. The balance between the coupled redox reactions that comprise the toxicity- and ROS signal-generating modules of Dox bioactivation determines the sensitivity-phenotype of leukemia cells and phenotypic changes in the Dox-sensitivity of leukemia cells can be induced by the successful modulation of the Dox bioactivation network through the pharmacological inhibition of NADPH in a concentration- and cell type-dependent manner. This study highlights the importance of the intracellular redox network in controlling chemotherapy-induced ROS. The unequal distribution in antioxidant burden across the various intracellular antioxidant enzymes suggests a significant role for NADPH supply, as controlled by the enzyme glucose-6-phosphate dehydrogenase (G6PD), to the intracellular ROS buffering capacity of cells during instances of oxidative stress. Changes in G6PD activity were shown to promote protein-S-glutathionylation during oxidative stress conditions, thereby implicating G6PD in the modulation of redox-sensitive signal transduction pathways. The intracellular glutathione redox balance, a measure of the intracellular redox environment, can effectively regulate Dox-induced NF-κB signal transduction in leukemia cells. The systematic modulation of intracellular glutathione redox balance in leukemia cells by N-acetylcysteine (NAC) revealed an important role for protein S-glutathionylation mechanisms in the control of NF-κB signal transduction induced by Dox treatment. These findings identify the glutathione redox network as a potential therapeutic target for the systematic modulation of Dox sensitivity in cancer cells and elucidate the complex role that antioxidants such as NAC can play in modulating the effectiveness of Dox chemotherapy treatment regimens. Lastly, this study highlights the need for and the capacity of computational models to accurately describe the complex redox-reactions that contribute to Dox metabolism in leukemia cells. This study is groundbreaking in its use of computational modeling to analyze reversible electron transfer events between proteins using mass-action kinetics. The models developed in this study can accurately explain cytosolic doxorubicin bioactivation, intracellular hydrogen peroxide clearance, and kinase-specific S-glutathionylation, thereby showing that the use of comprehensive and/or relatively simple computational models can provide semi-quantitative predictions about the behavior of redox systems in mammalian cells as they relate to Dox-induced toxicity and Dox-induced cell signaling.

Despite the common usage of high-dose vitamin C among breast cancer patients, the published medical literature is not in agreement as to how mega-dose vitamin C may interact with conventional therapy to affect clinical outcomes. The purpose of this study was to investigate the interaction of mega-dose vitamin C with radiation therapy and with doxorubicin in the treatment of breast cancer. Cultures of EMT6 mouse mammary tumor cells were treated concurrently with varying dose of vitamin C and either radiation or doxorubicin. A clonogenic assay was then performed to determine the surviving fraction of the cells. The surviving fractions of cells in cultures receiving different doses of vitamin C were compared among themselves as well as with controls and dose response curves were generated. Results show that ascorbic acid administered in concentrations of 1 mM or 10 mM 4 hours before x-irradiation protected the cells from radiation-induced cytotoxicity. The dose-modifying factors for 1 mM and 10 mM ascorbic acid as compared to controls were 1.23 and 1.37 respectively. These results support the hypothesis that mega dose vitamin C, when taken concurrently with radiation therapy, protects cancer cells from the cytotoxic effects of ionizing radiation. No evidence was found to suggest that mega-dose vitamin C alters the antineoplastic effects of doxorubicin.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 128-159).

Rationale: In the breast cancer setting, anti-cancer therapies, including Doxorubicin (DOX) and Trastuzumab (TRZ), are associated with an increased risk of cardiotoxicity. There is a need to develop prophylactic cardioprotective agents to mitigate the cardiotoxic side effects of these common anti-cancer drugs. Objective: To investigate whether the anti-oxidant, N-acetylcysteine amide (NACA), can attenuate the drug-induced heart failure caused by DOX+TRZ in a murine model. Methods: A total of 100 female mice received one of the following drug regimens: i) saline; ii) NACA; iii) DOX; iv) TRZ; v) DOX+TRZ; vi) NACA+DOX; vii) NACA+TRZ; and viii) NACA+DOX+TRZ. Serial echocardiography was performed over a 10-day study period, after which the mice were euthanized for histological and biochemical analyses. Results: In mice receiving DOX, left ventricular ejection fraction (LVEF) decreased from 73±4% to 43±2% at day 10. In mice receiving DOX+TRZ, LVEF decreased from 72±3% to 32±2% at day 10. Prophylactic administration of NACA to mice receiving DOX or DOX+TRZ was cardio-protective with an LVEF of 62±3% and 55±3% at day 10, respectively. Histological and biochemical analyses demonstrated loss of cellular integrity, increased oxidative stress (OS), and increased cardiac apoptosis in mice treated with DOX+TRZ which was attenuated by the prophylactic administration of NACA. Conclusion: NACA attenuates the cardiotoxic side effects of DOX+TRZ in a murine model of chemotherapy induced cardiac dysfunction by decreasing OS and apoptosis. / October 2015