The human gut microbiota contributes to host metabolic processes. Diverse microbial metabolic enzymes can affect therapeutic agents, resulting in chemical modifications that alter drug efficacy and toxicology. These interactions may result in ineffective treatments and dose-limiting side effects, as shown by bacterial modifications of the cardiac drug digoxin and chemotherapy drug irinotecan, respectively. Yet, few drug-microbiome interactions have been characterized. Here, a platform is developed to screen for drug-microbiome interactions, validated by the isolation of a gut bacterium capable of inactivating the antineoplastic drug doxorubicin. Two hundred gut strains isolated from a healthy patient fecal sample were cultured in the presence of antibiotic and antineoplastic drugs to enrich for resistance and possible inactivation. Raoultella planticola was identified for its ability to inactivate doxorubicin anaerobically through whole cell and crude lysate assays. This activity was also observed in other Enterobacteriaceae and resulted in doxorubicin inactivation by the removal of its daunosamine sugar, likely mediated by a molybdopterin-dependent enzyme. Other potential drug-microbiome interactions were identified in this screen and can be analyzed further. This platform enables the identification of drug-microbiome interactions that can be used to study drug pharmacology, improve the efficacy of therapeutic treatments, and advance personalized medicine. / Thesis / Bachelor of Science (BSc) / The collection of microbes in the human intestinal tract, referred to as the gut microbiome, can modify therapeutic agents and change the efficacy of drug treatments. Identifying these interactions between drugs and the microbiome will help the study of drug metabolism, provide explanations for treatment failure, and enable more personalized health care. For this project, a platform was developed to isolate gut bacteria from human fecal samples and characterize bacteria that are capable of inactivating various antibiotics and anticancer drugs. Through this platform, the gut bacterium Raoultella planticola was found to inactivate doxorubicin, a commonly used anticancer drug. These results suggest that doxorubicin may be inactivated in the gut and demonstrates how this platform can be used to identify drug-microbiome interactions.

Doxorubicin (DOX) is an incessantly used chemotherapeutic drug that can cause detrimental dose-dependent effects such as cardiotoxicity and congestive heart failure. Studies have focused on therapeutic strategies such as exosomes derived from embryonic stem cell (ES-Exos) and antioxidants for example resveratrol; however, the function of mesenchymal stem cell-derived exosomes (MSC-Exos) have never been examined in DOX-induced pyroptosis. MSC-Exos maintains the therapeutic potential of exosome therapy without the ethical concerns. Hence, the current study focuses on determining whether MSC-Exos has the potential to ameliorate inflammation-induced cell death pyroptosis in our established in vitro DOX-induced cardiotoxicity (DIC) model. Rat embryonic cardiomyocytes (H9c2) were first exposed to DOX to stimulate pyroptosis, followed by subsequent treatment with MSC-Exos, with further analysis performed through immunocytochemistry, western blotting, and RT-PCR. We evaluated the therapeutic potential of MSC-Exos by investigating the pyroptotic initiator HMGB1 which binds to TLR4 resulting in the formation of the NLRP3 inflammasome that initiates pyroptosis by activating the pyroptotic markers, caspase-1, IL-1β and IL-18, and the pyroptotic executioner GSDMD. Our data depicted that treatment with MSC-Exos significantly (p

20 svenska barn diagnostiseras årligen med barntumören neuroblastom (NB). Läkemedelsresistens och intratumoral heterogenitet försvårar behandlingen och 50-60% av hög-risk NB-patienter drabbas av återfall. Subpopulationer av resistenta celler har identifierats i hög-risk NB-cellinjer, vilket belyser behovet av nya behandlingsalternativ. Dessa celler har visats vara känsliga mot antimetaboliter, som är S-fas specifika läkemedel. Syftet med detta projekt är att utvärdera antimetaboliter som singelbehan-dling eller i kombination med kemoterapi som en ny behandlingsmetod i resistenta NB-celler. Cellvia-bilitiet, cellåterväxt efter långtidsbehandling och cellcykelarrest samt cellcykelns förlopp analyserades på en panel bestående av de fem TP53-muterade ultra-högrisk NB-cellinjerna Kelly, SK-N-DZ, SK-N-AS, BE(2)-C och SK-N-FI. Läkemedlena som utvärderades var kemoterapierna doxorubicin (doxo) och cisplatin samt antimetaboliterna cytarabine (Ara-C), gemcitabine, 5-fluorouracil (5-FU) och hy-droxyurea (HU). Resultaten indikerar att Kelly och SK-N-FI var de cellinjer som var mest känsliga mot kombinationer av doxo tillsammans med antimetaboliter. Detta styrktes ytterligare av analysen av cellcykelarrest som visade att dessa cellinjer föredrar att arrestera i S-fas. Cellinjerna SK-N-DZ, SK-N-AS och BE(2)-C var mindre känsliga mot kombinationsbehandlingarna och visade preferens för att arrestera i andra faser av cellcykeln. Eftersom cellinjerna visade variationer i cellcykelarrest så föreslår vi en utvärdering av trippelkombinationer med läkemedel som är G2/M-fas specifika för att ytterligare eliminera resistenta subpopulationer. / Every year, 20 Swedish children are diagnosed with the pediatric tumor neuroblastoma (NB). Therapy resistance and intratumor heterogeneities complicate treatment of NB and ultimately, 50-60% of high- risk NB patients relapse. Subpopulations of resistant cells have been identified in high-risk NB cell lines, which elucidates the need for novel treatment options. These cells have been suggested to be sensitive to the S-phase specific drugs antimetabolites. The objective of this project is to evaluate antimetabolites as monotherapy and in combination with chemotherapy as a novel treatment option in high-risk NB. Analyses of cell viability, cell regrowth following long-term treatment, and cell cycle progression and mitotic arrest were performed in a panel of five TP53 mutated ultra-high risk NB cell lines, Kelly, SK- N-DZ, SK-N-AS, BE(2)-C, and SK-N-FI. The evaluated drugs were the chemotherapies doxorubicin (doxo) and cisplatin, and the antimetabolites cytarabine (Ara-C), gemcitabine, 5-fluorouracil (5-FU), and hydroxyurea (HU). Obtained results indicated that out of the tested cell lines, Kelly and SK-N-FI are the most sensitive to combinations of doxo with antimetabolites. This was further corroborated via analysis of cell cycle progression and mitotic arrest which demonstrated that Kelly and SK-N-FI have a preference for S-phase arrest. Cell lines SK-N-DZ, SK-N-AS, and BE(2)-C were less sensitive to combination treatments and showed preference for arrest in other phases of the cell cycle. Since resistant cell lines show variations in mitotic arrest, we suggest evaluating triple combinations with targeted treatments for G2/M-phase, in order to further eliminate resistant subpopulations.

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No / Objective: To investigate the use of the National Cancer Institute's hollow fiber assay (HFA) to evaluate and prioritize novel treatment strategies for clinical trials in the Ewing's sarcoma family of tumors (ESFT). Study design: The growth and morphology of ESFT cell lines in hollow fibers (HFs) was characterized in vitro and in vivo. Reliability and reproducibility were evaluated using doxorubicin. Results: The numbers of viable cells in all 6 ESFT cell lines increased with time in vitro (0 to 96 hours). The SKES-1 and A673 cell lines grew exponentially after implantation of HFs in nude mice at subcutaneous and intraperitoneal sites. ESFT cells formed highly organized distinctive morphology within the HFs in vitro and in vivo. The number of viable ESFT cells within the HFs decreased in a time-dependent (24 to 96 hours) and dose-dependent (1 to 10 mg/kg) manner after treatment with doxorubicin in vivo. Conclusions: The HFA is a versatile short-term in vivo model that may be exploited to predict efficacy of potential anticancer agents in ESFT cells. Tumor markers and pharmacodynamic endpoints may be quantified in the pure population of ESFT cells from within the HFs.

No / Inadequate drug delivery to tumours is now recognised as a key factor that limits the efficacy of anticancer drugs. Extravasation and penetration of therapeutic agents through avascular tissue are critically important processes if sufficient drug is to be delivered to be therapeutic. The purpose of this study is to develop an in silico model that will simulate the transport of the clinically used cytotoxic drug doxorubicin across multicell layers (MCLs) in vitro. Three cell lines were employed: DLD1 (human colon carcinoma), MCF7 (human breast carcinoma) and NCI/ADR-Res (doxorubicin resistant and P-glycoprotein [Pgp] overexpressing ovarian cell line). Cells were cultured on transwell culture inserts to various thicknesses and doxorubicin at various concentrations (100 or 50 microM) was added to the top chamber. The concentration of drug appearing in the bottom chamber was determined as a function of time by HPLC-MS/MS. The rate of drug penetration was inversely proportional to the thickness of the MCL. The rate and extent of doxorubicin penetration was no different in the presence of NCI/ADR-Res cells expressing Pgp compared to MCF7 cells. A mathematical model based upon the premise that the transport of doxorubicin across cell membrane bilayers occurs by a passive "flip-flop" mechanism of the drug between two membrane leaflets was constructed. The mathematical model treats the transwell apparatus as a series of compartments and the MCL is treated as a series of cell layers, separated by small intercellular spaces. This model demonstrates good agreement between predicted and actual drug penetration in vitro and may be applied to the prediction of drug transport in vivo, potentially becoming a useful tool in the study of optimal chemotherapy regimes.

Yes / Anthracyclines such as doxorubicin (Dox), widely used to treat various types of tumours, may result in induced testicular toxicity and oxidative stress. The present investigation was designed to determine whether exposure of isolated and purified mouse germ cells to Dox induces DNA damage in the form of strand breaks (presumably) resulting in apoptosis and to investigate the relative sensitivity of specific cell types. DNA damage was assessed using the Comet assay and the presence of apoptosis was determined by TUNEL assay. Isolated mouse germ cells were treated with different concentrations (0.05, 0.5 and 1 mM, respectively) of Dox, and fixed 1 h after treatment. The incidences of both DNA damage shown by single cell gel-electrophoresis and of apoptosis increased significantly in each specific cell type in a concentration-dependent manner. The DNA damage and apoptosis incidences gradually increased with concentration from 0.05 to 1 mM with Dox. Our results indicate that apoptosis plays a vital role in the induction of germ cell phase-specific toxicity caused by Dox with pre-meiotically and meiotically dividing spermatogonia and spermatocytes respectively as highly susceptible target cells. / Higher Education Funding Council for England (HEFCE)

Yes / Multidrug resistance (MDR) limits the beneficial outcomes of conventional breast cancer chemotherapy. Ras-related nuclear protein (Ran-GTP) plays a key role in these resistance mechanisms, assisting cancer cells to repair damage to DNA. Herein, we investigate the co-delivery of Ran-RCC1 inhibitory peptide (RAN-IP) and doxorubicin (DOX) to breast cancer cells using liposomal nanocarriers. A liposomal delivery system, co-encapsulating DOX, and RAN-IP, was prepared using a thin-film rehydration technique. Dual-loaded liposomes were optimized by systematic modification of formulation variables. Real-Time-Polymerase Chain Reaction was used to determine Ran-GTP mRNA expression. In vitro cell lines were used to evaluate the effect of loaded liposomes on the viability of breast and lung cancer cell lines. In vivo testing was performed on a murine Solid Ehrlich Carcinoma model. RAN-IP reversed the Ran-expression-mediated MDR by inhibiting the Ran DNA damage repair function. Co-administration of RAN-IP enhanced sensitivity of DOX in breast cancer cell lines. Finally, liposome-mediated co-delivery with RAN-IP improved the anti-tumor effect of DOX in tumor-bearing mice when compared to single therapy. This study is the first to show the simultaneous delivery of RAN-IP and DOX using liposomes can be synergistic with DOX and lead to tumor regression in vitro and in vivo.