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New preclinical strategies for characterization and development of anticancer drugs
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala university.ORCID iD: 0000-0003-3901-383X
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

Increased understanding of the molecular mechanisms underlying cancer development has shifted drug discovery towards target driven drug development the last decades, but the development of effective cancer drugs has been hampered by the lack of predictive preclinical models. 3-D cultures, considered to more accurately reflect solid tumors in vivo, have been proposed as one way to increase the predictability of clinical efficacy in cancer drug discovery and development.

The aims of this thesis were to improve preclinical models for cancer drug development, with focus on colorectal cancer (CRC) and use of multicellular tumor spheroids (MCTS), and also to mechanistically characterize some potentially new anticancer drugs (papers I – IV). The most important technical improvement was the development of direct measurement of green fluorescent protein (GFP) marked cells in spheroids, simplifying live collection of viability data and enabling high-throughput screening (HTS) in the MCTS model (paper I). In paper III and IV, the 3-D model was adapted to enable studies on the interaction between drugs and radiation. Two potentially new anticancer drugs, VLX50 and VLX60, were mechanistically characterized. VLX60, a novel copper containing thiosemicarbazone, induced reactive oxygen species (ROS) formation, was selectively active against BRAF mutated colon cancer cells and exhibited anticancer activity in vivo (paper II). Furthermore, two potentially new anticancer drugs were found suitable for further development for use in combination with radiation (papers III and IV). In paper III, synergy with radiation in spheroids compared to monolayer cultured colon cancer cells was shown with the novel iron-chelating inhibitor of oxidative phosphorylation, VLX600. In paper IV, the antiprotozoal drug nitazoxanide was shown to sensitize quiescent clonogenic colon cancer cells to radiation.

In conclusion, introduction of measurement of fluorescence of GFP marked cells in spheroids makes clinically relevant 3-D models feasible for HTS experiments and characterization of candidate drugs and radiosensitizers in early cancer drug discovery and development. VLX60 has several characteristics suitable for further development into a cancer drug, notably against BRAF mutated colorectal cancer cells. VLX600 and nitazoxanide show radiosensitizing properties making them promising for further development for use as cancer drugs in combination with radiation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1378
Keyword [en]
colorectal cancer, spheroids, green fluorescent protein, VLX50, VLX60, VLX600, nitazoxanide, radiation, radiosensitizer
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
URN: urn:nbn:se:uu:diva-330999ISBN: 978-91-513-0101-3 (print)OAI: oai:DiVA.org:uu-330999DiVA: diva2:1148072
Public defence
2017-12-01, Enghoffsalen, Ing 50 bv, Akademiska sjukhuset, Sjukhusvägen, Uppsala, 14:00 (Swedish)
Opponent
Supervisors
Available from: 2017-11-09 Created: 2017-10-09 Last updated: 2017-11-09
List of papers
1. Loss of cancer drug activity in colon cancer HCT-116 cells during spheroid formation in a new 3-D spheroid cell culture system
Open this publication in new window or tab >>Loss of cancer drug activity in colon cancer HCT-116 cells during spheroid formation in a new 3-D spheroid cell culture system
2012 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 318, no 13, 1577-1585 p.Article in journal (Refereed) Published
Abstract [en]

Clinically relevant in vitro methods are needed to identify new cancer drugs for solid tumors. We report on a new 3-D spheroid cell culture system aimed to mimic the properties of solid tumors in vivo. The colon cancer cell lines HCT-116 wt and HCT-116 wt/GFP were grown as monolayers and for 3 or 6 days on 96-well NanoCulture (R) plates to form spheroids. Expression of surface markers, genes and hypoxia were assessed to characterize the spheroids and drug induced cytotoxicity was evaluated based on fluorescein diacetate (FDA) conversion by viable cells to fluorescent fluorescein or by direct measurement of fluorescence of GFP marked cells after a 72 h drug incubation. The cells reproducibly formed spheroids in the NanoCulture (R) plates with tight cell-attachment after 6 days. Cells in spheroids showed geno- and phenotypical properties reminiscent of hypoxic stem cells. Monolayer cultured cells were sensitive to standard and investigational drugs, whereas the spheroids gradually turned resistant. Similar results for cytotoxicity were observed using simplified direct measurement of fluorescence of GFP marked cells compared with FDA incubation. In conclusion, this new 3-D spheroid cell culture system provides a convenient and clinically relevant model for the identification and characterization of cancer drugs for solid tumors.

Keyword
Tumor cell, Cell culture, Monolayer, Spheroid, Cancer drug, Colon cancer
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-177550 (URN)10.1016/j.yexcr.2012.03.026 (DOI)000305591800011 ()
Available from: 2012-07-16 Created: 2012-07-16 Last updated: 2017-10-09Bibliographically approved
2. Mechanistic characterization of a copper containing thiosemicarbazone with potent antitumor activity
Open this publication in new window or tab >>Mechanistic characterization of a copper containing thiosemicarbazone with potent antitumor activity
Show others...
2017 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 8, no 18, 30217-30234 p.Article in journal (Refereed) Published
Abstract [en]

Background: The thiosemicarbazone CD 02750 (VLX50) was recently reported as a hit compound in a phenotype-based drug screen in primary cultures of patient tumor cells. We synthesized a copper complex of VLX50, denoted VLX60, and characterized its antitumor and mechanistic properties.

Materials and Methods: The cytotoxic effects and mechanistic properties of VLX60 were investigated in monolayer cultures of multiple human cell lines, in tumor cells from patients, in a 3-D spheroid cell culture system and in vivo and were compared with those of VLX50.

Results: VLX60 showed >= 3-fold higher cytotoxic activity than VLX50 in 2-D cultures and, in contrast to VLX50, retained its activity in the presence of additional iron. VLX60 was effective against non-proliferative spheroids and against tumor xenografts in vivo in a murine model. In contrast to VLX50, gene expression analysis demonstrated that genes associated with oxidative stress were considerably enriched in cells exposed to VLX60 as was induction of reactive oxygen. VLX60 compromised the ubiquitin-proteasome system and was more active in BRAF mutated versus BRAF wild-type colon cancer cells.

Conclusions: The cytotoxic effects of the copper thiosemicarbazone VLX60 differ from those of VLX50 and shows interesting features as a potential antitumor drug, notably against BRAF mutated colorectal cancer.

Place, publisher, year, edition, pages
IMPACT JOURNALS LLC, 2017
Keyword
cancer drug, thiosemicarbazone, spheroid, VLX60, BRAF
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-323035 (URN)10.18632/oncotarget.16324 (DOI)000400456200055 ()28415818 (PubMedID)
Funder
Swedish Cancer SocietySwedish Foundation for Strategic Research
Available from: 2017-06-01 Created: 2017-06-01 Last updated: 2017-10-09Bibliographically approved
3. A novel tumor spheroid model identifies selective enhancement of radiation by an inhibitor of oxidative phosphorylation
Open this publication in new window or tab >>A novel tumor spheroid model identifies selective enhancement of radiation by an inhibitor of oxidative phosphorylation
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(English)In: Article in journal (Other academic) Submitted
Place, publisher, year, edition, pages
Uppsala:
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-330981 (URN)
Available from: 2017-10-09 Created: 2017-10-09 Last updated: 2017-10-09
4. Selective radiosensitization by nitazoxanide of quiescent clonogenic colon cancer tumor cells
Open this publication in new window or tab >>Selective radiosensitization by nitazoxanide of quiescent clonogenic colon cancer tumor cells
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-330980 (URN)
Available from: 2017-10-09 Created: 2017-10-09 Last updated: 2017-10-09

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