Novel NBQ-48 as marker of hypoxic cells in 2D and 3D colon cancer cells

Abstract

This study presents the applicability of a novel nitro-substituted heterocyclic compound NBQ48, member of the family of compounds identified as 3 nitrobenzazolo[3, 2-a] quinolinium chloride salts (NBQS) as a screening tool to identify hypoxic tumor cells. The applicability was tested on COLO 205 colon cancer cells 2D and 3D cultures treated with NBQ48 to assess the formation of a bio-reduction fluorescent metabolite under hypoxic conditions in contrast, to those under aerobic environment. Hypoxic environment was created applying a controlled hypoxic gas chamber. Prior to testing the applicability of NBQ48 as a hypoxic fluorescent marker, cytotoxic studies where performed to identify a low-toxicity dose at 24 hours under aerobic and hypoxic environments that would allow the bio-reduction process with little toxicity. The differences in fluorescence emission after treatment was measured by fluorometer and fluorescence microscopy. Results indicated that cell treatments up to 24 hours with NBQ48 under aerobic environment did not reach an IC50 dose in COLO205 cells, however under hypoxic environment the IC50 reached at 100μM. The significant fluorescence increment after 24 and 48 hrs in 2D and 3D cultures treated with NBQ48 (75uM) at hypoxic in contrast to aerobic environments clearly demonstrated the need of a low oxygen content for the bio-reduction of the parent NBQ48. This study confirms the applicability of NBQ48 as markers for hypoxia in metabolically active 2D and 3D cultures. This hydrophilic hypoxic marker could additionally aid researchers in testing hypoxia activated pro-drugs for therapeutic applications in cancer as well as on other diseases where cellular hypoxia is a significant risk factor.

Keywords: 3D cultures; NBQ quinolinium salt; colon cancer; fluorescent marker; hypoxia.

Figure 1

NBQ48 Structures and proposed bio-reduction of NBQ48 under hypoxic conditions to form a fluorescent metabolite. The fluorescent microscopy image is an actual data from colon cancer cells after treatment with NBQ-48 as describe in the result section.

Figure 2

Fluorescence emission of 2D COLO205 cells exposed to NBQ48 (75μM) for 24 hours under hypoxia versus aerobic environment. A one way ANOVA with Dunnett’s multiple comparison test was performed to determine significance. Significant difference was observed in the fluorescence emission (adjusted p-value<0.001) of cultures treated with NBQ48 at hypoxic environment versus aerobic cultures.

Figure 3

Fluorescence emission of 2D COLO205 cells exposed to NBQ48 (75μM) for 48 hours under hypoxia versus aerobic environment. A one way ANOVA with Dunnett’s multiple comparison test was performed to determine significance. Significant difference was observed in the fluorescence emission (adjusted p-value <0.001) of cultures treated with NBQ48 at hypoxic environment versus aerobic cultures.

Figure 4

Fluorescent microscopy analysis of COLO 205 cancer cells treated with NBQ48 to detect fluorescent emission after 24 hours resulting from bio-reduction of NBQ48 into a fluorescent metabolite. Cell nucleus stained with Hoechst stain (blue) for imaging clarity of each cell.A) Aerobic cells w/o NBQ48; B) Aerobic cells with NBQ48; C) Hypoxic cells w/o NBQ48; D) Hypoxic cells with NBQ48.The greenish/fluorescent cells on D, indicates the hypoxic formed metabolite.

Figure 5

Comparison of fluorescence emission after enzymatic reduction of NBQ48 by Cytochrome P450.Average fluorescence values (FSU) per sample as follows: Blank 111.40FSU, Aerobic negative sample 158.96FSU, positive ABQ spike 2476.73FSU and NBQ48 experimental of 1486.68FSU.

Figure 6

Fluorescence emission of 3D COLO205 cells exposed to NBQ48 (75μM) for 24 hours under hypoxia versus aerobic environment. A one way ANOVA with Dunnett’s multiple comparison test was performed to determine significance. Significant difference was observed in the fluorescence emission (adjusted p-value <0.001) of cultures treated with NBQ48 at hypoxic environment versus aerobic cultures.

Figure 7

Fluorescent microscopy analysis of 3D COLO 205 cancer cells treated with NBQ48 to detect fluorescent emission after 24 hours resulting from bio-reduction of NBQ48 into a fluorescent metabolite. A) Aerobic cells w/o NBQ48; B) Aerobic cells with NBQ48; C) Hypoxic cells w/o NBQ48; D) Hypoxic cell with NBQ48.The greenish/fluorescent cells on D, indicates the hypoxic formed metabolite. Minimum auto fluorescence (background) of colon cells is also observed in images of aerobic samples A, B.