CNOB/ChrR6, a new prodrug enzyme cancer chemotherapy

Abstract

We report the discovery of a new prodrug, 6-chloro-9-nitro-5-oxo-5H-benzo(a)phenoxazine (CNOB). This prodrug is efficiently activated by ChrR6, the highly active prodrug activating bacterial enzyme we have previously developed. The CNOB/ChrR6 therapy was effective in killing several cancer cell lines in vitro. It also efficiently treated tumors in mice with up to 40% complete remission. 9-Amino-6-chloro-5H-benzo(a)phenoxazine-5-one (MCHB) was the only product of CNOB reduction by ChrR6. MCHB binds DNA; at nonlethal concentration, it causes cell accumulation in the S phase, and at lethal dose, it induces cell surface Annexin V and caspase-3 and caspase-9 activities. Further, MCHB colocalizes with mitochondria and disrupts their electrochemical potential. Thus, killing by CNOB involves MCHB, which likely induces apoptosis through the mitochondrial pathway. An attractive feature of the CNOB/ChrR6 regimen is that its toxic product, MCHB, is fluorescent. This feature proved helpful in in vitro studies because simple fluorescence measurements provided information on the kinetics of CNOB activation within the cells, MCHB killing mechanism, its generally efficient bystander effect in cells and cell spheroids, and its biodistribution. The emission wavelength of MCHB also permitted its visualization in live animals, allowing noninvasive qualitative imaging of MCHB in mice and the tumor microenvironment. This feature may simplify exploration of barriers to the penetration of MCHB in tumors and their amelioration.

Figure 1

Effect of MCHB on cell cycle and AnnexinV. a) Treatment with MCHB (0.1µM) results in increasing accumulation of cells in the S-phase with time. HCT 116 cells were stained with 7-AAD for cell cycle analysis by flow cytometry. b) Induction of AnnexinV as determined by AnnexinV-PE staining of the cells: red graph, untreated; blue graph, MCHB (1.0 µM; 1h)-treated cells.

Figure 2

Effect of MCHB on caspases, and mitochondria. a) Induction of caspase activities upon treatment of HCT 116 with 1 µM MCHB, relative to untreated cells or doxycycline (known to induce apoptosis through activation of caspase-8 and caspase-3) (*p<0.05); (b) Co-localization of MCHB with mitochondria of HCT 116 (stained with MitoTracker™ green FM) as visualized by confocal microscopy (60× magnification). c) MCHB effect on mitochondrial membrane potential: JC-1 staining of MCHB treated HCT 116 cells Increase in green staining (X-axis) and decrease in red staining (Y-axis) indicate mitochondrial depolarization.

Figure 3

Correlation between MCHB production from CNOB and JC cell killing. The in vitro GDEPT system involving different doses of GFP-expressing SL7838-chrR6, or SL7838 (control) bacteria was used to infect the JC cells exposed to CNOB. Relative fluorescence units (RFU) of GFP (indicative of the extent of cell infection by the bacteria) and MCHB (indicative of the extent of CNOB reduction) (panel a) and cell killing (panel b) were measured as described in Materials and Methods. Symbols: Panel a): ▲ and ■, JC cells infection indicated by GFP levels by SL7838-chrR6 and SL7838, respectively – note that cancer cells were infected by both strains to the same extent; △ and □ MCHB generation by cells infected with SL7838-chrR6 and SL7838, respectively. Panel b) ▲ and ■, killing of SL7838-chrR6- and SL7838-infected cells, respectively. Note that despite equivalent degree of infection by the two bacterial strains, SL7838-chrR6 infected cells exhibit markedly more MCHB generation and cell killing.

Figure 4

In vitro imaging of MCHB within JC cancer cells. (a) Kinetics of CNOB reduction as visualized by the generation of MCHB fluorescence in JC cells infected with GFP-expressing SL7838-chrR6 at MOI of 10/cell. Cells were imaged by confocal microscopy at the indicated times (minutes) after CNOB addition (4×magnification). See Materials and Methods for details. The ‘GFP-30’ panel shows GFP fluorescence from bacterial cells at 30 minutes from monolayers to which no CNOB was added. Panel ‘C-30’ represents a control plate of cells treated with CNOB, but not SL7838-chrR6, imaged 30 minutes after the addition of CNOB. (b) An equivalent experiment was run and CNOB conversion to MCHB was quantified at the indicated times following CNOB addition in a fluorescence plate reader for cells with or without CNOB addition (■ and ▲, respectively). (c) The experiment of Fig. 3a was repeated using an MOI of 1.0 CFU/cell, resulting in approximately 20% of cells becoming infected. The representative image shown was taken 30 minutes after CNOB addition (20×magnification). (d) JC cells were used to form multicellular tumor spheroids. SL7838-chrR6 expressing GFP was added at an MOI of approximately 1000 CFU/spheroid and incubated for 30 minutes to permit infection. Images were taken by 2-photon microscope 1 hour after CNOB addition. Z-stack images were reconstructed into a three-dimensional model using Velocity software and snapshots were taken from different angles. Note that the image represents a cross section through the center of the spheroid and 95 µm depth.

Figure 5

a) Visualization of MCHB generation (red fluorescence) in JC implanted tumors in living mice by noninvasive imaging at 6 hours after CNOB administration. Lux-expressing SL7838-chrR6 bacteria were injected intratumorally, and CNOB (upper panel mouse only) intravenously 24 hours later. Luc and Lux luminescence represent the location of tumors and the bacteria, respectively. (The Luc signal includes Lux, but since the former was more than 50 fold greater, the latter is negligible; see Materials and Methods.) Note the absence of red fluorescence in tumors of mice not injected with bacteria but administered CNOB (lower panels). b) MCHB levels (quantified from fluorescence measurements) in blood samples collected by bleeding mice at indicated time points: ■) CNOB/SL7838-chrR6 treated mice; △) CNOB only treated mice. c) Qualitative visualization of MCHB generation in tumor in a living mouse (intravital microscopy) in relation to vasculature (white) and the ChrR6-generating bacteria (green). CNOB and SL7838-chrR6 were administered as above.

Figure 6

In vivo Efficacy of CNOB/ChrR6 treatment. Survival (Kaplan-Meier) curves of: a) Mice implanted with 4T1 tumors (50–100 mm³) endogenously expressing humanized ChrR6 (HChrR6) and treated with PBS (◆); 3.3 mg/kg CNOB (■); or 10 mg/kg CNOB (▲) (n=5 animals/group) (*p= 0.0018). b) Mice implanted with non-transfected 4T1 tumors (50–100 mm³) not expressing ChrR6 and treated with PBS (○); CNOB alone (●); SL7838-ChrR6 alone (▲); SL7838 and CNOB (▼); or SL7838-ChrR6 and CNOB 10 mg/kg (♦) (n=8 animals/group). (SL7838 and CNOB compared to SL7838-ChrR6 and CNOB, p=0.0012.) Bacteria (1×10⁵ CFU) were administered via intratumoral injection on day 0; CNOB (3.3mg/kg each time) was given on days 1, 3 and 5 via intravenous injection. (* p<0.05).