Preclinical Evaluation of 1,2-Diamino-4,5-Dibromobenzene in Genetically Engineered Mouse Models of Pancreatic Cancer

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to standard chemo- and radiotherapy. Recently, a new class of non-platinum-based halogenated molecules (called FMD compounds) was discovered that selectively kills cancer cells. Here, we investigate the potential of 1,2-Diamino-4,5-dibromobenzene (2Br-DAB) in combination with standard chemotherapy and radiotherapy in murine and human PDAC. Methods: Cell viability and colony formation was performed in human (Panc1, BxPC3, PaTu8988t, MiaPaCa) and three murine LSL-Kras^G12D/+;LSL-Trp53^R172H/+;Pdx-1-Cre (KPC) pancreatic cancer cell lines. In vivo, preclinical experiments were conducted in LSL-Kras^G12D/+;p48-Cre (KC) and KPC mice using 2Br-DAB (7 mg/kg, i.p.), +/- radiation (10 × 1.8 Gy), gemcitabine (100 mg/kg, i.p.), or a combination. Tumor growth and therapeutic response were assessed by high-resolution ultrasound and immunohistochemistry. Results: 2Br-DAB significantly reduced cell viability in human and murine pancreatic cancer cell lines in a dose-dependent manner. In particular, colony formation in human Panc1 cells was significantly decreased upon 25 µM 2Br-DAB + radiation treatment compared with vehicle control (p = 0.03). In vivo, 2Br-DAB reduced tumor frequency in KC mice. In the KPC model, 2Br-DAB or gemcitabine monotherapy had comparable therapeutic effects. Furthermore, the combination of gemcitabine and 2Br-DAB or 2Br-DAB and 18 Gy irradiation showed additional antineoplastic effects. Conclusions: 2Br-DAB is effective in killing pancreatic cancer cells in vitro. 2Br-DAB was not toxic in vivo, and additional antineoplastic effects were observed in combination with irradiation.

Keywords: 1,2-Diamino-4,5-dibromobenzene; GEMMs; chemoresistance; femtomedicine compounds; pancreatic cancer; radiation therapy; radiosensitizer.

Figure 1

(A): Chemical structure of 2Br-DAB and cisplatin showing an aromatic ring system (2Br-DAB) or a central platin ion (cisplatin). Both molecules coordinate two halogenids and two amino residues [19]. (B–H): In vitro cell viability assays conducted in four different human ((B–E): Panc1, BxPC3, MiaPaca, PaTu8988t) and three murine (F–H) pancreatic cancer cell lines. After 72 h treatment with 2Br-DAB in various concentrations, cell viability was obtained by thiazolyl blue tetrazolium bromide (MTT) assay. Each graph represents results of at least two independent experiments. KPC—LSL-Kras^G12D/+;LSL-Trp53^R172H/+;Pdx-1-Cre.

Figure 2

(A): Weight development in LSL-Kras^G12D/+;p48-1-Cre (KC)-mice weekly treated by intraperitoneal injection with either 2Br-DAB at 7 mg/kg bodyweight (blue curve) or vehicle (red curve). Depicted are mean values of n = 16 2Br-DAB-treated and n = 15 vehicle-treated mice. (B): Survival analysis upon treatment start of 2Br-DAB (n = 16) and vehicle (n = 15)-treated KC-mice shows comparable survival times for KC-mice (244 days versus 281 days, p = 0.84, log-rank-test). (C): Tumor frequency determined for 2Br-DAB (n = 16; tumor frequency 50%) and vehicle (n = 15; tumor frequency 66%) treated KC-mice (p = 0.34, Chi square test). (D): H&E-staining for KC-mice showing histologic appearance of pancreatic ductal adenocarcinoma (PDAC) and pancreatic intraepithelial neoplasia (PanIN) lesions found in 2Br-DAB and vehicle-treated animals.

Figure 3

(A): High-resolution ultrasound system for tumor detection and volume quantification. a: working stage with anaesthetic nose cone. b: 4 MHz scan head. c: induction chamber. d: isoflurane vaporizer. (B): Anatomical structures during ultrasound examination. P: normal pancreatic tissue. D: duodenum with typical ultrasound extinction. S: part of spleen T: pancreatic tumor. (C): Absolute pre-enrollment tumor volumes obtained prior to treatment start for vehicle (n = 7), gemcitabine (n = 7), 2Br-DAB (n = 7), and gemcitabine + 2Br-DAB (n = 7). No significant difference between cohorts was seen. (D,E): Tumor volume increase at day 7 and day 10 by treatment cohorts. (F,G): Quantification of Ki67 and γH2AX immunohistochemistry of all treatment cohorts. For each mouse, at least seven high power fields (HPFs) were acquired at 40x magnification and manually quantified using ImageJ (Mann–Whitney U Test). (H): Representative images of γH2AX immunohistochemistry.

Figure 4

(A): Colony forming assay for pancreatic cancer cell lines. Panc1 cells were seeded in six-well plates (30,000 cells/µL), incubated with 25 µM DAB for 24 h, irradiated (2 Gy), and brought out in triplicates on 100 mm dishes (3000 cells/dish). Quantification was done manually 14 d later. (B): Quantification of manually counted colonies. For the Panc1 cell line, 2Br-DAB and irradiation led to a significant reduction of colony formation (results of two independent biologic experiments) (Mann–Whitney U test). (C): Colony-forming assay for murine pancreatic cancer cell lines (experimental design as mentioned in (A)).

Figure 5

(A): Experimental set-up for radiotherapy of KPC mice. a: Irradiation chamber b: control table c: anesthetic induction chamber d: working stage including mouse fixation system and 25 mm thick lead plates. e: oxygen supply and isoflurane vaporizer. (B,C). Quantification of Ki67 and γH2AX immunohistochemistry. Ki67 positivity is significantly decreased upon combination of 2Br-DAB and 18 Gy (p = 0.02; Mann–Whitney U Test). (D): Representative images of γH2AX and Ki67 immunohistochemistry for all three treatment cohorts.

Figure 5

(A): Experimental set-up for radiotherapy of KPC mice. a: Irradiation chamber b: control table c: anesthetic induction chamber d: working stage including mouse fixation system and 25 mm thick lead plates. e: oxygen supply and isoflurane vaporizer. (B,C). Quantification of Ki67 and γH2AX immunohistochemistry. Ki67 positivity is significantly decreased upon combination of 2Br-DAB and 18 Gy (p = 0.02; Mann–Whitney U Test). (D): Representative images of γH2AX and Ki67 immunohistochemistry for all three treatment cohorts.