While PBOX 6 is a strong apoptotic PBOX, in the rat R2C Leydig cell line it has no general harmful effects. PBOX 6 (0-25 μM, 16 h) induces apoptosis in HL-60 cells in a dose- and time-dependent manner. At 10 μM, it also fragments DNA. PBOX 6 (10 μM) activates caspase 3-like protease in HL-60 cells, causing them to undergo apoptosis. PBOX 6 (10 μM) causes apoptosis and cytochrome accumulation in the cytoplasm; however, oxidative stress is not responsible for this impact, nor is it dependent on NF-κB or peripheral benzodiazepine receptors (PBR)[1]. By activating caspase-7, PBOX 6 (25 μM) causes MCF-7 cells to undergo apoptosis[2]. In K562 cells, PBOX 6 (10 μM) causes cypA to be redistributed from the nucleus to the cytoplasm. Trypsin-like serine proteases must be activated upstream in order for PBOX 6 (10 μM) to cause the nucleocytoplasmic translocation of cypA and pin1 in a JNK-dependent manner. This result is linked to G2/M phase arrest in K562 cells [3].

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In MCF-7 cells, PBOX-6 induced apoptosis in a dose-dependent manner (0-50 µM for 16 h) and time-dependent manner (25 µM for 4-72 h), with apoptosis measured by cytospin and RapiDiff staining. [2]
In MCF-7 cells, PBOX-6 (25 µM, 24 h) induced DNA fragmentation as confirmed by TUNEL staining and flow cytometry (sub-G1 apoptotic peak: 22% vs <3% in control). [2]
In MCF-7 cells, PBOX-6 (25 µM, 24 h) activated caspase-7 (DEVDase activity) but not caspase-6 (DEIDase activity). Pretreatment with caspase-7 inhibitor z-DEVD-cmk (200 µM) significantly reduced both caspase-7 activity and the morphological signs of apoptosis. [2]
In K562 CML cells, PBOX-6 (10 µM) induced time-dependent (1-8 h) and dose-dependent (0.1-10 µM) nucleocytoplasmic redistribution of cyclophilin A (cypA) and pin1 from the nucleus to the cytosol, as quantified by immunofluorescence. [3]
In K562 cells, PBOX-6 (10 µM) induced G2/M arrest in a time- and dose-dependent manner (up to ~60% at 8 h), correlating with the redistribution of cypA and pin1, and preceding apoptosis (which appeared after 24 h). [3]
In K562 cells, PBOX-6-induced redistribution of cypA and pin1 was blocked by the JNK inhibitor CEP-11004 (5 µM, 1 h pretreatment) and by the trypsin-like serine protease inhibitor TLCK (200 µM, 1 h pretreatment), but not by the chymotrypsin-like inhibitor TPCK (20 µM). [3]
In various hematopoietic cell lines (KYO.1, LAMA 84, Jurkat, HL-60) and solid tumor cells (HeLa, H1299), PBOX-6 (10 µM, 16 h) induced nucleocytoplasmic redistribution of cypA and pin1, though baseline localization varied by cell type. [3]
CypA and pin1 were found to be overexpressed (4- to 10-fold) in CML cell lines (KYO.1, LAMA 84, K562) compared to normal PBMCs, as determined by Western blot and densitometry. [3]

For caspase activity assay: Cytosolic extracts (50 µg protein) from PBOX-6-treated cells were incubated with 100 mM HEPES pH 7.5 containing 10% (w/v) sucrose, 0.1% (w/v) CHAPS, 10 mM dithiothreitol, and 20 µM fluorogenic substrate (Ac-DEVD-AMC for caspase-7 or Ac-DEID-AMC for caspase-6) in a total volume of 3 ml. After 60 min at 25°C, fluorescence was monitored at excitation 380 nm and emission 460 nm, and AMC release was quantified using a standard curve. [2]
For mass spectrometry identification of cypA: Plasma membrane extracts from K562 cells treated with PBOX-6 (10 µM, 45 min) were separated by SDS-PAGE and silver-stained. The protein band (~16 kDa) was excised, destained, digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. Peptide mass fingerprints were searched against the SwissProt database using Mascot; the protein was identified as human cyclophilin A (accession P05092) with a Mascot probability score of 60 and 55% sequence coverage. [3]

For apoptosis induction: MCF-7 cells were seeded at 3×10^5 cells/ml and treated with PBOX-6 (0-50 µM) for 16 h or 25 µM for 4-72 h. An aliquot (100 µl) was cytocentrifuged onto poly-L-lysine-coated slides, stained with RapiDiff kit, and apoptosis/necrosis determined by light microscopy counting ~300 cells per sample. [2]
For DNA fragmentation (TUNEL): MCF-7 cells treated with PBOX-6 (25 µM, 48 h) were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and labeled with terminal transferase and horseradish peroxidase. Colorimetric detection was performed with diaminobenzidine. [2]
For cell cycle analysis: MCF-7 or K562 cells treated with PBOX-6 were harvested, fixed in 70% ethanol overnight at 4°C, then resuspended in PBS containing RNase A (0.5 mg/ml) and propidium iodide (200 µM), incubated at 37°C for 30 min, and analyzed by flow cytometry. Sub-G1 peak indicated DNA fragmentation. [2][3]
For immunofluorescence: Cells (3×10^5/ml) treated with PBOX-6 (10 µM, 16 h) were cytocentrifuged, fixed in 3% paraformaldehyde for 30 min, permeabilized in 0.1% Triton X-100 for 3 min, blocked in 2% BSA for 30 min, then incubated with anti-cypA (1:50 or 1:100) or anti-pin1 (1:10 or 1:100) primary antibodies for 1 h, followed by Alexa Fluor 594 or 488 secondary antibodies (1:200) for 1 h. Nuclei were stained with DAPI (1 µg/ml). Images were captured by fluorescence microscopy. [3]
For Western blot: Whole-cell extracts (30 µg protein) from normal PBMCs or CML cells (KYO.1, LAMA 84, K562) were separated by 15% SDS-PAGE, transferred to PVDF membranes, blocked in 5% nonfat dry milk, probed with anti-cypA or anti-pin1 primary antibodies (1:1000), then HRP-conjugated secondary antibody (1:1000), and visualized by ECL. Membranes were stripped and reprobed with anti-β-actin as loading control. Densitometry was performed using Image J software. [3]

[1]. Pyrrolo-1,5-benzoxazepines induce apoptosis in HL-60, Jurkat, and Hut-78 cells: a new class of apoptotic agents. J Pharmacol Exp Ther. 2000 Apr;293(1):48-59.

[2]. Caspase-3 is not essential for DNA fragmentation in MCF-7 cells during apoptosis induced by the pyrrolo-1,5-benzoxazepine, PBOX-6. FEBS Lett. 2002 Mar 27;515(1-3):66-70.

[3]. The microtubule-targeting agents, PBOX-6 [pyrrolobenzoxazepine 7-[(dimethylcarbamoyl)oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine] and paclitaxel, induce nucleocytoplasmic redistribution of the peptidyl-prolyl isomerases, cycloph.

PBOX-6 is a microtubule-depolymerizing agent that binds tubulin and disrupts microtubule dynamics, leading to G2/M arrest and apoptosis. It has been studied in various cancer cell lines including MCF-7 (breast carcinoma), K562 (CML), KYO.1, LAMA 84, Jurkat (T-lymphoma), HL-60 (promyelocytic leukemia), HeLa (cervical cancer), and H1299 (non-small cell lung carcinoma). [2][3]
The mechanism of PBOX-6-induced apoptosis involves activation of c-Jun N-terminal kinase (JNK) and a trypsin-like serine protease, and it does not require caspase-3. In caspase-3-deficient MCF-7 cells, PBOX-6 induces DNA fragmentation via caspase-7 activation. [2][3]
PBOX-6 causes nucleocytoplasmic redistribution of peptidyl-prolyl isomerases cyclophilin A and pin1, which are overexpressed in hematological malignancies. This redistribution correlates with mitotic arrest and precedes cell death. [3]

C25H20N2O3

396.437906265259

396.147

290814-68-5

9865511

White to off-white solid powder

1.2±0.1 g/cm3

580.6±50.0 °C at 760 mmHg

305.0±30.1 °C

0.0±1.6 mmHg at 25°C

1.640

5.69

0

3

3

30

675

0

SOIZAFVNIXAZFQ-UHFFFAOYSA-N

InChI=1S/C25H20N2O3/c1-26(2)25(28)30-24-21-14-8-16-27(21)20-13-5-6-15-22(20)29-23(24)19-12-7-10-17-9-3-4-11-18(17)19/h3-16H,1-2H3

(6-naphthalen-1-ylpyrrolo[2,1-d][1,5]benzoxazepin-7-yl) N,N-dimethylcarbamate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~33.33 mg/mL (~84.07 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)