Rat Brain PKC (IC50 = 158 nM)
PKC-α (IC50 = 53 nM)
PKC-βI (IC50 = 195 nM)
PKC-βII (IC50 = 163 nM)
PKC-γ (IC50 = 213 nM)
PKC-ε (IC50 = 175 nM)

Bisindolylmaleimide VIII (Bis VIII) has been previously shown to enhance Fas-mediated apoptosis through a protein kinase C-independent mechanism. In the present study, we examined the effect of Bis VIII on apoptosis induced by DR5 (TRAIL-R2), using an agonistic anti-human DR5 monoclonal antibody, TRA-8. Our results demonstrated that Bis VIII was able to enhance the apoptosis-inducing activity of TRA-8 both in vitro and in vivo. The combination of TRA-8 and Bis VIII led to a synergistic and sustained activation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase, which was mediated by MAPK kinase 4 and was caspase-8-dependent. The mitochondrial pathway is involved in the synergistic induction of apoptosis by Bis VIII and TRA-8. Bis VIII alone induced the loss of mitochondrial membrane potential in a caspase-independent fashion without subsequent release of cytochrome c. However, in the presence of Bis VIII, TRA-8 induced more profound loss of mitochondrial membrane potential and release of cytochrome c. These results suggest that the enhanced and persistent activation of the JNK/p38 and the decreased mitochondrial membrane potential play a crucial role in synergistic induction of the death receptor-mediated apoptosis by Bis VIII. The unique ability of Bis VIII to enhance DR5-mediated apoptosis signal transduction discloses a potential utility of this compound in combination with anti-DR5 antibody in cancer therapy. [2]

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In a time-dependent and TRA-8 dose-dependent manner, bisindolylmaleimide VIII acetate (Ro 31-7549 acetate; 5 μM; 8, 12 hours) markedly accelerated TRA-8-induced cell death[2]. Following simultaneous treatment with TRA-8, procaspase-8 was greatly decreased at 4 hours and totally gone at 6 hours with bisindolylmaleimide VIII acetate (5 μM; 6 hours) [2].

Tumor regression was almost entirely achieved when bisindolylmaleimide VIII acetate (Ro 31-7549 acetate; 100 μg; i.p.; every other day for three doses) was coupled with TRA-8. Significant tumor regression is not achieved with bisindolylmaleimide VIII acetate treatment alone [2].
Since TRA-8 has a tumoricidal activity in 1321N1 cells in vivo, we further examined the in vivo effect of co-administration of Bisindolylmaleimide VIII/Bis VIII with TRA-8 on tumor growth. Compared with control-treated mice, the treatment with Bis VIII alone did not induce significant tumor regression (Fig. 1 D). Although TRA-8 alone significantly inhibited tumor growth, complete tumor regression was never achieved. However, the treatment with combined TRA-8 and Bis VIII resulted in nearly complete tumor regression. These results indicate that similar to its effect on Fas-induced cell death, Bis VIII has a synergistic effect on DR5-mediated cell death both in vitro and in vivo [2].

Assay of PKC activity [1]
Assay mixtures contained 0.2 mg/ml peptide-y, 10 ,M MgCl2, 0.6 mM CaCl2, 10l,M [y-32P]ATP, 1.25 mg/ml phosphatidylserine and 1.25 ng/ml phorbol 12-myristate 13-acetate in 20 mM Hepes (pH 7.5), 2 mM EDTA, 1 mM dithiothreitol and 0.02 % (w/v) Triton X-100. Peptide-y is a synthetic peptide, GPRPLFCRKGSLRQKW, resembling the PKC-y pseudosubstrate site, except that a serine residue replaces the pseudosubstrate alanine, converting the peptide from an inhibitor into a substrate. The assays were started by the addition of 2.5 m-units of enzyme, incubated at 30 °C for 10 min and terminated by spotting on to P81 paper, followed by extensive washing in 75 mM orthophosphoric acid. The papers were then washed in ethanol, dried, and incorporated radioactivity was determined by liquidscintillation spectroscopy [1].

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The protein kinase C (PKC) family of isoenzymes is believed to mediate a wide range of signal-transduction pathways in many different cell types. A series of bisindolylmaleimides such as Bisindolylmaleimide VIII have been evaluated as inhibitors of members of the conventional PKC family (PKCs-alpha, -beta, -gamma) and of a representative of the new, Ca(2+)-independent, PKC family, PKC-epsilon. In contrast with the indolocarbazole staurosporine, all the bisindolylmaleimides investigated showed slight selectivity for PKC-alpha over the other isoenzymes examined. In addition, bisindolylmaleimides bearing a conformationally restricted side-chain were less active as inhibitors of PKC-epsilon. Most noticeable of these was Ro 32-0432, which showed a 10-fold selectivity for PKC-alpha and a 4-fold selectivity for PKC-beta I over PKC-epsilon.[1]

Apoptosis analysis [2]
Cell Types: 1321N1 Cell
Tested Concentrations: 5 μM
Incubation Duration: 8, 12 hrs (hours)
Experimental Results: TRA-8-induced cell death was Dramatically increased in a time-dependent and TRA-8 dose-dependent manner.

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Western Blot Analysis[2]
Cell Types: 1321N1 Cell
Tested Concentrations: 5 μM
Incubation Duration: 6 hrs (hours)
Experimental Results: procaspase-8 was Dramatically diminished at 4 hrs (hours) and completely disappeared at 6 hrs (hours).

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Cell Viability Assays [2]
For cell viability assay, cells (1–2 × 103cells/well) were seeded onto a 96-well plate in a volume of 50 μl. The caspase inhibitors were added 1 h before the addition of stimulants. After incubating the cells for the indicated periods, cell viability was determined using the ATPLite kit according to the manufacturer's instructions. For trypan blue dye exclusion assays, the medium was removed, and the adherent cells were detached with 10 mm EDTA in phosphate-buffered saline for 2 min at room temperature. Both the medium and adherent cells were placed in a tube, and the cells were collected by centrifugation. An aliquot of the cells resuspended in the medium was mixed with 0.4% dye at a 1:1 ratio. Live and dead cells were quantitated with a hemocytometer.

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Western Blot Analysis [2]
After the required treatments, cells (1–3 × 106) were washed once with phosphate-buffered saline and lysed in the sample buffer (100–120 μl) for SDS-polyacrylamide gel electrophoresis (PAGE) and immediately boiled for 4 min. To measure the release of cytochrome c from mitochondria, cytosolic and mitochondrial fractions were prepared as described. Each sample was subjected to 7.5, 10, or 12.5% SDS-PAGE, and the proteins separated in the gel were subsequently electrotransferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat dry milk in TBS-T (20 mm Tris-HCl (pH 7.4), 8 g/liter NaCl, and 0.1% Tween 20) for 1–2 h at room temperature. The membrane was then incubated with the indicated primary antibodies in TBS-T containing either 5% nonfat dry milk or 5% bovine serum albumin at 4 °C overnight. The membrane was washed three times with TBS-T and probed with peroxidase-conjugated secondary antibodies at room temperature for 1.5–2 h. After washing four times with TBS-T, the protein was visualized using the ECL Plus Western blotting detection system according to the manufacturer's instructions. Proteins were quantified by densitometric analysis using the Quantify One program. Kinase activities for JNK and p38 in cell extracts were determined using SAPK/JNK and p38 MAPK assay kits, respectively.

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Caspase Activity Assay [2]
After the required treatments, cells (2.5 × 106) were harvested and resuspended in 100 μl of lysis buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mmdithiothreitol, and 0.2% Nonidet P-40). The cells were lysed for 20 min on ice followed by vigorous mixing. After centrifugation at 4 °C for 15 min at 14,000 rpm, the protein concentration in the supernatant was determined using a Bio-Rad protein assay kit. For assaying caspase-3 activation, 20 μg of each cell extract were incubated at 37 °C in assay buffer (20 mm HEPES, pH 7.4, 100 mm NaCl, 10 mm dithiothreitol, 1 mmEDTA, 0.1% CHAPS, and 10% sucrose) containing 200 μmAc-DEVD-p-nitroanilide substrate. Caspase-3-mediated cleavage of Ac-DEVD-p-nitroanilide into p-nitroanilide was measured using a plate reader at 405 nm.

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DNA Fragmentation and Nuclear Staining Assay [2]
After the required treatments of cells (1 × 106), both adherent and detached cells were collected as described above, washed once with phosphate-buffered saline, and lysed in 100 μl of TE-T buffer containing 10 mm Tris-HCl (pH 7.4), 10 mm EDTA, and 0.5% Triton X-100. Lysates were centrifuged at 14,000 rpm for 5 min at 4 °C, and supernatants were then subjected to digestion with ribonuclease A (0.2 mg/ml) for 1 h at 37 °C followed by incubation with proteinase K (0.2 mg/ml) for 1 h at 37 °C. DNA in the sample was precipitated by centrifugation at 14,000 rpm for 15 min at 4 °C after treatment with 50% isopropyl alcohol and 0.5m NaCl overnight at −20 °C. DNA was resuspended in 30 μl of TE buffer and analyzed by electrophoresis on a 2% agarose gel in the presence of 0.2 μg/ml ethidium bromide. For fluorescent microscopic analysis of apoptotic cells, Hoechst 33342 (300 ng/ml) was added in culture medium, and the cells were incubated for 20 min at room temperature before microscopic observation.

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Determination of Mitochondrial Membrane Potential [2]
The mitochondrial membrane potential was assessed by using JC-1, a lipophilic cation that can selectively enter into mitochondria. JC-1 was dissolved in dimethyl sulfoxide to give a 1 mg/ml solution. This was further diluted to 20 μg/ml in a fluorescence-activated cell sorting buffer containing 5% FCS and 0.1% NaN3 in phosphate-buffered saline and filtered using a 0.45-μm filter. After the required treatments of cells (2 × 105), both adherent and detached cells were collected as described above and resuspended in 125 μl of the fluorescence-activated cell sorting buffer. The cell suspension was incubated for 20 min at room temperature with 250 μl of the filtered working solution of JC-1. Both red and green fluorescence emissions were analyzed with a flow cytometer. A minimum of 10,000 cells per sample were acquired in list mode and analyzed using Winmdi software. The decrease in mitochondrial membrane potential was determined by a decrease in the ratio of red to green fluorescence intensities.

Animal/Disease Models: 6-8 weeks old female NOD/SCID (severe combined immunodeficient) mouse [2].
Doses: 100 μg
Route of Administration: IP; every other day for three doses
Experimental Results: Combined with TRA-8, tumor regression was almost complete.

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Analysis of Tumoricidal Activity in Vivo [2]
6–8-week-old female NOD/SCID mice were inoculated subcutaneously with 1321N1 cells (1 × 107). Seven days after inoculation, mice were intraperitoneally injected with 100 μg of TRA-8 and/or 100 μg of Bisindolylmaleimide VIII/Bis VIII every other day for three doses. Seven days after treatment, the growth of the 1321N1 cells was determined by the weight of the tumor mass.

[1]. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J. 1993 Sep 1;294 ( Pt 2):335-7.

[2]. Bisindolylmaleimide VIII enhances DR5-mediated apoptosis through the MKK4/JNK/p38 kinase and the mitochondrial pathways. J Biol Chem. 2002 Aug 9;277(32):29294-303.

The protein kinase C (PKC) family of isoenzymes is believed to mediate a wide range of signal-transduction pathways in many different cell types. A series of bisindolylmaleimides have been evaluated as inhibitors of members of the conventional PKC family (PKCs-alpha, -beta, -gamma) and of a representative of the new, Ca(2+)-independent, PKC family, PKC-epsilon. In contrast with the indolocarbazole staurosporine, all the bisindolylmaleimides investigated showed slight selectivity for PKC-alpha over the other isoenzymes examined. In addition, bisindolylmaleimides bearing a conformationally restricted side-chain were less active as inhibitors of PKC-epsilon. Most noticeable of these was Ro 32-0432, which showed a 10-fold selectivity for PKC-alpha and a 4-fold selectivity for PKC-beta I over PKC-epsilon. [1]

---

Bisindolylmaleimide VIII (Bis VIII) has been previously shown to enhance Fas-mediated apoptosis through a protein kinase C-independent mechanism. In the present study, we examined the effect of Bis VIII on apoptosis induced by DR5 (TRAIL-R2), using an agonistic anti-human DR5 monoclonal antibody, TRA-8. Our results demonstrated that Bis VIII was able to enhance the apoptosis-inducing activity of TRA-8 both in vitro and in vivo. The combination of TRA-8 and Bis VIII led to a synergistic and sustained activation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase, which was mediated by MAPK kinase 4 and was caspase-8-dependent. The mitochondrial pathway is involved in the synergistic induction of apoptosis by Bis VIII and TRA-8. Bis VIII alone induced the loss of mitochondrial membrane potential in a caspase-independent fashion without subsequent release of cytochrome c. However, in the presence of Bis VIII, TRA-8 induced more profound loss of mitochondrial membrane potential and release of cytochrome c. These results suggest that the enhanced and persistent activation of the JNK/p38 and the decreased mitochondrial membrane potential play a crucial role in synergistic induction of the death receptor-mediated apoptosis by Bis VIII. The unique ability of Bis VIII to enhance DR5-mediated apoptosis signal transduction discloses a potential utility of this compound in combination with anti-DR5 antibody in cancer therapy. [2]

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In conclusion, we propose a model in which a positive interaction between the JNK/p38 pathway and caspase-8 leads to enhanced apoptosis signal transduction. This finding suggests that any stress assaults against cancer cells through the JNK/p38 pathway might be able to enhance the tumoricidal activity of TRA-8 or TRAIL. The identification of the unique property of Bisindolylmaleimide VIII/Bis VIII in enhancement of DR5-mediated apoptosis by targeting both the JNK/p38 pathway and the mitochondrial pathway might be helpful in the design and screening of better chemotherapy drugs to synergize cancer cells to death receptor-mediated apoptosis.[2]

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PKC-o, -/?1, -/3w, -y, -8, -E and -C have all been identified as present in the rat brain, although the relative amount of each isoenzyme is not known precisely. The effects of the bisindolylmaleimides on PKC-& and, perhaps more interestingly, the atypical PKC family member PKC-C have yet to be investigated.[1]
Furthermore, there may well be other, as yet unidentified, PKC isoenzymes present in the rat brain preparation. The presence of these additional isoenzymes may explain why a comparison of the IC50 results obtained against the individual isoenzymes with those obtained for each compound against rat brain PKC does not always result in a constant ratio. However, the temptation to draw too many conclusions from these data should be avoided, since the variations may be largely accounted for by the standard deviations associated with these measurements. These compounds show little selectivity for the limited range of isoenzymes examined, and, therefore, are not suitable as tools to probe the role of particular isoenzymes in cell function. However, the small differences in IC50 values between different isoenzymes may explain variations in the potency of Ro 31-7549 observed against a range of phorbol-ester-induced cellular responses, although again these differences may not be marked enough to be translated into cellular systems. IC50 values would be expected to differ if the various processes are mediated through distinct isoenzymes. The indolocarbazole, K252a has been reported to show a high degree of selectivity for the conventional PKC isoenzymes (PKCs-a, -/3, -y) over PKC4 and PKC-e. Although K252a, unlike the bisindolylmaleimides described in this paper, is not selective for PKC over other serine/threonine kinases, this observation suggests that there is a potential for the design ofhighly selective bisindolylmaleimide PKC inhibitors of protein kinase C that will also show a high degree of selectivity for particular PKC isoenzymes.

C26H26N4O4

458.50904

458.195

C, 68.11; H, 5.72; N, 12.22; O, 13.96

138516-31-1

125313-65-7

9868770

Brown to reddish brown solid powder

693ºC at 760 mmHg

372.9ºC

4.7E-19mmHg at 25°C

4.169

3

5

5

34

766

0

CN1C=C(C2=C(C(NC2=O)=O)C3=CN(C4=CC=CC=C34)CCCN)C5=CC=CC=C51.CC(O)=O

VEOXVBTXROWDAH-UHFFFAOYSA-N

InChI=1S/C24H22N4O2.C2H4O2/c1-27-13-17(15-7-2-4-9-19(15)27)21-22(24(30)26-23(21)29)18-14-28(12-6-11-25)20-10-5-3-8-16(18)20;1-2(3)4/h2-5,7-10,13-14H,6,11-12,25H2,1H3,(H,26,29,30);1H3,(H,3,4)

3-(1-(3-aminopropyl)-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione acetate

Ro 31-7549 acetate;bisindolylmaleimide viii; 138516-31-1; Bisindolylmaleimide VIII acetate; Bisindolylmaleimide VIII (acetate); Bisindolylmaleimide VIII acetate salt; 1H-Pyrrole-2,5-dione, 3-[1-(3-aminopropyl)-1H-indol-3-yl]-4-(1-methyl-1H-indol-3-yl)-, acetate (1:1); 3-(1-(3-Aminopropyl)-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione acetate; Bisindolylmaleimide VIII Acetic Acid Salt; acetic acid;3-[1-(3-aminopropyl)indol-3-yl]-4-(1-methylindol-3-yl)pyrrole-2,5-dione; Ro-31-7549; 138516-31-1; Ro 31-7549; Bis VIII

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~250 mg/mL (~545.24 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.54 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.54 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: Formulation 1: ≥ 2.1 mg/mL (4.5 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 21 mg/mL of DMSO stock solution and add tO + 400 μL of PEG300, mix well (clear solution); Then add 50 μL of Tween 80 to the above solution, mix well (clear solution); Finally, add 450 μL of saline to the above solution, mix well (clear solution).
Formulation 2: ≥ 2.1 mg/mL (4.5 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline)
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 21 mg/mL of DMSO stock solution and add to 900 μL of 20% SBE-β-CD in saline, mix well (clear solution)

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