MGMT/AGT (O6-alkylguanine-DNA alkyltransferase; DNA repair enzyme)

L3.6pl cells exhibit dose- and time-dependent sensitivity to O6-Benzylguanine (24-72 hours). 50 μg (48 hours) is the IC50[2]. O6-Benzylguanine (50 μg; 48 hours) dramatically lowers MGMT protein activity in L3.6pl and changes the p53 target downstream [2].
O(6)-benzyl guanine (O6BG) sensitized pancreatic cancer cells to gemcitabine. Protein and mRNA expression of MGMT, cyclin B1, cyclin B2, cyclin A, and ki-67 were significantly decreased in the presence of O(6)BG. In sharp contrast, protein expression and mRNA message of p21(cip1) were significantly increased. Interestingly, O(6)BG increases p53-mediated p21(cip1) transcriptional activity and suppresses cyclin B1. In addition, our results indicate that p53 is recruited to p21 promoter. Furthermore, an increase in p21(cip1) and a decrease in cyclin transcription are p53 dependent [2].

In response to gemcitabine (100 mg/kg), O6-benzylguanine (100 μg; i.p.; once daily for 35 days) suppresses pancreatic development and raises pancreatic cell weight [2].
Researchers next determined the in vivo effects of O(6)-benzyl guanine (O6BG) (alone or in combination) with gemcitabine. Tumors harvested from the different treatment groups were processed for routine histologic and immunohistochemical analysis. Tumors from mice treated with O6BG alone or in combination with gemcitabine exhibited a significant decrease in MGMT, cyclin B1, cyclin B2, and cyclin A as compared with tumors treated with gemcitabine alone or the control group. In sharp contrast, the expression of p21 was significantly increased in tumors from mice treated with O6BG.

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O(6)-benzyl guanine (O6BG) inhibits pancreatic cancer cell proliferation and induces tumor cell apoptosis in vivo [2]
The induction of apoptosis (TUNEL-positive stain) of tumor cells was inversely correlated with proliferation (ki-67 positivity). The TUNEL-positive stain revealed that many tumor cells underwent apoptosis in mice treated with O6BG (alone or in combination with gemcitabine) compared with mice treated with gemcitabine alone or control (Fig. 5). Tumor cell proliferation was decreased with O6BG alone or in combination with gemcitabine as compared with control mice or gemcitabine-treated mice.

Reporter assays [2]
For reporter assays, L3.6pl cells (5 × 10~4) were seeded in a 12-well plate and transfected with MGMT-luc construct. After 5 to 6 h the cells were treated with or without O(6)-benzyl guanine (O6BG) (50μg). At 24 h posttreatment the cells were harvested and lysed, and luciferase activity was measured by using dual luciferase reporter assay system following the manufacturer's protocol. LipofectAMINE 2000 was used to transfect the cells, and the manufacturer's protocol for transfections was followed. In another experiment, L3.6pl cells (5 × 104) were transfected with MGMT-luc in the presence or absence of wt p53 construct (5 μg) and 24 h later the cells were harvested. In a final set of experiments, L3.6pl cells (5 × 10~4) were seeded in a 12-well plate and transfected with p21-luc construct and cyclin B1-luc construct in presence or absence of O(6)-benzyl guanine (O6BG) (50 μg) and gemcitabine (1 μmol/L), and 24 h posttreatment the cells were harvested.

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Chromatin immunoprecipitation assays [2]
L3.6pl cells were plated (2 × 10~5) and 24 h later the cells were transfected with nonspecific and p53 siRNA, both at 20 nmol/L. After 48 h the cells were treated with or without O(6)-benzyl guanine (O6BG), and 24 h posttreatment the cells were harvested and samples were used for chromatin immunoprecipitation assays (ChIP). ChIP assays were done as per the manufacturer's instructions with minor modifications. L3.6pl cells were plated (2 × 10~5) and the next day were transfected with nonspecific (20 nmol/L) and p53 siRNA (20 nmol/L) and 24 h later cells were treated with or without O(6)-benzyl guanine (O6BG). At 48 h posttreatment cells were collected. Cell lysates (400μL) were sonicated 25 times and each time a 10-s pulse and 20-s gap were given. After centrifugation, 50 μL of the supernatant were used for checking DNA fragmentation as well as input, and the remaining 350 μL were used for chromatin immuoprecipitation.

Western Blot Analysis[2]
Cell Types: L3 .6pl and PANC1 Cell
Tested Concentrations: 50 μg
Incubation Duration: 48 hrs (hours)
Experimental Results: O6 methylguanine DNA methyltransferase (MGMT), cyclin B1, cyclin B2, The expression of cyclin A, p53, and ki-67 was diminished, while p21 was increased. Cellular C and caspase 9 levels increased, while PARP1 protein levels diminished.

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RT-PCR[2]
Cell Types: L3.6pl Cell
Tested Concentrations: 50 μg
Incubation Duration: 48 hrs (hours)
Experimental Results: MGMT transcriptional activity was diminished in L3.6pl.

Animal/Disease Models: Male athymic nude mice (NCI-nu) (carrying human pancreatic cancer L3.6pl cells) [2]
Doses: 100 μg
Route of Administration: ip; cell proliferation and induction of pancreatic cells [2]. one time/day for 35 days
Experimental Results: Median tumor volume and weight were Dramatically diminished.

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Therapy of established human pancreatic carcinoma tumors growing in the pancreas of nude mice [2]
Seven days after injection of tumor cells into the pancreas, five mice were sacrificed to confirm the presence of tumor lesions. At that time, the median tumor volume was 18 mm3. Histologic examination confirmed that the lesions were actively growing pancreatic cancer. The mice were randomized into four groups (n = 10) as follows: (a) daily (M-F) i.p injections of 1× PBS in control groups; (b) twice weekly (T, Th) i.p injections of gemcitabine 100 mg/k; (c) daily (M-F) i.p injections of O(6)-benzyl guanine (O6BG) (100 μg); and (d) i.p injections of combination of gemcitabine (twice) and O(6)-benzyl guanine (O6BG) (5 d/wk). Tumor volumes were calculated by using the following formula: 0.5 × (length) × (width)2. Treatments were continued for 5 wk and the mice were sacrificed and subjected to necropsy.

[1]. Rabik CA, Njoku MC, Dolan ME. Inactivation of O6-alkylguanine DNA alkyltransferase as a means to enhance chemotherapy. Cancer Treat Rev. 2006;32(4):261‐276.

[2]. Blockade of MGMT expression by O6 benzyl guanine leads to inhibition of pancreatic cancer growth and induction of apoptosis. Clinical cancer research : an official journal of the American Association for Cancer Research vol. 15,19 (2009): 6087-95.

6-O-Benzylguanine has been used in clinical trials for the treatment of various diseases, including HIV infection, adult glioma, adult glioblastoma, stage I adult Hodgkin lymphoma, and stage II adult Hodgkin lymphoma. 6-O-Benzylguanine is a guanine analog with antitumor activity. It binds to the DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase (AGT), transferring a benzyl group to the active site cysteine residue, thereby inhibiting AGT-mediated DNA repair. Combining this drug with other DNA-damaging chemotherapeutic agents can enhance its efficacy. (NCI04) Objective: We aimed to determine whether the optimal biological dose of the MGMT blocker 6-O-Benzylguanine (O(6)BG), used alone or in combination with gemcitabine, could inhibit the growth of human pancreatic cancer cells. Experimental Design: Human pancreatic cancer L3.6pl and PANC1 cells were treated with O(6)BG alone or in combination with gemcitabine, and the therapeutic effects and bioactivity of these drug combinations were investigated. Results: O(6)BG enhanced the sensitivity of pancreatic cancer cells to gemcitabine. In the presence of O(6)BG, the protein and mRNA expression of MGMT, cyclin B1, cyclin B2, cyclin A, and Ki-67 were significantly reduced. In stark contrast, the protein and mRNA expression of p21(cip1) were significantly increased. Interestingly, O(6)BG enhanced p53-mediated p21(cip1) transcriptional activity and inhibited cyclin B1. Furthermore, our results indicate that p53 can be recruited to the p21 promoter. Moreover, the increase in p21(cip1) and the decrease in cyclin transcription were both dependent on p53. The volume of pancreatic tumors in mice treated with gemcitabine alone decreased by 27%, the volume of pancreatic tumors in mice treated with O(6)BG alone decreased by 47%, and the volume of pancreatic tumors in mice treated with the combination of the two drugs decreased by 65%. Immunohistochemical analysis showed that O(6)BG inhibited the expression of MGMT and cyclin and increased the expression of p21 (cip1). In addition, tumor cell proliferation was significantly reduced and tumor cell apoptosis was increased. Conclusion: In summary, our results indicate that decreased MGMT expression is associated with p53 activation and significantly inhibits the growth of primary pancreatic tumors. These findings suggest that the use of O(6)BG alone or in combination with gemcitabine may provide a new and effective approach for the treatment of human pancreatic cancer. [2]

C12H11N5O

241.25

241.096

C, 59.74; H, 4.60; N, 29.03; O, 6.63

19916-73-5

100994-97-6 (sodium); 19916-73-5 (free)

4578

White to off-white solid powder

1.4±0.1 g/cm3

621.4±63.0 °C at 760 mmHg

193(dec.)

329.6±33.7 °C

0.0±1.8 mmHg at 25°C

1.743

1.95

2

5

3

18

271

0

C1=CC=C(C=C1)COC2=NC(=NC3=C2NC=N3)N

KRWMERLEINMZFT-UHFFFAOYSA-N

InChI=1S/C12H11N5O/c13-12-16-10-9(14-7-15-10)11(17-12)18-6-8-4-2-1-3-5-8/h1-5,7H,6H2,(H3,13,14,15,16,17)

6-phenylmethoxy-7H-purin-2-amine

CCRIS9383; CCRIS 9383; o6-benzylguanine; 19916-73-5; 6-O-Benzylguanine; 6-(Benzyloxy)-7H-purin-2-amine; O(6)-Benzylguanine; 2-Amino-6-(benzyloxy)purine; 6-(benzyloxy)-9H-purin-2-amine; 2-amino-6-benzyloxypurine; O(6)-Benzylguanine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~110 mg/mL (~455.96 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (11.40 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 27.5 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: ≥ 2.75 mg/mL (11.40 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 27.5 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.)