Maytansinoids

Combination therapy, in which two or more agents are applied, is more effective than single therapies for combating cancer. For this reason, combinations of chemotherapy with radiation are being explored in clinical trials, albeit with an empirical approach. We developed a screen to identify, from the onset, molecules that act in vivo in conjunction with radiation, using Drosophila as a model. Screens through two small molecule libraries from the NCI Developmental Therapeutics Program yielded microtubule poisons; this class of agents is known to enhance the effect of radiation in mammalian cancer models. Here we report an analysis of one microtubule depolymerizing agent, maytansinol isobutyrate (NSC292222; maytansinol), in Drosophila and in human cancer cells. We find that the effect of maytansinol is p53 dependent in Drosophila cells and human cancer cells, that maytansinol enhances the effect of radiation in both systems, and that the combinatorial effect of drug and radiation is additive. We also uncover a differential sensitivity to maytansinol between Drosophila cells and Drosophila larvae, which illustrates the value of studying cell behavior in the context of a whole organism. On the basis of these results, we propose that Drosophila might be a useful model for unbiased screens through new molecule libraries to find cancer drugs for combination therapy [1].

Maytaninol (2 μ M. 10 μ M. Adding it to food for 1-2 hours can cause microtubule depolymerization in fruit flies. Maytaninol (0.5-2 μ M. Adding it to food for 10 days reduced the survival rate of wild-type and p53 mutant larvae. Maytaninol (1 or 2 μ M. Adding to food for 24-26 hours can induce apoptosis in wild-type Drosophila cells [1].

The growth inhibitory effects of maytansinol with radiation were evaluated using a modified tetrazolium salt (MTT) assay (Carmichael et al., 1988). In the MTT assay, 1000–2000 viable cells were plated in 100 μl of growth medium in 96-well plates (Corning, Ithaca, NY). Following an overnight incubation, maytansinol was added at varying concentrations and the plates were irradiated on the same day (co-treatment) or 24 hours later (pre-treatment) and incubated for 6–7 days. The tetrazolium salt was added at a concentration of 0.4 mg/ml to each well following the 6- to 7-day treatment. The plates were incubated with the salt for 4 hours at 37°C. At 4 hours, the medium was aspirated off, leaving the dark blue formazan product at the bottom of the wells. The reduced MTT product was solubilized by adding 100 μl of 0.2 N HCl in 75% isopropanol, 23% MilliQ water to each well. Thorough mixing was done using a Titertek multichannel pipetman. The absorbency of each well was measured using an automated plate reader (Molecular Devices, Sunnyvale, CA). All experiments were performed in triplicate [1].

Fly stocks [1]
Wild-type flies were of the Sevelin stock. p535A-1-4 results from targeted deletion of the gene (Rong et al., 2002).[1]

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Irradiation[1]
Feeding-stage third instar larvae were irradiated as previously described (Jaklevic et al., 2006). Briefly, 120-hour-old larvae were rinsed to remove food and passed through sizing sieves to obtain animals of uniform size. Larvae were placed in a Petri dish and irradiated using a TORREX X-ray generator, set at 115 kV and 5 mA (producing 2.4 Rads/second). Irradiated larvae were then cultured on cornmeal-agar media (Jaklevic et al., 2006) containing drug or DMSO carrier. Human cells in 96-well plates were irradiated with a RS2000 Biological Irradiator (Rad Source Technologies) delivering 1 Gy/minute.[1]

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AO staining[1]
Larvae were dissected in PBS. Imaginal discs were incubated for 5 minutes in PBS + 0.5 mM AO (Sigma) at room temperature, washed once with PBS, mounted in PBS, and imaged immediately using a Leica DMR fluorescence compound microscope, a Sensicam CCD camera and Slidebook software (Intelligent Imaging). Images were compiled using Photoshop software.[1]

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Antibody staining[1]
To detect phosphorylated Histone H3, larval imaginal discs were extirpated in PBS, fixed for 10 minutes in PBT (PBS with 0.2% Tween) containing 10% formaldehyde and washed three times with PBT. Samples were incubated with primary antibodies in blocking solution, which is PBT + 3% normal goat serum, for 2 hours at room temperature or overnight at 4°C. Primary antibodies were rabbit polyclonal anti-phospho-Histone-H3 antibody (Upstate Biotechnology) diluted at 1:1000 and mouse monoclonal anti-β-Tubulin antibody (Developmental Hybridoma Bank) diluted at 1:100. Samples were then washed three times with PBT and incubated for 2–4 hours at room temperature with secondary antibody conjugated to rhodamine or fluorescein, diluted to 1:500 in blocking solution (Jackson ImmunoResearch). Samples were washed three times with PBT, stained with 10 μg/ml Hoechst 33258 in PBT for 2 minutes, and washed three times with PBT before mounting onto slides with Fluoromount G. Samples were imaged on a Leica DMR fluorescence microscope using a Sensicam CCD camera and Slidebook software (Intelligent Imaging). Images at different focal planes were combined using ImageJ (http://rsb.info.nih.gov/ij/), displayed using Photoshop software and the number of mitotic cells was counted manually. [1].

[1]. Combinatorial effect of maytansinol and radiation in Drosophila and human cancer cells. Dis Model Mech. 2011 Jul;4(4):496-503.

[2]. New insights into the anticancer therapeutic potential of maytansine and its derivatives. Biomed Pharmacother. 2023 Sep;165:115039.

Combination therapy, in which two or more agents are applied, is more effective than single therapies for combating cancer. For this reason, combinations of chemotherapy with radiation are being explored in clinical trials, albeit with an empirical approach. We developed a screen to identify, from the onset, molecules that act in vivo in conjunction with radiation, using Drosophila as a model. Screens through two small molecule libraries from the NCI Developmental Therapeutics Program yielded microtubule poisons; this class of agents is known to enhance the effect of radiation in mammalian cancer models. Here we report an analysis of one microtubule depolymerizing agent, maytansinol isobutyrate (NSC292222; maytansinol), in Drosophila and in human cancer cells. We find that the effect of maytansinol is p53 dependent in Drosophila cells and human cancer cells, that maytansinol enhances the effect of radiation in both systems, and that the combinatorial effect of drug and radiation is additive. We also uncover a differential sensitivity to maytansinol between Drosophila cells and Drosophila larvae, which illustrates the value of studying cell behavior in the context of a whole organism. On the basis of these results, we propose that Drosophila might be a useful model for unbiased screens through new molecule libraries to find cancer drugs for combination therapy.[1]

C28H37CLN2O8

565.0550

564.223

C, 59.52; H, 6.60; Cl, 6.27; N, 4.96; O, 22.65

57103-68-1

57103-68-1;

9915934

White to yellow solid powder

Microbes such as Bacillus megaterium IFO 12108, Streptomyces coelicolor IFO 3807, Streptomyces castaneus IFO 13670 and Streptomyces minutiscleroticus IFO 13361

3.73

3

8

2

39

993

7

C[C@@H]1[C@@H]2C[C@]([C@@H](/C=C/C=C(/CC3=CC(=C(C(=C3)OC)Cl)N(C(=O)C[C@@H]([C@]4([C@H]1O4)C)O)C)\C)OC)(NC(=O)O2)O

QWPXBEHQFHACTK-RZKXNLMUSA-N

InChI=1S/C28H37ClN2O8/c1-15-8-7-9-22(37-6)28(35)14-20(38-26(34)30-28)16(2)25-27(3,39-25)21(32)13-23(33)31(4)18-11-17(10-15)12-19(36-5)24(18)29/h7-9,11-12,16,20-22,25,32,35H,10,13-14H2,1-6H3,(H,30,34)/b9-7+,15-8+/t16-,20+,21+,22-,25+,27+,28+/m1/s1

(1S,2R,3S,5S,6S,16E,18E,20R,21S)-11-chloro-6,21-dihydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaene-8,23-dione

Maytansinol; maytansine derivative; Ansamitocin P 0; Antibiotic C 15003P 0; MAYTANSINOL; 57103-68-1; ZWJ9A2AJ2U; UNII-ZWJ9A2AJ2U; NSC-239386; 3-o-De(2-(acetylmethylamino)-1-oxopropyl)maytansine; Maytansine, 3-o-de(2-(acetylmethylamino)-1-oxopropyl)-; (14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14,4-dihydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-diene-12,6-dione; Ansamitocin P-0; NSC 239386; NSC239386; NSC-239386

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: ≥ 35 mg/mL (~61.9 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.68 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 (3.68 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: ≥ 2.08 mg/mL (3.68 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 20.8 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.)