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Purity: ≥98%
(±)-10-Hydroxycamptothecin, a naturally occuring indole alkaloid isolated from Camptotheca acuminate, is a cell-permeable and potent inhibitor of topoisomerase I. Its anticancer properties are wide ranging, and it primarily inhibits the phosphorylation of histones H1 and H3, with less of an impact on other histones. The growth of Colo 205 cells is significantly suppressed by (±)-10-hydroxycamptothecin in a manner that is dependent on dosage. (±)-10-Hydroxycamptothecin inhibits Colo 205 cells in the G2 phase of the cell cycle at concentrations of 5–20 nM and initiates apoptosis via a caspase-3-dependent mechanism.
| Targets |
Topoisomerase I
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| ln Vitro |
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| ln Vivo |
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| Enzyme Assay |
DNA topoisomerase I (Topo I) can exist in several different molecular weight forms in human leukemic cells. The Mr 98,000 form of Topo I was inhibited by several nucleoside triphosphates and their analogues at a 500 microM concentration in the order: dideoxy-GTP greater than 2-bromo-dATP greater than dideoxy-ATP greater than dideoxy-CTP greater than 2-fluoro-dATP greater than 2-chloro-dATP. The same concentration of these nucleoside triphosphates also inhibited the Mr 32,000 and the Mr 35,000 Topo I forms in the order: 2-bromo-dATP greater than dideoxy-GTP greater than 2-fluoro-dATP greater than dideoxy-ATP; however, dideoxy-CTP and 2-chloro-dATP did not inhibit these forms. ATP inhibited both the large and the small molecular weight forms of Topo I at a concentration of 8 mM. DNA topoisomerase II (Topo II) isolated from human leukemic cells requires ATP for its activity. Of the nucleoside triphosphates examined, only dATP could substitute for ATP. In the presence of 500 microM ATP, equimolar concentrations of 2-bromo-dATP, dideoxy-ATP, 2-chloro-dATP, 2-fluoro-dATP, and dideoxy-GTP nucleotide analogues inhibited the unknotting activity of the Topo II enzyme. When the nucleotide analogue concentration was decreased to 250 microM, only 2-bromo-dATP still had a significant inhibitory effect on Topo II. With the exception of 2-bromo-dATP, the analogues studied appeared to inhibit the nicking step of both the Topo I and Topo II enzyme activity. These results differ from previously described mechanisms of inhibition by camptothecin of Topo I and etoposide of Topo II. These enzymatic studies suggest the inhibition of Topo I and Topo II activities could contribute to the cytotoxicity of the respective nucleoside analogues in cell culture, particularly when high concentrations of these nucleoside analogues accumulate as triphosphates inside the cells[1].
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| Cell Assay |
After being seeded in 25T flasks for an overnight period, Colo 205 cells (5 × 105) (ATCC: CCL-222) are treated with 5, 10, 15, or 20 nM of (±)-10-Hydroxycamptothecin, respectively, as a control. Trypsin-EDTA is used to harvest the cells after 24-120 hours of treatment, and they are then centrifuged for 5 minutes at 4,500 rpm and 4˚C. A hemocytometer is used to count the viable cells in the cell pellet after it has been resuspended in culture medium containing 0.04% trypan blue[2].
To study whether 10-hydroxycamptothecin (HPT) can induce apoptosis in bladder cancer cell and establish methods for detecting apoptotic cells. Methods: Human urinary bladder cancer cell line (T24) was exposed in vitro to different concentrations of 10-hydroxycamptothein for various lengths of time. Flow cytometry, Hochest 33258 and Hematoxylin staining were used to determine the induction of apoptosis after use of HPT. DNA gel analysis was also carried out to detect DNA fragmentation. Results: Cell shrinage, nuclear fragmentation and condensed chromosomes showed that apoptosis can be induced by HPT within the concentration of 0.01 - 10 microg/ml. The flow cytometry analysis showed that the percentage of apoptotic cells were related to the concentration and the time of induction. T24 cell line exposed to HPT experienced internucleosomal DNA fragmentation by producing a typical ladder pattern on agarose gel electrophoresis. The detection of minimum exposure time for HPT-induced apoptosis in T24 cells showed that 3 hours of exposure to HPT were enough to trigger internucleosomal DNA fragmentation. Compared to Hochest 33258 staining, Hematoxylin staining was more easy, rapid and accurate to detect apoptosis. Conclusions: The induction of apoptosis exposed to HPT in T24 human urinary bladder cancer cells is a good model for further studying urinary bladder cancer. Hematoxylin staining is a useful method for detecting apoptosis[3]. |
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| Animal Protocol |
Sterilized conditions and a controlled 12-hour light and 12-hour dark cycle are provided for BALB/c-nu mice living in a laminar flow room at 25 ± 2˚C. RPMI-1640 medium without serum is used to harvest and resuspend the Colo 205 cells. A volume of 0.1 mL is subcutaneously injected into the mice's flanks after the cell density is adjusted to 1 × 107 cells/mL. Three to seven tumor-bearing mice per experimental group were used. Therapy commences when the tumor size reaches 3-5 mm and is administered with a solution of (±)-10-hydroxycamptothecin dissolved in propylene glycol. (±)-10-Hydroxycamptothecin is injected intraperitoneally (i.e., once every two or four days) at 1, 2.5, 5, 7.5, and 1 mg/kg (0.1 mL/20 g of body weight for the injection volume). Every two days, a propylene glycol vehicle is given to the control group. Throughout the experiment, body weight and tumor size are recorded twice a week. A vernier caliper is used to measure the size of the tumor. The formula for calculating tumor volume (V) is V (mm3) = 0.4AB2, where A and B represent the longest and shortest diameters, respectively. Upon completion of the experiment, CO2 gas is used to kill every mouse. For pathological analysis, tumors, livers, kidneys, and lungs are removed, preserved, embedded, and stained with hematoxylin and eosin[2].
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| References |
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| Additional Infomation |
10-hydroxycamptothecin is under investigation in clinical trial NCT00956787 (Study of AR-67 (DB-67) in Myelodysplastic Syndrome (MDS)).
See also: 10-Hydroxycamptothecin (annotation moved to). |
| Molecular Formula |
C20H16N2O5
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| Molecular Weight |
364.35
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| Exact Mass |
364.105
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| Elemental Analysis |
C, 65.93; H, 4.43; N, 7.69; O, 21.96
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| CAS # |
64439-81-2
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| Related CAS # |
(S)-10-Hydroxycamptothecin;19685-09-7
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| PubChem CID |
4330531
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| Appearance |
Light yellow to yellow solid
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
820.7±65.0 °C at 760 mmHg
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| Melting Point |
230-237°C
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| Flash Point |
450.1±34.3 °C
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| Vapour Pressure |
0.0±3.1 mmHg at 25°C
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| Index of Refraction |
1.777
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| LogP |
1.32
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
27
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| Complexity |
774
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O1C(C(C([H])([H])C([H])([H])[H])(C2C([H])=C3C4=C(C([H])=C5C([H])=C(C([H])=C([H])C5=N4)O[H])C([H])([H])N3C(C=2C1([H])[H])=O)O[H])=O
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| InChi Key |
HAWSQZCWOQZXHI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H16N2O5/c1-2-20(26)14-7-16-17-11(5-10-6-12(23)3-4-15(10)21-17)8-22(16)18(24)13(14)9-27-19(20)25/h3-7,23,26H,2,8-9H2,1H3
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| Chemical Name |
19-ethyl-7,19-dihydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa-1(21),2(11),3,5,7,9,15(20)-heptaene-14,18-dione
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (6.86 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7446 mL | 13.7231 mL | 27.4461 mL | |
| 5 mM | 0.5489 mL | 2.7446 mL | 5.4892 mL | |
| 10 mM | 0.2745 mL | 1.3723 mL | 2.7446 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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