Topoisomerase I
DNA topoisomerase I. No IC50, Ki, EC50, or DC50 values are reported in the specified literature. [2]

(±)-10-Hydroxycamptothecin (10-OH-camptothecin) is a topo I inhibitor[1]. The proliferation of Colo 205 cells is significantly inhibited in a dose-dependent manner by (±)-10-Hydroxycamptothecin (10-HCPT, 5-20 nM). By stopping Colo 205 cells in the G2 phase of the cell cycle, (±)-10-hydroxycamptothecin (5–20 nM) initiates apoptosis via a caspase-3-dependent mechanism[2]. ± The human urinary bladder cancer cell line (T24) is exposed to -10-Hydroxycamptothecin (HPT, 0.01–10 μg/mL), which results in nuclear fragmentation, condensed chromosomes, and cell shrinage[3].

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(±)-10-Hydroxycamptothecin significantly repressed the proliferation of human colon cancer Colo 205 cells at relatively low concentrations (5-20 nM) in a dose- and time-dependent manner. At 20 nM, the cell number at 120 h was similar to that at 24 h, indicating strong growth suppression. [2]
Flow cytometry analysis (propidium iodide staining) after 48 h treatment showed that (±)-10-Hydroxycamptothecin induced a dose-dependent increase in the G2/M phase cell population and an increase in the sub-G0/G1 phase (apoptotic) population, with a corresponding decrease in G0/G1 phase. [2]
Western blot analysis revealed that (±)-10-Hydroxycamptothecin dose-dependently increased the expression of p21, cyclin E, CDK2, cyclin B1, and cdc25c, while decreasing p27 expression. p53 expression was also detected. PCNA level was unchanged. The increase in p21 contributed to G2/M arrest despite increased cyclin B1 and cdc25c. [2]
DNA fragmentation assay showed that (±)-10-Hydroxycamptothecin induced DNA ladder formation in a concentration-dependent manner after 48 h treatment, confirming apoptosis. [2]
Caspase activity assay using DEVD-pNA substrate demonstrated that (±)-10-Hydroxycamptothecin induced caspase-3 activation in a dose-dependent manner, while IETD-pNA (caspase-8) and LEHD-pNA (caspase-9) substrates showed only low activation, indicating apoptosis proceeds through a caspase-3-dependent pathway. [2]

(±)-10-Hydroxycamptothecin (10-HCPT, 2.5-7.5 mg/kg/2 days, p.o.) dramatically inhibits tumor growth in mouse xenografts. (±)-10-Hydroxycamptothecin (1-7.5 mg/kg, p.o., once per 2 or 4 days) does not appear to cause any acute toxicity in nude mice[2].
Moreover, following oral administration at doses of 2.5-7.5 mg/kg/2 days, significant suppression of tumor growth by 10-HCPT was observed in mouse xenografts. No acute toxicity was observed after an oral challenge of 10-HCPT in BALB/c-nude mice every 2 days. The results of this study suggest that a relatively low dose of 10-HCPT (p.o.) is able to inhibit the growth of colon cancer, facilitating the development of a new protocol of human trials with this anticancer drug.[2]

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In a BALB/c-nu mouse xenograft model with human colon cancer Colo 205 cells, oral administration of (±)-10-Hydroxycamptothecin at doses of 2.5, 5, or 7.5 mg/kg every 4 days resulted in tumor volumes of 273.1±33.7, 228.9±45.3, and 182.9±43.9 mm^3 respectively at day 20, with significant (p<0.05) reduction at 5 and 7.5 mg/kg compared to control (300.5±22.3 mm^3). [2]
When administered every 2 days at doses of 1, 2.5, 5, or 7.5 mg/kg, (±)-10-Hydroxycamptothecin significantly (p<0.05) reduced tumor volumes to 221.5±20.3, 85.9±12.3, 60.8±15.4, and 7.2±2.8 mm^3 respectively at day 20, showing marked antitumor efficacy. [2]

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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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].

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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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Trypan blue exclusion assay: Colo 205 cells (5×10^5) were seeded in 25T flasks overnight and treated without (control) or with 5, 10, 15, or 20 nM of (±)-10-Hydroxycamptothecin for 24-120 h. Cells were harvested, resuspended in culture medium containing 0.04% trypan blue, and viable cells were counted using a hemocytometer. [2]
Cell cycle distribution analysis: After treatment, cells were trypsinized, resuspended in 70% ethanol, incubated on ice for ≥1 h, then resuspended in cell cycle assay buffer (0.38 mM sodium citrate, 0.5 mg/ml RNase A, 0.01 mg/ml propidium iodide) at 5×10^5 cells/ml. Samples were stored in the dark at 4°C and analyzed by flow cytometry using ModFit LT 2.0 software. [2]
Western blotting: Colo 205 cells were cultured in RPMI-1640 with 10% FBS. Cells were seeded in 6-well plates and incubated with various concentrations of (±)-10-Hydroxycamptothecin for 48 h. Cells were lysed in ice-cold reporter lysis buffer, protein quantified, and 15 μg lysate protein resolved by 10% SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against topoisomerase I, p53, p27, phosphorylated p27, cyclin E, CDK2, PCNA, p21, and α-tubulin. Protein was visualized using a chemiluminescence blotting kit and exposed to X-ray film. [2]
Caspase-3 activity assay: N-Acetyl-Asp-Glu-Val-Asp-pNA (DEVD-pNA) cleavage activity was measured using a colorimetric assay kit. 10-HCPT-treated cells (3×10^6) were harvested, lysed in chilled lysis buffer, and centrifuged. 100 μg of cell lysate was mixed with 2X reaction buffer containing 10 mM DTT and 200 μM DEVD-pNA, incubated at 37°C for 1 h, and p-nitroanilide formation was detected at 405 nm using a microtiter plate reader. [2]
DNA fragmentation assay: Treated cells (2×10^7) were pelleted and resuspended in NP-40 lysis buffer (1% NP-40 in 20 mM EDTA, 50 mM Tris-HCl, pH 7.5). Supernatants were collected after centrifugation, treated with 1% SDS and 2.5 μg/μl RNase for 2 h at 56°C, followed by proteinase K (2.5 μg/μl) for 2 h at 37°C. DNA was precipitated with ammonium acetate and ethanol, collected by centrifugation, and electrophoresed on 1.5% agarose gel with ethidium bromide staining. [2]

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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BALB/c-nu mice were obtained and housed under sterilized conditions. Colo 205 cells (1×10^7 cells/ml, 0.1 ml) were injected subcutaneously into the flank region. When tumors reached 3-5 mm in size, treatment began. (±)-10-Hydroxycamptothecin was dissolved in propylene glycol and administered orally (p.o.) once every two days or once every four days at doses of 1, 2.5, 5, or 7.5 mg/kg (injection volume: 0.1 ml/20 g body weight). The control group received propylene glycol vehicle every two days. Tumor size (measured with a vernier caliper) and body weight were monitored twice weekly. Tumor volume was calculated as V (mm^3) = 0.4 × A × B^2, where A is the longest diameter and B is the shortest diameter. At the end of the experiment, mice were sacrificed by CO2 gas, and tumors, livers, kidneys, and lungs were collected, fixed, embedded, and stained with hematoxylin and eosin for pathological analysis. [2]

No acute toxicity was observed after oral challenge of (±)-10-Hydroxycamptothecin at doses of 1-7.5 mg/kg every 2 or 4 days in BALB/c-nude mice. No significant reduction in body weight was found except in the 7.5 mg/kg/2 days-treated group. Histopathological examination of liver, lung, and kidney tissue slices stained with hematoxylin and eosin showed no tissue damage. [2]

[1]. Interaction of several nucleoside triphosphate analogues and 10-hydroxycamptothecin with human DNA topoisomerases. Cancer Res. 1989 Mar 15;49(6):1366-70.

[2]. Anticancer effects of low-dose 10-hydroxycamptothecin in human colon cancer. Oncol Rep. 2006 May;15(5):1273-9.

[3]. Detection of apoptosis exposed to 10-hydroxycamptothecin in T24 human urinary bladder cancer cells. Zhonghua Wai Ke Za Zhi. 1999 Jan;37(1):57-9.

10-Hydroxycamptothecin is being investigated in the clinical trial NCT00956787 (Study of AR-67 (DB-67) for the treatment of myelodysplastic syndromes (MDS)). See also: 10-Hydroxycamptothecin (note moved to).

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(±)-10-Hydroxycamptothecin is a topoisomerase I inhibitor that traps the topoisomerase I-DNA cleavage complex, preventing relegation and generating DNA damage that leads to apoptosis. It induces G2/M cell cycle arrest and caspase-3-dependent apoptosis in colon cancer cells. The study demonstrates that low-dose, long-term oral administration is effective and safe, suggesting a potential clinical protocol for colon cancer treatment via the oral route. [2]

C20H16N2O5

364.35

364.105

C, 65.93; H, 4.43; N, 7.69; O, 21.96

64439-81-2

(S)-10-Hydroxycamptothecin;19685-09-7

4330531

Light yellow to yellow solid

1.6±0.1 g/cm3

820.7±65.0 °C at 760 mmHg

230-237°C

450.1±34.3 °C

0.0±3.1 mmHg at 25°C

1.777

1.32

2

6

1

27

774

0

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

HAWSQZCWOQZXHI-UHFFFAOYSA-N

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

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

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)

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.

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