Bax; Bad; Bcl-2; Bcl-XL; caspase-9; Microtubule; tubulin polymerization; tubulin stabilizer

Paclitaxel, a natural product originally isolated from Taxus brevifolia, belongs to the most successful anticancer drugs. Nevertheless, its poor water solubility represents a considerable disadvantage in clinical use, and novel derivatives with improved pharmacological features are required. We isolated 7-xylosyl-10-deacetylpaclitaxel from Taxus chinensis, which reveals higher water solubility than paclitaxel. This compound induced mitotic cell cycle arrest and apoptosis as measured by flow cytometry, DNA laddering, and transmission electron microscopy. Pro-apoptotic Bax and Bad protein expression was up-regulated and anti-apoptotic Bcl-2 and Bcl-XL expression down-regulated, which lead to a disturbance of the mitochondrial membrane permeability and to the activation of caspase-9. In turn, caspase-9 activated downstream caspases-3 and -6, but not caspase-8. Bid was also activated by caspase-3. Reversely, treatment with a caspase-10-specific inhibitor could not protect PC-3 cells from 7-xylosyl-10-deacetyl-paclitaxel-triggered apoptosis. Moreover, 7-xylosyl-10-deacetylpaclitaxel had no effect on the expression of CD95 and NF-kappaB proteins, indicating that apoptosis was induced through the mitochondrial-dependent pathway in PC-3 cells[1].

Assessment of caspase activity. [1]
The activity of caspases was determined by a colorimetric assay based on the ability of caspase-3, -6, -8 and -9 to convert acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA), acetyl-Val-Glu-Ile-Asp p-nitroanilide (Ac-VEID-pNA), acetyl-Ile-Glu-Thr-Asp p-nitroanilide (Ac-IETD-pNA) and acetyl-Leu-Glu-His-Asp p-nitroanilide (Ac-LEHD-pNA), respectively, into a yellow formazan product [p-nitroaniline (pNA)]. An increase in absorbance at 405 nm was used to quantify the activation of caspases. After exposure for 0, 6, 12 and 24 h, PC-3 cells were collected and rinsed with cold PBS, and then lysed by lysis buffer (40 μl) for 15 min on ice. Cell lysates were centrifuged at 18,000 rpm for 10 min at 4˚C. Activities of caspase-3, -6, -8 and -9 in the supernatant were assayed using a commercial kit. The caspase activities were expressed as percentage of enzyme activity compared to control. Control groups received 0.1% dimethyl sulfoxide. All the experiments were carried out in triplicate.

Cytotoxicity assay.[1]
PC-3 cells were plated in 96-well plates at a density of 1x104 cells per well. After incubation with medium for 24 h, 5 μM 7-xylosyl-10-deacetylpaclitaxel and paclitaxel, respectively, was applied for 48 h. Afterwards, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide was added to cell cultures at a final concentration of 0.5 mg/ml and incubated at 37˚C for 4 h. Then, the adherent cells were solubilized with 200 μl DMSO. Absorbance at 492 nm was measured using a multi-plate reader.

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Cell cycle analysis.[1]
PC-3 cells were treated with or without 5 μM 7-xylosyl-10-deacetylpaclitaxel. After incubation for 24 h, cells were collected and stained with 50 μg/ml DAPI. The cell cycle distribution of 10,000 cells was collected by using a flow cytometry. Cell cycle analysis was performed using FloMax software. Determination of apoptosis. Apoptosis was measured by Annexin-V staining according to manufacturer's instructions. After incubation with or without 5 μM 7-xylosyl- 10-deacetylpaclitaxel for 48 h, cells were spun at 1200 x g for 5 min, and the supernatant was decanted. The cell pellet was re-suspended with 100 μl of Annexin-V binding buffer and 5 μl of Annexin-V dye, and was left in dark at room temperature for 15 min. Following incubation, an additional 400 μl of Annexin-V binding buffer was added to each sample.

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Determination of levels of Fas (CD95).[1]
PC-3 cells were treated with 7-xylosyl-10-deacetyltaxol (5 μM) for 24 h or left untreated. Cells were spun at 1200 x g for 5 min, and the supernatant was decanted. The cell pellet was re-suspended in 10 μl anti-Fas (CD95) antibody, and was left in dark at room temperature for 15 min.

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Morphology analysis by transmission electron microscopy.[1]
PC-3 cells treated with or without 5 μM 7-xylosyl-10- deacetylpaclitaxel were washed with PBS, centrifuged, and pre-fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer at 4˚C for 2 h. The cells were then rinsed thoroughly in phosphate buffer and post-fixed in 1% OsO4 at 4˚C for 30 min. After being fixed, the cells were pelleted in 2% agar. Then, cell blocks were prepared, dehydrated through a graded ethanol series, and embedded in Epon 812. The ultra-structure of cells was analyzed in ultra-thin sections in a transmission electron microscope after the sections were stained with uranyl acetate and lead citrate.

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DNA fragmentation assay. [1]
PC-3 cells were exposed to 5 μM 7-xylosyl-10-deacetylpaclitaxel for 24 and 48 h or left untreated. Then, cells were collected by centrifugation. Total DNA was purified with a DNA isolation kit according to the manufacturer's instructions. The DNA was separated in 2% agarose gel and visualized by ultraviolet illumination after staining with ethidium bromide.

[1]. Activation of the mitochondria-driven pathway of apoptosis in human PC-3 prostate cancer cells by a novel hydrophilic paclitaxel derivative, 7-xylosyl-10-deacetylpaclitaxel. Int J Oncol. 2008 Jul;33(1):103-11.

C50H57NO17

943.9841

943.362

C, 63.62; H, 6.09; N, 1.48; O, 28.81

90332-63-1

90332-63-1

9811678

White to off-white solid powder

1.5±0.1 g/cm3

1068.4±65.0 °C at 760 mmHg

599.9±34.3 °C

0.0±0.3 mmHg at 25°C

1.660

7.18

7

17

14

68

1940

15

O([C@@]12CO[C@@H]1C[C@H](O[C@@H]1OC[C@@H](O)[C@H](O)[C@H]1O)[C@]1(C([C@@H](C3=C(C)[C@@H](OC(=O)[C@H](O)[C@H](C4C=CC=CC=4)NC(C4C=CC=CC=4)=O)C[C@]([C@H]([C@H]21)OC(C1C=CC=CC=1)=O)(O)C3(C)C)O)=O)C)C(=O)C

ORKLEZFXASNLFJ-ODBGVJAMSA-N

InChI=1S/C50H57NO17/c1-25-31(65-45(61)38(56)35(27-15-9-6-10-16-27)51-43(59)28-17-11-7-12-18-28)22-50(62)42(67-44(60)29-19-13-8-14-20-29)40-48(5,41(58)37(55)34(25)47(50,3)4)32(21-33-49(40,24-64-33)68-26(2)52)66-46-39(57)36(54)30(53)23-63-46/h6-20,30-33,35-40,42,46,53-57,62H,21-24H2,1-5H3,(H,51,59)/t30-,31+,32+,33-,35+,36+,37-,38-,39-,40-,42+,46+,48-,49+,50-/m1/s1

[(1S,2S,3S,4S,7R,9S,10S,12R,15S)-4-acetyloxy-15-[(2R,3S)-3-benzamido-2-hydroxy-3-phenylpropanoyl]oxy-1,12-dihydroxy-10,14,17,17-tetramethyl-11-oxo-9-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxy-6-oxatetracyclo[11.3.1.03,10.04,7]heptadec-13-en-2-yl] benzoate

10-deacetyl-7-xylosyl Paclitaxel; 10-Deacetyl-7-xylosyltaxol; Benzenepropanoic acid, b-(benzoylamino)-a-hydroxy-,(2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-12b-(acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-6,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-4-(b-D-xylopyranosyloxy)-7,11-methano-1H-cyclodeca[3,4]benz[1,2-b]oxet-9-yl ester, (aR,bS)-; [(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4-acetyloxy-15-[(2R,3S)-3-benzamido-2-hydroxy-3-phenylpropanoyl]oxy-1,12-dihydroxy-10,14,17,17-tetramethyl-11-oxo-9-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxy-6-oxatetracyclo[11.3.1.03,10.04,7]heptadec-13-en-2-yl] benzoate; 7-xylosyl-10-deacetyl taxol; 10-Deacetylpaclitaxel 7-Xyloside; 10-deacetyl-7-xylosyl paclitaxel (62%);

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: 33.3~100 mg/mL (35.3~105.9 mM)
H2O: ~1 mg/mL (~1.06 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.65 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 25.0 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: ≥ 2.5 mg/mL (2.65 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 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.65 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 25.0 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.)