No specific molecular target was identified. Chitosan exerts its antitumor effects through modulation of cell cycle regulatory proteins (p21, p27, PCNA) and apoptosis-related proteins (Bax, Bcl-2, caspase-3, caspase-8, caspase-9). [1,2,3]

Antiproliferative activity against melanoma cells: Coating culture surfaces with chitosan (2 mg/mL) produced distinct effects on three human melanoma cell lines. In A375 primary melanoma cells, chitosan reduced adhesion (from 90% to 73%) but did not affect proliferation or induce apoptosis. In SKMEL28 primary melanoma cells, chitosan increased doubling time from 29.0 h to 30.5 h, reduced proliferation rate by 15.1%, and induced low-level caspase-3 activation. In RPMI7951 metastatic melanoma cells, chitosan increased doubling time from 30.8 h to 34.4 h, induced strong caspase-3 activation, and increased apoptosis from 20% to 56% over 72 h. [1]
Mechanism of apoptosis in melanoma cells: In RPMI7951 cells, chitosan induced apoptosis through the mitochondrial pathway, as confirmed by inhibition with caspase-9 inhibitor Ac-LEHD-CHO (which reduced apoptosis) but not with caspase-8 inhibitor Ac-IETD-CHO. Western blot analysis showed that chitosan treatment upregulated pro-apoptotic Bax and downregulated anti-apoptotic Bcl-2 and Bcl-XL. The Bax/Bcl-2 ratio increased 3-fold. Chitosan also increased CD95/Fas receptor expression on the cell surface, sensitizing cells to FasL-induced apoptosis. [1]
Antiproliferative activity against gastric and colon cancer cells: Water-soluble chitosan oligomer (WSCO, with 3–10 saccharide residues) at 1,000 μg/mL significantly suppressed proliferation of AGS gastric cancer cells and COLO 205 colon cancer cells after 72 hours, but had no effect on T24 bladder cancer cells or Jurkat leukemia cells. [3]
Cell cycle effects in gastric cancer cells: WSCO (1,000 μg/mL) treatment of synchronized AGS cells reduced the percentage of cells in S phase from 24.2% to 16.8% at 48 hours, as measured by BrdU incorporation and flow cytometry. Western blot analysis showed that WSCO decreased PCNA expression and increased p21/Cip1 and p27/Kip1 expression, with no effect on cyclins A, B, E, cdc-2, p19, CDK2, or CDK4. [3]
Antimicrobial activity: Chitosan and its derivatives exhibit antimicrobial activity against various bacteria, fungi, and viruses. The minimum inhibitory concentrations (MICs) vary by target: against E. coli (8–16 μg/mL), S. aureus (8–16 μg/mL), C. albicans (5–100 μg/mL), and A. fumigatus (1 μg/mL). The anti-HIV activity of QMW-chitosan oligomers showed IC₅₀ values of 48.14–68.13 μg/mL for inhibition of cytopathic effects and p24 production. [2]
Antioxidant activity: Low-molecular-weight chitosan oligosaccharides scavenge free radicals (hydroxyl, superoxide, alkyl, DPPH) in a concentration-dependent manner, with activity correlated to degree of deacetylation and molecular weight. [2]

Colon cancer prevention in mice: Male ICR mice treated with azoxymethane (AOM, 5 mg/kg, i.p., twice weekly for 2 weeks) to induce aberrant crypt foci (ACF) were fed a diet containing 2% high-molecular-weight chitosan (HMWC, 327.9 kDa, 83.1% deacetylated) or low-molecular-weight chitosan (LMWC, 227.9 kDa, 87.3% deacetylated). After 2 weeks of co-treatment, HMWC significantly reduced ACF formation from 6.2 ± 3.7 to 3.8 ± 1.7 (P < 0.05). After 6 weeks of co-treatment, both LMWC and HMWC significantly reduced ACF formation (from 8.1 ± 7.6 to 2.9 ± 2.2 and 5.1 ± 4.5, respectively; P < 0.05). No significant reduction was observed in therapeutic experiments where chitosan was administered after AOM. [3]
No effect on body weight: Chitosan supplementation did not significantly affect body weight in any treatment group. [3]
Antitumor activity in sarcoma 180-bearing mice: Low-molecular-weight chitosan enhanced natural killer cell activity in intestinal intraepithelial lymphocytes, contributing to antitumor effects. [2]

Cell viability assay (trypan blue exclusion): Cells were seeded in 24-well plates and treated with various concentrations of chitosan (100–1,000 μg/mL) for 1–3 days. Medium with or without chitosan was changed daily. Viable cells were counted using trypan blue exclusion. [3]
Caspase activity measurement: Melanoma cells were lysed after culture on chitosan-coated surfaces (2 mg/mL) for various times. Caspase-3, -8, and -9 activities were measured using fluorogenic substrates, with fluorescence units normalized to protein concentration. [1]
Western blot analysis: Cells were lysed in RIPA buffer, and proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against Bax, Bcl-2, Bcl-XL, p21, p27, PCNA, PARP, and actin. Bands were visualized using HRP-conjugated secondary antibodies and chemiluminescence. [1,3]
Flow cytometry for apoptosis: RPMI7951-DsRed cells were cultured on chitosan-coated plates, and apoptosis was quantified by measuring DsRed fluorescence in supernatants versus attached cells. Caspase inhibitors (Ac-IETD-CHO for caspase-8, Ac-LEHD-CHO for caspase-9) were added to determine pathway involvement. [1]
BrdU incorporation assay: Synchronized AGS cells were treated with WSCO (1,000 μg/mL) and pulsed with BrdU for 1 hour before harvest. Cells were fixed, DNA denatured, and stained with anti-BrdU antibody and propidium iodide, then analyzed by flow cytometry. [3]
Cell cycle analysis by flow cytometry: Cells were harvested, fixed in 70% ethanol, stained with propidium iodide (50 μg/mL), and analyzed by flow cytometry to determine cell cycle distribution. [3]

Colon cancer prevention study (AOM model): Male ICR mice (10–12 weeks, 30–35 g) were given azoxymethane (AOM, 5 mg/kg) via intraperitoneal injection twice weekly for 2 weeks. Chitosan (HMWC or LMWC) was mixed into standard chow at 2% (w/w). In preventive experiments, mice received chitosan diet simultaneously with AOM for 2 or 6 weeks. In therapeutic experiments, chitosan diet was given for 6 weeks after AOM treatment. Mice were sacrificed at 2, 6, or 8 weeks, and colons were excised, fixed, stained with methylene blue, and aberrant crypt foci (ACF) were counted under light microscopy. [3]
Melanoma cell adhesion study: Human melanoma cells were plated on 12-well plates coated with chitosan (2 mg/mL in 0.1% acetic acid) or control (0.1% acetic acid). Non-adherent cells were collected at 2, 4, 6, and 8 hours, and counted using a Coulter counter. Adherent cells were also counted after washing. [1]
Chitosan coating preparation: Chitosan was dissolved in 0.1% acetic acid at 2 mg/mL. Culture wells were coated with this solution overnight at 4°C, then aspirated before cell seeding. [1]

Cytotoxicity to normal cells: Human dermal fibroblasts cultured on chitosan-coated surfaces (2 mg/mL) showed no significant caspase-3 activation and did not undergo apoptosis, though proliferation was reduced at higher concentrations (4 mg/mL). Fibroblasts maintained viability on chitosan-coated plates for up to 6 days. [1]
No effect on body weight: Mice fed chitosan-supplemented diet (2% w/w) for up to 8 weeks showed no significant difference in body weight compared to control animals. [3]
General safety: Chitosan is considered biocompatible, non-toxic, and non-immunogenic, with low allergenicity and good biodegradability. [2]

[1]. Anticancer properties of chitosan on human melanoma are cell line dependent. Int J Biol Macromol. 2015 Jan;72:370-9.

[2]. Chitosan: An Update on Potential Biomedical and Pharmaceutical Applications. Mar Drugs. 2015 Aug 14;13(8):5156-86.

[3]. Chitosan prevents the development of AOM-induced aberrant crypt foci in mice and suppressed the proliferation of AGS cells by inhibiting DNA synthesis. J Cell Biochem. 2007 Apr 15;100(6):1573-80.

Chitosan has reportedly been found in Didymella pinodes, and relevant data is available. Deacetylated chitosan, a linear polysaccharide composed of deacetylated β-1,4-D-glucosamine, is used in hydrogels and wound healing. See also: Polydextrose (note moved here).

C56H103N9O39

1526.4539

1525.635

9012-76-4

71853

White to off-white solid powder

1.75g/cm3

88ºC

1.7

-21.4

29

47

27

104

2630

45

COC(=O)N[C@@H]1[C@H]([C@@H]([C@H](O[C@H]1O[C@@H]2[C@H](O[C@H]([C@@H]([C@H]2O)N)O[C@@H]3[C@H](O[C@H]([C@@H]([C@H]3O)N)O)CO)CO)CO)O[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)CO)O[C@H]5[C@@H]([C@H]([C@@H]([C@H](O5)CO)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O[C@H]7[C@@H]([C@H]([C@@H]([C@H](O7)CO)O[C@H]8[C@@H]([C@H]([C@@H]([C@H](O8)CO)O[C@H]9[C@@H]([C@H]([C@@H]([C@H](O9)CO)O)O)N)O)N)O)N)O)N)O)N)O)N)O

FLASNYPZGWUPSU-SICDJOISSA-N

InChI=1S/C56H103N9O39/c1-87-56(86)65-28-38(84)46(19(10-74)96-55(28)104-45-18(9-73)95-49(27(64)37(45)83)97-39-12(3-67)88-47(85)20(57)31(39)77)103-54-26(63)36(82)44(17(8-72)94-54)102-53-25(62)35(81)43(16(7-71)93-53)101-52-24(61)34(80)42(15(6-70)92-52)100-51-23(60)33(79)41(14(5-69)91-51)99-50-22(59)32(78)40(13(4-68)90-50)98-48-21(58)30(76)29(75)11(2-66)89-48/h11-55,66-85H,2-10,57-64H2,1H3,(H,65,86)/t11-,12-,13-,14-,15-,16-,17-,18-,19-,20-,21-,22-,23-,24-,25-,26-,27-,28-,29-,30-,31-,32-,33-,34-,35-,36-,37-,38-,39-,40-,41-,42-,43-,44-,45-,46-,47-,48+,49+,50+,51+,52+,53+,54+,55+/m1/s1

methyl N-[(2S,3R,4R,5S,6R)-5-[(2S,3R,4R,5S,6R)-3-amino-5-[(2S,3R,4R,5S,6R)-3-amino-5-[(2S,3R,4R,5S,6R)-3-amino-5-[(2S,3R,4R,5S,6R)-3-amino-5-[(2S,3R,4R,5S,6R)-3-amino-5-[(2S,3R,4R,5S,6R)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-2-[(2R,3S,4R,5R,6S)-5-amino-6-[(2R,3S,4R,5R,6R)-5-amino-4,6-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-4-hydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-4-hydroxy-6-(hydroxymethyl)oxan-3-yl]carbamate

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)

H2O : < 0.1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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