In combination studies, the IC50 value was lower than that of the ATRA group when cells were treated with both C-phycocyanin and all-trans retinoic acid (ATRA). However, the more C-phycocyanin used, the less ATRA used under the same IC50. The outcomes demonstrated that ATRA and C-phycocyanin together could considerably lower CDK-4 mRNA levels (P<0.05). When comparing the C-phycocyanin+ATRA combination group to the mutant, C-phycocyanin or ATRA treatment, the combined group's integrated optical density (IOD) increased, the difference was statistically significant (P<0.05), and the difference in the combined group further expanded (P<0.01). The combination index (CI) value of the group was 0.852, which was less than 1. While the combination group had the highest expression of caspase-3, the C-phycocyanin group had the biggest expression, which was comparable to that of the ATRA group [1].

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C-Phycocyanin alone inhibited the proliferation of A549 lung cancer cells in a concentration-dependent manner. The IC₅₀ value of C-Phycocyanin alone was 176.62 ± 6.38 μg/l after 48 h of treatment. [1]
Combination of C-Phycocyanin (88 μg/l) with all-trans retinoic acid (0.102 mM) showed synergistic antitumor effects with a combination index (CI) of 0.852 (CI < 1 indicates synergy). The dosage of ATRA could be reduced when combined with C-Phycocyanin. [1]
C-Phycocyanin alone decreased CDK-4 mRNA level in A549 cells as determined by in situ hybridization. The integrated optical density (IOD) of CDK-4 mRNA was significantly lower in the C-Phycocyanin-treated group compared to the control group (P < 0.05). [1]
C-Phycocyanin alone induced apoptosis in A549 cells as shown by TUNEL assay. The IOD value of apoptotic cells was increased in the C-Phycocyanin group compared to the control group (P < 0.05). [1]
C-Phycocyanin alone upregulated caspase-3 protein expression in A549 cells as determined by Western blot. The grayscale ratio of caspase-3/β-actin was higher than that of the control group (P < 0.05). [1]

The C-phycocyanin or ATRA-treated groups' average tumor weights were significantly lower than those of the tray group, and they were even lower in the C-phycocyanin+ATRA synergistic group. The findings indicate that mice's liver weight can be raised by a single C-phycocyanin, and that C-phycocyanin can enhance immune function and encourage the development of mice's immune organs. When administered separately, C-phycocyanin and ATRA can both considerably decrease the production of Cyclin D1, and when combined, the inhibitory impact is more pronounced [1].

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In NU/NU mice bearing A549 tumor xenografts, treatment with C-Phycocyanin alone (0.2 ml of 320 mg/ml injected into the tumor area daily for 10 days) significantly reduced tumor weight compared to the control group (4.538 ± 0.043 g vs. 6.057 ± 0.033 g, P < 0.05). [1]
C-Phycocyanin alone increased spleen weight in tumor-bearing mice (1.456 ± 0.055 g) compared to the control (1.034 ± 0.11 g) (P < 0.05). [1]
C-Phycocyanin alone enhanced T lymphocyte activities as determined by the specific rosette forming test (SRFC) (20,254 ± 352 per 10⁶ lymphocytes) compared to the control (16,724 ± 321) (P < 0.05). The MTT assay also showed increased optical density (1.425 ± 0.108 vs. 0.978 ± 0.087, P < 0.05). [1]
C-Phycocyanin alone increased TNF content in the supernatant of spleen cells compared to the control group (P < 0.05). [1]
C-Phycocyanin alone reduced Bcl-2 protein expression in tumor tissues as determined by immunohistochemistry (P < 0.05). [1]
C-Phycocyanin alone inhibited Cyclin D1 protein expression in tumor tissues as determined by immunofluorescence (P < 0.05). [1]

A549 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum at 37°C in a 5% CO₂ atmosphere. Exponentially growing cells were used for all experiments. [1]
For the MTT proliferation assay, cells (1×10⁴ cells/well) were seeded into 96-well plates in 100 μl of medium and cultured overnight. Then, various concentrations of C-Phycocyanin in 100 μl of medium were added and incubated for 48 h. Subsequently, 20 μl of MTT solution was added to each well and incubated for 4 h at 37°C. The medium was discarded, formazan crystals were dissolved in 100 μl of DMSO, and the absorbance was read at 490 nm using a spectrophotometer. Absorbance values were expressed as a percentage of the treated group versus the untreated control group. The half-maximal inhibitory concentration (IC₅₀) was calculated. [1]
For in situ hybridization (ISH) detection of CDK4 mRNA, A549 cells were seeded on poly-L-lysine-coated coverslips in 6-well plates, treated with drugs for 48 h, then fixed in freshly prepared 4% paraformaldehyde solution for 30 min at room temperature. Endogenous peroxidase was inactivated by 3% H₂O₂. Binding sites were exposed by digestion with pepsin (diluted in 3% citric acid) for 2 min at 37°C. Prehybridization was carried out at 38°C for 4 h, followed by hybridization at 38°C for 12 h with a digoxin-labeled oligonucleotide probe for human CDK4 mRNA (5'-ACCTT TAACC CACAT AAGCG AATCT CTGCC TT-3'). The coverslips were then washed sequentially with 2× SSC for 5 min, 0.5× SSC for 15 min, and 0.2× SSC for 15 min at 37°C. Cells were incubated in blocking buffer for 30 min and then with mouse anti-digoxin IgG for 60 min at 37°C. After washing with PBS, the coverslips were immersed in streptavidin-biotin complex (SABC) for 20 min and then in biotinylated peroxidase for 30 min at 37°C. Finally, the coverslips were stained with diaminobenzidine (DAB) and observed under a light microscope. The integrated optical density (IOD) of each group was calculated by Image-Pro Plus software and used as an index for CDK4 mRNA levels. [1]
For TUNEL apoptosis determination, A549 cells were washed with PBS and fixed in 4% paraformaldehyde. DNA breaks were labeled sequentially with TdT, bromodeoxyuridine triphosphate (BrdUTP), and DIG-labeled anti-BrdU antibody. Then the cells were incubated in blocking buffer, biotinylated anti-digoxin IgG, SABC, and DAB in turn. IOD of each group was calculated by Image-Pro Plus software and used as an index of apoptosis levels. [1]
For Western blot detection of caspase-3 protein, A549 cells were harvested, washed twice with cold PBS, lysed for 2 h on ice, and centrifuged at 4°C to remove insoluble materials. The lysate supernatant was separated using SDS-PAGE gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Subsequently, the membrane was blocked with TBS (containing 5% skim milk powder, pH 7.4) and then incubated overnight with rabbit anti-human caspase-3 antibody and rabbit anti-human β-actin antibody, followed by HRP-conjugated goat anti-rabbit secondary antibody for 2 h. Finally, the caspase-3 protein was detected by DAB. The bands were scanned and analyzed by Image J software. Caspase-3 levels were normalized with respect to β-actin levels, and the grayscale ratio of caspase-3/β-actin was calculated. [1]

A total of 40 adult NU/NU mice (20 males and 20 females, 20-22 g) were used. Mouse tumor models were established by subcutaneous injection of 2×10⁶ A549 cells near the armpit area. Fifteen days later, the mice were randomly divided into four groups. For C-Phycocyanin treatment alone, mice received 0.2 ml of C-Phycocyanin at a concentration of 320 mg/ml injected directly into the tumor area once daily for 10 days. For the combination group, mice received 0.2 ml of C-Phycocyanin (320 mg/ml) together with 0.2 ml of all-trans retinoic acid (10 mM) injected into the tumor area at the same time daily for 10 days. Two days after drug withdrawal, the mice were euthanized, and tumors and spleens were removed. All mice survived. All studies were approved by the institutional animal care and use committee. Animals were housed under standard conditions with ad libitum food and water and a 12:12 light:dark cycle. [1]

C-Phycocyanin is a natural component of edible Spirulina platensis and has no toxic side effects. When combined with ATRA, C-Phycocyanin reduced the toxicity of ATRA and ameliorated the ATRA-induced decrease in spleen weight and T lymphocyte activity in tumor-bearing mice. [1]

[1]. The synergistic antitumor effects of all-trans retinoic acid and C-phycocyanin on the lung cancer A549 cells in vitro and in vivo. Eur J Pharmacol. 2015 Feb 15;749:107-14.

C-Phycocyanin is extracted from Spirulina platensis and has been widely used for 100 years as an excellent nutrient supplement for human beings. In this study, C-Phycocyanin combined with all-trans retinoic acid showed synergistic antitumor effects on A549 lung cancer cells both in vitro and in vivo, reducing the required dosage and toxicity of ATRA. The combination inhibited tumor growth by inhibiting cell cycle progression (decreasing CDK-4 and Cyclin D1), inducing apoptosis (upregulating caspase-3 and downregulating Bcl-2), and enhancing body immunity (increasing T lymphocyte activity, spleen weight, and TNF secretion). [1]

0

11016-15-2

Light blue to blue liquid

[C-Phycocyanin]

Spirulina extract

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)