Oxychloroaphine (1-256 μM; 24 h) induces damage to cell membranes, increasing apoptosis and lactate dehydrogenase leakage, as well as increasing the formation of cytochrome c protein. The IC50 values for cytotoxic agents against A549, HeLa, and SW480 cancer cell lines range from 32 to 40 μM[2]. Cycle arrest at G1 phase and induction of sub-G phase are caused by oxychloroaphine (32 μM; A549 and SW480 cells) [2]. After 48 hours, oxychloroaphine (A549 cells) causes the proapoptotic protein caspase-3 to become activated, which in turn causes the cleavage of PARP[2]. This results in the downregulation of the antiapoptotic Bcl-2 protein.

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Oxychlororaphine exhibited cytotoxicity in a dose-dependent manner on A549, HeLa, and SW480 cancer cell lines, with 50% inhibitory concentration (IC50) values between 32 and 40 μM. [2]
Treatment with Oxychlororaphine caused significant increase in lactate dehydrogenase (LDH) leakage into the culture medium, indicating cancer cell membrane destruction. The LDH levels were much lower than those induced by 0.10% Triton X-100, confirming that cell death was due to apoptosis rather than necrosis. [2]
Oxychlororaphine treatment resulted in increased production of cytochrome c protein in all three cancer cell lines, as measured by ELISA. [2]
Phase-contrast microscopy of Oxychlororaphine-treated cells showed dead and detached cells compared to compact control cells. Fluorescence microscopy with acridine orange/ethidium bromide staining revealed hallmarks of apoptosis including nuclear fragmentation, nuclear condensation, cell shrinkage, and formation of apoptotic bodies. [2]
Oxychlororaphine induced G1 cell cycle arrest and increased the sub-G phase (apoptotic phase) as shown by cell cycle analysis. [2]
Western blot analysis of Oxychlororaphine-treated cells showed downregulation of antiapoptotic proteins Bcl-2, Bcl-xL, and Bcl-w, upregulation of proapoptotic protein Bax, increased expression of p53 (phosphorylated S15), activation of caspase-3 (cleaved), and cleavage of PARP. [2]
RT-PCR analysis revealed that Oxychlororaphine treatment increased p53 mRNA expression (lower Ct values compared to control) and decreased mRNA expression of antiapoptotic Bcl-w and Bcl-xL (higher Ct values). [2]

Caspase-3 activity was measured in Oxychlororaphine-treated cancer cells. After treatment, cells were washed twice with ice-cold PBS and lysed in a hypotonic buffer solution. The lysates were homogenized, kept on ice for 30 minutes, and centrifuged at 12,500 × g for 20 minutes. The collected supernatants were used to measure caspase-3 activity using 200 μM Ac-DEVD-AMC fluorogenic substrate in an assay buffer containing 100 mM HEPES, 10% sucrose, 10 mM dithiothreitol, and 0.1% CHAPS. [2]

Cell Viability Assay[2]
Cell Types: A549, HeLa, and SW480 cancer cell lines
Tested Concentrations: 1, 2, 4, 8, 16, 32, 64, 128, and 256 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibited cell proliferative in a dose-dependent manner.

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MTT cytotoxicity assay: Cancer cells (A549, HeLa, SW480) were plated in 96-well plates at 5,000 cells per well and incubated for 24 hours at 37°C in 5% CO2. Oxychlororaphine was dissolved in DMSO, diluted with culture medium, and added at final concentrations of 1, 2, 4, 8, 16, 32, 64, 128, and 256 μM. After 48 hours incubation, cells were treated with 10% (v/v) MTT dye solution (5 mg/mL) for 4 hours. The MTT media solution was then replaced with DMSO solution, and absorbance was measured at 490 nm to determine cell viability and IC50 values. [2]
LDH leakage assay: Oxychlororaphine-treated cancer cells were centrifuged at 1,000 × g for 2 minutes, and LDH in the culture medium was measured using an in vitro toxicology assay kit. Triton X-100 (0.05%) was used as a positive control for membrane damage. [2]
Cytochrome c assay: Cytochrome c levels in Oxychlororaphine-treated cancer cells were determined using a cytochrome c ELISA kit, following the manufacturer’s instructions. [2]
RT-PCR for apoptotic genes: Total RNA was isolated from normal and Oxychlororaphine-treated cancer cell lines using an RNA isolation kit. Reverse transcription PCR was carried out with a first-strand cDNA synthesis kit following the manufacturer’s instructions to analyze p53, Bcl-w, and Bcl-xL mRNA expression. [2]
Caspase-3 activity assay: After Oxychlororaphine treatment, cells were lysed as described in Enzyme Assay. Caspase-3 activity was measured fluorometrically using Ac-DEVD-AMC substrate. [2]
Western blot analysis: The three cancer cell lines were treated with IC50 concentrations of Oxychlororaphine for 48 hours. Cell pellets were washed with PBS and lysed in lysis buffer (20 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 10 μg/mL aprotinin) for 30 minutes at 4°C, then centrifuged at 10,000 rpm for 20 minutes at 4°C. The supernatant (100 μg protein) was separated on 10% SDS-PAGE, transferred onto a nitrocellulose membrane, blocked with 1% bovine serum albumin for 1 hour at room temperature, and probed with antibodies against Bcl-2, Bcl-xL, Bcl-w, Bax, p53 phosphorylated S15, cleaved caspase-3, and cleaved PARP. Gene expression was quantified by band intensity using ImageJ software. [2]
Clonogenic assay: Mid-log-phase cells were trypsinized and placed in 60 mm Petri dishes at 150 cells per dish in triplicate. After treatment, cells were fixed with ethanol and stained with 1% methylene blue before counting. Cloning efficiency was 90–95% for A549 and HeLa, and about 60% for SW480. [2]

Oxychlororaphine caused increased LDH leakage in a concentration-dependent manner, indicating cell membrane damage. However, LDH levels were very low compared to those induced by 0.10% Triton X-100, confirming that the inhibition of cancer cells by Oxychlororaphine was due to apoptosis and not necrosis. [2]

[1]. Comparative metabolomics and transcriptomics analyses provide insights into the high-yield mechanism of phenazines biosynthesis in Pseudomonas chlororaphis GP72. J Appl Microbiol. 2022 Nov;133(5):2790-2801.

[2]. Isolation of Bioactive Phenazine-1-Carboxamide from the Soil Bacterium Pantoea agglomerans and Study of Its Anticancer Potency on Different Cancer Cell Lines. J AOAC Int. 2016 Sep;99(5):1233-9.

Phenazine-1-carboxamide is an aromatic amide with a structure in which phenazine is substituted at the C-1 position with a carbamoyl group. It belongs to the phenazine class of compounds and is both an aromatic amide and a monocarboxylic acid amide. Phenazine-1-carboxamide has been reported to exist in Streptomyces, and relevant data are available for reference.

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Oxychlororaphine (phenazine-1-carboxamide) is a natural phenazine compound isolated from Pantoea agglomerans present in soil. Phenazines are heterocyclic nitrogenous compounds also found in genera such as Pseudomonas, Streptomyces, Burkholderia, Pelagiobacter, and marine Vibrio spp. They are considered promising biological compounds with broad-spectrum antibiotic and antitumor activities. Due to their small molecule size, phenazines can easily permeate tissues and organs and have been associated with anticancer activities since 1959. The study concluded that Oxychlororaphine acts as an effective anticancer compound by inducing apoptosis through the mitochondrial pathway, involving p53 upregulation, Bax activation, cytochrome c release, and caspase-3/PARP cleavage. [2]

C13H9N3O

223.23

223.075

550-89-0

120282

Light yellow to brown solid powder

1.371g/cm3

526.1ºC at 760 mmHg

242ºC

272ºC

1.76

2.582

1

3

1

17

307

0

KPZYYKDXZKFBQU-UHFFFAOYSA-N

InChI=1S/C13H9N3O/c14-13(17)8-4-3-7-11-12(8)16-10-6-2-1-5-9(10)15-11/h1-7H,(H2,14,17)

phenazine-1-carboxamide

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)

DMSO : 10 mg/mL (44.80 mM)

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.)