Ca2+/Mn2+; mannose/glucose

Concanavalin A  (ConA), a Ca(2+)/Mn(2+)-dependent and mannose/glucose-binding legume lectin, has drawn a rising attention for its remarkable anti-proliferative and anti-tumor activities to a variety of cancer cells. ConA induces programmed cell death via mitochondria-mediated, P73-Foxo1a-Bim apoptosis and BNIP3-mediated mitochondrial autophagy. Through IKK-NF-κB-COX-2, SHP-2-MEK-1-ERK, and SHP-2-Ras-ERK anti-angiogenic pathways, ConA would inhibit cancer cell survival. In addition, ConA stimulates cell immunity and generates an immune memory, resisting to the same genotypic tumor. These biological findings shed light on new perspectives of ConA as a potential anti-neoplastic agent targeting apoptosis, autophagy and anti-angiogenesis in pre-clinical or clinical trials for cancer therapeutics. [1]

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Photodynamic therapy (PDT) is considered a very promising therapeutic modality for antimicrobial therapy. Although several studies have demonstrated that Gram-positive bacteria are very sensitive to PDT, Gram-negative bacteria are more resistant to photodynamic action. This difference is due to a different cell wall structure. Gram-negative bacteria have an outer cell membrane containing lipopolysaccharides (LPS) that hinder the binding of photosensitizer molecules, protecting the bacterial cells from chemical attacks. Combination of the lipopolysaccharides-binding activity of Concanavalin A  (ConA) with the photodynamic properties of Rose Bengal (RB) holds the potential of an innovative protein platform for targeted photodynamic therapy against Gram-negative bacteria. A ConA-RB bioconjugate was synthesized and characterized. Approximately 2.4 RB molecules were conjugated per ConA monomer. The conjugation of RB to ConA determines a decrease of the singlet oxygen generation and an increase of superoxide and peroxide production. The photokilling efficacy of the ConA-RB bioconjugate was demonstrated in a planktonic culture of E. coli. Irradiation with white light from a LED lamp produced a dose-dependent photokilling of bacteria. ConA-RB conjugates exhibited a consistent improvement over RB (up to 117-fold). The improved uptake of the photosensitizer explains the enhanced PDT effect accompanying increased membrane damages induced by the ConA-RB conjugate. The approach can be readily generalized (i) using different photo/sonosensitizers, (ii) to target other pathogens characterized by cell membranes containing lipopolysaccharides (LPS) [2].

Pharmacologic Relevance. Resveratrol, an antioxidant derived from grapes, has been reported to modulate the inflammatory process. In this study, we investigated the effects of resveratrol and its mechanism of protection on Concanavalin A - (ConA-) induced liver injury in mice. Materials and Methods. Acute autoimmune hepatitis was induced by ConA (20 mg/kg) in Balb/C mice; mice were treated with resveratrol (10, 20, and 30 mg/kg) daily by oral gavage for fourteen days prior to a single intravenous injection of ConA. Eight hours after injection, histologic grading, proinflammatory cytokine levels, and hedgehog pathway activity were determined. Results. After ConA injection, the cytokines IL-2, IL-6, and TNF-α were increased, and Sonic hedgehog (Shh), Glioblastoma- (Gli-) 1, and Patched (Ptc) levels significantly increased. Pretreatment with resveratrol ameliorated the pathologic effects of ConA-induced autoimmune hepatitis and significantly inhibited IL-2, IL-6, TNF-α, Shh, Gli-1, and Ptc. The effects of resveratrol on the hedgehog pathway were studied by western blotting and immunohistochemistry. Resveratrol decreased Shh expression, possibly by inhibiting Shh expression in order to reduce Gli-1 and Ptc expression. Conclusion. Resveratrol protects against ConA-induced autoimmune hepatitis by decreasing cytokines expression in mice. The decreases seen in Gli-1 and Ptc may correlate with the amelioration of hedgehog pathway activity. [3]

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Resveratrol Pretreatment Ameliorates Concanavalin A /ConA-Induced Hepatitis [3]
We performed an assay using a ConA-induced hepatitis model and assessed liver function by measuring serum ALT and AST levels. Mice liver tissue of each group was assessed by pathologic examination and Knodell scoring. ALT and AST levels increased after injection of ConA and peak levels decreased in the high-dose resveratrol group. As shown in Table 1, ALT and AST levels clearly increased after ConA injection compared with the saline group at 8 hours (p < 0.05); resveratrol pretreatment significantly attenuated the ConA injection-induced elevation of serum ALT and AST (p < 0.05). This same result was demonstrated in the histopathologic study and Knodell scores. As shown in Table 1, the Knodell scores of the resveratrol group gradually decreased and there was statistical significance demonstrated between the different concentrations of resveratrol (p < 0.01). As shown in Figure 1, we found massive areas of necrosis in the ConA-induced group. In contrast, the resveratrol-treated group showed minor liver damage, indicating that resveratrol pretreatment significantly reduced liver necrosis. Resveratrol administered at 30 mg/kg was more effective. According to the results as analyzed with Image pro Plus 6.0, statistical significance was clearly demonstrated among groups. Thus, resveratrol pretreatment was shown to attenuate ConA-induced autoimmune hepatitis in mice.

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Resveratrol Pretreatment Inhibits the Release of Cytokines during Concanavalin A /ConA-Induced Hepatitis and Expression of Hh Signal Pathway [3]
ConA-induced hepatitis is associated with changes in the levels of inflammatory cytokines. We demonstrated with immunoblotting that TNF-α, IL-2, and IL-6 expression was significantly increased in the ConA model group compared to the saline control group. Resveratrol reduced TNF-α, IL-2, and IL-6 production in the ConA-induced mice liver, which is consistent with the degree of liver injury. Next, we examined the effect of resveratrol on Shh, Gli-1, and Ptc expression in the ConA-induced mice liver. Resveratrol pretreatment significantly attenuated Gli-1 and Ptc expression compared to the ConA model group (p < 0.05), which was consistent with changes in immunohistochemistry (Figure 2).

ConA-RB Synthesis [2]
Rose Bengal disodium salt was dissolved in DMSO to obtain a concentration 10 mM, then solid EDC and Sulfo-NHS were added under stirring, to obtain a final concentration of 27.5 mM and 15 mM respectively. After 5 h this solution was mixed, under vigorous stirring, to Concanavalin A 0.05 mM in sodium carbonate buffer 100 mM (pH 9) with a ten-fold excess of activated RB (concentration 0.5 mM). The reaction was incubated overnight under mild stirring condition.

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ConA-BR Purification [2]
Once the cross-linking reaction was completed, the ConA-RB bioconjugate was purified by dialysis. Unreacted RB and small molecular weight byproducts of the crosslinking reaction were removed by dialysis in sodium carbonate buffer 10 mM (pH 9) using a membrane with a 14,000 KD cut off. Dialysis was repeated until the UV–vis signal at 550 nm typical of the RB disappeared completely from the dialyzing solution. ConA-RB synthesis and purification are carried out at room temperature.

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ABMDMA Singlet Oxygen Assay [2]
Isoabsorbing solutions at 555 nm of RB and ConA-RB were prepared in deuterated water. ABMDMA was added to the solutions to reach a final concentration of 15 μM of the sensitizer and 25 μM of ABMDMA. The solutions were stirred vigorously to ensure air saturation. The solutions were irradiated at 555 nm and the bleaching of the absorption band of ABMDMA at 401 nm was monitored. The singlet oxygen quantum yield (ΦΔ) was determined using Rose Bengal (RB) as the reference with a yield of 0.76 in PBS. The ΦΔ of ConA-RB was calculated by the following equation where K is the slope of the photodegradation rate of ABMDMA, S represents the sample (ConA-RB), R means the reference (RB), and ΦΔR is the ΦΔ of the reference (RB).

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Amplex Red Peroxides Quantification [2]
1 ml of phosphate buffered saline 50 mM pH 7.4 was added to 10 μl of Amplex Red 50 mM in DMSO. Then 10 μl of HRP 0.4 mg/ml in PBS 50 mM pH 7.4 was added to the Amplex Red solution to obtain the final working solution. 90 μl of the solutions under investigation, containing different concentrations (0.025 μM, 0.05 μM, 0.25 μM, 0.5 μM, 2.5 μM and 5 μM) of RB or ConA-RB in PBS 50 mM pH 7.4, isoabsorbing in the visible range, were irradiated for 45 min with visible light (white LED Valex 30 W lamp at 30 cm distance from the cell-plate, irradiation power density on the cell plate = 2 mW/cm2; measured with the photo-radiometer Delta Ohm LP 471 RAD), on microtiter plates and 10 μl of Amplex Red working solution was added to each sample immediately after irradiation. Solutions were incubated 30 min at room temperature and the absorbance of the samples is read at 560 nm. The absorbance values were converted to the concentration of H2O2 generated upon irradiation, using a calibration curve generated using standard solutions of H2O2.

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Nitroblue Tetrazolium (NBT) Assay to Determine the Production of Superoxide Anion [2]
3 ml of isoabsorbing solutions of RB and ConA-RB (2.5 μM) in PBS 50 mM pH 7.4 were prepared. NBT was added to the solutions to reach a final concentration of 0.24 mM. Control solutions of NBT in milli-Q water and NBT in ConA were also prepared. The solutions were irradiated for 45 min with visible light (white LED Valex 30 W lamp at 30 cm distance from the cuvette, irradiation power density on the cell plate = 2 mW/cm2; measured with the photo-radiometer Delta Ohm LP 471 RAD).

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Terephthalate (TPA) Assay to Determine the Production of Hydroxyl Radical [2]
A 200 mM stock solution of terephthalic acid (TPA) was prepared using NaOH. 2.5 μM solutions of RB and ConA-RB in PBS 50 mM pH 7.4 were prepared. TPA was added to the solutions to reach a final concentration of 500 μM. The solutions were irradiated at 555 nm where RB and ConA-RB are isoabsorbing. The changes in fluorescence at 425 nm (excitation 315 nm), due to the production of 2-hydroxyterephthalate (HTPA), was measured using a Edinburgh Analytical Instruments FLS920 spectrofluorometer. All fluorescence measurements were carried out at room temperature.

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Photodynamic Antimicrobial Activity Assay [2]
The antimicrobial activity of ConA-adducts was tested on E. coli (strain DH5α) during exponential growth. Bacteria were resuspended in PBS 1× to obtain a final concentration of 2·106 cfu/ml in each well of a 96-well microtiter plate. Different concentrations of RB and ConA-RB were used, in a sensitizer concentration range between 5 and 0.025 μM. The plate with bacteria and photosensitizers was incubated for 30 min in the dark and subsequently irradiated for 45 min with white LED lamp producing 2 mW/cm2 at the cell plate, at room temperature. These conditions were optimized to provide the maximum non-lethal light fluency on untreated DH5α control cultures.

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Photosensitizer Uptake by Bacteria [2]
2·105 bacteria grown in LB to exponential phase were pelleted by centrifugation, washed once in 1×PBS and then resuspended in 200 μl 1×PBS containing RB or the ConA-RB adduct (both at 2.5 μM sensitizer). After 30-min incubation in the dark, bacteria were pelleted and the supernatant, containing unbound RB or ConA-RB, was collected. Bacterial pellets were then extensively washed 3 times and resuspended in 1X PBS. All fractions were collected for fluorescence readout and read in an EnSpire multimode plate reader (PerkinElmer; 560/575 nm excitation/emission).

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Membrane Damage Assay [2]
A fluorimetric permabilization assay was used to investigate the light-dependent damage induced by RB and ConA-RB on the bacterial membrane. This assay is based on Propidium Iodide (PI), a widely used DNA intercalating fluorescent stain unable to permeate intact, undamaged cell membranes. Briefly, 5·108 bacterial cells grown to late exponential phase (A600 = 0.9) were pelleted, and resuspended in 1× PBS containing the ConA-RB adduct or RB alone (2.5 μM sensitizer for both) in the wells of two microtiter plates. The first was irradiated as previously described, while the second was kept in the dark (control). Following irradiation, bacteria were pelleted and cross-linked for 5 min in 2% formaldehyde 1× PBS to fix the cells. The reaction was then blocked with 125 mM glycine for 10 min. After two rounds of washing in 1× PBS, bacteria were stained in a solution containing 2 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) and 0.3 μg/ml PI in 1× PBS. After a last washing step in PBS, the fluoresence signal of PI (535/617 nm) and DAPI (358/461 nm) was recorded in a plate reader. DAPI was used to normalize the PI fluorescent signal since it similarly stains the DNA of both permeabilized and non-permeabilized cells. The permabilization index, defined as the PI/DAPI signal ratio, was normalized to the control samples kept in the dark (n = 6).

ConA/Concanavalin A was dissolved in pyrogen-free physiological saline and intravenously injected at a dose of 20 mg/kg to induce hepatitis.
Male Balb/c mice (6–8 weeks old, 20 ± 2 g) were housed in plastic cages with controlled light and dark cycles and fed a standard diet with water in a controlled temperature (25 ± 1°C) and humidity (50 ± 5%) environment. Hepatic injury was elicited in 6–8-week-old male mice by injecting Concanavalin A/ConA (20 mg/kg body weight or bw) into the tail vein. Resveratrol was given according to dose into three groups (high, medium, and low dose). The mice were randomly divided into six groups of ten mice as follows: (1) saline control group, (2) resveratrol-alone group, (3) ConA-induced model group, (4) low-dose resveratrol + ConA group, (5) medium-dose resveratrol + ConA group, and (6) high-dose resveratrol + ConA group. For the first two weeks, the mice in the resveratrol group received 0.1 mL/10 gbw/day by oral administration. The remaining mice received 0.5% carboxymethyl cellulose solution at 0.1 mL/10 gbw/day. At the fourteenth day, one hour after oral administration, Concanavalin A (0.05 mL/10 g) was injected into the caudal veins of mice, except for the saline and resveratrol-alone groups, which received saline (0.05 mL/10 g). After 8 hours, animals were sacrificed to obtain eye blood. The left hepatic lobes were stored at −80°C until the IL-2, IL-6, and TNF-α assays were performed. The right hepatic lobes were fixed in 4% paraformaldehyde at 4°C for hematoxylin-eosin (HE) and immunohistochemical staining [3].

Adverse Effects
Occupational hepatotoxin - Secondary hepatotoxins: the potential for toxic effect in the occupational setting is based on cases of poisoning by human ingestion or animal experimentation.

[1]. Concanavalin A: a potential anti-neoplastic agent targeting apoptosis, autophagy and anti-angiogenesis for cancer therapeutics. Biochem Biophys Res Commun. 2011 Oct 22;414(2):282-6.

[2]. Concanavalin A-Rose Bengal bioconjugate for targeted Gram-negative antimicrobial photodynamic therapy. J Photochem Photobiol B. 2020 Mar 13;206:111852.

[3]. The Protective Effect of Resveratrol on Concanavalin-A-Induced Acute Hepatic Injury in Mice. Gastroenterol Res Pract. 2015;2015:506390.

Concanavalin A is a mannose-binding lectin originally isolated from jack-bean, Canavalia ensiformis. Concanavalin A is a potent lymphocyte mitogen and a stimulator of matrix metalloproteinases, thereby exhibiting immunostimulatory effects. It is used in the characterization and purification of glycoproteins.
A MANNOSE/GLUCOSE binding lectin isolated from the jack bean (Canavalia ensiformis). It is a potent mitogen used to stimulate cell proliferation in lymphocytes, primarily T-lymphocyte, cultures.

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ConA/Concanavalin A has exhibited the potential as an anti-tumor agent to diversified kinds of cells (e.g., PU-1.8, A375, fibroblasts, HepG2, hepatoma, U87 glioblastoma and p53-null cells) with concomitant toxicity that triggers acute hepatitis in pre-clinical and clinical trials. As discussed previously, ConA has been shown to induce cancer cell death by mitochondria-mediated, p73-Foxo1a-Bim apoptosis, BNIP3-mediated mitochondrial autophagy, as well as IKK-NF-κB-COX-2, SHP-2-MEK-1-ERK, SHP-2-Ras-ERK anti-angiogenesis, ConA also brings about tumor cell death via immunomodulatory. As the molecular mechanisms of ConA-induced cell death becoming much clearer, new possible therapeutic strategies would be further developed and tested, such as those based on programmed cell death and anti-angiogenesis activities. Included in pre-clinical and clinical trials, more in-depth researches aimed at the machinery of PCD and anti-angiogenesis on the molecular level help cancer biologists and clinicians to gain intimate knowledge of therapeutic effects of ConA. The elucidation of the molecular mechanism of lectin-induced cell death has opened a new perspective field for novel agent and drug developments. [1]

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Conjugation of RB with Concanavalin A/ConA increases i) the uptake of the photosensitizer, ii) its photokilling efficacy and iii) membrane damage after PDT treatment. A modulation between type I and type II mechanisms in the ROS generation is exerted by the protein conjugation. Even if the current set of experiments is not sufficient to validate ConA-RB bioconjugates as a novel targeted-photosensitizer with clinical significance, they may pave the way to additional in-vivo PK/PD, therapeutic and toxicity studies needed for their translational application. The developed procedure of conjugation of RB to ConA is general and may be used for any photosensitizer in targeted Gram-negative antimicrobial photodynamic therapy, improving the therapeutic efficiency of PDT. [2]

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We aimed to determine whether resveratrol could inhibit the expression and release of cytokines such as TNFα, IL-2, and IL-6 in Concanavalin A/ConA-induced autoimmune hepatitis. We found the following: (1) resveratrol attenuated ConA-induced autoimmune hepatitis in mice, (2) resveratrol decreases TNFα, IL-2, and IL-6 expression in vivo, and (3) resveratrol may inhibit the release of Gli-1 and Ptc through modulation of the hedgehog signal pathway. Although there are further mechanisms to be explored, our study provides a new approach for the treatment of acute hepatitis in the clinic. [3]

11028-71-0

155486958

White to light yellow solid powder

39 °C

3

11

10

38

858

0

CONCANAVALIN A; 11028-71-0; Concanavaline A; Ricintoxin con A; Ricin-toxin con A; Con-A; CON A; DTXSID3037202;

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 : ~50 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.)