STAT3

MC0704 (0–50 μM; 72 h) is cytotoxic, with IC50 values of 3.11, 33.25, 2.98, 3.24, and 2.87 μM against HEK-293, lung epithelial cells (MRC-5), MDA–MB 231, MDA–MB-231 paclitaxel-resistant (MDA–MB-231-PTR), and MDA–MB– 231 docetaxel-resistant (MDA–MB-231-DTR) [1]. The STAT3 activity is inhibited by MC0704 (0, 0.4, 2, 10 and 50 μM; 24 h) with an IC50 value of 2.13 μM [1]. MC0704 (5 μM; 24 h) prevents STAT3 from being activated [1]. MC0704 (0, 2.5, 5 and 10 μM; 36-48 h) causes apoptosis and enhances the cell cycle arrest of MDA-MB-231-DTR cells in the G2/M phase [1].

In mice transplanted with MDA-MB-231-DTR cells, MC0704 (10 mg/kg, intraperitoneal injection, once day, for 12 days) suppresses tumor growth [1].

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Antitumor Activity of MC0704 in Nude Mouse Xenograft Models Implanted with MDA-MB-231-DTR Cells [1]
The in vivo antitumor activity of MC0704 was evaluated in MDA-MB-231-DTR cell-implanted nude mouse xenograft models. When the tumor volume reached about 100 mm3, vehicle (DMSO/cremophor/normal saline = 10:10:80), docetaxel (5 mg/kg body weight), paclitaxel (5 mg/kg body weight), MC0704 (10 mg/kg body weight), or a combination of docetaxel (5 mg/kg body weight) and MC0704 (10 mg/kg body weight) was intraperitoneally treated daily for 12 days. Tumor volume and body weight change were measured every 2 days during the experiments. Previous studies have reported that docetaxel or paclitaxel effectively inhibits tumor growth in an MDA-MB-231 cell-implanted mouse model. In the present study, however, the antitumor activity of docetaxel and paclitaxel was negligible in MDA-MB-231-DTR cell-implanted models. These data suggested that the acquired resistance to docetaxel was still maintained during tumor formation and that cross-resistance occurred for paclitaxel, a taxane-type compound. Treatment with MC0704 (10 mg/kg), however, significantly inhibited tumor growth without any overt toxicity (Figure 9A). Tumor weight on the last day of the experiments was also diminished by MC0704 treatment. In addition, consistent with the in vitro combination effect on antiproliferation, the combination treatment of docetaxel and MC0704 exhibited significantly enhanced antitumor activity (tumor volume and tumor weight) compared to the administration of either docetaxel or MC704 alone (Figure 9A–C). Furthermore, immunohistochemical analysis of tumor tissues revealed that p-STAT3 (activated-STAT3), Ki-67 (a well-known proliferation marker), and vimentin (a mesenchymal marker) was considerably upregulated but that the expression of E-cadherin (an epithelial marker) was downregulated in the resistant MDA-MB-231-DTR cell-implanted tumor tissues compared to the parental MDA-MB-231 cell-implanted tumor tissues. The expression of these biomarker proteins was effectively reversed by MC0704 treatment in MDA-MB-231-DTR cell-implanted tumor tissues.

Human Phospho-Kinase Array[1]
MDA-MB-231-DTR cells were treated with the 10 μM of MC0704 for 24 h, and the levels of protein expression were determined using human phospho-kinase array kit according to the manufacturer’s protocol.

Cell Cytotoxicity Assay[1]
Cell Types: HEK-293 and MRC-5 cell lines
Tested Concentrations: 0-50 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: demonstrated cytotoxicity to HEK-293 and MRC-5 cells.

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Western Blot Analysis[1]
Cell Types: MDA-MB-231 TNBC cell line
Tested Concentrations: 5 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibited the expression of p-STAT3.

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Cell Cycle Analysis [1]
Cell cycle dynamics were measured using flow cytometry. MDA-MB-231-DTR cells were cultured in serum-free medium for 24 h for synchronization. After synchronization, the cells were treated with either vehicle (DMSO) or MC0704 for the indicated times in complete medium. After incubation, all adherent and floating cells were collected, washed twice with PBS, and subsequently fixed in 70% cold ethanol overnight at −20 °C. The fixed cells were washed with cold PBS and resuspended in 100 μg/mL RNase A in a shaker for 30 min. Cells were then stained with 50 μg/mL PI in the dark. The fluorescence-activated cells were then sorted, and the cellular DNA content was analyzed using a flow cytometer. Data were calculated using CellQuest 3.0.1 software, and the distributions of cells in each phase of the cell cycle are displayed as histograms.

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Apoptosis Detection Assay (Annexin V-Fluorescein Isothiocyanate and PI Double Staining)[1]
MDA-MB-231-DTR cells were treated with MC0704 for 48 h in complete medium and then stained with Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and PI using the Annexin V-FITC apoptosis detection kit according to the manufacturer’s recommendations. Briefly, the incubated cells were harvested, washed twice in cold PBS, resuspended in 1× binding buffer, and treated with Annexin V-FITC and PI in the dark for 15 min. The stained cells were resuspended in 1× binding buffer and immediately analyzed using a flow cytometer.

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Transwell Cell Invasion Assay[1]
In brief, 24-well Transwell membrane inserts (diameter, 6.5 mm; pore size, 8 μm) were coated with 10 μL of type I collagen (0.5 mg/mL) and 20 μL of a 1:20 mixture of Matrigel in PBS. After treatment with MC0704 for 24 h, MDA-MB-231 human TNBC cells (parent or docetaxel-resistant) were harvested, resuspended in serum-free medium, and plated (3 × 105 cells/chamber) in the upper chamber of the Matrigel-coated Transwell insert. Medium containing 30% FBS was used as the chemoattractant in the lower chamber. After 24 h of incubation, cells that had invaded the outer surfaces of the lower chambers were fixed and stained using the Diff-Quik Staining Kit and imaged using the Vectra 3.0 Automated Quantitative Pathology Imaging System. Representative images from three separate experiments were evaluated, and the number of invasive cells was semiquantified using ImageJ 1.52a software.

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Cell Migration Assay (Wound-Healing Assay)[1]
MDA-MB-231 human TNBC cells (parent or docetaxel-resistant) were grown to 90% confluence in a six-well plate. Subsequently, the cell monolayer was artificially wounded using the SPL Scar Scratcher, and detached cells were removed after washing with PBS. Wounded cell cultures were then incubated with media containing 1% FBS and various concentrations of MC0704 for 24 h. Wounds were photographed at 0 and 24 h under an inverted microscope. Wound areas were quantified using ImageJ 1.52a software and presented as percent cell migration (%) relative to the wound area at 0 h.

Animal/Disease Models: Female BALB/c-nu (nude) mice with MDA-MB-231-DTR cells transplant[1]
Doses: 10 mg/kg
Route of Administration: intraperitoneal (ip)injection; 10 mg/kg, one time/day for 12 days
Experimental Results: Dramatically inhibited tumor growth without overt toxicity, and reversed the mesenchymal marker vimentin and epithelial marker E-cadherin expression changes.

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In Vivo Tumor Xenograft Model [1]
Female nude mice (BALB/c-nu, aged 5–6 weeks, and weighing 18 g) were housed under pathogen-free conditions with a 12 h light–dark schedule. MDA-MB-231 or MDA-MB-231-DTR cells were injected subcutaneously into the flanks of mice (4 × 106 cells in 200 μL of PBS), and tumors were allowed to grow for 10 days until the tumor volume reached approximately 100 mm3. The mice were randomized into vehicle control and treatment groups (n = 6). Vehicle control (DMSO/cremophor/normal saline = 10:10:80), docetaxel (5 mg/kg body weight), paclitaxel (5 mg/kg body weight), MC0704 (10 mg/kg body weight), or a combination of docetaxel (5 mg/kg body weight) and MC0704 (10 mg/kg body weight) was administered intraperitoneally (i.p.) daily for 12 days, and mice were euthanized 3 days later. Tumors were excised, weighed, and frozen for biochemical analysis. Tumor volume was measured using an electronic caliper according to the following formula: tumor volume (mm3) = 3.14 × length × width × height/6. Toxicity was evaluated based on body weight loss.

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Ex Vivo Immunohistochemical Analysis 1]
Tumor tissues fixed in 10% neutral buffered formalin solution were embedded in paraffin, sectioned, mounted onto slides, deparaffinized with xylene, rehydrated with an ethanol series, and subjected to antigen retrieval. The slides were incubated with the indicated antibodies, which were detected using the LSAB2 System-HRP kit. Stained sections were observed and imaged using the Vectra 3.0 Automated Quantitative Pathology Imaging System

[1]. Design, Synthesis, and Biological Activity of Marinacarboline Analogues as STAT3 Pathway Inhibitors for Docetaxel-Resistant Triple-Negative Breast Cancer. J Med Chem. 2023 Feb 23;66(4):3106-3133.

Metastatic triple-negative breast cancer (mTNBC) is a fatal type of breast cancer (BC), and signal transducer and activator of transcription 3 (STAT3) has emerged as an effective target for mTNBC. In the present study, compound MC0704 was found to be a novel synthetic STAT3 pathway inhibitor, and its potential antitumor activity was demonstrated using in vitro and in vivo models in docetaxel-resistant TNBC cells. Based on marinacarboline (MC), a series β-carboline derivatives were synthesized and investigated for their antitumor activities against docetaxel-resistant MDA-MB-231 (MDA-MB-231-DTR) cells. Combining antiproliferation and STAT3 inhibitory activities, MC0704 was selected as the most promising β-carboline compound. MC0704 effectively impeded the metastatic potential of MDA-MB-231-DTR cells in vitro, and the combination of MC0704 and docetaxel exhibited potent antitumor activities in a xenograft mouse model. These findings suggested that MC0704 can be a lead candidate as a target therapeutic agent for TNBC patients with docetaxel resistance. [1]

C29H21BRN4O2

537.41

536.0847

C, 64.81; H, 3.94; Br, 14.87; N, 10.43; O, 5.95

3034176-57-0

Typically exists as solid at room temperature

6.4

3

3

6

36

802

0

C1=CC=C2C(=C1)C3=CC(=NC(=C3N2)C(=O)C4=CC=C(C=C4)Br)C(=O)NCCC5=CNC6=CC=CC=C65

DLOFNWDJTXTZPF-UHFFFAOYSA-N

InChI=1S/C29H21BrN4O2/c30-19-11-9-17(10-12-19)28(35)27-26-22(21-6-2-4-8-24(21)33-26)15-25(34-27)29(36)31-14-13-18-16-32-23-7-3-1-5-20(18)23/h1-12,15-16,32-33H,13-14H2,(H,31,36)

1-(4-bromobenzoyl)-N-[2-(1H-indol-3-yl)ethyl]-9H-pyrido[3,4-b]indole-3-carboxamide

MC0704; MC 7404; 74V87G4R62; MC-0704; 1-(4-bromobenzoyl)-N-[2-(1H-indol-3-yl)ethyl]-9H-pyrido[3,4-b]indole-3-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)

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.)