Doxycycline (hydrochloride)

Cat. No.: HY-N0565AG: Data Sheet Handling Instructions
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Doxycycline hydrochloride GMP is Doxycycline (hydrochloride) (HY-N0565A) produced by using GMP guidelines. GMP small molecules works appropriately as an auxiliary reagent for cell therapy manufacture. Doxycycline hydrochloride is an orally active highly lipophilic, tissue-permeable MMP inhibitor with broad-spectrum antibacterial activity. Doxycycline hydrochloride is also a semi-synthetic antibiotic with chelating properties, which blocks bacterial protein synthesis and inhibits extracellular matrix degradation through interactions with zinc and calcium atoms. Doxycycline hydrochloride also inhibits mitochondrial biogenesis, translation, and the expression of respiratory chain proteins. Doxycycline hydrochloride induces apoptosis, inhibits autophagy and EMT, downregulates stem cell markers, and activates the PI3K-AKT pathway, thereby effectively inhibiting the viability and proliferation of cancer cells such as breast cancer cells. Doxycycline hydrochloride also promotes the survival and self-renewal of embryonic stem cells and neural stem cells, and reduces the frequency of medium changes in culture. Doxycycline hydrochloride has been applied in studies related to breast cancer, prostate cancer, bladder cancer, and other cancers.

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Doxycycline (hydrochloride) Chemical Structure

CAS No. : 10592-13-9

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Biological Activity
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Description

In Vitro

Doxycycline (11.39 μM + MCF-7; 7.13 μM + MDA-MB-468; 72 h) hydrochloride GMP significantly reduces the population of CD44⁺/CD24^-/low breast cancer stem cells in MCF-7 and MDA-MB-468 cells^[2].
Doxycycline (11.39 μM+MCF-7; 7.13 μM+MDA-MB-468; 72 h) hydrochloride GMP significantly downregulates the mRNA and protein expression of stem cell markers (Nanog, Oct4, Sox2, c-Myc, CD44) in MCF-7 and MDA-MB-468 breast cancer cells^[2].
Doxycycline (1 μg/mL; 5 d) hydrochloride GMP increases the colony formation efficiency of undifferentiated human embryonic stem cells (H9, HSF6, H1, H7, HUES6) and human induced pluripotent stem cells (retrovirus-derived, lentivirus-derived, protein-induced lines), and elevates the number of colonies expressing undifferentiated markers under various culture conditions^[3].
Doxycycline (1 μg/mL; 5 d) hydrochloride GMP promotes the survival and proliferation of H9 human embryonic stem cells (hESC) more effectively than Y-27632 (HY-10071), resulting in larger cell colonies, a higher proportion of undifferentiated AP⁺ cells, as well as downregulated expression of pro-apoptotic genes and upregulated expression of pluripotency-related genes^[3].
Doxycycline (1 μg/mL; 6 d; passage 12) hydrochloride GMP reduces cell apoptosis, increases S-phase cell accumulation, supports the long-term expansion of H9 human embryonic stem cells (H9 hESCs) and Lenti-1 induced pluripotent stem cells (Lenti-1 hiPSCs) cultured in clusters on MEF feeder layers, and maintains the pluripotency and normal karyotype of the cells during repeated passaging^[3].
Doxycycline (1 μg/mL; 7 d) hydrochloride GMP maintains the viability, proliferation and expression of undifferentiated markers of H9 human embryonic stem cells for up to 7 days without medium change, and reduces apoptotic cell death of newly seeded dissociated cells and preformed cell clusters within 3 days without medium change^[5].
Doxycycline (1 μg/mL; 30-72 d) hydrochloride GMP supports long-term subculture of H9, HSF6 human embryonic stem cells as well as Retro-1, Lenti-1, Pro-1 human induced pluripotent stem cells. The culture medium is replaced every 3 days, and the growth rate remains consistent with that under the standard daily medium change condition during passages 5 to 12^[5].
Doxycycline (1 μg/mL; 72 d) hydrochloride GMP maintains normal karyotype, expression of undifferentiated markers and pluripotency (the ability to differentiate into all cells of the three germ layers in vitro) of H9 human embryonic stem cells that are subcultured 12 times with medium replacement every 3 days^[5].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Cell Viability Assay^[2]

Cell Line:	MCF-7 human breast cancer monolayer cells, MDA-MB-468 human breast cancer monolayer cells, BCSC-enriched mammosphere cultures derived from MCF-7 and MDA-MB-468 cell lines
Concentration:	1-250 μM; 37.5 μM (MCF-7 mammospheres); 29.1 μM (MDA-MB-468 mammospheres)
Incubation Time:	72 h
Result:	Inhibited breast cancer cell viability in a dose-dependent manner, with IC₅₀ values of 11.39 μM for MCF-7 monolayer cells and 7.13 μM for MDA-MB-468 monolayer cells. Reduced viability of BCSC-enriched mammosphere cultures with IC₅₀ values 3-fold higher than their corresponding monolayer cultures (37.5 μM for MCF-7 mammospheres, 29.1 μM for MDA-MB-468 mammospheres).

Cell Proliferation Assay^[5]

Cell Line:	human embryonic stem cell lines H9, HSF6; human induced pluripotent stem cell lines Retro-1, Lenti-1, Pro-1
Concentration:	1 μg/mL
Incubation Time:	30 days (5 passages, media changes every 3 days); 72 days (12 passages, media changes every 3 days)
Result:	Supported H9 hESCs expansion 3684-fold over 5 passages, with an average population doubling level (PDL) of 3.18 per passage, comparable to cultures with daily media changes and doxycycline-free medium. Enabled HSF6, Retro-1, Lenti-1, and Pro-1 lines to show identical growth (PDL values) between doxycycline-supplemented cultures with 3-day media changes and doxycycline-free cultures with daily media changes. Maintained consistent PDL values (3.17 at passage 2 vs. 2.96 at passage 12) for H9 hESCs cultured for 12 passages in doxycycline-supplemented mTESR™-1 or PluriSTEM™ medium. Supported steady accumulation of cell numbers over passages for H9 hESCs cultured for 12 passages.

In Vivo

Doxycycline (25-50 mg/kg; p.o.; twice daily; 10 days) hydrochloride GMP exerts dose-dependent toxic effects on male Wistar rats: a dosage of 50 mg/kg twice daily for 10 consecutive days causes 30% mortality, cardiomyopathy, and significant elevation of biomarkers for muscle and cardiac injury; whereas a dosage of 25 mg/kg twice daily for 10 consecutive days only induces mild skeletal muscle injury without cardiac toxicity^[1].
Doxycycline (60 mg/kg; intraperitoneal injection; once daily for 15 consecutive days) hydrochloride GMP significantly inhibits the growth of CD44v9-expressing prostate cancer xenografts by reducing the proliferation of CD44v9-positive cells^[4].
Supplementary culture of H9 human embryonic stem cells (H9 hESCs) with Doxycycline (1 μg/mL; medium changed every 3 days; up to passage 12) hydrochloride GMP maintains their pluripotency and enables in vivo teratoma formation, which contains all three embryonic germ layers^[5].

Note:
Please do not refer to only one article to determine the experimental conditions. It is recommended to determine the optimal experimental conditions (animal strain, age, dosage, frequency and cycle, detection time and indicators, etc.) through preliminary experiments before the formal experiment.

Doxycycline (oral gavage; 200 or 800 mg/kg; once daily; 3 months) reduces MMP-9 activity in untreated HT mice in a dose-dependent manner^[3].
Doxycycline and Tetracycline (HY-A0107), act systemically after absorption from the upper gastrointestinal tract. The main advantage of Doxycycline over Tetracycline is its longer activity, and it can be taken twice or once a day. The peak concentration of both agents is similar, but in the case of Doxycycline the time to peak concentration is shorter, and half life is significantly longer^[6].

Doxycycline (Dox) is often used as an inducer in molecular biology studies to induce gene expression. In cells or model animals that have constructed tetracycline induced expression systems (Tet-On/Tet-Off systems), the expression of target genes can be precisely controlled by adding or removing Dox^[7]^[8].
Dose reference for Dox induction^[7]^[8]:
(1) Model animal: male Sprague-Dawley rats
Tet regulatory system: 20-3000 ppm of Dox is supplied in diet.
(2) Model animal: Cags mice
Tet regulatory system: 625 ppm of Dox is supplied in diet.

Induction of Modeling ON-OFF System (Gene expression regulation)^[6]^[7]^[8]

Background

Doxycycline is often used as an inducer in molecular biology research to induce gene expression. In cells or model animals that have constructed a Tetracycline (Tet; HY-A0107) inducible expression (Tet-ON/Tet-OFF) system, the expression of the target gene can be precisely controlled by adding or removing Doxycycline. Doxycycline can act as an inhibitor of transcriptional activation in the Tetracycline (Tc)-controlled transactivation (tTA) system, and as an inducer of transcriptional activation in the "reverse tTA' system. Doxycycline and Tetracycline both act systemically after being absorbed by the upper gastrointestinal tract. In comparison, the main advantage of Doxycycline is that it has a longer activity and can be taken twice or once a day. Although the peak concentrations of the two are similar, Doxycycline takes a shorter time to reach peak concentration and has a significantly longer half-life.

Specific Modeling Methods

Rat^[8]: Sprague-Dawley rats • male • adult middle-aged (12-month-old)
Administration: (for GDNF as targeted gene) 3g/kg (dietary with regular food) • po • once daily for 6 days

Note

(1) Recommend use the recombinant adeno-associated virus (rAAV)-based bicistronic tetracycline (tet)-OFF construct was used for dynamic control of GDNF (target gene) expression during long-term expression^[7].
(2) 3 g/kg dietary DOX produced DOX serum levels equivalent to 1mg/ml DOX in drinking water.

Modeling Indicators

Molecular changes: The expression level of the target gene decreases.

Phenotype changes: The positively correlated phenotype corresponding to the target gene is alleviated.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Model:	Wistar rats with Cardiomyopathy (8-week-old male, 220 g)^[1]
Dosage:	25 mg/kg; 50 mg/kg
Administration:	i.g.; twice daily; 10 consecutive days
Result:	Caused minimal skeletal muscle injury (individual myocyte necrosis with mononuclear cell infiltration), slight but significant increases in creatine kinase (CK) and aspartate aminotransferase (AST) compared to controls, and maintained histologically normal cardiac muscle with normal troponin I (cTnI) levels at 25 mg/kg. Resulted in 30% mortality, dullness, loss of appetite, dyspnoea, congested cava and pulmonary vein with scanty oedematous fluid in abdominal and pleural cavities, multifocal skeletal muscle degeneration/necrosis, mild to moderate epicardial lymphocytic infiltration with focal atrial myocarditis and myocardial necrosis, ventricular muscle degeneration/necrosis with myositis, pulmonary lesions including thickened interalveolar septa, alveolar histiocytosis, and haemosiderin-laden macrophages (heart failure cells), significant increases in alanine aminotransferase (ALT), AST, CK, and cTnI compared to control and 25 mg/kg groups, and a significant decrease in blood calcium levels at 50 mg/kg.

Molecular Weight

480.90

Formula

C₂₂H₂₅ClN₂O₈

CAS No.

10592-13-9

SMILES

O=C(C(C1=O)=C(O)[C@@H](N(C)C)[C@]2([H])[C@@H](O)[C@]3([H])[C@@H](C)C4=C(C(C3=C(O)[C@@]21O)=O)C(O)=CC=C4)N.Cl

Shipping

Room temperature in continental US; may vary elsewhere.

Storage

Please store the product under the recommended conditions in the Certificate of Analysis.

Purity & Documentation

Handling Instructions (2659 KB)

References

[1]. El-Neweshy MS, et al. Experimental doxycycline overdose in rats causes cardiomyopathy. Int J Exp Pathol. 2013;94(2):109-114. [Content Brief]

[2]. Zhang L, et al. Doxycycline inhibits the cancer stem cell phenotype and epithelial-to-mesenchymal transition in breast cancer. Cell Cycle. 2017;16(8):737-745. [Content Brief]

[3]. Chang M Y, Rhee Y H, Yi S H, et al. Doxycycline enhances survival and self-renewal of human pluripotent stem cells[J]. Stem cell reports, 2014, 3(2): 353-364.

[4]. Matsumoto T, Uchiumi T, Monji K, et al. Doxycycline induces apoptosis via ER stress selectively to cells with a cancer stem cell-like properties: importance of stem cell plasticity[J]. Oncogenesis, 2017, 6(11): 397.

[5]. Chang MY, et al. Doxycycline supplementation allows for the culture of human ESCs/iPSCs with media changes at 3-day intervals. Stem Cell Res. 2015;15(3):608-613. [Content Brief]

[6]. Manfredsson FP, et al. Tight Long-term dynamic doxycycline responsive nigrostriatal GDNF using a single rAAV vector. Mol Ther. 2009 Nov;17(11):1857-67. [Content Brief] [Content Brief]

[7]. Kistner A, et al. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10933-8. [Content Brief]

[8]. Niv Y. Doxycycline in Eradication Therapy of Helicobacter pylori--a Systematic Review and Meta-Analysis. Digestion. 2016;93(2):167-73. [Content Brief]