RIP140 inhibits glycolysis-dependent proliferation of breast cancer cells by regulating GLUT3 expression through transcriptional crosstalk between hypoxia induced factor and p53.

Glycolysis is essential to support cancer cell proliferation, even in the presence of oxygen. The transcriptional co-regulator RIP140 represses the activity of transcription factors that drive cell proliferation and metabolism and plays a role in mammary tumorigenesis. Here we use cell proliferation and metabolic assays to demonstrate that RIP140-deficiency causes a glycolysis-dependent increase in breast tumor growth. We further demonstrate that RIP140 reduces the transcription of the glucose transporter GLUT3 gene, by inhibiting the transcriptional activity of hypoxia inducible factor HIF-2α in cooperation with p53. Interestingly, RIP140 expression was significantly associated with good prognosis only for breast cancer patients with tumors expressing low GLUT3, low HIF-2α and high p53, thus confirming the mechanism of RIP140 anti-tumor activity provided by our experimental data. Overall, our work establishes RIP140 as a critical modulator of the p53/HIF cross-talk to inhibit breast cancer cell glycolysis and proliferation.

Expression of Progesterone Receptor A as an Independent Negative Prognosticator for Cervical Cancer.

The role of progesterone receptor A (PRA) for the survival outcome of cervical cancer patients is ambiguous. In mouse models, it has been shown that PRA plays a rather protective role in cancer development. The aim of this study was to assess its expression by immunohistochemistry in 250 cervical cancer tissue samples and to correlate the results with clinicopathological parameters including patient survival. PRA expression was positively correlated with the International Federation of Gynecology and Obstetrics (FIGO) classification scores. PRA was significantly overexpressed in adenocarcinomas compared to squamous epithelial carcinoma subtypes. Correlation analyses revealed a trend association with the HPV virus protein E6, a negative correlation with p16 and a positive correlation with EP3. PRA expression was also associated with the expression of RIP140, a transcriptional coregulator that we previously identified as a negative prognostic factor for survival in cervical cancer patients. Univariate survival analyses revealed PRA as a negative prognosticator for survival in patients with cervical adenocarcinoma. Multivariate analyses showed that simultaneous expression of RIP140 and PRA was associated with the worst survival, whereas with negative RIP140, PRA expression alone was associated with the best survival. We can therefore assume that the effect of nuclear PRA on overall survival is dependent upon nuclear RIP140 expression.

The Transcription Coregulator RIP140 Inhibits Cancer Cell Proliferation by Targeting the Pentose Phosphate Pathway.

Cancer cells switch their metabolism toward glucose metabolism to sustain their uncontrolled proliferation. Consequently, glycolytic intermediates are diverted into the pentose phosphate pathway (PPP) to produce macromolecules necessary for cell growth. The transcription regulator RIP140 controls glucose metabolism in tumor cells, but its role in cancer-associated reprogramming of cell metabolism remains poorly understood. Here, we show that, in human breast cancer cells and mouse embryonic fibroblasts, RIP140 inhibits the expression of the gene-encoding G6PD, the first enzyme of the PPP. RIP140 deficiency increases G6PD activity as well as the level of NADPH, a reducing cofactor essential for macromolecule synthesis. Moreover, G6PD knock-down inhibits the gain of proliferation observed when RIP140 expression is reduced. Importantly, RIP140-deficient cells are more sensitive to G6PD inhibition in cell proliferation assays and tumor growth experiments. Altogether, this study describes a novel role for RIP140 in regulating G6PD levels, which links its effect on breast cancer cell proliferation to metabolic rewiring.

Cytoplasmic PPARγ is a marker of poor prognosis in patients with Cox-1 negative primary breast cancers.

BACKGROUND: The aim of this study was to investigate the expression of the nuclear receptor PPARγ, together with that of the cyclooxygenases Cox-1 and Cox-2, in breast cancer (BC) tissues and to correlate the data with several clinicobiological parameters including patient survival. METHODS: In a well characterized cohort of 308 primary BC, PPARγ, Cox-1 and Cox-2 cytoplasmic and nuclear expression were evaluated by immunohistochemistry. Correlations with clinicopathological and aggressiveness features were analyzed, as well as survival using Kaplan-Meier analysis. RESULTS: PPARγ was expressed in almost 58% of the samples with a predominant cytoplasmic location. Cox-1 and Cox-2 were exclusively cytoplasmic. Cytoplasmic PPARγ was inversely correlated with nuclear PPARγ and ER expression, but positively with Cox-1, Cox-2, and other high-risk markers of BC, e.g. HER2, CD133, and N-cadherin. Overall survival analysis demonstrated that cytoplasmic PPARγ had a strong correlation with poor survival in the whole cohort, and even stronger in the subgroup of patients with no Cox-1 expression where cytoplasmic PPARγ expression appeared as an independent marker of poor prognosis. In support of this cross-talk between PPARγ and Cox-1, we found that Cox-1 became a marker of good prognosis only when cytoplasmic PPARγ was expressed at high levels. CONCLUSION: Altogether, these data suggest that the relative expression of cytoplasmic PPARγ and Cox-1 may play an important role in oncogenesis and could be defined as a potential prognosis marker to identify specific high risk BC subgroups.

Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor.

Breast cancer (BC) remains the primary cause of death from cancer among women worldwide. Cholesterol-5,6-epoxide (5,6-EC) metabolism is deregulated in BC but the molecular origin of this is unknown. Here, we have identified an oncometabolism downstream of 5,6-EC that promotes BC progression independently of estrogen receptor α expression. We show that cholesterol epoxide hydrolase (ChEH) metabolizes 5,6-EC into cholestane-3ß,5α,6ß-triol, which is transformed into the oncometabolite 6-oxo-cholestan-3ß,5α-diol (OCDO) by 11ß-hydroxysteroid-dehydrogenase-type-2 (11ßHSD2). 11ßHSD2 is known to regulate glucocorticoid metabolism by converting active cortisol into inactive cortisone. ChEH inhibition and 11ßHSD2 silencing inhibited OCDO production and tumor growth. Patient BC samples showed significant increased OCDO levels and greater ChEH and 11ßHSD2 protein expression compared with normal tissues. The analysis of several human BC mRNA databases indicated that 11ßHSD2 and ChEH overexpression correlated with a higher risk of patient death, highlighting that the biosynthetic pathway producing OCDO is of major importance to BC pathology. OCDO stimulates BC cell growth by binding to the glucocorticoid receptor (GR), the nuclear receptor of endogenous cortisol. Interestingly, high GR expression or activation correlates with poor therapeutic response or prognosis in many solid tumors, including BC. Targeting the enzymes involved in cholesterol epoxide and glucocorticoid metabolism or GR may be novel strategies to prevent and treat BC.

Cytoplasmic and Nuclear Forms of Thyroid Hormone Receptor ß1 Are Inversely Associated with Survival in Primary Breast Cancer.

The aim of this study was to investigate the expression of thyroid hormone receptor ß1 (THRß1) by immunohistochemistry in breast cancer (BC) tissues and to correlate the results with clinico-biological parameters. In a well-characterized cohort of 274 primary BC patients, THRß1 was widely expressed with a predominant nuclear location, although cytoplasmic staining was also frequently observed. Both nuclear and cytoplasmic THRß1 were correlated with high-risk BC markers such as human epidermal growth factor receptor 2 (HER2), Ki67 (also known as MKI67), prominin-1 (CD133), and N-cadherin. Overall survival analysis demonstrated that cytoplasmic THRß1 was correlated with favourable survival (p = 0.015), whereas nuclear THRß1 had a statistically significant correlation with poor outcome (p = 0.038). Interestingly, in our cohort, nuclear and cytoplasmic THRß1 appeared to be independent markers either for poor (p = 0.0004) or for good (p = 0.048) prognosis, respectively. Altogether, these data indicate that the subcellular expression of THRß1 may play an important role in oncogenesis. Moreover, the expression of nuclear THRß1 is a negative outcome marker, which may help to identify high-risk BC subgroups.

The Prognostic Impact of the Aryl Hydrocarbon Receptor (AhR) in Primary Breast Cancer Depends on the Lymph Node Status.

Increasing evidence implicates the aryl hydrocarbon receptor (AhR) as a possible regulator of mammary carcinogenesis. This study aims to clarify its prognostic impact in breast cancer (BC). Meta-analyses performed at the mRNA level demonstrated that the predictive value of AhR expression in BC depends on the lymph node (LN) status. AhR expression and sub-cellular location were then analyzed by immunohistochemistry in 302 primary BC samples. AhR was expressed in almost 90% of cases with a predominant nuclear location. Nuclear and cytoplasmic AhR levels were significantly correlated and associated with the expression of RIP140 (receptor-interacting protein of 140 kDa), an AhR transcriptional coregulator and target gene. Interestingly, total and nuclear AhR levels were only significantly correlated with short overall survival in node-negative patients. In this sub-group, total and nuclear AhR expression had an even stronger prognostic impact in patients with low RIP140-expressing tumors. Very interestingly, the total AhR prognostic value was also significant in luminal-like BCs and was an independent prognostic marker for LN-negative patients. Altogether, this study suggests that AhR is a marker of poor prognosis for patients with LN-negative luminal-like BCs, which warrants further evaluation.

Prognostic relevance of RIP140 and ERß expression in unifocal versus multifocal breast cancers: a preliminary report.

The aim of this study was to investigate the expression of two nuclear receptor transcriptional coregulators, namely RIP140 (receptor-interacting protein of 140 kDa) and LCoR (ligand-dependent corepressor) in unifocal versus multifocal breast cancers. The expression of these two proteins was analyzed by immunohistochemistry in a matched-pair cohort of 21 unifocal and 21 multifocal breast tumors. The expression of the two estrogen receptors (ERα and ERß) was studied in parallel. RIP140 and LCoR levels appeared lower in unifocal tumors compared to multifocal samples (decreased of immune-reactive scores and reduced number of high expressing cells). In both tumor types, RIP140 and LCoR expression was correlated with each other and with expression of ERß. Very interestingly, the expression of RIP140, LCoR, and ERß was inversely correlated with overall survival only for the unifocal group. The negative correlation with overall and recurrence free survival was more pronounced in patients whose unifocal tumors expressed high levels of both RIP140 and ERß. Altogether, this preliminary report indicates that the ERß/RIP140 signaling is altered in unifocal breast cancers and correlated with patient outcome. Further investigation is needed to decipher the molecular mechanisms and the biological relevance of this deregulation.

A Dominant Mutation in Nuclear Receptor Interacting Protein 1 Causes Urinary Tract Malformations via Dysregulation of Retinoic Acid Signaling.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of CKD in the first three decades of life. However, for most patients with CAKUT, the causative mutation remains unknown. We identified a kindred with an autosomal dominant form of CAKUT. By whole-exome sequencing, we identified a heterozygous truncating mutation (c.279delG, p.Trp93fs*) of the nuclear receptor interacting protein 1 gene (NRIP1) in all seven affected members. NRIP1 encodes a nuclear receptor transcriptional cofactor that directly interacts with the retinoic acid receptors (RARs) to modulate retinoic acid transcriptional activity. Unlike wild-type NRIP1, the altered NRIP1 protein did not translocate to the nucleus, did not interact with RARα, and failed to inhibit retinoic acid-dependent transcriptional activity upon expression in HEK293 cells. Notably, we also showed that treatment with retinoic acid enhanced NRIP1 binding to RARα RNA in situ hybridization confirmed Nrip1 expression in the developing urogenital system of the mouse. In explant cultures of embryonic kidney rudiments, retinoic acid stimulated Nrip1 expression, whereas a pan-RAR antagonist strongly reduced it. Furthermore, mice heterozygous for a null allele of Nrip1 showed a CAKUT-spectrum phenotype. Finally, expression and knockdown experiments in Xenopus laevis confirmed an evolutionarily conserved role for NRIP1 in renal development. These data indicate that dominant NRIP1 mutations can cause CAKUT by interference with retinoic acid transcriptional signaling, shedding light on the well documented association between abnormal vitamin A levels and renal malformations in humans, and suggest a possible gene-environment pathomechanism in this disease.

The emerging role of the transcriptional coregulator RIP140 in solid tumors.

RIP140 is a transcriptional coregulator (also known as NRIP1) which plays very important physiological roles by finely tuning the activity of a large number of transcription factors. Noticeably, the RIP140 gene has been shown to be involved in the regulation of energy expenditure, in mammary gland development and intestinal homeostasis as well as in behavior and cognition. RIP140 is also involved in the regulation of various oncogenic signaling pathways and participates in the development and progression of solid tumors. This short review aims to summarize the role of this transcription factor on nuclear estrogen receptors, E2F and Wnt signaling pathways based on recent observations focusing on breast, ovary, liver and colon tumors.

Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes.

Bisphenol A (BPA) is an industrial compound and a well known endocrine-disrupting chemical with estrogenic activity. The widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development, and metabolism. Here we report that the mechanisms by which BPA and two congeners, bisphenol AF and bisphenol C (BPC), bind to and activate estrogen receptors (ER) α and ß differ from that used by 17ß-estradiol. We show that bisphenols act as partial agonists of ERs by activating the N-terminal activation function 1 regardless of their effect on the C-terminal activation function 2, which ranges from weak agonism (with BPA) to antagonism (with BPC). Crystallographic analysis of the interaction between bisphenols and ERs reveals two discrete binding modes, reflecting the different activities of compounds on ERs. BPA and 17ß-estradiol bind to ERs in a similar fashion, whereas, with a phenol ring pointing toward the activation helix H12, the orientation of BPC accounts for the marked antagonist character of this compound. Based on structural data, we developed a protocol for in silico evaluation of the interaction between bisphenols and ERs or other members of the nuclear hormone receptor family, such as estrogen-related receptor γ and androgen receptor, which are two known main targets of bisphenols. Overall, this study provides a wealth of tools and information that could be used for the development of BPA substitutes devoid of nuclear hormone receptor-mediated activity and more generally for environmental risk assessment.

Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors.

Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERß), whereas the zebrafish genome encodes three ERs, zfERα and two zfERßs (zfERß1 and zfERß2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28°C as compared to 37°C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERß selective agonists displayed greater potency for zfERα as compared to zfERßs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology.

Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 µM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 µM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis.

RIP140 regulates transcription factor HES1 oscillatory expression and mitogenic activity in colon cancer cells.

The transcription factor receptor-interacting protein 140 (RIP140) regulates intestinal homeostasis and tumorigenesis through Wnt signaling. In this study, we investigated its effect on the Notch/HES1 signaling pathway. In colorectal cancer (CRC) cell lines, RIP140 positively regulated HES1 gene expression at the transcriptional level via a recombining binding protein suppressor of hairless (RBPJ)/neurogenic locus notch homolog protein 1 (NICD)-mediated mechanism. In support of these in vitro data, RIP140 and HES1 expression significantly correlated in mouse intestine and in a cohort of CRC samples, thus supporting the positive regulation of HES1 gene expression by RIP140. Interestingly, when the Notch pathway is fully activated, RIP140 exerted a strong inhibition of HES1 gene transcription controlled by the level of HES1 itself. Moreover, RIP140 directly interacts with HES1 and reversed its mitogenic activity in human CRC cells. In line with this observation, HES1 levels were associated with a better patient survival only when tumors expressed high levels of RIP140. Our data identify RIP140 as a key regulator of the Notch/HES1 signaling pathway, with a dual effect on HES1 gene expression at the transcriptional level and a strong impact on colon cancer cell proliferation.

[The Warburg effect: from theory to therapeutic applications in cancer]. / L'effet Warburg - De la théorie du cancer aux applications thérapeutiques en cancérologie.

Cancer cell metabolism described by Otto Warburg in the thirties became a cancer specific hallmark, also called "Warburg effect". Cancer cells use essentially glucose as fuel, through glycolysis, in order to meet their energy and biomass needs to insure their cell proliferation. Recent advances describe Warburg effect regulation by oncogenes and tumor suppressor genes. Moreover, mutations in some glycolysis enzymes are found in various cancers, highlighting the role of cell metabolism in cancer. In this review, we describe the mechanisms responsible for the Warburg effect at the molecular and cellular level, the role of cell signalling along with the implication of different transcription factors. As a cause or a consequence of tumorigenesis, the Warburg effect is now considered as a promising therapeutic target in the fight against cancer.

Gelatin methacrylate hydrogel with drug-loaded polymer microspheres as a new bioink for 3D bioprinting.

3D bioprinted hydrogel constructs are advanced systems of a great drug delivery application potential. One of the bioinks that has recently gained a lot of attention is gelatin methacrylate (GelMA) hydrogel exhibiting specific properties, including UV cross-linking possibility. The present study aimed to develop a new bioink composed of GelMA and gelatin modified by addition of polymer (polycaprolactone or polyethersulfone) microspheres serving as bioactive substance carriers. The prepared microspheres suspension in GelMA/gelatin bioink was successfully bioprinted and subjected to various tests, which showed that the addition of microspheres and their type affects the physicochemical properties of the printouts. The hydrogel stability and structure was examined using scanning electron and optical microscopy, its thermal properties with differential scanning calorimetry and thermogravimetric analysis and its biocompatibility on HaCaT cells using viability assay and electron microscopy. Analyses also included tests of hydrogel equilibrium swelling ratio and release of marker substance. Subsequently, the matrices were loaded with ampicillin and the antibiotic release was validated by monitoring the antibacterial activity on Staphylococcus aureus and Escherichia coli. It was concluded that GelMA/gelatin bioink is a good and satisfying material for potential medical use. Depending on the polymer used, the addition of microspheres improves its structure, thermal and drug delivery properties.

Cytoplasmic Localization of Thyroid Hormone Receptor (TR) Alpha and Nuclear Expression of Its Isoform TRα2 Determine Survival in Breast Cancer in Opposite Ways.

The aim of this retrospective study was to assess the respective prognostic values of cytoplasmic and nuclear TRα, TRα1, and TRα2 expression in breast cancer (BC) tissue samples and correlate the results with clinico-pathological parameters. In 249 BC patients, the expression patterns of general TRα and the α1 and α2 isoforms were evaluated via immuno-histochemistry. Prognosis-determining aspects were calculated via univariate, as well as multivariate, analysis. Univariate Cox-regression analysis revealed no association between nuclear TRα expression and overall survival (OS) (p = 0.126), whereas cytoplasmic TRα expression was significantly correlated with a poor outcome for both OS (p = 0.034) and ten-year survival (p = 0.009). Strengthening these results, cytoplasmic TRα was found to be an independent marker of OS (p = 0.010) when adjusted to fit clinico-pathological parameters. Analyses of the TRα-subgroups revealed that TRα1 had no prognostic relevance, whereas nuclear TRα2 expression was positively associated with OS (p = 0.014), ten-year survival (p = 0.029), and DFS (p = 0.043). Additionally, nuclear TRα2 expression was found to be an independent positive prognosticator (p = 0.030) when adjusted to fit clinico-pathological parameters. Overall, our results support the hypothesis that subcellular localization of TRα and its isoforms plays an important role in the carcinogenesis and prognosis of breast cancer. Cytoplasmic TRα expression correlates with more aggressive disease progression, whereas nuclear TRα2 expression appears to be a protective factor. These data may help us to prioritize high-risk BC subgroups for possible targeted tumor therapy.

Prognostic Relevance of Tumor-Infiltrating Immune Cells in Cervix Squamous Cell Carcinoma.

There exists a variety of studies about tumor-infiltrating immune cells (TIICs) in cervical cancer, but their prognostic value in correlation with the histopathological subtype has never been investigated. Therefore, the aim of this study was to quantify TIICs in a panel of 238 sporadic cervical cancers and investigate the correlation with cervical cancer subtype and patient survival. TIICs levels were significantly increased in the subgroup of CSCC (191 samples) in comparison to CAC (47 samples). In CSCC, TIICs' infiltration showed a negative correlation with age, FIGO stage and with the histone protein modification H3K4me3. Moreover, in CAC, it was positively correlated with p16 and with the glucocorticoid receptor and inversely correlated with the MDM2 protein and with H3K4me3. Interestingly, immune infiltration was an independent positive prognosticator for disease-free survival (DFS) in patients with CSCC, those bearing tumors with the strongest TIICs infiltration showing the better DFS. Altogether, the present study provides a differentiated overview of the relations between TIIC levels and prognosis in patients with CSCC vs. patients with CAC.

Prognostic significance of TRIM24/TIF-1α gene expression in breast cancer.

In this study, we have analyzed the expression of TRIM24/TIF-1α, a negative regulator of various transcription factors (including nuclear receptors and p53) at the genomic, mRNA, and protein levels in human breast tumors. In breast cancer biopsy specimens, TRIM24/TIF-1α mRNA levels (assessed by Real-Time Quantitative PCR or microarray expression profiling) were increased as compared to normal breast tissues. At the genomic level, array comparative genomic hybridization analysis showed that the TRIM24/TIF-1α locus (7q34) exhibited both gains and losses that correlated with mRNA levels. By re-analyzing a series of 238 tumors, high levels of TRIM24/TIF-1α mRNA significantly correlated with various markers of poor prognosis (such as the molecular subtype) and were associated with worse overall survival. By using a rabbit polyclonal antibody for immunochemistry, the TRIM24/TIF-1α protein was detected in nuclei of normal luminal epithelial breast cells, but not in myoepithelial cells. Tissue microarray analysis confirmed that its expression was increased in epithelial cells from normal to breast infiltrating duct carcinoma and correlated with worse overall survival. Altogether, this work is the first study that shows that overexpression of the TRIM24/TIF-1α gene in breast cancer is associated with poor prognosis and worse survival, and it suggests that this transcription coregulator may play a role in mammary carcinogenesis and represent a novel prognostic marker.

Development of a new 3D bioprinted antibiotic delivery system based on a cross-linked gelatin-alginate hydrogel.

3D bioprinting uses bioink deposited directly on a collector to create any previously designed 3D model. One of the most common and the easiest to operate bioinks is gelatin-alginate hydrogel. The present study aimed to combine 3D bioprinting with different cross-linking techniques to develop a new stable and biodegradable gelatin-alginate hydrogel matrix for drug delivery applications. The matrix-building biopolymers were crosslinked by ionotropic gelation with Ca2+ ions, chemical crosslinking with GTA or a combination of the two crosslinkers at various concentrations. The influence of the crosslinking method on the hydrogel properties, stability and structure was examined using scanning electron and optical microscopy, differential scanning calorimetry and thermogravimetric analysis. Analyses included tests of hydrogel equilibrium swelling ratio and release of marker substance. Subsequently, biological properties of the matrices loaded with the antibiotic chlorhexidine were studied, including cytotoxicity on HaCAT cells and antibacterial activity on Staphylococcus aureus and Escherichia coli bacteria. The conducted study confirmed that the 3D bioprinted cross-linked drug-loaded alginate-gelatin hydrogel is a good and satisfying material for potential use as a drug delivery system.