Publications by authors named "Jolanta Saczko"

101 Publications

Testing Lab-on-a-Chip Technology for Culturing Human Melanoma Cells under Simulated Microgravity.

Cancers (Basel) 2021 Jan 22;13(3). Epub 2021 Jan 22.

Department of Molecular and Cellular Biology, Wroclaw Medical University, Borowska 211A, 50-556 Wroclaw, Poland.

The dynamic development of the space industry makes space flights more accessible and opens up new opportunities for biological research to better understand cell physiology under real microgravity. Whereas specialized studies in space remain out of our reach, preliminary experiments can be performed on Earth under simulated microgravity (). Based on this concept, we used a 3D-clinostat (3D-C) to analyze the effect of short exposure to on human keratinocytes HaCaT and melanoma cells A375 cultured on all-glass Lab-on-a-Chip (LOC). Our preliminary studies included viability evaluation, mitochondrial and caspase activity, and proliferation assay, enabling us to determine the effect of on human cells. By comparing the results concerning cells cultured on LOCs and standard culture dishes, we were able to confirm the biocompatibility of all-glass LOCs and their potential application in microgravity research on selected human cell lines. Our studies revealed that HaCaT and A375 cells are susceptible to simulated microgravity; however, we observed an increased caspase activity and a decrease of proliferation in cancer cells cultured on LOCs in comparison to standard cell cultures. These results are an excellent basis to conduct further research on the possible application of LOCs systems in cancer research in space.
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http://dx.doi.org/10.3390/cancers13030402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866167PMC
January 2021

Investigating the photodynamic efficacy of chlorin e6 by millisecond pulses in metastatic melanoma cells.

Bioelectrochemistry 2021 Apr 29;138:107728. Epub 2020 Dec 29.

Department of Physical and Quantum Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology, Poland.

Melanoma is considered the most aggressive type of skin cancer, still without effective treatment. Thus, alternative therapeutic methods are still in demand, and electroporation-supported photodynamic therapy (EP-PDT) of cancer cells seems a promising approach. New developments in EP-PDT aim at enhanced tumor selectivity and biocompatibility by applying a second-generation photosensitizer, i.e., Chlorin e6 (Ce6). We have verified the improved photodynamic effect of Ce6 on skin cancer melanoma (Me45) cells and control (CHO-K1) cells. In this study, we applied 1 or 5 pulses of 10 ms duration and assessed the EP-PDT effect with a variety of tests, such as singlet oxygen scavenger (ABMDMA) photooxidation, oxidoreductive potential measurements, kinetic measurements with fluorescent microscopy, photosensitizer uptake studies, lipid peroxidation level, and actin fibers organization. The optimization of photosensitizer uptake as a function of temperature was also performed. Our results indicated efficient Ce6 delivery into Me45 cells and good photodynamic efficiency enhanced by the electroporation of cancer cells.
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http://dx.doi.org/10.1016/j.bioelechem.2020.107728DOI Listing
April 2021

Oxidative Effects during Irreversible Electroporation of Melanoma Cells-In Vitro Study.

Molecules 2020 Dec 31;26(1). Epub 2020 Dec 31.

Department of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, 50-556 Wroclaw, Poland.

Irreversible electroporation (IRE) is today used as an alternative to surgery for the excision of cancer lesions. This study aimed to investigate the oxidative and cytotoxic effects the cells undergo during irreversible electroporation using IRE protocols. To do so, we used IRE-inducing pulsed electric fields (PEFs) (eight pulses of 0.1 ms duration and 2-4 kV/cm intensity) and compared their effects to those of PEFs of intensities below the electroporation threshold (eight pulses, 0.1 ms, 0.2-0.4 kV/cm) and the PEFs involving elongated pulses (eight pulses, 10 ms, 0.2-0.4 kV/cm). Next, to follow the morphology of the melanoma cell membranes after treatment with the PEFs, we analyzed the permeability and integrity of their membranes and analyzed the radical oxygen species (ROS) bursts and the membrane lipids' oxidation. Our data showed that IRE-induced high cytotoxic effect is associated both with irreversible cell membrane disruption and ROS-associated oxidation, which is occurrent also in the low electric field range. It was shown that the viability of melanoma cells characterized by similar ROS content and lipid membrane oxidation after PEF treatment depends on the integrity of the membrane system. Namely, when the effects of the PEF on the membrane are reversible, aside from the high level of ROS and membrane oxidation, the cell does not undergo cell death.
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http://dx.doi.org/10.3390/molecules26010154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796376PMC
December 2020

Electrochemotherapy with Calcium Chloride and 17β-Estradiol Modulated Viability and Apoptosis Pathway in Human Ovarian Cancer.

Pharmaceutics 2020 Dec 24;13(1). Epub 2020 Dec 24.

Department of Molecular and Cellular Biology, Wroclaw Medical University, 50-556 Wroclaw, Poland.

Estrogens (Es) play a significant role in the carcinogenesis and progression of ovarian malignancies. Depending on the concentration, Es may have a protective or toxic effect on cells. Moreover, they can directly or indirectly affect the activity of membrane ion channels. In the presented study, we investigated in vitro the effectiveness of the ovarian cancer cells (MDAH-2774) pre-incubation with 17β-estradiol (E; 10 µM) in the conventional chemotherapy (CT) and electrochemotherapy (ECT) with cisplatin or calcium chloride. We used three different protocols of electroporation including microseconds (µsEP) and nanoseconds (nsEP) range. The cytotoxic effect of the applied treatment was examined by the MTT assay. We used fluorescent staining and holotomographic imaging to observe morphological changes. The immunocytochemical staining evaluated the expression of the caspase-12. The electroporation process's effectiveness was analyzed by a flow cytometer using the Yo-Pro™-1 dye absorption assay. We found that pre-incubation of ovarian cancer cells with 17β-estradiol may effectively enhance the chemo- and electrochemotherapy with cisplatin and calcium chloride. At the same time, estradiol reduced the effectiveness of electroporation, which may indicate that the mechanism of increasing the effectiveness of ECT by E is not related to the change of cell membrane permeability.
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http://dx.doi.org/10.3390/pharmaceutics13010019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823502PMC
December 2020

Cepharanthine induces ROS stress in glioma and neuronal cells via modulation of VDAC permeability.

Saudi Pharm J 2020 Nov 4;28(11):1364-1373. Epub 2020 Sep 4.

Department of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, Wroclaw, Poland.

Cepharanthine (CEP) is a bisbenzylisoquinoline alkaloid. Molecular dynamics studies show that CEP interacts with Voltage-dependent anion channel (VDAC), inducing the voltage-independent channel narrowing. In the new conformation, transport between mitochondria and cytoplasm is altered, which leads to the dose-dependent cytotoxicity. The biological effects of the interaction were investigated on glioblastoma multiforme (SNB-19) and neuronal (PC-12 + NGF) cell lines. The cytotoxic potential of cepharanthine was determined by MTT assay and flow cytometry apoptosis/necrosis studies. T-type calcium channel and VDAC were labelled by the immunocytochemical method. Additionally, fluorescent labelling of reactive oxygen species and mitochondria was performed. Changes in the pore size of VDAC were calculated as well. Molecular dynamics simulations were carried out to examine the interactions of cepharanthine with VDAC. The obtained results prove that cepharanthine enhances the apoptosis in glioma and neuronal cells by the release of reactive oxygen species. Cepharanthine alters the mitochondria-to-cytoplasm transport and thus induces the cytotoxicity with no selectivity.
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http://dx.doi.org/10.1016/j.jsps.2020.08.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679435PMC
November 2020

In Vitro Study of Calcium Microsecond Electroporation of Prostate Adenocarcinoma Cells.

Molecules 2020 Nov 19;25(22). Epub 2020 Nov 19.

Department of Molecular and Cellular Biology, Wroclaw Medical University, 50-556 Wroclaw, Poland.

Electroporation, applied as a non-thermal ablation method has proven to be effective for focal prostate treatment. In this study, we performed pre-clinical research, which aims at exploring the specific impact of this so-called calcium electroporation on prostate cancer. First, in an in-vitro study of DU 145 cell lines, microsecond electroporation (μsEP) parameters were optimized. We determined hence the voltage that provides both high permeability and viability of these prostate cancer cells. Subsequently, we compared the effect of μsEP on cells' viability with and without calcium administration. For high-voltage pulses, the cell death's mechanism was evaluated using flow-cytometry and confocal laser microscopy. For lower-voltage pulses, the influence of electroporation on prostate cancer cell mobility was studied using scratch assays. Additionally, we applied calcium-binding fluorescence dye (Fluo-8) to observe the calcium uptake dynamic with the fluorescence microscopy. Moreover, the molecular dynamics simulation visualized the process of calcium ions inflow during μsEP. According to our results calcium electroporation significantly decreases the cells viability by promoting apoptosis. Furthermore, our data shows that the application of pulsed electric fields disassembles the actin cytoskeleton and influences the prostate cancer cells' mobility.
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http://dx.doi.org/10.3390/molecules25225406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699241PMC
November 2020

Effect of Interaction between 17β-Estradiol, 2-Methoxyestradiol and 16α-Hydroxyestrone with Chromium (VI) on Ovary Cancer Line SKOV-3: Preliminary Study.

Molecules 2020 Nov 9;25(21). Epub 2020 Nov 9.

Department of Toxicology, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211, 50-556 Wroclaw, Poland.

Ovarian cancer is the leading cause of death from gynecologic malignancies. Some estrogens, as well as xenoestrogens, such as chromium (VI) (Cr(VI)), are indicated as important pathogenic agents. The objective of this study was to evaluate the role of estradiol and some its metabolites upon exposure to the metalloestrogen Cr(VI) in an in vitro model. The changes in cell viability of malignant ovarian cancer cells (SKOV-3 resistant to cisplatin) exposed to 17β-estradiol (E2) and its two metabolites, 2-methoxyestradiol (2-MeOE2) and 16α-hydroxyestrone (16α-OHE1), upon exposure to potassium chromate (VI) and its interactions were examined. The single and mixed models of action, during short and long times of incubation with estrogens, were applied. The different effects (synergism and antagonism) of estrogens on cell viability in the presence of Cr(VI) was observed. E2 and 16α-OHE1 caused a synergistic effect after exposure to Cr(VI). 2-MeOE2 showed an antagonistic effect on Cr(VI). The examined estrogens could be ranked according to the most protective effect or least toxicity in the order: 2-MeOE2 > E2 > 16α-OHE1. Early pre-incubation (24 h or 7 days) of cells with estrogens caused mostly an antagonistic effect-protective against the toxic action of Cr(VI). The beneficial action of estrogens on the toxic effect of Cr(VI), in the context of the risk of ovarian cancer, seems to be important and further studies are needed.
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http://dx.doi.org/10.3390/molecules25215214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665134PMC
November 2020

Effects of curcumin based PDT on the viability and the organization of actin in melanotic (A375) and amelanotic melanoma (C32) - in vitro studies.

Biomed Pharmacother 2020 Dec 28;132:110883. Epub 2020 Oct 28.

Department of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, Wroclaw, Poland. Electronic address:

Curcumin is a turmeric, antioxidative compound, well-known of its anti-cancer properties. Nowadays more and more effort is made in the field of enhancing the efficiency of the anticancer therapies. Combining the photoactive properties of curcumin with the superficial localization of melanoma and photodynamic therapy (PDT) seems to be a promising treatment method. The research focused on the evaluation of the curcumin effectiveness as an anticancer therapeutic agent in the in vitro treatment of melanotic (A375) and amelanotic (C32) melanoma cell lines. Keratinocytes (HaCat) and fibroblasts (HGF) were used to assess the impact of the therapy on the skin tissue. The aim of the study was to investigate the cell death after exposure to light irradiation after preincubation with curcumin. Additionaly the authors analized the interactions between curcumin and the actin cytoskeleton. The cytotoxic effect initiated by curcumin and increased by irradiation confirm the usefulness of the flavonoid in the PDT approach. Depending on curcumin concentration and incubation time, melanoma cells survival rate ranged from: 93.68 % (C32 cell line, 10 μM, 24 h) and 83.47 % (A375 cell line, 10 μM, 24 h) to 8.98 % (C32 cell line, 50 μM, 48 h) and 12.42 % (A375 cell line, 50 μM, 48 h). Moreover, photodynamic therapy with curcumin increased the number of apoptotic and necrotic cells in comparison to incubation with curcumin without irradiation. The study demonstrated that PDT induced caspase-3 overexpression and DNA cleavage in the studied cell lines. The cells revealed decreased proliferation after the therapy due to the actin cytoskeleton rearrangement. Although effective, the therapy remains not selective towards melanoma cells.
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http://dx.doi.org/10.1016/j.biopha.2020.110883DOI Listing
December 2020

Immunomodulatory activity of IR700-labelled affibody targeting HER2.

Cell Death Dis 2020 10 20;11(10):886. Epub 2020 Oct 20.

Division of Radiotherapy and Imaging, The Institute of Cancer Research, London, UK.

There is an urgent need to develop therapeutic approaches that can increase the response rate to immuno-oncology agents. Photoimmunotherapy has recently been shown to generate anti-tumour immunological responses by releasing tumour-associated antigens from ablated tumour cell residues, thereby enhancing antigenicity and adjuvanticity. Here, we investigate the feasibility of a novel HER2-targeted affibody-based conjugate (Z-IR700) selectively to induce cancer cell death in vitro and in vivo. The studies in vitro confirmed the specificity of Z-IR700 binding to HER2-positive cells and its ability to produce reactive oxygen species upon light irradiation. A conjugate concentration- and light irradiation-dependent decrease in cell viability was also demonstrated. Furthermore, light-activated Z-IR700 triggered all hallmarks of immunogenic cell death, as defined by the translocation of calreticulin to the cell surface, and the secretion of ATP, HSP70/90 and HMGB1 from dying cancer cells into the medium. Irradiating a co-culture of immature dendritic cells (DCs) and cancer cells exposed to light-activated Z-IR700 enhanced DC maturation, as indicated by augmented expression of CD86 and HLA-DR. In SKOV-3 xenografts, the Z-IR700-based phototherapy delayed tumour growth and increased median overall survival. Collectively, our results strongly suggest that Z-IR700 is a promising new therapeutic conjugate that has great potential to be applicable for photoimmunotherapy-based regimens.
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http://dx.doi.org/10.1038/s41419-020-03077-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576828PMC
October 2020

Effect of diallyl disulfide and garlic oil on different human astrocytoma cell lines.

Biomed Rep 2020 Oct 31;13(4):32. Epub 2020 Jul 31.

Department of Histology and Embryology, Faculty of Medicine, Wroclaw Medical University, 50-368 Wroclaw, Poland.

Gliomas are a group of malignant brain tumors. Despite significant efforts to optimize treatment options for patients with high-grade glioma, the prognosis of the overwhelming majority of patients remain poor. This bleak prognosis despite treatment of the glioma, is partly due to the tendency of therapeutics to diffusely penetrate into the neighboring brain tissues, but also due to the innate resistance of these tumors to chemotherapy and radiation. Garlic contains water-soluble and oil-soluble sulfur compounds. The oil-soluble compounds, including diallyl sulfide, diallyl disulfide (DADS), diallyl trisulfide and ajoene, are more effective potential anti-cancer treatments than the water-soluble compounds. There are several studies examining the effects of oil-soluble compounds on various types of cancer cells, although, to the best of our knowledge, there are no studies examining the effects of these compounds on glioma cells. The aim of the present study was to investigate the potential anti-glioma properties of DAD and garlic oil on proliferation and induction of apoptosis in four different types of glioma cell lines representative of different grades of the disease. The results showed that garlic oil exhibits favorable anti-cancer potential towards gliomas of various degrees of differentiation.
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http://dx.doi.org/10.3892/br.2020.1339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412714PMC
October 2020

Electroporation-Based Treatments in Urology.

Cancers (Basel) 2020 Aug 7;12(8). Epub 2020 Aug 7.

Department of Molecular and Cellular Biology, Wroclaw Medical University, 50-556 Wroclaw, Poland.

The observation that an application of a pulsed electric field (PEF) resulted in an increased permeability of the cell membrane has led to the discovery of the phenomenon called electroporation (EP). Depending on the parameters of the electric current and cell features, electroporation can be either reversible or irreversible. The irreversible electroporation (IRE) found its use in urology as a non-thermal ablative method of prostate and renal cancer. As its mechanism is based on the permeabilization of cell membrane phospholipids, IRE (as well as other treatments based on EP) provides selectivity sparing extracellular proteins and matrix. Reversible EP enables the transfer of genes, drugs, and small exogenous proteins. In clinical practice, reversible EP can locally increase the uptake of cytotoxic drugs such as cisplatin and bleomycin. This approach is known as electrochemotherapy (ECT). Few in vivo and in vitro trials of ECT have been performed on urological cancers. EP provides the possibility of transmission of genes across the cell membrane. As the protocols of gene electrotransfer (GET) over the last few years have improved, EP has become a well-known technique for non-viral cell transfection. GET involves DNA transfection directly to the cancer or the host skin and muscle tissue. Among urological cancers, the GET of several plasmids encoding prostate cancer antigens has been investigated in clinical trials. This review brings into discussion the underlying mechanism of EP and an overview of the latest progress and development perspectives of EP-based treatments in urology.
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http://dx.doi.org/10.3390/cancers12082208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465806PMC
August 2020

Apoptosis as the main type of cell death induced by calcium electroporation in rhabdomyosarcoma cells.

Bioelectrochemistry 2020 Dec 2;136:107592. Epub 2020 Jul 2.

Faculty of Pharmacy, Department of Molecular and Cellular Biology, Wroclaw Medical University, Poland. Electronic address:

Calcium electroporation (CaEP) has been previously reported as an effective method of rhabdomyosarcoma cells reduction. CaEP causes temporary cell membrane permeabilization with simultaneous calcium ions influx. A rapid influx of calcium ions leads to mitochondrial overload by Ca, loss of mitochondrial membrane potential causing cytochrome c release, caspase cascade activation and, as a consequence, cell death. This study was conducted on two cell lines: normal muscle cells (C2C12) and rhabdomyosarcoma cells (RD), which showed different cellular responses to CaEP. Our study defined apoptosis as the main cell death type occurring after CaEP in RD cells. Increased activity of caspase 3/7, Parp-1 and cleaved Parp-1 were proven in the case of RD cells. RD cells compartment rearrangement was observed in the time-lapse by holotomographic microscopy (HTM). C2C12 cells were less sensitive to electroporation and increased Ca concentration, and viability was maintained at the level of control cells, only slight changes in pro-apoptotic factors were observed. The results reveal CaEP as a promising therapeutic approach in cancers which develop from muscle tissue.
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http://dx.doi.org/10.1016/j.bioelechem.2020.107592DOI Listing
December 2020

Cytotoxic Effect of Vanicosides A and B from Reynoutria sachalinensis Against Melanotic and Amelanotic Melanoma Cell Lines and in silico Evaluation for Inhibition of BRAFV600E and MEK1.

Int J Mol Sci 2020 Jun 29;21(13). Epub 2020 Jun 29.

Department of Dental Surgery, Wroclaw Medical University, 50425 Wroclaw, Poland.

Vanicosides A and B are the esters of hydroxycinnamic acids with sucrose, occurring in a few plant species from the Polygonaceae family. So far, vanicosides A and B have not been evaluated for anticancer activity against human malignant melanoma. In this study, we tested these two natural products, isolated from Reynoutria sachalinensis rhizomes, against two human melanoma cell lines (amelanotic C32 cell line and melanotic A375 cell line, both bearing endogenous BRAFV600E mutation) and two normal human cell lines-keratinocytes (HaCaT) and the primary fibroblast line. Additionally, a molecular docking of vanicoside A and vanicoside B with selected targets involved in melanoma progression was performed. Cell viability was studied using an MTT assay. A RealTime-Glo™ Annexin V Apoptosis and Necrosis assay was used for monitoring programmed cell death (PCD). Vanicoside A demonstrated strong cytotoxicity against the amelanotic C32 cell line (viability of the C32 cell line was decreased to 55% after 72 h incubation with 5.0 µM of vanicoside A), significantly stronger than vanicoside B. This stronger cytotoxic activity can be attributed to an additional acetyl group in vanicoside A. No significant differences in the cytotoxicity of vanicosides were observed against the less sensitive A375 cell line. Moreover, vanicosides caused the death of melanoma cells at concentrations from 2.5 to 50 µM, without harming the primary fibroblast line. The keratinocyte cell line (HaCaT) was more sensitive to vanicosides than fibroblasts, showing a clear decrease in viability after incubation with 25 µM of vanicoside A as well as a significant phosphatidylserine (PS) exposure, but without a measurable cell death-associated fluorescence. Vanicosides induced an apoptotic death pathway in melanoma cell lines, but because of the initial loss of cell membrane integrity, an additional cell death mechanism might be involved like permeability transition pore (PTP)-mediated necrosis that needs to be explored in the future. Molecular docking indicated that both compounds bind to the active site of the BRAFV600E kinase and MEK-1 kinase; further experiments on their specific inhibitory activity of these targets should be considered.
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http://dx.doi.org/10.3390/ijms21134611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370030PMC
June 2020

The effect of dental gel formulation on human primary fibroblasts - an in vitro study.

Folia Histochem Cytobiol 2020 22;58(2):156-161. Epub 2020 Jun 22.

Department of Dental Surgery, Wroclaw Medical University, Wroclaw, Poland.

Introduction: In ordinary dental practice, the dentist often meets with patients suffering from ulcers, aphtha with edema, bleeding gums, bothersome burning, and dry mouth. These are prosthetic, orthodontic patients, after surgery, in various age ranges. Protefix® gel is a soothing and regenerating preparation aimed at patients with mucosal problems. The aim of our study was to evaluate the protective properties and the safety of Protefix® gel application after dental procedures in vitro.

Material And Methods: Human gingival fibroblasts (HGFs) were isolated from normal gingival tissues, cultured to full monolayer and exposed to Protefix® gel in the concentration from 1 to 100%. The viability of cells was examined by MTT assays. Cell migration as a response of treated cells was assessed. The expression of collagen III was estimated by immunocytochemistry after 20 minutes or 24 hours incubation with Protefix® gel.

Results: The obtained results indicated that the verified gel significantly stimulated fibroblasts' proliferation, and mitochondrial activity determined by MTT assay increased almost two-fold for lower gel concentrations. The immunohistochemical detection of collagen III revealed an increased expression after incubation with 5% gel.

Conclusions: The results proved that the gel is safe for cells derived from human gingiva and moreover has regenerative properties, which can be of great importance in the treatment of gingivitis after retraction and surgical procedures, or even ordinary daily injuries of oral cavity.
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http://dx.doi.org/10.5603/FHC.a2020.0013DOI Listing
June 2020

Caffeic Acid Phenethyl Ester Assisted by Reversible Electroporation-In Vitro Study on Human Melanoma Cells.

Pharmaceutics 2020 May 24;12(5). Epub 2020 May 24.

Department of Molecular and Cellular Biology, Wroclaw Medical University, 50-556 Wroclaw, Poland.

Melanoma is one of the most serious skin cancers. The incidence of this malignant skin lesion is continuing to increase worldwide. Melanoma is resistant to chemotherapeutic drugs and highly metastatic. Surgical resection can only be used to treat melanoma in the early stages, while chemotherapy is limited due to melanoma multi-drug resistance. The overexpression of glutathione -transferase (GST) may have a critical role in this resistance. Caffeic acid phenethyl ester (CAPE) is a natural phenolic compound, which occurs in many plants. Previous studies demonstrated that CAPE suppresses the growth of melanoma cells and induces reactive oxygen species generation. It is also known that bioactivation of CAPE to its corresponding quinone metabolite by tyrosinase would lead to GST inhibition and selective melanoma cell death. We investigated the biochemical toxicity of CAPE in combination with microsecond electropermeabilization in two human melanoma cell lines. Our results indicate that electroporation of melanoma cells in the presence of CAPE induced high oxidative stress, which correlates with high cytotoxicity. Moreover, it can disrupt the metabolism of cancer cells by inducing apoptotic cell death. Electroporation of melanoma cells may be an efficient CAPE delivery system, enabling the application of this compound, while reducing its dose and exposure time.
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http://dx.doi.org/10.3390/pharmaceutics12050478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284363PMC
May 2020

Polish Propolis-Chemical Composition and Biological Effects in Tongue Cancer Cells and Macrophages.

Molecules 2020 May 22;25(10). Epub 2020 May 22.

Department of Engineering and Technology of Chemical Processes, Wroclaw University of Science and Technology, 50-370 Wroclaw, Poland.

The purpose of this study was to compare the chemical composition and biological properties of Polish propolis. Ethanol, ethanol-hexane, hexane and hexane-ethanol extracts of propolis from three different regions of Poland were prepared. On the basis of the evaluation of their chemical composition as well as the extraction yield and free radical scavenging activity, the ethanol and hexane-ethanol extractions were proposed as the most effective methods. Subsequently, the biological properties of the extracts were evaluated to investigate the selectivity of an anticancer effect on tongue cancer cells in comparison to normal gingival fibroblasts. The obtained products demonstrated anticancer activity against tongue cancer cells. Additionally, when the lowest extract concentration (100 µg/mL) was applied, they were not cytotoxic to gingival fibroblasts. Finally, a possible anti-inflammatory potential of the prepared products was revealed, as reduced mitochondrial activity and proliferation of macrophages exposed to the extracts were observed. The results obtained indicate a potential of Polish propolis as a natural product with cancer-selective toxicity and anti-inflammatory effect. However, further studies are still needed to thoroughly explain the molecular mechanisms of its action and to obtain the promising health benefits of this versatile natural product.
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http://dx.doi.org/10.3390/molecules25102426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7287845PMC
May 2020

Ultrathin glass fiber microprobe for electroporation of arbitrary selected cell groups.

Bioelectrochemistry 2020 Oct 13;135:107545. Epub 2020 May 13.

Department of Glass, Institute of Electronic Materials Technology, Wolczyńska 133, 01-919, Warsaw, Poland; Faculty of Physics, Warsaw University, Pasteura 7, 02-093 Warsaw Poland. Electronic address:

A new type of ultrathin fiber microprobe for selective electroporation is reported. The microprobe is 10 cm long and has a diameter of 350 µm. This microprobe is a low cost tool, which allows electroporation of an arbitrary selected single cell or groups of cells among population with use of a standard microscope and cell culture plates. The microprobe in its basic form contains two metal microelectrodes made of a silver-copper alloy, running along the fiber, each with a diameter of 23 µm. The probe was tested in vitro on a population of normal and cancer cells. Successful targeted electroporation was observed by means of accumulation of trypan blue (TB) dye marker in the cell. The electroporation phenomenon was also verified with propidium iodide and AnnexinV in fluorescent microscopy.
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http://dx.doi.org/10.1016/j.bioelechem.2020.107545DOI Listing
October 2020

Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes.

J Biol Chem 2020 07 21;295(28):9567-9582. Epub 2020 May 21.

Wroclaw University of Science and Technology, Department of Chemical Biology and Bioimaging, Wroclaw, Poland

Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate.
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http://dx.doi.org/10.1074/jbc.RA120.013204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363135PMC
July 2020

3PO as a Selective Inhibitor of 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 in A375 Human Melanoma Cells.

Anticancer Res 2020 May;40(5):2613-2625

Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland

Background/aim: The occurrence of BRAF mutation causes an up-regulation of the B-raf kinase activity leading to the stabilization of hypoxia-inducible factor 1-alpha (HIF-1α) - the promoter of the 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) enzyme. The aim of the study was to examine the effect of the (2E)-3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), as an inhibitor of PFKFB3, on human melanoma cells (A375) with endogenous BRAF mutation.

Materials And Methods: A375 cells were exposed to different concentrations of 3PO and the following tests were performed: docking, cytotoxicity assay, immunocytochemistry staining glucose uptake, clonogenic assay, holotomography imaging, and flow cytometry.

Results: Our studies revealed that 3PO presents a dose-dependent and time-independent cytotoxic effect and promotes apoptosis of A375 cells. Furthermore, the obtained data indicate that 3PO induces cell cycle arrest in G1/0 and glucose uptake reduction.

Conclusion: Taking all together, our research demonstrated a here should be proapoptotic and antiproliferative effect of 3PO on A375 human melanoma cells.
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http://dx.doi.org/10.21873/anticanres.14232DOI Listing
May 2020

Proapoptotic activity induced by photodynamic reaction with novel cyanine dyes in caspase-3-deficient human breast adenocarcinoma cell lines (MCF/WT and MCF/DX).

Photodiagnosis Photodyn Ther 2020 Jun 21;30:101775. Epub 2020 Apr 21.

Wroclaw Medical University, Department of Molecular and Cellular Biology, Wroclaw, Poland.

Photodynamic therapy (PDT) is currently one of the cancer treatment options. PDT requires the application of a photosensitizer (such as: porphyrins, chlorines, and phthalocyanines) that selectively targets malignant cells. It is a dilemma to find a proper photosensitizer. In our study, we have tested a new in-vitro group of cyanine dyes. These dyes are widely applied in biotechnology as fluorescent markers. Two malignant adenocarcinoma cell lines (MCF-7/WT and MCF-7/DOX) were investigated using photodynamic reaction (PDR) with four cyanine dyes (KF-570, HM-118, FBF-749, and ER-139). KF-570 and HM-118 were irradiated with red light (630 nm), whereas FBF-749 and ER-139 with green light (435 nm). To evaluate PDR efficiency, a clonogenic test was conducted. Apoptosis was investigated by TUNEL and NCA (neutral comet) assays. Proteins selected as indicators of the apoptotic pathway (AIF, sPLA2, Smac/Diablo) and intracellular response markers (SOD-1 and GST-pi) were detected using western blot. The highest number of apoptotic cells (ca. 100%) was observed after PDR with HM-118 and KF-570 in both conducted tests, in both cell lines. The results showed that HM-118 and KF-570 cyanine dyes demonstrated a major phototoxic effect causing apoptosis in doxorubicin-resistant and sensitive cell lines.
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http://dx.doi.org/10.1016/j.pdpdt.2020.101775DOI Listing
June 2020

Extract Enriched in Mangiferin Protects PC12 Cells against a Neurotoxic Agent-3-Nitropropionic Acid.

Int J Mol Sci 2020 Apr 4;21(7). Epub 2020 Apr 4.

Department of Molecular and Cellular Biology, Wroclaw Medical University, 211A 50556 Borowska, Poland.

The rhizome of Bunge, used in Traditional Chinese Medicine as a brain function-improving herb, is a promising source of neuroprotective substances. The aim of this study was to evaluate the protective action of xanthones from rhizomes on the PC12 cell line exposed to the neurotoxic agent-3-nitropropionic acid (3-NP). The xanthone-enriched fraction of the ethanolic extract of (abbreviated from now on as XF, for the Xanthone Fraction), rich in polyphenolic xanthone glycosides, in concentrations from 5 to 100 μg/mL, and 3-NP in concentrations from 2.5 to 15 mM, were examined. After 8, 16, 24, 48, and 72 h of exposure of cells to various combinations of 3-NP and XF, the MTT viability assay was performed and morphological changes were estimated by confocal fluorescence microscopy. The obtained results showed a significant increase in the number of cells surviving after treatment with XF with exposure to neurotoxic 3-NP and decreased morphological changes in PC12 cells in a dose and time dependent manner. The most effective protective action was observed when PC12 cells were pre-incubated with the XF. This effect may contribute to the traditional indications of this herb for neurological and cognitive complaints. However, a significant cytotoxicity observed at higher XF concentrations (over 10 µg/mL) and longer incubation time (48 h) requires caution in future research and thorough investigation into potential adverse effects.
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http://dx.doi.org/10.3390/ijms21072510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177269PMC
April 2020

effects of vasoconstrictive retraction agents on primary human gingival fibroblasts.

Exp Ther Med 2020 Mar 20;19(3):2037-2044. Epub 2020 Jan 20.

Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw 50-556, Poland.

The biological activity of chemical retraction/displacement agents in surrounding periodontal tissues is of unquestionable importance, but the activity of these agents has not been completely elucidated. In the present study, we aimed to evaluate the effects of vasoconstrictive retraction agents on primary human gingival fibroblasts (HGFs). A total of six commercial adrenergic solutions (0.05 and 0.01% HCl-epinephrine, two based on 0.05% HCl-tetrahydrozoline, 0.05% HCl-oxymetazoline, and 10% HCl-phenylephrine) and three experimental gel formulations (EG-1, EG-2, and EG-3) were used to treat primary HGFs. The biological effect of the retraction treatment on the expression of collagen types I and III was detected by performing immunocytochemical analysis. The generation of reactive oxygen species triggered by the retraction agents were evaluated by using the dichlorofluorescein (DCF) fluorescent probe. The effect of retraction agents on the expression of fibronectin was visualized by confocal laser scanning microscopy. According to the results, experimental retraction gels did not limit the expression of collagen types I and III. EG-3 even induced the synthesis of both types of collagen. The DCF assay indicated oxidative stress similar to the control cells for most of the selected retraction agents. Experimental gels did not cause degradation of the cellular shape and morphology of the primary HGFs. The proposed experimental retraction gels in the present study demonstrated higher biocompatibility with primary HGFs, suggesting their use as clinical vasoconstrictive agents for the application of gingival retraction with minimal damage to periodontal tissues.
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http://dx.doi.org/10.3892/etm.2020.8462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027151PMC
March 2020

Electropermeabilization does not correlate with plasma membrane lipid oxidation.

Bioelectrochemistry 2020 Apr 15;132:107433. Epub 2019 Dec 15.

Frank Reidy Research Center for Bioelectrics, Old Dominion University, 4211 Monarch Way, Norfolk, VA 23508, USA.

The permeabilized condition of the cell membrane after electroporation can last minutes but the underlying mechanisms remain elusive. Previous studies suggest that lipid peroxidation could be responsible for the lasting leaky state of the membrane. The present study aims to link oxidation within the plasma membrane of live cells to permeabilization by electric pulses. We have introduced a method for the detection of oxidation by ratiometric fluorescence measurements of BODIPY-C11 dye using total internal reflection fluorescence (TIRF) microscopy, limiting the signal to the cell membrane. CHO-K1 cells were cultured on glass coverslips coated with an electroconductive indium tin oxide (ITO) layer, which enabled electroporation with micro- and submicrosecond pulses. No oxidation was observed with the electric field directed towards the ITO (cathode), even at field strengths much higher than that needed for permeabilization. Oxidation was readily detectable with the opposite polarity of pulses, but with the threshold higher than the permeabilization threshold. Moreover, a decrease in the medium conductance had opposite effects on permeabilization and lipid oxidation (it enhanced the former and suppressed the latter). We conclude that lipid oxidation can indeed occur at the plasma membrane after electric pulses, but it is not the cause of lasting membrane permeabilization.
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http://dx.doi.org/10.1016/j.bioelechem.2019.107433DOI Listing
April 2020

Effects of Photosensitization of Curcumin in Human Glioblastoma Multiforme Cells.

In Vivo 2019 Nov-Dec;33(6):1857-1864

Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland

Background/aim: There is no satisfactory treatment of glioblastoma multiforme, a highly invasive brain tumor. The aim of this study was to analyze the cytotoxic effects of curcumin (CUR) alone and as a photosensitizer on glioblastoma cells.

Materials And Methods: The SNB-19 cells where incubated for 2 and 24 h with 5-200 mM of CUR. The cells were radiated with blue light (6 J/cm) and compared to non-irradiated ones. The effects of treatment were assessed by measuring mitochondrial activity with the MTT method and apoptosis progression by flow cytometry. To investigate CUR uptake, fluorescence imaging of cells was performed.

Results: Photosensitization of CUR decreased the EC 6.3 times when the incubation time was 2 h and over 90% of cells underwent apoptosis. The study of the uptake of CUR showed that during the 2 h, CUR was placed in the entire cytoplasm, and over time, its amount decreased and localized in the subcellular compartments.

Conclusion: CUR is a promising medicament that can be used as a photosensitizer in photodynamic therapy for glioma treatment.
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http://dx.doi.org/10.21873/invivo.11679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899084PMC
March 2020

Correction: Calcium electroporation for treatment of sarcoma in preclinical studies.

Oncotarget 2019 Aug 20;10(49):5118. Epub 2019 Aug 20.

Department of Medical Biochemistry, Wroclaw Medical University, Wroclaw, Poland.

[This corrects the article DOI: 10.18632/oncotarget.24352.].
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http://dx.doi.org/10.18632/oncotarget.27158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707949PMC
August 2019

Electropermeabilization of metastatic chondrosarcoma cells from primary cell culture.

Biotechnol Appl Biochem 2019 Nov 18;66(6):945-954. Epub 2019 Sep 18.

Department of Thoracic Surgery, Wroclaw Medical University, Wroclaw, Poland.

Primary cell cultures are challenging, but reliable model reflecting tumor response in vitro. The study was designed to examine if the increased electropermeabilization can overcame initial drug insensitivity in chondrosarcoma cells from lung metastasis. We established a primary cell culture and evaluated the cytotoxic impact of four drugs-cisplatin (CDDP), camptothecin, 2-methoxyestradiol, and leucovorin calcium (LeuCa). After determination of parameters allowing for electropermeabilization, we performed electrochemotherapy in vitro with the least toxic drugs-CDDP and LeuCa. Although combining CDDP and leucovorin together increased their toxicity and supported apoptosis, application of pulsed electric fields (PEFs) brought no advantage for their efficacy. The study emphasizes the need for introduction of primary cell cultures into studies on pulse electric fields as model frequently less sensitive to PEF-based treatments than continuous cell lines.
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http://dx.doi.org/10.1002/bab.1809DOI Listing
November 2019

A New Betulin Derivative Stimulates the Synthesis of Collagen in Human Fibroblasts Stronger than its Precursor.

In Vivo 2019 Jul-Aug;33(4):1087-1093

Faculty of Pharmacy, Department of Molecular and Cellular Biology, Wroclaw Medical University, Wroclaw, Poland.

Background/aim: The exploration of substances that stimulate collagen synthesis and retard the aging process of the skin is an active field of current research. The natural environment and plants used in traditional medicine have been a source of such substances. The aim of this study was to compare the stimulatory effect of betulin (BE), betulinic acid (BA) and the new derivative - betulin ester with diaminobutyl acid (BE-Dab-NH) on collagen synthesis in human normal fibroblasts.

Materials And Methods: Primary fibroblast cultures were obtained from the gums of a healthy patient. The effect of the above-mentioned compounds was assessed by Sircol collagen assay, immunocytochemistry, and proliferation test.

Results: Fibroblasts cultured in the presence of BE-Dab-NH produced 6.85 times more collagen than control cells, 7.85 times more than those cultured in the presence of BA and 6.31 times more than those cultured in the presence of BE. An intense immunocytochemical reaction for collagen type I and III was found in fibroblasts cultured in the presence of BE-Dab-NH Conclusion: BE-Dab-NH stimulates significantly more collagen synthesis in normal human fibroblasts than its precursor.
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http://dx.doi.org/10.21873/invivo.11577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689372PMC
December 2019

Cisplatin and Vinorelbine -Mediated Electrochemotherapeutic Approach Against Multidrug Resistant Small Cell Lung Cancer (H69AR) .

Anticancer Res 2019 Jul;39(7):3711-3718

Department of Thoracic Surgery, Wroclaw Medical University, Wroclaw, Poland.

Background/aim: Small cell lung cancer (SCLC) originates from neuroendocrine branchial cells (15-20%). It is regarded as distinct from other lung cancers due to its biological and clinical features. In most cases of SCLC, surgery or radiotherapy alone is not an effective cure. The aim of our study was to examine the cytotoxic effects of chemotherapy supported by electroporation (EP) on a resistant SCLC model, in vitro.

Material And Methods: The multidrug resistant small lung cell line H69AR was used to evaluate the cytotoxic effects of cisplatin (CPPD) and vinorelbine (Navirel®; NAV) at lower doses when used with EP. Cells were treated with different concentrations of CPPD and NAV, alone or in combination with the following EP parameters: 400-1200 V/cm, 8 pulses of 100 μs duration, at 1Hz. The cell viability was estimated by MTT assay after 24 and 48 h. Apoptotic cells were detected by neutral comet assay and immunofluorescence assay with PARP-6.

Results: CPPD and NAV alone showed a dose-dependent effect on cell viability. Cytostatic drugs combined with EP revealed increased anticancer activity. Lower doses of CPPD or NAV delivered by EP were as effective as higher doses of these drugs without EP. The electrochemotherapeutic protocols increased the number of apoptotic cells and increased immunoreactivity of PARP-6. Our results indicated higher sensitivity of H69AR cells to NAV supported by EP.

Conclusion: In SCLC cells, an increased anticancer activity was potentiated by exposure of cells to high intensity electric pulses and low drug doses. It is suggested that this method could be effectively applied in the treatment of lung cancer.
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http://dx.doi.org/10.21873/anticanres.13519DOI Listing
July 2019

In Search of Panacea-Review of Recent Studies Concerning Nature-Derived Anticancer Agents.

Nutrients 2019 Jun 25;11(6). Epub 2019 Jun 25.

Department of Molecular and Cellular Biology, Wroclaw Medical University, Borowska 211A, 50-25 556 Wroclaw, Poland.

Cancers are one of the leading causes of deaths affecting millions of people around the world, therefore they are currently a major public health problem. The treatment of cancer is based on surgical resection, radiotherapy, chemotherapy or immunotherapy, much of which is often insufficient and cause serious, burdensome and undesirable side effects. For many years, assorted secondary metabolites derived from plants have been used as antitumor agents. Recently, researchers have discovered a large number of new natural substances which can effectively interfere with cancer cells' metabolism. The most famous groups of these compounds are topoisomerase and mitotic inhibitors. The aim of the latest research is to characterize natural compounds found in many common foods, especially by means of their abilities to regulate cell cycle, growth and differentiation, as well as epigenetic modulation. In this paper, we focus on a review of recent discoveries regarding nature-derived anticancer agents.
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http://dx.doi.org/10.3390/nu11061426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627480PMC
June 2019

Chemical Composition of East Asian Invasive Knotweeds, their Cytotoxicity and Antimicrobial Efficacy Against Cariogenic Pathogens: An In-Vitro Study.

Med Sci Monit 2019 May 4;25:3279-3287. Epub 2019 May 4.

Department of Pharmaceutical Biology and Botany, Wrocław Medical University, Wrocław, Poland.

BACKGROUND Giant knotweeds originating from East Asia, such as Reynoutria japonica, and Reynoutria sachalinensis, and their hybrid such as Reynoutria x bohemica, are invasive plants in Europe and North America. However, R. japonica is also a traditional East Asian drug (Polygoni cuspidati rhizoma) used in Korean folk medicine to improve oral hygiene. The aim of this study was to evaluate the antibacterial activity of acetone extracts of Reynoutria species against dominant caries pathogen such as Streptococcus mutans and alternative pathogens, as well as characterize the phytochemical composition of extracts and examine their cytotoxicity. MATERIAL AND METHODS Ultrasonic extraction was used to obtain polyphenol-rich extracts. The extracts were characterized by HPLC-DAD-ESI-MS. To test bacterial viability, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against S. mutans, S. salivarius, S. sanguinis, and S. pyogenes were determined. The cytotoxicity of the extracts to human fibroblasts derived from gingiva was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RESULTS The R. japonica extract had the highest bacteriostatic and bactericidal activity against pathogens causing caries, mainly dominant caries pathogen S. mutans (mean MIC 1000 μg/mL and MBC 2000 μg/mL), which was most likely associated with a higher content of stilbene aglycons and anthraquinone aglycons in the extract. Moreover, the R. japonica extract demonstrated the lowest cytotoxic effect on human fibroblasts and exhibited cytotoxic activity only at the concentration causing the death of all S. mutans. CONCLUSIONS The results indicate that the R. japonica acetone extract can be considered as a natural, antimicrobial agent for caries control.
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http://dx.doi.org/10.12659/MSM.913855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512351PMC
May 2019