Modulation of heat shock protein expression and cytokine levels in MCF-7 cells through photodynamic therapy.

In this study, we assess the impact of photodynamic therapy (PDT) using aluminum phthalocyanine tetrasulfonate (AlPcS4) on the viability and cellular stress responses of MCF-7 breast cancer cells. Specifically, we investigate changes in cell viability, cytokine production, and the expression of stress-related genes. Experimental groups included control cells, those treated with AlPcS4 only, light-emitting diode (LED) only, and combined PDT. To evaluate these effects on cell viability, cytokine production, and the expression of stress-related genes, techniques such as 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, enzyme-linked immunosorbent assays (ELISA), and real-time quantitative PCR (RT‒qPCR) were employed. Our findings reveal how PDT with AlPcS4 modulates mitochondrial activity and cytokine responses, shedding light on the cellular pathways essential for cell survival and stress adaptation. This work enhances our understanding of PDT's therapeutic potential and mechanisms in treating breast cancer.

Specific nanomarkers fluorescence: in vitro analysis for EGFR overexpressed cells in triple-negative breast cancer and malignant glioblastoma.

BACKGROUND: Epidermal Growth Factor Receptor (EGFR receptor) is encoded by the EGFR gene. EGFR receptor signaling pathways are activated by EGF protein, regulating cell actions. Overexpression of EGFR receptor may be linked to malignancies with a poor prognosis. As a result, EGFR receptor is being studied for a variety of tumor diagnostics, spurring the development of innovative approaches to increase quality and efficiency. Nanomaterials can recognize cancer cells by specifically targeting of molecular pathways, underscoring the importance of nanomedicine. In this study, we synthesized EGFR-specific nanomarkers by functionalizing EGF protein and Chlorin e6 in gold nanoparticles. These nanoparticles use active targeting to deliver EGF protein to EGFR receptor, and Chlorin e6 serves as a fluorescent marker molecule METHODS: Nanomarkers were examined in vitro in MDA-MB-468 and M059J cell lines. Confocal microscopy and flow cytometry were used to examine the distribution, uptake, internalization, and fluorescence intensity of nanomarkers in vitro RESULTS: The results show that both lines examined accumulate nanomarkers. However, MDA-MB-468 had the highest intensity due to its EGFR receptor overexpression properties CONCLUSION: The findings point to ideal properties for detecting EGFR receptor overexpressed cells.

Photodynamic effect of protoporphyrin IX in gliosarcoma 9l/lacZ cell line.

Photodynamic Therapy (PDT) is an oncologic treatment, producing reactive oxygen species (ROS) to induce the death of cancer cells. This study aimed to evaluate the action of PDT on gliosarcoma cells, using protoporphyrin IX as PS by incubation with the precursor aminolevulinic acid (ALA). An LED device was used with a light dose of 10 J/cm². The success of the therapy proved to be dependent on the concentration of ALA, and an incubation time of 4 h required for an effective response. Cell death was prevalent due to necrosis when assessed 18 h post-PDT. ALA proved to be an option to PDT in cells of the 9 L/lacZ, with the protocol tested.

Neuroprotective and restorative properties of the GLP-1/GIP dual agonist DA-JC1 compared with a GLP-1 single agonist in Alzheimer's disease.

The sister incretins glucagon-like peptide-1 (GLP-1) and glucagon dependent insulinotropic polypeptide (GIP) are growth factors responsible for re-sensitizing insulin signalling. Interestingly, their analogues, originally developed to treat type 2 diabetes (T2D), have demonstrated a range of neuroprotective and neurorestorative properties. Novel peptide GLP-1/GIP dual agonist (DA) shows good effects in diabetic patients, superior to the effects demonstrated by single GIP or GLP-1 mimetics. Furthermore, novel DAs have shown considerable neuroprotection in neurodegenerative models. Here, we investigated the neuroprotective and restorative involvement of the DA DA-JC1 and liraglutide (Lg), a single GLP-1 receptor analogue, in vitro using human neuroblastoma (SH-SY5Y) against oxidative stress induced by oxygen peroxide (H2O2), and in vivo, in a mouse model of Alzheimer's disease (AD), APP/PS1. First, we determined the ideal concentration of the peptides and demonstrated that DA-JC1 protects cells against oxidative stress more than Lg, improving cell viability, normalizing reactive oxygen species (ROS) and attenuating DNA damage generated by H2O2. Moreover, in 10-to-12-months-old APP/PS1 animals treated for 4 weeks, both Lg and DA-JC1 were very efficient in stimulating neurogenesis and reducing some important hallmarks of AD, but DA-JC1 was better than Lg in attenuating crucial neuroinflammatory markers, especially reactive astrocyte, in both wild-type (WT) and APP/PS1 hippocampal regions. Altogether, this study suggests an interactive role of GLP-1 and GIP receptors, enhancing the efficiency of single GLP-1 analogues, especially in attenuating oxidative stress and neuroinflammation. We confirm that combining GLP-1 and GIP results in a variety of beneficial effects, providing key evidences for the development of a promising therapeutic strategy for AD.

Genotoxic effects of photodynamic therapy in laryngeal cancer cells - An in vitro study.

IMPACT STATEMENT: Recently, the use of photodynamic therapy grows as an alternative treatment for cancer, since it has a noninvasive characteristic and affinity to the tumor tissue. Accordingly, understanding the therapy's foci of action is important for the technique improvement. This work aims to understand the genotoxic effect triggered by the therapy action, thus evidencing the permanent changes caused to the genetic material of the tumor cell after the treatment. Therefore, to increase the knowledge in this study field, the methodology of the comet assay and count of micronucleus formed after the therapy was adopted in order to understand if the damage caused to the DNA of tumor cell makes its replication process unfeasible in future generations. The study allows a better therapeutic approach to the cancer treatment, making the process of association between therapies a more effective option during the disease treatment.

Prolonged Drug-Releasing Fibers Attenuate Alzheimer's Disease-like Pathogenesis.

Delivering drugs and agents to the brain is a huge challenge, especially for chronic neurodegenerative disorders, such as Alzheimer's disease (AD). For this, prolonged and sustained release methods to increase brain uptake represent an impacting concept. The bioresorbable polymer poly-lactic acid (PLA) has high potential for medical implants; at the same time, glucagon-like peptide-1 (GLP-1) analogues have considerable neuroprotective attributes and represent a therapeutic strategy for AD. Here, a biodevice is produced by electrospinning PLA with a GLP-1 analogue (liraglutide, LG), coated with a thin layer of gelatin. The biodevice is subcutaneously implanted in a transgenic mouse model of AD and LG is released for 14 days in mice serum. After 4 weeks of implantation, crucial hallmarks of the AD are highly diminished: hippocampal senile amyloid ß plaque load and neuroinflammatory markers. Furthermore, neurogenesis is enhanced in the subventricular zone, an important neurogenic niche in the brain. The designed biodevice holds great promise for being an affordable candidate to act as a prolonged drug provider, promoting LG mission through increasing its lifetime, constituting a relevant approach for old and impaired brain.

Alteration of Surface Glycoproteins After Photodynamic Therapy.

BACKGROUND: Cell membranes have been identified as an important intracellular cancer treatment target, since the glycoconjugates present on the cell surface are involved in numerous cell functions. Photodynamic therapy (PDT) is a therapeutic modality employed in the treatment of tumors that uses visible light to activate a photosensitizer. OBJECTIVE: This study analyzed the expression of surface carbohydrates after PDT with two different photosensitizers, 5-aminolevulinic acid (ALA) and Photosan-3. METHODS: Mice were injected subcutaneously with 2 × 105 B16 cells. After 7-10 days, the presence of a tumor with a diameter of 3.6 mm was observed. Photosan-3® and 5-aminolevulinic acid-ALA were used in the PDT treatment. Control animals (not submitted to either laser treatment or photosensitizer injection) and treated animals were euthanized 15 days post-treatment. The tumors were irradiated with a red diode laser, λ = 655 nm, energy density of 10 J.cm-2, and power density of 45 mW.cm-2. After 2 weeks of treatment with PDT, the mice were euthanized, the tumors were collected, and the cell surfaces were labeled with lectins concanavalin A (ConA) and wheat germ agglutinin (WGA). RESULTS: Fluorescence microscopy analysis of the cell surfaces with lectins ConA and WGA showed the presence of α-mannose and α-glucose. CONCLUSIONS: The combined effects of either Photosan-3 or ALA and red laser light on melanoma suggest an inhibitory glycosylation action from PDT on the surface of B16-F10 cells.

Methylene blue internalization and photodynamic action against clinical and ATCC Pseudomonas aeruginosa and Staphyloccocus aureus strains.

Bacterial infections have been a major challenge to health. Increasing resistance to antimicrobial agents, according to World Health Organization, could be the major cause of death until 2050. Photodynamic therapy emerges as an alternative in microbial inactivation, due to its selectivity and to decreasing or dismissing antibiotic use. This study aimed at evaluating, in vitro, the internalization of the Methylene Blue and its photodynamic activity against a clinical and ATCC strain of Pseudomonas aeruginosa and Staphyloccocus aureus. Thus, the strains were incubated with MB in concentrations of 100, 300 e 500 µg/ml and then irradiated with a LED (±660 nm) at fluence of 10 and 25 J/cm2. The MB internalization was evaluated using a confocal microscope (Zeiss LSM 700), to capture the MB and the DAPI (for DNA staining). It was possible to observe that the MB was internalized by the bacterial cells, in all concentrations tested. The CFU/ml count demonstrated significant reduction (p ≤ 0,01) at the average 5.0 logs comparing with control group for the two species in all the tested concentrations. In conclusion, the strains tested were capable of internalizing the MB. PDT with MB was able to decrease the growth of the tested strains in vitro, being a promising alternative to the future treatment of infections caused by these species.

Biological activities of Rosmarinus officinalis L. (rosemary) extract as analyzed in microorganisms and cells.

R. officinalis L. is an aromatic plant commonly used as condiment and for medicinal purposes. Biological activities of its extract were evaluated in this study, as antimicrobial effect on mono- and polymicrobial biofilms, cytotoxicity, anti-inflammatory capacity, and genotoxicity. Monomicrobial biofilms of Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and Pseudomonas aeruginosa and polymicrobial biofilms composed of C. albicans with each bacterium were formed in microplates during 48 h and exposed for 5 min to R. officinalis L. extract (200 mg/mL). Its cytotoxic effect was examined on murine macrophages (RAW 264.7), human gingival fibroblasts (FMM-1), human breast carcinoma cells (MCF-7), and cervical carcinoma cells (HeLa) after exposure to different concentrations of the extract, analyzed by MTT, neutral red (NR), and crystal violet (CV) assays. The anti-inflammatory activity was evaluated on RAW 264.7 non-stimulated or stimulated by lipopolysaccharide (LPS) from Escherichia coli and treated with different concentrations of the extract for 24 h. Interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) were quantified by ELISA. Genotoxicity was verified by the frequency of micronuclei (MN) at 1000 cells after exposure to concentrations of the extract for 24 h. Data were analyzed by T-Test or ANOVA and Tukey Test ( P ≤ 0.05). Thus, significant reductions in colony forming units per milliliter (CFU/mL) were observed in all biofilms. Regarding the cells, it was observed that concentrations ≤ 50 mg/mL provided cell viability of above 50%. Production of proinflammatory cytokines in the treated groups was similar or lower compared to the control group. The MN frequency in the groups exposed to extract was similar or less than the untreated group. It was shown that R. officinalis L. extract was effective on mono- and polymicrobial biofilms; it also provided cell viability of above 50% (at ≤ 50 mg/mL), showed anti-inflammatory effect, and was not genotoxic. Impact statement Rosmarinus officinalis L. extract effectively contributed to in vitro control of important species of microorganisms such as Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans, and Pseudomonas aeruginosa in mono- and polymicrobial biofilms that are responsible for several infections in oral cavity as in other regions of the body. Furthermore, this extract promoted also cell viability above 50% at concentrations ≤ 50 mg/mL, excellent anti-inflammatory effect, showing inhibition or reduction of the synthesis of proinflammatory cytokines, being also non-genotoxic to cell lines studied. Thus, this extract may be a promising therapeutic agent that can be added in some medical and dental formulations such as toothpastes, mouthwashes, irrigating root canals, ointments, soaps, in order to control pathogenic microorganisms and biofilms, with anti-inflammatory effect and absence of cytotoxic and genotoxic.

A Novel Bioresorbable Device as a Controlled Release System for Protecting Cells from Oxidative Stress from Alzheimer's Disease.

Bioresorbable electrospun fibres have highly functional features that can preserve drug efficacy, avoiding premature degradation, and control drug release rates over long periods. In parallel, it is known that Alzheimer's disease (AD) has been linked to impaired insulin signalling in the brain. Glucagon-like peptide 1 (GLP-1) analogues have beneficial effects on insulin release and possess exceptional neuroprotective properties. Herein, we describe for the first time the incorporation of a GLP-1 analogue, liraglutide, into electrospun poly (lactic acid) (PLA) fibres with in situ gelatin capsules, in order to provide the controlled release of liraglutide, improving neuroprotective properties. In this study, PLA, a bioresorbable polymer in which degradation products have neurogenesis characteristics, was electrospun and loaded with liraglutide. Moreover, PLA/liraglutide fibres were encapsulated with gelatin and were shown to have better properties than the non-encapsulated fibres in terms of the controlled release of liraglutide, which was accomplished in the present study for up to 60 days. We observed that this biodevice was completely encapsulated with gelatin, which made the material more hydrophilic than PLA fibres alone and the biodevice was able to enhance fibroblast interaction and reduce mitochondrial stress in a neuroblastoma cell line. In this manner, this study introduces a new material which can improve neuroprotective properties from AD oxidative stress via the sustained long-lasting release of liraglutide. Graphical Abstract ᅟ.

High doses of alcohol during pregnancy cause DNA damages in osteoblasts of newborns rats.

BACKGROUND: Alcohol exerts teratogenic effects and its consumption during pregnancy can cause deficit of bone development. The aim of the current study was to evaluate the genotoxic effects of prenatal exposure to ethanol on newborn rat osteoblasts. METHODS: Wistar rats were initially divided into two groups: Ethanol group which received Ethanol 20% V/V in liquid diet and solid diet ad libitum, and Control group, which received solid diet and water ad libitum. Each group received a specific diet for 8 weeks before breeding and throughout three weeks of gestation and the treatment was finished on the day the pups were killed. On the fifth day of life, the pups from each group were killed for removal of the calvaria and isolation of osteogenic cells by sequential enzymatic digestion. The cells were cultured for a maximum period of 14 days. The detection of genotoxic effects of alcohol was investigated by the comet and the micronucleus assay. RESULTS: Micronucleus and comet assay showed significant increases in DNA damage at 7 days in Ethanol group (p = 0.0302, p = 0.0446, respectively). However, at 14 days both assay showed no significant difference between the groups (p = 0.6194, p = 0.8326, respectively). CONCLUSION: Our results showed that prenatal exposure to ethanol induced DNA damage in osteoblasts, as shown by micronucleus formation and higher percentage of DNA in the comet tail. It can be concluded that prenatal exposure to ethanol damages osteoblast DNA in newborns exposed to high doses of ethanol during pregnancy, suggesting that prenatal ethanol consumption has a direct effect on fetal osteoblasts.

Evaluation of the photobiomodulation in L929 cell culture.

Low-level laser therapy has become an important tool for bio-modulation process. It can induce stimulatory or inhibitory effects according to cell behavior at specific irradiation. Our objective was to determine L929 cell line response to irradiation at λ 685 and 830 nm, concentrations of 5 and 10% fetal bovine serum and different energy densities of 0.1, 0.5, 1, 2, 3, 5, 7, 10, 20, and 30 J/cm(2). Thus, cells were plated at 1 × 10(5) cells/mL and irradiated with semiconductor laser As-Ga-Al. Twenty-four hours after irradiation, cells were subjected to MTT, neutral red, crystal violet tests, and cell staining was performed using the kit Alexa Fluor 488 Annexin V-FITC and propidium iodide. Our results showed that low-level laser therapy stimulates effect when the energy density is 5 to 3030 J/cm(2) and inhibits effects on energy density 0.1 to 3 J/cm(2). This inhibitory effect was evidenced by the absence of dead cells labeled, decreased cell density, and by the absorption of neutral red in intact cells. The study also demonstrated that fetal bovine serum, at different concentrations, did not affect response of the cells after irradiation.

Evaluation of photodynamic therapy in adhesion protein expression.

Photodynamic therapy (PDT) is a treatment modality that has clinical applications in both non-neoplastic and neoplastic diseases. PDT involves a light-sensitive compound (photosensitizer), light and molecular oxygen. This procedure may lead to several different cellular responses, including cell death. Alterations in the attachment of cancer cells to the substratum and to each other are important consequences of photodynamic treatment. PDT may lead to changes in the expression of cellular adhesion structure and cytoskeleton integrity, which are key factors in decreasing tumor metastatic potential. HEp-2 cells were photosensitized with aluminum phthalocyanine tetrasulfonate and zinc phthalocyanine, and the proteins ß1-integrin and focal adhesion kinase (FAK) were assayed using fluorescence microscopy. The verification of expression changes in the genes for FAK and ß1 integrin were performed by reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that HEp-2 cells do not express ß-integrin or FAK 12 h following PDT. It was concluded that the PDT reduces the adhesive ability of HEp-2 cells, inhibiting their metastatic potential. The present study aimed to analyze the changes in the expression and organization of cellular adhesion elements and the subsequent metastatic potential of HEp-2 cells following PDT treatment.

Photodynamic therapy in the cattle protozoan Tritrichomonas foetus cultivated on superhydrophilic carbon nanotube.

Superhydrophilic vertically aligned carbon nanotubes (VACNT-O2) were used for the first time as scaffolds for photodynamic therapy (PDT) to induce inhibition of cell division in eukaryotic cells. VACNT-O2 scaffolds were produced on Ti substrates using plasma enhanced chemical vapor deposition technique and functionalized by oxygen plasma. Scanning electron microscopy (SEM) analysis was performed to characterize the surface changes of the protozoan and interaction with VACNT-O2. Characterization of lipid and total protein expression was performed with protozoa that were or not treated with PDT. Quantification of protein was conducted using Qubit fluorometer and separated on a polyacrylamide gel. SEM analysis showed the release of lipid vesicles by protozoa after the PDT. These vesicles were characterized by the PKH26 fluorescent probe. The results demonstrated a greater amount of protein released after PDT than in the control. When analyzing the protein material in polyacrylamide gel, a significant protein expression of approximately 65 kDa was found. A model identified the programmed death of Tritrichomonas foetus after the PDT was also proposed.

Effect of Spilanthes acmella hydroethanolic extract activity on tumour cell actin cytoskeleton.

Numerous natural products have pharmacological activity such that many biologically active compounds have led to the development of cancer chemotherapy drugs. Spilanthes acmella (Asteraceae) is widely cultivated in the State of Pará, Brazil, being employed in folk medicine for its anti-inflammatory, antimicrobial, antioxidant, analgesic, insecticide, and larvicidal properties. However, its cytotoxicity and influence on actin cytoskeleton organisation in tumour cell lines are practically nonexistent. We have verified the cytotoxicity of a hydroethanolic extract of the inflorescence of S. acmella, and examined its effects on the cytoskeleton of tumour cells. Decreasing concentrations of the extract (250, 500 and 1,000 µg/mL) were given to cultures of neoplastic cells (HEp-2). Cytotoxicity was assessed by the MTT test, and the influence on cytoskeleton organisation was examined by fluorescence microscopy. The IC50 of the hydroethanolic extract was 513 µg/mL, confirming the data obtained from the MTT assay that gave high cytotoxicity. The actin cytoskeleton arrangement of HEp2 cells at 500 and 1,000 µg/mL showed depolymerisation of the filaments, causing loss of morphology and consequently compromising cell adhesion.

A novel function for CUGBP2 in controlling the pro-inflammatory stimulus in H9c2 cells: subcellular trafficking of messenger molecules.

Accumulating evidence demonstrates that chronic inflammation plays an important role in heart hypertrophy and cardiac diseases. However, the fine-tuning of cellular and molecular mechanisms that connect inflammatory process and cardiac diseases is still under investigation. Many reports have demonstrated that the overexpression of the cyclooxygenase-2 (COX-2), a key enzyme in the conversion of arachidonic acid to prostaglandins and other prostanoids, is correlated with inflammatory processes. Increased level of prostaglandin E2 was also found in animal model of left ventricle of hypertrophy. Based on previous observations that demonstrated a regulatory loop between COX-2 and the RNA-binding protein CUGBP2, we studied cellular and molecular mechanisms of a pro-inflammatory stimulus in a cardiac cell to verify if the above two molecules could be correlated with the inflammatory process in the heart. A cellular model of investigation was established and H9c2 was used. We also demonstrated a regulatory connection between COX-2 and CUGBP2 in the cardiac cells. Based on a set of different assays including gene silencing and fluorescence microscopy, we describe a novel function for the RNA-binding protein CUGBP2 in controlling the pro-inflammatory stimulus: subcellular trafficking of messenger molecules to specific cytoplasmic stress granules to maintain homeostasis.

Biomineralization of superhydrophilic vertically aligned carbon nanotubes.

Vertically aligned carbon nanotubes (VACNT) promise a great role for the study of tissue regeneration. In this paper, we introduce a new biomimetic mineralization routine employing superhydrophilic VACNT films as highly stable template materials. The biomineralization was obtained after VACNT soaking in simulated body fluid solution. Detailed structural analysis reveals that the polycrystalline biological apatites formed due to the -COOH terminations attached to VACNT tips after oxygen plasma etching. Our approach not only provides a novel route for nanostructured materials, but also suggests that COOH termination sites can play a significant role in biomimetic mineralization. These new nanocomposites are very promising as nanobiomaterials due to the excellent human osteoblast adhesion.

Fast preparation of hydroxyapatite/superhydrophilic vertically aligned multiwalled carbon nanotube composites for bioactive application.

A method for the electrodeposition of hydroxyapatite films on superhydrophilic vertically aligned multiwalled carbon nanotubes is presented. The formation of a thin homogeneous film with high crystallinity was observed without any thermal treatment and with bioactivity properties that accelerate the in vitro biomineralization process and osteoblast adhesion.

Cellular changes after photodynamic therapy on HEp-2 cells using the new ZnPcBr(8) phthalocyanine.

OBJECTIVE: This study investigated the effects of photodynamic therapy (PDT) by using the new photosensitizer Octal-Bromide Zinc Phthalocyanine (ZnPcBr(8)) on the nucleus, mitochondria, and cytoskeleton of HEp-2 cells. BACKGROUND DATA: PDT has been widely used as a therapeutic method for tumor-selective treatment and for other diseases. The therapy requires a photosensitizer, molecular oxygen, and visible light. Different studies have demonstrated that cellular organelles are potential targets for PDT, and the results are dependent on the photosensitizer used in the treatment. In this study, we investigated changes in the nucleus, mitochondria, and cytoskeleton of HEp-2 cells after PDT with the new ZnPcBr(8) phthalocyanine. MATERIAL AND METHODS: HEp-2 cells were cultivated under standard conditions, and then incubated with ZnPcBr(8) (1 micromol/L) for 1 h, and subsequently irradiated with a diode laser light (676 nm, 30 mW, 4.5 J/cm(2)). The cells were further cultured for 1 and 24 h at 37 degrees C in a 5% CO(2) and analyzed with fluorescence microscopy by using specific probes for the investigated organelles. RESULTS: Before PDT, the photosensitizer showed a cytoplasmic diffuse distribution. After PDT, cells showed multinucleation, a punctuated mitochondrial distribution in the perinuclear region, and cellular retraction due to the cytoskeleton changes. All those cellular alterations disrupted homeostasis, contributing to cellular death, which is the major goal of PDT. CONCLUSION: Based on our results and the characteristics of the new ZnPcBr(8) phthalocyanine, mechanistic and biochemical studies must be performed, but it is tempting to consider the chemical as a promising agent for PDT.

Laser biomodulation on L 929 cell culture.

OBJECTIVE: The aim of the present study was to analyze the effects of photobiomodulation using a 904-nm diode laser at two energy densities (6 J/cm(2) and 50 mJ/cm(2)) on L929 fibroblast cells. BACKGROUND: Low-power laser irradiation (LPLI) is a non-pharmacological resource that induces important in vitro photobiomodulation on cell cultures and tissues. METHODS: Irradiation was performed for three days at 24-h intervals. After each interval, the cells were stained with MitoTracker Orange and DioC6 dyes to assess the photobiomodulatory effects of irradiation on mitochondrial activity and changes in the endoplasmic reticulum. The MTT assay [3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide] was used to evaluate cell proliferation. RESULTS AND CONCLUSIONS: The fluorescence microscopy assessment of mitochondria and endoplasmic reticulum in cells irradiated with 6 J/cm(2) and 50 mJ/cm(2) demonstrated intense mitochondrial activity, which was confirmed by DioC6 staining. Reticular activity was observed stemming from increased protein synthesis. Photobiomodulation with 50 mJ/cm(2) was slightly higher than with 6 J/cm(2), as demonstrated by fluorescence microscopy results. Photobiomodulation was also time-dependent, with better results 72-h after irradiation.