Assessment of Chemopreventive Potential of the Plant Extracts against Liver Cancer Using HepG2 Cell Line.

The edible parts of the plants Camellia sinensis, Vitis vinifera and Withania somnifera were extensively used in ancient practices such as Ayurveda, owing to their potent biomedical significance. They are very rich in secondary metabolites such as polyphenols, which are very good antioxidants and exhibit anti-carcinogenic properties. This study aims to evaluate the anti-cancerous properties of these plant crude extracts on human liver cancer HepG2 cells. The leaves of Camellia sinensis, Withania somnifera and the seeds of Vitis vinifera were collected and methanolic extracts were prepared. Then, these extracts were subjected to DPPH, α- amylase assays to determine the antioxidant properties. A MTT assay was performed to investigate the viability of the extracts of HepG2 cells, and the mode of cell death was detected by Ao/EtBr staining and flow cytometry with PI Annexin- V FITC dual staining. Then, the protein expression of BAX and BCl2 was studied using fluorescent dye to determine the regulation of the BAX and BCl2 genes. We observed that all the three extracts showed the presence of bioactive compounds such as polyphenols or phytochemicals. The W. somnifera bioactive compounds were found to have the highest anti-proliferative activity on human liver cancer cells.

Assessment of Betulinic Acid Cytotoxicity and Mitochondrial Metabolism Impairment in a Human Melanoma Cell Line.

Melanoma represents one of the most aggressive and drug resistant skin cancers with poor prognosis in its advanced stages. Despite the increasing number of targeted therapies, novel approaches are needed to counteract both therapeutic resistance and the side effects of classic therapy. Betulinic acid (BA) is a bioactive phytocompound that has been reported to induce apoptosis in several types of cancers including melanomas; however, its effects on mitochondrial bioenergetics are less investigated. The present study performed in A375 human melanoma cells was aimed to characterize the effects of BA on mitochondrial bioenergetics and cellular behavior. BA demonstrated a dose-dependent inhibitory effect in both mitochondrial respiration and glycolysis in A375 melanoma cells and at sub-toxic concentrations (10 µM) induced mitochondrial dysfunction by eliciting a decrease in the mitochondrial membrane potential and changes in mitochondria morphology and localization. In addition, BA triggered a dose-dependent cytotoxic effect characterized by apoptotic features: morphological alterations (nuclear fragmentation, apoptotic bodies) and the upregulation of pro-apoptotic markers mRNA expression (Bax, Bad and Bak). BA represents a viable therapeutic option via a complex modulatory effect on mitochondrial metabolism that might be useful in advanced melanoma or as reliable strategy to counteract resistance to standard therapy.

Assessment of Apoptosis Pathway in Peripheral Blood of Autistic Patients.

Autism spectrum disorder (ASD) includes a number of severe neurodevelopmental disorders known by defects in social interaction, impaired verbal and non-verbal interactions, and stereotypic activities and limited interests. Dysregulation of apoptotic pathways have been demonstrated in brain tissues of affected individuals. In the present study, we evaluated expression levels of apoptosis-related genes and miRNAs in peripheral blood of ASD patients compared with healthy subjects. Transcript levels of BCL2, CASP8, and hsa-29c-3p were significantly lower in total ASD patients compared with total normal children (P values = 0.003, 0.002, and 0.01 respectively). When sex of study participants was considered in the analysis, the difference in transcript levels of these genes was significant only in male subjects. Peripheral expression of BCL2 and hsa-29c-3p had 100% sensitivity 92% specificity in ASD diagnosis. The diagnostic power of combination of transcript levels of these genes was estimated to be 78% based on the calculated AUC value. The present study provides evidences for dysregulation of apoptotic pathways in peripheral blood of ASD patients and suggests certain apoptosis-related genes as biomarkers in this regard.

A whole-food approach to the in vitro assessment of the antitumor activity of gazpacho.

Gazpacho is a traditional cold soup of the Mediterranean diet consisting of a main base of fresh pureed tomato and other vegetables. Tomato and tomato products have demonstrated chemopreventive activity against several types of cancer through in vitro studies, and in animal and clinical research. Here we have applied a whole-food approach for the preclinical assessment of the antitumor potential of gazpacho. Colon cancer cells (HT-29) were exposed to growing concentrations of gazpacho previously digested in vitro to simulate the delivery of bioactive molecules to colon cells after food consumption. The cytotoxicity of gazpacho ingredients was also tested in independent experiments. Programmed cell death by apoptosis was detected by using a multiparametric analysis that combines image-based bright-field and fluorescence cytometry, intracellular ATP level determination and enzymatic activity of caspase-3/7. Modulation of gene expression of key regulatory genes (p53, Bcl-2, BAX, and cyclin D1) was also investigated. Our cytotoxicity data showed that in vitro digestion of samples allowed the delivery of bioactive levels of antitumor phytochemicals to cultured cells. Controlled experiments showed significant repetitive dose and time-response cytotoxicity of gazpacho. Gazpacho digestates caused net cell death of cultures suggesting synergic activity among phytochemicals from its vegetable ingredients. Multiparametric and genetic analyses showed that gazpacho digestates can trigger colon cancer cells death by apoptosis through the activation of caspase cascade. Our results show that coupled in vitro methodology employed can be applied to investigate the antitumor potential of complex food matrixes or combinations of foods in the diet.

Assessment of Dynamic BCL-2 Protein Shuttling Between Outer Mitochondrial Membrane and Cytosol.

BCL-2 proteins control stress-dependent commitment to the programmed cell death apoptosis. In nonapoptotic cells the proapoptotic BCL-2 proteins BAX and BAK but also prosurvival family members, like BCL-xL or MCL-1, translocate to the outer mitochondrial membrane (OMM) and retrotranslocate from the mitochondria back into the cytosol. The resulting equilibrium produces a broad range of localization pattern observed for BAX and BAK in human cells and shows correlation between relative BAX and BAK localizations and cellular predisposition to apoptosis. The retrotranslocation of BCL-2 proteins from the OMM can be measured using fluorescence-labeled protein in intact cells or endogenous protein from isolated heavy membrane fractions.

Hyperactivated PI3Kδ promotes self and commensal reactivity at the expense of optimal humoral immunity.

Gain-of-function mutations in the gene encoding the phosphatidylinositol-3-OH kinase catalytic subunit p110δ (PI3Kδ) result in a human primary immunodeficiency characterized by lymphoproliferation, respiratory infections and inefficient responses to vaccines. However, what promotes these immunological disturbances at the cellular and molecular level remains unknown. We generated a mouse model that recapitulated major features of this disease and used this model and patient samples to probe how hyperactive PI3Kδ fosters aberrant humoral immunity. We found that mutant PI3Kδ led to co-stimulatory receptor ICOS-independent increases in the abundance of follicular helper T cells (TFH cells) and germinal-center (GC) B cells, disorganized GCs and poor class-switched antigen-specific responses to immunization, associated with altered regulation of the transcription factor FOXO1 and pro-apoptotic and anti-apoptotic members of the BCL-2 family. Notably, aberrant responses were accompanied by increased reactivity to gut bacteria and a broad increase in autoantibodies that were dependent on stimulation by commensal microbes. Our findings suggest that proper regulation of PI3Kδ is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome.

Assessment of dose-response relationship of 5-fluorouracil to murine intestinal injury.

5-Fluorouracil (5-FU) is the most frequently prescribed anti-tumor drug, but has been reported to result in intestinal injury. Although some progress has been made in understanding the intestinal toxicity of 5-FU, confusion remains about animal models of 5-FU-induced intestinal injury, especially the dosage of 5-FU. This study aims to assess the dose-response relationship between the severity of intestinal injury and different doses of 5-FU, and to determine a proper dosing for the murine model. We found that mice in the 5-FU groups gradually lost body weight over time. Increasing doses of 5-FU resulted in more severe diarrhea, with a concomitant increase in mortality. Histopathological damage was more severe in mice that received higher doses of 5-FU. In addition, plasma diamine oxidase (DAO) activity decreased in experimental mice with intestinal injury in a dose-dependent way. TUNEL and western blot analysis showed cell apoptosis in the ileum and colon related to 5-FU dosage. However, administration of 200 and 400 mg/kg 5-FU caused extremely high mortality, severe diarrhea and histopathological damage, but 25 mg/kg 5-FU did not result in significant intestinal injury. The severity of intestinal injury induced by 5-FU appeared to be dose-dependent and we concluded that the proper dosage of 5-FU to induce a murine model with intestinal mucositis ranged from 50 mg/kg to 100 mg/kg.

Assessment of thymoquinone effects on apoptotic and oxidative damage induced by morphine in mice heart.

Opioids bind to specific receptors that are located in the central nervous system (CNS) and many other organs such as cardiovascular tissue. Morphine binds to opioid receptors and can induce oxidative stress under some certain conditions. Thymoquinone (TQ) has shown many therapeutic effects such as anti-inflammatory, antioxidant and immunomodulatory ones. Considering the oxidative effects of morphine, antioxidant effects of TQ and effects of oxidative damage in various types of biomolecules, the present study was conducted to determine the effect of morphine plus TQ on the expression of apoptotic genes in the heart of male mice. Hence we used real-time PCR to identify alterations in mRNA expression of genes involved in apoptotic pathway, including p53, Bax and Bcl-2 between the morphine-treated and TQ plus morphine-treated mice. Serum nitric oxide (NO) (Griess assay) and total antioxidant capacity (TAC) were analyzed and compared. In the morphine group, compared to control group, a significant increase in P53 and Bax mRNA expression and a significant decrease in Bcl-2 mRNA expression were observed (p < 0.01). In TQ plus morphine groups, NO was decreased (P <0 .001) and TAC levels were increased significantly (P < .001). Interestingly, TQ (9 and 18 mg/kg) plus morphine caused a significant decrease in p53 and Bax and a significant increase in Bcl2 mRNA expression, compared to morphine-treated group (p < 0.01). Collectively, the results of this study indicated that TQ, as an antioxidant, can improve the apoptotic effects induced by morphine in the heart tissue of mice.

Assessment of aflatoxin B1 myocardial toxicity in rats: mitochondrial damage and cellular apoptosis in cardiomyocytes induced by aflatoxin B1.

Objective The number of deaths from heart disease is increasing worldwide. Aflatoxin B1 (AFB1), a toxin produced by the fungi Aspergillus flavus and Aspergillus parasiticus, is frequently detected in improperly processed/stored human food products. While AFB1 hepatotoxicity and carcinogenic properties have been well addressed, its myocardial toxicity is poorly documented. This study aimed to investigate myocardial toxic activity of AFB1. Methods Ten rats were fed with AFB1 at a dose that did not result in acute toxic reactions for 30 days and 10 vehicle-fed rats served as controls. Transmission electron microscopy was used to assess mitochondrial damage in cardiomyocytes. The terminal deoxynucleotidyl transferase-mediated UTP nick-end labelling assay was performed to detect apoptosis of cardiomyocytes. Western blotting was performed to measure apoptotic proteins (i.e., active caspase-3, Bax, and Bcl-2) in heart tissue. Results AFB1 treatment resulted in mitochondrial membrane disruption and disorganization of cristae, which are indicators of mitochondrial damage. Myocardial cell apoptosis was significantly higher after AFB1 treatment (22.07% ± 3.29%) compared with controls (6.27% ± 2.78%, P < 0.05). AFB1 treatment enhanced expression of active caspase-3, Bax, and Bcl-2 in cardiac tissue. Conclusion Various adverse effects are exerted by AFB1 on the heart, indicating AFB1 myocardial toxicity.

Assessment of global ischemic/reperfusion and Tacrolimus administration on CA1 region of hippocampus: gene expression profiles of BAX and BCL2 genes.

BACKGROUND: It is well known that hippocampus has a pivotal role in learning, formation and consolidation of memory. Global cerebral ischemia causes severe damage to pyramidal neurons of the CA1 region and usually results in residual neurological deficits following a recovery from ischemia. Scientists investigate to find the molecular mechanism of apoptosis and to use this cell death for clinical treatment. OBJECTIVE: In this investigation, we evaluated the molecular mechanism of FK-506 in apoptosis using gene expression quantification of BAX and BCL-2 genes in hippocampus following global ischemic/reperfusion. MATERIALS AND METHODS: In the present experimental study, adult male Wistar rats were obtained and housed under standard conditions. Each experimental group consisted of five rats and was equally distributed in the normal control, ischemia/reperfusion, ischemia/reperfusion followed by FK-506. Global ischemia was induced for animals in ischemia and drug groups. In the drug group, moreover, two doses of FK-506 were injected as IV injection and intra-peritoneal (IP) injection after 48 h. Then, hippocampus tissue was removed. Consequently, RNA isolated, cDNA was synthesized and Real-Time PCR was performed. Finally, the obtained data was analyzed statistically (p<0.05). RESULTS: The quantitative results showed the BAX expression ratio increased approximately 3-times in ischemia/reperfusion (3.11 ± 0.28) group compared to the untreated (NR) and the drug group (p<0.001). The statistical analysis showed a significant difference for BCL-2 expression between the experimental groups (p<0.001). The mRNA level of BCL-2 decreased in the ischemia/reperfusion group, while it was without alteration in the other groups. CONCLUSION: The results showed that global cerebral ischemia/reperfusion induced BAX as pro-apoptotic gene and tacrolimus a neuroprotective drug inhibited BAX gene expression and induced BCL-2 gene expression as anti-apoptotic gene (Tab. 2, Fig. 3, Ref. 21).

Effect of Pomegranate Hull Extract on Liver Neoplastic Changes in Rats: More than an Antioxidant.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide. The current work was designed to elucidate the molecular mechanisms underlying the antitumorigenic effect of pomegranate hull extract (PHE) in livers of rats exposed to the hepatocarcinogen diethyl nitrosamine (DENA) with emphasis on oxidative stress, proliferation, and apoptosis. Male albino rats were divided into three groups: normal control, DENA group, and PHE group. PHE was given to rats orally 3 times weekly for 10 wk, 4 wk before and 6 wk after DENA (200 mg/kg, single i.p. dose). The results indicated a prophylactic effect of PHE against neoplastic changes in the liver, which was evidenced by the decrease of tumor size, liver index, and the anti-apoptotic protein Bcl-2; and the increase of glutathione. PHE group also showed decreased expression of liver cyclin D1 and ß-catenin genes compared with DENA group. It is proved that PHE has antitumorigenic effect and could be a candidate for anticancer drugs.

Comprehensive Assessment and Classification of High-Grade B-cell Lymphomas.

High-grade B-cell lymphomas (HGBCLs) are a heterogeneous group of neoplasms that include subsets of diffuse large B-cell lymphoma, Burkitt lymphoma, and lymphomas with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma. Morphologically indistinguishable HGBCLs may demonstrate variable clinical courses and responses to therapy. The morphologic evaluation and classification of these neoplasms must be followed by further genetic and immunophenotypic work-up. These additional diagnostic modalities lead to a comprehensive stratification of HGBCL that determines the prognosis and optimal therapy. This article reviews the well-established and emerging biomarkers that are most relevant to the clinical management of HGBCL.

Assessment of pro-apoptotic activity of doxorubicin-transferrin conjugate in cells derived from human solid tumors.

Conjugates of anthracyclines are a new possibility for anticancer agent delivery, which seems to be a very promising alternative to the currently used cancer treatment strategies. In our study, we investigated the ability of a doxorubicin-transferrin (DOX-TRF) conjugate to induce cell death in two solid tumor cell lines: non-small cell lung cancer (A549) and hepatocellular liver carcinoma (HepG2). The observed effects of the DOX-TRF conjugate on these cell cultures were compared with those of free doxorubicin (DOX), a widely used antineoplastic therapeutic agent. Our results provided direct evidence that the investigated conjugate is considerably more cytotoxic to the examined human cancer cell lines than is DOX alone. Moreover, we confirmed that the antitumor efficacy of DOX-TRF conjugate is related to its apoptosis-inducing ability, which was shown during measurements of typical features of programmed cell death. In solid tumor cell lines, the DOX-TRF conjugate induced changes in cellular morphology, mitochondrial membrane potential and caspases-3 and -9 activities. Furthermore, all of the analyzed hallmarks of apoptosis were confirmed by the oligonucleosomal DNA fragmentation assay and by a real-time PCR quantitative study, which displayed the superiority of the conjugate-induced programmed cell death over free drug-triggered cell death.

Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs.

BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.

Assessment of cricopharyngeal muscle aging with apoptotic markers.

OBJECTIVES: The aims of this study were to evaluate histochemical markers of apoptosis in the cricopharyngeus muscle, which is the gatekeeper of the pharyngoesophageal region during the swallowing process; to investigate the effects of primary aging on this muscle; and to determine whether a relationship exists with gastroesophageal reflux disease. MATERIALS AND METHODS: The study included 30 fresh cadavers with a time of death of 12 hours or less obtained from the Turkish Ministry of Justice Forensic Medicine Unit. All cadavers were dissected with routine postmortem skin incisions to extract specimens from the cricopharyngeus muscle and the esophagocardiac junction mucosa. Muscle degeneration and primary aging were demonstrated by immunodetection of Bax, Bcl-2, and Caspase-3 proteins as markers of the apoptosis. Esophageal specimens were examined for the presence of reflux esophagitis. RESULTS: The mean age was 41.5 (14-74) years, and the study included 18 male and 9 female cadavers. Three of them were excluded because of fixation artifacts. The mean Bax, Bcl-2, and Caspase scores showed no statistically significant relationship with age (P = 0.94). The right and left sides of the muscle were investigated separately, and the Bax scores of the right side of the cricopharyngeus muscle showed a statistically significant decrease with age (P = 0.026), whereas the Bax and Bcl-2 scores were increased with age (P = 0.035 and 0.049, respectively) on the left side. Evaluation of the 23 esophagus specimens revealed 10 cases of esophagitis. No relationship was found between the mean of each apoptotic marker and esophagitis. CONCLUSIONS: It is histopathologically not possible to demonstrate muscle death due to either primary aging or reflux. This might be attributable to the defensive capability of this unique muscle to maintain the feeding process.

The effect of etoricoxib on kidney ischemia-reperfusion injury in rats: a biochemical and immunohistochemical assessment.

The purpose of this study was to investigate the effect of etoricoxib on oxidative injury induced with ischemia-reperfusion (I/R) in rat kidney tissue in terms of biochemistry and immunohistochemistry. Male Albino Wistar rats were divided into renal I/R (RIR), 50 mg/kg etoricoxib+RIR (ETO-50), 100 mg/kg etoricoxib+RIR (ETO-100) and sham operation (SG) groups. Animals in the ETO-50 and ETO-100 groups were given etoricoxib by the oral route at dosages of 50 and 100 mg/kg, respectively. The RIR and SG groups were given distilled water as solvent. One hour after drug administration, 1h of ischemia and 3h of reperfusion were applied to the left kidneys of all rats (apart from SG) under 25 mg/kg thiopental sodium anesthesia. At the end of that time, kidneys were extracted and biochemical and immunohistochemical analyses were performed. Etoricoxib reduced, in a dose-dependent manner, levels of MDA, MPO and COX-2 that normally rise with I/R in rat kidney tissues. Etorixicob did not alter COX-1 activity at 50 and 100 mg/kg doses, but significantly prevented loss of tGSH in tissues with I/R. In addition, Bcl-2' gene expression inhibited with I/R was prevented in renal tubular and glomerular cells. Furthermore, etoricoxib significantly decreased the caspase-3 gene expression which increased with I/R. Etoricoxib significantly prevented I/R injury in a dose-dependent manner. The results of this study show that etoricoxib treatment could decrease kidney injury during IR.

Assessment of the roles of P-glycoprotein and cytochrome P450 in triptolide-induced liver toxicity in sandwich-cultured rat hepatocyte model.

Triptolide (TP), a main bioactive component of Tripterygium wilfordii Hook F., is a promising agent for treatment of autoimmune diseases. However, a high incidence of dose-limiting hepatotoxicity was observed in the clinic. Sandwich-cultured rat hepatocyte model was used in this study to identify the involvement of P-glycoprotein (P-gp) in TP disposition and to evaluate TP-induced hepatotoxicity after modulation of P-gp by the known inhibitors, ritonavir and tariquidar, and known inducers, phenobarbital, quercetin, and H(2)O(2). Our data showed that biliary clearance of TP reduced 73.7% and 84.2% upon treatment of ritonavir (25 µM) and tariquidar (5 µM), respectively. In contrast, increases of 346%, 280%, and 273% in biliary clearance of TP were observed with treatment of phenobarbital (1.0 mM), quercetin (20 µM), and H(2)O(2) (0.5 mM), respectively. The TP-induced hepatotoxicity increased by twofold when CYP activity was blocked by 1-aminobenzotriazole, suggesting that CYP and P-gp may both contribute to the detoxification of TP in the SCRH model. In addition, hepatotoxicity and the expression of apoptosis proteins Bax and Bcl-2 were correlated qualitatively with the TP exposure duration and its intracellular concentration, which, in turn, can be modulated by P-gp inhibitors or inducers. Our results for the first time demonstrated that in addition to CYP-mediated metabolism, P-gp also plays an important role in the disposition of TP and TP-induced hepatotoxicity. Thus, the modulation of canalicular P-gp has a potential to cause drug-drug interaction between TP and the coadministered P-gp inhibitors or inducers in the clinic.

Oral follicular lymphomas. A short report of 8 cases with assessment of the IGH/BCL2 gene fusion with fluorescence in situ hybridization.

OBJECTIVE: To present the clinicopathologic features and confirm the presence of the IGH/BCL2 gene fusion in an oral follicular lymphoma (OFL) series. STUDY DESIGN: Cases of OFLs were retrieved from a data base of non-Hodgkin lymphomas (NHL). Fluorescence in situ hybridization (FISH) was performed to confirm the IGH/BCL2 fusion. RESULTS: Eight (8.7%) of 92 NHL were OFLs. Six (75%) patients were male and two female (mean age: 73.4 ± 14.8). The most frequent site was the palate. Five of the 8 patients are alive and without disease. Five (three grade 1 and two grade 2) of six successfully hybridized cases revealed the IGH/BCL2 gene fusion. The sixth case, a grade 3 follicular lymphoma (FL), demonstrated multiple BCL2 signals without IGH/BCL2 fusion. CONCLUSIONS: OFLs exhibit an indolent clinical behavior. In the present study, 5/6 cases in which FISH was successful had an IGH/BCL2 fusion as would result from the t(14; 18)(q32; q21) translocation commonly seen in FL of extraoral sites.

Clinical, dermoscopic and immunohistochemical assessment of actinic keratoses and evaluation of the effectiveness of diclofenac therapy with immunohistochemical analysis.

Actinic keratoses (AKs) is a keratinocytic neoplasm that typically develops on the face of elderly patients. Little is known regarding the clinical, dermatoscopic and immunohistochemical assessments of AK using topical diclofenac therapy. We sought to determine these assessments and evaluate the efficacy of topical diclofenac gel in AK. In this prospective, open-label study, 44 patients with 66 AKs were treated for 12 weeks with topically applied diclofenac (3% gel in 2.5% hyaluronic acid). Immunohistopathologic analyses were performed before and after diclofenac treatment using epidermal stem cell markers such as Cytokeratin 15 (CK15), Cytokeratin 19 (CK19) and p63, in addition to proliferation markers (Bcl-2, Ki-67). Diclofenac gel was found to be effective in AK, including the hyperkeratotic type. Surprisingly, complete remission was observed at a significantly higher rate in Grade 3 lesions (p = 0.017). However, imunohistochemical and histopathologic examinations revealed that 12-week treatment periods may not be sufficient to fully cure AK. The immunohistochemical analyses revealed no change in the expression levels of CK15, CK19 and Bcl-2 following diclofenac therapy. However, the expression of Ki-67 (p = 0.042) and p63 (p = 0.030) exhibited a significant decrease after therapy. Dermatoscopy is an effective method for diagnosis of AK, and topical diclofenac sodium gel was found as an effective additional treatment modality. Since positive histopathological findings were detected in some patients even with significant remission, a 12-week treatment period should be extended even in patients presenting with positive clinical response. Importantly, anti-proliferative effects of diclofenac were demonstrated by decreased Ki-67 and p63 expression levels.