Targeting the Melanocortin 1 Receptor in Melanoma: Biological Activity of α-MSH-Peptide Conjugates.

Malignant melanoma is one of the most aggressive and resistant tumor types, with high metastatic properties. Because of the lack of suitable chemotherapeutic agents for treatment, the 5-year survival rate of melanoma patients with regional and distant metastases is lower than 10%. Targeted tumor therapy that provides several promising results might be a good option for the treatment of malignant melanomas. Our goal was to develop novel melanoma-specific peptide-drug conjugates for targeted tumor therapy. Melanocortin-1-receptor (MC1R) is a cell surface receptor responsible for melanogenesis and it is overexpressed on the surface of melanoma cells, providing a good target. Its native ligand, α-MSH (α-melanocyte-stimulating hormone) peptide, or its derivatives, might be potential homing devices for this purpose. Therefore, we prepared three α-MSH derivative-daunomycin (Dau) conjugates and their in vitro and in vivo antitumor activities were compared. Dau has an autofluorescence property; therefore, it is suitable for preparing conjugates for in vitro (e.g., cellular uptake) and in vivo experiments. Dau was attached to the peptides via a non-cleavable oxime linkage that was applied efficiently in our previous experiments, resulting in conjugates with high tumor growth inhibition activity. The results indicated that the most promising conjugate was the compound in which Dau was connected to the side chain of Lys (Ac-SYSNleEHFRWGK(Dau=Aoa)PV-NH2). The highest cellular uptake by melanoma cells was demonstrated using the compound, with the highest tumor growth inhibition detected both on mouse (38.6% on B16) and human uveal melanoma (55% on OMC-1) cells. The effect of the compound was more pronounced than that of the free drug.

Shikonin Causes an Apoptotic Effect on Human Kidney Cancer Cells through Ras/MAPK and PI3K/AKT Pathways.

(1) Background: Shikonin, the main ingredient in Chinese herbal medicine, is described as a novel angiogenesis inhibitor, and its anticancer effects have already been studied. Shikonin and its derivatives induce apoptosis and suppress metastasis, which further enhance the effectiveness of chemotherapy. However, their mechanism of function has not been completely elucidated on human renal cancer cells. (2) Methods: In our study, CAKI-2 and A-498 cells were treated with increasing concentrations (2.5-40 µM) of shikonin, when colony formation ability and cytotoxic activity were tested. The changes in the expression of the main targets of apoptotic pathways were measured by RT-qPCR and Western blot. The intracellular levels of miR-21 and miR-155 were quantified by RT-qPCR. (3) Results: Shikonin exerted a dose-dependent effect on the proliferation of the cell lines examined. In 5 µM concentration of shikonin in vitro elevated caspase-3 and -7 levels, the proteins of the Ras/MAPK and PI3K/AKT pathways were activated. However, no significant changes were detected in the miR-21 and miR-155 expressions. (4) Conclusions: Our findings indicated that shikonin causes apoptosis of renal cancer cells by activating the Ras/MAPK and PI3K/AKT pathways. These effects of shikonin on renal cancer cells may bear important potential therapeutic implications for the treatment of renal cancer.

Development and Biochemical Characterization of Self-Immolative Linker Containing GnRH-III-Drug Conjugates.

The human gonadotropin releasing hormone (GnRH-I) and its sea lamprey analogue GnRH-III specifically bind to GnRH receptors on cancer cells and can be used as targeting moieties for targeted tumor therapy. Considering that the selective release of drugs in cancer cells is of high relevance, we were encouraged to develop cleavable, self-immolative GnRH-III-drug conjugates which consist of a p-aminobenzyloxycarbonlyl (PABC) spacer between a cathepsin B-cleavable dipeptide (Val-Ala, Val-Cit) and the classical anticancer drugs daunorubicin (Dau) and paclitaxel (PTX). Alongside these compounds, non-cleavable GnRH-III-drug conjugates were also synthesized, and all compounds were analyzed for their antiproliferative activity. The cleavable GnRH-III bioconjugates revealed a growth inhibitory effect on GnRH receptor-expressing A2780 ovarian cancer cells, while their activity was reduced on Panc-1 pancreatic cancer cells exhibiting a lower GnRH receptor level. Moreover, the antiproliferative activity of the non-cleavable counterparts was strongly reduced. Additionally, the efficient cleavage of the Val-Ala linker and the subsequent release of the drugs could be verified by lysosomal degradation studies, while radioligand binding studies ensured that the GnRH-III-drug conjugates bound to the GnRH receptor with high affinity. Our results underline the high value of GnRH-III-based homing devices and the application of cathepsin B-cleavable linker systems for the development of small molecule drug conjugates (SMDCs).

Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variants in Primary Human Endometrial Carcinomas: Novel Therapeutic Approaches.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various tumors, including endometrial carcinomas (EC). However, tumoral receptors that mediate the antiproliferative effects of GHRH antagonists in human ECs have not been fully characterized. In this study, we investigated the expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors (GHRH-R) in 39 human ECs and in 7 normal endometrial tissue samples using RT-PCR. Primers designed for the PCR amplification of mRNA for the full length GHRH-R and SVs were utilized. The PCR products were sequenced, and their specificity was confirmed. Nine ECs cancers (23%) expressed mRNA for SV1, three (7.7%) showed SV2 and eight (20.5%) revealed mRNA for SV4. The presence of SVs for GHRH-Rs could not be detected in any of the normal endometrial tissue specimens. The presence of specific, high affinity GHRH-Rs was also demonstrated in EC specimens using radioligand binding studies. Twenty-four of the investigated thirty-nine tumor samples (61.5%) and three of the seven corresponding normal endometrial tissues (42.9%) expressed mRNA for GHRH ligand. Our findings suggest the possible existence of an autocrine loop in EC based on GHRH and its tumoral SV receptors. The antiproliferative effects of GHRH antagonists on EC are likely to be exerted in part by the local SVs and GHRH system.

Expression of Somatostatin Receptor Subtypes (SSTR-1-SSTR-5) in Pediatric Hematological and Oncological Disorders.

Hematological and oncological disorders represent leading causes of childhood mortality. Neuropeptide somatostatin (SST) has been previously demonstrated in various pediatric tumors, but limited information exists on the expression and characteristics of SST receptors (SSTR) in hematological and oncological disorders of children. We aimed to investigate the expression of mRNA for SSTR subtypes (SSTR-1-5) in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of SSTRs were further studies by ligand competition assay. Our results show that the pediatric tumor samples highly expressed mRNA for the five SSTR subtypes with various patterns. The mRNA for SSTR-2 was detected in all specimens independently of their histological type. A Hodgkin lymphoma sample co-expressed mRNA for all five SSTR subtypes. SSTR-3 and SSTR-5 were detected only in malignant specimens, such as rhabdomyosarcoma, Hodgkin lymphoma, acute lymphoblastic leukemia, and a single nonmalignant condition, hereditary spherocytosis. The incidence of SSTR-1 and SSTR-4 was similar (60%) in the 15 specimens investigated. Radioligand binding studies demonstrated the presence of specific SSTRs and high affinity binding of SST analogs in pediatric solid tumors investigated. The high incidence of SSTRs in hematological and oncological disorders in children supports the merit of further investigation of SSTRs as molecular targets for diagnosis and therapy.

Evaluation of the mechanical properties of the anterior lens capsule following femtosecond laser capsulotomy at different pulse energy settings.

PURPOSE: To evaluate and compare the mechanical properties of anterior capsule opening performed with femtosecond laser capsulotomy at different energy settings in ex vivo porcine anterior lens capsule specimens. METHODS: Twenty-five fresh porcine eyes per group were included in the study. Femtosecond laser capsulotomy was performed with three different pulse energy levels: 2 µJ (low energy group), 5 µJ (intermediate energy group), and 10 µJ (high energy group). The capsule openings were stretched with universal testing equipment until they ruptured. The morphologic profile of the cut capsule edges was evaluated using scanning electron microscopy. RESULTS: The high energy group had significantly lower rupture force (108 ± 14 mN) compared to the intermediate energy group (118 ± 10 mN) (P < .05) and low energy group (119 ± 11 mN) (P < .05), but the difference between the intermediate energy and low energy groups was not significant (P = .9479). The high energy group had significantly lower circumference stretching ratio (144% ± 3%) compared to the intermediate energy group (148% ± 3%) (P < .05) and low energy group (148% ± 3%) (P < .05), but the difference between the intermediate energy group and low energy group was not significant (P = .9985). Scanning electron microscopy images showed that the edge was only serrated with low and intermediate energy, but additional signs of collagen melting and denaturation were observed at high energy. CONCLUSIONS: Anterior capsule openings created at a high energy level were slightly weaker and less extensible than those created at low or intermediate levels, possibly due to the increased thermal effect of photo-disruption.

Analysis of planned and postoperatively measured flap thickness after LASIK using the LenSx multifunctional femtosecond laser system.

PURPOSE: To evaluate LASIK corneal flaps using a multifunctional femtosecond laser suitable for cataract and corneal surgery (LenSx; Alcon Laboratories, Inc., Aliso Viejo, CA) and to compare the planned and postoperatively measured flap thickness using an anterior segment optical coherence tomography device (AS-OCT). METHODS: Twenty patients (38 eyes) diagnosed as having myopia and myopic astigmatism were enrolled. LASlK was performed using the LenSx femtosecond laser for intracorneal flaps and the Wavelight Allegretto 400 excimer laser (Alcon Laboratories, Inc.) for intra- stromal photoablation. Desired flap thickness and diameter were 140.0 + 0.0 pm and 8.5 + 0.0 mm, respectively, whereas mean ablation depth and diameter of the excimer laser treatment were 67.9 ± 24.18 pm and 6.5 ± 0.08 mm, respectively. Entered data of the LenSx femtosecond laser were used to determine desired flap thickness, whereas AS-OCT (RTVue; Optovue, Inc., Fremont, CA) was used to measure flap thickness postoperatively. The Wilcoxon signed-rank test, dependent paired t test, and Friedman test were used for comparison of dependent and repeated measures. RESULTS: There was no statistically significant difference in the planned and postoperatively measured flap thickness (140.0 ± 0.0 vs 140.28 _ 8.0 pm; P = .4067). Interfaces of the flaps had even surfaces according to the images and calculations on the AS-OCT device (P = .058). CONCLUSIONS: Application of this multifunctional femtosecond laser performing LASIK proved to be a safe and effective method regarding predictability of flap thickness.

Changes of corneal topography indices after CXL in progressive keratoconus assessed by Scheimpflug camera.

PURPOSE: To evaluate the accuracy of Scheimpflug camera topography indices in detecting the therapeutic effect of corneal collagen cross-linking (CXL) on progressive keratoconus in the long term. METHODS: Fifty eyes of 25 patients with keratoconus were enrolled. CXL was performed in 25 eyes with progressive keratoconus (CXL group) and 25 fellow eyes with nonprogressive keratoconus served as controls. Thinnest corneal thickness, anterior keratometry (flat, steep), and keratoconus indices were measured with Scheimpflug camera before and 12 to 25 months after CXL. Regression analysis was used to evaluate the influence of corneal thickness and follow-up time on flattening effect of CXL. RESULTS: At baseline, steep keratometric values were significantly higher and thinnest corneal thickness values were lower in the CXL group (P = .027, .034), parallel with increased values of keratoconus indices: index of surface variance (P = .013), index of vertical asymmetry (P = .038), keratoconus index (P = .019), center keratoconus index (P = .039), index of height asymmetry (P = .037), index of height decentration (P = .0016), and radius minimum (P = .008). After adjustment for thinnest corneal thickness and follow-up time, CXL showed significant flattening effect expressed by changes in radius minimum (P < .001), index of surface variance (P = .03), keratoconus index (P = .006), center keratoconus index (P = .03), and index of height asymmetry (P = .026). Thinnest corneal thickness had significant influence on changes of index of surface variance (P = .049), index of vertical asymmetry (P = .01), and center keratoconus index (P = .03). Follow-up time showed no significant influence in any models (P > .05). CONCLUSIONS: Topographic indices indicate corneal flattening after CXL in the long term. Monitoring keratoconus index and index of height asymmetry should be the preferred choice in daily clinical practice because changes in values of these indices are independent from initial corneal thickness.

Comparison of the mechanical properties of the anterior lens capsule following manual capsulorhexis and femtosecond laser capsulotomy.

PURPOSE: To evaluate and compare the mechanical properties of anterior capsule openings performed with the continuous curvilinear capsulorhexis (CCC) technique and femtosecond laser capsulotomy (FLC) in ex vivo porcine lens capsule specimens. METHODS: Fresh porcine eyes were included in the study (CCC group, n = 50; FLC group, n = 30). The capsule openings were stretched with universal testing equipment until they ruptured. The rupture force and circumference stretching ratio were evaluated. The morphologic profile of the cut capsule edges was evaluated using scanning electron microscopy (SEM). RESULTS: The average rupture force was higher in the CCC group (median: 155 mN; interquartile range [IQR]: 129 to 201 mN; range: 71 to 294 mN) than in the FLC group (median: 119 mN; IQR: 108 to 128 mN; range: 91 to 142 mN) (P < .01, Mann-Whitney U test). The average circumference stretching ratio in the CCC group was greater (median: 150%; IQR: 146% to 156%; range: 136% to 161%) than in the FLC group (median: 148%; IQR: 145% to 150%; range: 141% to 154%) (P = .0468, Mann-Whitney U test). When less than 71 mN, no capsular tear occurred in either group. When less than 91 mN, no capsular tear occurred in the FLC group, whereas at 91 mN, the probability of capsular tears was 9% for the CCC group. SEM examination found that the CCC group had smooth edges, whereas those of the FLC group were gently serrated. CONCLUSIONS: According to the current results in a porcine eye model, FLC had less average resistance to capsule tear than CCC, but the weakest openings were seen in the CCC group.

Cell migration or cytokinesis and proliferation?--revisiting the "go or grow" hypothesis in cancer cells in vitro.

The mortality of patients with solid tumors is mostly due to metastasis that relies on the interplay between migration and proliferation. The "go or grow" hypothesis postulates that migration and proliferation spatiotemporally excludes each other. We evaluated this hypothesis on 35 cell lines (12 mesothelioma, 13 melanoma and 10 lung cancer) on both the individual cell and population levels. Following three-day-long videomicroscopy, migration, proliferation and cytokinesis-length were quantified. We found a significantly higher migration in mesothelioma cells compared to melanoma and lung cancer while tumor types did not differ in mean proliferation or duration of cytokinesis. Strikingly, we found in melanoma and lung cancer a significant positive correlation between mean proliferation and migration. Furthermore, non-dividing melanoma and lung cancer cells displayed slower migration. In contrast, in mesothelioma there were no such correlations. Interestingly, negative correlation was found between cytokinesis-length and migration in melanoma. FAK activation was higher in melanoma cells with high motility. We demonstrate that the cancer cells studied do not defer proliferation for migration. Of note, tumor cells from various organ systems may differently regulate migration and proliferation. Furthermore, our data is in line with the observation of pathologists that highly proliferative tumors are often highly invasive.

Selective use of radiological staging in node-positive stage II breast cancer patients may have a role in treatment.

BACKGROUND: Advanced-staging radiography is used inconsistently for patients with early-stage (stage I + II) breast cancer. However, accurate and appropriate staging of newly diagnosed breast cancer may significantly impact treatment decisions. METHODS: Four hundred and ninety-nine patients with stages II and III breast cancer were seen in the breast service at Waitemata DHB, New Zealand from 2013 to 2018 were enrolled in the study and audited for radiological staging. RESULTS: One hundred and two stage II patients had computed tomography (CT) at baseline; 88 of 102 (86%) of stage II patients were node-positive (≥N1) with six patients (6.8%) having distant metastatic disease, and were upstaged to stage IV. Fifty-two stage III patients out of 72 (72%) had baseline staging CTs. Nine out of 52 patients (17%) of stage III patients were upstaged to stage IV. Despite guideline recommendations, baseline staging for T4 disease (stage IIIB) was poor, with only 7 out of 13 patients with stage IIIB disease radiologically staged. CONCLUSION: Consideration for baseline radiological staging should be given to stages II and III, cN1 breast cancer patients, in which diagnosis of distant metastatic disease would change the treatment plan. Regional guidelines for baseline radiological staging for breast cancer patients may have an impact on patient management in breast cancer patients.

Signaling mechanism of growth hormone-releasing hormone receptor.

The hypothalamic peptide growth hormone-releasing hormone (GHRH) stimulates the secretion of growth hormone (GH) from the pituitary through binding and activation of the pituitary type of GHRH receptor (GHRH-R), which belongs to the family of G protein-coupled receptors with seven potential membrane-spanning domains. Splice variants of GHRH-Rs (SV) in human tumors and other extra pituitary tissues were identified and their cDNA was sequenced. Among the SVs, splice variant 1 (SV1) possesses the greatest similarity to the full-length GHRH-R and remains functional by eliciting cAMP signaling and mitogenic activity upon GHRH stimulation. A large body of work have evaluated potential clinical applications of agonists and antagonists of GHRH in diverse fields, including endocrinology, oncology, cardiology, diabetes, obesity, metabolic dysfunctions, Alzheimer's disease, ophthalmology, wound healing and other applications. In this chapter, we briefly review the expression and potential function of GHRH-Rs and their SVs in various tissues and also elucidate and summarize the activation, molecular mechanism and signalization pathways of these receptors. Therapeutic applications of GHRH analogs are also discussed.

Potential Role of VHL, PTEN, and BAP1 Mutations in Renal Tumors.

The genetic profiling of renal tumors has revealed genomic regions commonly affected by structural changes and a general genetic heterogeneity. The VHL, PTEN, and BAP1 genes are often mutated in renal tumors. The frequency and clinical relevance of these mutations in renal tumors are still being researched. In our study, we investigated VHL, PTEN, and BAP1 genes and the sequencing of 24 samples of patients with renal tumors, revealing that VHL was mutated at a noticeable frequency (25%). Six of the investigated samples showed mutations, and one genetic polymorphism (rs779805) was detected in both heterozygote and homozygote forms. PTEN gene mutation was observed in only one sample, and one specimen showed genetic polymorphism. In the case of the BAP1 gene, all of the samples were wild types. Interestingly, VHL mutation was detected in two female patients diagnosed with AML and in one with oncocytoma. We assume that VHL or PTEN mutations may contribute to the development of human renal cancer. However, the overall mutation rate was low in all specimens investigated, and the development and prognosis of the disease were not exclusively associated with these types of genetic alterations.

Assessment of Visual Quality Improvement as a Result of Spectacle Personalization.

Personalized spectacles customized according to an individual's facial anatomy were developed to provide enhanced visual performance and overall comfort when compared to standard spectacles. In this comparative crossover trial, each subject was randomly assigned to wear either personalized spectacles or standard spectacles for two weeks and then tried the second pair for another two weeks. Visual acuity and reading speed were measured, and visual quality and comfort were assessed using specific questionnaires. The correlation of the wearing parameters with the subjects' satisfaction was calculated. According to our results, the subjects wearing personalized glasses reported significantly less experience of swaying and significantly higher overall satisfaction compared to those wearing the control spectacles. At the end of the study, 62% of subjects preferred the personalized spectacles, and visual quality was the primary reason for their spectacle preference followed by wearing comfort. The difference from the ideal cornea-vertex distance was significantly lower when wearing the personalized spectacles compared to the control frames. In addition, the absolute value of the difference from the ideal cornea-vertex distance was significantly correlated with patient satisfaction. These results suggest that personalized spectacles, customized according to an individual's facial anatomy for the ideal wearing parameters, result in both visual and comfort advantages for wearers.

[Medical aspects of the open access initiative]. / A nyílt hozzáférés kezdeményezésének orvostudományi vonatkozásai.

The main condition of successful medical attendance and development of medical research is the use of scientific literature. The aim of our article is to briefly introduce the medical open access publishers and to call attention to the publishing possibilities. Hereby we want to incite Hungarian doctors to publish in these types of journals. Our internet research shows that since the millennium the number of open access publications has dynamically increased. A very promising fact is that most of the medical open access journals have high impact factor that guarantee high professional level. We hope that by the effect of our research more Hungarian doctors will publish in this way.

[Content informatics and professional analysis of Hungarian health-related websites]. / Magyar egészségügyi honlapok informatikai es szakmai elemzése.

INTRODUCTION: The number of health-related homepages is increasing and their content is exceeding. The visitor, let him/her be a private visitor or an expert, a patient or relative would like to access relevant data, trust the accuracy and up-to-date state of the web content. It is in the nature of these kind of services, that visitors would share their question and remarks with the authors specialized in a particular topic. Among others these circumstances led to the format and content-related regulation of websites. OBJECTIVE: In a nationwide research the authors examined, to what extent the Hungarian sites meet the requirements of the Health on Net codification and the criteria of the European Union Committee. METHOD: By studying the quality criteria-related websites, the authors examined each Hungarian website to see how the Hungarian pages live up to the regulations. The work concentrates on the content, but it does not neglect functional analysis either. CONCLUSION: The authors conclude, that in spite of shortcomings, home webpages aim to keep the directives of the European Union.

Accuracy of machine learning classifiers using bilateral data from a Scheimpflug camera for identifying eyes with preclinical signs of keratoconus.

PURPOSE: To describe the topographic and tomographic characteristics of normal fellow eyes of unilateral keratoconus cases and to evaluate the accuracy of machine learning classifiers in discriminating healthy corneas from the normal fellow corneas. SETTING: Department of Ophthalmology, Semmelweis University, Budapest, Hungary. DESIGN: Retrospective case-control study. METHODS: Patients with bilateral keratoconus (keratoconus group), clinically and according to the keratoconus indices of the Pentacam HR Scheimpflug camera; normal fellow eyes of patients with unilateral keratoconus (fellow-eye group); and eyes of refractive surgery candidates (control group) were compared. Tomographic data, topographic data, and keratoconus indices were measured in both eyes using the Scheimpflug camera. Receiver operating characteristic (ROC) analysis was used to assess the performance of automated classifiers trained on bilateral data as well as individual parameters to discriminate fellow eyes of patients with keratoconus from control eyes. RESULTS: Keratometry, elevation, and keratoconus indices values were significantly higher and pachymetry values were significantly lower in keratoconus eyes than in fellow eyes of unilateral keratoconus cases (P < .001). These fellow eyes had significantly higher keratometry, elevation, and keratoconus index values and significantly lower pachymetry values than control eyes (P < .001). Automated classifiers trained on bilateral data of index of height decentration had higher accuracy than the unilateral single parameter in discriminating fellow eyes of patients with keratoconus from control eyes (area under ROC 0.96 versus 0.88). CONCLUSION: Automatic classifiers trained on bilateral data were better than single parameters in discriminating fellow eyes of patients with unilateral keratoconus with preclinical signs of keratoconus from normal eyes. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Sensitivity of Melanoma Cells to EGFR and FGFR Activation but Not Inhibition is Influenced by Oncogenic BRAF and NRAS Mutations.

BRAF and NRAS are the two most frequent oncogenic driver mutations in melanoma and are pivotal components of both the EGF and FGF signaling network. Accordingly, we investigated the effect of BRAF and NRAS oncogenic mutation on the response to the stimulation and inhibition of epidermal and fibroblast growth factor receptors in melanoma cells. In the three BRAF mutant, two NRAS mutant and two double wild-type cell lines growth factor receptor expression had been verified by qRT-PCR. Cell proliferation and migration were determined by the analysis of 3-days-long time-lapse videomicroscopic recordings. Of note, a more profound response was found in motility as compared to proliferation and double wild-type cells displayed a higher sensitivity to EGF and FGF2 treatment when compared to mutant cells. Both baseline and induced activation of the growth factor signaling was assessed by immunoblot analysis of the phosphorylation of the downstream effectors Erk1/2. Low baseline and higher inducibility of the signaling pathway was characteristic in double wild-type cells. In contrast, oncogenic BRAF or NRAS mutation did not influence the response to EGF or FGF receptor inhibitors in vitro. Our findings demonstrate that the oncogenic mutations in melanoma have a profound impact on the motogenic effect of the activation of growth factor receptor signaling. Since emerging molecularly targeted therapies aim at the growth factor receptor signaling, the appropriate mutational analysis of individual melanoma cases is essential in both preclinical studies and in the clinical trials and practice.