Multifaceted approach toward mapping out the anticancer properties of small molecules via in vitro evaluation on melanoma and nonmelanoma skin cancer cells, and in silico target fishing.

Melanoma and nonmelanoma skin cancers are among the most prevalent and most lethal forms of skin cancers. To identify new lead compounds with potential anticancer properties for further optimization, in vitro assays combined with in-silico target fishing and docking have been used to identify and further map out the antiproliferative and potential mode of action of molecules from a small library of compounds previously prepared in our laboratory. From screening these compounds in vitro against A375, SK-MEL-28, A431, and SCC-12 skin cancer cell lines, 35 displayed antiproliferative activities at the micromolar level, with the majority being primarily potent against the A431 and SCC-12 squamous carcinoma cell lines. The most active compounds 11 (A431: IC50 = 5.0 µM, SCC-12: IC50 = 2.9 µM, SKMEL-28: IC50 = 4.9 µM, A375: IC50 = 6.7 µM) and 13 (A431: IC50 = 5.0 µM, SCC-12: IC50 = 3.3 µM, SKMEL-28: IC50 = 13.8 µM, A375: IC50 = 17.1 µM), significantly and dose-dependently induced apoptosis of SCC-12 and SK-MEL-28 cells, as evidenced by the suppression of Bcl-2 and upregulation of Bax, cleaved caspase-3, caspase-9, and PARP protein expression levels. Both agents significantly reduced scratch wound healing, colony formation, and expression levels of deregulated cancer molecular targets including RSK/Akt/ERK1/2 and S6K1. In silico target prediction and docking studies using the SwissTargetPrediction web-based tool suggested that CDK8, CLK4, nuclear receptor ROR, tyrosine protein-kinase Fyn/LCK, ROCK1/2, and PARP, all of which are dysregulated in skin cancers, might be prospective targets for the two most active compounds. Further validation of these targets by western blot analyses, revealed that ROCK/Fyn and its associated Hedgehog (Hh) pathways were downregulated or modulated by the two lead compounds. In aggregate, these results provide a strong framework for further validation of the observed activities and the development of a more comprehensive structure-activity relationship through the preparation and biological evaluation of analogs.

Anthracycline cardiotoxicity is exacerbated by global p38ß genetic ablation in a sexually dimorphic manner but unaltered by cardiomyocyte-specific p38α loss.

Severe cardiotoxic effects limit the efficacy of doxorubicin (DOX) as a chemotherapeutic agent. Activation of intracellular stress signaling networks, including p38 mitogen-activated protein kinase (MAPK), has been implicated in DOX-induced cardiotoxicity (DIC). However, the roles of the individual p38 isoforms in DIC remain incompletely elucidated. We recently reported that global p38δ deletion protected female but not male mice from DIC, whereas global p38γ deletion did not significantly modulate it. Here we studied the in vivo roles of p38α and p38ß in acute DIC. Male and female mice with cardiomyocyte-specific deletion of p38α or global deletion of p38ß and their wild-type counterparts were injected with DOX. Survival and health were tracked for 10 days postinjection. Cardiac function was assessed by echocardiography and electrocardiography and fibrosis by Picrosirius red staining. Expression and activation of signaling proteins and inflammatory markers were measured by Western blot, phosphorylation array, and chemokine/cytokine array. Global p38ß deletion significantly aggravated DIC and worsened cardiac electrical and mechanical function deterioration in female mice. Mechanistically, DIC in p38ß-null female mice correlated with increased autophagy, sustained hyperactivation of proapoptotic JNK signaling, as well as remodeling of a myocardial inflammatory environment. In contrast, cardiomyocyte-specific deletion of p38α improved survival of DOX30-treated male mice 5 days posttreatment but did not influence cardiac function in DOX-treated male or female mice. Our data highlight the sex- and isoform-specific roles of p38α and p38ß MAPKs in DOX-induced cardiac injury and suggest a novel in vivo function of p38ß in protecting female mice from DIC.NEW & NOTEWORTHY We show that p38α and p38ß have distinct in vivo functions in a murine model of acute DIC. Specifically, although conditional cardiomyocyte-specific p38α deletion exhibited mild cardioprotective effects in male mice, p38ß deletion exacerbated the DOX cardiotoxicity in female mice. Our findings caution against employing pyridinyl imidazole inhibitors that target both p38α and p38ß isoforms as a cardioprotective strategy against DIC. Such an approach could have undesirable sex-dependent effects, including attenuating p38ß-dependent cardioprotection in females.

Synthesis, in silico modelling, and in vitro biological evaluation of substituted pyrazole derivatives as potential anti-skin cancer, anti-tyrosinase, and antioxidant agents.

Twenty-five azole compounds (P1-P25) were synthesised using regioselective base-metal catalysed and microwave-assisted approaches, fully characterised by high-resolution mass spectrometry (HRMS), nuclear magnetic resonance (NMR), and infrared spectra (IR) analyses, and evaluated for anticancer, anti-tyrosinase, and anti-oxidant activities in silico and in vitro. P25 exhibited potent anticancer activity against cells of four skin cancer (SC) lines, with selectivity for melanoma (A375, SK-Mel-28) or non-melanoma (A431, SCC-12) SC cells over non-cancerous HaCaT-keratinocytes. Clonogenic, scratch-wound, and immunoblotting assay data were consistent with anti-proliferative results, expression profiling therewith implicating intrinsic and extrinsic apoptosis activation. In a mushroom tyrosinase inhibition assay, P14 was most potent among the compounds (half-maximal inhibitory concentration where 50% of cells are dead, IC50 15.9 µM), with activity greater than arbutin and kojic acid. Also, P6 exhibited noteworthy free radical-scavenging activity. Furthermore, in silico docking and absorption, distribution, metabolism, excretion, and toxicity (ADMET) simulations predicted prominent-phenotypic actives to engage diverse cancer/hyperpigmentation-related targets with relatively high affinities. Altogether, promising early-stage hits were identified - some with multiple activities - warranting further hit-to-lead optimisation chemistry with further biological evaluations, towards identifying new skin-cancer and skin-pigmentation renormalising agents.

High Prevalence of SARS-CoV-2 Omicron Infection Despite High Seroprevalence, Sweden, 2022.

We performed 2 surveys during 2022 to estimate point prevalences of SARS-CoV-2 infection compared with overall seroprevalence in Sweden. Point prevalence was 1.4% in March and 1.5% in September. Estimated seroprevalence was >80%, including among unvaccinated children. Continued SARS-CoV-2 surveillance is necessary for detecting emerging, possibly more pathogenic variants.

Evidence of sex differences in cancer-related cardiac complications in mouse models of pancreatic and liver cancer.

Abnormal heart rate variability (HRV) is commonly observed in cancer patients who have undergone targeted therapy and/or surgery, yet the effects of cancer itself on cardiac function remain underexplored. Specifically, there is limited knowledge about sex-specific manifestations of HRV in cancer patients. Transgenic mouse models are widely used to study different types of cancer. Here, we aimed to investigate the sex-specific effects of cancer on cardiac function using transgenic mouse models of pancreatic and liver cancers. This study used male and female transgenic mice with cancer and wild-type controls. Cardiac function was assessed by recording electrocardiograms in conscious mice. RR intervals were detected to determine HRV using time and frequency domain analyses. Histological analysis with Masson's trichrome staining was performed to determine structural changes. In females, increased HRV was observed in both pancreatic and liver cancer-bearing mice. In contrast, in males, increased HRV was observed only in the liver cancer group. Male pancreatic cancer mice demonstrated autonomic balance shift showing an increase in parasympathetic to sympathetic tone. The heart rate (HR) was higher in control and liver cancer male mice groups than in females. Histological analysis did not show significant sex differences but suggested a higher degree of remodeling in liver cancer mice than in control, specifically in the right atrium and left ventricle. This study revealed sex differences in cancer's HR modulation. Specifically, female cancer mice had lower median HR and higher HRV. These findings indicate that sex must be considered when using HRV as a cancer biomarker.

Open thoracic surgical implantation of cardiac pacemakers in rats.

Genetic engineering and implantable bioelectronics have transformed investigations of cardiovascular physiology and disease. However, the two approaches have been difficult to combine in the same species: genetic engineering is applied primarily in rodents, and implantable devices generally require larger animal models. We recently developed several miniature cardiac bioelectronic devices suitable for mice and rats to enable the advantages of molecular tools and implantable devices to be combined. Successful implementation of these device-enabled studies requires microsurgery approaches that reliably interface bioelectronics to the beating heart with minimal disruption to native physiology. Here we describe how to perform an open thoracic surgical technique for epicardial implantation of wireless cardiac pacemakers in adult rats that has lower mortality than transvenous implantation approaches. In addition, we provide the methodology for a full biocompatibility assessment of the physiological response to the implanted device. The surgical implantation procedure takes ~40 min for operators experienced in microsurgery to complete, and six to eight surgeries can be completed in 1 d. Implanted pacemakers provide programmed electrical stimulation for over 1 month. This protocol has broad applications to harness implantable bioelectronics to enable fully conscious in vivo studies of cardiovascular physiology in transgenic rodent disease models.

Investigating Cutaneous Squamous Cell Carcinoma in vitro and in vivo: Novel 3D Tools and Animal Models.

Cutaneous Squamous Cell Carcinoma (cSCC) represents the second most common type of skin cancer, which incidence is continuously increasing worldwide. Given its high frequency, cSCC represents a major public health problem. Therefore, to provide the best patients' care, it is necessary having a detailed understanding of the molecular processes underlying cSCC development, progression, and invasion. Extensive efforts have been made in developing new models allowing to study the molecular pathogenesis of solid tumors, including cSCC tumors. Traditionally, in vitro studies were performed with cells grown in a two-dimensional context, which, however, does not represent the complexity of tumor in vivo. In the recent years, new in vitro models have been developed aiming to mimic the three-dimensionality (3D) of the tumor, allowing the evaluation of tumor cell-cell and tumor-microenvironment interaction in an in vivo-like setting. These models include spheroids, organotypic cultures, skin reconstructs and organoids. Although 3D models demonstrate high potential to enhance the overall knowledge in cancer research, they lack systemic components which may be solved only by using animal models. Zebrafish is emerging as an alternative xenotransplant model in cancer research, offering a high-throughput approach for drug screening and real-time in vivo imaging to study cell invasion. Moreover, several categories of mouse models were developed for pre-clinical purpose, including xeno- and syngeneic transplantation models, autochthonous models of chemically or UV-induced skin squamous carcinogenesis, and genetically engineered mouse models (GEMMs) of cSCC. These models have been instrumental in examining the molecular mechanisms of cSCC and drug response in an in vivo setting. The present review proposes an overview of in vitro, particularly 3D, and in vivo models and their application in cutaneous SCC research.

Differential cardiotoxic electrocardiographic response to doxorubicin treatment in conscious versus anesthetized mice.

INTRODUCTION: Doxorubicin (DOX), an anticancer drug used in chemotherapy, causes significant cardiotoxicity. This study aimed to investigate the effects of DOX on mouse cardiac electrophysiology, in conscious versus anesthetized state. METHODS: Male and female C57BL/6 mice were injected with saline, 20 or 30 mg/kg DOX. ECGs were recorded 5 days post-injection in conscious and isoflurane anesthetized states. ECGs were analyzed using a custom MATLAB software to determine P, PR, QRS, QTc, and RR intervals as well as heart rate variability (HRV). RESULTS: ECGs from the same mouse demonstrated P wave and QTc shortening as well as PR and RR interval prolongation in anesthetized versus conscious saline-treated mice. ECG response to DOX was also modulated by anesthesia. DOX treatment induced significant ECG modulation in female mice alone. While DOX20 treatment caused decrease in P and QRS durations, DOX30 treatment-induced QTc and RR interval prolongation in anesthetized but not in conscious female mice. These data suggest significant sex differences and anesthesia-induced differences in ECG response to DOX. HRV measured in time and frequency domains, a metric of arrhythmia susceptibility, was increased in DOX20-treated mice compared to saline. CONCLUSIONS: This study for the first time identifies that the ECG response to DOX is modulated by anesthesia. Furthermore, this response demonstrated stark sex differences. These findings could have significant implications in clinical diagnosis of DOX cardiotoxicity.

Fully implantable and bioresorbable cardiac pacemakers without leads or batteries.

Temporary cardiac pacemakers used in periods of need during surgical recovery involve percutaneous leads and externalized hardware that carry risks of infection, constrain patient mobility and may damage the heart during lead removal. Here we report a leadless, battery-free, fully implantable cardiac pacemaker for postoperative control of cardiac rate and rhythm that undergoes complete dissolution and clearance by natural biological processes after a defined operating timeframe. We show that these devices provide effective pacing of hearts of various sizes in mouse, rat, rabbit, canine and human cardiac models, with tailored geometries and operation timescales, powered by wireless energy transfer. This approach overcomes key disadvantages of traditional temporary pacing devices and may serve as the basis for the next generation of postoperative temporary pacing technology.

p38δ genetic ablation protects female mice from anthracycline cardiotoxicity.

The efficacy of an anthracycline antibiotic doxorubicin (DOX) as a chemotherapeutic agent is limited by dose-dependent cardiotoxicity. DOX is associated with activation of intracellular stress signaling pathways including p38 MAPKs. While previous studies have implicated p38 MAPK signaling in DOX-induced cardiac injury, the roles of the individual p38 isoforms, specifically, of the alternative isoforms p38γ and p38δ, remain uncharacterized. We aimed to determine the potential cardioprotective effects of p38γ and p38δ genetic deletion in mice subjected to acute DOX treatment. Male and female wild-type (WT), p38γ-/-, p38δ-/-, and p38γ-/-δ-/- mice were injected with 30 mg/kg DOX and their survival was tracked for 10 days. During this period, cardiac function was assessed by echocardiography and electrocardiography and fibrosis by Picro Sirius Red staining. Immunoblotting was performed to assess the expression of signaling proteins and markers linked to autophagy. Significantly improved survival was observed in p38δ-/- female mice post-DOX relative to WT females, but not in p38γ-/- or p38γ-/-δ-/- male or female mice. The improved survival in DOX-treated p38δ-/- females was associated with decreased fibrosis, increased cardiac output and LV diameter relative to DOX-treated WT females, and similar to saline-treated controls. Structural and echocardiographic parameters were either unchanged or worsened in all other groups. Increased autophagy, as suggested by increased LC3-II level, and decreased mammalian target of rapamycin activation was also observed in DOX-treated p38δ-/- females. p38δ plays a crucial role in promoting DOX-induced cardiotoxicity in female mice by inhibiting autophagy. Therefore, p38δ targeting could be a potential cardioprotective strategy in anthracycline chemotherapy.NEW & NOTEWORTHY This study for the first time identifies the sex-specific roles of the alternative p38γ and p38δ MAPK isoforms in promoting doxorubicin (DOX) cardiotoxicity. We show that p38δ and p38γ/δ systemic deletion was cardioprotective in female but not in male mice. Cardiac structure and function were preserved in DOX-treated p38δ-/- females and autophagy marker was increased.

Cell Type-Specific p38δ Targeting Reveals a Context-, Stage-, and Sex-Dependent Regulation of Skin Carcinogenesis.

Activation and/or upregulated expression of p38δ are demonstrated in human skin malignancies including cutaneous squamous cell carcinoma, suggesting a role for p38δ in skin carcinogenesis. We previously reported that mice with germline deletion of the p38δ gene are significantly protected from chemical skin carcinogenesis. Here, we investigated the effects of cell-selective targeted ablation of p38δ in keratinocytes and in immune (myeloid) cells on skin tumor development in a two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical mouse skin carcinogenesis model. Conditional keratinocyte-specific p38δ ablation (p38δ-cKO∆K) did not influence the latency, incidence, or multiplicity of chemically-induced skin tumors, but led to increased tumor volume in females during the TPA promotion stage, and reduced malignant progression in males and females relative to their wild-type counterparts. In contrast, conditional myeloid cell-specific p38δ deletion (p38δ-cKO∆M) inhibited DMBA/TPA-induced skin tumorigenesis in male but not female mice. Thus, tumor onset was delayed, and tumor incidence, multiplicity, and volume were reduced in p38δ-cKO∆M males compared with control wild-type males. Moreover, the percentage of male mice with malignant tumors was decreased in the p38δ-cKO∆M group relative to their wild-type counterparts. Collectively, these results reveal that cell-specific p38δ targeting modifies susceptibility to chemical skin carcinogenesis in a context-, stage-, and sex-specific manner.

Managing Seborrheic Keratosis: Evolving Strategies and Optimal Therapeutic Outcomes.

Seborrheic keratosis (SK) is the most common skin tumor seen by dermatologists in everyday practice. Although the lesions are mostly benign, many patients still elect to have asymptomatic SK removed. The historical standards of treatment are cryosurgery and electrocautery, two surgical options that are effective at lesion removal but have high rates of postoperative adverse events such as treatment-site scarring and pigmentary alterations. The cosmetic outcomes of SK treatment modalities are of keen interest to dermatologists, as the American population becomes increasingly more diverse. In this article, the inclusion of darker Fitzpatrick skin types into clinical studies investigating post-treatment side effects of SK therapy is reviewed. The recent approval of a 40% hydrogen peroxide topical formulation is discussed in light of these issues, and several non-invasive topical treatments that optimize cosmetic outcomes of SK lesion removal are highlighted. Finally, treatment strategies aimed at reducing cost and minimizing the burden of adverse sequelae are provided. J Drugs Dermatol. 2018;17(9):933-940.

Keratinocyte p38δ loss inhibits Ras-induced tumor formation, while systemic p38δ loss enhances skin inflammation in the early phase of chemical carcinogenesis in mouse skin.

p38δ expression and/or activity are increased in human cutaneous malignancies, including invasive squamous cell carcinoma (SCC) and head and neck SCC, but the role of p38δ in cutaneous carcinogenesis has not been well-defined. We have reported that mice with germline loss of p38δ exhibited a reduced susceptibility to skin tumor development compared with wild-type mice in the two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical skin carcinogenesis model. Here, we report that p38δ gene ablation inhibited the growth of tumors generated from v-ras(Ha) -transformed keratinocytes in skin orthografts to nude mice, indicating that keratinocyte-intrinsic p38δ is required for Ras-induced tumorigenesis. Gene expression profiling of v-ras(Ha) -transformed p38δ-null keratinocytes revealed transcriptional changes associated with cellular responses linked to tumor suppression, such as reduced proliferation and increased differentiation, cell adhesion, and cell communications. Notably, a short-term DMBA/TPA challenge, modeling the initial stages of chemical skin carcinogenesis treatment, elicited an enhanced inflammation in p38δ-null skin compared with skin of wild-type mice, as assessed by measuring the expression of pro-inflammatory cytokines, including IL-1ß, IL-6, IL-17, and TNFα. Additionally, p38δ-null skin and p38δ-null keratinocytes exhibited increased p38α activation and signaling in response to acute inflammatory challenges, suggesting a role for p38α in stimulating the elevated inflammatory response in p38δ-null skin during the initial phases of the DMBA/TPA treatment compared with similarly treated p38δ(+/+) skin. Altogether, our results indicate that p38δ signaling regulates skin carcinogenesis not only by keratinocyte cell-autonomous mechanisms, but also by influencing the interaction between between the epithelial compartment of the developing skin tumor and its stromal microenvironment.

Delayed skin wound repair in proline-rich protein tyrosine kinase 2 knockout mice.

Proline-rich protein tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family. We used Pyk2 knockout (Pyk2-KO) mice to study the role of Pyk2 in cutaneous wound repair. We report that the rate of wound closure was delayed in Pyk2-KO compared with control mice. To examine whether impaired wound healing of Pyk2-KO mice was caused by a keratinocyte cell-autonomous defect, the capacities of primary keratinocytes from Pyk2-KO and wild-type (WT) littermates to heal scratch wounds in vitro were compared. The rate of scratch wound repair was decreased in Pyk2-KO keratinocytes compared with WT cells. Moreover, cultured human epidermal keratinocytes overexpressing the dominant-negative mutant of Pyk2 failed to heal scratch wounds. Conversely, stimulation of Pyk2-dependent signaling via WT Pyk2 overexpression induced accelerated scratch wound closure and was associated with increased expression of matrix metalloproteinase (MMP)-1, MMP-9, and MMP-10. The Pyk2-stimulated increase in the rate of scratch wound repair was abolished by coexpression of the dominant-negative mutant of PKCδ and by GM-6001, a broad-spectrum inhibitor of MMP activity. These results suggest that Pyk2 is essential for skin wound reepithelialization in vivo and in vitro and that it regulates epidermal keratinocyte migration via a pathway that requires PKCδ and MMP functions.

Serum response factor controls transcriptional network regulating epidermal function and hair follicle morphogenesis.

Serum response factor (SRF) is a transcription factor that regulates the expression of growth-related immediate-early, cytoskeletal, and muscle-specific genes to control growth, differentiation, and cytoskeletal integrity in different cell types. To investigate the role for SRF in epidermal development and homeostasis, we conditionally knocked out SRF in epidermal keratinocytes. We report that SRF deletion disrupted epidermal barrier function leading to early postnatal lethality. Mice lacking SRF in epidermis displayed morphogenetic defects, including an eye-open-at-birth phenotype and lack of whiskers. SRF-null skin exhibited abnormal morphology, hyperplasia, aberrant expression of differentiation markers and transcriptional regulators, anomalous actin organization, enhanced inflammation, and retarded hair follicle (HF) development. Transcriptional profiling experiments uncovered profound molecular changes in SRF-null E17.5 epidermis and revealed that many previously identified SRF target CArG box-containing genes were markedly upregulated in SRF-null epidermis, indicating that SRF may function to repress transcription of a subset of its target genes in epidermis. Remarkably, when transplanted onto nude mice, engrafted SRF-null skin lacked hair but displayed normal epidermal architecture with proper expression of differentiation markers, suggesting that although keratinocyte SRF is essential for HF development, a cross-talk between SRF-null keratinocytes and the surrounding microenvironment is likely responsible for the barrier-deficient mutant epidermal phenotype.

Role of Pyk2 in cardiac arrhythmogenesis.

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor protein kinase regulated by intracellular Ca(2+), CaMK, and PKC and can be activated by different stress signals involved in heart failure. However, Pyk2 has not been investigated in the human heart, and the functional role of Pyk2 signaling at the whole heart level has not been elucidated. We hypothesize that Ca(2+)-dependent activation of Pyk2 is involved in cardiac electrophysiology. We examined the expression of Pyk2 in nonfailing versus ischemic and nonischemic failing human hearts (n = 6 hearts/group). To investigate Pyk2 function, we optically mapped perfused hearts from wild-type (WT; n = 7) and knockout (Pyk2(-/-); n = 8) mice during autonomic stimulation. Experiments were done in control mice and after 1 wk of transverse aortic constriction. We used the Illumina beadarray approach for transcriptional profiling of WT and Pyk2(-/-) mouse ventricles. Western blot analysis revealed a doubling of Pyk2 activation in nonischemic failing versus nonfailing human hearts. In mouse hearts, we observed a much higher probability of ventricular tachyarrhythmia during ACh perfusion in Pyk2(-/-) versus WT mice. Parasympathetic stimulation resulted in a dose-dependent decrease of atrial action potential duration (APD) in both WT and Pyk2(-/-) mice, whereas in ventricles it induced APD shortening in Pyk2(-/-) mice but not in WT mice. Deficiency of Pyk2 abolished ACh-induced prolongation of atrioventricular delay in Pyk2(-/-) mouse hearts but did not affect heart rate. Lower mRNA and protein levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and higher mRNA levels of Na(+)/Ca(2+) exchanger 1 were detected in Pyk2(-/-) hearts compared with WT hearts. The transverse aortic constriction protocol did not change the phenotype. In conclusion, our results indicate a protective role of Pyk2 with respect to ventricular tachyarrhythmia during parasympathetic stimulation by regulation of gene expression related to Ca(2+) handling. We hypothesize that activation of Pyk2 in the human heart during heart failure may contribute to protection against arrhythmia.