An Optimized Model of Hypertrophic Preconditioning Confers Cardioprotection in the Mouse.

BACKGROUND: Conventional models of hypertrophic preconditioning (C-HP) can be established surgically through transverse aortic constriction (TAC) → deconstriction (De-TAC) → reconstriction (Re-TAC) characterized by dynamic afterload while it exerts technical difficulty on operators and poses high mortality during perioperative period in mice. We aimed to introduce an optimized method for obtaining a hypertrophic preconditioning (O-HP) model for further study on cardiac hypertrophy. METHODS: Ninety mice were divided into four groups: sham, TAC, C-HP, and O-HP. The sham group was exerted on three-time thoracotomies. The TAC group experienced twice thoracotomies and one TAC operation. C-HP and O-HP groups were given TAC, De-TAC, and Re-TAC operation at day 0, day 3, and day 7 in conventional and optimized method, respectively. We optimized the operating procedure in O-HP mice compared with the C-HP group by (1) leaving a ∼3-cm suture fixed in the subcutaneous layer after aortic constriction in TAC surgery (2) using two small forceps to untie the constriction knot instead of cutting it in the De-TAC operation. Ultrasound biomicroscopy was used for hemodynamics and cardiac function detection. Four weeks after the third surgery, all mice were sacrificed and pathology was analyzed among four groups. RESULTS: Four weeks after Re-TAC, the survival of O-HP mice was 63.3% while that of C-HP was 26.7%. Ultrasound biomicroscopy showed a successful establishment of HP models. C-HP and O-HP mice had improved cardiac structure and function indicated by left ventricular end-systolic diameter, left ventricular end-systolic posterior wall thickness, left ventricular ejection fraction, and left ventricular fractional shortening than the TAC group. Pathological analysis showed O-HP as well as C-HP had less hypertrophy than the TAC mice. CONCLUSIONS: Our results provide a rapid, safe, efficient, and reproducible method for optimized establishment of the HP model, which will facilitate studies for early intervention and prevention of left ventricular hypertrophy and heart failure.

NADPH oxidase 4 is a critical mediator in Ataxia telangiectasia disease.

Ataxia telangiectasia (A-T), a rare autosomal recessive disorder characterized by progressive cerebellar degeneration and a greatly increased incidence of cancer among other symptoms, is caused by a defective or missing ataxia telangiectasia mutated (ATM) gene. The ATM protein has roles in DNA repair and in the regulation of reactive oxygen species (ROS). Here, we provide, to our knowledge, the first evidence that NADPH oxidase 4 (NOX4) is involved in manifesting A-T disease. We showed that NOX4 expression levels are higher in A-T cells, and that ATM inhibition leads to increased NOX4 expression in normal cells. A-T cells exhibit elevated levels of oxidative DNA damage, DNA double-strand breaks and replicative senescence, all of which are partially abrogated by down-regulation of NOX4 with siRNA. Sections of degenerating cerebelli from A-T patients revealed elevated NOX4 levels. ATM-null mice exhibit A-T disease but they die from cancer before the neurological symptoms are manifested. Injecting Atm-null mice with fulvene-5, a specific inhibitor of NOX4 and NADPH oxidase 2 (NOX2), decreased their elevated cancer incidence to that of the controls. We conclude that, in A-T disease in humans and mice, NOX4 may be critical mediator and targeting it will open up new avenues for therapeutic intervention in neurodegeneration.

Pro-Apoptotic Activity of New Honokiol/Triphenylmethane Analogues in B-Cell Lymphoid Malignancies.

Honokiol and triphenylmethanes are small molecules with anti-tumor properties. Recently, we synthesized new honokiol analogues (HAs) that possess common features of both groups. We assessed the anti-tumor effectiveness of HAs in B-cell leukemia/lymphoma cells, namely in chronic lymphocytic leukemia (CLL) cells ex vivo and in pre-B-cell acute lymphoblastic leukemia (Nalm-6), Burkitt lymphoma (BL; Raji), diffuse large B-cell lymphoma (DLBCL; Toledo) and multiple myeloma (MM; RPMI 8226) cell lines. Four of these compounds appeared to be significantly active against the majority of cells examined, with no significant impact on healthy lymphocytes. These active HAs induced caspase-dependent apoptosis, causing significant deregulation of several apoptosis-regulating proteins. Overall, these compounds downregulated Bcl-2 and XIAP and upregulated Bax, Bak and survivin proteins. In conclusion, some of the HAs are potent tumor-selective inducers of apoptosis in ex vivo CLL and in BL, DLBCL and MM cells in vitro. Further preclinical studies of these agents are recommended.

Honokiol inhibits androgen receptor activity in prostate cancer cells.

BACKGROUND: We have shown previously that honokiol (HNK), a bioactive component of the medicinal plant Magnolia officinalis, inhibits growth of human prostate cancer cells in vitro and in vivo. However, the effect of HNK on androgen receptor (AR) signaling has not been studied. METHODS: LNCaP, C4-2, and TRAMP-C1 cells were used for various assays. Trypan blue dye exclusion assay or clonogenic assay was performed for determination of cell viability. The effects of HNK and/or its analogs on protein levels of AR and its target gene product prostate specific antigen (PSA) were determined by western blotting. RNA interference of p53 was achieved by transient transfection. Reverse transcription-polymerase chain reaction was performed for mRNA expression of AR. Nuclear level of AR was visualized by microscopy. Apoptosis was quantified by DNA fragmentation assay or flow cytometry after Annexin V-propidium iodide staining. RESULTS: HNK and its dichloroacetate analog (HDCA) were relatively more effective in suppressing cell viability and AR protein level than honokiol epoxide or biseugenol. Nuclear translocation of AR stimulated by a synthetic androgen (R1881) was markedly suppressed in the presence of HNK. Downregulation of AR protein resulting from HNK exposure was attributable to transcriptional repression as well as proteasomal degradation. HNK-mediated suppression of AR protein was maintained in LNCaP cells after knockdown of p53 protein. HNK-induced apoptosis was not affected by R1881 treatment. CONCLUSIONS: The present study demonstrates, for the first time, that HNK inhibits activity of AR in prostate cancer cells regardless of the p53 status.

Targeting NADPH oxidases for the treatment of cancer and inflammation.

NADPH oxidases are a family of oxidases that utilize molecular oxygen to generate hydrogen peroxide and superoxide, thus indicating physiological functions of these highly reactive and short-lived species. The regulation of these NADPH oxidases (nox) enzymes is complex, with many members of this family exhibiting complexity in terms of subunit composition, cellular location, and tissue-specific expression. While the complexity of the nox family (Nox1-5, Duox1, 2) is daunting, the complexity also allows for targeting of NADPH oxidases in disease states. In this review, we discuss which inflammatory and malignant disorders can be targeted by nox inhibitors, as well as clinical experience in the use of such inhibitors.

Honokiol activates AMP-activated protein kinase in breast cancer cells via an LKB1-dependent pathway and inhibits breast carcinogenesis.

INTRODUCTION: Honokiol, a small-molecule polyphenol isolated from magnolia species, is widely known for its therapeutic potential as an antiinflammatory, antithrombosis, and antioxidant agent, and more recently, for its protective function in the pathogenesis of carcinogenesis. In the present study, we sought to examine the effectiveness of honokiol in inhibiting migration and invasion of breast cancer cells and to elucidate the underlying molecular mechanisms. METHODS: Clonogenicity and three-dimensional colony-formation assays were used to examine breast cancer cell growth with honokiol treatment. The effect of honokiol on invasion and migration of breast cancer cells was evaluated by using Matrigel invasion, scratch-migration, spheroid-migration, and electric cell-substrate impedance sensing (ECIS)-based migration assays. Western blot and immunofluorescence analysis were used to examine activation of the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) axis. Isogenic LKB1-knockdown breast cancer cell line pairs were developed. Functional importance of AMPK activation and LKB1 overexpression in the biologic effects of honokiol was examined by using AMPK-null and AMPK-wild type (WT) immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown cell line pairs. Finally, mouse xenografts, immunohistochemical and Western blot analysis of tumors were used. RESULTS: Analysis of the underlying molecular mechanisms revealed that honokiol treatment increases AMP-activated protein kinase (AMPK) phosphorylation and activity, as evidenced by increased phosphorylation of the downstream target of AMPK, acetyl-coenzyme A carboxylase (ACC) and inhibition of phosphorylation of p70S6kinase (pS6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). By using AMPK-null and AMPK-WT (MEFs), we found that AMPK is required for honokiol-mediated modulation of pACC-pS6K. Intriguingly, we discovered that honokiol treatment increased the expression and cytoplasmic translocation of tumor-suppressor LKB1 in breast cancer cells. LKB1 knockdown inhibited honokiol-mediated activation of AMPK and, more important, inhibition of migration and invasion of breast cancer cells. Furthermore, honokiol treatment resulted in inhibition of breast tumorigenesis in vivo. Analysis of tumors showed significant increases in the levels of cytoplasmic LKB1 and phospho-AMPK in honokiol-treated tumors. CONCLUSIONS: Taken together, these data provide the first in vitro and in vivo evidence of the integral role of the LKB1-AMPK axis in honokiol-mediated inhibition of the invasion and migration of breast cancer cells. In conclusion, honokiol treatment could potentially be a rational therapeutic strategy for breast carcinoma.

Posterior lumbar vein off the retrohepatic inferior vena cava: a novel anatomical variant with surgical implications.

PURPOSE: Resection of tumors involving the inferior vena cava requires vascular control of posteriorly draining lumbar veins to ensure a bloodless field. Surgical texts and atlases assert that lumbar veins do not insert into the inferior vena cava superior to the renal hilum. However, at our institution we have encountered patients undergoing inferior vena cava tumor thrombectomy who have a posterior lumbar vein cephalad to the renal veins. Since this represents an unrecognized source of hemorrhage, we investigated the frequency of a superior lumbar vein in cadaveric dissection. MATERIALS AND METHODS: Retroperitoneal cadaveric dissection of the inferior vena cava was done to assess the frequency of a lumbar vein draining into the inferior vena cava cephalad to the renal veins. RESULTS: Of the 49 cadaveric dissections performed 19 (38.8%) showed a single posterior lumbar vein between the diaphragm and the renal hilum. Of these 19 cadavers 15 (78.9%) were male. This vein was located an average ± SD of 7.4 ± 0.6 cm cephalad to the right renal vein and it was 3.7 ± 1.6 cm in diameter. In all cadavers this vein inserted within 30 degrees to the left or right of the posterior (also termed dorsal) aspect of the inferior vena cava. CONCLUSIONS: The identification of a lumbar vein between the renal hilum and the diaphragm represents an important anatomical variant that occurs in a significant percent of individuals. Surgeons will benefit from the knowledge of this variant of inferior vena cava vasculature and should anticipate the presence of this vein to prevent unnecessary morbidity and mortality secondary to unexpected hemorrhage, particularly in male patients.

Correction: da Costa et al. Establishment of a Temperature-Sensitive Model of Oncogene-Induced Senescence in Angiosarcoma Cells. Cancers 2020, 12, 395.

The authors wish to make the following corrections to this paper [...].

Endophilin A2 deficiency protects rodents from autoimmune arthritis by modulating T cell activation.

The introduction of the CTLA-4 recombinant fusion protein has demonstrated therapeutic effects by selectively modulating T-cell activation in rheumatoid arthritis. Here we show, using a forward genetic approach, that a mutation in the SH3gl1 gene encoding the endocytic protein Endophilin A2 is associated with the development of arthritis in rodents. Defective expression of SH3gl1 affects T cell effector functions and alters the activation threshold of autoreactive T cells, thereby leading to complete protection from chronic autoimmune inflammatory disease in both mice and rats. We further show that SH3GL1 regulates human T cell signaling and T cell receptor internalization, and its expression is upregulated in rheumatoid arthritis patients. Collectively our data identify SH3GL1 as a key regulator of T cell activation, and as a potential target for treatment of autoimmune diseases.

Establishment of a Temperature-Sensitive Model of Oncogene-Induced Senescence in Angiosarcoma Cells.

: Lesions with driver mutations, including atypical nevi and seborrheic keratoses, are very common in dermatology, and are prone to senescence. The molecular events that prevent senescent lesions from becoming malignant are not well understood. We have developed a model of vascular proliferation using a temperaturesensitive, large T antigen and oncogenic HRas. By elevating the temperature to 39 °C, we can turn off large T antigen and study the molecular events in cells with the Ras driver mutation. To assess the signaling events associated with the switch from a proliferative to a nonproliferative state in the constant presence of a driver oncogene, SVR cells were cultivated for 24 and 48 hours and compared with SVR cells at 37 °C. Cells were evaluated by Western Blot (WB) gene chip microarray (GC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Upon evaluation, a novel phenotype was observed in endothelial cells after switching off the large T antigen. This phenotype was characterized by Notch activation, downregulation of p38 phosphorylation, downregulation of the master immune switch IRF7, and downregulation of hnRNP A0 . Switching off proliferative signaling may result in immune privilege and Notch activation, which may account, in part, for the survival of common skin lesions.

Tris DBA palladium is an orally available inhibitor of GNAQ mutant uveal melanoma in vivo.

Uveal melanoma is a rare but often lethal malignancy and is the leading cause of death due to an ophthalmic condition. Uveal melanoma is often diagnosed at a late stage and has a strong propensity to hepatic metastasis. Recently, the most common driver mutations in uveal melanoma have been identified, predominantly in the G-proteins GNAQ. This pattern differs from that of cutaneous melanoma in which Braf and Nras predominate. There are no current clinically used agents that target GNAQ mutations, unlike the use of Braf inhibitors in cutaneous melanoma. We tested the novel agent Tris DBA palladium and found that it was markedly more effective against GNAQ mutant melanomas than wild type uveal melanomas. Given that ARF6 has recently been discovered as a node in GNAQ mutations, we evaluated the efficacy of Tris DBA palladium on ARF6 signaling and found that it was effective in inhibiting ARF6 activation. Finally, Tris DBA palladium was orally effective against GNAQ mutant melanoma in vivo. Tris DBA Palladium deserves further evaluation as a systemic agent for uveal melanoma.

Selenium unmasks protective iron armor: A possible defense against cutaneous inflammation and cancer.

BACKGROUND: A link between selenium deficiency and inflammatory skin diseases have been noted by many, but this link is still not well understood. We have previously studied the efficacy of ceramide analogs, based on the fire ant venom Solenopsin A, against our psoriasis animal model. Treatment of animals with solenopsin analogs resulted in significantly improved skin as well as in a coordinate downregulation of selenoproteins, namely Glutathione Peroxidase 4 (GPX4). We thus hypothesize that ferroptosis may be a physiologic process that may protect the skin from both inflammatory and neoplastic processes. METHODS: We analyze and compare gene expression profiles in the GEO database from clinical skin samples taken from healthy patients and psoriasis patients (both involved and noninvolved skin lesions). We validated the gene expression results against a second, independent, cohort from the GEO database. RESULTS: Significant reduction in gene expression of GPX4, elevated expression of Nrf2 downstream targets, and expression profiles mirroring erastin-inhibition of Cystine/Glutamate Antiporter-System XC activity in psoriatic skin lesions, compared to both noninvolved skin and healthy patient samples, suggest an innately inducible mechanism of ferroptosis. CONCLUSIONS: We present data that may indicate selenoproteins, particularly GPX4, in resolving inflammation and skin cancer, including the novel hypothesis that the human organism may downregulate GPX4 and reactive oxygen (REDOX) regulating proteins in the skin as a way of resolving psoriasis and nonmelanoma skin cancer through increased reactive oxygen species. Further studies are needed to investigate ferroptosis as a possible physiologic mechanism for eliminating inflammatory and malignant tissues. GENERAL SIGNIFICANCE: This study provides a fresh framework for understanding the seemingly contradictory effects of selenium supplementation. In addition, it offers a novel explanation of how physiologic upregulation of ferroptosis and downregulation of selenoprotein synthesis may mediate resolution of inflammation and carcinogenesis. This is of therapeutic significance.

Targeting the duality of cancer.

Cancer is the second leading cause of death in the United States, and is an increasing cause of death in the developing world. While there is great heterogeneity in the anatomic site and mutations involved in human cancer, there are common features, including immortal growth, angiogenesis, apoptosis evasion, and other features, that are common to most if not all cancers. However, new features of human cancers have been found as a result of clinical use of novel "targeted therapies," angiogenesis inhibitors, and immunotherapies, including checkpoint inhibitors. These findings indicate that cancer is a moving target, which can change signaling and metabolic features based upon the therapies offered. It is well-known that there is significant heterogeneity within a tumor and it is possible that treatment might reduce the heterogeneity as a tumor adapts to therapy and, thus, a tumor might be synchronized, even if there is no major clinical response. Understanding this concept is important, as concurrent and sequential therapies might lead to improved tumor responses and cures. We posit that the repertoire of tumor responses is both predictable and limited, thus giving hope that eventually we can be more effective against solid tumors. Currently, among solid tumors, we observe a response of 1/3 of tumors to immunotherapy, perhaps less to angiogenesis inhibition, a varied response to targeted therapies, with relapse and resistance being the rule, and a large fraction being insensitive to all of these therapies, thus requiring the older therapies of chemotherapy, surgery, and radiation. Tumor phenotypes can be seen as a continuum between binary extremes, which will be discussed further. The biology of cancer is undoubtedly more complex than duality, but thinking of cancer as a duality may help scientists and oncologists discover optimal treatments that can be given either simultaneously or sequentially.

Honokiol protects skin cells against inflammation, collagenolysis, apoptosis, and senescence caused by cigarette smoke damage.

BACKGROUND: Pollution, especially cigarette smoke, is a major cause of skin damage. OBJECTIVES: To assess the effects of the small molecule polyphenol, honokiol, on reversing cigarette smoke-induced damage in vitro to relevant skin cells. METHODS: Keratinocytes (HaCat) cultures were exposed to cigarette smoke and, after 48 hours, IL-1α and IL-8 were measured in cell supernatants. Moreover, TIMP-2 production, apoptosis rate, and senescence ß-galactosidase expression were evaluated in primary human foreskin fibroblasts (HFF-1) cultures. RESULTS: Honokiol at 10 µm reduced IL-1α production by 3.4 folds (P < 0.05) and at 10 and 20 µm reduced IL-8 by 23.9% and 53.1% (P < 0.001), respectively, in HaCat keratinocytes. In HFF-1, honokiol restored TIMP-2 production by 96.9% and 91.9% (P < 0.001), respectively, at 10 and 20 µm, as well as reduced apoptosis by 47.1% (P < 0.001) and 41.3% (P < 0.01), respectively. Finally, honokiol reduced senescence-associated ß-galactosidase expression in HFF-1. CONCLUSION: Honokiol protects both HFF-1 and HaCat against cigarette smoke-induced inflammation, collagenolysis, apoptosis, and senescence.

Evidence for biochemical barrier restoration: Topical solenopsin analogs improve inflammation and acanthosis in the KC-Tie2 mouse model of psoriasis.

Psoriasis is a chronic inflammatory skin disease affecting 2.5-6 million patients in the United States. The cause of psoriasis remains unknown. Previous human and animal studies suggest that patients with a susceptible genetic background and some stimulus, such as barrier disruption, leads to a coordinated signaling events involving cytokines between keratinocytes, endothelial cells, T cells, macrophages and dendritic cells. Ceramides are endogenous skin lipids essential for maintaining skin barrier function and loss of ceramides may underlie inflammatory and premalignant skin. Ceramides act as a double-edged sword, promoting normal skin homeostasis in the native state, but can be metabolized to sphingosine-1-phosphate (S1P), linked to inflammation and tumorigenesis. To overcome this difficulty, we synthesized solenopsin analogs which biochemically act as ceramides, but cannot be metabolized to S1P. We assess their in vivo bioactivity in a well-established mouse model of psoriasis, the KC-Tie2 mouse. Topical solenopsin derivatives normalized cutaneous hyperplasia in this model, decreased T cell infiltration, interleukin (IL)-22 transcription, and reversed the upregulation of calprotectin and Toll-like receptor (TLR) 4 in inflamed skin. Finally, they stimulated interleukin (IL)-12 production in skin dendritic cells. Thus suggesting barrier restoration has both a biochemical and physical component, and both are necessary for optimal barrier restoration.

Seborrheic Keratoses: The Rodney Dangerfield of Skin lesions, and Why They Should Get Our Respect.

Neel et al. have demonstrated that seborrheic keratosis, the most common of all skin tumors, is dependent on acutely transforming retrovirus AKT8 in rodent T-cell lymphoma signaling. The authors found that these lesions are hypersensitive to Akt inhibitors which bind to the ATP binding site of Akt. Cutaneous squamous cell carcinoma is resistant to Akt inhibitors. The implications of this study are not limited to seborrheic keratosis. The presence of wild type p53 (seborrheic keratosis) or mutant p53 (cutaneous squamous cell carcinoma) appears to dictate whether a lesion is sensitive to Akt inhibition or not.