Physachenolide C induces complete regression of established murine melanoma tumors via apoptosis and cell cycle arrest.

Melanoma is an aggressive skin cancer that metastasizes to other organs. While immune checkpoint blockade with anti-PD-1 has transformed the treatment of advanced melanoma, many melanoma patients fail to respond to anti-PD-1 therapy or develop acquired resistance. Thus, effective treatment of melanoma still represents an unmet clinical need. Our prior studies support the anti-cancer activity of the 17ß-hydroxywithanolide class of natural products, including physachenolide C (PCC). As single agents, PCC and its semi-synthetic analog demonstrated direct cytotoxicity in a panel of murine melanoma cell lines, which share common driver mutations with human melanoma; the IC50 values ranged from 0.19-1.8 µM. PCC treatment induced apoptosis of tumor cells both in vitro and in vivo. In vivo treatment with PCC alone caused the complete regression of established melanoma tumors in all mice, with a durable response in 33% of mice after discontinuation of treatment. T cell-mediated immunity did not contribute to the therapeutic efficacy of PCC or prevent tumor recurrence in YUMM2.1 melanoma model. In addition to apoptosis, PCC treatment induced G0-G1 cell cycle arrest of melanoma cells, which upon removal of PCC, re-entered the cell cycle. PCC-induced cycle cell arrest likely contributed to the in vivo tumor recurrence in a portion of mice after discontinuation of treatment. Thus, 17ß-hydroxywithanolides have the potential to improve the therapeutic outcome for patients with advanced melanoma.

The Arp2/3 Inhibitory Protein Arpin Is Required for Intestinal Epithelial Barrier Integrity.

The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.

Hydroxyl radical scavenging of the compatible solute ectoine generates two N-acetimides.

Living cells employ various defence mechanisms against reactive oxygen species and free radicals. Besides protecting enzymes such as superoxide dismutase, catalase and peroxidase, non-enzymatic antioxidant molecules also play an important role as radical scavengers. Within bacteria the amino acid derivative ectoine (2-methyl-3,4,5,6-tetrahydropyrimidine-4-carboxylate) is the most abundant compatible solute and stress protectant. Although this compound is already produced commercially for applications as moisturizer and skin-care product, it has been a matter of debate whether ectoine also has radical-scavenging activity. Here we report on its hydroxyl radical scavenging activity in comparison to other compatible solutes and describe the reaction products obtained when ectoine is exposed to hydroxyl radicals generated by the Fenton reaction. In a sodium salicylate scavenging test system this compatible solute performed as well as mannitol. As a consequence of its reaction with hydroxyl radicals, ectoine was converted into two major products: N-acetimide aspartate and N-acetimide-ß-alanine. We propose a reaction mechanism in which the heterocycle of the compatible solute ectoine is cleaved and further oxidized at the C-terminus. The proven radical scavenging ability of ectoine will help to explain observed effects as anti-inflammatory compound in skin, lung and bowel disease.

Homoectoine Protects Against Colitis by Preventing a Claudin Switch in Epithelial Tight Junctions.

BACKGROUND: Inflammatory bowel diseases (IBD) are multifactorial disorders affecting millions of people worldwide with alarmingly increasing incidences every year. Dysfunction of the intestinal epithelial barrier is associated with IBD pathogenesis, and therapies include anti-inflammatory drugs that enhance intestinal barrier function. However, these drugs often have adverse side effects thus warranting the search for alternatives. Compatible solutes such as bacterial ectoines stabilize cell membranes and proteins. AIM: To unravel whether ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and homoectoine (4,5,6,7-tetrahydro-2-methyl-1H-(1,3)-diazepine-4-carboxylic acid), a synthetic derivative of ectoine, have beneficial effects during dextran sulfate sodium (DSS)-induced colitis in mice. METHODS/RESULTS: We found that the disease activity index was significantly reduced by both ectoines. DSS-induced edema formation, epithelial permeability, leukocyte recruitment and tissue damage were reduced by ectoine and homoectoine, with the latter having stronger effects. Interestingly, the claudin switch usually observed during colitis (decreased expression of claudin-1 and increased expression of the leaky claudin-2) was completely prevented by homoectoine, whereas ectoine only reduced claudin-2 expression. Concomitantly, only homoectoine ameliorated the drop in transepithelial electrical resistance induced by IFN-γ and TNF-α in Caco-2 cells. Both ectoines inhibited loss of ZO-1 and occludin and prevented IFN-γ/TNF-α-induced increased paracellular flux of 4 kDa FITC-dextran in vitro. Moreover, both ectoines reduced expression of pro-inflammatory cytokines and oxidative stress during colitis. CONCLUSION: While both ectoine and homoectoine have protective effects on the epithelial barrier during inflammation, only homoectoine completely prevented the inflammatory claudin switch in tight junctions. Thus, homoectoine may serve as diet supplement in IBD patients to reach or extend remission.

Hematopoietic cell-specific lyn substrate (HCLS1 or HS1): A versatile actin-binding protein in leukocytes.

Leukocytes are constantly produced in the bone marrow and released into the circulation. Many different leukocyte subpopulations exist that exert distinct functions. Leukocytes are recruited to sites of inflammation and combat the cause of inflammation via many different effector functions. Virtually all of these processes depend on dynamic actin remodeling allowing leukocytes to adhere, migrate, phagocytose, and release granules. However, actin dynamics are not possible without actin-binding proteins (ABP) that orchestrate the balance between actin polymerization, branching, and depolymerization. The homologue of the ubiquitous ABP cortactin in hematopoietic cells is hematopoietic cell-specific lyn substrate-1, often called hematopoietic cell-specific protein-1 (HCLS1 or HS1). HS1 has been reported in different leukocytes to regulate Arp2/3-dependent migration. However, more evidence is emerging that HS1 functions go far beyond just being a direct actin modulator. For example, HS1 is important for the activation of GTPases and integrins, and mediates signaling downstream of many receptors including BCR, TCR, and CXCR4. In this review, we summarize current knowledge on HS1 functions and discuss them in a pathophysiologic context.