Functional Validation of the Putative Oncogenic Activity of PLAU.

Plasminogen activator, urokinase (PLAU) is involved in cell migration, proliferation and tissue remodeling. PLAU upregulation is associated with an increase in aggressiveness, metastasis, and invasion of several cancer types, including breast cancer. In patients, this translates into decreased sensitivity to hormonal treatment, and poor prognosis. These clinical findings have led to the examination of PLAU as a biomarker for predicting breast cancer prognosis and therapy responses. In this study, we investigated the functional ability of PLAU to act as an oncogene in breast cancers by modulating its expression using CRISPR-deactivated Cas9 (CRISPR-dCas9) tools. Different effector domains (e.g., transcription modulators (VP64, KRAB)) alone or in combination with epigenetic writers (DNMT3A/3L, MSssI) were fused to dCas9 and targeted to the PLAU promoter. In MDA-MB-231 cells characterized by high PLAU expression downregulation of PLAU expression by CRISPR-dCas9-DNMT3A/3L-KRAB, resulted in decreased cell proliferation. Conversely, CRISPR-dCas9-VP64 induced PLAU upregulation in low PLAU expressing MCF-7 cells and significantly increased aggressiveness and invasion. In conclusion, modulation of PLAU expression affected metastatic related properties of breast cancer cells, thus further validating its oncogenic activity in breast cancer cells.

Antiproliferative, Antiangiogenic, and Antimetastatic Therapy Response by Mangiferin in a Syngeneic Immunocompetent Colorectal Cancer Mouse Model Involves Changes in Mitochondrial Energy Metabolism.

In spite of the current advances and achievements in cancer treatments, colorectal cancer (CRC) persists as one of the most prevalent and deadly tumor types in both men and women worldwide. Drug resistance, adverse side effects and high rate of angiogenesis, metastasis and tumor relapse remain one of the greatest challenges in long-term management of CRC and urges need for new leads of anticancer drugs. We demonstrate that CRC treatment with the phytopharmaceutical mangiferin (MGF), a glucosylxanthone present in Mango tree stem bark and leaves (Mangifera Indica L.), induces dose-dependent tumor regression and decreases lung metastasis in a syngeneic immunocompetent allograft mouse model of murine CT26 colon carcinoma, which increases overall survival of mice. Antimetastatic and antiangiogenic MGF effects could be further validated in a wound healing in vitro model in human HT29 cells and in a matrigel plug implant mouse model. Interestingly, transcriptome pathway enrichment analysis demonstrates that MGF inhibits tumor growth, metastasis and angiogenesis by multi-targeting of mitochondrial oxidoreductase and fatty acid ß-oxidation metabolism, PPAR, SIRT, NFκB, Stat3, HIF, Wnt and GP6 signaling pathways. MGF effects on fatty acid ß-oxidation metabolism and carnitine palmitoyltransferase 1 (CPT1) protein expression could be further confirmed in vitro in human HT29 colon cells. In conclusion, antitumor, antiangiogenic and antimetastatic effects of MGF treatment hold promise to reduce adverse toxicity and to mitigate therapeutic outcome of colorectal cancer treatment by targeting mitochondrial energy metabolism in the tumor microenvironment.

Phosphocatalytic Kinome Activity Profiling of Apoptotic and Ferroptotic Agents in Multiple Myeloma Cells.

Through phosphorylation of their substrate proteins, protein kinases are crucial for transducing cellular signals and orchestrating biological processes, including cell death and survival. Recent studies have revealed that kinases are involved in ferroptosis, an iron-dependent mode of cell death associated with toxic lipid peroxidation. Given that ferroptosis is being explored as an alternative strategy to eliminate apoptosis-resistant tumor cells, further characterization of ferroptosis-dependent kinase changes might aid in identifying novel druggable targets for protein kinase inhibitors in the context of cancer treatment. To this end, we performed a phosphopeptidome based kinase activity profiling of glucocorticoid-resistant multiple myeloma cells treated with either the apoptosis inducer staurosporine (STS) or ferroptosis inducer RSL3 and compared their kinome activity signatures. Our data demonstrate that both cell death mechanisms inhibit the activity of kinases classified into the CMGC and AGC families, with STS showing a broader spectrum of serine/threonine kinase inhibition. In contrast, RSL3 targets a significant number of tyrosine kinases, including key players of the B-cell receptor signaling pathway. Remarkably, additional kinase profiling of the anti-cancer agent withaferin A revealed considerable overlap with ferroptosis and apoptosis kinome activity, explaining why withaferin A can induce mixed ferroptotic and apoptotic cell death features. Altogether, we show that apoptotic and ferroptotic cell death induce different kinase signaling changes and that kinome profiling might become a valid approach to identify cell death chemosensitization modalities of novel anti-cancer agents.

Ferroptosis Induction in Multiple Myeloma Cells Triggers DNA Methylation and Histone Modification Changes Associated with Cellular Senescence.

Disease relapse and therapy resistance remain key challenges in treating multiple myeloma. Underlying (epi-)mutational events can promote myelomagenesis and contribute to multi-drug and apoptosis resistance. Therefore, compounds inducing ferroptosis, a form of iron and lipid peroxidation-regulated cell death, are appealing alternative treatment strategies for multiple myeloma and other malignancies. Both ferroptosis and the epigenetic machinery are heavily influenced by oxidative stress and iron metabolism changes. Yet, only a limited number of epigenetic enzymes and modifications have been identified as ferroptosis regulators. In this study, we found that MM1 multiple myeloma cells are sensitive to ferroptosis induction and epigenetic reprogramming by RSL3, irrespective of their glucocorticoid-sensitivity status. LC-MS/MS analysis revealed the formation of non-heme iron-histone complexes and altered expression of histone modifications associated with DNA repair and cellular senescence. In line with this observation, EPIC BeadChip measurements of significant DNA methylation changes in ferroptotic myeloma cells demonstrated an enrichment of CpG probes located in genes associated with cell cycle progression and senescence, such as Nuclear Receptor Subfamily 4 Group A member 2 (NR4A2). Overall, our data show that ferroptotic cell death is associated with an epigenomic stress response that might advance the therapeutic applicability of ferroptotic compounds.

Covalent Cysteine Targeting of Bruton's Tyrosine Kinase (BTK) Family by Withaferin-A Reduces Survival of Glucocorticoid-Resistant Multiple Myeloma MM1 Cells.

Multiple myeloma (MM) is a hematological malignancy characterized by plasma cells' uncontrolled growth. The major barrier in treating MM is the occurrence of primary and acquired therapy resistance to anticancer drugs. Often, this therapy resistance is associated with constitutive hyperactivation of tyrosine kinase signaling. Novel covalent kinase inhibitors, such as the clinically approved BTK inhibitor ibrutinib (IBR) and the preclinical phytochemical withaferin A (WA), have, therefore, gained pharmaceutical interest. Remarkably, WA is more effective than IBR in killing BTK-overexpressing glucocorticoid (GC)-resistant MM1R cells. To further characterize the kinase inhibitor profiles of WA and IBR in GC-resistant MM cells, we applied phosphopeptidome- and transcriptome-specific tyrosine kinome profiling. In contrast to IBR, WA was found to reverse BTK overexpression in GC-resistant MM1R cells. Furthermore, WA-induced cell death involves covalent cysteine targeting of Hinge-6 domain type tyrosine kinases of the kinase cysteinome classification, including inhibition of the hyperactivated BTK. Covalent interaction between WA and BTK could further be confirmed by biotin-based affinity purification and confocal microscopy. Similarly, molecular modeling suggests WA preferably targets conserved cysteines in the Hinge-6 region of the kinase cysteinome classification, favoring inhibition of multiple B-cell receptors (BCR) family kinases. Altogether, we show that WA's promiscuous inhibition of multiple BTK family tyrosine kinases represents a highly effective strategy to overcome GC-therapy resistance in MM.

Physical plasma-derived oxidants sensitize pancreatic cancer cells to ferroptotic cell death.

Despite modern therapeutic advances, the survival prospects of pancreatic cancer patients remain poor, due to chemoresistance and dysregulated oncogenic kinase signaling networks. We applied a novel kinome activity-mapping approach using biological peptide targets as phospho-sensors to identify vulnerable kinase dependencies for therapy sensitization by physical plasma. Ser/Thr-kinome specific activity changes were mapped upon induction of ferroptotic cell death in pancreatic tumor cells exposed to reactive oxygen and nitrogen species of plasma-treated water (PTW). This revealed a broad kinome activity response involving the CAMK, the AGC and CMGC family of kinases. This systems-level kinome network response supports stress adaptive switches between chemoresistant anti-oxidant responses of Kelch-like ECH-associated protein 1 (KEAP1)/Heme Oxygenase 1 (HMOX1) and ferroptotic cell death sensitization upon suppression of Nuclear factor (erythroid derived 2)-like 2 (NRF2) and Glutathione peroxidase 4 (GPX4). This is further supported by ex vivo experiments in the chicken chorioallantoic membrane assay, showing decreased GPX4 and Glutathione (GSH) expression as well as increased lipid peroxidation, along with suppressed BxPC-3 tumor growth in response to PTW. Taken all together, we demonstrate that plasma treated water-derived oxidants sensitize pancreatic cancer cells to ferroptotic cell death by targeting a NRF2-HMOX1-GPX4 specific kinase signaling network.

Tackling Chronic Inflammation with Withanolide Phytochemicals-A Withaferin a Perspective.

Chronic inflammatory diseases are considered to be one of the biggest threats to human health. Most prescribed pharmaceutical drugs aiming to treat these diseases are characterized by side-effects and negatively affect therapy adherence. Finding alternative treatment strategies to tackle chronic inflammation has therefore been gaining interest over the last few decades. In this context, Withaferin A (WA), a natural bioactive compound isolated from Withania somnifera, has been identified as a promising anti-cancer and anti-inflammatory compound. Although the majority of studies focus on the molecular mechanisms of WA in cancer models, recent evidence demonstrates that WA also holds promise as a new phytotherapeutic agent against chronic inflammatory diseases. By targeting crucial inflammatory pathways, including nuclear factor kappa B (NF-kB) and nuclear factor erythroid 2 related factor 2 (Nrf2) signaling, WA suppresses the inflammatory disease state in several in vitro and preclinical in vivo models of diabetes, obesity, neurodegenerative disorders, cystic fibrosis and osteoarthritis. This review provides a concise overview of the molecular mechanisms by which WA orchestrates its anti-inflammatory effects to restore immune homeostasis.

In Vitro Anti-Inflammatory, Anti-Oxidant, and Cytotoxic Activities of Four Curcuma Species and the Isolation of Compounds from Curcuma aromatica Rhizome.

The genus Curcuma is part of the Zingiberaceae family, and many Curcuma species have been used as traditional medicine and cosmetics in Thailand. To find new cosmeceutical ingredients, the in vitro anti-inflammatory, anti-oxidant, and cytotoxic activities of four Curcuma species as well as the isolation of compounds from the most active crude extract (C. aromatica) were investigated. The crude extract of C. aromatica showed 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity with an IC50 value of 102.3 µg/mL. The cytotoxicity effect of C. aeruginosa, C. comosa, C. aromatica, and C. longa extracts assessed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay at 200 µg/mL were 12.1 2.9, 14.4 4.1, 28.6 4.1, and 46.9 8.6, respectively. C. aeruginosa and C. comosa presented apoptosis cells (57.7 3.1% and 32.6 2.2%, respectively) using the CytoTox-ONE™ assay. Different crude extracts or phytochemicals purified from C. aromatica were evaluated for their anti-inflammatory properties. The crude extract of C. aromatica showed the highest potential to inhibit NF-κB activity, followed by C. aeruginosa, C. comosa, and C. longa, respectively. Among the various purified phytochemicals curcumin, germacrone, curdione, zederone, and curcumenol significantly inhibited NF-κB activation in tumor necrosis factor (TNF) stimulated HaCaT keratinocytes. Of all compounds, curcumin was the most potent anti-inflammatory.

Chemical Composition of Essential Oils from Different Parts of Zingiberkerrii Craib and Their Antibacterial, Antioxidant, and Tyrosinase Inhibitory Activities.

The essential oils of the fresh rhizomes; flowers; and leaves of Zingiber kerrii Craib were investigated using different extraction techniques; including solid-phase microextraction (SPME), hydrodistillation (HD), and organic solvent (OS), and characterized by gas chromatography-mass spectrometry (GC-MS). A total of 37 SPME; 19 HD; and 36 OS compounds were identified from the rhizome extract of Z. kerrii; with the major components being α-pinene; ß-pinene; and terpinen-4-ol; respectively. From the flower extract; 16 SPME; 2 HD; and 10 OS compounds were identified; (E)-caryophyllene was found as a major compound by these techniques. The leaf extract exhibited 20 SPME; 13 HD; and 14 OS compounds; with α-pinene; (E)-caryophyllene; and n-hexadecanoic acid being the major compounds; respectively. The rhizome extract showed tyrosinase inhibitory activity of 71.60% and a total phenolic content of 22.4 mg gallic acid/g. The IC50 values of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays were 25.2 µg/mL and 153.6 µg/mL; respectively; and the ferric ion reducing antioxidant power (FRAP) assay value was 318.5 µM ascorbic acid equivalent (AAE)/g extract. The rhizome extract showed weak antibacterial activity. This extract showed no adverse toxicity in human keratinocyte (HaCaT) cell lines at concentrations below 200 µg/mL.

Withaferin A: From ayurvedic folk medicine to preclinical anti-cancer drug.

Despite the recent successes of targeted cancer immuno-therapies, drug resistance and disease relapse remain a huge burden in cancer patient treatment. This has fueled renewed interest in natural product discovery to identify new pharmacophores for innovative cancer drug development. Reverse pharmacology approaches of Withania somnifera leaves and roots (alternatively also called Ashwagandha or Indian ginseng in traditional Ayurvedic and Unani folk medicine) have identified Withaferin A (WA) as the most bioactive compound for treatment of inflammatory ailments, supporting traditional use of their corresponding extracts in indigenous medicine. In this review we summarize preclinical in vivo evidence for therapeutic cancer applications of WA and provide a biochemical framework of its polypharmaceutical effects against cancer hallmarks.

Anti-angiogenic effects of mangiferin and mechanism of action in metastatic melanoma.

Advanced metastatic melanoma, one of the most aggressive skin malignancies, is currently without reliable therapy. The process of angiogenesis is crucial for progression and metastasis of the majority of solid tumors including melanomas. Therefore, new therapies are urgently needed. Mangiferin is a naturally occurring glucosylxanthone which exerts many pharmacological activities against cancer-inflammation. However, the effect of mangiferin on metastasis and tumor growth of metastatic melanoma remains unclear. In this study, we demonstrate that mangiferin interferes with inflammation, lipid and calcium signaling which selectively inhibits multiple NFkB target genes including interleukin-6, tumor necrosis factor, interferon gamma, vascular endothelial growth factor receptor 2, plasminogen activator urokinase, matrix metalloprotease 19, C-C Motif Chemokine Ligand 2 and placental growth factor. This abrogates angiogenic and invasive processes and capillary tube formation of metastatic melanoma cells as well as human placental blood vessel explants in-vitro and blocks angiogenesis characteristic of the chicken egg chorioallantoic membrane assay and in melanoma syngeneic studies in vivo. The results obtained in this research illustrate promising anti-angiogenic effects of the natural glucosylxanthone mangiferin for further (pre)clinical studies in melanoma cancer patients.

Connecting the Dots in the Neuroglobin-Protein Interaction Network of an Unstressed and Ferroptotic Cell Death Neuroblastoma Model.

Neuroglobin is a heme protein of which increased levels provide neuroprotection against amyloid proteinopathy and hemorrhagic damage. These cellular stressors involve the promotion of ferroptosis-an iron-dependent, lipid peroxide-accreting form of cell death. Hence, we questioned whether neuroglobin could oppose ferroptosis initiation. We detected human neuroglobin (hNgb)-EGFP-expressing SH-SY5Y cells to be significantly more resistant to ferroptosis induction, identifying 0.68-fold less cell death. To elucidate the underlying pathways, this study investigated hNgb-protein interactions with a Co-IP-MS/MS approach both under a physiological and a ferroptotic condition. hNgb binds to proteins of the cellular iron metabolism (e.g., RPL15 and PCBP3) in an unstressed condition and shows an elevated binding ratio towards cell death-linked proteins, such as HNRNPA3, FAM120A, and ABRAXAS2, under ferroptotic stress. Our data also reveal a constitutive interaction between hNgb and the longevity-associated heterodimer XRCC5/XRCC6. Disentangling the involvement of hNgb and its binding partners in cellular processes, using Ingenuity Pathway Analysis, resulted in the integration of hNgb in the ubiquitination pathway, mTOR signaling, 14-3-3-mediated signaling, and the glycolysis cascade. We also detected a previously unknown strong link with motor neuropathies. Hence, this study contributes to the elucidation of neuroglobin's involvement in cellular mechanisms that provide neuroprotection and the upkeep of homeostasis.

Regulation of NGF Signaling by an Axonal Untranslated mRNA.

Neurons are extraordinarily large and highly polarized cells that require rapid and efficient communication between cell bodies and axons over long distances. In peripheral neurons, transcripts are transported along axons to growth cones, where they are rapidly translated in response to extrinsic signals. While studying Tp53inp2, a transcript highly expressed and enriched in sympathetic neuron axons, we unexpectedly discovered that Tp53inp2 is not translated. Instead, the transcript supports axon growth in a coding-independent manner. Increasing evidence indicates that mRNAs may function independently of their coding capacity; for example, acting as a scaffold for functionally related proteins. The Tp53inp2 transcript interacts with the nerve growth factor (NGF) receptor TrkA, regulating TrkA endocytosis and signaling. Deletion of Tp53inp2 inhibits axon growth in vivo, and the defects are rescued by a non-translatable form of the transcript. Tp53inp2 is an atypical mRNA that regulates axon growth by enhancing NGF-TrkA signaling in a translation-independent manner.