In Model, In Vitro and In Vivo Killing Efficacy of Antitumor Peptide RDP22 on MUG-Mel2, a Patient Derived Cell Line of an Aggressive Melanoma Metastasis.

The host defense derived peptide was assessed in different model systems with increasing complexity employing the highly aggressive NRAS mutated melanoma metastases cell line MUG-Mel2. Amongst others, fluorescence microscopy and spectroscopy, as well as cell death studies were applied for liposomal, 2D and 3D in vitro models including tumor spheroids without or within skin models and in vivo mouse xenografts. Summarized, MUG-Mel2 cells were shown to significantly expose the negatively charged lipid phosphatidylserine on their plasma membranes, showing they are successfully targeted by RDP22. The peptide was able to induce cell death in MUG-Mel2 2D and 3D cultures, where it was able to kill tumor cells even inside the core of tumor spheroids or inside a melanoma organotypic model. In vitro studies indicated cell death by apoptosis upon peptide treatment with an LC50 of 8.5 µM and seven-fold specificity for the melanoma cell line MUG-Mel2 over normal dermal fibroblasts. In vivo studies in mice xenografts revealed effective tumor regression upon intratumoral peptide injection, indicated by the strong clearance of pigmented tumor cells and tremendous reduction in tumor size and proliferation, which was determined histologically. The peptide RDP22 has clearly shown high potential against the melanoma cell line MUG-Mel2 in vitro and in vivo.

Identification of Insulin-Mimetic Plant Extracts: From an In Vitro High-Content Screen to Blood Glucose Reduction in Live Animals.

Type 2 diabetes mellitus (T2DM) is linked to insulin resistance and a loss of insulin sensitivity, leading to millions of deaths worldwide each year. T2DM is caused by reduced uptake of glucose facilitated by glucose transporter 4 (GLUT4) in muscle and adipose tissue due to decreased intracellular translocation of GLUT4-containing vesicles to the plasma membrane. To treat T2DM, novel medications are required. Through a fluorescence microscopy-based high-content screen, we tested more than 600 plant extracts for their potential to induce GLUT4 translocation in the absence of insulin. The primary screen in CHO-K1 cells resulted in 30 positive hits, which were further investigated in HeLa and 3T3-L1 cells. In addition, full plasma membrane insertion was examined by immunostaining of the first extracellular loop of GLUT4. The application of appropriate inhibitors identified PI3 kinase as the most important signal transduction target relevant for GLUT4 translocation. Finally, from the most effective hits in vitro, four extracts effectively reduced blood glucose levels in chicken embryos (in ovo), indicating their applicability as antidiabetic pharmaceuticals or nutraceuticals.

ß,ß-Dimethylacrylshikonin Induces Apoptosis in Melanoma Cell Lines by NOXA Upregulation.

Melanoma is the most aggressive form of skin cancer, with high metastasis rates and poor prognosis. Survival rates and possible therapies depend on the state of the tumor and its mutational profile. BRAF and NRAS are the most frequent driver mutations. Currently, there is no efficient therapy for NRAS-mutated or late-stage melanoma. In this study, the therapeutic potential of ß,ß-dimethylacrylshikonin (DMAS) was investigated on melanoma. The influence of DMAS was determined in five different melanoma cell lines with different mutational profiles. The effects of this compound on cell viability, apoptosis, and gene and protein expression were examined. The results obtained were validated in vivo. DMAS significantly reduced the viability of several melanoma cell lines in a concentration- and time-dependent manner. Furthermore, DMAS induced caspase-3-dependent apoptosis via NOXA upregulation, as confirmed by NOXA knockdown experiments. This is the first time that NOXA-dependent apoptosis was shown with respect to a shikonin derivative and melanoma. Additionally, tumor regression and necrosis under DMAS treatment were demonstrated in vivo. Importantly, BRAF as well as NRAS-mutated metastatic human melanoma cell lines were treated successfully in vitro and in vivo. Taken together, DMAS showed promising results and is worthy of further study.

Pharmacologic IL-6Rα inhibition in cholangiocarcinoma promotes cancer cell growth and survival.

Biliary tract cancer (BTC) represents a malignant tumor of the biliary tract including cholangiocarcinoma (CCA) and the carcinoma of the gallbladder (GBC) with a 5-year survival rate between 5 and 18% due to late diagnosis and rapid disease progression. Chronic inflammation is one of the main risk factors for CCA and GBC in particular. IL-6, as a mediator of inflammation, can act through a membrane-bound receptor alpha-chain (mIL-6R, "IL-6 classic signaling") or via soluble forms (sIL-6R, "IL-6 trans-signaling"). However, little is known about the impact on cellular responses of IL-6 trans-signaling on BTC. We analyzed primary tumors as whole sections and as tissue microarrays, and also searched The Cancer Genome Atlas database. Compared to non-neoplastic, non-inflamed gallbladder tissue, IL-6Rα was downregulated in GBC, and this correlated with the patients' overall survival. Furthermore, different CCA cell lines and compounds for activation (IL-6 and Hyper-IL-6) or inhibition (Tocilizumab and sgp130Fc) of IL-6 classic signaling and trans-signaling were used to determine their effects on cellular processes between the two modes of IL-6 signaling. Inhibition of IL-6 trans-signaling by sgp130Fc reduced CCA cell line viability and apoptosis, whereas migration and proliferation were increased. We conclude that IL-6Rα expression is a good prognostic marker for GBC, and that the blocking of IL-6 trans-signaling and activation of IL-6 classic signaling have tumor promoting activity. These findings warrant the exclusion of patients with GBC or other malignancies associated with bile metabolism from IL-6R inhibitor therapy.

Synthesis of Novel Shikonin Derivatives and Pharmacological Effects of Cyclopropylacetylshikonin on Melanoma Cells.

Despite much research in the last centuries, treatment of malignant melanoma is still challenging because of its mostly unnoticeable metastatic spreading and aggressive growth rate. Therefore, the discovery of novel drug leads is an important goal. In a previous study, we have isolated several shikonin derivatives from the roots of Onosma paniculata Bureau & Franchet (Boraginaceae) which evolved as promising anticancer candidates. ß,ß-Dimethylacrylshikonin (1) was the most cytotoxic derivative and exhibited strong tumor growth inhibitory activity, in particular, towards melanoma cells. In this study, we synthesized eighteen novel shikonin derivatives in order to obtain compounds which exhibit a higher cytotoxicity than 1. We investigated their cytotoxic potential against various melanoma cell lines and juvenile skin fibroblasts. The most active compound was (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclopropylacetate (cyclopropylacetylshikonin) (6). It revealed significant stronger tumor growth inhibitory activity towards two melanoma cell lines derived from metastatic lesions (WM164 and MUG-Mel2). Further investigations have shown that 6 induced apoptosis caspase-dependently, increased the protein levels of cleaved PARP, and led to double-stranded DNA breaks as shown by phosphorylation of H2AX. Cell membrane damage and cell cycle arrest were not observed.

Comparative Gene Expression Analysis in WM164 Melanoma Cells Revealed That ß-ß-Dimethylacrylshikonin Leads to ROS Generation, Loss of Mitochondrial Membrane Potential, and Autophagy Induction.

Skin cancer is currently diagnosed as one in every three cancers. Melanoma, the most aggressive form of skin cancer, is responsible for 79% of skin cancer deaths and the incidence is rising faster than in any other solid tumor type. Previously, we have demonstrated that dimethylacrylshikonin (DMAS), isolated from the roots of Onosma paniculata (Boraginaceae), exhibited the lowest IC50 values against different tumor types out of several isolated shikonin derivatives. DMAS was especially cytotoxic towards melanoma cells and led to apoptosis and cell cycle arrest. In this study, we performed a comprehensive gene expression study to investigate the mechanism of action in more detail. Gene expression signature was compared to vehicle-treated WM164 control cells after 24 h of DMAS treatment; where 1192 distinct mRNAs could be identified as expressed in all replicates and 89 were at least 2-fold differentially expressed. DMAS favored catabolic processes and led in particular to p62 increase which is involved in cell growth, survival, and autophagy. More in-depth experiments revealed that DMAS led to autophagy, ROS generation, and loss of mitochondrial membrane potential in different melanoma cells. It has been reported that the induction of an autophagic cell death represents a highly effective approach in melanoma therapy.

Influence of silibinin and ß-ß-dimethylacrylshikonin on chordoma cells.

BACKGROUND: Chordoma, slow growing bone tumours originating from remnants of the notochord, leave affected patients with a median survival of six years. The high recurrence rate of chordoma, together with limited treatment options and bad overall prognosis, make the development of new treatment options urgently necessary. PURPOSE: In this study, the potential of two natural products, silibinin and ß-ß-dimethylacrylshikonin (DMAS), was tested on clival (MUG-CC1 and UM-Chor1) as well as sacral (MUG-Chor1 and U-CH2) chordoma cell lines. The treatment was administered both as single- and combined therapy. METHODS: For investigation of cell viability, the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit was used. Apoptosis induction was studied by flow cytometry, (Annexin V/SYTOX Green, caspase-3) and RT-qPCR. Pathway analyses were performed by western blot. RESULTS: Both drugs were found to reduce cell viability alone as well as in combination in a dose dependent manner, with DMAS being more efficient than silibinin. The mode of cell death was mainly apoptosis in DMAS treated samples, while the combination therapy led to apoptosis as well as late-apoptosis/necrosis. Silibinin therapy alone, although reducing cell viability, did not lead to significant apoptotic effects in the performed assays. Focussing on the molecular mechanism of DMAS induced apoptosis, it was found that major genes of the mitochondrial apoptosis pathway, like NOXA and PUMA were overexpressed. Additionally, western blot experiments showed a decrease of ERK/pERK, STAT3/pSTAT3 (Tyr705) and AKT/pAKT expression/activation levels under DMAS treatment. CONCLUSION: DMAS is a promising new candidate for chordoma therapy, while silibinin or a combination of both is less favourable.