[Clinicopathological study of epithelioid and spindle cell rhabdomysarcoma with EWSR1/FUS-TFCP2 fusion].

Objective: To investigate the clinicopathological and genetic features of epithelioid and spindle cell rhabdomysarcoma with EWSR1-TFCP2 or FUS-TFCP2 fusion. Methods: The clinical, morphological and immunohistochemical features of 14 cases of epithelioid and spindle cell rhabdomysarcoma with EWSR1-TFCP2 or FUS-TFCP2 fusion diagnosed from January 2019 to December 2022 in the Department of Pathology, Foshan Traditional Chinese Medicine Hospital, Foshan, China were retrospectively analyzed. The cases were all subject to FISH or next generation sequencing for analysis of molecular genetic features. The literature was reviewed. Results: There were 5 males and 9 females, with the age at presentation ranging from 6 to 36 years (mean, 22 years). Tumors occurred in the head and neck (9 cases), pelvic region (2 cases), bladder (one case), right humerus (one case), and the abdominal wall, humerus and pubic at the same time (one case). Presenting symptoms varied by location but often included pain or discomfort. Most of the patients showed aggressive radiographic features with soft tissue extension. The tumors had a median size of 6.6 cm (range, 2-23 cm). The tumors were poorly defined and irregularly shaped. Microscopic examination showed diffuse proliferation of spindle or epithelioid cells. While morphologically high-grade tumors displayed obvious cytological atypia, a high mitotic count and tumor necrosis, low-grade tumors grew in sheets and fascicles composed of spindle, epithelioid cells with moderate or abundant amounts of eosinophilic cytoplasm, without pronounced cytological atypia. The tumor cells expressed Desmin, MyoD1, and Myogenin, as well as ALK, EMA, and CKpan. EWSR1/FUS-TFCP2 gene fusion was detected in 14 cases with next generation sequencing and confirmed by FISH. Six cases had EWSR1-TFCP2 fusions and 8 cases showed FUS-TFCP2 fusions. Follow-up information was available in 13 patients, ranged from 5 to 37 months. At the end of follow-up period, 7 patients died of the disease. Six patients were alive:two cases had local recurrences and metastases, two cases of recurrences, one case of metastasis and one case without recurrences and metastasis. Conclusions: Epithelioid and spindle cell rhabdomysarcomas with EWSR1-TFCP2 or FUS-TFCP2 fusion show a very aggressive clinical course, and more commonly occur in the head and neck. Their genetic hallmark is the presence of EWSR1/FUS-TFCP2 fusions. Familiarity with its clinicopathological characteristics is helpful in avoiding misdiagnoses.

The modulation of iron metabolism affects the Rhabdomyosarcoma tumor growth in vitro and in vivo.

Rhabdomyosarcoma (RMS) is an aggressive rare neoplasm that derives from mesenchymal cells, which frequently develops resistance to the current therapies and the formation of metastases. Thus, new therapies are needed. The alteration of iron metabolism in cancer cells was effective in reducing the progression of many tumors but not yet investigated in RMS. Here we investigated the effect of iron modulation in RMS both in vitro and in vivo. We first characterized the most used RMS cell lines representing the most common subtypes, embryonal (ERMS, RD cells) and alveolar (ARMS, RH30 cells), for their iron metabolism, in basal condition and in response to its modulation. Then we investigated the effects of both iron overload and chelation strategies in vitro and in vivo. RMS cell lines expressed iron-related proteins, even if at lower levels compared to hepatic cell lines and they are correctly modulated in response to iron increase and deprivation. Interestingly, the treatment with different doses of ferric ammonium citrate (FAC, as iron source) and with deferiprone (DFP, as iron chelator), significantly affected the cell viability of RD and RH30. Moreover, iron supplementation (in the form of iron dextran) or iron chelation (in the form of DFP) were also effective in vivo in inhibiting the tumor mass growth both derived from RD and RH30 with iron chelation treatment the most effective one. All the data suggest that the iron modulation could be a promising approach to overcome the RMS tumor growth. The mechanism of action seems to involve the apoptotic cell death for both iron supplementation and chelation with the concomitant induction of ferroptosis in the case of iron supplementation.

Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model.

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time imaging and acoustic energy modulation, this modality enables precise temperature control within a defined area. Many thermal applications are being explored with this noninvasive, nonionizing technology, such as hyperthermia generation, to release drugs from thermosensitive liposomal carriers. These drugs can include chemotherapies such as doxorubicin, for which targeted release is desired due to the dose-limiting systemic side effects, namely cardiotoxicity. Doxorubicin is a mainstay for treating a variety of malignant tumors and is commonly used in relapsed or recurrent rhabdomyosarcoma (RMS). RMS is the most common solid soft tissue extracranial tumor in children and young adults. Despite aggressive, multimodal therapy, RMS survival rates have remained the same for the past 30 years. To explore a solution for addressing this unmet need, an experimental protocol was developed to evaluate the release of thermosensitive liposomal doxorubicin (TLD) in an immunocompetent, syngeneic RMS mouse model using MRgHIFU as the source of hyperthermia for drug release.

Triggered release from thermosensitive liposomes improves tumor targeting of vinorelbine.

Triggered drug delivery strategies have been shown to enhance drug accumulation at target diseased sites in comparison to administration of free drug. In particular, many studies have demonstrated improved targetability of chemotherapeutics when delivered via thermosensitive liposomes. However, most studies continue to focus on encapsulating doxorubicin while many other drugs would benefit from this targeted and localized delivery approach. The proposed study explores the therapeutic potential of a thermosensitive liposome formulation of the commonly used chemotherapy drug vinorelbine in combination with mild hyperthermia (39-43 °C) in a murine model of rhabdomyosarcoma. Rhabdomyosarcoma, the most common soft tissue sarcoma in children, is largely treated using conventional chemotherapy which is associated with significant adverse long-term sequelae. In this study, mild hyperthermia was pursued as a non-invasive, non-toxic means to improve the efficacy and safety profiles of vinorelbine. Thorough assessment of the pharmacokinetics, biodistribution, efficacy and toxicity of vinorelbine administered in the thermosensitive liposome formulation was compared to administration in a traditional, non-thermosensitive liposome formulation. This study shows the potential of an advanced formulation technology in combination with mild hyperthermia as a means to target an untargeted therapeutic agent and result in a significant improvement in its therapeutic index.

Cytotoxic Effect of Rosmarinus officinalis Extract on Glioblastoma and Rhabdomyosarcoma Cell Lines.

Rosmarinus officinalis is a well-studied plant, known for its therapeutic properties. However, its biological activity against several diseases is not known in detail. The aim of this study is to present new data regarding the cytotoxic activity of a hydroethanolic extract of Rosmarinus officinalis on glioblastoma (A172) and rhabdomyosarcoma (TE671) cancer cell lines. The chemical composition of the extract is evaluated using liquid chromatography combined with time-of-flight mass spectrometry, alongside its total phenolic content and antioxidant activity. The extract showed a promising time- and dose-dependent cytotoxic activity against both cell lines. The lowest IC50 values for both cell lines were calculated at 72 h after treatment and correspond to 0.249 ± 1.09 mg/mL for TE671 cell line and 0.577 ± 0.98 mg/mL for A172 cell line. The extract presented high phenolic content, equal to 35.65 ± 0.03 mg GAE/g of dry material as well as a strong antioxidant activity. The IC50 values for the antioxidant assays were estimated at 12.8 ± 2.7 µg/mL (DPPH assay) and 6.98 ± 1.9 µg/mL (ABTS assay). The compound detected in abundance was carnosol, a phenolic diterpene, followed by the polyphenol rosmarinic acid, while the presence of phenolic compounds such as rhamnetin glucoside, hesperidin, cirsimaritin was notable. These preliminary results suggest that R. officinalis is a potential, alternative source of bioactive compounds to further examine for abilities against glioblastoma and rhabdomyosarcoma.

Alternative splicing of Apoptosis Stimulating Protein of TP53-2 (ASPP2) results in an oncogenic isoform promoting migration and therapy resistance in soft tissue sarcoma (STS).

BACKGROUND: Metastatic soft tissue sarcoma (STS) are a heterogeneous group of malignancies which are not curable with chemotherapy alone. Therefore, understanding the molecular mechanisms of sarcomagenesis and therapy resistance remains a critical clinical need. ASPP2 is a tumor suppressor, that functions through both p53-dependent and p53-independent mechanisms. We recently described a dominant-negative ASPP2 isoform (ASPP2κ), that is overexpressed in human leukemias to promote therapy resistance. However, ASPP2κ  has never been studied in STS.  MATERIALS AND METHODS: Expression of ASPP2κ was quantified in human rhabdomyosarcoma tumors using immunohistochemistry and qRT-PCR from formalin-fixed paraffin-embedded (FFPE) and snap-frozen tissue. To study the functional role of ASPP2κ in rhabdomyosarcoma, isogenic cell lines were generated by lentiviral transduction with short RNA hairpins to silence ASPP2κ expression. These engineered cell lines were used to assess the consequences of ASPP2κ silencing on cellular proliferation, migration and sensitivity to damage-induced apoptosis. Statistical analyses were performed using Student's t-test and 2-way ANOVA. RESULTS: We found elevated ASPP2κ mRNA in different soft tissue sarcoma cell lines, representing five different sarcoma sub-entities. We found that ASSP2κ mRNA expression levels were induced in these cell lines by cell-stress. Importantly, we found that the median ASPP2κ expression level was higher in human rhabdomyosarcoma in comparison to a pool of tumor-free tissue. Moreover, ASPP2κ levels were elevated in patient tumor samples versus adjacent tumor-free tissue within individual patients. Using isogenic cell line models with silenced ASPP2κ expression, we found that suppression of ASPP2κ enhanced chemotherapy-induced apoptosis and attenuated cellular proliferation. CONCLUSION: Detection of oncogenic ASPP2κ in human sarcoma provides new insights into sarcoma tumor biology. Our data supports the notion that ASPP2κ promotes sarcomagenesis and resistance to therapy. These observations provide the rationale for further evaluation of ASPP2κ as an oncogenic driver as well as a prognostic tool and potential therapeutic target in STS.

Increasing the efficiency of hyperthermic intraperitoneal chemotherapy (HIPEC) by combination with a photosensitive drug in pediatric rhabdomyosarcoma in an animal model.

BACKGROUND: Cytoreductive surgery (CRS) in combination with hyperthermic intraperitoneal chemotherapy (HIPEC) is an option in advanced peritoneal sarcomatosis. Nevertheless, CRS and HIPEC are not successful in all patients. An enhancement of HIPEC using photodynamic therapy (PDT) might be beneficial. Therefore, a combination of the photosensitizer hypericin (HYP) with HIPEC was evaluated in an animal model. PROCEDURE: An established HIPEC animal model for rhabdomyosarcoma (NOD/LtSz-scid IL2Rγnullmice, n = 80) was used. All groups received HYP (100 µg/200 µl) intraperitoneally with and without cisplatin-based (30 or 60 mg/m2 ) HIPEC (37°C or 42°C, for 60 minutes) (five groups, each n = 16). Peritoneal cancer index (PCI) was documented visually and by HYP-based photodynamic diagnosis (PDD). HYP-based PDT of the tumor was performed. Tissue samples were evaluated regarding proliferation (Ki-67) and apoptosis (TUNEL). RESULTS: HYP uptake was detected even in smallest tumor nodes (<1 mm) with improved tumor detection during PDD (PCI with PDD vs. PCI without PDD: 8.5 vs. 7, p < .001***). Apoptotic effects after PDT without HIPEC were limited to the tumor surface, whereas PDT after HIPEC (60 mg/m2 , 42°C) showed additional reduction of tumor proliferation in the top nine to 11 cell layers (50 µm). CONCLUSION: HYP as fluorescent photosensitizer offers an intraoperative diagnostic advantage detecting intraperitoneal tumor dissemination. The combination of HYP and cisplatin-based HIPEC was feasible in vivo, showing enhanced effects on tumor proliferation and apoptosis induction across the tumor surface. Further studies combining HYP and HIPEC will follow to establish a clinical application.

The Effectiveness of the Nimali Variety of Sri Lankan Punica granatum L. Fruit Extracts on Rhabdomyosarcoma (RD) Cells Concerning the Apoptotic Signaling Pathway.

OBJECTIVE: Pomegranate ,a polyphenol-rich fruit, has been considered as one of the ancient fruits with anticancer effect. Cell cycle arrest is considered as an ordinary factor in human cancer, and apoptosis is the frequent drug target. This study aimed to evaluate the effectiveness of the Nimali variety of Sri Lankan Punica granatum L. fruit extracts on rhabdomyosarcoma (RD) cells concerning the apoptotic signaling pathway. METHODS: Antiproliferative activity of aqueous extracts of pomegranate peel, pericarp, was assessed using multiple extraction methods (sonication, microwaving, sonication followed by microwaving, keeping in a waterbath, and boiling at 100ºC). Total protein content, nitric oxide production, LDH, and caspase-8 and caspase-3 activities were analyzed in peel extracts prepared by sonicated or microwave methods. RT-qPCR was performed with intact RNA to explore the apoptotic pathway and gene expression. RESULTS: Peel extracts expressed minimum cell viability in a dose-dependent manner, induced cell death on RD cells. However, sonicated peel extract (SPL) indicated the lowest IC50 of 14.8±2.2 µg/mL comparative to healthy VERO cells (>1,000 µg/mL). A decrease of nitrite content in the supernatant was visualized in the graph plotted against concentration. Furthermore, SPL upregulated caspase-8 and caspase-3 signaling pathways and expression of p21 and p53 genes. CONCLUSION: The findings highlighted the promising therapeutic potential of SPL to inhibit RD growth and progression and to modulate the caspase-8 and caspase-3, p53, and p21 dependent pathway.

Study of synergistic effects of Ficus Carica leaves extract mediated chemo-photodynamic therapy on rhabdomyosarcoma cells.

BACKGROUND: Chemotherapy for rhabdomyosarcoma (RD) is effective, but it has critical side effects and unavoidable challenges. Photodynamic therapy (PDT) is an approach to treating cancer with relatively moderate side effects. Plant products are a rich source of polyphenols, which have potent antioxidant and anticancer activities. Therefore, their research has become an emerging field in recent decades. PURPOSE: This work aimed to evaluate the potential of hydrophobic extract of Ficus Carica (FC) to determine whether FC in the presence of low dose chemo and Aluminium Phthalocyanine (Photosense®) mediated photodynamic therapy synergistically enhances the treatment efficacy of RD cells. METHOD: FC with and without combination with individual therapeutic modalities like photosense mediated photodynamic therapy, chemotherapy, and their combinations were studied for cell viability and morphological changes in invitro RD cells. A semiconductor diode laser (630 nm) was used as a light source in PDT. The cytotoxic effect of FC on cell viability and cellular morphological changes were investigated by MTT reagent and a camera attached to an inverted visible light microscope. The effect of FC, followed by di-combination with low dose chemo (doxorubicin-HCl, and dacarbazine), Photosense® mediated PDT and chemo-Photosense® mediated PDT (tri-combination) at 630 nm diode laser and 10 J/cm2 fluency were also investigated by MTT reagent. The combination index method is used to identify the synergistic effect of combination therapy by using CompuSyn software based on the Chou-Talalay method. RESULTS: The dose-dependent effect of FC on cell viability and cellular morphological changes were observed in the RD cell line. It was found that the pre incubation of FC potentiated the anticancer effect as a neoadjuvant agent for doxorubicin-HCl and decarbazine based chemotherapy, Photosense® mediated PDT and chemo-PDT (tri-combination) with synergistic effect (CI<1). CONCLUSION: These results suggest a possible thread that the low dose combination of the aforementioned therapeutic modalities in the presence of FC remarkably enhances the treatment efficacy of RD in comparison with a single-agent treatment modality. The proposed sequence of FC with chemo and PDT might present better therapeutic outcomes in RD therapies and may provide result for RD metastasis. FC may also be used in the application of phyto-PDT to cancer in the future.

Vincetoxicum arnottianum modulates motility features and metastatic marker expression in pediatric rhabdomyosarcoma by stabilizing the actin cytoskeleton.

BACKGROUND: Prevention of metastatic invasion is one of the main challenges in the treatment of alveolar rhabdomyosarcoma. Still the therapeutic options are limited. Therefore, an anti-tumor screening was initiated focusing on the anti-metastatic and anti-invasion properties of selected medicinal plant extracts and phytoestrogens, already known to be effective in the prevention and treatment of different cancer entities. METHODS: Treatment effects were first evaluated by cell viability, migration, invasion, and colony forming assays on the alveolar rhabdomyosarcoma cell line RH-30 in comparison with healthy primary cells. RESULTS: Initial anti-tumor screenings of all substances analyzed in this study, identified the plant extract of Vincetoxicum arnottianum (VSM) as the most promising candidate, harboring the highest anti-metastatic potential. Those significant anti-motility properties were proven by a reduced ability for migration (60%), invasion (99%) and colony formation (61%) under 48 h exposure to 25 µg/ml VSM. The restricted motility features were due to an induction of the stabilization of the cytoskeleton - actin fibers were 2.5-fold longer and were spanning the entire cell. Decreased proliferation (PCNA, AMT, GCSH) and altered metastasis (e. g. SGPL1, CXCR4, stathmin) marker expression on transcript and protein level confirmed the significant lowered tumorigenicity under VSM treatment. Finally, significant alterations in the cell metabolism were detected for 25 metabolites, with levels of uracil, N-acetyl serine and propanoyl phosphate harboring the greatest alterations. Compared to the conventional therapy with cisplatin, VSM treated cells demonstrated a similar metabolic shutdown of the primary cell metabolism. Primary control cells were not affected by the VSM treatment. CONCLUSIONS: This study revealed the VSM root extract as a potential, new migrastatic drug candidate for the putative treatment of pediatric alveolar rhabdomyosarcoma with actin filament stabilizing properties and accompanied by a marginal effect on the vitality of primary cells.

Epigenetic regulator BMI1 promotes alveolar rhabdomyosarcoma proliferation and constitutes a novel therapeutic target.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma. There are two main subtypes of RMS, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma. ARMS typically encompasses fusion-positive rhabdomyosarcoma, which expresses either PAX3-FOXO1 or PAX7-FOXO1 fusion proteins. There are no targeted therapies for ARMS; however, recent studies have begun to illustrate the cooperation between epigenetic proteins and the PAX3-FOXO1 fusion, indicating that epigenetic proteins may serve as targets in ARMS. Here, we investigate the contribution of BMI1, given the established role of this epigenetic regulator in sustaining aggression in cancer. We determined that BMI1 is expressed across ARMS tumors, patient-derived xenografts, and cell lines. We depleted BMI1 using RNAi and inhibitors (PTC-209 and PTC-028) and found that this leads to a decrease in cell growth/increase in apoptosis in vitro, and delays tumor growth in vivo. Our data suggest that BMI1 inhibition activates the Hippo pathway via phosphorylation of LATS1/2 and subsequent reduction in YAP levels and YAP/TAZ target genes. These results identify BMI1 as a potential therapeutic vulnerability in ARMS and warrant further investigation of BMI1 in ARMS and other sarcomas.

Antitumor effects of curcumin in pediatric rhabdomyosarcoma in combination with chemotherapy and phototherapy in vitro.

Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, has an unfavorable outcome in advanced tumor stages with less than 30% failure­free survival. Curcumin (CUR) is a promising drug in complementary oncology with few side effects but proven efficacy in various adult oncological entities. The present study analyzed the effects of CUR on pediatric (RMS) cell lines in vitro. RMS cell lines (RD and RH30), and skeletal muscle cells (SKMC) were treated with different doses of CUR (1.5­30 µM) alone, with phototherapy (PDT, 488 nm) or in combination with vincristine (VCR) or dactinomycin (DAC). MTT assays were used for analysis of RMS tumor cell viability. Clonal cell growth was assessed via colony forming assays and migration of the cells was analyzed with scratch tests. Annexin V staining was used to determine apoptosis in flow cytometry. Possible RMS resistance towards CUR after long­term treatment was analyzed with MTT assays. CUR decreased cell viability in all assessed RMS cell lines in a concentration­dependent manner with IC50=14­20 µM. CUR enhanced the effects of the cytotoxic drugs VCR or DAC, and led to reduced migration and increased cell apoptosis. In combination with PDT, CUR decreased the cell viability in minute quantities with up to a 10­fold lower IC50 than without PDT. CUR effectively inhibited the malignant properties of pediatric RMS cells and should be focused on as a useful additional agent in standard chemotherapy of RMS in children.

Ginsenoside Rb1 is an immune-stimulatory agent with antiviral activity against enterovirus 71.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the theory of traditional Chinese medicine, the main pathogenesis of severe hand, foot and mouth disease (HFMD) is that the heat and wet poisons are deeply trapped in the viscera, which causes the deficiency of Qi and Yin in the patient's body. Ginsenoside Rb1 (Rb1) is the most abundant triterpenoid saponin in Panax quinquefolius L., which has the function of Qi-invigorating and Yin-nourishing. Enterovirus 71 (EV71) is one of the causative pathogens of HFMD, especially the form associated with some lethal complications. Therefore, the therapeutic effect of Rb1 on this disease caused by EV71 infection is worth exploring. AIM OF THE STUDY: We explored the effective antiviral activities of Rb1 against EV71 in vitro and in vivo and investigated its preliminary antiviral mechanisms. MATERIAL AND METHODS: EV71-infected two-day-old suckling mice model was employed to detect the antiviral effects of Rb1 in vivo. To detect the antiviral effects of Rb1 in vitro, cytopathic effect (CPE) reduction assay was performed in EV71-infected Rhabdomyosarcoma (RD) cells. Interferon (IFN)-ß interference experiment was employed to detect the antiviral mechanism of Rb1. RESULTS: In this paper, we first found that Rb1 exhibited strong antiviral activities in EV71-infected suckling mice when compared to those of ribavirin. Administration of Rb1 reduced the CPE of EV71-infected RD cells in a dose-dependent manner. Moreover, EV71-induced viral protein-1 (VP-1) expression was significantly reduced by Rb1 administration in vitro and in vivo. Furthermore, Rb1 treatment could induce high cellular and humoral immune responses in vivo. Meanwhile, Rb1 contributed to the enhanced Type I IFN responses and IFN-ß knockdown reversed the antiviral activity of Rb1 in vitro. CONCLUSION: In summary, our findings suggest that Rb1 is an immune-stimulatory agent and provide an insight into therapeutic potentials of Rb1 for the treatment of EV71 infection.

The common diabetes drug metformin can diminish the action of citral against Rhabdomyosarcoma cells in vitro.

Rhabdomyosarcoma (RMS) is a rare type of soft tissue sarcoma most commonly found in pediatric patients. Despite progress, new and improved drug regimens are needed to increase survival rates. Citral, a natural product plant oil can induce cell death in cancer cells. Another compound, metformin, isolated originally from French lilac and used by diabetics, has been shown to reduce the incidence of cancer in these patients. Application of citral to RMS cells showed increase in cell death, and RD and RH30 cells showed half maximal inhibitory concentration (IC50 ) values as low as 36.28 µM and 62.37 µM, respectively. It was also shown that the citral initiated cell apoptosis through an increase in reactive oxygen species (ROS) and free calcium. In comparison, metformin only showed moderate cell death in RMS cell lines at a very high concentration (1,000 µM). Combinatorial experiments, however, indicated that citral and metformin worked antagonistically when used together. In particular, the ability of metformin to quench the ROS induced by citral could lead to the suppression of activity. These results clearly indicate that while clinical use of citral is a promising anti-tumor therapy, caution should be exercised in patients using metformin for diabetes.

Magnoflorine-Isolation and the Anticancer Potential against NCI-H1299 Lung, MDA-MB-468 Breast, T98G Glioma, and TE671 Rhabdomyosarcoma Cancer Cells.

Magnoflorine (MGN) is a quaternary aporphine alkaloid that exhibits numerous therapeutic properties, including neuropsychopharmacological, anti-anxiety, immunomodulatory, anti-inflammatory, antioxidant, or antifungal activities. The aim of the present study was an investigation of the influence of MGN on viability, proliferation, induction of apoptosis, and cell cycle arrest in NCI-H1299 lung, MDA-MB-468 breast, T98G glioma, and TE671 rhabdomyosarcoma cancer cells. MGN was isolated from the roots of Berberis cretica L. by counter-current partition chromatography (CPC). Cell viability and proliferation assessments were performed by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and 5-bromo-2'-deoxyuridine (BrDU) assays, respectively. The induction of apoptosis and cell cycle progression was measured using fluorescence-activated cell sorting analysis. MGN in high doses inhibits proliferation, induces apoptosis, and inhibits cell cycle in S/G2 phases in a dose-dependent manner. MGN seems to be a promising anti-cancer compound in therapy of some types of lung, breast, glioma, and rhabdomyosarcoma cancers, for which current standard therapies are limited or have severe strong side effects.

Targeting Hippo-Dependent and Hippo-Independent YAP1 Signaling for the Treatment of Childhood Rhabdomyosarcoma.

Rhabdomyosarcoma is the most common childhood soft-tissue sarcoma, yet patients with metastatic or recurrent disease continue to do poorly, indicating a need for new treatments. The SRC family tyrosine kinase YES1 is upregulated in rhabdomyosarcoma and is necessary for growth, but clinical trials using single agent dasatinib, a SRC family kinase inhibitor, have failed in sarcomas. YAP1 (YES-associated protein) is highly expressed in rhabdomyosarcoma, driving growth and survival when the upstream Hippo tumor suppressor pathway is silenced, but efforts to pharmacologically inhibit YAP1 have been unsuccessful. Here we demonstrate that treatment of rhabdomyosarcoma with DNA methyltransferase inhibitor (DNMTi) upregulates Hippo activators RASSF1 and RASSF5 by promoter demethylation, activating canonical Hippo signaling and increasing inactivation of YAP1 by phosphorylation. Treatment with DNMTi decreased rhabdomyosarcoma cell growth and increased apoptosis and differentiation, an effect partially rescued by expression of constitutively active YAP (S127A), suggesting the effects of DNMTi treatment are, in part, due to Hippo-dependent inhibition of YAP1. In addition, YES1 and YAP1 interacted in the nucleus of rhabdomyosarcoma cells, and genetic or pharmacologic suppression of YES1 resulted in cytoplasmic retention of YAP1 and decreased YAP1 target gene expression, suggesting YES1 regulates YAP1 in a Hippo-independent manner. Combined treatment with DNMTi and dasatinib targeted both Hippo-dependent and Hippo-independent regulation of YAP1, ablating rhabdomyosarcoma cell growth in vitro and trending toward decreased tumor growth in vivo. These results show that the mechanisms regulating YAP1 in rhabdomyosarcoma can be inhibited by combinatorial therapy of DNMTi and dasatinib, laying the groundwork for future clinical investigations. SIGNIFICANCE: This study elucidates the signaling pathways that regulate the oncogenic protein YAP1 and identifies a combination therapy to target these pathways in the childhood tumor rhabdomyosarcoma.

Imperatorin as a Promising Chemotherapeutic Agent Against Human Larynx Cancer and Rhabdomyosarcoma Cells.

Naturally occurring coumarins are bioactive compounds widely used in Asian traditional medicine. They have been shown to inhibit proliferation, induce apoptosis, and/or enhance the cytotoxicity of currently used drugs against a variety of cancer cell types. The aim of our study was to examine the antiproliferative activity of different linear furanocoumarins on human rhabdomyosarcoma, lung, and larynx cancer cell lines, and dissolve their cellular mechanism of action. The coumarins were isolated from fruits of Angelica archangelica L. or Pastinaca sativa L., and separated using high-performance counter-current chromatography (HPCCC). The identity and purity of isolated compounds were confirmed by HPLC-DAD and NMR analyses. Cell viability and toxicity assessments were performed by means of methylthiazolyldiphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, respectively. Induction of apoptosis and cell cycle progression were measured using flow cytometry analysis. qPCR method was applied to detect changes in gene expression. Linear furanocoumarins in a dose-dependent manner inhibited proliferation of cancer cells with diverse activity regarding compounds and cancer cell type specificity. Imperatorin (IMP) exhibited the most potent growth inhibitory effects against human rhabdomyosarcoma and larynx cancer cell lines owing to inhibition of the cell cycle progression connected with specific changes in gene expression, including CDKN1A. As there are no specific chemotherapy treatments dedicated to laryngeal squamous cell carcinoma and rhabdomyosarcoma, and IMP seems to be non-toxic for normal cells, our results could open a new direction in the search for effective anti-cancer agents.

Systemic antitumor effect by regional hyperthermia combined with low-dose chemotherapy and immunologic correlates in an adolescent patient with rhabdomyosarcoma - a case report.

Introduction: An abscopal effect is a clinical observation whereby a local treatment is associated with regression of metastatic cancer at a site distant from the primary location of treatment. Here, we describe the clinical systemic effect induced by regional hyperthermia combined with low-dose chemotherapy and provide immunologic correlates.Case presentation: A 15-year-old patient had been diagnosed with alveolar rhabdomyosarcoma (ARMS). All previous treatment options failed in the patient including haploidentical stem cell transplantation and donor lymphocyte infusion. The patient presented with local and metastatic disease, and upon admission, underwent regional hyperthermia combined with low-dose chemotherapy. Immediately following therapy severe skin reactions were observed. Skin biopsies revealed an intraepithelial lymphocytic infiltration dominated by CD3+/CD8+ T cells with a regular network of dendritic cells. Clinical images compared before and during sequential treatment cycles showed complete metabolic response of the local tumor for more than 10 months of therapy. In addition, metastases completely regressed although they were not direct targets of regional hyperthermia. The systemic effect was associated with enhanced frequency of NK cells and T cells expressing the lectin-like natural-killer group 2 D activating receptor (NKG2D), an increase of the CD56bright subset of NK cells, as well as an increase of effector/memory and effector CD8+ and CD4+ T cells in the blood while the percentage of CD25+FOXP3+ regulatory T cells declined.Conclusions: Regional hyperthermia combined with low-dose chemotherapy had the potential to create a systemic effect which was associated with activation of NK cells and T cells.

Do not Forget Burkitt´s Lymphoma! Unusual Case of Primary Malignant Lymphoma of the Prostate in a Young Patient.

Primary Burkitt´s lymphoma (BL) of the prostate is rare in the adolescent population. The etiology remains poorly understood. There has been some proposed associations to Epstein-Barr virus and HIV. Clinical and histopathologic data of a 17-year-old patient who underwent transurethral resection of the prostate was obtained. We report the first case of primary malignant BL of the prostate in a 17-year-old Caucasian male who presented with hematuria, lower urinary tract symptoms. Differential diagnosis of a prostatic mass in adolescent patients must be primary or secondary lymphoma of the prostate, including BL as described for the first time in this article.

Assessment of Intratumoral Doxorubicin Penetration after Mild Hyperthermia-Mediated Release from Thermosensitive Liposomes.

In solid tumors, rapid local intravascular release of anticancer agents, e.g., doxorubicin (DOX), from thermosensitive liposomes (TSLs) can be an option to overcome poor extravasation of drug nanocarriers. The driving force of DOX penetration is the drug concentration gradient between the vascular compartment and the tumor interstitium. In this feasibility study, we used fibered confocal fluorescence microscopy (FCFM) to monitor in real-time DOX penetration in the interstitium of a subcutaneous tumor after its intravascular release from TSLs, Thermodox®. Cell uptake kinetics of the released DOX was quantified, along with an in-depth assessment of released-DOX penetration using an evolution model. A subcutaneous rat R1 rhabdomyosarcoma xenograft was used. The rodent was positioned in a setup including a water bath, and FCFM identification of functional vessels in the tumor tissue was applied based on AngioSense. The tumor-bearing leg was immersed in the 43°C water for preheating, and TSLs were injected intravenously. Real-time monitoring of intratumoral (i.t.) DOX penetration could be performed, and it showed the progressing DOX wave front via its native fluorescence, labeling successively all cell nuclei. Cell uptake rates (1/k) of 3 minutes were found (n=241 cells), and a released-DOX penetration in the range of 2500 µm2·s-1 was found in the tumor extravascular space. This study also showed that not all vessels, identified as functional based on AngioSense, gave rise to local DOX penetration.