A new valid rhabdomyosarcoma spheroid culture model for in vitro evaluation of hypericin-based photodynamic therapy.

BACKGROUND: Advanced stages of pediatric alveolar rhabdomyosarcoma (RMA) are associated with an unfavorable outcome at established therapeutic strategies, accentuating the need for novel treatment options. Photodynamic therapy (PDT) with hypericin (HYP) has shown strong cytotoxic effects in two-dimensional (2D) cell culture. In order to more accurately mimic in vivo tissue architecture and better predict pharmaceutical response, the aim of this study was to establish a spheroid culture model by which PDT efficacy could be assessed in a three-dimensional (3D) context. MATERIALS AND METHODS: 3D multicellular tumor spheroids were generated using various scaffold-based and scaffold-free techniques. On two reproducible methods, HYP-PDT was performed varying spheroid sizes, photosensitizer concentrations, and illumination times. The ability for HYP uptake within the spheroid was analyzed assessing the substrate's autofluorescence. Antitumorigenic treatment effects were evaluated investigating cell viability, spheroid morphology, proliferative activity, and induction of apoptosis. RESULTS: Magnetic spheroid printing and orbital shaking methods were established as reproducible culturing systems producing uniform spheroids. Within assessed incubation times, HYP showed good penetration depth in spheroids containing 50,000 cells. PDT was causing metabolic and molecular impairment of RMA cells, resulting in viability decrease, reduction of cell proliferation, and induction of apoptosis. CONCLUSION: Assessing HYP-based PDT in a 3D culture model, we were able to gain an insight on how parameters like photosensitizer, oxygen, and light distribution contribute to the phototoxic effect. Compared to 2D cell culture, a higher treatment resistance was detected, which can be related to spheroid structure and mechanisms of intercellular communication, signal transduction, and gene expression.

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.

Establishment of a new valid animal model for the evaluation of hyperthermic intraperitoneal chemotherapy (HIPEC) in pediatric rhabdomyosarcoma.

BACKGROUND: Cytoreductive surgery in combination with hyperthermic intraperitoneal chemotherapy has been established as a novel treatment approach for peritoneal sarcomatosis. Despite promising clinical reports, there is still a lack of knowledge regarding optimal drug usage and local effects. Therefore, we intended to establish a murine animal model for further evaluation. PROCEDURE: Alveolar rhabdomyosarcoma cells were xenotransplanted into NOD/LtSz-scid IL2Rγnullmice (n = 100). The mice received a continuous intraperitoneal lavage with isotonic saline solution as control or with cisplatin (30 or 60 mg/m2 ) as treatment group for 60 minutes at 37°C or 42°C (6 subgroups, each n = 16). Tumor spread was documented by an adapted peritoneal cancer index and MRI (n = 4). Tumor and tissue samples, harvested at the end of the perfusion, were evaluated regarding morphology, proliferation, and apoptosis (H&E-, Ki-67-, cleaved caspase 3-staining, TUNEL assay). RESULTS: Extensive peritoneal sarcomatosis in over 91% of the cases was observed. HIPEC was feasible without acute side effects. Ki-67 staining revealed concentration- or temperature-dependent effects of cisplatin-based HIPEC on the tumors. Although cleaved caspase-3 showed only sporadic apoptotic effects. TUNEL assay detected concentration- or temperature-dependent apoptotic effects at the outer tumor surface. MRI scans confirmed the observed tumor dissemination. CONCLUSION: This is the first animal model for evaluation of HIPEC in pediatric RMS in mice. Cisplatin-based HIPEC had early effects on the proliferation whereas circumscribed apoptotic effects could be detected at the tumor surface. This model allows further insights on the possible efficiency of HIPEC in RMS. Further studies using other drug combinations and treatment will follow.

Antitumor evaluation of two selected Pakistani plant extracts on human bone and breast cancer cell lines.

BACKGROUND: The medicinal plants Vincetoxicum arnottianum (VSM), Berberis orthobotrys (BORM), Onosma hispida (OHRM and OHAM) and Caccinia macranthera (CMM) are used traditionally in Pakistan and around the world for the treatment of various diseases including cancer, dermal infections, uterine tumor, wounds etc. The present study focuses on the investigation of the selected Pakistani plants for their potential as anticancer agents on human bone and breast cancer cell lines in comparison with non-tumorigenic control cells. METHODS: The antitumor evaluation was carried out on human bone (MG-63, Saos-2) and breast cancer cell lines (MCF-7, BT-20) in contrast to non-tumorigenic control cells (POB, MCF-12A) via cell viability measurements, cell cycle analysis, Annexin V/PI staining, microscopy based methods as well as migration/invasion determination, metabolic live cell monitoring and western blotting. RESULTS: After the first initial screening of the plant extracts, two extracts (BORM, VSM) revealed the highest potential with regard to its antitumor activity. Both extracts caused a significant reduction of cell viability in the breast and bone cancer cells in a concentration dependent manner. The effect of VSM is achieved primarily by inducing a G2/M arrest in the cell cycle and the stabilization of the actin stress fibers leading to reduced cell motility. By contrast BORM's cytotoxic properties were caused through the lysosomal-mediated cell death pathway indicated by an upregulation of Bcl-2 expression. CONCLUSIONS: The antitumor evaluation of certain medicinal plants presented in this study identified the methanolic root extract of Berberis orthobotrys and the methanolic extract of Vincetoxicum arnottianum as promising sources for exhibiting the antitumor activity. Therefore, the indigenous use of the herbal remedies for the treatment of cancer and cancer-related diseases has a scientific basis. Moreover, the present study provides a base for phytochemical investigation of the plant extracts.

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.

SGPL1321 mutation: one main trigger for invasiveness of pediatric alveolar rhabdomyosarcoma.

Sphingosine-1-phosphate (S1P), a sphingolipid with second messenger properties, is a main regulator of various cellular processes including lymphocyte cell trafficking, angiogenesis, cell proliferation, and survival. High S1P concentrations and deficiencies in S1P degradation have been associated with cancer cell progression, their directed chemoattraction and promotion of chemo-resistance mechanism. The endoplasmic reticulum (ER) membrane localized enzyme sphingosine-1-phosphate lyase (SGPL1) has a key role in prevention of S1P overstimulation in tumor cells by its irreversible S1P degradation activity. In this paper we demonstrated a SGPL1 overexpression and mislocalization in pediatric alveolar rhabdomyosarcoma (RMA) cells. Moreover, a homozygous point mutation from A to G at position 321 in the coding sequence was obvious, which interferes with the S1P degradation activity and correct localization in the ER-membrane. By complementation with the native SGPL1 variant, the ER localization was restored in RMA cells. More importantly, the SGPL1 restauration prevents the S1P induced migration and colony formation of RMA cells, significantly. This observation opens new highways for the treatment of pediatric RMA by gene therapeutic SGPL1 renewal and recommends the detection of specific SGPL1 mutations as pathological, molecular metastasis marker.

Hypericin and its radio iodinated derivatives - A novel combined approach for the treatment of pediatric alveolar rhabdomyosarcoma cells in vitro.

BACKGROUND: Alveolar rhabdomyosarcoma (RMA) is a highly malignant soft tissue tumor in children with poor prognosis and failure of established therapies in advanced stages. Therefore, novel treatment options are required. Photodynamic therapy (PDT) has been found useful for the treatment of different tumor entities and might represent such a novel treatment option. A major limitation of PDT remains the restriction to superficial tumor cell layers as illumination with light is essential for the generation of reactive oxygen species. Current research focusses on the development of modified Hypericin (HYP)-based photosensitizers, as well as combining PDT and targeted internal radiotherapy with 131I, to generate an additive anti-tumor effect. METHODS: A standardized protocol for in vitro Hypericin-PDT was established in RMA cells. The anti-tumor properties of this photosensitizer were analyzed on molecular and metabolic levels. Changes in cell morphology were visualized using bright field-, fluorescence- and scanning-electron microscopy. Iodinated Hypericin derivatives with both radioactive and non-radioactive isotopes 131I/127I were employed to establish a targeted radionuclide therapy and investigate the potential of a combined treatment with PDT. RESULTS: In vitro photodynamic treatment with Hypericin showed a strong anti-tumor efficiency with favorable cellular uptake and compromised cancer cells on metabolic and molecular levels. Iodination of the photosensitizer did not impair the photosensitizer´s properties. Targeted radiotherapy with 131I-HYP led to distinct reductions of tumor viability. A simultaneously performed PDT leads to a reduction of cell viability that begins earlier in time. However, an additive enhancement of the cell viability was not observed in the selected dose range. CONCLUSION: In this in vitro study, we got a first insight of a possible potential of Hypericin for the treatment of pediatric soft tissue sarcoma. By coupling with radioiodine, we developed a novel approach for a combined anti-tumor treatment. The in vitro experiments lay the foundation for further in vivo experiments, which are needed to study the effects of a sequential administration of 131I-HYP and HYP.

Berberis orthobotrys - A promising herbal anti-tumorigenic candidate for the treatment of pediatric alveolar rhabdomyosarcoma.

ETHNOPHARMACOLOGICAL RELEVANCE: Berberis orthobotrys (BORM) is a medical plant with a long history in traditional usage for the treatment of wounds, cancer, gastrointestinal malady and several other diseases. Our previous studies identified the endemic Pakistani plant Berberis orthobotrys Bien. ex Aitch. as promising source for the treatment of breast cancer and osteosarcoma. AIM OF THE STUDY: The present study was aimed to evaluate the anti-cancer properties of 26 plant derived extracts and compounds including the methanolic root extract of Berberis orthobotrys (BORM) on pediatric alveolar rhabdomyosarcoma (RMA), which is known to develop drug resistance, metastatic invasion and potential tumor progression. MATERIALS AND METHODS: The main anti-tumor activity of BORM was verified by focusing on morphological, cell structural and metabolic alterations via metabolic profiling, cell viability measurements, flow cytometry, western blotting and diverse microscopy-based methods using the human RMA cell line Rh30. RESULTS: Exposure of 25 µg/ml BORM exerts an influence on the cell stability, the degradation of oncosomes as well as the shutdown of the metabolic activity of RMA cells, primarily by downregulation of the energy metabolism. Therefore glycyl-aspartic acid and N-acetyl serine decreased moderately, and uracil increased intracellularly. On healthy, non-transformed muscle cells BORM revealed very low metabolic alterations and nearly no cytotoxic impact. Furthermore, BORM is also capable to reduce Rh30 cell migration (~50%) and proliferation (induced G2/M cycle arrest) as well as to initiate apoptosis confirmed by reduced Bcl-2, Bax and PCNA expression and induced PARP-1 cleavage. CONCLUSIONS: The study provides the first evidence, that BORM treatment is effective against RMA cells with low side effects on healthy cells.

GCSH antisense regulation determines breast cancer cells' viability.

Since it is known that cancer cells exhibit a preference for increased glycine consumption, the respective glycine metabolizing enzymes are in focus of many research projects. However, no cancer associated studies are available for the Glycine Cleavage System Protein H (GCSH) to date. Our initial analysis revealed a GCSH-overexpression of the protein-coding transcript variant 1 (Tv1) in breast cancer cells and tissue. Furthermore, a shorter (391 bp) transcript variant (Tv*) was amplified with an increased expression in healthy breast cells and a decreased expression in breast cancer samples. The Tv1/Tv* transcript ratio is 1.0 in healthy cells on average, and between 5-10 in breast cancer cells. Thus, a GCSH-equilibrium at the transcript level is likely conceivable for optimal glycine degradation. A possible regulative role of Tv* was proven by Tv1-Tv*-RNA-binding and overexpression studies which consequently led to serious physiological alterations: decreased metabolic activity, release of the lactate dehydrogenase, increased extracellular acidification, and finally necrosis as a result of impaired plasma membranes. In contrast, Tv1-overexpression led to an additional increase in cellular vitality of the tumor cells, primarily due to the acceleration of the mitochondrial glycine decarboxylation activity. Ultimately, we provide the first evidence of a sensitive GCSH-antisense regulation which determines cancerous cell viability.

First evidence of SGPL1 expression in the cell membrane silencing the extracellular S1P siren in mammary epithelial cells.

The bioactive lipid sphingosine-1-phosphate (S1P) is a main regulator of cell survival, proliferation, motility, and platelet aggregation, and it is essential for angiogenesis and lymphocyte trafficking. In that S1P acts as a second messenger intra- and extracellularly, it might promote cancer progression. The main cause is found in the high S1P concentration in the blood, which encourage cancer cells to migrate through the endothelial barrier into the blood vessels. The irreversible degradation of S1P is solely caused by the sphingosine-1-phosphate lyase (SGPL1). SGPL1 overexpression reduces cancer cell migration and therefore silences the endogenous S1P siren, which promotes cancer cell attraction-the main reason for metastasis. Since our previous metabolomics studies revealed an increased SGPL1 activity in association with successful breast cancer cell treatment in vitro, we further investigated expression and localization of SGPL1. Expression analyses confirmed a very low SGPL1 expression in all breast cancer samples, regardless of their subtype. Additionally, we were able to prove a novel SGPL expression in the cytoplasm membrane of non-tumorigenic breast cells by fusing three independent methods. The general SGPL1 downregulation and the loss of the plasma membrane expression resulted in S1P dependent stimulation of migration in the breast cancer cell lines MCF-7 and BT-20. Not only S1P stimulated migration could be repressed by overexpressing the natural SGPL1 variant not but also more general migratory activity was significantly reduced. Here, for the first time, we report on the SGPL1 plasma membrane location in human, non-malignant breast epithelial cell lines silencing the extracellular S1P siren in vitro, and thereby regulating pivotal cellular functions. Loss of this plasma membrane distribution as well as low SGPL1 expression levels could be a potential prognostic marker and a viable target for therapy. Therefore, the precise role of SGPL1 for cancer treatment should be evaluated.

Synergistic Action of Genistein and Calcitriol in Immature Osteosarcoma MG-63 Cells by SGPL1 Up-Regulation.

BACKGROUND: Phytoestrogens such as genistein, the most prominent isoflavone from soy, show concentration-dependent anti-estrogenic or estrogenic effects. High genistein concentrations (>10 µM) also promote proliferation of bone cancer cells in vitro. On the other hand, the most active component of the vitamin D family, calcitriol, has been shown to be tumor protective in vitro and in vivo. The purpose of this study was to examine a putative synergism of genistein and calcitriol in two osteosarcoma cell lines MG-63 (early osteoblast), Saos-2 (mature osteoblast) and primary osteoblasts. METHODS: Thus, an initial screening based on cell cycle phase alterations, estrogen (ER) and vitamin D receptor (VDR) expression, live cell metabolic monitoring, and metabolomics were performed. RESULTS: Exposure to the combination of 100 µM genistein and 10 nM calcitriol reduced the number of proliferative cells to control levels, increased ERß and VDR expression, and reduced extracellular acidification (40%) as well as respiratory activity (70%), primarily in MG-63 cells. In order to identify the underlying cellular mechanisms in the MG-63 cell line, metabolic profiling via GC/MS technology was conducted. Combined treatment significantly influenced lipids and amino acids preferably, whereas metabolites of the energy metabolism were not altered. The comparative analysis of the log2-ratios revealed that after combined treatment only the metabolite ethanolamine was highly up-regulated. This is the result: a strong overexpression (350%) of the enzyme sphingosine-1-phosphate lyase (SGPL1), which irreversibly degrades sphingosine-1-phosphate (S1P), thereby, generating ethanolamine. S1P production and secretion is associated with an increased capability of migration and invasion of cancer cells. CONCLUSION: From these results can be concluded that the tumor promoting effect of high concentrations of genistein in immature osteosarcoma cells is reduced by the co-administration of calcitriol, primarily by the breakdown of S1P. It should be tested whether this anti-metastatic pathway can be stimulated by combined treatment also in metastatic xenograft mice models.