Inhibition of Chemoresistance in Primary Tumor Cells by Camellia sinensis non fermentatum Extract Noviphenone (NPE®).

BACKGROUND/AIM: Despite improvements in cancer therapy, life expectancy after tumor recurrence remains low. Relapsed cancer is characterized by drug resistance, often mediated through overexpression of multidrug resistance (MDR) genes. Camellia sinensis non fermentatum extract is known for its anticancer properties in several cancer cell lines and might improve cancer therapy outcome after tumor recurrence. MATERIALS AND METHODS: Embryonal rhabdomyosarcoma cell lines, alveolar rhabdomyosarcoma cell lines and primary rhabdomyosarcoma MAST139 cells were used to test NPE® effects on cell viability in combination with chemotherapeutic agents. Cell viability was measured by the WST-1 assay and CV staining. Gene expression levels of chemotherapy-induced efflux pumps and their activity was assessed upon NPE® treatment by measuring doxorubicin retention through evaluation of the autofluorescence signal. RESULTS: Administration of increasing doxorubicin concentrations triggered immediate adaptation to the drug, which was surprisingly overcome by the addition of NPE®. Investigating the mechanism of immediate adaptation, MDR1 gene overexpression was observed upon doxorubicin treatment. Although NPE® did not alter pump gene expression, it was able to reduce pump activity, thus allowing the chemotherapeutic agent to stay inside the cells to exert its full anticancer activity. CONCLUSION: NPE® might improve chemotherapeutic treatment by re-sensitizing relapsed tumors to anticancer drugs. Fighting MDR represents the key to overcome tumor relapse and improve the overall survival of cancer patients.

Probabilistic modeling of personalized drug combinations from integrated chemical screen and molecular data in sarcoma.

BACKGROUND: Cancer patients with advanced disease routinely exhaust available clinical regimens and lack actionable genomic medicine results, leaving a large patient population without effective treatments options when their disease inevitably progresses. To address the unmet clinical need for evidence-based therapy assignment when standard clinical approaches have failed, we have developed a probabilistic computational modeling approach which integrates molecular sequencing data with functional assay data to develop patient-specific combination cancer treatments. METHODS: Tissue taken from a murine model of alveolar rhabdomyosarcoma was used to perform single agent drug screening and DNA/RNA sequencing experiments; results integrated via our computational modeling approach identified a synergistic personalized two-drug combination. Cells derived from the primary murine tumor were allografted into mouse models and used to validate the personalized two-drug combination. Computational modeling of single agent drug screening and RNA sequencing of multiple heterogenous sites from a single patient's epithelioid sarcoma identified a personalized two-drug combination effective across all tumor regions. The heterogeneity-consensus combination was validated in a xenograft model derived from the patient's primary tumor. Cell cultures derived from human and canine undifferentiated pleomorphic sarcoma were assayed by drug screen; computational modeling identified a resistance-abrogating two-drug combination common to both cell cultures. This combination was validated in vitro via a cell regrowth assay. RESULTS: Our computational modeling approach addresses three major challenges in personalized cancer therapy: synergistic drug combination predictions (validated in vitro and in vivo in a genetically engineered murine cancer model), identification of unifying therapeutic targets to overcome intra-tumor heterogeneity (validated in vivo in a human cancer xenograft), and mitigation of cancer cell resistance and rewiring mechanisms (validated in vitro in a human and canine cancer model). CONCLUSIONS: These proof-of-concept studies support the use of an integrative functional approach to personalized combination therapy prediction for the population of high-risk cancer patients lacking viable clinical options and without actionable DNA sequencing-based therapy.

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.

Synergistic Antitumour Properties of viscumTT in Alveolar Rhabdomyosarcoma.

Aqueous mistletoe extracts from the European mistletoe (Viscum album) contain mainly mistletoe lectins and viscotoxins as cytotoxic compounds. Lipophilic triterpene acids, which do not occur in conventional mistletoe preparations, were solubilised with ß-cyclodextrins. The combination of an aqueous extract (viscum) and a triterpene-containing extract (TT) recreated a whole mistletoe extract (viscumTT). These extracts were tested on rhabdomyosarcoma in vitro, ex vivo, and in vivo with regard to anticancer effects. Viscum and viscumTT inhibited cell proliferation and induced apoptosis effectively in a dose-dependent manner in vitro and ex vivo, whereas TT showed only moderate inhibitory effects. viscumTT proved to be more effective than the single extracts and displayed a synergistic effect in vitro and a stronger effect in vivo. viscumTT induced apoptosis via the extrinsic and intrinsic pathways, evidenced by the loss of mitochondrial membrane potential and activation of CASP8 and CASP9. CASP10 inhibitor inhibited apoptosis effectively, emphasising the importance of CASP10 in viscumTT-induced apoptosis. Additionally, viscumTT changed the ratio of apoptosis-associated proteins by downregulation of antiapoptotic proteins such as XIAP and BIRC5, thus shifting the balance towards apoptosis. viscumTT effectively reduced tumour volume in patient-derived xenografts in vivo and may be considered a promising substance for rhabdomyosarcoma therapy.

Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer exhibiting skeletal-muscle differentiation. New therapeutic targets are required to improve the dismal prognosis for invasive or metastatic alveolar rhabdomyosarcoma. Protein kinase C iota (PKCι) has been shown to have an important role in tumorigenesis of many cancers, but little is known about its role in rhabdomyosarcoma. Our gene-expression studies in human tumor samples revealed overexpression of PRKCI. We confirmed overexpression of PKCι at the mRNA and protein levels using our conditional mouse model that authentically recapitulates the progression of rhabdomyosarcoma in humans. Inhibition of Prkci by RNA interference resulted in a dramatic decrease in anchorage-independent colony formation. Interestingly, treatment of primary cell cultures using aurothiomalate (ATM), which is a gold-containing classical anti-rheumatic agent and a PKCι-specific inhibitor, resulted in decreased interaction between PKCι and Par6, decreased Rac1 activity and reduced cell viability at clinically relevant concentrations. Moreover, co-treatment with ATM and vincristine (VCR), a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a combination index of 0.470-0.793 through cooperative accumulation of non-proliferative multinuclear cells in the G2/M phase, indicating that these two drugs synergize. For in vivo tumor growth inhibition studies, ATM demonstrated a trend toward enhanced VCR sensitivity. Overall, these results suggest that PKCι is functionally important in alveolar rhabdomyosarcoma anchorage-independent growth and tumor-cell proliferation and that combination therapy with ATM and microtubule inhibitors holds promise for the treatment of alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma of the extremity and nodal metastasis: Is the in-transit lymphatic system at risk?

Alveolar rhabdomyosarcoma (RMS) of the extremity is not infrequently associated with regional node metastasis. Knowledge of lymphatic drainage of extremity RMSs is important to determine radiotherapy fields. In this report we describe two patients with alveolar RMS of the lower extremity with inguinal metastasis at presentation. Both the distal lower extremity and inguinal region received local therapy consisting of surgery and postoperative radiotherapy. Both patients later developed in-transit lymphatic metastasis outside of the irradiated field. The in-transit lymphatics can be a site of failure in children with alveolar RMS of the extremity and nodal involvement.

In vitro assessment of novel transcription inhibitors and topoisomerase poisons in rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Current chemotherapy regimes include the topoisomerase II poison etoposide and the transcription inhibitor actinomycin D. Poor clinical response necessitate identification of new agents to improve patient outcomes. METHODS: We assessed the in vitro cytotoxicity (MTT assay) of DNA intercalating agents in five established human RMS cell lines. These include novel classes of transcription inhibitors and topoisomerase poisons, previously shown to have potential as anti-cancer agents. RESULTS: Amongst the former agents, bisintercalating bis(9-aminoacridine-4-carboxamides) linked through the 9-position, and bis(phenazine-1-carboxamides) linked via their side chains, are compared with established transcription inhibitors. Amongst the latter, monofunctional acridine-4-carboxamides related to N-[2-(dimethylamino)ethyl]acridine-4-carboxamide, DACA, are compared with established topoisomerase poisons. CONCLUSIONS: Our findings specifically highlight the topoisomerase poison 9-amino-DACA, its 5-methylsulphone derivative, AS-DACA, and the bis(phenazine-1-carboxamide) transcription inhibitor MLN944/XR5944, currently in phase I trial, as candidates for further research into new agents for the treatment of RMS.

Near-precise interchromosomal recombination and functional DNA topoisomerase II cleavage sites at MLL and AF-4 genomic breakpoints in treatment-related acute lymphoblastic leukemia with t(4;11) translocation.

We analyzed the der(11) and der(4) genomic breakpoint junctions of a t(4;11) in the leukemia of a patient previously administered etoposide and dactinomycin by molecular and biochemical approaches to gain insights about the translocation mechanism and the relevant drug exposure. The genomic breakpoint junctions were amplified by PCR. Cleavage of DNA substrates containing the normal homologues of the MLL and AF-4 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha and etoposide, etoposide catechol, etoposide quinone, or dactinomycin. The der(11) and der(4) genomic breakpoint junctions both involved MLL intron 6 and AF-4 intron 3. Recombination was precise at the sequence level except for the overall gain of a single templated nucleotide. The translocation breakpoints in MLL and AF-4 were DNA topoisomerase II cleavage sites. Etoposide and its metabolites, but not dactinomycin, enhanced cleavage at these sites. Assuming that DNA topoisomerase II was the mediator of the breakage, processing of the staggered nicks induced by DNA topoisomerase II, including exonucleolytic deletion and template-directed polymerization, would have been required before ligation of the ends to generate the observed genomic breakpoint junctions. These data are inconsistent with a translocation mechanism involving interchromosomal recombination by simple exchange of DNA topoisomerase II subunits and DNA-strand transfer; however, consistent with reciprocal DNA topoisomerase II cleavage events in MLL and AF-4 in which both breaks became stable, the DNA ends were processed and underwent ligation. Etoposide and/or its metabolites, but not dactinomycin, likely were the relevant exposures in this patient.