Activity-guided fractionation of a petroleum ether-soluble extract of the roots of Onosma paniculata, which has been shown to affect the cell cycle and to induce apoptosis in melanoma cells, led to the isolation of several shikonin derivatives, namely, β-hydroxyisovalerylshikonin (1), acetylshikonin (2), dimethylacrylshikonin (3), and a mixture of α-methylbutyrylshikonin and isovalerylshikonin (4+5). All compounds exhibited strong cytotoxicity against eight cancer cell lines and MRC-5 lung fibroblasts, with 3 found to possess the most potent cytotoxicity toward four melanoma cell lines (SBcl2, WM35, WM9, and WM164). Furthermore, 3 and the mixture of 4+5 were found to interfere with cell-cycle progression in these cell lines and led to an increasing number of cells in the subG1 region as well as to caspase-3/7 activation, indicating apoptotic cell death.

Abstract. Chordomas are rare, low to intermediate grade malignant bone tumors of the axial skeleton. Current treatment options are limited to surgical procedures, as chordomas are largely resistant to conventional radiation and chemotherapy. Cell lines are valuable tools for exploring molecular mechan isms involved in tumorigenesis and they have a fundamental impact on the development of new anticancer agents. To date, only two chordoma cell lines exist world-wide. In the present study we report a third chordoma cell line, MUG-Chor1, as well as corresponding cultured fibroblasts established from a recur rent morphologically 'classic' sacrococcygeal chordoma of a 58-year-old Caucasian female. The cells are brachyury-positive and have the characteristics of chordoma. The genetic profile of the primary chordoma and the established chordoma cell line was investigated during the culturing period (early and late passage). MUG-Chor1 is karyotypically, <2n>43-47,XX,del(3) (q1?) [11], +7,del(9)(p1?),der(9;15)(q10;q10),-10,+der(12)t(9;12) (p2?;q1?),der (12)t(12;19)(p;p)t(17;19)(q;q),-15,der(17;21) (q10;q10),der (20)t(10;20) (q25?26?;q11?12?),-21,-22[20]/ idemx2[5] and displays known, chordoma-typical genetic changes, such as chromosomal gains at T/brachyury locus (6q27), losses at 9p24.3-p13.1 (includes the CDKN2a/CDKN2b locus), 10p15.3-q23.32 (includes the PTEN locus) and losses of 10q25.2 (includes the PDCD4 locus). MUG-Chor1 bears a marked resemblance to chordomas in vivo and is, therefore, an optimal in vitro chordoma model.

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Resistance to BRAF and MEK inhibition is a common phenomenon in melanoma. Cytokines and transcription factors have been attributed to contribute to the loss of sensitivity towards these inhibitors. Here, we show that transforming growth factor (TGF)-β1 if combined with PLX4032, a BRAF inhibitor, or GSK1120212, a MEK inhibitor, substantially increased cell death in BRAF-mutant melanoma cell lines. This increase was based on the combined regulatory decrease in Twist1, an antiapoptotic protein. Overexpression or silencing of Twist1 attenuated or aggravated induction of apoptosis through PLX4032 or GSK1120212, respectively. Exposure to tumour necrosis factor (TNF)-α, however, led to increased Twist1 levels and oppositional decrease in cell death if exposed to PLX4032 or GSK1120212. This increase in drug resistance again depended on Twist1 levels. Our studies suggest that Twist1 as a common downstream target of multiple signalling cascades plays a crucial role in mediating drug resistance to BRAF- and MEK-targeted molecular inhibitors.

Chordomas mainly arise along the axial skeleton and are characterized by their slow but destructive growth. Prognosis and quality of life are poor because treatment options are mainly limited to surgery and radiotherapy. Survivin, a member of the apoptosis inhibitor protein family, functions as a key regulator of mitosis and programmed cell death, and is overexpressed in many tumor types. The aim of this study was to determine the role of survivin in chordomas. Survivin expression was investigated in 50 chordoma samples and three chordoma cell lines using immunohistochemistry. The intensity of immunostaining was evaluated in regard to the development of recurrences. The immunohistochemical results were correlated with clinical parameters like gender, age, tumor size, and location and were performed in primary chordomas as well as in recurrent lesions. Furthermore, survivin knockdown experiments on chordoma cell lines were performed. YM155 decreased the growth behavior of chordoma cells dose-and time dependently. Transient knockdown of survivin led to a G2/M arrest, decreased proliferation, consistently induced an increase of polyploidy and morphological changes, and induced apoptosis. The resultant data from this study suggest that survivin plays a cell cycle-progressive role in chordomas. Hence, regulation of survivin by YM155 is a promising new target for the development of new therapeutic drugs. ß