Bcl-xL inhibition enhances Dinaciclib-induced cell death in soft-tissue sarcomas.

Soft-tissue sarcomas (STS) are an uncommon and heterogeneous group of malignancies that result in high mortality. Metastatic STS have very bad prognosis due to the lack of effective treatments. Dinaciclib is a model drug for the family of CDK inhibitors. Its main targets are cell cycle regulator CDK1 and protein synthesis controller CDK9. We present data supporting Dinaciclib ability to inactivate in vitro different STS models at nanomolar concentrations. Moreover, the different rhythms of cell death induction allow us to further study into the mechanism of action of the drug. Cell death was found to respond to the mitochondrial pathway of apoptosis. Anti-apoptotic Bcl-xL was identified as the key regulator of this process. Already natural low levels of pro-apoptotic proteins BIM and PUMA in tolerant cell lines were insufficient to inhibit Bcl-xL as this anti-apoptotic protein showed a slow decay curve after Dinaciclib-induced protein synthesis disruption. Combination of Dinaciclib with BH3-mimetics led to quick and massive apoptosis induction in vitro, but in vivo assessment was prevented due to liver toxicity. Additionally, Bcl-xL inhibitor A-1331852 also synergized with conventional chemotherapy drugs as Gemcitabine. Thus, Bcl-xL targeted therapy arises as a major opportunity to the treatment of STS.

EphA2 receptor is a key player in the metastatic onset of Ewing sarcoma.

Ewing sarcoma (ES) is the second most common bone malignancy affecting children and young adults with poor prognosis due to high metastasis incidence. Our group previously described that EphA2, a tyrosine kinase receptor, promotes angiogenesis in Ewing sarcoma (ES) cells via ligand-dependent signaling. Now we wanted to explore EphA2 ligand-independent activity, controlled upon phosphorylation at S897 (p-EphA2S897 ), as it has been linked to metastasis in several malignancies. By reverse genetic engineering we explored the phenotypic changes after EphA2 removal or reintroduction. Gene expression microarray was used to identify key players in EphA2 signaling. Mice were employed to reproduce metastatic processes from orthotopically implanted engineered cells. We established a correlation between ES cells aggressiveness and p-EphA2S897 . Moreover, stable overexpression of EphA2 in low EphA2 expression ES cells enhanced proliferation and migration, but not a non-phosphorylable mutant (S987A). Consistently, silencing of EphA2 reduced tumorigenicity, migration and invasion in vitro, and lung metastasis incidence in experimental and spontaneous metastasis assays in vivo. A gene expression microarray revealed the implication of EphA2 in cell signaling, cellular movement and survival. ADAM19 knockdown by siRNA technology strongly reproduced the negative effects on cell migration observed after EphA2 silencing. Altogether, our results suggest that p-EphA2S897 correlates with aggressiveness in ES, so blocking its function may be a promising treatment.

Improved selectivity and cytotoxic effects of irinotecan via liposomal delivery: A comparative study on Hs68 and HeLa cells.

Irinotecan (CPT-11) is an effective chemotherapeutic agent widely used to treat different cancers. Otherwise, the liposomal delivery of anti-tumor agents has been shown to be a promising strategy. The aim of this study has been to analyze the effect of liposomal CPT-11 (CPT-11lip) on two human cell lines (Hs68 and HeLa) to establish the suitability of this CPT-11 nanocarrier. We have demonstrated the highest uptake of CPT-11lip in comparison with that of CPT-11sol, in lactate buffer, and that CPT-11lip was internalized in the cells through an endocytic process whereas CPT-11sol does so by passive diffusion. CPT-11lip was not cytotoxic to normal fibroblast Hs68 cells, but induced a massive apoptosis accompanied by cell senescence in HeLa cells. CPT-11lip treatment modified the morphology of HeLa cells, induced different cell cycle alterations and accumulated into lysosomes in both cell lines. In particular, CPT-11lip treatment showed that surviving HeLa cells remained in a state of senescence whereas only a temporal growth arrest was induced in Hs68 cells. Results of RT-PCR indicated that the different responses in Hs68 (survival) and HeLa cells (apoptotic death), seemed to be induced by a p53- and p53- independent mechanism, respectively. An analysis of DNA damage also determined that released CPT-11 from liposomes was able to reach the nucleus and exert a genotoxic effect in both cell lines, which was repaired in Hs68 but not in HeLa cells. All results indicate that phospholipid-cholesterol liposomes possess optimum properties for CPT-11 delivery, being biocompatible and selectively cytotoxic against HeLa tumorigenic cells.

Metallo-naphthalocyanines as photothermal sensitisers for experimental tumours: in vitro and in vivo studies.

BACKGROUND AND OBJECTIVES: Photothermal sensitisation has been recently proposed as a novel approach for the treatment of solid tumours through the development of a therapeutic modality named photothermal therapy (PTT). The technique involves the use of high power pulsed laser irradiation and photosensitising agents with especially short lifetime (in the subnanosecond range) in the electronically excited states. This study aims to investigate the molecular features of the photosensitiser which optimise the photothermal activity. STUDY DESIGN/MATERIALS AND METHODS: Two octabutoxy-naphthalocyanines centrally coordinated with Pd(II) or Pt(II) ions were prepared by chemical synthesis and tested for their affinity and photothermal sensitisation activity toward a selected tumour cell line, namely B78H1 amelanotic melanoma. Irradiations were performed by using a Ti:sapphire laser operated in a pulsed regime (10 Hz, 30 nanosecond pulses, 120 mJ) at 809 nm (Pt) or 826 nm (Pd). The subcellular distribution pattern of the photosensitiser was also assessed by optical microscopy, while the nature of the photoinduced cell damage was determined by scanning electron microscopy. The results thus obtained provided a basis for subsequent in vivo studies, aimed at defining the phototherapeutic efficiency of the two metallo-naphthalocyanines: the photosensitisers were i.v. injected into C57BL/6 mice bearing a subcutaneously transplanted amelanotic melanoma and at 24 hours post-injection the tumour area was irradiated by the Ti:sapphire laser using the same protocol as above detailed. RESULTS: Both naphthalocyanines exhibited a high affinity for the amelanotic melanoma cells. The subcellular distribution pattern was modulated by the incubation time: after 48 hours incubation with 7.7 microM Pd- and Pt derivatives, the naphthalocyanine appeared to localise in specific sites with a gradual formation of aggregated clusters. Subsequent irradiation of the naphthalocyanine-loaded cells caused an extensive cell death; the photoinduced damage, as observed at the scanning electron microscope, mainly consisted in the formation of large endocellular holes consequent to the loss of cytoplasmic material. This scenario is typical of photothermal sensitisation processes. Lastly, both metallo-naphthalocyanines, and in particular the Pd(II) derivative, promoted an important response by the amelanotic melanoma, when the neoplastic tissue was irradiated by the pulsed Ti:sapphire laser. In certain cases, the photothermal treatment appeared to be curative. In all cases, the in vivo photodamage was confined within the tumour area with no detectable involvement of the perilesional tissues. CONCLUSION: PTT appears to act very efficiently at least on subcutaneous tumours. The technique can be used either in combination with photodynamic therapy (PDT) or as an alternative to PDT in those cases where the latter modality displays a limited efficacy, such as in the treatment of pigmented or poorly vascularised tumours.