Proteasome inhibition potentiates Kv1.3 potassium channel expression as therapeutic target in drug-sensitive and -resistant human melanoma cells.

Primary and acquired therapy resistance is a major problem in patients with BRAF-mutant melanomas being treated with BRAF and MEK inhibitors (BRAFI, MEKi). Therefore, development of alternative therapy regimes is still required. In this regard, new drug combinations targeting different pathways to induce apoptosis could offer promising alternative approaches. Here, we investigated the combination of proteasome and Kv1.3 potassium channel inhibition on chemo-resistant, BRAF inhibitor-resistant as well as sensitive human melanoma cells. Our experiments demonstrated that all analyzed melanoma cell lines were sensitive to proteasome inhibitor treatment at concentrations that are not toxic to primary human fibroblasts. To further reduce proteasome inhibitor-associated side effects, and to foster apoptosis, potassium channels, which are other targets to induce pro-apoptotic effects in cancer cells, were blocked. In support, combined exposure of melanoma cells to proteasome and Kv1.3 channel inhibitor resulted in synergistic effects and significantly reduced cell viability. On the molecular level, enhanced apoptosis correlated with an increase of intracellular Kv1.3 channels and pro-apoptotic proteins such as Noxa and Bak and a reduction of anti-apoptotic proteins. Thus, use of combined therapeutic strategies triggering different apoptotic pathways may efficiently prevent the outgrowth of drug-resistant and -sensitive BRAF-mutant melanoma cells. In addition, this could be the basis for an alternative approach to treat other tumors expressing mutated BRAF such as non-small-cell lung cancer.

The potential role of T-cells and their interaction with antigen-presenting cells in mediating immunosuppression following trauma-hemorrhage.

OBJECTIVE: Trauma-hemorrhage results in depressed immune responses of antigen-presenting cells (APCs) and T-cells. Recent studies suggest a key role of depressed T-cell derived interferon (IFN)-g in this complex immune cell interaction. The aim of this study was to elucidate further the underlying mechanisms responsible for dysfunctional T-cells and their interaction with APCs following trauma-hemorrhage. DESIGN: Adult C3H/HeN male mice were subjected to trauma-hemorrhage (3-cm midline laparotomy) followed by hemorrhage (blood pressure of 35 +/- 5 mmHg for 90 min and resuscitation) or sham operation. At 24 h thereafter, spleens were harvested and T-cells (by Microbeads) and APCs (via adherence) were Isolated. Co-cultures of T-cells and APCs were established for 48 h and stimulated with concanavalin A and lipopolysaccharide. T-Cell specific cytokines known to affect APC function (i.e. interleukin(IL)-2, IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF)) were measured in culture supernatants by Multiplex assay. The expression of MHC class II as well as co-stimulatory surface molecules on T-cells and APCs was determined by flow cytometry. RESULTS: The release of IL-4 and GM-CSF by T-cells was suppressed following trauma-hemorrhage, irrespective of whether sham or trauma-hemorrhage APCs were present. Antigen-presenting cells from animals subjected to trauma-hemorrhage did not affect T-cell derived cytokine release by sham T-cells. In contrast, T-cells from trauma-hemorrhage animals depressed MHC class II expression of CD11c(+) cells, irrespective of whether APCs underwent sham or trauma-hemorrhage procedure. Surprisingly, co-stimulatory molecules on APCs (CD80, CD86) were not affected by trauma-hemorrhage. CONCLUSIONS: These results suggest that beside IFN-g other T-cell derived cytokines contribute to immunosuppression following trauma-hemorrhage causing diminished MHC II expression on APCs. Thus, T-cells appear to play an important role in this interaction at the time-point examined. Therapeutic approaches should aim at maintenance of T-cell function and their interaction with APCs to prevent extended immunosuppression following trauma-hemorrhage.

Adenoassociated virus type 2-induced inhibition of the human papillomavirus type 18 promoter in transgenic mice.

The epithelium of the cervix uteri has been reported to be frequently coinfected with both human papillomaviruses (HPV) and helper virus-dependent adenoassociated viruses (AAV). Seroepidemiological data suggest that AAV infection could inhibit cervical cancer that is caused by specific ("high-risk") types of papillomaviruses. In vitro, infection with AAV type 2 (AAV-2) or transfection of AAV-2 early (rep) genes has been shown to inhibit transformation by papillomaviruses. To analyze the effects of AAV on HPV in vivo, we studied the influence of AAV-2 infection on the promoter activity of high-risk HPV type 18 (HPV-18) in mice, transgenic for sequences of the upstream regulatory region (URR) of HPV-18 controlling transcription of the reporter gene, lacZ. Transgenic animals (or tongue cells thereof, explanted and grown in culture) were treated with dexamethasone to induce the HPV-18 promoter. Simultaneously they were (i) infected with AAV, (ii) inoculated with AAV virus-like particles (VLPs; empty capsids), or (iii) mock infected. Inoculation with AAV-2 or VLPs inhibited activation of the HPV-18 promoter. In vitro, in baby hamster kidney cells transfected with the HPV-18-lacZ construct, tissue extracts from AAV-infected animals suppressed the HPV-18 URR to a similar extent as AAV infection did. Down-regulation of the HPV-18 promoter was less efficient with extracts from animals inoculated with VLPs and was not observed with extracts from uninfected or dexamethasone-treated animals. This indicates that AAV induces cellular factor(s) in vivo capable of mediating down-regulation of the HPV-18 promoter also in cells in vitro. In contrast, promoters of the low-risk HPV types (HPV-6, HPV-11) were not influenced by AAV infection as opposed to promoters of the high-risk types (HPV-18 and HPV-16).