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Background: Previous works have shown that the expression of Class-II-Transactivator (CIITA) in tumor cells reduces the growth of glioblastoma (GB) in animal models, but immune effects cannot solely explain this. Here, we searched for immune-independent effects of CIITA on the proliferation of GB. Methods: Murine GL261 and human U87, GM2 and GM3 malignant glioma cells were transfected with CIITA. NSG (immunodeficient) and nude (athymic) mice were injected in the striatum with GL261-wildtype (-WT) and -CIITA, and tumor growth was assessed by immunohistology and luminescence reporter genes. Clonogenic, sphere-formation, and 3D Matrigel-based in vitro growth assays were performed to compare the growth of WT versus CIITA-expressing murine and human cells. Bulk RNA sequencing and RT2 qRT-PCR profiler arrays were performed on these four cell lines to assess RNA expression changes following CIITA transfection. Western blot analysis on several proliferation-associated proteins was performed. Results: The intracerebral growth of murine GL261-CIITA cells was drastically reduced both in immunodeficient and athymic mice. Tumor growth was reduced in vitro in three of the four cell types. RNA sequencing and RT2 profiler array experiments revealed a modulation of gene expression in the PI3-Akt, MAPK- and cell-cycle regulation pathways following CIITA overexpression. Western blot analysis showed an upregulation of p27 in the growth-inhibited cells following this treatment. PDGFR-beta was downregulated in all cells. We did not find consistent regulation of other proteins involved in GB proliferation. Conclusions: Proliferation is drastically reduced by CIITA in GB, both in vivo and in vitro, notably in association with p27-mediated inhibition of cell-cycle pathways.

Background: Chikungunya virus (CHIKV) is a re-emerging pathogen that has caused widespread outbreaks affecting millions of people around the globe. Currently, there is no specific therapeutic drug against CHIKV, with symptomatic treatment only to manage the disease. Pi3-akt signaling has been implicated in infection of several viruses including that of CHIKV. Effect of Pi3-akt signaling inhibitors on CHIKV replication was evaluated in this study. Methods: Human primary dermal fibroblast cells were treated with inhibitors of the Pi3-akt signaling pathway. Suppression of CHIKV replication was evaluated as reduction in virus titer in cell supernatants. Effect of miltefosine (MF) on CHIKV replication was evaluated in pre and post treatment regimen. Inhibition of virus replication was determined by cell growth, virus titer and western blot. Results: Inhibition of Akt-phosphorylation significantly inhibited CHIKV replication. No effect on CHIKV replication was observed after treatment with Pi3-kinase and mTOR activation inhibitors. Further, MF, an FDA-approved Akt-inhibitor, inhibited CHIKV replication in pre- and post-infection treatment regimens. Conclusion: Data suggests that Akt-phosphorylation can be an amenable target of therapy against CHIKV infection. This is the first study to show inhibition of CHIKV replication by MF, and presents a case for further development of MF as an anti-CHIKV drug.

The pyrimidine analogue gemcitabine is an established effective agent in the treatment of non-small-cell lung cancer (NSCLC). The present study investigates whether gemcitabine would be synergistic with the topoisomerase I inhibitor topotecan against the NSCLC A549 and Calu-6 cells. Cells were treated with gemcitabine and topotecan for 1 h and the type of drug interaction was assessed using the combination index (CI). Cell cycle alterations were analysed by flow cytometry, while apoptosis was examined by the occurrence of DNA internucleosomal fragmentation, nuclear condensation and caspase-3 activation. Moreover, the possible involvement of the PI3K-Akt signalling pathway was investigated by the measurement of Akt phosphorylation. Finally, quantitative, real-time PCR (QRT-PCR) was used to study modulation of the gemcitabine-activating enzyme deoxycytidine kinase (dCK) and the cellular target enzyme ribonucleotide reductase (RR). In results, it was found that simultaneous and sequential topotecan → gemcitabine treatments were synergistic, while the reverse sequence was antagonistic in both cell lines. DNA fragmentation, nuclear condensation and enhanced caspase-3 activity demonstrated that the drug combination markedly increased apoptosis in comparison with either single agent, while cell cycle analysis showed that topotecan increased cells in S phase. Furthermore, topotecan treatment significantly decreased the amount of the activated form of Akt, and enhanced the expression of dCK (+155.0 and +115.3% in A549 and Calu-6 cells, respectively), potentially facilitating gemcitabine activity. In conclusion, these results indicate that the combination of gemcitabine and topotecan displays schedule-dependent activity in vitro against NSCLC cells. The gemcitabine → topotecan sequence is antagonistic while drug synergism is obtained with the simultaneous and the sequential topotecan → gemcitabine combinations, which are associated with induction of decreased Akt phosphorylation and increased dCK expression.