FTY720 increases paclitaxel efficacy in cisplatin-resistant oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma has high recurrence and cisplatin resistance. As cancer stem cells, autophagy, and sphingolipids have been appointed as associated with chemotherapy resistance, we tested combined treatments targeting autophagy and/or sphingolipid metabolism with paclitaxel using cisplatin-resistant oral squamous cell carcinoma cells. METHODS: Cisplatin-resistant oral squamous cell carcinoma cells were maintained under exposition to FTY720 and chloroquine combined with paclitaxel and submitted to viability, clonogenicity, and spheres formation assays. The xenograft tumor model using cisplatin-resistant CAL27 cells was adopted to examine the drug combinations' potential antitumoral efficacy. Using an animal model, sphingolipids profiles from plasma and tissue samples were obtained by liquid chromatography coupled to mass spectrometry to identify potential lipids associated with drug response. RESULTS AND DISCUSSION: Our results showed higher autophagic flux in cisplatin-resistant Ooral squamous cell carcinoma (CAL27 and SCC9) cells than in parental cells. The combinations of an autophagy inhibitor (chloroquine) or an autophagy inducer/sphingosine kinase 1 antagonist (FTY720) with paclitaxel (PTX) had a synergistic antitumor effect. Treated CisR cells lost clonogenicity and tumor sphere abilities and reduced proteins associated with proliferation, survival, and cancer stem cells. FTY720 plus PTX had higher antitumor efficacy than PTX against CAL27 CisR xenograft tumor formation. Additionally, increases in glucosylceramide, dehydroglucosylceramide, and sphingomyelin were presented in responsive tumors. CONCLUSION: FTY720 sensitizes cisplatin-resistant oral squamous cell carcinoma cells for paclitaxel.

Generation of a Three-Dimensional in Vitro Ovarian Cancer Co-Culture Model for Drug Screening Assays.

In vitro 3D culture models have emerged in the cancer field due to their ability to recapitulate characteristics of the in vivo tumor. Herein, we described the establishment and characterization of 3D multicellular spheroids using ovarian cancer cells (SKOV-3) in co-culture with mesenchymal cells (MUC-9) or fibroblasts (CCD27-Sk). We demonstrated that SKOV-3 cells in co-culture were able to form regular and compact spheroids with diameters ranging from 300 to 400 µm and with a roundness close to 1.0 regardless of the type of stromal cell used. In the 3D culture an increase was not observed in spheroid diameter nor was there significant cell growth. What is more, the 3D co-cultures presented an up regulation of genes related to tumorigenesis, angiogenesis and metastases (MMP2, VEGFA, SNAI1, ZEB1 and VIM) when compared with 2D and 3D monoculture. As expected, both 3D cultures (mono and co-cultures) exhibited a higher Paclitaxel chemoresistance when compared to 2D condition. Although we did not observe differences in the Paclitaxel resistance between the 3D mono and co-cultures, the gene expression results indicate that the presence of mesenchymal cells and fibroblasts better recapitulate the in vivo tumor microenvironment, being able, therefore, to more accurately evaluate drug efficacy for ovarian cancer therapy.

Toxicogenomic Analysis.

The biological functions of a cell may change in response to exposure to toxic agents. Toxicogenomics employs the recent developments in genomics, transcriptomics, and proteomics to study how a chemical impacts gene/protein expression and cell functions. We describe a method for transcriptomic analysis by RNA sequencing based on Illumina HiSeq, NextSeq, or NovaSeq Systems followed by real-time qPCR validation. We also depict a method for proteomic analysis by "one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis" (1D SDS-PAGE) and a sample preparation procedure for "liquid chromatography in tandem with mass spectrometry" (LC-MS/MS), and we present some generic points to consider during LC-MS/MS.

SET protein modulates H4 histone methylation status and regulates miR-137 level in oral squamous cell carcinoma.

Aim: Histone acetylation and methylation control gene expression. We investigated the impact of SET knockdown on histone methylation status and the consequences for the miRNAs levels in oral squamous cell carcinoma (OSCC). Methods: OSCC cells with and without SET knockdown were analyzed by quantitative real-time PCR to determine miRNA levels, and by immunoreactions to histone modifications. Results: The knockdown of SET increased the levels of histone H4K20me2 and miR-137. Still, SET protein binds to the miR-137 promoter region. The transfection of miR-137 mimic reduced the KI67 and Rb proteins and proliferation of OSCC cells. Conclusion: Our results show for the first time a relationship between SET and histone methylation associated with the control of miRNA expression and KI67 and Rb as targets of miR-137 in OSCC.

SET protein accumulation prevents cell death in head and neck squamous cell carcinoma through regulation of redox state and autophagy.

Molecular alterations in cell death pathways and imbalances in regulators of up- or downstream signaling pathways can lead to resistance to cell death, which is one of the hallmarks of cancer. These signaling modifications are strategies that tumor cells use to resist chemotherapy and that contribute to the high recurrence rate of head and neck squamous cell carcinoma (HNSCC). The SET oncoprotein is a PP2A inhibitor that accumulates in HNSCC and represents a promising therapeutic target. Here we report the role that SET protein plays in resistance to death of two HNSCC cell lines: Cal 27 and HN13. SET protein regulated intracellular redox balance by controlling cellular localization of APE 1 - an endonuclease that is part of the SET complex and regulates antioxidant gene transcription. SET protein knockdown (siSET) associated with tert-butyl hydroperoxide-induced oxidative stress sensitized Cal 27 and HN13 cells to apoptosis via the extrinsic and intrinsic pathways, respectively. SET protein upregulated autophagy in HNSCC cells in a PP2A-dependent manner and apparently regulated ULK1 expression. The fact that siSET lowered Bcl-2 phosphorylation levels indicated that SET protein interfered with an alternative pathway that modulated autophagy in HNSCC cells. Overall, SET protein regulated intracellular redox state and sustained autophagy in HNSCC cells, which may explain resistance to death of HNSCC cells. Altogether, the findings reported herein support SET protein as therapeutic target for HNSCC.

Anti-cancer activity of a new dihydropyridine derivative, VdiE-2N, in head and neck squamous cell carcinoma.

This study aims to examine the effects of a new 1,4-dihydropyridine derivative, VdiE-2N, on cell signaling pathways and mitochondrial events in head and neck squamous cell carcinoma (HNSCC) cells, and on a mice model of xenograft tumor growth/cell proliferation. Four HNSCC cell lines (HN13, HN12, HN6, and CAL27), HEK293 cells (human embryonic kidney 293 cells), and human oral healthy mucosa fibroblasts (OHMF) were used for in vitro assessment of cell viability (resazurin assay) and invasion capacity (modified Boyden chamber assay), and mitochondrial membrane potential (JC-1 fluorescence assay), morphology (transmission electron microscopy), and number of mitochondria (MitoTracker® imaging). SET and pDRP1 proteins were analyzed by immunofluorescence, and proteins involved in cell death/survival pathways were analyzed by Western blotting. HN12 xenograft tumors were established in the flank of Balb/c nude mice, and their characteristics and sensitivity to VdiE-2N were determined by immunohistochemistry and histology. VdiE-2N decreased cell viability in HNSCC cells (IC50 = 9.56 and 22.45µM for HN13 and HN12 cells, respectively) more strongly than it decreased cell viability in OHMF and HEK293 cells (IC50 = 32.90 and > 50µM, respectively). In HN13 cells, VdiE-2N dissipated mitochondrial membrane potential and altered the mitochondria size, shape, and number in a concentration-dependent manner, as well as it induced apoptosis and reduced their invasion capacity. Treatment of mice bearing xenograft tumors with VdiE-2N significantly diminished proliferation of cancer cells. Therefore, VdiE-2N induces HNSCC cell death in vitro through mitochondria-mediated apoptotic pathways and dampens tumor growth in vivo, thus supporting a potential anti-cancer effect.

Heterologous expression of mitochondrial nicotinamide adenine dinucleotide transporter (Ndt1) from Aspergillus fumigatus rescues impaired growth in Δndt1Δndt2 Saccharomyces cerevisiae strain.

Our understanding of nicotinamide adenine dinucleotide mitochondrial transporter 1 (Ndt1A) in Aspergillus fumigatus remains poor. Thus, we investigated whether Ndt1A could alter fungi survival. To this end, we engineered the expression of an Ndt1A-encoding region in a Δndt1Δndt2 yeast strain. The resulting cloned Ndt1A protein promoted the mitochondrial uptake of nicotinamide adenine dinucleotide (NAD+), generating a large mitochondrial membrane potential. The NAD+ carrier utilized the electrochemical proton gradient to drive NAD+ entrance into mitochondria when the mitochondrial membrane potential was sustained by succinate. Its uptake has no impact on oxidative stress, and Ndt1A expression improved growth and survival of the Δndt1Δndt2 Saccharomyces cerevisiae strain.