The effects of enoxaparin treatment in a xenograft mouse model of oral squamous cell carcinoma: A pilot study.

BACKGROUND: Recent studies suggest that enoxaparin may have therapeutic effects on oral squamous cell carcinoma. We aimed to assess this effect utilizing xenograft mouse model through evaluations of proliferation and angiogenesis markers at the RNA and protein levels. METHODS: Mice were divided into enoxaparin treatment (n = 4), positive control (n = 4) and negative control (n = 3) groups. Immunohistochemical analyses were performed utilizing Bcl-2, Bax and Ki-67 antibodies. Expression levels of proliferation and apoptosis related genes were calculated utilizing qRT-PCR. Time-dependent proliferation assays were performed in OSC-19 and HEK293 cell-lines. RESULTS: Bax antibody showed positive staining in the cytoplasm and nuclei of tumor cells, while Bcl-2 antibody displayed staining only in the cytoplasm. A proliferation index of 15%-20% was found in all groups with the Ki-67 marker indicating no metastasis. Enoxaparin treatment caused decrease in BCL2, BAX and CCNB1 genes' expressions. Compared to HEK293, proliferation assays demonstrated higher division rates in OSC-19 with a significant decrease in viability after 96 h. CONCLUSION: Reduced BCL-2 expression indicates a regression of tumor growth, but reduced BAX expression is not correlated with increased apoptosis. Despite the aggressive nature of OSC-19, our results showed a low cell viability with a high division rate when compared with the control HEK293. This paralleled our in vivo findings that showed absence of lymph node metastasis across all mice groups. This discrepancy with the literature suggests that further investigations of the underlying mechanisms and protein-level analyses are needed to draw definitive conclusions about the effect of enoxaparin on OSC-19 behavior.

Combined evaluation of proliferation and apoptosis to calculate IC50 of VPA-induced PANC-1 cells and assessing its effect on the Wnt signaling pathway.

Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly cancers. Since most patients develop resistance to conventional treatments, new approaches are in urgency. Valproic acid (VPA) was shown to induce apoptosis and reduce proliferation in PANC-1 cells. Wnt signaling pathway is known to be involved in apoptosis and PDAC onset. However, VPA-induced apoptosis and its impact on Wnt signaling in PDACs are not linked, yet. We aimed to calculate IC50 of VPA-induced PANC-1 cells by combined analyses of proliferation and apoptosis, while assessing its effect on Wnt signaling pathway. PANC-1 was induced with increased VPA doses and time points. Three independent proliferation and apoptosis assays were performed utilizing carboxyfluorescein succinimidyl ester and Annexin V/PI staining, respectively. Flow cytometry measurements were analyzed by CellQuest and NovoExpress. Taqman hydrolysis probes and SYBR Green PCR Mastermix were assessed in expression analyses of Wnt components utilizing 2-ΔΔCt method. Cell proliferation was inhibited by 50% at 2.5 mM VPA that evoked a significant apoptotic response. Among the screened Wnt components and target genes, only LEF1 exhibited significant four-fold upregulation at this concentration. In conclusion, cancer studies mostly utilize MTT or BrdU assays in estimating cell proliferation and calculating IC50 of drugs, which provided conflicting VPA dosages utilizing PANC-1 cells. Our novel combined approach enabled specific, accurate and reproducible IC50 calculation at single cell basis with no apparent effect on Wnt signaling components. Future studies are needed to clarify the role of LEF1 in this model.

Screening of AIP Gene Variations in a Cohort of Turkish Patients with Young-Onset Sporadic Hormone-Secreting Pituitary Adenomas.

Aims: Aryl hydrocarbon receptor-interacting protein (AIP) gene mutations have long been associated with apparently sporadic pituitary adenomas (PAs) with a prevalence range of 0-12%. The aim of this study was to evaluate the frequency of germline AIP variations in a large cohort of apparently sporadic PAs diagnosed before the age of 40 years, who did not exhibit hypercalcemia and/or MEN1 syndrome components during long-term follow-up. Materials and Methods: A total of 97 patients, diagnosed with functional PAs ≤40 years old, composed of somatotropinoma (n = 55), prolactinoma (n = 25), and corticotrophinoma (n = 17), were recruited for this study. Fifty-one of these patients [somatotropinoma (n = 30), prolactinoma (n = 15), and corticotrophinoma (n = 11)] were previously reported as AIP mutation-negative by Sanger sequencing. The entire coding sequence of the AIP gene, along with exon/intron boundaries and the untranslated regions of 41 newly recruited patients, were sequenced for germline variations. In addition, all patients were subjected to multiplex ligation-dependent probe amplification to detect copy number variations in the AIP gene. Results: The AIP c.911G>A: p.Arg304Gln (rs104894190) variant was detected in only two patients with functional PA: one with somatotropinoma [in 1/55 (1.8%)] and one with prolactinoma [in 1/25 (4%)]. None of the corticotrophinomas revealed AIP gene alterations. Thus, the overall prevalence of AIP variation was 2.1% in our cohort. Conclusions: Germline AIP gene variations among Turkish patients with apparently sporadic PAs are relatively rare among patients ≤40 years old. None of the patients in our cohort revealed any obviously pathogenic AIP variants.