Fernanda Visioli

The Unfolded Protein Response Induces the Angiogenic Switch in Human Tumor Cells through the PERK/ATF4 Pathway

Neovascularization is a limiting factor in tumor growth and progression. It is well known that changes in the tumor microenvironment, such as hypoxia and glucose deprivation (GD), can induce VEGF production. However, the mechanism linking GD to tumor growth and angiogenesis is unclear. We hypothesize that GD induces the angiogenic switch in tumors through activation of the unfolded protein response (UPR). We report that UPR activation in human tumors results in elevated expression of proangiogenic mediators and a concomitant decrease in angiogenesis inhibitors. cDNA microarray results showed that GD-induced UPR activation promoted upregulation of a number of proangiogenic mediators (VEGF, FGF-2, IL-6, etc.) and downregulation of several angiogenic inhibitors (THBS1, CXCL14, and CXCL10). In vitro studies revealed that partially blocking UPR signaling by silencing protein kinase RNA-like ER kinase (PERK) or activating transcription factor 4 (ATF4) significantly reduced the production of angiogenesis mediators induced by GD. However, suppressing the alpha subunit of hypoxia-inducible factors had no effect on this process. Chromatin immunoprecipitation (ChIP) confirmed binding of ATF4 to a regulatory site in the VEGF gene. In vivo results confirmed that knockdown of PERK in tumor cells slows down tumor growth and decreases tumor blood vessel density. Collectively, these results show that the PERK/ATF4 arm of UPR mediates the angiogenic switch and is a potential target for antiangiogenic cancer therapy.

Cisplatin Induces Bmi-1 and Enhances the Stem Cell Fraction in Head and Neck Cancer

Recent evidence has unveiled a subpopulation of highly tumorigenic, multipotent cells capable of self-renewal in head and neck squamous cell carcinomas (HNSCCs). These unique cells, named here cancer stem cells (CSCs), proliferate slowly and might be involved in resistance to conventional chemotherapy. We have shown that CSCs are found in perivascular niches and rely on endothelial cell-secreted factors [particularly interleukin-6 (IL-6)] for their survival and self-renewal in HNSCC. Here, we hypothesized that cisplatin enhances the stem cell fraction in HNSCC. To address this hypothesis, we generated xenograft HNSCC tumors with University of Michigan-squamous cell carcinoma 22B (UM-SCC-22B) cells and observed that cisplatin treatment increased (P = .0013) the fraction of CSCs [i.e., aldehyde dehydrogenase activity high and cluster of differentiation 44 high (ALDH(high)CD44(high))]. Cisplatin promoted self-renewal and survival of CSCs in vitro, as seen by an increase in the number of orospheres in ultralow attachment plates and induction in B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) and octamer-binding transcription factor 4 expression. Cisplatin-resistant cells expressed more Bmi-1 than cisplatin-sensitive cells. IL-6 potentiated cisplatin-induced orosphere formation generated when primary human HNSCC cells were sorted for ALDH(high)CD44(high) immediately after surgery and plated onto ultralow attachment plates. IL-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation (indicative of stemness) was unaffected by treatment with cisplatin in UM-SCC-22B cells, whereas IL-6-induced extracellular signal-regulated kinase (ERK) phosphorylation (indicative of differentiation processes) was partially inhibited by cisplatin. Notably, cisplatin-induced Bmi-1 was inhibited by interleukin-6 receptor blockade in parental and cisplatin-resistant cells. Taken together, these results demonstrate that cisplatin enhances the fraction of CSCs and suggest a mechanism for resistance to cisplatin therapy in head and neck cancer.

Exposure to alcohol or tobacco affects the pattern of maturation in oral mucosal cells: a cytohistological study

Objective: To assess the maturation pattern of oral mucosal cells of patients exposed to tobacco and alcohol.

Glucose-Regulated Protein 78 (Grp78) Confers Chemoresistance to Tumor Endothelial Cells under Acidic Stress

Objectives This study was designed to investigate the activation of the unfolded protein response (UPR) in tumor associated endothelial cells (TECs) and its association with chemoresistance during acidic pH stress. Materials and Methods Endothelial cells from human oral squamous cell carcinomas (OSCC) were excised by laser capture microdissection (LCM) followed by analysis of UPR markers (Grp78, ATF4 and CHOP) using quantitative PCR. Grp78 expression was also determined by immunostaining. Acidic stress was induced in primary human dermal microvascular endothelial cells (HDMECs) by treatment with conditioned medium (CM) from tumor cells grown under hypoxic conditions or by adjusting medium pH to 6.4 or 7.0 using lactic acid or hydrochloric acid (HCl). HDMEC resistance to the anti-angiogenic drug Sunitinib was assessed with SRB assay. Results UPR markers, Grp78, ATF4 and CHOP were significantly upregulated in TECs from OSCC compared to HDMECs. HDMECs cultured in acidic CM (pH 6.0–6.4) showed increased expression of the UPR markers. However, severe acidosis led to marked cell death in HDMECs. Alternatively, HDMECs were able to adapt when exposed to chronic acidosis at pH 7.0 for 7 days, with concomittant increase in Grp78 expression. Chronic acidosis also confers drug resistance to HDMECs against Sunitinib. Knockdown of Grp78 using shRNA resensitizes HDMECs to drug treatment. Conclusions UPR induction in ECs under acidic pH conditions is related to chemoresistance and may contribute to therapeutic failures in response to chemotherapy. Targeting Grp78, the key component of the UPR pathway, may provide a promising approach to overcome ECs resistance in cancer therapy.

Intravenous vs intraperitoneal mesenchymal stem cells administration: What is the best route for treating experimental colitis?

AIM To investigate the therapeutic effects of mesenchymal stem cells (MSCs) transplanted intraperitoneally and intravenously in a murine model of colitis. METHODS MSCs were isolated from C57BL/6 mouse adipose tissue. MSC cultures were analyzed according to morphology, cellular differentiation potential, and surface molecular markers. Experimental acute colitis was induced in C57BL/6 mice by oral administration of 2% dextran sulfate sodium (DSS) in drinking water ad libitum from days 0 to 7. Colitis mice were treated with 1 × 10(6) MSCs via intraperitoneal or intravenous injection on days 2 and 5. The disease activity index was determined daily based on the following parameters: weight loss, stool consistency and presence of blood in the feces and anus. To compare morphological and functional differences in tissue regeneration between different MSC injection modalities, mice were euthanized on day 8, and their colons were examined for length, weight, and histopathological changes. Inflammatory responses were determined by measuring the levels of different serum cytokines using a CBA Th1/Th2/Th17 kit. Apoptotic rates were evaluated by terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end labeling assay. RESULTS Intravenous infusion of MSCs was more effective than intraperitoneal treatment (P < 0.001) in reducing the clinical and histopathologic severity of colitis, which includes weight loss, diarrhea and inflammation. An histological evaluation demonstrated decreased colonic inflammation based on reduced crypt loss and reduced infiltration of inflammatory cells. This therapeutic effect was most likely mediated by the down-regulation of pro-inflammatory cytokines [interleukin (IL)-6 and tumor necrosis factor (TNF)]; and by the up-regulation of anti-inflammatory cytokines (IL-10 and IL-4). Intravenous transplantation also induced high levels of IFN that lead to activation of the immunosuppressive activity of the MSCs, which did not occur with intraperitoneal transplantation (P = 0.006). An increase in apoptotic T cells was observed after intravenous, but not intraperitoneal, MSC infusion, suggesting that MSCs can induce apoptosis in resistant T cells in colonic inflammation (P = 0.027). CONCLUSION Our results demonstrate that intravenous treatment is a superior method for reducing colon inflammation compared with intraperitoneal therapy.

Peripheral clear cell variant of calcifying epithelial odontogenic tumor affecting 2 sites: report of a case

A case of clear cell variant of calcifying epithelial odontogenic tumor (CEOT) affecting 2 different extraosseous sites is described. A 43-year-old female patient presented with 2 gingival lesions (1 in the upper premolar and 1 in the lower incisor area), which were clinically diagnosed as inflammatory hyperplasia and surgically removed. Microscopically, both lesions were composed of polyhedral cells (some with clear cytoplasm); hyaline material and areas of calcification were also observed. The diagnostic hypotheses raised were clear cell variant of CEOT, hyalinizing clear cell carcinoma, and renal metastasis. The hyaline material was positive for Congo red, crystal violet, and Lugols iodine stains, but negative for Coomassie blue; the clear cells showed positively stained granules with PAS stain. Based on these results, the conclusive diagnosis for both lesions was clear cell variant of CEOT. No evidence of recurrence was observed after 1 year of follow-up.

Metronomic Dosing of BH3 Mimetic Small Molecule Yields Robust Antiangiogenic and Antitumor Effects

Bcl-2 is an antiapoptotic protein that has also been found to function as a proangiogenic signaling molecule. Improvements in antiangiogenic therapy can be engendered by metronomic dosing. Thus, we hypothesized that BH3-mimetic drugs that antagonize Bcl-2 family proteins may exert a greater efficacy when dosed metronomically. To examine this hypothesis, we employed AT101, an orally available and well-tolerated BH3-mimetic drug that has been established as effective. In a mouse xenograft model of human squamous cell carcinomas (SCC) that includes a humanized vasculature, we explored the effects of docetaxel in combination with either daily (metronomic) or weekly (bolus) doses of AT101. In addition, we explored the effect of single or combination therapy on angiogenesis and survival of endothelial or SCC cells in vitro. Metronomic AT101 therapy increased mouse survival, decreased tumor mitotic index, and decreased tumor microvessel density, compared with bolus therapy. Therapeutic potentiation was achieved by similar overall drug exposure and without altering systemic toxicities. Combinations of AT101 and docetaxel produced additive toxicity in both endothelial and SCC tumor cells. Notably, subapoptotic concentrations of AT101 potently inhibited the angiogenic potential of endothelial cells. Taken together, our findings unveil the efficacious benefits that can be achieved by metronomic delivery of BH3-mimetic drugs, in particular suggesting that SCC patients with might benefit from low-dose continuous administration of these drugs.

Expression of the cell cycle regulation proteins p53 and p21WAF1 in different types of non-dysplastic leukoplakias

Objectives The aim of this study was to analyze the immunolabeling of two cell cycle protein regulators, p53 and p21WAF1, in non-dysplastic leukoplakias with different epithelial alterations: acanthosis, hyperkeratosis and acanthosis combined with hyperkeratosis, and compare them with dysplastic leukoplakias. Material and Methods This was a prospective cohort study involving 36 patients with oral homogeneous leukoplakias. Excisional biopsies were performed and the patients remain under clinical follow-up. The leukoplakias were divided into four groups: 6 acanthosis, 9 hyperkeratosis, 10 acanthosis combined with hyperkeratosis, and 11 epithelial dysplasias. Paraffin-embebeded sections were immunostained for p53 and p21WAF1. Five hundred cells from the basal layer and 500 from the parabasal layer were counted to determine the percentage of positive cells. A qualitative analysis was also carried out to determine the presence or absence of immunohistochemical staining in the intermediate and superficial layers. Groups were compared with ANOVA (p<0.05). Pearsons correlation coefficient was used to test for associations between the two markers, p53 and p21WAF1. Results No leukoplakia recurred and no malignant transformation was observed whitin a follow-up period of 3-6 years. The mean percentage of p53 staining in the basal and parabasal layers was similar in all groups. p21WAF1 staining differed between layers was as follows: in the basal, only 3 to 4% of cells were stained, while in the parabasal, between 16 and 28% of the epithelial cells were stained in the four different studied groups with no statistically significant difference (p>0.05). Conclusions Our findings failed to differentiate the non-dysplastic lesions by means of p53 and p21WAF1 immunostaining, notwithstanding similar profiles between non-dysplastic and dysplastic leukoplakias were observed.

Is nevoid basal cell carcinoma syndrome really so rare? Proposal for an investigative protocol based on a case series.

Because the initial clinical suspicion of NBCCS isoften triggered by oral manifestations, the presence ofKOT, and other features of the syndrome (cleft palate,maxillary hypoplasia, mandibular hyperplasia, higharched palate, etc), oral surgeons play an importantrole in recognizing and diagnosing them.

Tufted Angioma in Children: Report of Two Cases and a Review of the Literature

Tufted angioma (TA) is a benign vascular tumor with endothelial origin. It is extremely rare in oral mucosa; only seven cases have been reported in the literature so far. Here, we describe two cases of tufted angioma observed in children and we also present a review of the literature about this pathology, concerning the differential diagnosis and management of this lesion in children.