Anticancer effects of putative and validated BH3-mimetic drugs in head and neck squamous cell carcinomas: An overview of current knowledge.

The purpose of this review was to summarise available literature concerning the anticancer effects of both putative and validated BH3-mimetics in head and neck squamous cell carcinomas. A literature search was performed and studies assessing malignant cell lines, xenograft models, and/or humans were considered eligible. A total of 501 studies were identified, of which 40 were included. One phase-II clinical trial assessing gossypol (combined with docetaxel) was found. The remaining 39 preclinical studies investigated cell lines and/or xenograft models involving the use of six validated BH3-mimetics (A-1210477, A-1331852, ABT-737, navitoclax, S63845, venetoclax) and six putative BH3-mimetics (ApoG2, gossypol, obatoclax, sabutoclax, TW-37, and YC137). In preclinical settings, most validated BH3-mimetics were capable of inducing apoptosis (in-vitro) and tumour growth inhibition (in-vivo). The majority of putative BH3-mimetics were also capable of inducing cell death, although important off-target effects, such as autophagy induction, were also described. Combinations with conventional anticancer drugs, ionising radiation, or multiple BH3-mimetics generally resulted in enhanced anticancer effects, such as increased sensitivity to apoptotic stimuli, especially considering some cell lines that showed resistance to either treatment alone. In conclusion, although clinical data are still insufficient to evaluate the anticancer effects of BH3-mimetics in head and neck squamous cell carcinomas, promising results in preclinical settings were observed concerning induction of cell death and inhibition of tumour growth. Therefore, further clinical trials are highly encouraged.

"Just Engage in It or Not, You Get Out What You Put In": Student and Staff Experiences of Feedback and Feedforward in Workplace-Based Learning Environments.

Feedback is central to student learning in the veterinary workplace. Feedforward, a related concept, is used to describe the way information about a student's performance may be used to improve their future performance. Feedback and feedforward practices are diverse, with varied student and staff understandings of the nature and purpose of feedback (feedback literacy). This study compared the practices of feedback and feedforward in a range of programs in one institution during student transitions from the classroom to workplace-based learning environments. The study adopted a broad inter-professional approach to include health care programs and social work and theater and performance studies. Profession-specific focus groups were conducted with contribution from 28 students and 31 staff from five different professions. Thematic analysis revealed that students and staff shared an understanding of the feedback and feedforward concepts, and both groups recognized the importance of emotional and relational aspects of the process. Students and staff across all professions recognized the impact of time constraints on the feedback process, although this was particularly highlighted in the health science professions. Social work and theater and performance studies students demonstrated a more nuanced understanding of the emotional and relational aspects of feedback and feedforward. Overall, the approach highlights similarities and differences in practices and experiences in different workplace contexts, creating opportunities for cross-disciplinary learning, which may have relevance more widely in higher education programs with workplace-based elements. The study underpinned the development of the LeapForward feedback training resource (https://bilt.online/the-leapforward-project/).

Real-World Experience with Peanut Oral Immunotherapy: Lessons Learned From 270 Patients.

BACKGROUND: Peanut allergy (PA) is a significant and increasing problem, interfering with psychological development and family life. The standard recommendation to avoid peanut products and have access to injectable epinephrine is often inadequate. Oral immunotherapy for PA has been formally studied. Clinical observations of allergen immunotherapy have repeatedly enhanced patient care. OBJECTIVE: The purpose of this study was to report observations on the treatment of 270 patients with PA over 8.5 years. METHODS: This is a retrospective record review of patients beginning peanut oral immunotherapy between January 1, 2009, and June 1, 2017, approved by the North Texas Institutional Review Board. RESULTS: A total of 270 patients aged 4 to 18 years comprising 107 girls and 163 boys were treated. A total of 214 of 270 patients (79%) completed immunotherapy escalation. Age (P < .001) and peanut IgE (P < .001) correlate inversely with completing dose escalation. Epinephrine-treated reactions in 63 and isolated gastrointestinal symptoms in 101 patients, respectively, were the most common adverse reactions to treatment but did not preclude success. Peanut IgE decreased by 65% after 3 years of maintenance treatment and 14 of 214 patients (6.5%) were able to achieve sustained unresponsiveness. CONCLUSIONS: In an allergy office practice setting, 79% of patients are able to complete a peanut desensitization protocol and maintain the desensitized state indefinitely with daily dosing. With appropriate planning and precautions, peanut oral immunotherapy may be performed in an allergy office. Careful observations of clinical treatment can contribute to the development of effective treatment strategies.

The PI3K/Akt Pathway in Tumors of Endocrine Tissues.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is a key driver in carcinogenesis. Defects in this pathway in human cancer syndromes such as Cowden's disease and Multiple Endocrine Neoplasia result in tumors of endocrine tissues, highlighting its importance in these cancer types. This review explores the growing evidence from multiple animal and in vitro models and from analysis of human tumors for the involvement of this pathway in the following: thyroid carcinoma subtypes, parathyroid carcinoma, pituitary tumors, adrenocortical carcinoma, phaeochromocytoma, neuroblastoma, and gastroenteropancreatic neuroendocrine tumors. While data are not always consistent, immunohistochemistry performed on human tumor tissue has been used alongside other techniques to demonstrate Akt overactivation. We review active Akt as a potential prognostic marker and the PI3K pathway as a therapeutic target in endocrine neoplasia.

Association of differential ß-catenin expression with Oct-4 and Nanog in oral squamous cell carcinoma and their correlation with clinicopathological factors and prognosis.

BACKGROUND: The re-expression of pluripotent markers (Oct-4 and Nanog) and the reactivation of stem cell-related pathways in oral carcinoma have been well researched. However, the relationship between the stem cell signaling molecule ß-catenin and pluripotent markers Oct-4 and Nanog in oral cancer is yet to be studied in detail. Therefore, we have investigated the correlation among Oct-4, Nanog, and ß-catenin in oral squamous cell carcinoma, which, in turn, could provide valuable insight into its prognostic significance. METHODS: The immunohistochemical analysis was performed for 60 cases of oral cancer to study the expression pattern of Oct-4, Nanog, and ß-catenin. Whereas immunofluorescence analysis was used to investigate the co-localization of ß-catenin with Oct-4 and Nanog in oral carcinoma tissues and H314 cell line. Finally, co-immunoprecipitation analysis was used to study the possible interaction between ß-catenin and Oct-4 in oral carcinoma cells. RESULTS: ß-catenin, Oct-4, and Nanog showed significant correlation with lymph node metastasis, stage, grade, and prognosis in oral squamous cell carcinoma. Interestingly, a significant positive correlation was found among the expression of Oct-4, Nanog, and ß-catenin. Moreover, the interaction between ß-catenin and Oct-4 was observed in oral cancer. CONCLUSION: The positive correlation among Oct-4, Nanog, and ß-catenin suggests their coordinated role in maintaining proliferation in oral carcinoma cells. The interaction between ß-catenin and Oct-4 may be a crucial event in oral carcinogenesis. On the other hand, ß-catenin, Oct-4, and Nanog could be used as independent prognostic markers of oral squamous cell carcinoma.

Oral immunotherapy for peanut allergy: multipractice experience with epinephrine-treated reactions.

BACKGROUND: Peanut allergy creates the risk of life-threatening anaphylaxis that can disrupt psychosocial development and family life. The avoidance management strategy often fails to prevent anaphylaxis and may contribute to social dysfunction. Peanut oral immunotherapy may address these problems, but there are safety concerns regarding implementation in clinical practice. OBJECTIVE: The purpose of this report is to communicate observations about the frequency of epinephrine-treated reactions during peanut oral immunotherapy in 5 different allergy/immunology practices. METHODS: Retrospective chart review of peanut oral immunotherapy performed in 5 clinical allergy practices. RESULTS: A total of 352 treated patients received 240,351 doses of peanut, peanut butter, or peanut flour, and experienced 95 reactions that were treated with epinephrine. Only 3 patients received 2 doses of epinephrine, and no patient required more intensive treatment. A total of 298 patients achieved the target maintenance dose for a success rate of 85%. CONCLUSION: Peanut oral immunotherapy carries a risk of systemic reactions. In the context of oral immunotherapy, those reactions were recognized and treated promptly. Peanut oral immunotherapy may be a suitable therapy for patients managed by qualified allergists/immunologists.

Thioflavin S (NSC71948) interferes with Bcl-2-associated athanogene (BAG-1)-mediated protein-protein interactions.

The C-terminal BAG domain is thought to play a key role in BAG-1-induced survival and proliferation by mediating protein-protein interactions, for example, with heat shock proteins HSC70 and HSP70, and with RAF-1 kinase. Here, we have identified thioflavin S (NSC71948) as a potential small-molecule chemical inhibitor of these interactions. NSC71948 inhibited the interaction of BAG-1 and HSC70 in vitro and decreased BAG-1:HSC70 and BAG-1:HSP70 binding in intact cells. NSC71948 also reduced binding between BAG-1 and RAF-1, but had no effect on the interaction between two unrelated proteins, BIM and MCL-1. NSC71948 functionally reversed the ability of BAG-1 to promote vitamin D3 receptor-mediated transactivation, an activity of BAG-1 that depends on HSC70/HSP70 binding, and reduced phosphorylation of p44/42 mitogen-activate protein kinase. NSC71948 can be used to stain amyloid fibrils; however, structurally related compounds, thioflavin T and BTA-1, had no effect on BAG-1:HSC70 binding, suggesting that structural features important for amyloid fibril binding and inhibition of BAG-1:HSC70 binding may be separable. We demonstrated that NSC71948 inhibited the growth of BAG-1 expressing human ZR-75-1 breast cancer cells and wild-type, but not BAG-1-deficient, mouse embryo fibroblasts. Taken together, these data suggest that NSC71948 may be a useful molecule to investigate the functional significance of BAG-1 C-terminal protein interactions. However, it is important to recognize that NSC71948 may exert additional "off-target" effects. Inhibition of BAG-1 function may be an attractive strategy to inhibit the growth of BAG-1-overexpressing cancers, and further screens of additional compound collections may be warranted.

Bag-1 proteins in oral squamous cell carcinoma.

Bag-1 is an anti-apoptotic protein that exhibits altered expression in many malignancies, including oral squamous cell carcinoma. The bag-1 gene gives rise to different protein products with different subcellular localisations through alternative translational initiation sites. In oral squamous cell carcinoma, cytoplasmic expression has been associated with metastasis to regional lymph nodes and poor prognosis. In contrast, the longest Bag-1 isoform is nuclear and may regulate differentiation in oral epithelium. In this review, the functions of the three isoforms of Bag-1 expressed in oral epithelial cells are discussed in relation to their contribution to oral carcinogenesis.

BAG-1 inhibits PPARgamma-induced cell death, but not PPARgamma-induced transcription, cell cycle arrest or differentiation in breast cancer cells.

BAG-1 is a pleiotropic protein that exists as multiple isoforms. BAG-1 overexpression in breast cancer is associated with outcome. BAG-1 modulates the function of various nuclear hormone receptors, including the oestrogen receptor, and BAG-1 can influence the in vitro action of anti-hormonal therapies such as cyproterone acetate in prostate cancer. Activation of PPARgamma, a nuclear hormone receptor important for lipid and glucose homeostasis, may present a new therapeutic approach for breast cancer, since PPARgamma agonists promote cell cycle arrest, differentiation and apoptosis in breast cancer cells. Here we determined whether BAG-1 also modulated PPARgamma function in MCF7 cells. 15-deoxy-Delta12, 14-prostaglandin J(2) (15dPGJ2), an agonistic ligand for PPARgamma, induced expression of HSP70, a BAG-1 binding partner, but did not alter BAG-1 isoform expression. Overexpression of BAG-1 isoforms did not alter PPARgamma-dependent transcription or interfere with 15dPGJ2-induced cell cycle arrest or differentiation. However, overexpression of BAG-1 isoforms did interfere with induction of cell death by 15dPGJ2. Thus, BAG-1 is unlikely to directly modulate PPARgamma function, but the overexpression of BAG-1 in some breast cancers may limit the efficacy of PPARgamma agonists as cancer therapies, by suppression of PPARgamma-induced cell death pathways.

BAG-1 is up-regulated in colorectal tumour progression and promotes colorectal tumour cell survival through increased NF-kappaB activity.

Although expression of the anti-apoptotic protein Bcl-2-associated athanogene-1 (BAG-1) has been reported as up-regulated in a number of malignancies, we show for the first time that BAG-1 is over-expressed in medium/large-sized colorectal adenomas and carcinomas compared with normal epithelium. To investigate whether expression of BAG-1 is important for colorectal tumour cell survival, microarray analysis was carried out on the HCT116 colorectal carcinoma cell line following transfection with BAG-1 small interfering RNA (siRNA). Analysis identified altered expression of a subset of potential nuclear factor-kappaB (NF-kappaB)-regulated genes. Furthermore, knock down of BAG-1 was shown to inhibit NF-kappaB transcriptional activity. Inhibition of NF-kappaB activity using BAG-1 siRNA or the NF-kappaB inhibitor BAY-117082 suppressed HCT116 cell yield and induced apoptosis; combined treatment had no additive effect, suggesting that the decrease in cell yield associated with knock down of BAG-1 expression is mediated via inhibition of NF-kappaB. Of clinical relevance, BAG-1 siRNA sensitized colorectal carcinoma cells to apoptosis induced by potential therapeutic agent TRAIL as well as tumour necrosis factor-alpha, both inducers of NF-kappaB activity. In summary, knock down of BAG-1 leads to inhibition of NF-kappaB, identifying BAG-1 as a novel regulator of NF-kappaB. It is proposed that, by inhibiting NF-kappaB, suppression of BAG-1 could represent a novel strategy to impede colorectal cancer cell survival and as an adjuvant increase sensitivity to current therapeutic regimes.

Cell motility assays.

This report summarises practical aspects to measuring cell motility in culture. The methods described here were discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop organised by John Masters and Gareth E Jones that was held at University College London on 19th April 2007.

Subcellular localisation of BAG-1 and its regulation of vitamin D receptor-mediated transactivation and involucrin expression in oral keratinocytes: implications for oral carcinogenesis.

In oral cancers, cytoplasmic BAG-1 overexpression is a marker of poor prognosis. BAG-1 regulates cellular growth, differentiation and survival through interactions with diverse proteins, including the vitamin D receptor (VDR), a key regulator of keratinocyte growth and differentiation. BAG-1 is expressed ubiquitously in human cells as three major isoforms of 50 kDa (BAG-1L), 46 kDa (BAG-1M) and 36 kDa (BAG-1S) from a single mRNA. In oral keratinocytes BAG-1L, but not BAG-1M and BAG-1S, enhanced VDR transactivation in response to 1alpha,25-dihydroxyvitamin D3. BAG-1L was nucleoplasmic and nucleolar, whereas BAG-1S and BAG-1M were cytoplasmic and nucleoplasmic in localisation. Having identified the nucleolar localisation sequence in BAG-1L, we showed that mutation of this sequence did not prevent BAG-1L from potentiating VDR activity. BAG-1L also potentiated transactivation of known vitamin-D-responsive gene promoters, osteocalcin and 24-hydroxylase, and enhanced VDR-dependent transcription and protein expression of the keratinocyte differentiation marker, involucrin. These results demonstrate endogenous gene regulation by BAG-1L by potentiating nuclear hormone receptor function and suggest a role for BAG-1L in 24-hydroxylase regulation of vitamin D metabolism and the cellular response of oral keratinocytes to 1alpha,25-dihydroxyvitamin D3. By contrast to the cytoplasmic BAG-1 isoforms, BAG-1L may act to suppress tumorigenesis.

Over-expression of TGF-beta1 in Smad4-deficient human oral carcinoma cells causes tumour regression in vivo by mechanisms that sensitize cells to apoptosis.

We have shown previously that transforming growth factor-beta (TGF-beta) is a potent tumour suppressor in Smad4-deficient human malignant oral keratinocytes but the mechanism by which this occurs is unknown. In the present study, we show that over-expression of TGF-beta1 causes regression of tumours derived from Smad4-deficient oral keratinocytes transplanted orthotopically to athymic mice. Further, tumour regression is associated with the induction of apoptosis without changes in cell proliferation. In vitro, TGF-beta1 did not induce apoptosis directly in these cells but sensitized cells to cisplatin, but not Fas, -induced cell death. The data suggest that TGF-beta1 induces tumour regression in vivo by Smad4-independent pathways that sensitize keratinocytes to mitochondrial-mediated apoptosis.

Caspase-3 expression is reduced, in the absence of cleavage, in terminally differentiated normal oral epithelium but is increased in oral squamous cell carcinomas and correlates with tumour stage.

Oral carcinomas are known to have a greater apoptotic index than normal oral epithelium, evident as shrinking cells with condensed chromatin. In this study, these morphologically apoptotic cells stained positively for cleaved (active) caspase-3. In normal oral epithelium, cleaved caspase-3 positive-cells were only rarely detected. The terminally differentiated surface epithelial layers did not express cleaved caspase-3. The caspase-3 pro-enzyme showed a gradient of expression in normal oral epithelium, decreasing with differentiation. No expression was detectable in surface epithelial layers. Lack of expression of the major 'executioner' caspase-3 may, at least in part, explain differences in morphology between terminally differentiated and apoptotic cells. In cancers of different tissue origins, caspase-3 pro-enzyme expression can be either increased or decreased compared with normal tissue counterparts. To determine how caspase-3 expression alters during oral carcinogenesis, caspase-3 expression was compared in 39 samples of normal oral epithelium and 54 oral squamous cell carcinomas. Squamous cell carcinomas had more intense caspase-3 staining than normal epithelium (p < 0.001). Moreover, within the oral squamous cell carcinoma series, there was significantly more intense nuclear and cytoplasmic staining with increasing STNMP stage (p = 0.017 and 0.03, respectively). This was a reflection of significant associations with site (S), palpable lymph nodes (N), and differentiation (P). Both caspase-3 staining intensity and the percentage of cells positive for caspase-3 were inversely associated with differentiation. Studies of the mechanisms by which high levels of caspase-3 expression are tolerated in oral carcinoma cells may identify targets that can be used to harness caspase-3 overexpression for therapeutic benefit.

The retinoblastoma protein interacts with Bag-1 in human colonic adenoma and carcinoma derived cell lines.

Although the retinoblastoma susceptibility gene RB1 is inactivated in a wide range of human tumours, overexpression in colonic carcinomas has been linked to the antiapoptotic function of the protein. In the current study we show that the Retinoblastoma susceptibility protein (Rb) protein interacts with Bag-1, an apoptotic regulator, in human colonic adenoma- and carcinoma-derived cell lines. Coimmunoprecipitation demonstrated that endogenous Rb and Bag-1 interact in both adenoma- and carcinoma-derived cell lines. The specificity of the interaction was demonstrated by expression of human Papillomavirus E7 oncoprotein, an inhibitor of Rb protein interactions, which disrupted the Rb/Bag-1 complex. We report that Bag-1 is predominantly localised in the nucleus of colorectal adenoma- and carcinoma-derived epithelial cells. Disruption of the Rb/Bag-1 complex through expression of E7 changes the subcellular distribution of Bag-1, decreasing nuclear localised Bag-1. Our work establishes that the Rb protein interacts with the Bag-1 apoptotic regulator protein, and introduces a novel function for Rb, involving modulation of the subcellular localisation of Bag-1 in human colonic epithelial cells.

The cyclooxygenase 2-selective inhibitor NS398 inhibits proliferation of oral carcinoma cell lines by mechanisms dependent and independent of reduced prostaglandin E2 synthesis.

PURPOSE: We investigated the potential of cyclooxygenase (COX)-2 as anappropriate chemopreventive and/or therapeutic target for oral cancer. EXPERIMENTAL DESIGN: Immunohistochemical analysis of COX-2 expression was carried out on 37 oral squamous cell carcinomas (OSCCs) and 23 normal oral epithelium samples. We investigated whether the COX-2-selective inhibitor NS398 induced growth inhibition in four human OSCC cell lines and whether this was COX-2 dependent. RESULTS: COX-2 staining was more intense in the carcinomas compared with normal epithelium (P < 0.001). Early-stage tumors (stages I and II) had significantly higher epithelial COX-2 staining than late-stage tumors (stages III and IV; P = 0.034), and overexpression of COX-2 was detected in hyperplastic and dysplastic epithelium. Treatment of OSCC cells with NS398 for 72 h at concentrations of 50 micro M and above resulted in growth inhibition accompanied by a reversible G(0)-G(1) arrest, but no apoptosis or terminal differentiation. However, a concentration of 10 micro M was sufficient to abolish secreted prostaglandin E(2) (PGE(2)) production. Over a longer treatment time, lower concentrations of NS398 were growth inhibitory. Growth inhibition of the OSCC cell line H357 was detected after treatment with 5 micro M NS398 as well as 100 micro M NS398 for 6-12 days. In cultures treated with 5 micro M NS398, but not in those treated with 100 micro M NS398, restoration of PGE(2) to control levels abrogated growth inhibition. CONCLUSIONS: NS398 inhibits the growth of OSCC cells by mechanisms that are dependent and independent of suppression of PGE(2) synthesis. Molecular targeting of COX-2, PGE(2) synthase, or PGE(2) receptors may be useful as a chemopreventive or therapeutic strategy for oral cancer.

Deregulated Bag-1 protein expression in human oral squamous cell carcinomas and lymph node metastases.

Bag-1 is an anti-apoptotic protein that promotes metastasis in some tumour cell types. To determine whether Bag-1 expression is altered in 64 oral squamous cell carcinomas, tumour samples were compared with 17 samples of normal oral epithelium. Normal oral epithelia had pronounced nuclear staining in the basal and maturation layers and weak cytoplasmic staining that was most pronounced in the basal and suprabasal layers. Oral squamous cell carcinomas demonstrated a tendency for reduced nuclear staining intensity (p=0.036). Cytoplasmic staining intensity was not significantly different between tumour and normal tissue. However, many tumours were observed to have less of a difference between nuclear staining intensity and cytoplasmic staining intensity than normal oral epithelium. Furthermore, in lymph node metastases, cytoplasmic Bag-1 staining was stronger in 8/13 cases than in corresponding primary tumours (p=0.021). Western blotting using nine oral primary carcinoma cell lines and four normal keratinocyte cultures showed that the isoforms Bag-1s, Bag-1M, and Bag-1L were expressed in normal and malignant oral epithelial cells. Bag-1L unique sequences were shown to adopt an exclusively nuclear, and predominantly nucleolar, localization by use of transiently transfected N-terminal Bag-1L-EGFP. However, levels of Bag-1L in carcinoma cells did not differ significantly from those of normal keratinocytes. Therefore the reduced nuclear staining observed in oral squamous cell carcinomas compared with normal epithelium may reflect changes in the localization of Bag-1 isoforms, rather than decreased expression of Bag-1L. Alterations in the relative proportions of Bag-1S, Bag-1M, and Bag-1L were detected in 6/9 oral carcinoma cell lines; 5/9 oral carcinoma cell lines had a significantly greater proportion of Bag-1M than normal keratinocytes and in another cell line, Bag-1L was significantly underrepresented. Overall, the results suggest that Bag-1 deregulation plays a role in oral carcinogenesis at two different stages: during primary carcinoma development and during lymph node metastasis.