CDKN2A-p53 mediated antitumor effect of Lupeol in head and neck cancer

Purpose

The tumor suppressor protein p53 is known to control cell cycle arrest and apoptosis. Lupeol is a phytochemical that has been found to induce apoptosis in different cancer types through the extrinsic pathway. As yet, however, its role in the induction of cell cycle arrest and apoptosis through the intrinsic pathway in head and neck cancer has not been investigated. Here, we aimed at understanding the mechanism underlying the antitumor effect of Lupeol in head and neck cancer.

Methods

The antitumor effect of Lupeol on oral and laryngeal carcinomas was assessed using two in vitro 2D cell line models (HEp-2, UPCI:SCC-131) and, subsequently, an ex vivo 3D tumor explant culture platform that maintains key features of the native tumor microenvironment. The mechanism underlying Lupeol-mediated antitumor responses was delineated using MTT, colony formation, flow cytometry, immunofluorescence, Western blotting and immunohistochemistry assays.

Results

We found that Lupeol induced an enhanced expression of p53 in both cell line models tested and, subsequently, cell cycle arrest at the G1 phase. In addition we found that, following Lupeol treatment, p53 induced Bax expression and activated the intrinsic apoptotic pathway (as measured by Caspase-3 cleavage). Interestingly, Lupeol was also found to trigger G1 cell cycle arrest through up-regulation of the expression of CDKN2A, but not p21, resulting in inhibition of CyclinD1. In an ex vivo platform Lupeol was found to impart a potent antitumor response as defined by inhibition of Ki67 expression, decreased cell viability and concomitant activation (cleavage) of Caspase-3. Finally, we found that Lupeol can re-sensitize primary head and neck squamous cell carcinoma (HNSCC) tumor samples that had clinically progressed under a Cisplatin treatment regimen.

Conclusion

Together, our data indicate that Lupeol may orchestrate a bifurcated regulation of neoplastic growth and apoptosis in head and neck cancers and may serve as a promising agent for the management of tumors that have progressed on a platinum-based treatment regimen.

     

Oropharyngeal true teratoma

Oropharyngeal teratoma in newborn is very rare. Here we report a case of oropharyngeal true teratoma where a 17-day-old female baby presented with a protruding mass from oropharynx with episodic respiratory distress and feeding difficulty complicated by aspiration pneumonia, and treated successfully with coordinated team approach.     

Correction: RRBP1 rewires cisplatin resistance in oral squamous cell carcinoma by regulating Hippo pathway

Background Chemoresistance is one of the major factors for treatment failure in OSCC. Identifying key resistance triggering molecules will be useful strategy for developing novel treatment methods.Methods To identify the causative factors of chemoresistance, we performed RNA sequencing and global proteomic profiling of human OSCC lines presenting with sensitive, early and late cisplatin-resistance patterns.Results From the common set of dysregulated genes from both the analysis, RRBP1 was identified to be upregulated in both early and late cisplatin-resistant cells with respect to the sensitive counterpart. Analysis of OSCC patient sample indicates that RRBP1 expression is upregulated in chemotherapy-non-responder tumours as compared to chemotherapy-responder tumours. Genetic (knockout) or pharmacological (Radezolid, represses expression of RRBP1) inhibition of RRBP1 restores cisplatin-mediated cell death in chemo-resistant OSCC. Mechanistically, RRBP1 regulates Yes-associated protein1 (YAP1), a key protein in the Hippo pathway to induce chemoresistance. The PDC xenograft data suggests that knockout of RRBP1 induces cisplatin-mediated cell death and facilitates a significant reduction of tumour burden.Conclusion Overall, our data suggests that (I) RRBP1 is a major driver of cisplatin-resistance in OSCC, (II) RRBP1 regulates YAP1 expression to mediate cisplatin-resistance, (III) Radezolid represses RRBP1 expression and (IV) targeting RRBP1 reverses cisplatin-induced chemoresistance in advanced OSCC.Subject terms: Oral cancer, Drug regulation     

Exposure to chewing tobacco promotes primary oral squamous cell carcinoma and regional lymph node metastasis by alterations of SDF1α/CXCR4 axis

A high incidence of oral squamous cell carcinoma (OSCC) is observed in South‐East Asian countries due to addictions such as chewing tobacco. Local invasion and distant metastases are primary causes of poor prognosis in OSCC. This study aimed to understand the alterations in metastasis biomarkers, such as stromal cell–derived factor‐1α (SDF‐1 or SDF1α) and its receptor C‐X‐C chemokine receptor type 4 (CXCR4), in OSCC patient samples that were stratified based on the history of addiction to chewing tobacco. Targeted immunohistochemical staining and Western blotting were performed on primary tumour and metastatic lymph node (LN) tissues in parallel. Overexpression of hepatocyte growth factor (HGF), activated form of its cognate receptor tyrosine kinase, c‐Met (p‐Met), GRB2‐associated‐binding protein 1 (Gab1), phospho‐protein kinase B (pAkt), nuclear factor kappa B (NF‐κB) and cyclooxygenase‐2 (COX‐2) were observed in primary tumour and metastatic lymph nodes in both chewer and non‐chewer cohorts. Variance analysis showed significant positive correlation between them (P < .0001) indicating upregulation of these biomarkers upon ligand‐induced activation of c‐Met in both tobacco chewers and non‐chewers. Significantly higher expressions of SDF1α and CXCR4 were observed in both primary tumours and metastatic lymph nodes of tobacco chewers (P < .0001) and coincided with overexpressed HGF. In contrast, no significant correlation was observed between expression of HGF and that of SDF1α and CXCR4 in non‐chewers. Together, our findings provide important insights into the association of HGF/c‐Met and the SDF1α/CXCR4 axis in lymph node metastasis and to an aetiological link with the habit of chewing tobacco.     

Dynamic antagonism between RNA-binding protein CUGBP2 and cyclooxygenase-2-mediated prostaglandin E2 in radiation damage

Damage to intestinal epithelium limits the use of ionizing radiation (IR) in cancer therapy. Prostaglandins (PGs), generated through the action of cyclooxygenase-1 (COX-1) and COX-2 protect the intestinal stem cells from IR. In previous studies, we demonstrated that the RNA-binding protein CUGBP2 regulates the stability and translation of COX-2 mRNA by interacting with AU-rich sequences in 3' UTR. Here, we demonstrate a dynamic antagonistic relationship between CUGBP2 and COX-2. Both CUGBP2 and COX-2 are rapidly induced after IR in intestinal crypt epithelial cells in mice, but CUGBP2 protein expression is observed immediately and COX-2 protein expression is delayed. In contrast, administration of bacterial lipopolysaccharide induced COX-2 expression and PGE(2), resulting in the inhibition of CUGBP2 expression and radioprotection of the intestine. These effects were reversed by NS398, a COX-2-specific inhibitor, suggesting that lipopolysaccharide-mediated inhibition of CUGBP2 is a PG-dependent mechanism. Furthermore, CUGBP2 expression is higher in COX-1(-/-) and COX-2(-/-) mice than wild-type controls at basal conditions, which is further increased after IR.     

An unusual foreign body in the urinary bladder

A coiled electric cable was removed from the urinary bladder of a 35-year-old male, electrician by profession. Psychiatric evaluation revealed normal childhood and no psychiatric illness. Proper management by both a urologist and a psychiatrist is required in these cases.     

Weaning age, social isolation, and gender, interact to determine adult explorative and social behavior, and dendritic and spine morphology in prefrontal cortex of rats

We tested the effect of weaning at 21 or 30 days, followed by individual or group housing, on explorative and social behavior in adult male and female rats, and in males, on dendritic length and spine density in prefrontal cortex. In the open field, rats weaned early were the most active, while those weaned late and group housed were the most explorative. In the social interaction test, behavior in adult females was relatively impervious to weaning age or rearing condition. Isolated males sought out social interaction, whereas, group-reared males tended to avoid it. Social behaviors in males weaned early or group-reared correlated with decreased dendrite length and spine density, whereas, non-social behaviors correlated with increased dendritic length. Such changes are consistent with neural pruning in the development of social behavior. Although our experimental manipulations were mild, and serve as standard rearing conditions in many laboratories, their effects on brain and behavior were marked, and differed by gender. Early rearing conditions may have few appreciable effects when studied in isolation, but their interactive effects on adult social behavior are significant and varied.     

Changes of spine density and dendritic complexity in the prefrontal cortex in offspring of mothers exposed to stress during pregnancy

Both chronic stress in adulthood and episodes of stress in the early postnatal period have been shown to interfere with neuronal development in limbic prefrontal cortical regions. The present study in rats showed for the first time that the development of layer II/III pyramidal neurons in the dorsal anterior cingulate (ACd) and orbitofrontal cortex (OFC) is significantly affected in offspring of mothers exposed to stress during pregnancy. In prenatally stressed (PS) male rat pups the ACd and OFC showed significantly lower spine densities on the apical dendrite (ACd, -20%; OFC, -25%), on basal dendrites reduced spine densities where found only in the OFC (-20% in PS males). Moreover, in both cortical areas a significant reduction of dendritic length was observed in PS males compared to control offspring, which was confined to the apical dendrites (ACd, -30%, OFC, -26%). Sholl analysis revealed that these alterations were accompanied by a significantly reduced complexity of the dendritic trees in both cortical regions. PS females displayed reductions of dendritic spine densities in the ACd and OFC on both the basal (ACd, -21%; OFC, -20%) and apical dendrites (ACd, -21%; OFC, -21%), however, in contrast to the findings in PS males, no dendritic atrophy was detected in the PS females. These findings demonstrate that gestational stress leads to significant alterations of prefrontal neuronal structure in the offspring of the stressed mothers in a sex-specific manner.     

EGFR tyrosine kinase inhibitors decrease VEGF expression by both hypoxia-inducible factor (HIF)-1-independent and HIF-1-dependent mechanisms

Epidermal growth factor receptor (EGFR) inhibitors can decrease vascular endothelial growth factor (VEGF) expression and tumor angiogenesis. In the current study, we investigate the molecular pathways by which this occurs using two drugs that have been used in the clinic, gefitinib (Iressa) and erlotinib (Tarceva). The decrease in VEGF expression by gefitinib in SQ20B squamous cell carcinoma cells was opposed by adenoviral expression of Akt in these cells. The hypoxia-inducible factor-1 (HIF-1) binding site located at approximately -1 kbp in the VEGF promoter was not required for down-regulation of promoter activity by gefitinib under normoxia. Furthermore, the drug decreased activity of a reporter containing the -88/+54 region. In a gel shift assay, gefitinib led to decreased retardation of a labeled DNA oligonucleotide probe corresponding to the -88/-66 region of the VEGF promoter, which contains Sp1 binding sites. These effects of gefitinib on VEGF promoter activity and DNA binding were both reversed by Akt expression. Phosphorylation of Sp1 was decreased in the presence of gefitinib. Gefitinib also decreases VEGF expression by decreasing HIF-1alpha expression. This occurs due to decreased protein translation without any change in the level of HIF-1alpha mRNA. Together, these results suggest that gefitinib decreases VEGF expression both by decreasing Sp1 binding to the proximal core VEGF promoter and by down-regulating HIF-1alpha expression. Similar results were obtained with erlotinib in SQ20B and gefitinib in HSC3 squamous carcinoma cells. These results indicate that there are at least two separate mechanisms by which EGFR inhibitors decrease VEGF expression.