Project description:Inhibitor of differentiation/DNA-binding (Id) proteins are helix-loop-helix (HLH) transcription factors. The Id protein family (Id1-Id4) mediates tissue homeostasis by regulating cellular processes including differentiation, proliferation, and apoptosis. Ids typically function as dominant negative HLH proteins, which bind other HLH proteins and sequester them away from DNA promoter regions. Previously, we have found that Id3 induced apoptosis in immortalized human keratinocytes upon UVB exposure, consistent with its role as a tumor suppressor. To investigate the role of Id3 in malignant squamous cell carcinoma (SCC) cells (A431), a tetracycline-regulated inducible system was used to induce Id3 in cell culture and mouse xenograft models. We found that upon Id3 induction, there was a decrease in cell number under low serum conditions, as well as in soft agar. Microarray, RT-PCR, immunoblot, siRNA, and inhibitor studies revealed that Id3 induced expression of Elk-1, an E-twenty-six (ETS)-domain transcription factor, inducing procaspase-8 expression and activation. Id3 deletion mutants revealed that 80 C-terminal amino acids, including the HLH, are important for Id3-induced apoptosis. In a mouse xenograft model, Id3 induction decreased tumor size by 30%. Using immunofluorescent analysis, we determined that the tumor size decrease was also mediated through apoptosis. Furthermore, we show that Id3 synergizes with 5-FU and cisplatin therapies for nonmelanoma skin cancer cells. Our studies have shown a molecular mechanism by which Id3 induces apoptosis in SCC, and this information can potentially be used to develop new treatments for SCC patients.
Project description:The role of phosphatidylcholine-specific phospholipase C (PC-PLC), the enzyme involved in cell differentiation and proliferation, has not yet been explored in tumor initiating cells (TICs). We investigated PC-PLC expression and effects of PC-PLC inhibition in two adherent (AD) squamous carcinoma cell lines (A431 and CaSki), with different proliferative and stemness potential, and in TIC-enriched floating spheres (SPH) originated from them.Compared with immortalized non-tumoral keratinocytes (HaCaT) A431-AD cells showed 2.5-fold higher PC-PLC activity, nuclear localization of a 66-kDa PC-PLC isoform, but a similar distribution of the enzyme on plasma membrane and in cytoplasmic compartments. Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content. Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 ?g/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%). In A431-SPH and CaSki-SPH D609 induced both cytostatic and cytotoxic effects at about 20 to 30-fold lower doses (IC50 ranging between 1.2 and 1.6 ?g/ml). Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).These data suggest that the inhibition of PC-PLC activity may represent a new therapeutic approach to selectively target the most aggressive and tumor promoting sub-population of floating spheres originated from squamous cancer cells possessing different proliferative and stemness potential.
Project description:Milk consumption may modify the risk of squamous cell carcinoma. The role of milk to modulate the gene expression in oral squamous cell carcinoma cells has not been investigated so far. Here, HSC2 oral squamous carcinoma cells were exposed to an aqueous fraction of human milk and a whole-genome array was performed. Among the genes that were significantly reduced by human and cow milk were the DNA-binding protein inhibitor 1 (ID1), ID3 and Distal-Less Homeobox 2 (DLX2) in HSC2 cells. Also, in TR146 oral squamous carcinoma cells, there was a tendency towards a decreased gene expression. Upon size fractionation, lactoperoxidase but not lactoferrin and osteopontin was identified to reduce ID1 and ID3 in HSC2 cells. Dairy products and hypoallergenic infant formula failed to decrease the respective genes. These data suggest that milk can reduce the expression of transcription factors in oral squamous carcinoma cells.
Project description:Previously it has been shown that Id3 can act as an apoptosis-inducer gene in immortalized human keratinocytes. To further investigate the role of Id3 in the progression of skin cancer, the role of Id3 in A431 cells is investigated through ectopic induction of Id3. Microarray analysis is used to guide us the genes and pathways that are significantly altered after Id3 induction. 4 groups are included in this study, each with one sample. There are two cell lines: A431 cells stably transfected with Id3 or control vector. In each cell line, there are two conditions: induced and un-induced with tetracycline.
Project description:Although there is increasing evidence that human bone marrow mesenchymal stem cells (hBM-MSCs) play an important role in cancer progression, the underlying mechanisms are poorly understood. Transforming growth factor ? (TGF-?) is an important pro-metastatic cytokine. We have previously shown that CD109, a glycosylphosphatidylinositol-anchored protein, is a TGF-? co-receptor and a strong inhibitor of TGF-? signalling. Moreover, CD109 can be released from the cell surface. In the current study, we examined whether hBM-MSCs regulate the malignant properties of squamous cell carcinoma cells, and whether CD109 plays a role in mediating the effect of hBM-MSCs on cancer cells. Here we show that hBM-MSC-conditioned medium decreases proliferation and induces apoptosis in human squamous carcinoma cell lines, A431 and FaDu. Importantly, hBM-MSC-conditioned medium markedly suppresses markers of epithelial-to-mesenchymal transition and stemness, and concomitantly decreases cell migration, invasion, and spheroid formation in A431 and FaDu cells. In addition, knockdown of CD109 in hBM-MSCs abrogates the anti-malignant activity of hBM-MSC-conditioned medium on A431 and FaDu cells. Furthermore, overexpression of CD109 in A431 cells decreases their malignant traits. Together, our findings suggest that hBM-MSCs inhibit the malignant traits of squamous cell carcinoma cells by a paracrine effect via released factors and that CD109 released from hBM-MSCs, at least partially, mediates these effects.
Project description:Bromodomain-containing protein 4 (BRD4) is a potential therapeutic target of skin squamous cell carcinoma (SCC). I-BET726 is a novel BRD4 inhibitor. Its potential effect in skin SCC cells was tested in the present study. We show that I-BET726 potently inhibited survival, proliferation, cell cycle progression, and migration in established (A431/SCC-9/SCC-12/SCC-13 lines) and primary human skin SCC cells. I-BET726 induced significant apoptosis activation in skin SCC cells. It was more efficient in inhibiting skin SCC cells than known BRD4 inhibitors (JQ1, CPI203, and AZD5153). I-BET726 not only downregulated BRD4-regulated proteins (c-Myc, Bcl-2, and cyclin D1), but also inhibited sphingosine kinase 1 (SphK1) and Akt signalings in SCC cells. Restoring Akt activation, by a constitutively active S473D mutant Akt1 ("caAkt1"), partially inhibited I-BET726-induced cytotoxicity in A431 cells. In vivo, I-BET726 oral administration potently inhibited A431 xenograft growth in severe combined immunodeficient mice. Downregulation of BRD4-regulated proteins and inhibition of the SphK1-Akt signaling were detected in I-BET726-treated A431 xenograft tumor tissues. Together, I-BET726 inhibits skin SCC cell growth in vitro and in vivo.
Project description:Cortical thickness has been investigated since the beginning of the 20th century, but we do not know how similar the cortical thickness profiles among humans are. In this study, the local similarity of cortical thickness profiles was investigated using sliding window methods. Here, we show that approximately 5% of the cortical thickness profiles are similarly expressed among humans while 45% of the cortical thickness profiles show a high level of heterogeneity. Therefore, heterogeneity is the rule, not the exception. Cortical thickness profiles of somatosensory homunculi and the anterior insula are consistent among humans, while the cortical thickness profiles of the motor homunculus are more variable. Cortical thickness profiles of homunculi that code for muscle position and skin stimulation are highly similar among humans despite large differences in sex, education, and age. This finding suggests that the structure of these cortices remains well preserved over a lifetime. Our observations possibly relativize opinions on cortical plasticity.
Project description:Administration of cetuximab (C-mab) in combination with paclitaxel (PTX) has been used for patients with head and neck squamous cell carcinoma (SCC) clinically. In this study, we attempted to clarify the molecular mechanisms of the enhancing anticancer effect of C-mab combined with PTX on oral SCC cells in vitro. We used two oral SCC cells (HSC4, OSC19) and A431 cells. PTX alone inhibited cell growth in all cells in a concentration-dependent manner. C-mab alone inhibited the growth of A431 and OSC19 cells at low concentrations, but inhibited the growth of HSC4 cells very weakly, even at high concentrations. A combined effect of the two drugs was moderate on A431 cells, but slight on HSC4 and OSC19 cells. A low concentration of PTX enhanced the antibody-dependent cellular cytotoxicity (ADCC) induced by C-mab in all of the cells tested. PTX slightly enhanced the anticancer effect of C-mab in this ADCC model on A431 and HSC4 cells, and markedly enhanced the anticancer effect of C-mab on OSC19 cells. These results indicated that PTX potentiated the anticancer effect of C-mab through enhancing the ADCC in oral SCC cells.
Project description:p38 mitogen-activated protein kinases (MAPKs) respond to a wide range of extracellular stimuli. While the inhibition of p38 signaling is implicated in the impaired capacity to repair ultraviolet (UV)-induced DNA damage-a primary risk factor for human skin cancers-its mechanism of action in skin carcinogenesis remains unclear, as both anti-proliferative and survival functions have been previously described. In this study, we utilized cultured keratinocytes, murine tumorigenesis models, and human cutaneous squamous cell carcinoma (SCC) specimens to assess the effect of p38 in this regard. UV irradiation of normal human keratinocytes increased the expression of all four p38 isoforms (?/?/?/?); whereas irradiation of p53-deficient A431 keratinocytes derived from a human SCC selectively decreased p38?, without affecting other isoforms. p38? levels are decreased in the majority of human cutaneous SCCs assessed by tissue microarray, suggesting a tumor-suppressive effect of p38? in SCC pathogenesis. Genetic and pharmacological inhibition of p38? and in A431 cells increased cell proliferation, which was in turn associated with increases in NAPDH oxidase (NOX2) activity as well as intracellular reactive oxygen species (ROS). These changes led to enhanced invasiveness of A431 cells as assessed by the matrigel invasion assay. Chronic treatment of p53-/-/SKH-1 mice with the p38 inhibitor SB203580 accelerated UV-induced SCC carcinogenesis and increased the expression of NOX2. NOX2 knockdown suppressed the augmented growth of A431 xenografts treated with SB203580. These findings indicate that in the absence of p53, p38? deficiency drives SCC growth and progression that is associated with enhanced NOX2 expression and ROS formation.