Cyclin D1 promotes radioresistance through regulation of RAD51 in melanoma.

Melanoma is a notoriously radioresistant type of skin cancer. Elucidation of the specific mechanisms underlying radioresistance is necessary to improve the clinical efficacy of radiation therapy. To identify the key factors contributing to radioresistance, five melanoma cell lines were selected for study and genes that were upregulated in relatively radioresistant melanomas compared with radiosensitive melanoma cells determined via RNA sequencing technology. In particular, we focused on cyclin D1 (CCND1), a well known cell cycle regulatory molecule. In radiosensitive melanoma, overexpression of cyclin D1 reduced apoptosis. In radioresistant melanoma cell lines, suppression of cyclin D1 with a specific inhibitor or siRNA increased apoptosis and decreased cell proliferation in 2D and 3D spheroid cultures. In addition, we observed increased expression of γ-H2AX, a molecular marker of DNA damage, even at a later time after γ-irradiation, under conditions of inhibition of cyclin D1, with a response pattern similar to that of radiosensitive SK-Mel5. In the same context, expression and nuclear foci formation of RAD51, a key enzyme for homologous recombination (HR), were reduced upon inhibition of cyclin D1. Downregulation of RAD51 also reduced cell survival to irradiation. Overall, suppression of cyclin D1 expression or function led to reduced radiation-induced DNA damage response (DDR) and triggered cell death. Our collective findings indicate that the presence of increased cyclin D1 potentially contributes to the development of radioresistance through effects on RAD51 in melanoma and could therefore serve as a therapeutic target for improving the efficacy of radiation therapy.

Integrin αvß3-Akt signalling plays a role in radioresistance of melanoma.

Melanoma is a deadly type of skin cancer that is particularly difficult to treat owing to its resistance to radiation therapy. Here, we attempted to determine the key proteins responsible for melanoma radioresistance, with the aim of improving disease response to radiation therapy. Two melanoma cell lines, SK-Mel5 and SK-Mel28, with different radiosensitivities were analysed via RNA-Seq (Quant-Seq) and target proteins with higher abundance in the more radioresistant cell line, SK-Mel28, identified. Among these proteins, integrin αvß3, a well-known molecule in cell adhesion, was selected for analysis. Treatment of SK-Mel28 cells with cilengitide, an integrin αvß3 inhibitor, as well as γ-irradiation resulted in more significant cell death than γ-irradiation alone. In addition, Akt, a downstream signal transducer of integrin αvß3, showed high basic activation in SK-Mel28 and was significantly decreased upon co-treatment with cilengitide and γ-irradiation. MK-2206, an Akt inhibitor, exerted similar effects on the SK-Mel28 cell line following γ-irradiation. Our results collectively demonstrate that the integrin αvß3-Akt signalling pathway contributes to radioresistance in SK-Mel28 cells, which may be manipulated to improve therapeutic options for melanoma.

Imatinib mesylate elicits extracellular signal-related kinase (ERK) activation and enhances the survival of γ-irradiated epithelial cells.

Imatinib mesylate, commercially known as Gleevec/Glivec, is the first targeted anticancer drug that inhibits activity of the tyrosine kinases, c-ABL, c-KIT, and PDGFR. A number of studies have shown that treatment with imatinib mesylate elicits extracellular signal-related kinase (ERK) activation, which, in turn, has been shown to confer radioresistance. Here, we investigated whether treatment with imatinib mesylate protects skin-derived epithelial cells, including normal keratinocytes, immortalized HaCaT and A431 cancer cell lines, from the effects of γ-radiation. ERK activation was detected 30 min after imatinib mesylate treatment in all three cell lines. In cells exposed to γ-irradiation in the presence of imatinib mesylate, this activation of ERK was associated with a reduction in radiation-induced apoptosis and enhanced cell survival. Similar effects of imatinib mesylate treatment were observed following γ-irradiation of a three-dimensional human skin culture system that reproduces a fully differentiated epithelium. Collectively, our findings provide the evidence of a protective effect of imatinib mesylate against the effects of γ-irradiation on epithelial-derived cells, regardless of their malignancy status.

A secretome analysis reveals that PPARα is upregulated by fractionated-dose γ-irradiation in three-dimensional keratinocyte cultures.

Studies have shown that γ-irradiation induces various biological responses, including oxidative stress and apoptosis, as well as cellular repair and immune system responses. However, most such studies have been performed using traditional two-dimensional cell culture systems, which are limited in their ability to faithfully represent in vivo conditions. A three-dimensional (3D) environment composed of properly interconnected and differentiated cells that allow communication and cooperation among cells via secreted molecules would be expected to more accurately reflect cellular responses. Here, we investigated γ-irradiation-induced changes in the secretome of 3D-cultured keratinocytes. An analysis of keratinocyte secretome profiles following fractionated-dose γ-irradiation revealed changes in genes involved in cell adhesion, angiogenesis, and the immune system. Notably, peroxisome proliferator-activated receptor-α (PPARα) was upregulated in response to fractionated-dose γ-irradiation. This upregulation was associated with an increase in the transcription of known PPARα target genes in secretome, including angiopoietin-like protein 4, dermokine and kallikrein-related peptide 12, which were differentially regulated by fractionated-dose γ-irradiation. Collectively, our data imply a mechanism linking γ-irradiation and secretome changes, and suggest that these changes could play a significant role in the coordinated cellular responses to harmful ionizing radiation, such as those associated with radiation therapy. This extension of our understanding of γ-irradiation-induced secretome changes has the potential to improve radiation therapy strategies.

Heat Shock Protein 90 Regulates Subcellular Localization of Smads in Mv1Lu Cells.

Heat shock protein 90 (HSP90) regulates the stability of various proteins and plays an essential role in cellular homeostasis. Many client proteins of HSP90 are involved in cell growth, survival, and migration; processes that are generally accepted as participants in tumorigenesis. HSP90 is also up-regulated in certain tumors. Indeed, the inhibition of HSP90 is known to be effective in cancer treatment. Recently, studies showed that HSP90 regulates transforming growth factor ß1 (TGF-ß1)-induced transcription by increasing the stability of the TGF-ß receptor. TGF-ß signaling also has been implicated in cancer, suggesting the possibility that TGF-ß1 and HSP90 function cooperatively during the cancer cell progression. Here in this paper, we investigated the role of HSP90 in TGF-ß1-stimulated Mv1Lu cells. Treatment of Mv1Lu cells with the HSP90 inhibitor, 17-allylamino-demethoxy-geldanamycin (17AAG), or transfection with truncated HSP90 (ΔHSP90) significantly reduced TGF-ß1-induced cell migration. Pretreatment with 17AAG or transfection with ΔHSP90 also reduced the levels of phosphorylated Smad2 and Smad3. In addition, the HSP90 inhibition interfered the nuclear localization of Smads induced by constitutively active Smad2 (S2EE) or Smad3 (S3EE). We also found that the HSP90 inhibition decreased the protein level of importin-ß1 which is known to regulate R-Smad nuclear translocation. These data clearly demonstrate a novel function of HSP90; HSP90 modulates TGF-ß signaling by regulating Smads localization. Overall, our data could provide a detailed mechanism linking HSP90 and TGF-ß signaling. The extension of our understanding of HSP90 would offer a better strategy for treating cancer.

TGF-ß1 accelerates the DNA damage response in epithelial cells via Smad signaling.

The evidence suggests that transforming growth factor-beta (TGF-ß) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-ß1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-ß1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-ß1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (γH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-ß1 pretreatment. However, they soon thereafter exhibited downregulation in TGF-ß1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-ß type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-ß1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-ß1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression.

Induction of galectin-1 by TGF-ß1 accelerates fibrosis through enhancing nuclear retention of Smad2.

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-ß1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expression level and nuclear accumulation of Gal-1. The TGF-ß1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-ß1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases.

Knockdown of TWIST1 enhances arsenic trioxide- and ionizing radiation-induced cell death in lung cancer cells by promoting mitochondrial dysfunction.

TWIST1 is implicated in the process of epithelial mesenchymal transition, metastasis, stemness, and drug resistance in cancer cells, and therefore is a potential target for cancer therapy. In the present study, we found that knockdown of TWIST1 by small interfering RNA (siRNA) enhanced arsenic trioxide (ATO)- and ionizing radiation (IR)-induced cell death in non-small-cell lung cancer cells. Interestingly, intracellular reactive oxygen species levels were increased in cells treated with TWIST1 siRNA and further increased by co-treatment with ATO or IR. Pretreatment of lung cancer cells with the antioxidant N-acetyl-cysteine markedly suppressed the cell death induced by combined treatment with TWIST1 siRNA and ATO or IR. Moreover, treatment of cells with TWIST1 siRNA induced mitochondrial membrane depolarization and significantly increased mitochondrial fragmentation (fission) and upregulated the fission-related proteins FIS1 and DRP1. Collectively, our results demonstrate that siRNA-mediated TWIST1 knockdown induces mitochondrial dysfunction and enhances IR- and ATO-induced cell death in lung cancer cells.

TGF-ß signaling plays an important role in resisting γ-irradiation.

Transforming growth factor-ß1 (TGF-ß1) regulates various biological processes, including differentiation, bone remodeling and angiogenesis, and is particularly important as a regulator of homeostasis and cell growth in normal tissue. Interestingly, some studies have reported that TGF-ß1 induces apoptosis through induction of specific genes, whereas others suggest that TGF-ß1 inhibits apoptosis and facilitates cell survival. Resolving these discrepancies, which may reflect differences in cellular context, is an important research priority. Here, using the parental mink lung epithelial cell line, Mv1Lu, and its derivatives, R1B and DR26, lacking TGF-ß receptors, we investigated the involvement of TGF-ß signaling in the effects of γ-irradiation. We found that canonical TGF-ß signaling played an important role in protecting cells from γ-irradiation. Introduction of functional TGF-ß receptors or constitutively active Smads into R1B and DR26 cell lines reduced DNA fragmentation, Caspase-3 cleavage and γ-H2AX foci formation in γ-irradiated cells. Notably, we also found that de novo protein synthesis was required for the radio-resistant effects of TGF-ß1. Our data thus indicate that TGF-ß1 protected against γ-irradiation, decreasing DNA damage and reducing apoptosis, and thereby enhanced cell survival.

Comparison of cellular responses to ionizing radiation in keratinocytes isolated from healthy donors and type II diabetes patients.

PURPOSE: Due to the expanding repertoire of treatment devices that use radiation, the possibility of exposure to both low-dose and high-dose radiation continues to increase. Skin is the outermost part of the body and thus directly exposed to radiation-induced damage. In particular, the skin of diabetes patients is fragile and easily damaged by external stimuli, such as radiation. However, damage and cellular responses induced by ionizing irradiation in diabetic skin have not been explored in detail. In this study, we investigated the effects of several irradiation dose on normal keratinocytes and those from type II diabetes patients, with particular focus on DNA damage. MATERIALS AND METHODS: Cellular responses to low-dose radiation (0.1 Gy) and high-dose radiation (0.5 and 2 Gy) were evaluated. Cell cycle analysis was conducted via flow cytometry and cell viability analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Proteins related to the DNA damage response (DDR) and repair signaling pathways and apoptosis were detected via immunoblot analysis. Apoptosis and cell differentiation were additionally examined in 3D skin organoids using immunohistochemistry. RESULTS: Compared to respective control groups, no significant changes were observed in cell cycle, DDR and repair mechanisms, cell survival, and differentiation in response to 0.1 Gy irradiation in both normal and diabetes type II keratinocytes. On the other hand, the cell cycle showed an increase in the G2/M phase in both cell types following exposure to 2 Gy irradiation. At radiation doses 2 Gy, activation of the DDR and repair signaling pathways, apoptosis, and cell differentiation were increased and viability was decreased in both cell types. Notably, these differences were more pronounced in normal than diabetes type II keratinocytes. CONCLUSIONS: Normal keratinocytes respond more strongly to radiation-induced damage and recovery than diabetes type II keratinocytes.

Brain type of creatine kinase induces doxorubicin resistance via TGF-ß signaling in MDA-MB-231 breast cancer cells.

Brain type of creatine kinase (CKB) regulates energy homeostasis by reversibly transferring phosphate groups between phosphocreatine and ATP at sites of high energy demand. Several types of cancer cells exhibit upregulated CKB expression, but the function of CKB in cancer cells remains unclear. In this study, we investigated the function of CKB in breast cancer by overexpressing CKB in MDA-MB-231 cells. The overexpression of CKB did not affect cell growth rate, cell cycle distribution, ATP level or key mediators of aerobic glycolysis and lactate dehydrogenase isoform levels. Meanwhile, CKB overexpression did increase resistance to doxorubicin. TGF-ß-induced Smad phosphorylation and Smad-dependent transcriptional activity were significantly up-regulated by CKB expression without changes in inhibitory Smad protein levels. Moreover, treatment with TGF-ß considerably enhanced cell viability during doxorubicin treatment and decreased doxorubicin-induced apoptosis in CKB-expressing MDA-MB-231 cells compared to control cells. These results suggest that CKB attenuates doxorubicin-induced apoptosis and potentiates resistance to doxorubicin by enhancing TGF-ß signaling in MDA-MB-231 cells.

CD24 enhances DNA damage-induced apoptosis by modulating NF-κB signaling in CD44-expressing breast cancer cells.

Cluster of differentiation 24 (CD24) is a small glycosylphosphatidylinositol-linked cell surface molecule that is expressed in a variety of human carcinomas, including breast cancer. To determine the role of CD24 in breast cancer cells, we expressed CD24 in CD24-negative/low and cluster of differentiation 44 (CD44)-positive MDA-MB-231 metastatic breast cancer cells. Forced expression of CD24 resulted in a decrease in c-Raf/mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase signaling and reduced cell proliferation. Apoptosis induced by DNA damage was greatly enhanced in MDA-MB-231 CD24 cells as compared with MDA-MB-231 vec cells. CD24 expression efficiently attenuated DNA damage-induced nuclear factor-kappaB (NF-κB) signaling in MDA-MB-231 cells. However, in CD24-positive and CD44-negative/low MCF-7 cells, knockdown of CD24 did not significantly affect DNA damage-induced apoptosis nor NF-κB signaling. Silencing of CD24 in CD24/CD44-double-positive MDA-MB-468 cells partially rescued DNA damage-induced apoptosis. Transient transfection studies with 293T cells also revealed that CD24 attenuated cell viability and NF-κB signaling only when CD44 was cotransfected. These data indicate that CD24 expression potentiated DNA-induced apoptosis by suppressing antiapoptotic NF-κB signaling in CD44-expressing cells.

Substance P stimulates the recovery of bone marrow after the irradiation.

The therapeutic use of ionizing radiation (e.g., X-rays and γ-rays) needs to inflict minimal damage on non-target tissue. Recent studies have shown that substance P (SP) mediates multiple activities in various cell types, including cell proliferation, anti-apoptotic responses, and inflammatory processes. The present study investigated the effects of SP on γ-irradiated bone marrow stem cells (BMSCs). In mouse bone marrow extracts, SP prolonged activation of Erk1/2 and enhanced Bcl-2 expression, but attenuated the activation of apoptotic molecules (e.g., p38 and cleaved caspase-3) and down-regulated Bax. We also observed that SP-decreased apoptotic cell death and stimulated cell proliferation in γ-irradiated mouse bone marrow tissues through TUNEL assay and PCNA analysis. To determine how SP affects bone marrow stem cell populations, mouse bone marrow cells were isolated and colony-forming unit (CFU) of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) was estimated. SP-pretreated ones showed higher CFUs of MSC and HSC than untreated ones. Furthermore, when SP was pretreated in cultured human MSC, it significantly decreased apoptotic cells at 48 and 72 h after γ-irradiation. Compared with untreated cells, SP-treated human MSCs showed reduced cleavage of apoptotic molecules such as caspase-8, -9, -3, and poly ADP-ribose polymerase (PARP). Thus, our results suggest that SP alleviates γ-radiation-induced damage to mouse BMSCs and human MSCs via regulation of the apoptotic pathway.

Redd1 inhibits the invasiveness of non-small cell lung cancer cells.

Redd1 acts as a negative regulator of mTOR in response to various stress conditions, but its specific physiological role is currently unclear. In the present study, we showed that Redd1 inhibits the invasive activity of non-small cell lung cancer (NSCLC) cells. Interestingly, expression of Redd1 was extremely low in H1299 cells displaying high invasiveness, compared with that in H460 cells with lower invasive activity. Overexpression of Redd1 inhibited the invasive activity of H1299 cells, while suppression with specific siRNAs enhanced the invasiveness of H460 cells. Knockdown of the mTOR downstream substrate, S6K, resulted in a decrease in the invasive property of H1299 cells. Our results provide preliminary evidence that Redd1 inhibits the invasive activity of NSCLC cells via suppression of the mTOR downstream pathway.

Autophagy-mediated anti-tumoral activity of imiquimod in Caco-2 cells.

Imiquimod (IMQ) is recognized as a topical immune response modifier compound that enhances immune responses with anti-viral and anti-tumoral activities. Its anti-tumoral effects have been previously demonstrated in a variety of cancer cells, and were identified as indirect responses mediated by the immune modulation of cutaneous dendritic cells. Recently, the pro-apoptotic activities of IMQ occurring via the modulation of bcl-2 family have been reported in several tumor cells. In this study, we first observed IMQ-initiated autophagy determined by vesicular organelle formation and the generation of LC3-II in Caco-2 human colonic adenocarcinoma cells, which expressing functional TLR7. Additionally, IMQ-induced autophagy resulted in cell death occurring independently of molecular changes of apoptotic markers. Loxoribine also induced autophagy and autophagy-induced cell death at less potent than IMQ. Moreover, the activation of autophagy by rapamycin induced enhanced cell death in TNF-alpha-treated Caco-2 cells, which were autophagy and cell death-resistant. Our results led us to conclude that IMQ exerts a direct effect on the anti-tumoral activity of Caco-2 cells via autophagy-induced cell death. In conclusion, the modulation of autophagy might be applied in a potential cancer therapy for the treatment of colon cancer cells.

Syndecan-2 overexpression regulates adhesion and migration through cooperation with integrin alpha2.

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is known to serve as an adhesion receptor, but details of the regulatory mechanism governing syndecan-2 cell adhesion and migration remain unclear. Here, we examined this regulatory mechanism, showing that overexpression of syndecan-2 enhanced collagen adhesion, cell migration and invasion of normal rat intestinal epithelial cells (RIE1), and increased integrin alpha2 expression levels. Interestingly, RIE1 cells transfected with either syndecan-2 or integrin alpha2 showed similar adhesion and migration patterns, and a function-blocking anti-integrin alpha2 antibody abolished syndecan-2-mediated adhesion and migration. Consistent with these findings, transfection of integrin alpha2 siRNA diminished syndecan-2-induced cell migration in HCT116 human colon cancer cells. Taken together, these results demonstrate a novel cooperation between syndecan-2 and integrin alpha2beta1 in adhesion-mediated cell migration and invasion. This interactive dynamic might be a possible mechanism underlying the tumorigenic activities of colon cancer cells.

Substance P accelerates intestinal tissue regeneration after gamma-irradiation-induced damage.

Radiation therapy causes varying degrees of damage to biological systems. Many groups are investigating the mechanism underlying radiation-induced cellular damage but there are limited therapeutic solutions for affected patients. Recent studies show that substance P (SP) participates in cell proliferation. In the present study, we characterized the mechanism underlying SP-induced cellular signaling in radiation-induced damage of the intestine. Exposure of Caco-2 cells to SP increases cell proliferation and Erk phosphorylation in a time- and dose-dependent manner. The proliferation of cells exposed to gamma-irradiation is also stimulated by exposure to SP, a phenomenon that may result from inhibition of apoptosis because SP activates Akt and inhibits the cleavage of caspase-3. The effect of SP on cell proliferation and protection was confirmed by investigations in mice. Proliferating cell nuclear antigen staining shows that cell proliferation in radiation-damaged mouse intestine increases significantly upon exposure to SP. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling assay reveals fewer cells stained in SP-treated mice compared with untreated controls. These findings show the potential for SP-induced acceleration of intestinal wound healing and reveal that the mechanism underlying this process involves activation of Erk and Akt and inhibition of caspase-3 cleavage.

Mechanisms of Energy Metabolism in Skeletal Muscle Mitochondria Following Radiation Exposure.

An understanding of cellular processes that determine the response to ionizing radiation exposure is essential for improving radiotherapy and assessing risks to human health after accidental radiation exposure. Radiation exposure leads to many biological effects, but the mechanisms underlying the metabolic effects of radiation are not well known. Here, we investigated the effects of radiation exposure on the metabolic rate and mitochondrial bioenergetics in skeletal muscle. We show that ionizing radiation increased mitochondrial protein and mass and enhanced proton leak and mitochondrial maximal respiratory capacity, causing an increase in the fraction of mitochondrial respiration devoted to uncoupling reactions. Thus, mice and cells treated with radiation became energetically efficient and displayed increased fatty acid and amino acid oxidation metabolism through the citric acid cycle. Finally, we demonstrate that radiation-induced alterations in mitochondrial energy metabolism involved adenosine monophosphate-activated kinase signaling in skeletal muscle. Together, these results demonstrate that alterations in mitochondrial mass and function are important adaptive responses of skeletal muscle to radiation.

Histone deacetylase inhibitor (HDACi) upregulates activin A and activates the Smad signaling pathway in melanomas.

BACKGROUND: Histone deacetylase (HDAC) is an enzyme that regulates gene expression, cell cycle arrest, apoptosis and modulation of various pathways. HDAC inhibitors (HDACis) can modulate these pathways by hyper-acetylating target proteins, thereby acting as cancer chemotherapeutic agents. OBJECTIVE: One of HDACis, suberoylanilide hydroxamic acid (SAHA), has been found to regulate the Smad signaling pathway, by an as yet unclear mechanism. This study therefore investigated the mechanism by which SAHA regulates Smad signaling in the melanoma cell line SK-Mel-5. METHODS: Cell proliferation was assessed by MTT assays and fluorescence activated cell sorter (FACS) analysis. The activation of Smad signaling pathway was assessed by western blots analysis. The transcriptions of target genes were checked by RT-PCR and dual luciferase assay. RESULTS: Treatment with SAHA inhibited the proliferation of SK-Mel-5 cells, enhanced the phosphorylation of R-Smad, and up-regulated p21 protein. Surprisingly, R-Smad was also activated by conditioned medium from SAHA-treated SK-Mel-5 cells. An analysis of the conditioned medium showed that activin A was responsible for the activation of R-Smad. SAHA treatment enhanced the level of activin A mRNA, increasing the level of activin A in the secretome. The activity of the SAHA-treated secretome could be eliminated by pre-incubation with antibody to activin A. In addition, activin A supplemented medium could mimic the effect of the SAHA-treated secretome. CONCLUSION: These findings indicate that the anti-cancer function of SAHA is mediated, at least in part, by the upregulation of activin A.

Transmembrane domain-dependent functional oligomerization of syndecans.

Cell surface adhesion receptors of the syndecan family initiate intracellular events through clustering of receptors. This crucial clustering occurs through receptor dimerization or oligomerization, which is mediated by receptor transmembrane domains. However, the exact role of the transmembrane domain during receptor activation is not fully understood. Researchers have not yet determined whether the transmembrane domain functions solely in the physical aspects of receptor clustering, or whether the domain has additional functional roles. Here we review recent advances in understanding the functionality of transmembrane domain-dependent oligomerization of syndecan cell adhesion receptor.