Betulin Accelerated the Functional Recovery of Injured Muscle in a Mouse Model of Muscle Contusion.

Muscle contusion is an injury to muscle fibers and connective tissues. It commonly happens in impact events, and could result in pain, swelling, and limited range of motion. Diclofenac is one of commonly used nonsteroidal anti-inflammatory drugs to alleviate pain and inflammation after injury. However, it can potentially cause some side effects including gastrointestinal complications and allergy. Betulin is a lupine-type pentacyclic triterpenoid. It is showed to have valuable pharmacological effects, but the physiological effect of betulin on muscle contusion has not been reported. This study aimed to explore the therapeutic effects of betulin on muscle contusion that produced by the drop-mass method in mice. C57BL/6 mice were randomly assigned to control (no injury), only drop-mass injury (Injury), diclofenac treatment (Injury+diclofenac), and betulin treatment (Injury+betulin) groups. Injury was executed on the gastrocnemius of the right hind limb, and then phosphate-buffered saline (PBS), diclofenac, or betulin were oral gavage administrated respectively for 7 days. Results revealed that betulin significantly restored motor functions based on locomotor activity assessments, rota-rod test, and footprints analysis. Betulin also attenuated serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels after muscle injury. Neutrophil infiltration was alleviated and desmin levels were increased after betulin treatment. Our data demonstrated that betulin attenuated muscle damage, alleviated inflammatory response, improved muscle regeneration, and restored motor functions after muscle contusion. Altogether, betulin may be a potential compound to accelerate the repair of injured muscle.

Sesamin and sesamolin potentially inhibit adipogenesis through downregulating the peroxisome proliferator-activated receptor γ protein expression and activity in 3T3-L1 cells.

Sesamin and sesamolin are major sesame lignans that have demonstrated anti-inflammatory, anticancer, and neuroprotective properties and potential benefits in the liver, cardiovascular diseases, and metabolic syndrome. However, despite previous research on their antiobesity effects and underlying mechanisms, a comprehensive investigation of these aspects is still lacking. In this study, we evaluated the regulatory effects of 20 to 80 µM sesamin and sesamolin on adipogenesis in vitro using 3T3-L1 cells as a model cell line. We hypothesized that the lignans would inhibit adipogenic differentiation in 3T3-L1 cells through the regulation of peroxisome proliferator-activated receptor γ (PPARγ). Our data indicate that sesamin and sesamolin inhibited the adipogenic differentiation of 3T3-L1 cells by dose-dependently decreasing lipid accumulation and triglyceride formation. Sesamin and sesamolin reduced the mRNA and protein expression of the adipogenesis-related transcription factors, PPARγ and CCAAT/enhancer-binding protein α, leading to the dose-dependent downregulations of their downstream targets, fatty acid binding protein 4, hormone-sensitive lipase, lipoprotein lipase, and glucose transporter 4. In addition, glucose uptake was dose-dependently attenuated by sesamin and sesamolin in both differentiated 3T3-L1 cells and HepG2 cells. Interestingly, our results suggested that sesamin and sesamolin might directly bind to PPARγ to inhibit its transcriptional activity. Finally, sesamin and sesamolin decreased the phosphorylation of 3 mitogen-activated protein kinase signaling components in differentiated 3T3-L1 cells. Taken together, our findings suggest that sesamin and sesamolin may exhibit antiobesity effects by potentially downregulating PPARγ and its downstream genes through the mitogen-activated protein kinase signaling pathway, offering important insights into the molecular mechanisms underlying the potential antiobesity effects of sesamin and sesamolin.

DNA repair proteins as the targets for paroxetine to induce cytotoxicity in gastric cancer cell AGS.

To evaluate the potential anticancer effects of 1175 FDA-approved drugs, cell viability screening was performed using 25 human cancer cell lines covering 14 human cancer types. Here, we focus on the action of paroxetine, which demonstrated greater toxicity toward human gastric adenocarcinoma cell-line AGS cells compared with the other FDA-approved drugs, exhibiting an IC50 value lower than 10 µM. Evaluation of the underlying novel mechanisms revealed that paroxetine can enhance DNA damage in gastric cancer cells and involves downregulation of Rad51, HR23B and ERCC1 expression and function, as well as nucleotide shortage. Enhancement of autophagy counteracted paroxetine-induced apoptosis but did not affect paroxetine-induced DNA damage. Paroxetine also enhanced ROS generation in AGS cells, but a ROS scavenger did not improve paroxetine-mediated DNA damage, apoptosis, or autophagy, suggesting ROS might play a minor role in paroxetine-induced cell toxicity. In contrast, paroxetine did not enhance DNA damage, apoptosis, or autophagy in another insensitive gastric adenocarcinoma cell-line MKN-45 cells. Interestingly, co-administration of paroxetine with conventional anticancer agents sensitized MKN-45 cells to these agents: co-treated cells showed increased apoptosis relative to MKN-45 cells treated with the anticancer agent alone. Unequivocally, these data suggest that for the first time that paroxetine triggers cytotoxicity and DNA damage in AGS cells at least partly by reducing the gene expression of Rad51, HR23B, and ERCC1. Our findings also suggest that paroxetine is a promising candidate anticancer agent and/or chemosensitizing agent for use in combination with other anticancer drugs in cancer therapy. The molecular mechanisms underlying the anticancer activity of co-treatment with paroxetine and chemotherapy appear to be complex and are worthy of further investigation.

Capsaicin Potentiates Anticancer Drug Efficacy Through Autophagy-Mediated Ribophorin II Downregulation and Necroptosis in Oral Squamous Cell Carcinoma Cells.

The ability of capsaicin co-treatment to sensitize cancer cells to anticancer drugs has been widely documented, but the detailed underlying mechanisms remain unknown. In addition, the role of ribophorin II turnover on chemosensitization is still uncertain. Here, we investigated capsaicin-induced sensitization to chemotherapeutic agents in the human oral squamous carcinoma cell lines, HSC-3 and SAS. We found that capsaicin (200 µM) did not induce remarkable apoptotic cell death in these cell lines; instead, it significantly enhanced autophagy with a concomitant decrease of ribophorin II protein. This capsaicin-induced decrease in ribophorin II was intensified by the autophagy inducer, rapamycin, but attenuated by the autophagy inhibitors, ULK1 inhibitor and chloroquine, indicating that the autophagic process was responsible for the capsaicin-induced down-regulation of ribophorin II. Co-administration of capsaicin with conventional anticancer agents did, indeed, sensitize the cancer cells to these agents. In co-treated cells, the induction of apoptosis was significantly reduced and the levels of the necroptosis markers, phospho-MLKL and phospho-RIP3, were increased relative to the levels seen in capsaicin treatment alone. The levels of DNA damage response markers were also diminished by co-treatment. Collectively, our results reveal a novel mechanism by which capsaicin sensitizes oral cancer cells to anticancer drugs through the up-regulation of autophagy and down-regulation of ribophorin II, and further indicate that the induction of necroptosis is a critical factor in the capsaicin-mediated chemosensitization of oral squamous carcinoma cells to conventional anticancer drugs.

Imiquimod-induced ROS production disrupts the balance of mitochondrial dynamics and increases mitophagy in skin cancer cells.

BACKGROUND: Mitochondrial homeostasis is a highly dynamic process involving continuous fission and fusion cycles and mitophagy to maintain mitochondrial functionality. Imiquimod (IMQ), a Toll-like receptor (TLR) 7 ligand, is used to treat various skin malignancies. IMQ also induces apoptotic and autophagic cell death in various cancers through a TLR7-independent pathway. OBJECTIVE: To investigate whether IMQ-induced ROS production is involved in mitochondrial dysfunction, mitochondrial fragmentation and mitophagy in skin cancer cells. METHODS: BCC/KMC-1, B16F10 and A375 skin cancer cells, AGS gastric cancer cells and primary human keratinocytes were treated with 50 µg/mL IMQ. After 4 h, ROS were detected by CM-H2DCFDA, DHE, and MitoSOX Red staining. After 24 h, cell viability and the mitochondrial membrane potential were evaluated by a CCK-8 assay and JC-1 staining, respectively. Oxygen consumption was assessed with an Oroboros instrument. Mitochondrial morphology and mitophagy were evaluated by MitoTracker and LysoTracker staining. Mitochondrial dynamics markers, including MFN-1, DRP-1 and OPA1, and mitophagy markers, including LC3, S65-phosphorylated ubiquitin, PINK1 and TOM20, were detected by immunoblotting. RESULTS: IMQ not only induced severe ROS production but also resulted in increased mitochondrial membrane potential loss, mitochondrial fission and mitophagy and decreased oxygen consumption in skin cancer cells compared with normal keratinocytes. Pretreatment with the antioxidant NAC reduced IMQ-induced ROS production and attenuated IMQ-induced mitochondrial fission and mitophagy in skin cancer cells. CONCLUSIONS: IMQ-induced ROS might be associated with mitochondrial dysfunction, mitochondrial fission and mitophagy in cancer cells. Alleviating IMQ-induced ROS production would reduce mitochondrial fission-to-fusion skewing and further reduce IMQ-induced mitophagy.

MEK2 is a critical modulating mechanism to down-regulate GCIP stability and function in cancer cells.

Loss of tumor suppressor activity and upregulation of oncogenic pathways simultaneously contribute to tumorigenesis. Expression of the tumor suppressor, GCIP (Grap2- and cyclin D1-interacting protein), is usually reduced or lost in advanced cancers, as seen in both mouse tumor models and human cancer patients. However, no previous study has examined how cancer cells down-regulate GCIP expression. In this study, we first validate the tumor suppressive function of GCIP using clinical gastric cancer tissues and online database analysis. We then reveal a novel mechanism whereby MEK2 directly interacts with and phosphorylates GCIP at its Ser313 and Ser356 residues to promote the turnover of GCIP by ubiquitin-mediated proteasomal degradation. We also reveal that decreased GCIP stability enhances cell proliferation and promotes cancer cell migration and invasion. Taken together, these findings provide a more comprehensive view of GCIP in tumorigenesis and suggest that the oncogenic MEK/ERK signaling pathway negatively regulates the protein level of GCIP to promote cell proliferation and migration.

HR23A-knockdown lung cancer cells exhibit epithelial-to-mesenchymal transition and gain stemness properties through increased Twist1 stability.

The epithelial-mesenchymal transition is a major cause of cancer metastasis, and deregulation of the transcription factor, Twist1, is a critical molecular event in the epithelial-mesenchymal transition. The importance of Twist1 protein turnover in this process has not yet been defined. Here, we show that HR23A directly targets the Twist1 protein without changing its gene transcription. Our experiments reveal that: HR23A interacts with Twist1, and this promotes the ubiquitin-mediated proteasomal degradation of Twist1. Depletion of HR23A enhances Twist1 protein levels, epithelial-mesenchymal transition, cancer cell migration and various cancer stemness properties, including the expression of major pluripotency factors, the capacity for tumor-sphere formation in culture and the expression of cancer stem cell surface markers. The increases of these stemness properties are reversed by ectopic expression of HR23A or further knockdown of Twist1 in HR23A-depleted cells. Thus, HR23A-knockdown cells appear to undergo epithelial-mesenchymal transition and take on certain attributes of cancer stemness. Together, our findings indicate that HR23A importantly contributes to regulating Twist1 protein stability, and suggest that altering the stability of Twist1 by modulating HR23A may be a new avenue for therapeutic intervention in cancer.

Engagement with tNOX (ENOX2) to Inhibit SIRT1 and Activate p53-Dependent and -Independent Apoptotic Pathways by Novel 4,11-Diaminoanthra[2,3-b]furan-5,10-diones in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver and is among the top three causes of cancer-associated death worldwide. However, the clinical use of chemotherapy for HCC has been limited by various challenges, emphasizing the urgent need for novel agents with improved anticancer properties. We recently synthesized and characterized a series of 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives that exhibit potent apoptotic activity against an array of cancer cell lines, including variants with multidrug resistance. Their effect on liver cancer cells, however, was unknown. Here, we investigated three selected 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives (compounds 1⁻3) for their cytotoxicity and the underlying molecular mechanisms in wild-type or p53-deficient HCC cells. Cytotoxicity was determined by WST-1 assays and cell impedance measurements and apoptosis was analyzed by flow cytometry. The interaction between compounds and tumor-associated NADH oxidase (tNOX, ENOX2) was studied by cellular thermal shift assay (CETSA). We found that compound 1 and 2 induced significant cytotoxicity in both HepG2 and Hep3B lines. CETSA revealed that compounds 1 and 2 directly engaged with tNOX, leading to a decrease in the cellular NAD⁺/NADH ratio. This decreased the NAD⁺-dependent activity of Sirtuin 1 (SIRT1) deacetylase. In p53-wild-type HepG2 cells, p53 acetylation/activation was enhanced, possibly due to the reduction in SIRT1 activity, and apoptosis was observed. In p53-deficient Hep3B cells, the reduction in SIRT1 activity increased the acetylation of c-Myc, thereby reactivating the TRAIL pathway and, ultimately leading to apoptosis. These compounds thus trigger apoptosis in both cell types, but via different pathways. Taken together, our data show that derivatives 1 and 2 of 4,11-diaminoanthra[2,3-b]furan-5,10-diones engage with tNOX and inhibit its oxidase activity. This results in cytotoxicity via apoptosis through tNOX-SIRT1 axis to enhance the acetylation of p53 or c-Myc in HCC cells, depending on their p53 status.

Capsaicin-induced TRIB3 upregulation promotes apoptosis in cancer cells.

BACKGROUND: Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is one of the main pungent components of chili peppers and has been shown to exert various effects on numerous physiological processes. Recent studies have focused on the chemopreventive effects of capsaicin, which can combat growth in various human cancer cell systems. The tribbles-related protein 3 (TRIB3) is evolutionarily conserved from Drosophila to humans. In the latter, TRIB3 is a key determinant in numerous cellular processes, including apoptosis. PURPOSE: The aim of this study was to examine the importance of TRIB3 in the antitumor efficacy of capsaicin in human cancer cells, and further assess potential mechanism(s) underlying the capsaicin-induced upregulation of TRIB3. METHODS: Human cancer cell lines were treated with capsaicin, then evaluated for levels of TRIB3 and molecules related to apoptosis or signaling pathways. The impact of TRIB3 on capsaicin-induced apoptosis was investigated using si-RNA or overexpression of TRIB3. RESULTS: It is the first time to show that TRIB3 is targeted by capsaicin to promote apoptosis. Capsaicin promotes apoptotic cell death by upregulating TRIB3 expression in cancer cells. Overexpression of TRIB3 enhances capsaicin-induced apoptosis, and TRIB3 knockdown experiments demonstrate that the effect of capsaicin in apoptotic cell death is correlated with the induction of TRIB3 in cancer cells. Finally, enhancements in gene expression and protein stability are involved in the capsaicin-induced upregulation of TRIB3. CONCLUSION: Our results show that the capsaicin-induced upregulation of TRIB3 triggers apoptosis and thereby contributes to the suppression of cell growth in cancer cell lines.

Osteoblast-secreted WISP-1 promotes adherence of prostate cancer cells to bone via the VCAM-1/integrin α4ß1 system.

Bone metastasis is a frequent occurrence in prostate cancer (PCa) that is associated with severe complications such as fracture, bone pain and hypercalcemia. The cross-talk between metastatic cancer cells and bone is critical to the development and progression of bone metastases. In our previous data, we have described how the involvement of the Wnt-induced secreted protein-1/vascular cell adhesion molecule-1 (WISP-1/VCAM-1) system in this tumor-bone interaction contributes to human PCa cell motility. In this study, we found that WISP-1 regulates bone mineralization by inducing bone morphogenetic protein-2 (BMP2), BMP4 and osteopontin (OPN) expression in osteoblasts. We also found that WISP-1 inhibited RANKL-dependent osteoclastogenesis. Moreover, osteoblast-derived WISP-1 enhanced VCAM-1 expression in PCa cells and subsequently promoted the adherence of cancer cells to osteoblasts. Furthermore, endothelin-1 (ET-1) expression in PCa cells was regulated by osteoblast-derived WISP-1, which promoted integrin α4ß1 expression in osteoblasts via the MAPK pathway. Pretreatment of PCa cells with VCAM-1 antibody or osteoblasts with integrin α4ß1 antibody attenuated the adherence of PCa cells to osteoblasts, suggesting that integrin α4ß1 serves as a ligand that captures VCAM-1+ metastatic tumor cells adhering to osteoblasts. Our findings reveal that osteoblast-derived WISP-1 plays a key role in regulating the adhesion of PCa cells to osteoblasts via the VCAM-1/integrin α4ß1 system. Osteoblast-derived WISP-1 is a promising target for the prevention and inhibition of PCa-bone interaction.

TRIB3 downregulation enhances doxorubicin-induced cytotoxicity in gastric cancer cells.

TRIB3, which is a pseudokinase known to regulate multiple pro-survival pathways, appears to be a potential therapeutic target for the treatment of human tumors. However, its precise role in cancer is controversial, as TRIB3 protein levels have been associated with both good and poor prognosis in cancer patients. Here, we investigated the significance of TRIB3 expression in the survival of gastric cancer cells exposed to anticancer drugs. We found that the tested anticancer drug, doxorubicin, induced cytotoxicity by decreasing TRIB3 transcription, which was followed by apoptotic cell death. Moreover, TRIB3 siRNA knockdown appeared to enhance doxorubicin-induced apoptosis in gastric cancer cells, concurrently with altering the expression of downstream apoptotic factors. Conversely, overexpression of TRIB3 significantly protected cells against doxorubicin-induced apoptosis. Our results indicate that downregulation of TRIB3 appears to promote cell death and enhance doxorubicin-induced apoptosis, supporting the anti-apoptotic role of TRIB3. The inductions of three classes of MAPKs failed to affect doxorubicin-mediated TRIB3 downregulation, while TRIB3 overexpression did not affect doxorubicin-induced MAPK activation. In sum, our findings indicate that TRIB3 plays an anti-apoptotic role in doxorubicin-treated gastric cancer cell lines, perhaps indicating that the status of TRIB3 expression in response to anticancer drugs, such as doxorubicin, irinotecan or oxaliplatin, may reflect the efficiency for cancer therapy.

Biocompatibility assessment of nanomaterials for environmental safety screening.

In view of the extensive use of nanoparticles in countless applications, a fast and effective method for assessing their potential adverse effects on the environment and human health is extremely important. At present, in vitro cell-based assays are the standard approach for screening chemicals for cytotoxicity because of their relative simplicity, sensitivity, and cost-effectiveness compared with animal studies. Regrettably, such cell-based viability assays encounter limitations when applied to determining the biological toxicity of nanomaterials, which often interact with assay components and produce unreliable outcomes. We have established a cell-impedance-based, label-free, real-time cell-monitoring platform suitable for use in a variety of mammalian cell lines that displays results as cell index values. In addition to this real-time screening platform, other traditional cytotoxicity assays were employed to validate cytotoxicity assessments. We suggest that the cell impedance measurement approach is effective and better suited to determining the cytotoxicity of nanomaterials for environmental safety screening. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1170-1182, 2017.

Human Rad23A plays a regulatory role in autophagy.

Chemotherapeutic agents can upregulate autophagy which contributes to the acquisition of chemoresistance and the recurrence of cancer. The involvement of hHR23A in chemoresistance is unknown. In this study, we provide evidence suggesting that hHR23A may regulate autophagy. Knockdown of hHR23A decreased cell growth and increased the resistance in A549 cells to the DNA-damaging agents, cisplatin and oxaliplatin. Measurement of EGFP-LC3 puncta (a marker of autophagy) revealed that autophagy was increased in hHR23A-depleted cells. This effect was augmented by exposure to cisplatin or oxaliplatin. In contrast, the overexpression of hHR23A reversed the levels of autophagy-related proteins to control levels in hHR23A-knockdown cells. Moreover, we observed direct interactions among hHR23A, Beclin 1, and LC3. Finally, 3-methyladenine (3-MA)-induced inhibition of autophagy was found to reverse the sensitivity of hHR23A-knockdown cells to the tested DNA-damaging agents. These results collectively indicated that hHR23A-depleted cells exhibit enhanced autophagy when treated with DNA-damaging agents, perhaps suggesting a basis for the involvement of hHR23A in the acquired chemoresistance of cancer cells. Our study thus reveals a previously unrecognized autophagic function for hHR23A and suggests that it could be a potential therapeutic target for chemosensitizing resistant cancer cells.

Selective recognition and stabilization of new ligands targeting the potassium form of the human telomeric G-quadruplex DNA.

The development of a ligand that is capable of distinguishing among the wide variety of G-quadruplex structures and targeting telomeres to treat cancer is particularly challenging. In this study, the ability of two anthraquinone telomerase inhibitors (NSC749235 and NSC764638) to target telomeric G-quadruplex DNA was probed. We found that these ligands specifically target the potassium form of telomeric G-quadruplex DNA over the DNA counterpart. The characteristic interaction with the telomeric G-quadruplex DNA and the anticancer activities of these ligands were also explored. The results of this present work emphasize our understanding of the binding selectivity of anthraquinone derivatives to G-quadruplex DNA and assists in future drug development for G-quadruplex-specific ligands.

Discovery of a potent cyclooxygenase-2 inhibitor, S4, through docking-based pharmacophore screening, in vivo and in vitro estimations.

Cyclooxygenase (COX; EC: 1.14.99.1), the key enzyme in prostaglandin production in the human body, is a major pharmacological target for developing anti-inflammatory agents. Nonsteroidal anti-inflammatory drugs exhibit anti-inflammatory and analgesic activities when inhibiting COX-2 but cause gastrointestinal toxicity and other side effects because of concurrent inhibition of COX-1. Thus, potent and safe inhibitors against COX-2 are urgently required. We constructed a novel docking-based pharmacophore model for screening selective COX-2 inhibitors and discovered compounds S1, S2, S3, and S4, which apparently inhibit COX-2. Particularly, S4 inhibits COX-2 in vitro and shows a potent anti-inflammatory effect in vivo without cytotoxicity. Molecular docking analyses revealed that S4 interacted satisfactorily with the active site of COX-2 but not with that of COX-1. This reveals that S4 more specifically inhibits COX-2 and has potential for application in developing anti-inflammatory and anticancer agents.

hHR23A is required to control the basal turnover of Chk1.

Among other functions, the Chk1 protein plays an essential role in coordinating the cellular stress response by determining cell cycle arrest. The levels of Chk1 expression and activity are critical for its functions, especially in cell cycle progression, genomic integrity, cell viability and tissue development. Chk1 protein expression should therefore be tightly controlled both during normal growth and under stress situations. However, it is still unknown how Chk1 protein levels are regulated during normal cell cycle progression. In this study, we show that the effect of hHR23A on Chk1 protein turnover could impact the cell cycle progression observed in hHR23A-knockdown cells. Our results reveal that hHR23A associates with Chk1 through its UBA domains, and that knockdown of hHR23A increases and stabilizes the protein level of Chk1 and its phosphorylation at S347. Knockdown of hHR23A results in proliferation defects and S-phase accumulation. DNA damage reduces the interaction between Chk1 and hHR23A, releasing Chk1 from hHR23A and enhancing S-phase accumulation. Based on these novel findings, we propose that hHR23A acts as a carrier to promote Chk1 degradation through the Ubiquitin Proteasome System. These results strengthen the model in which DNA damage induces Chk1 phosphorylation on chromatin followed by releasing phospho-Chk1 from the chromatin into soluble nucleus and the cytoplasm where Chk1 activates the cell cycle checkpoints; and finally, Chk1 is degraded and checkpoint signaling is terminated.

Role of ribophorin II in the response to anticancer drugs in gastric cancer cell lines.

The identification of prognostic markers and establishing their value as therapeutic targets improves therapeutic efficacy against human cancers. Ribophorin II (RPN2) has been demonstrated to be a prognostic marker of human cancer, including breast and pancreatic cancers. The present study aimed to evaluate RPN2 expression in gastric cancer and to examine the possible correlation between RPN2 expression and the response of cells to clinical anticancer drugs, which has received little research attention at present. The gastric cancer AGS, TMC-1, SNU-1, TMK-1, SCM-1, MKN-45 and KATO III cell lines were used as a model to elucidate the role of RPN2 in the response of cells to six common chemotherapeutic agents, comprising oxaliplatin, irinotecan, doxorubicin, docetaxel, cisplatin and 5-fluorouricil. The functional role of RPN2 was assessed by silencing RPN2 using small interfering RNA (siRNA), and the cytotoxicity was determined by an MTS assay and analysis of apoptosis. Molecular events were evaluated by western blotting. All the anticancer drugs were found to exert a concentration-dependent decrease on the cell survival rate of each of the cell lines tested, although the RPN2 levels in the various cell lines were not directly correlated with responsiveness to clinical anticancer drugs, based on the calculated IC50 values. siRNA-mediated RPN2 downregulation enhanced cisplatin-induced apoptosis in AGS cells, but did not markedly decrease the cell survival rates of these cells in response to the tested drugs. Furthermore, RPN2 silencing in MKN-45 cells resulted in no additional increase in the cisplatin-induced apoptosis and survival rates. It was also found that RPN2 depletion increased anticancer drug-mediated cytotoxicity in gastric cancer cell lines. However, the predictive value of RPN2 expression in cancer therapy is questionable in gastric cancer models.

The S100A4 D10V polymorphism is related to cell migration ability but not drug resistance in gastric cancer cells.

Upregulation of the metastasis-promoting S100A4 protein has been linked to tumor migration and invasion, and clinical studies have demonstrated that significant expression of S100A4 in primary tumors is indicative of poor prognosis. However, the involvement of S100A4 in the drug responsiveness of gastric cancer remains unclear. In the present study, we used gastric cancer cell lines as a model to investigate the involvement of S100A4 in drug responsiveness. We overexpressed S100A4 in AGS and SCM-1 cells, which are characterized by relatively low-level expression of endogenous S100A4, and found that this significantly enhanced cell migration but did not affect cell survival in the presence of six common anticancer drugs. Moreover, in vitro cell proliferation was unchanged. Using RNA interference, we suppressed S100A4 expression in MKN-45 and TMK-1 cells (which are characterized by high-level expression of endogenous S100A4), and found that knockdown of S100A4 markedly attenuated cell motility but did not affect cell survival in the presence of six common anticancer drugs. Further study revealed that a single nucleotide polymorphism (SNP) of S100A4 (rs1803245; c.29A>T), which substitutes an Asp residue with Val (D10V), is localized within the conserved binding surface for Annexin II. Cells overexpressing S100A4D10V showed a significant reduction in cell migration ability, but no change in cell survival, upon anticancer drug treatment. Taken together, our novel results indicate that the expression level of S100A4 does not significantly affect cell survival following anticancer drug treatment. Thus, depending on the cell context, the metastasis-promoting effects of S100A4 may not be positively correlated with anticancer drug resistance in the clinic.

Rad23 interaction with the proteasome is regulated by phosphorylation of its ubiquitin-like (UbL) domain.

Rad23 was identified as a DNA repair protein, although a role in protein degradation has been described. The protein degradation function of Rad23 contributes to cell cycle progression, stress response, endoplasmic reticulum proteolysis, and DNA repair. Rad23 binds the proteasome through a UbL (ubiquitin-like) domain and contains UBA (ubiquitin-associated) motifs that bind multiubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. This property is shared by structurally similar proteins (Dsk2 and Ddi1) and is conserved among the human and mouse counterparts of Rad23. Despite much effort, the regulation of Rad23 interactions with ubiquitinated substrates and the proteasome is unknown. We report here that Rad23 is extensively phosphorylated in vivo and in vitro. Serine residues in UbL are phosphorylated and influence Rad23 interaction with proteasomes. Replacement of these serine residues with acidic residues, to mimic phosphorylation, reduced proteasome binding. We reported that when UbL is overexpressed, it can compete with Rad23 for proteasome interaction and can inhibit substrate turnover. This effect is not observed with UbL containing acidic substitutions, consistent with results that phosphorylation inhibits interaction with the proteasome. Loss of both Rad23 and Rpn10 caused pleiotropic defects that were suppressed by overexpressing either Rad23 or Rpn10. Rad23 bearing a UbL domain with acidic substitutions failed to suppress rad23Δ rpn10Δ, confirming the importance of regulated Rad23/proteasome binding. Strikingly, threonine 75 in human HR23B also regulates interaction with the proteasome, suggesting that phosphorylation is a conserved mechanism for controlling Rad23/proteasome interaction.