DAB-APT: a Fluorescence-Based Assay for Determining Aminopropyl Transferase Activity and Inhibition.

Polyamines are polycationic molecules that are crucial in a wide array of cellular functions. Their biosynthesis is mediated by aminopropyl transferases (APTs), promising targets in antimicrobial, antineoplastic and antineurodegenerative therapies. A major limitation, however, is the lack of high-throughput assays to measure their activity. We developed the first fluorescence-based assay, DAB-APT, for measurement of APT activity using 1,2-diacetyl benzene, which forms fluorescent conjugates with putrescine, spermidine and spermine with fluorescence intensity increasing with increasing carbon chain length. The assay has been validated using APT enzymes from S. cerevisiae and P. falciparum and is suitable for high-throughput screening of large chemical libraries. Given the importance of APTs in infectious diseases, cancer and neurobiology, our DAB-APT assay has broad applications, holding promise for advancing research and drug discovery efforts.

Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization.

BACKGROUND: Solid tumors promote tumor malignancy through interaction with the tumor microenvironment, resulting in difficulties in tumor treatment. Therefore, it is necessary to understand the communication between cells in the tumor and the surrounding microenvironment. Our previous study revealed the cancer malignancy mechanism of Bcl-w overexpressed in solid tumors, but no study was conducted on its relationship with immune cells in the tumor microenvironment. In this study, we sought to discover key factors in exosomes secreted from tumors overexpressing Bcl-w and analyze the interaction with the surrounding tumor microenvironment to identify the causes of tumor malignancy. METHODS: To analyze factors affecting the tumor microenvironment, a miRNA array was performed using exosomes derived from cancer cells overexpressing Bcl-w. The discovered miRNA, miR-6794-5p, was overexpressed and the tumorigenicity mechanism was confirmed using qRT-PCR, Western blot, invasion, wound healing, and sphere formation ability analysis. In addition, luciferase activity and Ago2-RNA immunoprecipitation assays were used to study the mechanism between miR-6794-5p and its target gene SOCS1. To confirm the interaction between macrophages and tumor-derived miR-6794-5p, co-culture was performed using conditioned media. Additionally, immunohistochemical (IHC) staining and flow cytometry were performed to analyze macrophages in the tumor tissues of experimental animals. RESULTS: MiR-6794-5p, which is highly expressed in exosomes secreted from Bcl-w-overexpressing cells, was selected, and it was shown that the overexpression of miR-6794-5p increased migratory ability, invasiveness, and stemness maintenance by suppressing the expression of the tumor suppressor SOCS1. Additionally, tumor-derived miR-6794-5p was delivered to THP-1-derived macrophages and induced M2 polarization by activating the JAK1/STAT3 pathway. Moreover, IL-10 secreted from M2 macrophages increased tumorigenicity by creating an immunosuppressive environment. The in vitro results were reconfirmed by confirming an increase in M2 macrophages and a decrease in M1 macrophages and CD8+ T cells when overexpressing miR-6794-5p in an animal model. CONCLUSIONS: In this study, we identified changes in the tumor microenvironment caused by miR-6794-5p. Our study indicates that tumor-derived miR-6794-5p promotes tumor aggressiveness by inducing an immunosuppressive environment through interaction with macrophage.

miR-765 as a promising biomarker for low-dose radiation-induced pulmonary fibrosis.

High-dose radiation (HDR) is widely used for cancer treatment, but the effectiveness of low-dose radiation (LDR) in the treatment of various diseases is controversial. Therefore, to safely utilize LDR for therapeutic purposes, further research on its numerous biological effects of LDR is required. Interest in the increased use of medical imaging devices or the effects of surrounding living environmental radiation on the human body, particularly on fibrosis, is rapidly increasing. Therefore, this study aimed to verify the relationship between LDR and pulmonary fibrosis by evaluating the changes in fibroblasts after LDR treatment and their associated signaling mechanisms. LDR increased the expression of fibrosis markers COL1A1 and α-SMA, cell proliferation, and migration by activating YAP1 and Twist in fibroblasts. Meanwhile, miRNA was employed as a tool to inhibit LDR-induced fibrosis and it was found that miR-765 simultaneously targeted COL1A1, α-SMA, and YAP1. At the cellular level, miR-765 reduced the proliferation and migration of fibroblasts by suppressing the expression of LDR-induced fibrosis factors COL1A1, α-SMA, and YAP1. The efficacy of miR-765 in vivo was confirmed using bleomycin (BLM)-induced fibrotic mouse model. The characteristics of pulmonary fibrosis were reduced after injection of miR-765-overexpressing cells into BLM-induced fibrotic mice. In addition, the suppression of miR-765 expression in the plasma of patients with pulmonary fibrosis confirmed the negative relationship between pulmonary fibrosis and miR-765 expression. Therefore, this study demonstrates that miR-765 is a potential novel diagnostic biomarker and major target for the development of therapeutic agents to inhibit pulmonary fibrosis.

Maturation of the malarial phosphatidylserine decarboxylase is mediated by high affinity binding to anionic phospholipids.

Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.

Targeting miR-5088-5p attenuates radioresistance by suppressing Slug.

Radiotherapy is widely used for cancer treatment, but paradoxically, it has been reported that surviving cancer cells can acquire resistance, leading to recurrence or metastasis. Efforts to reduce radioresistance are required to increase the effectiveness of radiotherapy. miRNAs are advantageous as therapeutic agents because it can simultaneously inhibit the expression of several target mRNAs. Therefore, this study discovered miRNA that regulated radioresistance and elucidated its signaling mechanism. Our previous study confirmed that miR-5088-5p was associated with malignancy and metastasis in breast cancer. As a study to clarify the relationship between radiation and miR-5088-5p identified as onco-miRNA, it was confirmed that radiation induced hypomethylation of the promoter of miR-5088-5p and its expression increased. On the other hand, miR-5088-5p inhibitors were confirmed to reduce radiation-induced epithelial-mesenchymal transition, stemness, and metastasis by reducing Slug. Therefore, this study showed the potential of miR-5088-5p inhibitors as therapeutic agents to suppress radioresistance.

miR-519d-3p suppresses tumorigenicity and metastasis by inhibiting Bcl-w and HIF-1α in NSCLC.

Bcl-w, a member of the Bcl-2 family, is highly expressed in various solid tumor, including lung cancer, suggesting that it is involved in cancer cell survival and carcinogenesis. Solid cancer-induced hypoxia has been reported to increase angiogenesis, growth factor, gene instability, invasion, and metastasis. Despite many studies on the treatment of non-small cell lung cancer (NSCLC) with a high incidence rate, the survival rate of patients has not improved because the cancer cells acquired resistance to treatment. This study investigated the correlation between Bcl-w expression and hypoxia in tumor malignancy of NSCLC. Meanwhile, microRNAs (miRNAs) are involved in a variety of key signaling mechanisms associated with hypoxia. Therefore, we discovered miR-519d-3p, which inhibits the expression of Bcl-w and hypoxia-inducing factor (HIF)-1α, and found that it reduces hypoxia-induced tumorigenesis. Spearman's correlation analysis showed that the expression levels of miR-519d-3p and Bcl-w/HIF-1α were negatively correlated, respectively. This showed that miR-519d-3p can be used as a diagnostic biomarker and target therapy for NSCLC.

Novel miR-5088-5p promotes malignancy of breast cancer by inhibiting DBC2.

Breast cancer is the most common female cancer in the world. Despite the active research on metastatic breast cancer, the treatment of breast cancer patients is still difficult because the mechanism is not well known. Therefore, research on new targets and mechanisms for diagnosis and treatment of breast cancer patients is required. On the other hand, microRNA (miRNA) has the advantage of simultaneously regulating the expression of many target genes, so it has been proposed as an effective biomarker for the treatment of various diseases including cancer. This study analyzed the role and mechanism of DBC2 (deleted in breast cancer 2), which is known to inhibit its expression in breast cancer, and proposed microRNA (miR)-5088-5p, which regulates its expression. It was revealed that the biogenesis of miR-5088-5p was upregulated by hypomethylation of its promoter, promoted by Fyn, and was involved in malignancy in breast cancer. With the use of the cellular level, clinical samples, and published data, we verified that the expression patterns of DBC2 and miR-5088-5p were negatively related, suggesting the potential as novel biomarkers for the diagnosis of breast cancer patients.

An improved and highly selective fluorescence assay for measuring phosphatidylserine decarboxylase activity.

Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis and a potential target for development of antimicrobial and anti-cancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, we described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, we report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/ß-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochemical methods. Model reactions examining adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent additions in 96- or 384-well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.

miR-340-5p Suppresses Aggressiveness in Glioblastoma Multiforme by Targeting Bcl-w and Sox2.

Glioblastoma multiforme (GBM), a particularly aggressive type of malignant brain tumor, has a high mortality rate. Bcl-w, an oncogene, is reported to enhance cell survival, proliferation, epithelial-mesenchymal transition (EMT), migratory and invasive abilities, and stemness maintenance in a variety of cancer cell types, including GBM. In this study, we confirmed that Bcl-w-induced conditional medium (CM) enhances tumorigenic phenotypes of migration, invasiveness, and stemness maintenance. Notably, platelet-derived growth factor-A (PDGF-A) expression, among other factors of the tumor environment, was increased by CM of Bcl-w-overexpressing cells, prompting investigation of the potential correlation between Bcl-w and PDGF-A and their effects on GBM malignancy. Bcl-w and PDGF-A levels were positively regulated and increased tumorigenicity by Sox2 activation in GBM cells. miR-340-5p was further identified as a direct inhibitor of Bcl-w and Sox2. Overexpression of miR-340-5p reduced mesenchymal traits, cell migration, invasion, and stemness in GBM through attenuating Bcl-w and Sox2 expression. Our novel findings highlight the potential utility of miR-340-5p as a therapeutic agent for glioblastoma multiforme through inhibitory effects on Bcl-w-induced PDGF-A and Sox2 activation.

High-throughput screening for phosphatidylserine decarboxylase inhibitors using a distyrylbenzene-bis-aldehyde (DSB-3)-based fluorescence assay.

Phosphatidylserine decarboxylases (PSDs) catalyze the decarboxylation of phosphatidylserine to generate phosphatidylethanolamine, a critical step in phospholipid metabolism in both prokaryotes and eukaryotes. Most PSDs are membrane-bound, and classical radioisotope-based assays for determining their activity in vitro are not suitable for high-throughput drug screening. The finding that the PkPSD from Plasmodium knowlesi can be purified in a soluble and active form and the recent development of a fluorescence-based distyrylbenzene-bis-aldehyde (DSB-3) assay to measure PSD activity in vitro have laid the groundwork for screening chemical libraries for PSD inhibitors. Using this assay, here we conducted a high-throughput screen of a structurally diverse 130,858-compound library against PkPSD. Further characterization of the hits identified in this screening yielded five PkPSD inhibitors with IC50 values ranging from 3.1 to 42.3 µm Lead compounds were evaluated against the pathogenic yeast Candida albicans in the absence or presence of exogenous ethanolamine, and YU253467 and YU254403 were identified as inhibiting both native C. albicans PSD mitochondrial activity and C. albicans growth, with an MIC50 of 22.5 and 15 µg/ml without ethanolamine and an MIC50 of 75 and 60 µg/ml with ethanolamine, respectively. Together, these results provide the first proof of principle for the application of DSB-3-based fluorescent readouts in high-throughput screening for PSD inhibitors. The data set the stage for future analyses to identify more selective and potent PSD inhibitors with antimicrobial or antitumor activities.

Volumetric change of the latissimus dorsi muscle after immediate breast reconstruction with an extended latissimus dorsi musculocutaneous flap.

BACKGROUND: In immediate breast reconstruction using an extended latissimus dorsi musculocutaneous (eLDMC) flap, the volume of the flap decreases, which causes a secondary deformity of the breast shape. Since little research has investigated this decrease in muscle volume, the authors conducted an objective study to characterize the decrease in muscle volume after breast reconstruction using an eLDMC flap. METHODS: Research was conducted from October 2011 to November 2016. The subjects included 23 patients who underwent mastectomy due to breast cancer, received immediate reconstruction using an eLDMC flap without any adjuvant chemotherapy or radiotherapy, and received a computed tomography (CT) scan from days 7 to 10 after surgery and 6 to 8 months postoperatively. In 10 patients, an additional CT scan was conducted 18 months postoperatively. Axial CT scans were utilized to measure the volumetric change of the latissimus dorsi muscle during the follow-up period. RESULTS: In the 23 patients, an average decrease of 54.5% was observed in the latissimus dorsi muscle volume between the images obtained immediately postoperatively and the scans obtained 6 to 8 months after surgery. Ten patients showed an average additional decrease of 11.9% from 6-8 months to 18 months after surgery. CONCLUSIONS: We studied changes in the volume of the latissimus dorsi muscle after surgery using an eLDMC flap performed after a mastectomy without adjuvant chemotherapy or radiotherapy. In this study, we found that immediate breast reconstruction using a latissimus dorsi muscle flap led to a decrease in muscle volume of up to 50%.

Hypermethylation of miR-205-5p by IR Governs Aggressiveness and Metastasis via Regulating Bcl-w and Src.

Although radiotherapy has been successfully applied to treat many cancer types, surviving cancer cells often acquire therapeutic resistance, leading to increased risk of local recurrence and distant metastases via modification of the tumor microenvironment. Previously, we reported that high expression of Bcl-w in cancer patients is significantly correlated with poor survival as well as malignant activity. However, the relationship between ionizing radiation (IR)-induced resistance and Bcl-w expression in cancer cells is currently unclear. We showed that IR-induced Bcl-w contributes to EMT (epithelial-mesenchymal transition), migration, angiogenesis, stemness maintenance, and metastasis by promoting the expression of factors related to these phenotypes, both in vitro and in vivo. Meanwhile, IR enhanced hypermethylation of miR-205-5p CpG islands through Src activation, leading to decreased miR-205-5p expression and, in turn, potentially stimulating Bcl-w-mediated malignant activity and metastasis. The clinical applicability of Bcl-w and miR-205-5p from cells or animal models was confirmed using tissues and plasma of breast carcinoma patients. Based on the collective findings, we propose that miR-205-5ps as important negative mediators of resistance in radiotherapy could serve as useful potential targets of concurrently applied genetic therapy aimed to inhibit tumor aggressiveness and enhance the efficiency of radiotherapy in cancer patients.

Interaction of two antitumor peptides with membrane lipids - Influence of phosphatidylserine and cholesterol on specificity for melanoma cells.

R-DIM-P-LF11-322 and DIM-LF11-318, derived from the cationic human host defense peptide lactoferricin show antitumor activity against human melanoma. While R-DIM-P-LF11-322 interacts specifically with cancer cells, the non-specific DIM-LF11-318 exhibits as well activity against non-neoplastic cells. Recently we have shown that cancer cells expose the negatively charged lipid phosphatidylserine (PS) in the outer leaflet of the plasma membrane, while non-cancer cells just expose zwitterionic or neutral lipids, such as phosphatidylcholine (PC) or cholesterol. Calorimetric and zeta potential studies with R-DIM-P-LF11-322 and cancer-mimetic liposomes composed of PS, PC and cholesterol indicate that the cancer-specific peptide interacts specifically with PS. Cholesterol, however, reduces the effectiveness of the peptide. The non-specific DIM-LF11-318 interacts with PC and PS. Cholesterol does not affect its interaction. The dependence of activity of R-DIM-P-LF11-322 on the presence of exposed PS was also confirmed in vitro upon PS depletion of the outer leaflet of cancer cells by the enzyme PS-decarboxylase. Further corresponding to model studies, cholesterol depleted melanoma plasma membranes showed increased sensitivity to R-DIM-P-LF11-322, whereas activity of DIM-LF11-318 was unaffected. Microscopic studies using giant unilamellar vesicles and melanoma cells revealed strong changes in lateral distribution and domain formation of lipids upon addition of both peptides. Whereas R-DIM-P-LF11-322 enters the cancer cell specifically via PS and reaches an intracellular organelle, the Golgi, inducing mitochondrial swelling and apoptosis, DIM-LF11-318 kills rapidly and non-specifically by lysis of the plasma membrane. In conclusion, the specific interaction of R-DIM-P-LF11-322 with PS and sensitivity to cholesterol seem to modulate its specificity for cancer membranes.

miR-93-5p suppresses cellular senescence by directly targeting Bcl-w and p21.

Cellular senescence, a distinctive type of irreversible growth arrest, develops in response to various stimuli. Bcl-w, an oncogene and member of the Bcl-2 family, has been reported to promote tumorigenicity in various cancer cells. Here, we sought to explore the potential role of Bcl-w in premature senescence, which has received relatively little research attention. Our findings demonstrate that Bcl-w enhances the activity of senescence-associated ß-galactosidase (SA-ß-gal) and promotes histone H3 tri-methylation at lysine 9 (H3K9me3) and expressions of p53, Notch2, p21, and p16-hallmarks of the senescent phenotype-in human U251 glioblastoma and H460 lung carcinoma cells. It is also known that microRNAs (miRNAs) regulate processes related to tumor development, such as cell proliferation, differentiation, survival, metabolism, inflammation, invasion, angiogenesis, and senescence. In this context, we found that miR-93-5p inhibited premature cellular senescence by directly suppressing Bcl-w and p21 expressions. Collectively, these findings suggest that targeting miR-93-5p-regulated Bcl-w may be a useful strategy for preventing premature senescence.

The efficacy of elongated axillary incision on extended latissimus dorsi flap for immediate breast reconstruction.

BACKGROUND: In performing extended latissimus dorsi (ELD) flap procedures, a skin paddle design on the bra line helps reduce visible scarring. This improves the patient's satisfaction with the outcome. However, such a design leads to a longer operation time and increased fatigue of the surgeon due to the narrow operative field. In this study, the authors propose a method that elongates the axillary incision line posteriorly by 1.5 cm from the lateral border of the latissimus dorsi muscle. We examined whether this method could shorten the operation time and compared the incidence of complications between patients who underwent this novel procedure and patients who underwent the traditional procedure. METHODS: In this study of patients who underwent ELD flap procedures for immediate breast reconstruction, 89 underwent surgery with the elongated axillary incision and 45 underwent surgery without the elongated incision. The total operation time and complications were retrospectively examined based on the patients' medical records, and we examined whether there was any statistically significant difference in the total operation time. RESULTS: In the experimental group with the elongated axillary incision, the operation time ranged from 125 to 255 minutes (median, 175 minutes). In contrast, in the control group without the elongated axillary incision, the operation time ranged from 142 and 340 minutes (median, 205 minutes). The operation time was statistically significantly different between the two groups, and no significant complications were observed in the experimental group. CONCLUSIONS: Elongation of the axillary incision alone may shorten the operation time of the ELD flap procedure without causing additional complications.

Podophyllotoxin acetate triggers anticancer effects against non-small cell lung cancer cells by promoting cell death via cell cycle arrest, ER stress and autophagy.

We previously reported that podophyllotoxin acetate (PA) radiosensitizes NCI-H460 cells. Here, we confirmed that PA treatment also induces cell death among two other non-small cell lung cancer (NSCLC) cell lines: NCI-H1299 and A549 cells (IC50 values = 7.6 and 16.1 nM, respectively). Our experiments further showed that PA treatment was able to induce cell death via various mechanisms. First, PA dose-dependently induced cell cycle arrest at G2/M phase, as shown by accumulation of the mitosis-related proteins, p21, survivin and Aurora B. This G2/M phase arrest was due to the PA-induced inhibition of microtubule polymerization. Together, the decreased microtubule polymerization and increased cell cycle arrest induced DNA damage (reflected by accumulation of γ-H2AX) and triggered the induction of intrinsic and extrinsic apoptotic pathways, as shown by the time-dependent activations of caspase-3, -8 and -9. Second, PA time-dependently activated the pro-apoptotic ER stress pathway, as evidenced by increased expression levels of BiP, CHOP, IRE1-α, phospho-PERK, and phospho-JNK. Third, PA activated autophagy, as reflected by time-dependent increases in the expression levels of beclin-1, Atg3, Atg5 and Atg7, and the cleavage of LC3. Collectively, these results suggest a model wherein PA decreases microtubule polymerization and increases cell cycle arrest, thereby inducing apoptotic cell death via the activation of DNA damage, ER stress and autophagy.

Podophyllotoxin acetate enhances γ-ionizing radiation-induced apoptotic cell death by stimulating the ROS/p38/caspase pathway.

To develop a new radiosensitizer against non-small cell lung cancer cells, we screened a natural product library for growth-inhibitory compounds. PA was found to be cytotoxic toward NCI-H460 cells, and its IC50 value was determined. The radiosensitizer effects of PA were tested at its IC50 value in clonogenic and cell-counting assays. The intracellular mechanism underlying this effect was determined by immunoblotting and by measuring propidium iodide uptake and ROS generation. The radiosensitizer activity of PA in vivo was tested in nude mice by treating with PA and IR, and measuring tumor volume and assessing apoptosis. PA, tested at its experimentally determined IC50 value (12 nM), enhanced IR-induced death of NCI-H460 cells by increasing apoptosis, yielding a mean calculated dose-enhancement ratio of 1.67. Combination with PA and IR also increased the production of ROS, which subsequently induced phosphorylation of p38, suppressed phosphorylation of ERK, and activated caspase-3, -8, and -9. Notably, inhibition of ROS production prevented p38 phosphorylation, and inhibition of ROS production or p38 activation blocked caspase activation and apoptosis. In a xenograft assay, combination with PA and IR delayed tumor growth by 11.4 days compared with controls, yielding an enhancement factor of 1.48. Collectively, these results indicate that PA functions as a radiosensitizer by enhancing apoptosis through activation of a ROS/p38/caspase pathway and suppression of ERK.

Luteolin acts as a radiosensitizer in non­small cell lung cancer cells by enhancing apoptotic cell death through activation of a p38/ROS/caspase cascade.

To improve radiation therapy, the development of effective radiosensitizer is required. Fifty percent inhibitory concentration (IC50) values of 3',4',5',7'­tetrahydroxyflavone (luteolin) against NCI­H460 and ­H1299 non­small cell lung cancer (NSCLC) cells were determined using 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assays. Radiosensitizing activity was assessed in vitro by treating cells with luteolin prior to irradiation of γ­ionizing radiation (IR), and performing cell count and clonogenic assays. Cell signaling pathways involved in the radiosensitizing effects of luteolin were examined using propidium iodide (PI) uptake, reactive oxygen species (ROS) detection and immunoblot assays, with or without specific chemical inhibitors. Apoptotic cell death was confirmed by PI uptake and immunoblot assays. In vivo radiosensitizing activity was tested using an NCI­H460 cell xenograft model in nude mice. Tumor size was measured and apoptosis was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in animals in four treatment groups: mock­treated control, luteolin only, IR only, and luteolin plus IR. Treatment with luteolin or IR induced NSCLC cell death in vitro, but the combination of luteolin pre­treatment and IR was more effective than either agent alone, yielding dose enhancement ratios (DERs) of 1.22 and 1.35 for NCI­H460 and ­H1299 cells, respectively. Combined treatment with luteolin and IR enhanced apoptotic cell death in association with downregulation of B­cell lymphoma 2 (Bcl­2) and activation of caspase­3, ­8, and ­9; it also induced phosphorylation of p38 mitogen­activated protein kinase (MAPK) and ROS accumulation. Inhibition of p38 MAPK decreased ROS production, and inhibition of either p38 MAPK or ROS production attenuated apoptotic cell death and activation of caspase­8 and ­9. In a xenograft model, tumor growth was delayed by 21.8 days in the luteolin/IR combination group compared with controls, and apoptotic cell death was increased. The enhancement factor of the luteolin and IR combination was 1.83. Collectively, these findings indicate that luteolin acts as a radiosensitizer by enhancing apoptotic cell death through activation of a p38/ROS/caspase cascade.

ICAM-3 endows anticancer drug resistance against microtubule-damaging agents via activation of the ICAM-3-AKT/ERK-CREB-2 pathway and blockage of apoptosis.

In a previous study, we showed that induction of ICAM-3 endows radioresistance in cervical cancer [1]. To ascertain whether ICAM-3 also promotes anticancer drug resistance, mock control (H1299/pcDNA3) or ICAM-3-expressing stable transfectants (H1299/ICAM-3) of the non-small cell lung cancer (NSCLC) cell line, NCI-H1299, were generated and treated with the microtubule-damaging agents, paclitaxel (TXL) and vincristine (VCS). TXL-/VCS-treated H1299/ICAM-3 cells showed significantly lower levels of apoptosis, activation of caspases-3, 8 or 9, and decrease in anti-apoptotic protein levels, compared to H1299/pcDNA3 cells. Our data clearly indicate that ICAM-3 promotes drug resistance via inhibition of apoptosis. We additionally showed that Akt, ERK, and CREB-2 are located downstream of ICAM-3, and activation of the ICAM-3-Akt/ERK-CREB-2 pathway induces resistance against TXL and VCS. ICAM-3-expressing stable NCI-H460/ICAM-3 transfectant cells and radioresistant SiHa cells endogenously overexpressing ICAM-3 additionally showed drug resistance against TXL and VCS via activation of the ICAM-3-Akt/ERK-CREB-2 pathway. The finding that ICAM-3 endows drug resistance as well as radioresistance supports its potential utility as a major therapeutic target against cancer.

Impact of Disturbed Wound Healing after Surgery on the Prognosis of Marjolin's Ulcer.

BACKGROUND: Marjolin's ulcer is known to present a high proportion of recurrence and poor prognosis compared to other kinds of skin cancer. Based on our experience, Marjolin's ulcer patients who have received reconstructive surgery present a higher proportion of recurrence when there was disturbed wound healing after surgery. The impact of disturbed wound healing after surgery on the prognosis was examined in this study. METHODS: A retrospective study was carried out on 26 patients who were diagnosed with Marjolin's ulcer and received surgery in this hospital from 1996 to 2011. Histologic grading, lymph node metastasis at diagnosis, and the wound healing process were evaluated and chi-squared analysis applied in order to determine the correlation with recurrence. RESULTS: The proportion of recurrence increases in patients with a low histologic grade or lymph node metastasis at diagnosis. The proportion of recurrence is even higher when the problem occurs during the wound healing process after surgery. CONCLUSIONS: Disturbed wound healing after surgery could be used as a sign to quickly identify the recurrence of carcinoma. Therefore, in the event a problem occurs in the wound healing process after surgery, one should keep in mind that this could be a sign of the possibility of recurrence and proceed with careful observation and active diagnosis through additional physical examinations, general X-ray tests, computed tomographys, magnetic resonance imagings, and so on, to obtain an early diagnosis of recurrence.