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One of the globally common cancers is colorectal cancer (CRC). At present, a surgical approach remains a good option for CRC patients; however, 20% of surgically treated CRC patients experience metastasis. Currently, even the first-line used drug, oxaliplatin, remains inadequate for treating metastatic CRC, and its side effect of neurotoxicity is a major problem when treating CRC. The Gene Omnibus GSE42387 database contains gene expression profiles of parental and oxaliplatin-resistant LoVo cell lines. Differentially expressed genes (DEGs) between parental and oxaliplatin-resistance LoVo cells, protein-protein interactions (PPIs), and a pathway analysis were determined to identify overall biological changes by an online DAVID bioinformatics analysis. The ability of DEGs to predict overall survival (OS) and disease-free survival (DFS) was validated by the SPSS 22.0, using liver metastasis CRC patient samples of GSE41258. The bioinformatics web tools of the GEPIA, the Human Protein Atlas, WebGestalt, and TIMER platforms were used. In total, 218 DEGs were identified, among which 105 were downregulated and 113 were upregulated. After mapping the PPI networks and pathways, 60 DEGs were identified as hub genes (with high degrees). Six genes (TGFB1, CD36, THBS1, FABP1, PCK1, and IRS1) were involved with malaria, PPAR signaling, and the adipocytokine signaling pathway. High expressions of CD36 and PCK1 were associated with the poor survival of CRC patients in the GSE41258 database. We predicted specific micro (mi)RNAs that targeted the 3 ′ untranslated region (UTR) of PCK1 by using miRWalk. It was found that three miRNAs, viz., miR-7-5p, miR-20a-3p, and miR-636, may be upstream targets of those genes. High expression levels of miR-7-5p, miR-20a-3p, and miR-636 were associated with poor OS of CRC patients, and the small-molecule compound, mersalyl, is a promising drug for treating oxaliplatin-resistant CRC. In conclusion, miR-7-5p miR-20a-3p, and miR-636 targeted the PCK1 biomarker in the PPAR signaling pathway, which is involved in oxaliplatin-resistant CRC. Meanwhile, mersalyl was identified as a potential drug for overcoming oxaliplatin resistance in CRC. Our findings may provide novel directions and strategies for CRC therapies.

The impacts of antidepressants on the pathogenesis of dementia remain unclear despite depression and dementia are closely related. Antidepressants have been reported may impair serotonin-regulated adaptive processes, increase neurological side-effects and cytotoxicity. An 'astroglio-centric' perspective of neurodegenerative diseases proposes astrocyte dysfunction is involved in the impairment of proper central nervous system functioning. Thus, defining whether antidepressants are harmful to astrocytes is an intriguing issue. We used an astrocyte cell line, primary cultured astrocytes and neuron cells, to identify the effects of 11 antidepressants which included selective serotonin reuptake inhibitors, a serotonin-norepinephrine reuptake inhibitor, tricyclic antidepressants, a tetracyclic antidepressant, a monoamine oxide inhibitor, and a serotonin antagonist and reuptake inhibitor. We found that treatment with 10 μM sertraline and 20 μM paroxetine significantly reduced cell viability. We further explored the underlying mechanisms and found induction of the [Ca2+]i level in astrocytes. We also revealed that sertraline and paroxetine induced mitochondrial damage, ROS generation, and astrocyte apoptosis with elevation of cleaved-caspase 3 and cleaved-PARP levels. Ultimately, we validated these mechanisms in primary cultured astrocytes and neuron cells and obtained consistent results. These results suggest that sertraline and paroxetine cause astrocyte dysfunction, and this impairment may be involved in the pathogenesis of neurodegenerative diseases.

Biofilms of Cutibacterium (C.) acnes (formerly Propionibacterium acnes) are responsible for the persistence and antibiotic resistance of acne vulgaris. In addition to the standard treatments for acne vulgaris, a common adjunctive treatment is the topical administration of nicotinamide (NAM). However, the effects of NAM on biofilms of C. acnes have never been explored. This study comprehensively investigates the effects of NAM against biofilms of C. acnes using in vitro and in vivo approaches. The results showed that NAM potentiated the efficacy of suboptimal dosing of tetracycline against C. acnes. Moreover, NAM alone decreased the formation and increased the degradation of biofilms in C. acnes. The antibiofilm effect of NAM against C. acnes was further enhanced in combination with deoxyribonuclease (DNase) I, an enzyme with known antibiofilm properties. The computational molecular docking, surface plasmon resonance analysis, and enzymatic kinetic assay demonstrated that NAM binds to DNase I and accelerated its reaction. In conclusion, NAM activates DNase I to attenuate biofilms of C. acnes. This offers valuable insights into the strategies against biofilms that are worth elaborating on in other biofilm-related chronic cutaneous infections in the future.

Use of the ablative laser has been approved to enhance topical drug penetration. Investigation into the usefulness of the non-ablative laser for assisting drug delivery is very limited. In this study, we explored the safety and efficacy of the non-ablative fractional erbium:glass (Er:glass) laser as an enhancement approach to promote drug permeation. Both pig and nude mouse skins were employed as transport barriers. We histologically examined the skin structure after laser exposure. The permeants of 5-aminolevulinic acid (ALA), imiquimod, tretinoin, peptide, dextrans and quantum dots (QD) were used to evaluate in vitro and in vivo skin passage. The fractional laser selectively created an array of photothermal dots deep into the dermis with the preservation of the stratum corneum and epidermis. The barrier function of the skin could be recovered 8-60h post-irradiation depending on the laser spot densities. The application of the laser caused no local infection of Staphylococcus aureus and Pseudomonas aeruginosa. Compared to intact skin, ALA flux was enhanced up to 1200-fold after laser exposure. The penetration enhancement level by the laser was decreased following the increase of permeant lipophilicity. The skin accumulation of tretinoin, an extremely lipophilic drug, showed only a 2-fold elevation by laser irradiation. The laser promoted peptide penetration 10-fold compared to the control skin. Skin delivery of dextrans with a molecular weight (MW) of at least 40kDa could be achieved with the Er:glass laser. QD with a diameter of 20nm penetrated into the skin with the assistance of the non-ablative laser. The confocal microscopic images indicated the perpendicular and lateral diffusions of dextrans and nanoparticles via laser-created microscopic thermal zones. Controlled Er:glass laser irradiation offers a valid enhancement strategy to topically administer the permeants with a wide MW and lipophilicity range.

Background/aim Recent studies implied a significant role of hypoxia-inducible factor-1α (HIF1α) in cell transformation. This study aimed to assess the effects of HIF1α on the epithelial-to-mesenchymal transition (EMT) and tumorigenesis of lung adenocarcinoma cells. Materials and methods Invasion, migration and colony formation assays were used to evaluate cell transformation. Expression of EMT-related markers were analyzed by western blot, reverse-transcription polymerase chain reaction or zymography. A luciferase assay was carried out to access the transcriptional activity of β-catenin. Results Hypoxia enhanced migration, invasion and transformation of A549 lung adenocarcinoma cells. Hypoxic stimulation promoted the expression of EMT-related markers in lung cancer cells. The expression of HIF1α was found to be involved in hypoxia-mediated modulation of expression of snail family transcriptional repressors 1 (SNAI1) and 2 (SLUG). Hypoxia enhanced nuclear accumulation and transcriptional activity of β-catenin. Conclusion β-Catenin promotes expression of EMT-related genes and eventually contributes to the metastatic process.

Cigarette smoking is associated with an increased risk of melanoma metastasis. Smokers show higher PD-L1 expression and better responses to PD-1/PD-L1 inhibitors than nonsmokers. Here, we investigate whether nicotine, a primary constituent of tobacco, induces PD-L1 expression and promotes melanoma cell proliferation and migration, which is mediated by the &alpha, 9 nicotinic acetylcholine receptor (&alpha, 9-nAChR). &alpha, 9-nAChR overexpression in melanoma using melanoma cell lines, human melanoma tissues, and assessment of publicly available databases. &alpha, 9-nAChR expression was significantly correlated with PD-L1 expression, clinical stage, lymph node status, and overall survival (OS). Overexpressing or knocking down &alpha, 9-nAChR in melanoma cells up- or downregulated PD-L1 expression, respectively, and affected melanoma cell proliferation and migration. Nicotine-induced &alpha, 9-nAChR activity promoted melanoma cell proliferation through stimulation of the &alpha, 9-nAChR-mediated AKT and ERK signaling pathways. In addition, nicotine-induced &alpha, 9-nAchR activity promoted melanoma cell migration via activation of epithelial-mesenchymal transition (EMT). Moreover, PD-L1 expression was upregulated in melanoma cells after nicotine treatment via the transcription factor STAT3 binding to the PD-L1 promoter. These results highlight that nicotine-induced &alpha, 9-nAChR activity promotes melanoma cell proliferation, migration, and PD-L1 upregulation. This study may reveal important insights into the mechanisms underlying nicotine-induced melanoma growth and metastasis through &alpha, 9-nAChR-mediated carcinogenic signals and PD-L1 expression.

Nilotinib (AMN), a second-generation tyrosine kinase inhibitor, induces apoptosis in various cancer cells, and our recent study showed that AMN effectively reduced the viability of human ovarian cancer cells via mitochondrion-dependent apoptosis. The effect of AMN in the melanogenesis of melanoma cells is still unclear. In the present study, we found that the addition of AMN but not imatinib (STI) significantly increased the darkness of B16F0 melanoma cells, and the absorptive value increased with the concentration of AMN. A decrease in the viability of B16F0 cells by AMN was detected in a concentration-dependent manner, accompanied by increased DNA ladders, hypodiploid cells and cleavage of the caspase-3 protein. An in vitro tyrosinase (TYR) activity assay showed that increased TYR activity by AMN was detected in a concentration-dependent manner; however, induction of TYR activity by STI at a concentration of 40 μmol/L was observed. Increased intracellular peroxide by AMN was detected in B16F0 cells, and application of the antioxidant, N-acetylcysteine (NAC), significantly reduced AMN-induced peroxide production which also reduced the darkness of B16F0 cells. Additionally, AMN induced c-Jun N-terminal kinase (JNK) protein phosphorylation in B16F0 cells, which was inhibited by the addition of NAC. AMN-induced melanogenesis of B16F0 cells was significantly inhibited by the addition of NAC and the JNK inhibitor, SP600125 (SP). Data of Western blotting showed that increased protein levels of melanogenesis-related enzymes of tyrosinase-related protein-1 (TRP1), TRP2 and TYR were observed in AMN-treated B16F0 cells which were inhibited by the addition of NAC and SP. Evidence is provided supporting AMN effectively inducing the melanogenesis of B16F0 melanoma cells via reactive oxygen species-dependent JNK activation.

Most of the investigations into laser-assisted skin permeation have used the intact skin as the permeation barrier. Whether the laser is effective in improving cutaneous delivery via barrier-defective skin is still unclear. In this study, ablative (Er:YAG) and non-ablative (Er:glass) lasers were examined for the penetration of peptide and siRNA upon topical application on in vitro skin with a healthy or disrupted barrier. An enhanced peptide flux (6.9 fold) was detected after tape stripping of the pig stratum corneum (SC). A further increase of flux to 11.7 fold was obtained after Er:YAG laser irradiation of the SC-stripped skin. However, the application of Er:glass modality did not further raise the flux via the SC-stripped skin. A similar trend was observed in the case of psoriasiform skin. Conversely, the flux was enhanced 3.7 and 2.6 fold after treatment with the Er:YAG and the Er:glass laser on the atopic dermatitis (AD)-like skin. The 3-D skin structure captured by confocal microscopy proved the distribution of peptide and siRNA through the microchannels and into the surrounding tissue. The fractional laser was valid for ameliorating macromolecule permeation into barrier-disrupted skin although the enhancement level was lower than that of normal skin.

Glioblastoma multiforme (GBM) is a highly malignant type of brain tumor found in humans. GBM cells reproduce quickly, and the median survival time for patients after therapy is approximately 1 year with a high relapse rate. Current therapies and diagnostic tools for GBM are limited; therefore, we searched for a more favorable therapeutic target or marker protein for both therapy and diagnosis. We used mass spectrometry (MS) analysis to identify GBM-associated marker proteins from human plasma and GBM cell cultures. Additional plasma and 52 brain tissues obtained from patients with gliomas were used to validate the association rate of serum amyloid A1 (SAA1) in different grades of gliomas and its distribution in tumors. Microarray database analysis further validated the coefficient of SAA1 levels in gliomas. The cellular mechanisms of SAA1 in GBM proliferation and infiltration were investigated in vitro. We analyzed the correlation between SAA1 and patients' medication requirement to demonstrate the clinical effects of SAA1 in GBM. SAA1 was identified from MS analysis, and its level was revealed to be correlated with the disease grade, clinical severity, and survival rate of patients with gliomas. In vitro cultures, including GBM cells and normal astrocytes, revealed that SAA1 promotes cell migration and invasion through integrin αVβ3 to activate the Erk signaling pathway. Magnetic resonance imaging and tumor region-specific microarray analysis identified a correlation between SAA1 and GBM cell infiltration in patients. In summary, our results demonstrate that SAA1 in combination with integrin αV and β3 can serve as an indicator of high glioblastoma risk. We also identified the cellular mechanisms of SAA1 contributing to GBM progression, which can serve as the basis for future GBM therapy.

The Ras/ERK (extracellular signal-regulated protein kinase) and cAMP/PKA (protein kinase A) pathways are essential for the transcriptional activities of CREB (cAMP response element binding protein) and MITF (microphthalmia-associated transcription factor) in melanogenesis and the progression of melanoma. However, the interaction between Ras/ERK and cAMP/PKA pathways in the melanogenesis and progression of melanoma is not fully known. Here, we report that CSE1L (chromosome segregation 1-like protein) regulates cAMP/PKA-induced CREB and MITF expressions as well as Ras-induced ERK1/2 phosphorylation. IBMX, a cAMP/PKA activator, treatment induced CSE1L phosphorylation and augmented Ras-induced ERK1/2 phosphorylation. CSE1L knockdown by CSE1L shRNA expression vectors inhibited Ras-induced ERK1/2 phosphorylation and melanogenesis in melanoma cells. CSE1L overexpression increased phospho-CREB expression; CSE1L knockdown also inhibited Ras-induced phospho-CREB, MITF, and tyrosinase expressions, regardless of the presence of IBMX. This study identifies CSE1L links and controls the Ras/ERK and cAMP/PKA pathways in the melanogenesis of melanoma cells. Melanomas frequently develop drug resistance via paradoxical activation of Ras/Raf/MEK/ERK or alternatively activated Ras/ERK and cAMP/PKA pathways. Thus CSE1L may be a potential target for treating melanomas that harbor Ras mutations or are resistant to drugs targeting Raf/MEK/ERK. © 2015 Wiley Periodicals, Inc.

// Kao-Hui Liu 1 , Shun-Tai Yang 2 , Yen-Kuang Lin 3 , Jia-Wei Lin 2 , Yi-Hsuan Lee 4 , Jia-Yi Wang 1,5 , Chaur-Jong Hu 6 , En-Yuan Lin 7 , Shu-Mei Chen 8 , Chee-Kin Then 9 and Shing-Chuan Shen 1 1 Taipei Medical University, College of Medicine, Graduate Institute of Medical Sciences, Taipei, Taiwan 2 Taipei Medical University-Shuang Ho Hospital, Department of Neurosurgery, Taipei, Taiwan 3 Taipei Medical University, Biostatistics Center, Taipei, Taiwan 4 National Yang-Ming University, Department and Institute of Physiology, Taipei, Taiwan 5 Taipei Medical University, College of Medicine, School of Medicine, Department of Physiology, Taipei, Taiwan 6 Taipei Medical University-Shuang Ho Hospital, School of Medicine, Department of Neurology, Taipei, Taiwan 7 Taipei Medical University Hospital, Department of Neurosurgery, Taipei, Taiwan 8 Taipei Medical University-Wan Fang Hospital, Department of Neurosurgery, Taipei, Taiwan 9 Taipei Medical University, College of Medicine, School of Medicine, Taipei, Taiwan Correspondence: Shing-Chuan Shen, email: // Keywords : glioblastoma, antidepressant, AMPA receptor, excitotoxicity Received : November 24, 2014 Accepted : December 26, 2014 Published : December 31, 2014 Abstract The efficacy of glioblastoma chemotherapy is not satisfactory; therefore, a new medication is expected to improve outcomes. As much evidence shows that antidepressants decrease cancer incidence and improve patients’ quality of life, we therefore attempted to explore the potential for fluoxetine to be used to treat GBM and its possible underlying mechanism. The expression level of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) was determined using immunohistochemical staining and PCR analysis. The mechanism of fluoxetine-induced apoptosis of gliomas was elucidated. Computer modeling and a binding assay were conducted to investigate the interaction of fluoxetine with the AMPAR. The therapeutic effect of fluoxetine was evaluated using an animal model. We found that fluoxetine directly bound to AMPAR, thus inducing transmembrane Ca 2+ influx. The rise in the intracellular calcium concentration ([Ca 2+ ] i ) causes mitochondrial Ca 2+ overload, thereby triggering apoptosis. AMPARs are excessively expressed in glioma tissues, suggesting that fluoxetine specifically executes glioma cells. Our in vivo study revealed that fluoxetine suppressed the growth of glioblastomas in brains of Nu/Nu mice, an effect similar to that produced by temozolomide. Our preclinical studies suggest fluoxetine, a commonly used antidepressant, might be selectively toxic to gliomas and could provide a new approach for managing this disease.

In this study, we aimed to illustrate the utility of fractional radiofrequency (RF) that generated microchannels in the skin, allowing delivery of peptide and siRNA via the skin. The mechanisms involved in the correlation between macromolecule permeation and skin structure were also elucidated. The morphology of the skin was examined by transmission electron microscopy (TEM), higher harmonic generation microscopy (HGM), and physiological factors. In vivo skin distribution of macromolecules was assessed by fluorescence and confocal microscopies. RF thermolysis selectively created an array of micropores deep into the epidermis without significant removal of the stratum corneum (SC). With energy of 30 mJ, a pore depth of 35 μm was achieved. The bipolar RF resulted in a 3-fold increase of transepidermal water loss (TEWL) compared with intact skin. The respective skin accumulation and flux of the peptide with a molecular weight (MW) of 2335 Da was 3- and 23-fold greater for the RF-treated group than for the non-treatment group. RF enhanced skin accumulation of siRNAs with MW of 10 and 15 kDa by 6.2- and 2.6-fold, respectively. Cutaneous penetration of the macromolecules with an MW of at least 40 kDa could be accomplished by RF. Confocal microscopy imaging revealed that RF could effectively deliver the peptide up to at least a 74-μm depth. The penetration depth of siRNA by RF irradiation was about 50 μm. The novel RF device efficiently delivered macromolecules into the skin while reserving SC layers to support some barrier functions. In this work, for the first time the assistance of fractional RF on peptide and siRNA transport was demonstrated.

Alopecia usually cannot be cured because of the available drug therapy being unsatisfactory. To improve the efficiency of treatment, erbium–yttrium–aluminum–garnet (Er–YAG) laser treatment was conducted to facilitate skin permeation of antialopecia drugs such as minoxidil (MXD), diphencyprone (DPCP), and peptide. In vitro and in vivo percutaneous absorption experiments were carried out by using nude mouse skin and porcine skin as permeation barriers. Fluorescence and confocal microscopies were used to visualize distribution of permeants within the skin. Laser ablation at a depth of 6 and 10 μm enhanced MXD skin accumulation twofold to ninefold depending on the skin barriers selected. DPCP absorption showed less enhancement by laser irradiation as compared with MXD. An ablation depth of 10 μm could increase the peptide flux from zero to 4.99 and 0.33 μg cm −2 h −1 for nude mouse skin and porcine skin, respectively. The laser treatment also promoted drug uptake in the hair follicles, with DPCP demonstrating the greatest enhancement (sixfold compared with the control). The imaging of skin examined by microscopies provided evidence of follicular and intercellular delivery assisted by the Er–YAG laser. Besides the ablative effect of removing the stratum corneum , the laser may interact with sebum to break up the barrier function, increasing the skin delivery of antialopecia drugs. The minimally invasive, well‐controlled approach of laser‐mediated drug permeation offers a potential way to treat alopecia. This study's findings provide the basis for the first report on laser‐assisted delivery of antialopecia drugs.

Ethnopharmacological relevance Phellinus linteus (Berkeley & Curtis), a well-known medical fungus, has long been used as a traditional medicine in Oriental countries to treat various diseases, and hispolon (HIS) is one of its bioactive components. HIS is known to possess potent antineoplastic and antiviral properties; however, its effect on inflammatory apoptosis is still undefined. Materials and methods RAW264.7 macrophages were incubated with HIS for 30 min followed by LPS, LTA, or PGN stimulation for 12 h. The expression of indicated proteins AP-1 and NF-κB transcriptional activities was examined by Western blotting using specific antibodies. Levels of NO and ROS were examined by Griess reaction, and DCHF-DA staining via flow cytometric analysis, respectively. AP-1 and NF-κB transcriptional activities were detected by luciferase reporter assay. Knockdown of HO-1 protein expression was performed by transfection of macrophages with HO-1 siRNA. Pharmacological inhibitors including ROS scavenger NAC, JNK inhibitor SP600125, NF-κB inhibitor BAY117082 were applied for mechanism study. Results HIS showed concentration-dependent inhibition of LPS, LTA, and PGN-induced iNOS protein expressions and NO production by RAW264.7 macrophages. Accordingly, HIS protected RAW264.7 cells from LPS-, LTA-, and PGN-induced apoptosis. Increased HO-1 by HIS was detected at both protein and mRNA levels along with an increase in intracellular peroxide, and this was inhibited by the translational inhibitor, cycloheximide (CHX), the transcriptional inhibitor, actinomycin D (Act D), and the reactive oxygen species scavenger, N-acetylcysteine (NAC). A mechanistic study indicated that inhibition of c-Jun N-terminal kinase (JNK) protein phosphorylation, and activator protein (AP)-1 and nuclear factor (NF)-κB activation were involved in the anti-inflammatory actions of HIS in macrophages. A structure-activity relationship analysis showed that HIS expressed the most potent effect of inhibiting iNOS and apoptosis elicited by LPS, LTA, and PGN with a significant increase in HO-1 protein in macrophages. Conclusions Evidence supporting HIS prevention of inflammatory apoptosis via blocking NO production and inducing HO-1 protein expression in macrophages is provided, and the hydroxyl at position C3 is a critical substitution for the anti-inflammatory actions of HIS.

The present study was conducted to evaluate the impacts of fractional erbium (Er):YAG and CO 2 lasers on skin permeation of small interfering (si)RNA and plasmid (p)DNA vectors. In vitro skin delivery was determined with a Franz diffusion cell. In vivo absorption was investigated by observing fluorescence and confocal microscopic imaging. Fractional laser-mediated ablation of the skin resulted in significant enhancement of dextran and siRNA penetration. Respective fluxes of dextran (10 kDa) and siRNA, which had similar molecular size, with Er:YAG laser irradiation at 5 J/cm 2 were 56- and 11-fold superior to that of intact skin. The respective permeation extents of dextran and siRNA by the CO 2 laser at 4 mJ/400 spots were 42- and 12-fold greater than that of untreated skin. Fluorescence and confocal images showed increased fluorescence intensities and penetration depths of siRNA and pDNA delivery. According to an examination of the follicular permeant amount and fluorescence microscopy, hair follicles were important deposition areas for fractional laser-assisted delivery, with the Er:YAG modality revealing higher follicular siRNA selectivity than the CO 2 modality. This is the first report of siRNA and pDNA penetrating the skin with a sufficient amount and depth with the assistance of fractional lasers.

Oxidative stress or excessive antioxidant levels-caused redox imbalance can alter apoptotic responses, and N-acetyl-L-cysteine (NAC) was able to inhibit H2O2-mediated cell death, but unable to prevent apoptosis induced by other chemicals such as etoposide. We now demonstrate that 10 and 20 mM NAC, non-toxic concentrations, can enhance fisetin (FIS)-mediated apoptosis in colon cancer cells COLO205. Compared to treatment with FIS alone, combination treatment with NAC increased the expression of cleaved caspase-3 and PAPR protein, and produced greater density of DNA ladders. NAC reduced the mitochondrial membrane potential of FIS-treated COLO205 cells with induction of caspase 9 protein cleavage. DNA ladders induced by FIS + NAC were diminished by adding the caspase 3 inhibitor, DEVD-FMK, and the caspase 9 inhibitor, YVAD-FMK. Combinatorial treatment COLO205 cells with NAC and FIS showed potent inhibition on ERK protein phosphorylation, compared with those from FIS or NAC-treated groups by Western blotting using specific antibodies. Addition of the chemical ERK inhibitors, PD98059 and U0126, significantly inhibited ERK protein phosphorylation, accompanied by induced DNA ladder formation, cleavage of caspase 3 and PARP protein in COLO205 cells. Furthermore, NAC showed an enhancement on a FIS-related chemical chrysin-induced apoptosis of COLO205 cells, and NAC sensitization of colon cancer cells to FIS-induced apoptosis was also identify in colonic cancer cells HCT-116, HT-29, and HCT-15 cells. The evidence to support NAC sensitizing human colon cancer cells to FIS-induced apoptosis was provided, and application of NAC and FIS as a strategy to treat colonic cancer deserved for further in vivo study. © 2013 Wiley Periodicals, Inc.

We revealed the cytotoxic effect of the flavonoid, fisetin (FIS), on human COLO205 colon cancer cells in the presence and absence of the HSP90 inhibitors, geldanamycin (GA) and radicicol (RAD). Compared to FIS treatment alone of COLO205 cells, GA and RAD significantly enhanced FIS-induced cytotoxicity, increased expression of cleaved caspase-3 and the PAPR protein, and produced a greater density of DNA ladder formation. GA and RAD also reduced the MMPs with induction of caspase-9 protein cleavage in FIS-treated COLO205 cells. Increased caspase-3 and -9 activities were detected in COLO205 cells treated with FIS+GA or FIS+RAD, and the intensity of DNA ladder formation induced by FIS+GA was reduced by adding the caspase-3 inhibitor, DEVD-FMK. A decrease in Bcl-2 but not Bcl-XL or Bax protein by FIS+GA or FIS+RAD was identified in COLO205 cells by Western blotting. A reduction in p53 protein with increased ubiquitin-tagged proteins was observed in COLO205 cells treated with FIS+GA or FIS+RAD. Furthermore, GA and RAD reduced the stability of the p53 protein in COLO205 cells under FIS stimulation. The evidence supports HSP90 inhibitors possibly sensitizing human colon cancer cells to FIS-induced apoptosis, and treating colon cancer by combining HSP90 inhibitors with FIS deserves further in vivo study.

To evaluate skin permeation enhancement mediated by fractional laser for different permeants, including hydroquinone, imiquimod, fluorescein isothiocyanate-labeled dextran (FD), and quantum dots.Skin received a single irradiation of a fractional CO(2) laser, using fluence of 2 or 4 mJ with densities of 100 ∼ 400 spots/cm(2). In vitro and in vivo skin penetration experiments were performed. Fluorescence and confocal microscopies for imaging delivery pathways were used.The laser enhanced flux of small-molecule drugs 2 ∼ 5-fold compared to intact skin. A laser fluence of 4 mJ with a 400-spot/cm(2) density promoted FD flux at 20 and 40 kDa from 0 (passive transport) to 0.72 and 0.43 nmol/cm(2)/h, respectively. Microscopic images demonstrated a significant increase in fluorescence accumulation and penetration depth of macromolecules and nanoparticles after laser exposure. Predominant routes for laser-assisted delivery may be intercellular and follicular transport. CO(2) laser irradiation produced 13-fold enhancement in follicular deposition of imiquimod. Laser-mediated follicular transport could deliver permeants to deeper strata. Skin barrier function as determined by transepidermal water loss completely recovered by 12 h after irradiation, much faster than conventional laser treatment (4 days).Fractional laser could selectively enhance permeant targeting to follicles such as imiquimod and FD but not hydroquinone, indicating the importance of selecting feasible drugs for laser-assisted follicle delivery.

Tumor-derived microvesicles are rich in metastasis-related proteases and play a role in the interactions between tumor cells and tumor microenvironment in tumor metastasis. Because shed microvesicles may remain in the extracellular environment around tumor cells, the microvesicle membrane protein may be the potential target for cancer therapy. Here we report that chromosome segregation 1–like (CSE1L) protein is a microvesicle membrane protein and is a potential target for cancer therapy. v-H-Ras expression induced extracellular signal–regulated kinase (ERK)-dependent CSE1L phosphorylation and microvesicle biogenesis in various cancer cells. CSE1L overexpression also triggered microvesicle generation, and CSE1L knockdown diminished v-H-Ras–induced microvesicle generation, matrix metalloproteinase (MMP)-2 and MMP-9 secretion and metastasis of B16F10 melanoma cells. CSE1L was preferentially accumulated in microvesicles and was located in the microvesicle membrane. Furthermore, anti-CSE1L antibody–conjugated quantum dots could target tumors in animal models. Our findings highlight a novel role of Ras-ERK signaling in tumor progression and suggest that CSE1L may be involved in the “early” and “late” metastasis of tumor cells in tumorigenesis. Furthermore, the novel microvesicle membrane protein, CSE1L, may have clinical utility in cancer diagnosis and targeted cancer therapy.

While laser skin resurfacing is expected to result in reduced barrier function and increased risk of drug absorption, the extent of the increment has not yet been systematically investigated. We aimed to establish the skin permeation profiles of tetracycline and sunscreens after exposure to the erbium:yttrium-aluminum-garnet (Er:YAG) laser during postoperative periods. Physiological and histopathological examinations were carried out for 5 days after laser treatment on nude mice. Percutaneous absorption of the permeants was determined by an in vitro Franz cell. Ablation depths varied in reaching the stratum corneum (10 μm, 2.5 J/cm 2 ) to approach the epidermis (25 μm, 6.25 J/cm 2 ) and upper dermis (40 μm, 10 J/cm 2 ). Reepithelialization evaluated by transepidermal water loss was complete within 2–4 days and depended on the ablation depth. Epidermal hyperplasia was observed in the 40-μm-treated group. The laser was sufficient to disrupt the skin barrier and allow the transport of the permeants into and across the skin. The laser fluence was found to play an important role in modulating skin absorption. A 25-μm ablation depth increased tetracycline flux 84-fold. A much smaller enhancement (3.3-fold) was detected for tetracycline accumulation within the skin. The laser with different fluences produced enhancement of oxybenzone skin deposition of 3.4–6.4-fold relative to the untreated group. No penetration across the skin was shown regardless of whether titanium dioxide was applied to intact or laser-treated skin. However, laser resurfacing increased the skin deposition of titanium dioxide from 46 to 109–188 ng/g. Tetracycline absorption had recovered to the level of intact skin after 5 days, while more time was required for oxybenzone absorption. The in vivo skin accumulation and plasma concentration revealed that the laser could increase tetracycline absorption 2–3-fold. The experimental results indicated that clinicians should be cautious when determining the dose for postoperative treatment.

In this study, QUE, but not the structurally related chemical, rutin, enhanced the cytotoxicity of arsenic trioxide (As(+3)) against the viability of normal human HaCaT keratinocytes via induction of apoptosis. QUE enhancement of As(+3)-mediated apoptosis was accompanied by increased intracellular peroxide production according to a DCFH-DA analysis, and DNA ladders induced by QUE/As(+3) were inhibited by adding the antioxidative compound, N-acetyl cysteine (NAC). A loss of the mitochondrial membrane potential by QUE/As(+3) was observed according to DiOC(6) staining in concert with increased Bax protein and cytosolic cytochrome (Cyt) c protein expression in HaCaT cells, which was prevented by the addition of NAC. A decrease in the p53 protein with increased protein ubiquitination was detected in QUE/As(+3)-treated HaCaT cells, and this was prevented by the addition of NAC. The decrease in the p53 protein by QUE/As(+3) was reversed by adding the proteasome inhibitor, MG132. L-Buthionine sulphoximine (BSO) enhanced the cytotoxicity of As(+3) against the viability of HaCaT cells with reduced p53 protein through inducing protein ubiquitination and reactive oxygen species (ROS) production, and disrupting the mitochondrial membrane potential in HaCaT cells. Additionally, QUE and BSO enhanced the cytotoxic effects of monomethylarsonous acid (MMA(+3)) but not other arsenic compounds in accordance with increased p53 protein ubiquitination in HaCaT cells. QUE plus As(+3) stimulation of apoptosis in human HaCaT keratinocytes via activating ROS-dependent p53 protein ubiquitination may offer a rationale for the use of QUE to improve the clinical efficacy of arsenics in treating psoriasis.

2-Methyl-1,4-naphthoquinone (menadione or vitamin K3; EPO) and K3-2,3-epoxide (EPO1), but not vitamin K3-3-OH (EPO2), exhibited cytotoxicity that caused DNA fragmentation and chromatin condensation in U87 and C6 cells. EPO1 showed more-potent cytotoxicity than EPO, and the IC50 values of EPO and EPO1 in U87 cells were 37.5 and 15.7 μM, respectively. Activation of caspase 3 enzyme activity with cleavage of caspase 3 protein was detected in EPO1-treated U87 and C6 cells, and the addition of the caspase 3 peptidyl inhibitor, DEVD-FMK, reduced the cytotoxic effect of EPO1. An increase in the intracellular ROS level by EPO1 was observed in the DCHF-DA analysis, and EPO1-induced apoptosis and caspase 3 protein cleavage were prevented by adding the antioxidant, N-acetyl-cysteine (NAC), with decreased ROS production elicited by EPO1. Activation of ERK and JNK, but not p38, via phosphorylation induction was identified in EPO1- but not EPO- or EPO2-treated U87 and C6 cells, and this was blocked by adding NAC. However, the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125, showed no effect on EPO1-induced cytotoxicity in either cell type. Our findings demonstrate that 2,3-epoxide substitution significantly potentiates the apoptotic effect of vitamin K3 via stimulating ROS production, which may be useful in the chemotherapy of glioblastoma cells.

Background Imatinib mesylate (STI571), a protein tyrosine kinase inhibitor, was shown to reduce the viability of several cancer cell lines via apoptosis induction; however, the role of reactive oxygen species (ROS) in STI571-induced melanoma cell apoptosis is still undefined. Objective In this study, we investigated the contribution of ROS to STI571-induced apoptosis in melanoma B16F0 cells, and the apoptotic mechanism elicited by STI571 was illustrated. Methods Using an in vitro cell culture system, the effects of STI571 on ROS production, cell cycle progression, caspase activation, and mitochondrial functions were examined via Western blotting, a flow cytometric analysis, an enzyme activity assay, and a DNA integrity assay. In pharmacological studies, the ROS scavenger, N-acetyl cysteine (NAC), the NADPH oxidase inhibitor, dipheylene iodide (DPI), and mitogen-activated protein kinase (MAPK) inhibitors (PD98059, SP600125, and SB203580) were applied to investigate the mechanism. Results STI571 reduced the viability of melanoma cells B16F0, but not human skin fibroblasts WS1, via apoptosis induction. Besides, apoptosis induced by STI571 was inhibited by the addition of NAC and DPI, and an increase in the intracellular peroxide level by STI571 was identified in melanoma B16F0 cells. Activation of caspases 3 and 9 enzyme activities accompanied by disrupting the mitochondria membrane potential in according with stimulating JNK and p38 protein phosphorylation was identified in STI571-treated B16F0 cells. STI571-mediated a ROS-dependent apoptosis potentiated by JNK inhibitor SP600125 was first identified in melanoma B16F0 cells. Conclusion Our results support the idea that ROS-dependent apoptosis in STI571-treated melanoma cells B16F0. The combination of a JNK inhibitor with STI571 for treating melanomas is suggested for further in vivo studies.

Tumor invasion and metastasis are the main cause of cancer mortality. CSE1L/CAS, the cellular apoptosis susceptibility protein, is the human homologue of the yeast chromosome segregation gene product, CSE1. Pathological studies show that CSE1L is highly expressed in various cancers, such as lung cancer, breast cancer, liver cancer, ovarian cancer, endometrial carcinomas, skin cancer, colorectal cancer, lymphomas, prostate cancers, nasopharyngeal carcinomas, medulloblastomas, and glioblastomas. The CSE1L gene is located on chromosome 20q13, a region that frequently harbors amplifications that correlate with cancer aggression. Experimental studies have shown that CSE1L regulates the invasion of cancer cells in vitro and in animal metastasis models. The results of our recent studies have revealed that CSE1L is a secretory protein present in sera from cancer patients. Significantly, there is a higher prevalence of secretory CSE1L in sera of patients with metastatic cancer. Here, we discuss the potential of CSE1L as a serum marker for the diagnosis of cancer.

Colorectal glands are important functional organs in colorectal tissue and are also the origin of colorectal carcinomas. Epithelial cell polarization of colorectal glands is related to structural integrity and physiological functions of colorectal glands as well as colorectal carcinoma formation. The cellular apoptosis susceptibility (CSE1L/CAS) protein has been shown to induce polarity formation of human colorectal cells in cell culture. E-cadherin expression in epithelial cells is crucial for the establishment and maintenance of epithelial cell polarity. In this study we examined the distributions of CSE1L and E-cadherin in the epithelial glands of normal and neoplastic colorectal epithelium and correlated these to polarity formation in the colorectal glands. Our results showed that CSE1L was differentially stained in the epithelial glands of neoplastic colorectal epithelium, and the staining was related to gland epithelial cell polarization and E-cadherin distribution. CSE1L was associated E-cadherin in GST pull-down experiments and immunoprecipitation assays. Basolateral staining of CSE1L and E-cadherin were seen in the polarized glands of normal and neoplastic colorectal epithelium. Absence of basolateral CSE1L staining in neoplastic epithelium glands was associated with loss of gland epithelial cell polarity, and this was parallel with E-cadherin staining. The non-polarized areas in epithelium glands showed a patchy staining for CSE1L and E-cadherin. These results indicate that examination of CSE1L and E-cadherin distribution in colorectal epithelium glands may be valuable for evaluating the malignance of colorectal disease.

In the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation, an increase in the migration/invasion of U87 glioblastoma cells was detected by a wound healing assay, transwell analysis, and spheroid formation assay by inducing matrix metalloproteinase-9 (MMP-9) enzyme activity via a gelatin zymographic analysis. A dose- and time-dependent increase in cyclooxygenase-2 (COX-2) gene expression with elevated prostaglandin E(2) (PGE(2)) production was identified in TPA- but not in 4alpha-TPA (a respective inactive compound)-treated U87 cells TPA-induced migration/invasion was significantly blocked by adding the COX-2-specific inhibitor, NS398, through a reduction in PGE(2) production. Data from the pharmacological studies using specific chemical inhibitors showed that activation of protein kinase C (PKC) and extracellular signal-regulated kinases (ERKs) was involved in TPA-induced migration/invasion, COX-2 protein expression, and MMP-9 activation. Stimulation of intracellular peroxide production by TPA was detected by a DCHF-DA assay, and the addition of superoxide dismutase (SOD) or tempol significantly inhibited TPA-induced migration/invasion and COX-2 protein expression accompanied by a decrease in peroxide production. An increase in NADPH oxidase activity by TPA was examined, and TPA-induced migration/invasion was blocked by adding DPI, an NADPH oxidase inhibitor. Additionally, the natural flavonoids quercetin (QE), baicalein (BE), and myricetin (ME) effectively blocked TPA-induced migration/invasion while simultaneously inhibiting COX-2/PGE(2) production, MMP-9 enzyme activity, and peroxide production in U87 cells. The contribution of ROS production to the migration/invasion of U87 glioblastoma cells via ERK-activated COX-2/PGE(2) and MMP-9 induction was first investigated here, and agents such as QE, BE, and ME with the ability to block these events possess the potential to be developed for use against migration/invasion by glioblastomas.

In the present study, the roles of telomerase and prostaglandin E(2) (PGE(2)) in platelet-derived growth factor (PDGF's) and fibroblast growth factor-2 (FGF-2's) effects against C(2)-ceramide-induced cell death were investigated. C(2)-ceramide reduced the viability of NIH3T3 cells in a condition without calf serum (CS) in accordance with decreasing telomerase activity according to the TRAP assay. The addition of CS significantly protected cells from C(2)-ceramide-induced apoptosis through increased telomerase activity, and the phosphorylations of PDGF and the FGF-2-like receptor in NIH3T3 cells were detected. Adding PDGF and FGF-2 decreased the cytotoxic effect elicited by C(2)-ceramide through stimulating telomerase activity, which was blocked by adding a telomerase inhibitor (TI). Activations of ERKs and JNKs were detected in PDGF- and FGF-2-treated NIH3T3 cells, and the telomerase activities induced by PDGF and FGF were respectively inhibited by the addition of the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125. Accordingly, induction of cyclooxygenase-2 (COX-2) protein expression and PGE(2) production was detected in PDGF- and FGF-2-treated NIH3T3 cells, and the telomerase activities stimulated by PDGF and FGF were reduced by adding a specific COX-2 inhibitor, NS398, through a decrease in PGE(2) production. Incubation of cells with PGE(2) or the EP1 agonist, 17-PT, but not the EP2 agonist, sulprostone, the EP3 agonist, butaprost, or the EP4 agonist, PGE(1) alcohol, significantly enhanced the telomerase activity of NIH3T3 cells. PGE(2) protection of NIH3T3 cells against C(2)-ceramide-induced cell death was identified by the MTT and LDH-release assays, and it was inhibited by adding the EP1 antagonist, SC-19220. Ceramide metabolites including ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P), and a standard control of exogenous ceramide C(2)-dihydroceramide show no effect on the telomerase activity and viability of NIH3T3 cells. The involvement of COX-2/PGE(2)-mediated telomerase activation by PDGF and FGF-2 against C(2)-ceramide-induced cell death is first demonstrated herein.

Quercetin (QUE; 3,5,7,3',4'-tetrahydroxyflavone) has been shown to possess several beneficial biological activities including antitumor, anti-inflammation and antioxidant properties; however, the effects of QUE in preventing invasion by breast carcinoma cells are still undefined. Increases in the protein, messenger RNA and enzyme activity levels of matrix metalloproteinase (MMP)-9 were observed in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 cells, and these were blocked by QUE, but not by quercitrin or rutin. A translocation of protein kinase C (PKC)delta from the cytosol to the membrane followed by activation of extracellular signal-regulated kinase (ERK) and c-Jun/activator protein-1 (AP-1) by TPA was demonstrated, and TPA-induced MMP-9 activation and migration were inhibited by the pan PKC inhibitor, GF109203X, the specific PKCdelta inhibitor, rottlerin, an ERK inhibitor (PD98059) and an AP-1 inhibitor (curcumin). Application of QUE significantly suppressed TPA-induced activation of the PKCdelta/ERK/AP-1-signaling cascade. To elucidate the importance of hydroxyl (OH) substitutions to QUE's inhibition of tumor migration, several structurally related flavones of QUE including 3',4'-diOH, 3',4'-diOCH(3), 3,5,7-triOH, 3,4',4'-triOH, 3,3',4'-triOCH(3), luteolin and fisetin were used. Results suggested that OH groups at both C3' and C4' play central roles in QUE's inhibition of TPA-induced MMP-9 activation and migration, and an additional OH at C3, C5 or C7 may increase the inhibitory potency of the 3',4'-diOH flavone against TPA-induced MMP-9 activity and migration. The antitumor invasion and migration effects of breast carcinoma cells induced by QUE with the structure-activity relationship analysis were identified.

We investigate the cytotoxic effect of metal protoporphyrins including ferric protoporphyrin (FePP; hemin), cobalt protoporphyrin (CoPP), and tin protoporphyrin (SnPP) in glioblastoma cells C6 and GBM8401. Data of MTT assay show that FePP and CoPP, but not SnPP, significantly reduce the viability of glioma cells C6 and GBM8401 in the absence of serum. In the condition with fetal bovine serum (FBS) or bovine serum albumin (BSA), the cytotoxic effect of FePP and CoPP was completely inhibited. Binding of FePP, CoPP, and SnPP with BSA was examined via spectrophotometer analysis, and the protective effect of serum against FePP and CoPP-induced cell death was abolished by BSA depletion. A loss in the integrity of DNA with an occurrence of apoptotic events including DNA ladders, caspase 3 and PARP protein cleavage, and chromatin-condensed cells is observed in FePP-treated or CoPP-treated C6 cells. An increase in intracellular peroxide level was examined in FePP, but not CoPP, -treated C6 cells, and N-acetyl-l-cysteine (NAC) addition significantly protected C6 cells from FePP, but not CoPP, -induced cell death with reducing FePP-stimulated reactive oxygen species (ROS) production. Activation of extracellular regulated kinases (ERKs) and c-Jun-N-terminal kinases (JNKs) with an increase in the heme oxygenase-1 (HO-1) protein was observed in FePP-treated or CoPP-treated C6 cells in the absence of FBS or BSA, and adding JNKs inhibitor SP600125 (SP), but not ERKs inhibitor PD98059 (PD), significantly attenuated FePP-induced or CoPP-induced HO-1 protein expression in accordance with reducing JNKs protein phosphorylation. However, PD98059, SP600125, or transfection of C6 cells with antisense HO-1 oligonucleotides show no effect on the cytotoxicity elicited by FePP and CoPP in C6 cells. Effect of serum and BSA on the cytotoxicity of metal protoporphyrins in glioma cells is first demonstrated in the present study, and the roles of ROS, MAPKs, and HO-1 were elucidated.

Because of their low oral bioavailabilities and short half-lives, it may be more feasible to administer narcotic analgesics via the skin. However, this delivery method is limited by the low permeability of the stratum corneum (SC). The aim of this study was to enhance the transdermal delivery of three narcotic drugs, including morphine, nalbuphine, and buprenorphine, with an erbium:yttrium-aluminum-garnet (Er:YAG) laser pretreatment. In an in vitro pig skin permeation experiment, Er:YAG laser pretreatment of the skin produced a 10~35-fold enhancement in drug permeation that was dependent on the laser fluence and the narcotic analgesic used. The permeation of morphine and nalbuphine showed higher enhancement with Er:YAG laser treatment as compared to that of buprenorphine. This may have been due to the higher lipophilicity and molecular mass of buprenorphine than the other two narcotic drugs. A photomechanical wave was generated by filtering laser radiation through a polystyrene target. The experimental results showed that a single photomechanical wave was sufficient to enhance morphine permeation by sevenfold. This enhancement was significantly lower than that produced by direct laser irradiation, indicating the predominant mechanism of SC ablation by the Er:YAG laser for transdermal drug delivery.

Among 13 different cell lines, gossypol (GOS) showed the most potent cytotoxic effect against human colorectal carcinoma cells including HT29, COLO205, COLO320HSR and COLO320DM cells according to an MTT assay. The cytotoxic effect of GOS was mediated by its induction of apoptosis as characterized by the occurrence of DNA ladders, apoptotic bodies and chromosome condensation in both COLO205 and HT29 cells. Activation of caspase 3, 6, 8 and 9, but not caspase 1, accompanied by the appearance of cleaved fragments of PARP (85 kDa), and caspase 3 (p17/p15), was identified in GOS-treated cells. Decreases in Bcl-xL and phosphorylated Bad proteins were found in GOS-treated cells. GOS induction of ROS production was detected by in vitro plasmid digestion, and an increase in the intracellular peroxide level was observed in GOS-treated COLO205 cells by the DCHF-DA assay. Antioxidants including N-acetyl-L-cysteine (NAC), catalase (CAT), tempol (TEM) and melatonin (MEL), but not allopurinol (ALL), pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited GOS-induced Reactive oxygen species (ROS) production through blocking the occurrence of apoptosis. GOS induced mitochondrial dysfunction characterized by a loss of the mitochondria membrane potential via DiOC6 staining, and the release of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) from mitochondria to the cytoplasm was observed. Removing mitochondria by ethidium bromide (EtBr) treatment significantly reduced the apoptotic effect of GOS in COLO205 cells. Furthermore, an intraperitoneal injection of GOS or gossypol acetic acid (GAA) significantly reduced the growth of colorectal carcinoma induced by a subcutaneous injection of COLO205 cells in nude mice. Results of the present study provide the first evidences demonstrating the in vitro and in vivo antitumor effects of GOS via an ROS-dependent mitochondrial apoptosis in colorectal carcinoma.

Background Although targeted therapies have improved the clinical outcomes of cancer treatment, tumors resistance to targeted drug are often detected too late and cause mortality. CSE1L is secreted from tumor and its phosphorylation is regulated by ERK1/2. ERK1/2 is located downstream of various growth factor receptors and kinases, the targets of most targeted drugs. Serum phospho-CSE1L may be a marker for monitoring the efficacy of targeted therapy. Methods We used mice tumor xenograft model to study the assay of serum phosphorylated CSE1L for early detecting the efficacy of targeted drugs. The phosphorylation status of CSE1L in vemurafenib and sorafenib treated tumor cells were assayed by immunoblotting with antibody against phosphorylated CSE1L. Results Ras activation increased phospho-CSE1L expression in B16F10 melanoma cells. Vemurafenib and sorafenib treatment did not significantly reduce the total CSE1L levels; however, they inhibited ERK1/2 and CSE1L phosphorylation in A375 melanoma cells and HT-29 colorectal cancer cells. In the melanoma xenograft model, serum phospho-CSE1L level declined 5 days after vemurafenib/sunitinib treatment and 3 days after sorafenib/lapatinib treatment in the HT-29 colon cancer xenograft model. Vemurafenib/sunitinib and sorafenib/lapatinib treatments resulted in tumor regression. Conclusions Our results indicated that serum phospho-CSE1L is useful for early detecting the efficacy of targeted therapy in initial treatment and for monitoring emerging secondary drug resistance to facilitate timely therapeutic decision making.

The Ras/ERK (extracellular signal-regulated protein kinase) and cAMP/PKA (protein kinase A) pathways are essential for the transcriptional activities of CREB (cAMP response element binding protein) and MITF (microphthalmia-associated transcription factor) in melanogenesis and the progression of melanoma. However, the interaction between Ras/ERK and cAMP/PKA pathways in the melanogenesis and progression of melanoma is not fully known. Here, we report that CSE1L (chromosome segregation 1-like protein) regulates cAMP/PKA-induced CREB and MITF expressions as well as Ras-induced ERK1/2 phosphorylation. IBMX, a cAMP/PKA activator, treatment induced CSE1L phosphorylation and augmented Ras-induced ERK1/2 phosphorylation. CSE1L knockdown by CSE1L shRNA expression vectors inhibited Ras-induced ERK1/2 phosphorylation and melanogenesis in melanoma cells. CSE1L overexpression increased phospho-CREB expression; CSE1L knockdown also inhibited Ras-induced phospho-CREB, MITF, and tyrosinase expressions, regardless of the presence of IBMX. This study identifies CSE1L links and controls the Ras/ERK and cAMP/PKA pathways in the melanogenesis of melanoma cells. Melanomas frequently develop drug resistance via paradoxical activation of Ras/Raf/MEK/ERK or alternatively activated Ras/ERK and cAMP/PKA pathways. Thus CSE1L may be a potential target for treating melanomas that harbor Ras mutations or are resistant to drugs targeting Raf/MEK/ERK. © 2015 Wiley Periodicals, Inc.

In the present study, we found that baicalein (BE), but not its glycoside baicalin (BI), induced apoptosis in human leukemia HL-60 and Jurkat cells, but not in primary murine peritoneal macrophages (PMs) or human polymorphonuclear (PMN) cells, by the MTT assay, LDH release assay, and flow cytometric analysis. Activation of the caspase 3, but not caspase 1, enzyme via inducing protein processing was detected in BE-induced apoptosis. The ROS-scavenging activity of BE was identified by the anti-DPPH radical, DCHF-DA, and in vitro plasmid digestion assay, and none of chemical antioxidants including allpurinol (ALL), N-acetyl-cystein (NAC), and diphenylene iodonium (DPI) affected BE-induced apoptosis in HL-60 cells. This suggests that apoptosis induced by BE is independent of the production of ROS in HL-60 cells. Interestingly, the apoptotic events such as DNA ladders formation and activation of the caspase 3 cascade were significantly blocked by TPA addition in the presence of membrane translocation of PKCalpha, and TPA-induced protection was reduced by adding the PKC inhibitors, GF-109203X and staurosporin. TPA addition induces the phosphorylation of JNKs and ERKs, but not p38, protein in HL-60 cells, and incubation of HL-60 cells with JNKs inhibitor SP600125, but not ERKs inhibitor, PD98059 or the p38 inhibitor SB203580, suppressed the protective effect of TPA against BE-induced apoptotic events including DNA ladders, apoptotic bodies, caspase 3 and D4-GDI protein cleavage in according with blocking JNKs protein phosphorylation. In addition, PKC inhibitor GF-109203X treatment blocks TPA-induced ERKs and JNKs protein phosphorylation, which indicates that activation of PKC locates at upstream of MAPKs activation in TPA-treated HL-60 cells. Additionally, a loss in mitochondrial membrane potential with a reduction in Bcl-2 protein expression, the induction of Bad protein phosphorylation, and translocation of cytochrome c from mitochondria to the cytosol were observed in BE-treated HL-60 cells, and these events were prevented by the addition of TPA. GF-109203X and SP600125 suppression of TPA against cytochrome c release induced by BE was identified. This suggests that activation of PKC and JNKs participate in TPA's prevention of BE-induced apoptosis via suppressing mitochondrial dysfunction in HL-60 cells.

Summary Background Yeast extracts have been shown to perform anti-inflammatory and cytoprotective activities. However, the effects of yeast extracts on lipopolysaccharide (LPS)-induced nitric oxide (NO) production and epidermal damages are still unclear. Objective To investigate the effect of Saccharomycopsis Ferment Filtrate (SFF) on LPS-induced NO production in RAW264.7 macrophages and epidermal damages. Method RAW264.7 cells are incubated with LPS (25 ng/mL) and different concentrations of SFF. The amount of NO production is detected by Griess reaction. Additionally, the expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) are detected by Western blotting. Artificial epidermis is also used to mimic the in vivo condition to investigate the protective effects of SFF on LPS- or ultraviolet radiation (UVR)-induced damages by histology and electron microscopy. Results The results show that SFF addition inhibits LPS-induced NO production and iNOS protein expression in a concentration-dependent manner without notable cytotoxicity in RAW264.7 cells, and induction of HO-1 protein expression by SFF was observed. Interestingly, both HO-1 inducers, hemin and CoCl 2 , significantly attenuated LPS-induced NO production and iNOS protein expression. The addition of CoCl 2 potentiated the inhibitory effect of SFF on LPS-induced NO production. It seems that HO-1 protein participates in SFF inhibition of LPS-induced NO production. Furthermore, SFF exhibits significant protective effect on LPS- or UVR-induced damages in the artificial epidermis via histological and electron microscopic observations. Conclusion This study provided the first evidence to indicate the beneficial effects of SFF in preventing NO production in macrophages and damages in epidermis, respectively. It suggests that SFF possesses potential to be further developed.

5-Aminolevulinic acid (ALA) is used as a precursor of protoporphyrin IX (PpIX) for photodynamic therapy (PDT) of superficial skin cancers and subcutaneous metastases of internal malignancies. The permeability of ALA across intact skin is always low, making it difficult to achieve the desired therapeutic benefits. Hence new methods for enhancing ALA permeation are urgently needed. The aim of this study was to determine the in vivo kinetics of PpIX generation in mouse tissues after topical ALA application enhanced by an erbium (Er):yttrium-aluminum-garnet (YAG) laser. The in vitro permeation of ALA was also used to screen the optimal method for the in vivo study. The efficacy of the improved drug delivery was determined as a function of various laser fluences and cancer models. ALA applied to laser-treated skin produced a higher accumulations of PpIX within superficial skin and subcutaneous tumors as compared to those of the non-treated group (t-test, p < 0.05). The enhancement ratios (ER) of laser-treated skin ranged from 1.7 to 4.9 times as compared to the control depending to the fluences used. The enhanced PpIX level of laser-treated skin was generally more pronounced in normal and lesional skin than in subcutaneous nodular tumors. Confocal laser scanning microscopy (CLSM) of laser-treated skin revealed intense red fluorescence within the epidermis and upper dermis, and a much-weaker fluorescence within the bottom layers of the skin. On the other hand, the fluorescence intensity of the control group was much lower than that of laser-treated group. The barrier properties of the skin irradiated by the laser had completely recovered within 3 days. Pretreatment of skin using an Er:YAG laser was useful in increasing the amount of Pp IX within skin tumors.

In the present study, we examine the protective mechanism of quercetin (QE) on oxidative stress-induced cytotoxic effect in RAW264.7 macrophages. Results of Western blotting show that QE but not its glycoside rutin (RUT) and quicitrin-induced HO-1 protein expression in a time- and dose-dependent manner, and HO-1 protein induced by QE was blocked by an addition of cycloheximide or actinomycin D. Induction of HO-1 gene expression by QE was accompanied by inducing ERKs, but not JNKs or p38, proteins phosphorylation. Addition of PD98059, but not SB203580 or SP600125, significantly attenuates QE-induced HO-1 protein and mRNA expression associated with blocking the expression of phosphorylated ERKs proteins. H(2)O(2) addition reduces the viability of cells by MTT assay, and appearance of DNA ladders, hypodiploid cells, and an increase in intracellular peroxide level was detected. Addition of QE, but not QI or RUT, significantly reduced the cytotoxic effect induced by H(2)O(2) associated with blocking the production of intracellular peroxide, DNA ladders, and hypodiploid cells. QE protection of cells from H(2)O(2)-induced apoptosis was significantly suppressed by adding HO inhibitor SnPP or ERKs inhibitor PD98059. Additionally, QE protects cells from H(2)O(2)-induced a decrease in the mitochondrial membrane potential and a release of cytochrome c from mitochondria to cytosol by DiOC6 and Western blotting assay, respectively. Activation of apoptotic proteins including the caspase 3, caspase 9, PARP, D4-GDI proteins was identified in H(2)O(2)-treated cells by Western blotting and enzyme activity assay, and that was significantly blocked by an addition of QE, but not RUT and QI. Furthermore, HO-1 catalytic metabolites carbon monoxide (CO), but not Fe(2+), Fe(3+), biliverdin or bilirubin, performed protective effect on cells from H(2)O(2)-induced cell death with an increase in HO-1 protein expression and ERKs protein phosphorylation. These data suggest that induction of HO-1 protein may participate in the protective mechanism of QE on oxidative stress (H(2)O(2))-induced apoptosis, and reduction of intracellular ROS production and mitochondria dysfunction with blocking apoptotic events were involved. Differential anti-apoptotic effect between QE and its glycosides RUT and QI via distinct HO-1 protein induction was also delineated.

The presence of prostaglandins (PGs) has been demonstrated in the processes of carcinogenesis and inflammation. In the present study, we found that 12-o-tetradecanoylphorbol 13-acetate (TPA) induced cyclooxygenase 2 (COX-2), but not COX-1, protein expression in HL-60 cells, and the addition of arachidonic acid (AA) in the presence or absence of TPA significantly reduced the viability of HL-60 cells, an effect that was blocked by adding the COX inhibitors, NS398 and aspirin. The AA metabolites, PGD(2) and PGJ(2), but not PGE(2) or PGF(2alpha), reduced the viability of the human HL60 and Jurkat leukemia cells according to the MTT assay and LDH release assay. Apoptotic characteristics including DNA fragmentation, apoptotic bodies, and hypodiploid cells were observed in PGD(2)- and PGJ(2)-treated leukemia cells. A dose- and time-dependent induction of caspase 3 protein procession, and PARP and D4-GDI protein cleavage with activation of caspase 3, but not caspase 1, enzyme activity was detected in HL-60 cells treated with PGD(2) or PGJ(2). Additionally, DNA ladders induced by PGD(2) and PGJ(2) were significantly inhibited by the caspase 3 peptidyl inhibitor, Ac-DEVD-FMK, but not by the caspase 1 peptidyl inhibitor, Ac-YVAD-FMK, in accordance with the blocking of caspase 3, PARP, and D4-GDI protein procession. An increase in intracellular peroxide levels by PGD(2) and PGJ(2) was identified by the DCHF-DA assay, and anti-oxidant N-acetyl cysteine (NAC), mannitol (MAN), and tiron significantly inhibited cell death induced by PGD(2) and PGJ(2) by reducing reactive oxygen species (ROS) production. The PGJ(2) metabolites, 15-deoxy-Delta(12,14)-PGJ(2) and Delta(12)-PGJ(2), exhibited effective apoptosis-inducing activity in HL-60 cells through ROS production via activation of the caspase 3 cascade. The proliferator-activated receptor-gamma (PPAR-gamma) agonists, rosiglitazone (RO), troglitazone (TR), and ciglitazone (CI), induced apoptosis in cells which was blocked by the addition of the PPAR-gamma antagonists, GW9662 and BADGE, via blocking of caspase 3 and PARP cleavage. However, neither GW9662 nor BADGE showed any protective effect on PGD(2)- and PGJ(2)-induced apoptosis. A differential apoptotic effect of PGs through ROS production, followed by activation of the caspase 3 cascade, was demonstrated.

Colorectal carcinoma is a leading cause of human mortality due to its high metastatic ability. Because the activation of matrix metalloproteinases (MMP) is a key factor in the metastatic process, agents with the ability to inhibit MMP activity have potential in the treatment of colorectal carcinoma. In the present study, among 36 flavonoids examined, myricetin was found to be the most potent inhibitor of MMP-2 enzyme activity in COLO 205 cells (IC50 = 7.82 μmol/L). Myricetin inhibition of MMP-2 enzyme activity was also found in the human colorectal carcinoma cell lines COLO 320HSR, COLO 320DM, HT 29, and COLO 205-X (IC50 = 11.18, 11.56, 13.25, and 23.51 μmol/L, respectively). In contrast, no inhibitory effect of MMP-2 protein expression or enzyme activity was observed in myricitrin (myricetin-3-rhamnoside)-treated cells. In 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated COLO 205 cells, an increase in MMP-2 protein expression and enzyme activity, as well as of protein kinase C (PKC) α protein translocation, extracellular signal-regulated kinase (ERK) 1/2 protein phosphorylation, and c-Jun protein expression was observed. ERK inhibitor (PD98059) and PKC inhibitors (GF-109203X and H-7), but not p38 inhibitor (SB203580) or c-jun-NH2-kinase inhibitor (SP600125), significantly inhibited TPA-induced MMP-2 protein expression, with reduced ERK phosphorylation and c-Jun protein expression. Addition of myricetin but not myricitrin suppressed TPA-induced MMP-2 protein expression in COLO 205 cells by blocking the TPA-induced events, including translocation of PKCα from cytosol to membrane, phosphorylation of ERK1/2 protein, and induction of c-Jun protein expression. Addition of PD98059 or GF-109203X significantly enhanced the inhibitory effect of myricetin on MMP-2 enzyme activity induced by TPA. Furthermore, myricetin, but not myricitrin, suppressed TPA-induced invasion of COLO 205 cells in an in vitro invasion assay using Engelbreth-Holm-Swarm sarcoma tumor extract Matrigel–coated Transwells. Results of the present study indicate that myricetin significantly blocked both endogenous and TPA-induced MMP-2 enzyme activity by inhibiting its protein expression and enzyme activity. The blockade involved suppression of PKC translocation, ERK phosphorylation, and c-Jun protein expression.

Previous studies demonstrated that hydroxyl groups play important roles in the antioxidative activities of flavonoids; however, the importance of structurally related hydroxylation in their apoptosis-inducing activities is still undefined. In the present study, flavanone with hydroxylation at C4′ and C6 had a significant cytotoxic effect in human leukemia HL-60 cells accompanied by the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, characteristics of apoptosis. The replacement of a hydroxyl group (OH) by a methoxyl (OCH3) group at C4′ or C6 attenuated the apoptotic effect in cells, and there was no significant cytotocity of flavanone or flavanone with OH or OCH3 in C7-treated HL-60 cells. Induction of enzyme activity of caspase-3 and -9, but not caspase-1 and -8, accompanied by release of cytocrome C from mitochondria to cytosol and the appearance of cleaved of PARP (85 kDa), D4-GDI (23 kDa), and caspase-3 (p17/p15) fragments, was identified in 4′-OH- or 6-OH- flavanone-treated HL-60 cells. Caspase-3 and -9 inhibitors Ac-DEVD-FMK and Ac-LEHD-FMK, but not caspase-1 and -8 inhibitors Ac-YVAD-FMK and Ac-LETD-FMK, attenuated 4′-OH- or 6-OH-flavanone-induced cell death. And, inhibition of capsase-9 activity by Ac-LEHD-FMK suppresses caspase-3 protein procession induced by 4′-OH- and 6-OH-flavanone, indicative of caspase-9 activation locating upstream of caspase-3. A decrease in the antiapoptotic protein Mcl-1 and increases in the pro-apoptotic proteins Bax and Bad were found in 4′-OH- or 6-OH-flavanone-treated HL-60 cells. Induction of endogenous ROS production was detected in 4′-OH- or 6-OH-flavanone-treated HL-60 cells by the DCHF-DA assay. Antioxidants such as N-acetylcysteine (NAC), catalase (CAT), superoxide dismutase (SOD), and allopurinol (ALL), but not pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited 4′-OH- or 6-OH-flavanone-induced ROS production, with blocking of the apoptosis induced by 4′-OH- or 6-OH-flavanone. The apoptosis-inducing activity of 4′-OH- or 6-OH-flavanone was also observed in another leukemia cell line (Jurkat), but was not found in mature monocytic cells (THP-1) and normal human polymorphonuclear neutrophils (PMNs). This suggests that hydroxylation at C4′ or C6 is important to the apoptosis-inducing activities of flavanone through ROS production, and that activation of the caspase-3 cascade, downstream of caspase-9 activation, is involved.

Lipopolysaccharide (LPS) from Gram-negative bacterial has been identified as an important molecule involved in the inflammatory process through inducing nitric oxide (NO) production. However, the effect of LPS in carcinogenesis is still undefined. In the present study, the biological effect of LPS was examined in 12-o-tetradecanoylphorbol 13-acetate (TPA)-treated rat glioma cells C6. Results of MTT assay showed that LPS and TPA exhibited no significant cytotoxicity in glioma C6 cells. Interestingly, transformation foci were found in LPS/TPA-treated glioma C6 cells, but not in LPS- or TPA-treated cells. The transformation foci induced by LPS/TPA were also observed in the absence of serum. It indicates that induction of transformation foci formation by LPS and TPA is independent on the serum in glioma C6 cells. Induction of iNOS gene expression and NO production was examined in LPS/TPA-treated cells, but not obvious in LPS- or TPA-treated cells. NO donor sodium nitroprusside (SNP) induces transformation in glioma C6 cells in according with elevating NO production. In addition, LPS/TPA induces metalloproteinase 9 (MMP9) activity by gelatin activity assay in gel. Wogonin and quercetin but not rutin, inhibitors of iNOS gene expression and NO production induced by LPS, showed the significant inhibition on LPS/TPA-induced transformation foci formation, accompanied by inhibiting iNOS gene expression, NO production and MMP9 activity. Results of the present study provide scientific evidences to link the inflammatory responses and carcinogenesis, and suggest that NO derived from inflammation may contribute to the progression of carcinogenesis; natural products with anti-inflammatory effects such as wogonin and quercetin possess the ability to block transformation induced by LPS/TPA.