Facile Synthesis and X-ray Attenuation Properties of Ultrasmall Platinum Nanoparticles Grafted with Three Types of Hydrophilic Polymers.

Ultrasmall platinum nanoparticles (Pt-NPs) grafted with three types of hydrophilic and biocompatible polymers, i.e., poly(acrylic acid), poly(acrylic acid-co-maleic acid), and poly(methyl vinyl ether-alt-maleic acid) were synthesized using a one-pot polyol method. Their physicochemical and X-ray attenuation properties were characterized. All polymer-coated Pt-NPs had an average particle diameter (davg) of 2.0 nm. Polymers grafted onto Pt-NP surfaces exhibited excellent colloidal stability (i.e., no precipitation after synthesis for >1.5 years) and low cellular toxicity. The X-ray attenuation power of the polymer-coated Pt-NPs in aqueous media was stronger than that of the commercial iodine contrast agent Ultravist at the same atomic concentration and considerably stronger at the same number density, confirming their potential as computed tomography contrast agents.

PF-3845, a Fatty Acid Amide Hydrolase Inhibitor, Directly Suppresses Osteoclastogenesis through ERK and NF-κB Pathways In Vitro and Alveolar Bone Loss In Vivo.

Alveolar bone loss, the major feature of periodontitis, results from the activation of osteoclasts, which can consequently cause teeth to become loose and fall out; the development of drugs capable of suppressing excessive osteoclast differentiation and function is beneficial for periodontal disease patients. Given the difficulties associated with drug discovery, drug repurposing is an efficient approach for identifying alternative uses of commercially available compounds. Here, we examined the effects of PF-3845, a selective fatty acid amide hydrolase (FAAH) inhibitor, on receptor activator of nuclear factor kappa B ligand (RANKL)-mediated osteoclastogenesis, its function, and the therapeutic potential for the treatment of alveolar bone destruction in experimental periodontitis. PF-3845 significantly suppressed osteoclast differentiation and decreased the induction of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and the expression of osteoclast-specific markers. Actin ring formation and osteoclastic bone resorption were also reduced by PF-3845, and the anti-osteoclastogenic and anti-resorptive activities were mediated by the suppression of phosphorylation of rapidly accelerated fibrosarcoma (RAF), mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase, (ERK) and nuclear factor κB (NF-κB) inhibitor (IκBα). Furthermore, the administration of PF-3845 decreased the number of osteoclasts and the amount of alveolar bone destruction caused by ligature placement in experimental periodontitis in vivo. The present study provides evidence that PF-3845 is able to suppress osteoclastogenesis and prevent alveolar bone loss, and may give new insights into its role as a treatment for osteoclast-related diseases.

Effects of Cognitive Training in Mild Cognitive Impairmentmeasured by Resting State Functional Imaging.

Mild cognitive impairment (MCI) is defined as an intermediate state of cognitive alteration between normal aging and dementia. In this study, we performed a functional network connectivity analysis using resting-state functional magnetic resonance imaging to investigate the association between changes in functional connectivity in the brain and the improvement in cognitive abilities after cognitive training. A computerized cognitive training program was used to improve the abilities of fifteen participants with MCI. The cognitive training program (Comcog), which consists of three weekly sessions totaling 90 min, was conducted with all participants over six weeks. The cognitive abilities before (pre-Comcog) and after (post-Comcog) the cognitive training process were measured using a neurocognitive function test. After the Comcog, the participants enhanced their visual and verbal memories, attention, and visuo-motor coordination. The functional connectivity between cingulo-opercular (CON) and default mode (DMN) showed significant improvements after Comcog training. Therefore, our study suggests that cognitive training may improve the cognitive abilities of participants. This improvement was associated with an increase in the functional connectivity between DMN and CON. The increase in functional connectivity after cognitive training was specifically associated with overall cognitive functions, including executive, memory, decision-making, and motivational functions.

Inhibitory effect of oolonghomobisflavan B on osteoclastogenesis by suppressing p38 MAPK activation.

Suppression of differentiation and/or function of osteoclasts is considered an effective therapeutic strategy for osteolytic bone diseases such as periodontitis and osteoporosis. Evidence regarding the health benefits of oolong tea consumption is accumulating, and tea polyphenols have various pharmacological properties such as anti-cancer and anti-diabetes effects. In this study, we investigated the effect of oolonghomobisflavan B (OFB), a polyphenolic compound in oolong tea, on osteoclast differentiation. OFB suppressed receptor activator of nuclear factor-κB (RANKL)-induced formation of tartate-resistant acid phosphatase-positive multinuclear cells without cytotoxicity. OFB also significantly attenuated p38 phosphorylation, which is essential for RANKL-induced osteoclastogenesis, and inhibited the expressions of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and osteoclast-specific target genes, including dendritic cell-specific transmembrane protein and cathepsin K. Our findings suggest that OFB exhibits an anti-osteoclastogenic activity by inhibiting RANKL-mediated p38 activation, which is useful for the prevention and treatment of osteolytic bone diseases.

Effect of Diquafosol on Hyperosmotic Stress-induced Tumor Necrosis Factor-α and Interleukin-6 Expression in Human Corneal Epithelial Cells.

PURPOSE: Diquafosol is a pharmaceutical drug used for dry eye treatment with a novel mechanism of action. It is a purinergic P2Y2 receptor agonist that promotes the secretion of tears and healing of corneal epithelial wounds. However, its inhibitory effect on hyperosmotic stress-induced inflammation in human corneal epithelial cells (HCECs) remains unclear. METHODS: A hyperosmotic stress model was established by transferring HCECs from isosmotic (312 mOsm/kg to hyperosmotic medium (500 mOsm/kg). HCECs were incubated with 500 mOsm/kg hyperosmotic medium for 30 minutes, and then treated with diquafosol (0.6-6 mg/mL) for 4 or 24 hours. Cells were then harvested and analyzed by western blot, immunocytochemistry, and real-time polymerase chain reaction to evaluate the expression of interleukin-6, tumor necrosis factor-alpha, and the phosphorylation status of nuclear factor-kappa B. RESULTS: Diquafosol significantly decreased the mRNA and protein expression of hyperosmotic stress-induced tumor necrosis factor-alpha and interleukin-6. These results were supported by immunofluorescence staining and quantitative real-time polymerase chain reaction analysis. Furthermore, diquafosol inhibits nuclear factor-kappa B activation by suppressing the phosphorylation and degradation of the inhibitor of кB. CONCLUSIONS: This study shows that diquafosol inhibits nuclear factor-kappa B signaling and inflammatory factors induced by hyperosmotic stress in HCECs. This suggests that using diquafosol for the improvement of dry eye syndrome could be effective in the treatment of inflammation-related corneal and conjunctival diseases.

Gadolinium Complex of 1,4,7,10-Tetraazacyclododecane-1,4,7-trisacetic Acid (DO3A)-Ethoxybenzyl (EOB) Conjugate as a New Macrocyclic Hepatobiliary MRI Contrast Agent.

We report the synthesis of a macrocyclic Gd chelate based on a 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid (DO3A) coordinationn cage bearing an ethoxybenzyl (EOB) moiety and discuss its use as a T1 hepatobiliary magnetic resonance imaging (MRI) contrast agent. The new macrocyclic liver agent shows high chelation stability and high r1 relaxivity compared with linear-type Gd chelates, which are the current clinically approved liver agents. Our macrocyclic, liver-specific Gd chelate was evaluated in vivo through biodistribution analysis and liver MRI, which demonstrated its high tumor detection sensitivity and suggested that the new Gd complex is a promising contrast agent for liver cancer imaging.

Manganese Complex of Ethylenediaminetetraacetic Acid (EDTA)-Benzothiazole Aniline (BTA) Conjugate as a Potential Liver-Targeting MRI Contrast Agent.

A novel manganese(II) complex based on an ethylenediaminetetraacetic acid (EDTA) coordination cage bearing a benzothiazole aniline (BTA) moiety (Mn-EDTA-BTA) was designed and synthesized for use as a liver-specific MRI contrast agent with high chelation stability. In addition to forming a hydrophilic, stable complex with Mn2+, this new Mn chelate was rapidly taken up by liver hepatocytes and excreted by the kidneys and biliary system. The kinetic inertness and R1 relaxivity of the complex were much higher than those of mangafodipir trisodium (MnDPDP), a clinically approved liver-specific MRI contrast agent. The diagnostic utility of this new Mn complex in MRI was demonstrated by high-sensitivity tumor detection in an animal model of liver cancer.

Inhibition of Pterygium Fibroblast Migration and Outgrowth by Bevacizumab and Cyclosporine A Involves Down-Regulation of Matrix Metalloproteinases-3 and -13.

We examined the connection between matrix metalloproteinase (MMP) expression/activity and pterygium fibroblast migration, and how these were affected by bevacizumab and/or cyclosporine A (CsA). Fibroblasts were obtained from 20 pterygia and 6 normal conjunctival specimens. Expression levels of MMP-3 and MMP-13 were examined after bevacizumab administration. Immunofluorescence staining was used to examine expression of both MMPs in fibroblasts migrating out from explanted pterygium tissues. Rates of cell migration from explant-cultured pterygia tissues and scratch-wounded confluent pterygium fibroblasts were examined in the presence of MMP-3 or MMP-13 inhibitors, as well as bevacizumab and/or CsA. A scratch wound healing migration assay was performed to determine the effects of bevacizumab and/or CsA. Protein expression of both MMPs in pterygium tissues and in cells migrating from organ-cultured pterygium tissues was greater than that observed in normal cells. Inhibition of the activities of both MMPs decreased their expression levels; these were also significantly reduced in bevacizumab-injected pterygium tissues. Bevacizumab significantly reduced the expression of both MMPs and cell migration. Pretreatment with CsA prior to bevacizumab exposure markedly inhibited cell migration and the expression of both MMPs. CsA enhanced the inhibitory effects of bevacizumab on pterygium fibroblast migration in vitro, possibly by inhibiting expression of both MMPs. These findings suggest that combined CsA and bevacizumab treatment may provide a potential therapeutic strategy for reducing the rate of pterygium recurrence.

The neuroprotective effect of staurosporine on mouse retinal ganglion cells after optic nerve injury.

This study aimed to investigate the neuroprotective properity of staurosporine (STS) and identify the neuroprotective mechanism of staurosporine in mouse retina ganglion cell after optic nerve injured. Mice (C57BL/6) were anaesthetised with a mixture of 5 mg/kg xylazine hydrochloride and 40 mg/kg tiletamine/zolazepam (Zoletil®). Optic nerves of the mice were crushed (Templeton JP et al., 2012). With micro-forceps, the bulbar conjunctiva was grasped and retracted, rotating the globe nasally. The exposed optic nerve was grasped approximately 1-3 mm from the globe with Dumont #N7 cross-action forceps for 10 s. One day after crushing, intravitreal injections of STS (500 nM) were administered using a Narishige IM-300 air pressure regulator. For analysing the change in ganglion cell number, the mice were allowed to live for 30 days, after which they were killed and the ganglion cell survival was assessed. A significant and marked loss of fluorescent spots was found after 30 days, with fewer 4',6-diamidino-2-phenylindole (DAPI)-expressing retinal ganglion cells (RGCs) remaining in the injured and phosphate buffered saline (PBS)-injected group than those in non-injured PBS-injected controls. However, RGC cell numbers dramatically increased in the STS intravitreal injection group. Moreover, degradation of nerve fibre (NF) was markedly reduced in the STS injection group compared with the injured and PBS-injected group by inducing astrocyte expression of Bcl-2. Our data suggested that injection of STS into the vitreous may have a potential therapeutic effect in retinal diseases such as glaucoma.

Cyclosporine A inhibits TGF-ß2-induced myofibroblasts of primary cultured human pterygium fibroblasts.

Cyclosporine A (CsA), an immunomodulatory drug, and is increasingly used to treat moderate dry eye syndrome and ocular surface inflammation. However, any inhibitory effect on differentiation of fibroblasts to myofibroblasts remains unclear. Here, we show that the inhibitory effect of CsA on transforming growth factor-beta2 (TGF-ß2)-induced myofibroblasts in primary cultured human pterygium fibroblasts. CsA significantly decreased mRNA and protein expression of myofibroblast-related markers including α-SMA, laminin, and fibronectin. These findings were supported by the results from immunofluorescence staining. Taken together, these results indicate the therapeutic potential of CsA against pterygium progression. Further studies are necessary to elucidate the precise intracellular signal mechanism responsible for CsA-induced downregulation of myofibroblast markers in pterygium fibroblasts.

Gadolinium Nanoparticles Conjugated with Therapeutic Bifunctional Chelate as a Potential T1 Theranostic Magnetic Resonance Imaging Agent.

This work is directed toward the synthesis of two types of gadolinium oxide nanoparticles (Gd-oxide NPs), abbreviated as Gd@SiO2-DO3A and Gd@SiO2-DO2A-BTA, with diameters of 50-60 nm. The synthesis involves sequential coating of Gd-oxide NPs with tetraethyl orthosilicate (TEOS) and (3-aminopropyl) triethoxysilane (APTES), followed by functionalization of the aminopropylsilane group with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid conjugates of benzothiazoles (DO3A-BTA). Gd@SiO2-DO3A and Gd@SiO2-DO2A-BTA exhibit high water solubility and colloidal stability. The r1 relaxivities of both Gd@SiO2-DO3A and Gd@SiO2-DO2A-BTA are higher than those of the corresponding low-molecular-weight magnetic resonance imaging contrast agents (MRI CAs), and their r2/r1 ratios are close to 1, indicating that both can be used as potential T1 MRI CAs. Biodistribution studies demonstrated that Gd@SiO2-DO2A-BTA was excreted via both hepatobiliary and renal pathways. Gd@SiO2-DO2A-BTA exhibits a strong intracellular uptake property in a series of tumor cell lines, and has significant anticancer characteristics against cell lines such as SK-HEP-1, MDA-MB-231, HeLa, and Hep-3B.

Comparison of the Efficacy of Fluorometholone With and Without Benzalkonium Chloride in Ocular Surface Disease.

PURPOSE: The purpose of this study was to compare the cytotoxicity and antiinflammatory effect of preserved and unpreserved 0.1% fluorometholone (FML). METHODS: Drug-induced morphological changes and cytotoxicity were examined in human corneal epithelial cells. Dry eye was induced in mice by treatment with 0.2% benzalkonium chloride (BAC) for the first 2 weeks, and then, the eyes (4 groups; Normal saline, BAC, preserved FML, and unpreserved FML) were treated thrice daily with each formulation for the next 2 weeks. Corneal tissues were embedded in paraffin and stained with hematoxylin and eosin for histopathological examination. Immunofluorescence staining was performed for tumor necrosis factor-α, interleukin-6, and human leukocyte antigen-DR. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed to evaluate drug-induced cytotoxicity. RESULTS: BAC and preserved FML caused cell shrinkage and detachment from the plate in a dose-dependent manner, and cell viability decreased significantly. However, cytotoxicity was reduced on treatment with unpreserved FML. Hematoxylin-eosin staining revealed surface desquamation, irregular surface, loss of cell borders, and stromal shrinkage in the group treated with BAC. On BAC exposure, tumor necrosis factor-α, interleukin-6, and human leukocyte antigen-DR were strongly detected, and cytotoxicity was markedly increased, as evidenced by a positive result in the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Ocular surface damage and inflammation were slightly reduced on treatment with preserved FML. In comparison, unpreserved FML did not induce morphological changes; moreover, decreased cell cytotoxicity and ocular surface inflammation were observed. CONCLUSIONS: The cytotoxicity of antiinflammatory eye drops evaluated in this study was induced by the preservative BAC. Accordingly, unpreserved FML is more effective than preserved eye drops in decreasing ocular inflammation.

In Vivo Effects of Preservative-free and Preserved Prostaglandin Analogs: Mouse Ocular Surface Study.

PURPOSE: Chronic use of topical hypotensive agents induces several side effects caused by preservatives. The purpose of this study was to evaluate the effects of prostaglandin analogs with varying concentrations of benzalkonium chloride (BAC), preservative-free (PF), and alternative preservatives on mouse corneal tissue. METHODS: Thirty-five, 8- to 10-week-old female C57BL/6 mice (five mice for each group) were used for this study. To the control group, we applied normal saline, and to each drug-treated group we applied 0.02% BAC, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), travoprost 0.004% (with 0.001% polyquad) or tafluprost 0.0015% with/without 0.001% BAC, once a day (9 p.m.) for 4 weeks. Corneal fluorescein staining was evaluated in all groups. After harvest, the corneal tissues were embedded in paraffin and then Hematoxylin-Eosin stain was performed for histopathological examination. Immunofluorescence staining was done against TNF-α, IL-6, HLA DR, pJNK, and pAkt. RESULTS: In corneal fluorescein staining, severe punctate epithelial keratitis was seen in the groups of 0.02% BAC, 0.02% BAC containing bimatoprost 0.01% and latanoprost 0.005%. The surface desquamation, irregular surface, loss of cell borders, anisocytosis and stromal shrinkage were observed in the groups of BAC-containing eye drops. Moreover, the groups treated with BAC-containing eye drops have high inflammatory markers, significantly decreased cell viability-related signal, pAkt, and higher apoptosis-inducing signal, pJNK, than the control group. On the other hand, travoprost 0.004% and PF tafluprost 0.0015% have less cellular morphologic changes, lower inflammation, and higher cellular viability than BAC-containing formulations. CONCLUSIONS: Corneal damage, increased inflammation and apoptosis and low cell viability were observed in BAC-containing groups. PF or alternatively preserved glaucoma medications seem to be a reasonable and viable alternative to those preserved with BAC.

Cyclosporine A Downregulates MMP-3 and MMP-13 Expression in Cultured Pterygium Fibroblasts.

PURPOSE: To investigate the regulation of matrix metalloproteinase (MMP)-3 and MMP-13 expression over time and in the presence of cyclosporine A (CsA) in primary cultured human pterygium fibroblasts. We also examined the effects of CsA on cultured human pterygium fibroblasts. METHODS: Primary cultured human pterygium fibroblasts subjected to scratch assays were exposed to 1 and 100 µg/mL of CsA for 3 or 10 minutes. Cells were washed with Dulbecco phosphate-buffered saline, and then incubated with serum-depleted Dulbecco modified Eagle medium/F-12 medium for 48 hours. Expression levels of MMP-3 and MMP-13 proteins and the corresponding mRNA transcripts were determined by western blotting and reverse transcription polymerase chain reaction assays, respectively. RESULTS: Migration of cultured pterygium fibroblast cells was suppressed by pretreatment with CsA compared with controls in a time-dependent and dose-dependent manner (3 minutes, 50.6% ± 1.1 in 1 µg/mL, 60.0% ± 1.2 in 100 µg/mL; 10 minutes, 59.8% ± 5.7 in 1 µg/mL, 60.5 ± 2.4 in 100 µg/mL, respectively, P < 0.01). Pretreatment with CsA also reduced the mRNA (P < 0.05) and protein expression levels (P < 0.05). CONCLUSIONS: CsA was actively involved in the migration of pterygium fibroblasts. Cell migration is inhibited in response to CsA through the inhibition of MMP-3 and MMP-13 expression. These findings reveal the therapeutic potential of CsA on pterygium progression. Further studies will be necessary to elucidate the precise intracellular signal mechanism responsible for CsA-induced downregulation of MMPs in pterygium fibroblasts.

Gadolinium complex of DO3A-benzothiazole aniline (BTA) conjugate as a theranostic agent.

A gadolinium complex of 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid (DO3A) and benzothiazole-aniline (BTA) of the type [Gd(DO3A-BTA)(H2O)] has been prepared for use as a single molecule theranostic agent. The kinetic inertness and r1 relaxivity (= 3.84 mM(-1) s(-1)) of the complex compare well with those of structurally analogous Gd-DOTA. The same complex is not only tumor-specific but also intracellular, enhancing MR images of cytosols and nuclei of tumor cells such as MCF-7, MDA-MB-231, and SK-HEP-1. Both DO3A-BTA and Gd(DO3A-BTA) reveal antiproliferative activities as demonstrated by GI50 and TGI values obtainable from the cell counting kit-8 (CCK-8) assays performed on these cell lines. Ex vivo and in vivo monitoring of tumor sizes provide parallel and supportive observations for such antiproliferative activities.

Expression of matrix metalloproteinase-13 (MMP-13) in the testes of growing and adult chicken.

Although several matrix metalloproteinases (MMPs) have been implicated in testis development, the presence of MMP-13 protein has not been directly substantiated in the male avian gonads. In this study, we examined the expression patterns of MMP-13 and MMP inhibitors, TIMP-1 and TIMP-2, in immature (4weeks), pre-pubertal (16weeks), and mature (1year) chicken testes. Using RT-PCR analysis, we observed that MMP-13 mRNA was expressed in immature testis. In Western blot analysis, the expression level of MMP-13 protein peaked in the immature testes during marked tissue remodeling, whereas it gradually decreased during testis maturation. High expression levels of TIMP-1 (34-kDa) and TIMP-2 (55-kDa) were detected only in immature and pre-pubertal testes and not in adult testis. Four different forms of TIMP-2 protein were differentially detected in the testes of growing and adult chicken. Using immunohistochemistry we localized both secreted and intracellular forms of MMP-13, TIMP-1, and TIMP-2 proteins. These proteins were temporally and spatially distributed in growing and adult testes, and all their expression levels were similar to the expression profile of Western blot results. These findings suggest that age-related changes of MMP-13 with balance of TIMPs act in concert to effect the controlled testicular remodeling and maturation.

ADAM10 mediates N-cadherin ectodomain shedding during retinal ganglion cell differentiation in primary cultured retinal cells from the developing chick retina.

Here, we examined the role of ADAM10 during retinal cell differentiation in retinal sections and in vitro cultures of developing chick retinal cells from embryonic day 6 (ED6). Immunohistochemistry showed that ADAM10 is abundantly expressed in the inner zone of neuroblastic layer at ED5, and it becomes more highly expressed in the ganglion cell layer at ED7 and ED9. Western blotting confirmed that ADAM10 was expressed as an inactive pro-form that was processed to a shorter, active form in control cultured cells, but in cultures treated with an ADAM10 inhibitor (GI254023X) and ADAM10-specific siRNA, the level of mature ADAM10 decreased. Phase-contrast microscopy showed that long neurite extensions were present in untreated cultures 24 h after plating, whereas cultures treated with GI254023X showed significant decreases in neurite extension. Immunofluorescence staining revealed that there were far fewer differentiated ganglion cells in ADAM10 siRNA and GI254023X-treated cultures compared to controls, whereas the photoreceptor cells were unaltered. The Pax6 protein was more strongly detected in the differentiated ganglion cells of control cultures compared to ADAM10 siRNA and GI254023X-treated cultures. N-cadherin ectodomain shedding was apparent in control cultures after 24 h, when ganglion cell differentiation was observed, but ADAM10 siRNA and GI254023X treatment inhibited these processes. In contrast, N-cadherin staining was strongly detected in photoreceptor cells regardless of ADAM10 siRNA and GI254023X treatment. Taken together, these data indicate that the inhibition of ADAM10 can inhibit Pax6 expression and N-cadherin ectodomain shedding in retinal cells, possibly affecting neurite outgrowth and ganglion cell differentiation.

Sulforaphane inhibits proliferation by causing cell cycle arrest at the G2/M phase in rabbit articular chondrocytes.

Sulforaphane (SFN), a natural compound extracted from cruciferous vegetables, exhibits potent anticancer activity in various types of tumor cells. However, the effect of SFN on the proliferation of chondrocytes is not well understood. In the present study, we addressed the mechanism of action by which SFN suppresses proliferation. We demonstrate that SFN causes an irreversible arrest in cell proliferation, as determined by trypan blue dye exclusion assay and flow cytometric analysis. SFN induced cell cycle arrest at the G2/M phase by downregulation of cyclin B, cdc2 and cdc25c and upregulation of p21WAF1/CIP1 and p53 in a dose- and time-dependent manner, as determined by western blot analysis. Our data suggest that SFN regulates cell cycle arrest at the G2/M phase in rabbit articular chondrocytes.

Suppression of tunicamycin-induced CD44v6 ectodomain shedding and apoptosis is correlated with temporal expression patterns of active ADAM10, MMP-9 and MMP-13 proteins in Caki-2 renal carcinoma cells.

CD44v6 has been shown to coordinate the activation of anti-apoptotic molecules as well as resistance to apoptosis. Here, we investigated CD44v6 ectodomain shedding in Caki-2 human renal carcinoma cells as well as its underlying mechanisms. Exposure of cells to tunicamycin (TM)-induced apoptosis was accompanied by cleavage of caspase-3, PARP-1 and CD44v6 ectodomain. TM-induced apoptosis was also closely associated with endoplasmic reticulum (ER) stress, as shown by increased expression of GRP-78 and CHOP proteins. Furthermore, induction of matrix metallo-proteinase (MMP)-13, MMP-9 and ADAM10 expression was highly stimulated by tunicamycin in a time- and dose-dependent manner. TM-induced PARP-1 cleavage was significantly inhibited by treatment with GM6001 (a broad spectrum MMP inhibitor), MMP-9/-13 inhibitor and GI254023X (specific ADAM10 inhibitor). In addition, inhibition of all examined MMPs resulted in reversal of TM-induced apoptosis as well as increased cell viability. When considering the functional implications of MMP-9 and ADAM10, it is likely that active MMP-9 and ADAM10 help regulate the cellular levels of CD44v6 through cleavage of CD44v6 ectodomain during TM-induced apoptosis of Caki-2 cells. Collectively, these findings suggest that multiple TM-induced MMPs may cooperate to induce apoptosis.

Protein kinase Cα protects against multidrug resistance in human colon cancer cells.

Multidrug resistance is the phenomenon by which, after exposure to a single chemotherapeutic agent, cancer cells evade the agent's cytotoxic effects as well as become resistant to several classes of diverse drugs. ATP-binding cassette (ABC) transporters are a family of transporter proteins that contribute to drug resistance via a n ATP - dependent drug efflux pump. P-glycoprotein (P-gp) is a prominent ABC superfamily protein encoded by the mdr gene which has the ability to mediate the cellular extrusion of xenobiotics and anticancer drugs from tumor cells. Exclusively expressed P-gp cells from the human colon cancer HCT15/DOX line showed resistance to doxorubicin while parental HCT15 cells treated with doxorubicin displayed typical signs of apoptosis. In order to verify the hypothesis that expression of MDR is controlled in part, by protein kinase C (PKC), expression patterns of different PKC isoforms were examined in both cell lines. Of the PKC isoforms evaluated, the membrane translocation and expression levels of PKCα were strikingly increased in HCT15/DOX cells. PKCα reversed doxorubicin-induced apoptosis through the scavenging of ROS as well as inhibition of PARP cleavage. In addition, inhibition of PKCα with Go6976, a specific inhibitor of classical PKC, led to reduced MDR expression and increased doxorubicin-induced apoptosis. Knockdown of PKCα by siRNA diminished the protective effects of PKCα for doxorubicin-induced apoptosis. These results suggested that over-expression and activity of PKCα is closely associated with the regulation of the MDR phenotype in human colon cancer HCT15 cells and provided insight into a new strategy for inhibiting doxorubicin resistance in human cancers.