Oligomannurarate sulfate inhibits CXCL12/SDF-1-mediated proliferation and invasion of human tumor cells in vitro

Aim:

JG6 is a novel marine-derived oligosaccharide that has shown to inhibit angiogenesis and tumor metastasis. In this study, we sought to identify the potential target responsible for the anti-cancer activity of JG6.

Methods:

Human liver cancer cell line Bel-7402 and human cervical cancer cell line HeLa were examined. CXCL12-stimulated cell proliferation and migration were determined using a CCK-8 kit and a transwell assay, respectively. Western blotting was performed to examine the changes in CXCL12/CXCR4 axis. Molecular docking and surface plasmon resonance (SPR) were performed to characterize the possible interaction between JG6 and the CXCL12/CXCR4 axis.

Results:

Treatment with CXCL12 potently stimulated the proliferation and migration in both Bel-7402 and HeLa cells. Co-treatment of the cells with JG6 (10, 50 and 100 μg/mL) dose-dependently impeded the CXCL12-stimulated cell proliferation and migration. Furthermore, CXCL12 rapidly induced phosphorylation of AKT, ERK, FAK and Paxillin in Bel-7402 and HeLa cells, whereas pretreatment with JG6 dose-dependently inhibited the CXCL12-induced phosphorylation of these proteins. The SPR assay showed that JG6 bound to CXCL12 with a high affinity. In molecular docking study, JG6 appeared to interact with CXCL12 via multiple polar interactions, including 6 ionic bonds and 7 hydrogen bonds.

Conclusion:

Inhibition of the CXCL12/CXCR4 axis by JG6 may account for its anticancer activity.     

Establishment of platform for screening insulin-like growth factor-1 receptor inhibitors and evaluation of novel inhibitors

Aim:

The insulin-like growth factor-1 receptor (IGF1R) is over-expressed in a wide variety of tumors and contributes to tumor cell proliferation, metastasis and drug resistance. The aim of this study was to establish a sensitive screening platform to identify novel IGF1R inhibitors.

Methods:

The catalytic domain of IGF1R was expressed using the Bac-to-Bac baculovirus expression system. The screening platform for IGF1R inhibitors was established based on ELISA. The binding profile of IGF1R with the inhibitors was predicted with molecular docking and then subjected to the surface plasmon resonance (SPR) approach. The growth inhibition of cancer cells by the inhibitors was assessed with MTT assay. Apoptosis was analyzed using flow cytometry and Western blotting.

Results:

A naturally occurring small molecule compound hematoxylin was identified as the most potent inhibitor (IC₅₀ value=1.8±0.1 μmol/L) within a library of more than 200 compounds tested. Molecular simulation predicted the possible binding mode of hematoxylin with IGF1R. An SPR assay further confirmed that hematoxylin bound directly to IGF1R with high binding affinity (Kd=4.2×10^-6 mol/L). In HL-60 cancer cells, hematoxylin inactivated the phosphorylation of IGF1R and downstream signaling and therefore suppressed cell proliferation. Mechanistic studies revealed that hematoxylin induced apoptosis in HL-60 cells via both extrinsic and intrinsic pathways.

Conclusion:

A simple, sensitive ELISA-based screening platform for identifying IGF1R inhibitors was established. Hematoxylin was identified as a promising IGF1R inhibitor with effective antitumor activity that deserves further investigation.     

Adenovirus-mediated expression of UHRF1 reduces the radiosensitivity of cervical cancer HeLa cells to γ-irradiation

Aim:

An in vitro study was carried out to determine the effect of UHRF1 overexpression on radiosensitivity in human cervical cancer HeLa cells using adenovirus-mediated UHRF1 gene transfer (Ad5-UHRF1).

Methods:

Cell survival was evaluated using the clonogenic survival assay and the MTT assay; apoptosis and cell cycle distribution were monitored by flow cytometry. Protein levels were measured by Western blotting. Silencing XRCC4 expression was performed by transfection of small interfering RNA (siRNA).

Results:

Increased expression of UHRF1 by Ad5-UHRF1 significantly reduced the radiosensitivity of HeLa cells. The UHRF1-mediated radioresistance was correlated with increased DNA repair capability and increased expression of the DNA damage repair protein, XRCC4. Knocking down XRCC4 expression in the cells using XRCC4 siRNA markedly reduced the UHRF1-mediated radioresistance.

Conclusion:

These results provide the first evidence for revealing a functional role of UHRF1 in human cervical cancer cells as a negative regulator of radiosensitivity.     

Mono-galloyl glucose derivatives are potent poly(ADP-ribose) glycohydrolase (PARG) inhibitors and partially reduce PARP-1-dependent cell death

Background and purpose:

Maintenance of poly(ADP-ribose) (PAR) polymers at homoeostatic levels by PAR glycohydrolase (PARG) is central in cell functioning and survival. Yet the pharmacological relevance of PARG inhibitors is still debated. Gallotannin, a complex mixture of hydrolysable tannins from oak gall, inhibits PARG but which of its constituents is responsible for the inhibition and whether the pharmacodynamic properties are due to its antioxidant properties, has not yet been established.

Experimental approach:

A structure–activity relationship study was conducted on different natural and synthetic tannins/galloyl derivatives as potential PARG inhibitors, using a novel in vitro enzymic assay. Cytotoxicity was assayed in cultured HeLa cells.

Key results:

Mono-galloyl glucose compounds were potent inhibitors of PARG, with activities similar to that of ADP-(hydroxymethyl) pyrrolidinediol, the most potent PARG inhibitor yet identified. When tested on HeLa cells exposed to the PAR polymerase (PARP)-1-activating compound 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), 3-galloyl glucose weakly inhibited PAR degradation. Conversely, the more lipophilic, 3-galloyl-1,2-O-isopropylidene glucose, despite being inactive on the pure enzyme, efficiently prolonged the half-life of the polymers in intact HeLa cells. Also, PARG inhibitors, but not radical scavengers, reduced, in part, cell death caused by MNNG.

Conclusions and implications:

Taken together, our findings identify mono-galloyl glucose derivatives as potent PARG inhibitors, and emphasize the active function of this enzyme in cell death.     

Bcl-2-dependent upregulation of autophagy by sequestosome 1/p62 in vitro

Aim:

To investigate whether sequestosome 1/p62 (p62), a key cargo adaptor protein involved in both the ubiquitin-proteasome system and the autophagy-lysosome system, could directly regulate autophagy in vitro.

Methods:

HEK 293 cells or HeLa cells were transfected with p62-expressing plasmids or siRNA targeting p62. The cells or the cell lysates were subsequently subjected to immunofluorescence assay, immunoprecipitation assay, or immunoblot analysis. In vitro pulldown assay was used to study the interaction of p62 with Bcl-2.

Results:

Overexpression of p62 significantly increased the basal level of autophagy in both HEK 293 cells and HeLa cells, whereas knockdown of p62 significantly decreased the basal level of autophagy. In vitro pulldown assay showed that p62 directly interacted with Bcl-2. It was observed in HeLa cells that p62 co-localized with Bcl-2. Furthermore, knockdown of p62 in HEK 293 cells significantly increased the amount of Beclin 1 that co-immunoprecipitated with Bcl-2.

Conclusion:

p62 induces autophagy by disrupting the association between Bcl-2 and Beclin 1.     

5-Lipoxygenase inhibitors induce potent anti-proliferative and cytotoxic effects in human tumour cells independently of suppression of 5-lipoxygenase activity

BACKGROUND AND PURPOSE

Certain 5-lipoxygenase (5-LO) inhibitors exhibit anti-carcinogenic activities against 5-LO overexpressing tumour types and cultured tumour cells. It has been proposed therefore that 5-LO products significantly contribute to tumour cell proliferation. To date, the relationship between the inhibitory mechanisms of 5-LO inhibitors, which vary widely, and tumour cell viability has not been evaluated. This study addresses the anti-proliferative and cytotoxic potency of a number of 5-LO inhibitors with different inhibitory mechanisms in 5-LO-positive and 5-LO-negative tumour cells.

EXPERIMENTAL APPROACH

Cell viability was measured by the WST-1 assay; cell proliferation was assessed using the bromodeoxyuridine (BrdU) incorporation assay. Cell death was analysed by annexin V staining, Western blot analysis of PARP (poly ADP-ribose polymerase) cleavage and a cytotoxicity assay. 5-LO product formation was quantified by a 5-LO activity assay.

KEY RESULTS

The common 5-LO inhibitors AA-861, Rev-5901 and MK-886 induced cytotoxic and anti-proliferative effects in 5-LO-positive Capan-2 pancreatic cancer cells; BWA4C and CJ-13,610 only caused anti-proliferative effects, while zileuton failed to impair cell viability. Moreover, the concentrations of the 5-LO inhibitors required to induce anti-proliferation and cytotoxicity highly exceeded those for suppression of 5-LO. Supplementation with mitogenic 5-LO products failed to protect Capan-2 cells from the effects of 5-LO inhibitors. Finally, the cytotoxic and anti-proliferative 5-LO inhibitors also potently reduced the viability of 5-LO-deficient tumour cell lines (HeLa, Panc-1 and U937).

CONCLUSIONS AND IMPLICATIONS

Certain 5-LO inhibitors cause cytotoxic and anti-proliferative effects independently of suppression of 5-LO activity. Thus, the role of 5-LO overexpression in tumour cell viability remains unclear and requires further elucidation.     

Essential role of autophagy in fucoxanthin-induced cytotoxicity to human epithelial cervical cancer HeLa cells

Aim： To investigate the effects and the molecular mechanisms of fucoxanthin, a major carotenoid found in edible seaweed, on HeLa cells. Methods： The cytotoxicity of fucoxanthin was evaluated using MTT assay. Cell cycle and apoptosis were evaluated using flow cytometric analysis. Autophagy was detected with acridine orange staining and transient transfection of the GFP-LC3 plasmid into the cells. Protein expression was detected with Western blotting. Results： Treatment of HeLa cells with fucoxanthin （10-80 μmol/L） for 48 h caused dose-dependent cytotoxicity with an IC50 value of 55.1±7.6 μmol/L. Fucoxanthin （10, 20, and 40 pmol/L） dose-dependently induced Go/G1 arrest, but did not change the apoptosis of HeLa cells. The same concentrations of fucoxanthin dose-dependently increased the protein expression of LC3 II （the autophagosome marker） and Beclin 1 （the initiation factor for autophagosome formation） in HeLa cells. Moreover, fucoxanthin dose-dependently decreased the levels of phosphorylated Akt and its downstream proteins p53, p7OS6K, and mTOR, and increases the expression of PTEN in HeLa cells. Pretreatment of HeLa cells with 3-methyladenine （5 mmol/L） blocked the cytotoxic effect of fucoxanthin as well as fucoxanthin-induced autophagy. Conclusion： Fucoxanthin exerts autophagy-dependent cytotoxic effect in HeLa cells via inhibition of Akt/mTOR signaling pathway.     

Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells

Aim:

Parkin has been shown to exert protective effects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in different models of Parkinson disease. In the present study we investigated the molecular mechanisms underlying the neuroprotective action of parkin in vitro.

Methods:

HEK293, HeLa and PC12 cells were transfected with parkin, parkin mutants, p62 or si-p62. Protein expression and ubiquitination were assessed using immunoblot analysis. Immunoprecipitation assay was performed to identify the interaction between parkin and scaffold protein p62. PC12 and SH-SY5Y cells were treated with 6-OHDA (200 μmol/L), and cell apoptosis was detected using PI and Hoechst staining.

Results:

In HEK293 cells co-transfected with parkin and p62, parkin was co-immunoprecipitated with p62, and parkin overexpression increased p62 protein levels. In parkin-deficient HeLa cells, transfection with wild-type pakin, but not with ligase activity-deficient pakin mutants, significantly increased p62 levels, suggesting that parkin stabilized p62 through its E3 ligase activity. Transfection with parkin or p62 significantly repressed ERK1/2 phosphorylation in HeLa cells, but transfection with parkin did not repress ERK1/2 phosphorylation in p62-knockdown HeLa cells, suggesting that p62 was involved in parkin-induced inhibition on ERK1/2 phosphorylation. Overexpression of parkin or p62 significantly repressed 6-OHDA-induced ERK1/2 phosphorylation in PC12 cells, and parkin overexpression inhibited 6-OHDA-induced apoptosis in PC12 and SH-SY5Y cells.

Conclusion:

Parkin protects PC12 cells against 6-OHDA-induced apoptosis via ubiquitinating and stabilizing scaffold protein p62, and repressing ERK1/2 activation.     

A novel sulfonamide agent,MPSP-001, exhibits potent activity against human cancer cells in vitro through disruption of microtubule

Aim:

To evaluate the anti-cancer effects of a new sulfonamide derivative, 2-(N-(3-chlorophenyl)-4-methoxyphenylsulfonamido)-N-hydroxypropanamide (MPSP-001).

Methods:

Human cancer cell lines (HepG2, THP-1, K562, HGC-27, SKOV3, PANC-1, SW480, Kba, HeLa, A549, MDA-MB-453, and MCF-7) were examined. The cytotoxicity of MPSP-001 was evaluated using the WST-8 assay. Cell cycle distribution was examined with flow cytometry. Mitotic spindle formation was detected using immunofluorescence microscopy. Apoptosis-related proteins were examined with Western blot using specific phosphorylated protein antibodies. Competitive tubulin-binding assay was performed to test whether the compound competitively bound to the colchicine site. Molecular docking was performed to explore the possible binding conformation.

Results:

MPSP-001 potently inhibited the growth of the 12 different types of human cancer cells with the IC₅₀ values ranging from 1.9 to 15.7 μmol/L. The compound exerted potent inhibition on the drug-resistant Kb/VCR and MCF-7/ADR cells, as on Kba and MCF-7 cells. In HeLa, HGC-27, A549, and other cells, the compound (5 μmol/L) caused cell cycle arrest at the G₂/M phase, and subsequently induced cell apoptosis. In Hela cells, it prevented the mitotic spindle formation. Furthermore, the compound dose-dependently inhibited polymerization of tubulin in vitro, and directly bound to the colchicine-site of β-tubulin. Molecular docking predicted that the compound may form two hydrogen bonds to the binding pocket. The compound showed synergistic effects with colchicine and taxol in blocking mitosis of HeLa cells.

Conclusion:

MPSP-001 shows a broad-spectrum of anti-tumor efficacy in vitro and represents a novel structure with anti-microtubule activity.     

L655,240, acting as a competitive BACE1 inhibitor,efficiently decreases β-amyloid peptide production in HEK293-APPswe cells

Aim:

To identify a small molecule L655,240 as a novel β-secretase (BACE1) inhibitor and to investigate its effects on β-amyloid (Aβ) generation in vitro.

Methods:

Fluorescence resonance energy transfer (FRET) was used to characterize the inhibitory effect of L655,240 on BACE1. Surface plasmon resonance (SPR) technology-based assay was performed to study the binding affinity of L655,240 for BACE1. The selectivity of L655,240 toward BACE1 over other aspartic proteases was determined with enzymatic assay. The effects of L655,240 on Aβ40, Aβ42, and sAPPβ production were studied in HEK293 cells stably expressing APP695 Swedish mutant^K595N/M596L (HEK293-APPswe cells). The activities of BACE1, γ-secretase and α-secretase were assayed, and both the mRNA and protein levels of APP and BACE1 were evaluated using real-time PCR (RT-PCR) and Western blot analysis.

Results:

L655,240 was determined to be a competitive, selective BACE1 inhibitor (IC₅₀=4.47±1.37 μmol/L), which bound to BACE1 directly (K_D=17.9±0.72 μmol/L). L655,240 effectively reduced Aβ40, Aβ42, and sAPPβ production by inhibiting BACE1 without affecting the activities of γ-secretase and α-secretase in HEK293-APPswe cells. L655,240 has no effect on APP and BACE1 mRNA or protein levels in HEK293-APPswe cells.

Conclusion:

The small molecule L655,240 is a novel BACE1 inhibitor that can effectively decreases Aβ production in vitro, thereby highlighting its therapeutic potential for the treatment of Alzheimer''s disease.     

Sulfated polymannuroguluronate inhibits Tat-induced SLK cell adhesion via a novel binding site, a KKR spatial triad

Aim:

Sulfated polymannuroguluronate (SPMG), a candidate anti-AIDS drug, inhibited HIV replication and interfered with HIV entry into host T lymphocytes. SPMG has high binding affinity for the transactivating factor of the HIV-1 virus (Tat) via its basic domain. However, deletion or substitution of the basic domain affected, but did not completely eliminated Tat-SPMG interactions. Here, we sought to identify other SPMG binding sites in addition to the basic domain.

Methods:

The potential SPMG binding sites were determined using molecular simulation and a surface plasmon resonance (SPR) based competitive inhibition assay. The effect of SPMG on Tat induced adhesion was evaluated using a cell adhesion assay.

Results:

The KKR domain, a novel high-affinity heparin binding site, was identified, which consisted of a triad of Lys12, Lys41, and Arg78. The KKR domain, spatially enclosed SPMG binding site on Tat, functions as another binding domain for SPMG. Further functional evaluation demonstrated that SPMG inhibits Tat-mediated SLK cell adhesion by directly binding to the KKR region.

Conclusion:

The KKR domain is a novel high-affinity binding domain for SPMG. Our findings provide important new insights into the molecular mechanisms of SPMG and a potential therapeutic intervention for Tat-induced cell adhesion.     

A Technology-Enhanced Patient Case Workshop

Objectives

To assess the impact of technology-based changes on student learning, skill development, and satisfaction in a patient-case workshop.

Design

A new workshop format for a course was adopted over a 3-year period. Students received and completed patient cases and obtained immediate performance feedback in class instead of preparing the case prior to class and waiting for instructors to grade and return their cases. The cases were designed and accessed via an online course management system.

Assessment

Student satisfaction was measured using end-of-course surveys. The impact of the technology-based changes on student learning, problem-solving, and critical-thinking skills was measured and compared between the 2 different course formats by assessing changes in examination responses. Three advantages to the new format were reported: real-life format in terms of time constraint for responses, a team learning environment, and expedient grading and feedback. Students overwhelmingly agreed that the new format should be continued. Students'' examination scores improved significantly under the new format.

Conclusion

The change in delivery of patient-case workshops to an online, real-time system was well accepted and resulted in enhanced learning, critical thinking, and problem-solving skills.     

Comparative amino acid decomposition analysis of potent type I p38α inhibitors

Background and purpose of the study

p38α is a member of mitogen-activated protein kinases (MAPK) considered as a prominent target in development of anti-inflammatory agents. Any abnormality in the phosphorylation process leads to the different human diseases such as cancer, diabetes and inflammatory diseases. Several small molecule p38α inhibitors have been developed up to now. In this regard, structural elucidation of p38 inhibitors needs to be done enabling us in rational lead development strategies.

Methods

Various interactions of three potent inhibitors with p38α active site have been evaluated in terms of binding energies and bond lengths via density function theory and MD simulations.

Results

Our comparative study showed that both ab initio and MD simulation led to the relatively similar results in pharmacophore discrimination of p38α inhibitors.

Conclusion

The results of the present study may find their usefulness in pharmacophore based modification of p38α inhibitors.     

α-Mangostin suppresses human gastric adenocarcinoma cells in vitro via blockade of Stat3 signaling pathway

Aim:

To investigate the anti-tumor effects of α-mangostin, a major xanthone identified in the pericarp of mangosteen (Garcinia mangostana Linn), against human gastric adenocarcinoma cells in vitro, and the mechanisms of the effects.

Methods:

Human gastric adenocarcinoma cell lines BGC-823 and SGC-7901 were treated with α-mangostin. The cell viability was measured with MTT assay, and cell apoptosis was examined using flow cytometry and TUNEL assay. The expression of the relevant proteins was detected using Western blot.

Results:

Treatment with α-mangostin (3–10 μg/mL) inhibited the viability of both BGC-823 and SGC-7901 cells in dose- and time-manners. Furthermore, α-mangostin (7 μg/mL) time-dependently increased the apoptosis index of the cancer cells, reduced the mitochondrial membrane potential of the cancer cells, and significantly increased the release of cytochrome c and AIF into cytoplasm. Moreover, the α-mangostin treatment markedly suppressed the constitutive Stat3 protein activation, and Stat3-regulated Bcl-xL and Mcl-1 protein levels in the cancer cells.

Conclusion:

The anti-tumor effects of α-mangostin against human gastric adenocarcinoma cells in vitro can be partly attributed to blockade of Stat3 signaling pathway.     

Discovery of novel PDE10 inhibitors by a robust homogeneous screening assay

Aim:

To develop a homogeneous assay for high-throughput screening (HTS) of inhibitors of phosphodiesterase 10 (PDE10).

Methods:

Purified human PDE10 enzyme derived from E coli, [³H]-cAMP and yttrium silicate microbeads were used to develop an HTS assay based on the scintillation proximity assay (SPA) technology. This method was applied to a large-scale screening campaign against a diverse compound library and subsequent confirmation studies. Preliminary structure-activity relationship (SAR) studies were initiated through limited structural modifications of the hits.

Results:

The IC₅₀ value of the control compound (papaverine) assessed with the SPA approach was comparable and consistent with that reported in the literature. Signal to background (S/B) ratio and Z'' factor of the assay system were evaluated to be 5.24 and 0.71, respectively. In an HTS campaign of 71 360 synthetic and natural compounds, 67 hits displayed reproducible PDE10 inhibition, of which, 8 were chosen as the scaffold for structural modifications and subsequent SAR analysis.

Conclusion:

The homogeneous PDE10 SPA assay is an efficient and robust tool to screen potential PDE10 inhibitors. Preliminary SAR studies suggest that potent PDE10 inhibitors could be identified and developed through this strategy.     

Inhibition of paracetamol glucuronidation by tyrosine kinase inhibitors

AIMS

We aimed to investigate the effects of tyrosine kinase inhibitors (TKIs) on paracetamol (acetaminophen) glucuronidation.

METHODS

The inhibition of nine small molecule TKIs on paracetamol glucuronidation was investigated in human liver microsomes (HLMs) and recombinant human UDP-glucuronosyltransferases (UGTs).

RESULTS

Sorafenib, dasatinib and imatinib exhibited mixed inhibition against paracetamol glucuronidation in pooled HLMs, and potent inhibition in UGT1A9 and UGT2B15. Dasatinib and imatinib also inhibited UGT1A1-mediated paracetamol glucuronidation. Axitinib, erlotinib, gefitinib, lapatinib, nilotinib and vandetanib exhibited weak inhibition of paracetamol glucuronidation activity in HLMs.

CONCLUSIONS

The inhibition of paracetamol glucuronidation by TKIs might be of particular concern when they are co-administered.     

LGH00031, a novel ortho-quinonoid inhibitor of cell division cycle 25B,inhibits human cancer cells via ROS generation

Aim:

To discover novel cell division cycle 25 (CDC25) B inhibitors and elucidate the mechanisms of inhibition in cancer cells.

Methods:

Cell growth inhibition was detected by MTT assay, the cell cycle was analyzed by flow cytometry, and protein expression and phosphorylation was examined by Western blot analysis.

Results:

LGH00031 inhibited CDC25B irreversibly in vitro in a dose-dependent manner, and impaired the proliferation of tumor cell lines. In synchronized HeLa cells, LGH00031 delayed the cell cycle progression at the G₂/M phase. LGH00031 increased cyclin-dependent kinase 1 (CDK1) tyrosine 15 phosphorylation and cyclin B1 protein level. The activity of LGH00031 against CDC25B in vitro relied on the existence of 1,4-dithiothreitol (DTT) or dihydrolipoic acid and oxygen. The oxygen free radical scavenger catalase and superoxide dismutase reduced the inactivation of CDC25 by LGH00031, confirming that reactive oxygen species (ROS) mediate the inactivation process in vitro. LGH00031 accelerated cellular ROS production in a dose-dependent manner, and N-acetyl cysteine (NAC) markedly decreased the ROS production induced by LGH00031. Correspondingly, the LGH00031-induced decrease in cell viability and cell cycle arrest, cyclin B1 protein level, and phosphorylation of CDK1 tyrosine 15 were also rescued by NAC that decreased ROS production.

Conclusion:

The activity of LGH00031 at the molecular and cellular level is mediated by ROS.     

Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury

BACKGROUND AND PURPOSE

Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells.

EXPERIMENTAL APPROACH

Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors.

KEY RESULTS

NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor α-glycyrrhetinic acid (α-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of α-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by α-GA.

CONCLUSION AND IMPLICATIONS

Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury.