AMP-activated protein kinase contributes to ROS-mediated p53 activation in cisplatin-induced nephrotoxicity.

OBJECTIVE: Cisplatin is a widely used anticancer drug that provokes various side effects. Nephrotoxicity is one of the well-known major side effects in the chemotherapeutic use of cisplatin. Reactive oxygen species (ROS) and p53 play important roles in cisplatin-induced nephrotoxicity. AMP-activated protein kinase (AMPK) is known to be sensitively activated by ROS and can directly activate p53. The present study investigated the role of AMPK on cisplatin-induced apoptosis in rat renal epithelial NRK-52E cells. MATERIALS AND METHODS: NRK-52E cells were treated with cisplatin in the absence or presence of specific ROS scavenger and AMPK inhibitor for indicated times under the serum-free condition. The expression and phosphorylation levels of proteins were evaluated by Western blot and densitometry analysis. RESULTS: Cisplatin induced apoptotic cell death through ROS-mediated p53 activation, which is associated with AMPK activation. AMPK inhibitor suppressed cisplatin-induced p53 activation, as well as AMPK activation. Interestingly, ROS scavenger also diminished cisplatin-induced p53 activation and AMPK activation. Furthermore, cisplatin induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which attenuated p53 activation, but did not affect the expression levels of total p53, cleaved caspase-3 and PARP. Meanwhile, inhibition of AMPK induced premature phosphorylation of eIF2α in cisplatin-treated cells. CONCLUSIONS: Taken together, these suggest that AMPK may be required for activation of p53 by oxidative stress in cisplatin-induced nephrotoxicity. Moreover, eIF2α phosphorylation may interrupt the AMPK-activated p53 in NRK-52E cells exposed to cisplatin, but does not critically affect cisplatin-induced nephrotoxicity because AMPK activation can be disrupted eIF2α phosphorylation.

Licorice and its active compound glycyrrhizic acid ameliorates cisplatin-induced nephrotoxicity through inactivation of p53 by scavenging ROS and overexpression of p21 in human renal proximal tubular epithelial cells.

OBJECTIVE: Nephrotoxicity is one of the major side effects that limit the use of cisplatin in cancer therapy. Cisplatin-induced apoptosis in renal cells is associated with reactive oxygen species (ROS)-mediated p53 activation. Licorice (Glycyrrhiza uralensis Fischer) is one of the most widely used medicinal herbs in Korea, China and Japan. The aim of the study was to evaluate the protective effects of licorice extract (LE) and its active compound glycyrrhizic acid (GA) against cisplatin-induced nephrotoxicity in human renal proximal tubular epithelial (HK-2) cells. MATERIALS AND METHODS: HK-2 cells were pretreated with LE or GA for 1 h and then treated with 40 µM of cisplatin for indicated times under the serum-free condition. Cell viability was evaluated by MTT assay. Apoptosis was evaluated by flow cytometric analysis and caspase-3 activity. The intracellular ROS levels were determined by DCFH-DA assay. The expression and phosphorylation levels of protein were evaluated by Western blot and densitometry analysis. RESULTS: When treating HK-2 cells with LE or GA, both of them alleviated cisplatin-induced cytotoxicity and apoptosis. LE and GA inhibited caspase-3 activity and polymerase (PARP) cleavage in cisplatin-treated cells. LE and GA also inhibited p53 expression and its phosphorylation as well as ROS production in cells exposed to cisplatin. Meanwhile, LE and GA enhanced cisplatin-induced p21 expression, which then led to S-phase arrest in cell cycle and limited cell growth. Presumably, increased p21 expression may contribute to cellular prevention from cisplatin-induced apoptosis, because p21 is the key molecule to cytoprotection during cisplatin-induced nephrotoxicity. CONCLUSIONS: These results suggest that LE and GA ameliorate cisplatin-induced apoptosis through reduction of ROS-mediating p53 activation and promotion of p21 expression in HK-2 cells.

[Establishment and characterization of an orthotopic murine model of mucosal malignant melanoma of the maxillary sinus].

Objective:To establish a stable and reliable orthotopic murine model of mucosal malignant melanoma of the maxillary sinus so as to provide animal models for further studying for pathogenesis and therapy of sinonasal malignancy.Method:B16 were implanted in the right maxillary sinus of male nude mice. After tumors appeared in right maxillary sinus, tumor growth and survival rate were recorded. The degree of tumor infiltration was observed through the MRI.Result:Mice with B16 implanted in right maxillary sinus developed primary tumors. The survival and the tumor volume were related to the number of tumor cells implanted. We found that the tumor can invade into nasal cavity, orbital cavity and the basilar region using MRI.Conclusion:We successfully established a model for mucosal malignant melanoma of the maxillary sinus. This model offers an experimental tool for further research on biological characteristics of sinonasal malignancy and the development of new therapy.

Chondroitinase administration and pcDNA3.1-BDNF-BMSC transplantation promote motor functional recovery associated with NGF expression in spinal cord-transected rat.

STUDY DESIGN: We evaluated whether combination of chondroitinase (chABC) administration and brain-derived neurotrophic factor (BDNF)-mesenchymal stem cell (MSC) transplantation could provide an optimal effect for the treatment of spinal cord injury (SCI) subjected to complete transection. OBJECTIVES: Behavioral assessments and DBA tracing were used to evaluate the effects of combination of chABC administration and BDNF-MSC transplantation on axonal regeneration and functional improvement in SCT rats. SETTING: Sichuan, ChinaMethods:Bone mesenchymal stem cells (BMSCs) were cultured and overexpressed BDNF recombinant vector was constructed into MSCs, then transplanted into the impaired spinal cord, together with chABC administration. Finally, the cortical spinal tract regeneration was detected by DBA tracing at 4 weeks post operation, and the expression of nerve growth factor (NGF), BDNF, neurotrophic factor (NT)-3, NT-4, fibroblast growth factor (FGF-2)-2, B cell lymphoma 2 (BCL-2) assaciated X protein (BAX) and BCL-2 in the caudal cord tissues was assessed by reverse transcription-PCR. RESULTS: Animals received both BDNF-BMSC transplantation and chABC administration presented the best functional recovery and obvious axonal regeneration. Moreover, NGF expression was significantly higher than that in the other groups. CONCLUSION: Co-treated strategy could effectively promote motor functional recovery and axonal regeneration in SCT rats associated with NGF upregulation.

Apigenin inhibits indoxyl sulfate-induced endoplasmic reticulum stress and anti-proliferative pathways, CHOP and IL-6/p21, in human renal proximal tubular cells.

OBJECTIVE: Indoxyl sulfate (IS) has been reported to induce endoplasmic reticulum (ER) stress in tubular cells and to inhibit the cell proliferation via ER stress and ERK/IL-6/p21 pathways. This study has investigated the effect of apigenin on IS-induced ER stress in immortalized human renal proximal tubular HK-2 cells. MATERIALS AND METHODS: Human Kidney 2 (HK-2) cells were treated with IS (5 mM) in the absence or presence of apigenin (10 µM) or salubrinal (20 µM) for indicated times under the serum-free condition. Cell viability was evaluated by MTT assay. The levels of protein expression and phosphorylation were evaluated by Western blot analysis. RESULTS: In HK-2 cells, apigenin completely inhibited IS-induced ER stress, as indicated by decreased expression of CHOP, ATF4 and GRP78, although the phosphorylated level of eIF2α did not decrease. IS-induced expression levels of IL-6 and p21 proteins were also inhibited by apigenin, with no significant changes in ERK activation. The suppression of cell proliferation by IS was abolished by salubrinal, an ER stress inhibitor, but not by apigenin. Apigenin inhibited the phosphorylation of Akt and GSK-3ß in IS-treated HK-2 cells. The phosphorylation of GSK-3ß, which was inhibited by apigenin, resulted in hypo-phosphorylation of retinoblastoma (Rb) protein, which was associated with the decrease in cyclin D1 expression. CONCLUSIONS: These results suggest that apigenin may inhibit IS-induced ER stress and expression of IL-6 and p21 proteins in HK-2 cells. It is most likely that apigenin, together with its inhibitory effect on ER stress, may also suppress the cell growth by inducing the loss of Rb phosphorylation, which was associated with the decrease in cyclin D1 expression by GSK-3ß activation through the inhibition of PI3K/Akt pathway.

Role of reactive oxygen species in p53 activation during cisplatin-induced apoptosis of rat mesangial cells.

BACKGROUND AND OBJECTIVES: Nephrotoxicity is one of the main side effects of the anticancer drug cisplatin, and one of its main therapeutic limitations. It has been suggested that p53 activation plays important roles in renal cell injury by cisplatin. However, the mechanism of p53 activation by cisplatin is unclear. This study examined whether reactive oxygen species (ROS) production by cisplatin would be linked to p53 activation in rat mesangial cells. MATERIALS AND METHODS: Renal cells were incubated with cisplatin in the absence or presence of pifithrin-a (PFT), N-acetyl-cysteine (NAC), or dimethylthiourea (DMT). Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazol yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was evaluated by caspase-3 activity and cleavage of poly (ADP-ribose) polymerase (PARP). The relative levels of ROS and p53 phosphorylation were determined by fluorometric assay and Western blot analysis, respectively. RESULTS: Cisplatin induced apoptotic cell death via caspase-3 activation and PARP cleavage, and also increased p53 activation and ROS production. The p53 inhibitor PFT inhibited cisplatin-induced apoptosis. NAC and DMT, two antioxidants, also inhibited cisplatin-induced apoptosis. Interestingly, NAC and DMT reduced ROS production and suppressed p53 activation in renal cells exposed to cisplatin. CONCLUSIONS: Our results suggest that the ability of cisplatin to induce apoptosis of rat mesangial cells requires ROS-dependent p53 activation, thus, supporting the potential therapeutic role of antioxidants in preventing the cisplatin nephrotoxicity.

Molecular cloning of kpcA gene encoding a Kex2p-like endoprotease from Aspergillus nidulans.

We cloned and sequenced a gene, kpcA (Kex2p-like proprotein convertase A), from a genomic library of Aspergillus nidulans. The kpcA gene encodes an 820-residue protein, named KpcA, which contains a putative subtilisin-like catalytic domain (residues 136-466) homologous to that of the subtilisin serine protease family. KpcA shows 56, 73, and 47% amino acid identities with Saccharomyces cerevisiae Kex2p, Aspergillus niger KexB, and mouse furin within the subtilisin-like catalytic domain, respectively. The sequences around the proposed active site Asp, His, and Ser residues of KpcA are similar to those of other Kex2p family members. The KpcA mRNA transcript with an expected size of approximately 2.8 kb was detected in A. nidulans. The substrate specificity of KpcA, expressed in CHO cells, is similar to that of A. niger KexB and yeast Kex2p. We conclude that KpcA is a resident Kex2p-like proprotein that processes endoprotease in A. nidulans.