Effect of Stemona curtisii root extract on P-glycoprotein and MRP-1 function in multidrug-resistant cancer cells

Multidrug resistance (MDR) is the result of overexpression of membrane bound proteins that efflux chemotherapeutic drugs from the cells. Two proteins, P-glycoprotein (P-gp) and multidrug-resistance associated protein-1 (MRP-1) efflux chemotherapeutic agents out of the cancer cell that decrease intracellular drug accumulation, thereby decreasing the effectiveness of many chemotherapeutic agents. In the present study, the ethanolic extract of the roots of Stemona curtisii Hook. was tested for the potential ability to modulate the MDR phenotype and function of P-gp and MRP-1. The S. curtisii extract reversed the resistance to putative chemotherapeutic agents, including vinblastine, paclitaxel and colchicine of KB-V1 cells (MDR human cervical carcinoma with high P-gp expression) in a dose-dependent manner, but not in KB-3-1 cells (drug sensitive human cervical carcinoma, which lack P-gp expression). The root extract also increased the intracellular uptake and retention of (3)[H]-vinblastine in KB-V1 cells dose dependently. The extract did not influence MDR phenotype-mediated MRP-1 in MRP1-HEK293 (human embryonic kidney cells stably transfected with pcDNA3.1-MRP1-H10 which show high MRP-1 expression) and pcDNA3.1-HEK293 (wild type). In summary, the S. curtisii root extract modulated P-gp activity but not MRP-1 activity. The result obtained from this study strongly indicated that S. curtisii extract may play an important role as a P-gp modulator as used in vitro and may be effective in the treatment of multidrug-resistant cancers. The purified form of the active components of S. curtisii extract should be investigated in more details in order to explain the molecular mechanisms involved in P-gp modulation. This is the first report of new biological activity in this plant, which could be a potential source of a new chemosensitizer.     

Expression of the hyperphosphorylated tau attenuates ER stress-induced apoptosis with upregulation of unfolded protein response

The neural dysfunction in Alzheimer's disease (AD) could arise from endoplasmic reticulum (ER) stress and deficits of the unfolded protein response (UPR). To explore whether tau hyperphosphorylation, a hallmark of AD brain pathologies, plays a role in ER stress-induced alterations of cell viability, we established cell lines with stable expression of human tau (HEK293/tau) or the vector (HEK293/vec) and treated the cells with thapsigargin (TG), an ER stress inducer. We observed that the HEK293/tau cells were more resistant than the HEK293/vec cells to the TG-induced apoptosis, importantly, a time dependent increase of tau phosphorylation at Thr205 and Thr231 sites was positively correlated with the inhibition of apoptosis. We also observed that expression of tau upregulated phosphorylation of PERK, eIF2 and IRE1 with an increased cleavage of ATF6 and ATF4. The potentiation of UPR was also detected in HEK293/tau cells treated with other ER stress inducers, including staurosporine, camptothecin and hydrogen peroxide, in which a suppressed apoptosis was also shown. Our data suggest that tau hyperphosphorylation could attenuate the ER stress-induced apoptosis with the mechanism involving upregulation of UPR system.     

Bone microenvironment-related growth factors, zoledronic acid and dexamethasone differentially modulate PTHrP expression in PC-3 prostate cancer cells.

Bone metastasis microenvironment-related growth factors such as insulin-like growth factor 1 (IGF-1), transforming growth factor beta 1 (TGF-beta1), basic fibroblast growth factor (bFGF) and interleukin 6 (IL-6) show survival factor activity, thereby inhibiting chemotherapy-induced apoptosis of PC-3 prostate cancer cells in vitro. Recently, zoledronic acid has been shown to induce apoptosis in PC-3 prostate cancer cells while overexpression of parathyroid hormone-related protein (PTHrP) inhibits serum deprivation-induced apoptosis in PC-3 cells. Consequently, we have investigated whether IGF-1, TGF-beta1, bFGF, IL-6, zoledronic acid and/or dexamethasone affect the expression of the PTHrP and type I PTH/PTHrP receptor (PTH.1R) in PC-3 prostate cancer cells using relative quantitative PCR and real-time PCR (expression at mRNA level) and immunocytochemical and immunofluorescence analysis (expression at protein level). Our data show that IGF-1, TGF-beta1, bFGF and IL-6 increase PTHrP mRNA expression and its perinuclear localization, while zoledronic acid (50 muM, 100 muM for 24 h and 48 h) and dexamethasone suppress PTHrP expression in PC-3 cells. We did not detect any appreciable change of the PTH.1R expression due to IGF-1, TGF- beta1, bFGF, IL-6, zoledronic acid or dexamethasone in PC-3 cells. Therefore, it is conceivable that bone metastasis microenvironment-related survival factor/anti-apoptotic activity and zoledronic acid anticancer action/pro-apoptotic activity on PC-3 cells is mediated, at least in part, by differential modulation of PTHrP expression.     

多核酶表达系统在HEK293细胞中对多药耐药相关蛋白（MRP1）的表达抑制作用

为了研究多核酶表达系统在HEK293细胞中对多药耐药相关蛋白表达抑制的作用．我们构建了含有20个可以自身切割的顺式作用核酶和10个靶向MRP1基因特定位点的反式作用核酶的多核酶表达系统。利用RT—PCR、Westem blot和MTT分析了多核酶系统分别与MRP1靶基因质粒和MRP1 全长基因质粒共转染的HEK293细胞。结果显示．多核酶表达系统能够明显降低荧光融合蛋白在HEK293细胞中的表达。RT—PCR分析表明．懈用靶mRNA降低程度与多核酶表达系统所含的反式作用核酶数目有关。Westernblot分析显示了与RT—PCR相似的结果。Mrrr分析表明,多核酶表达系统能够逆转由MRP1基因转染HEK293细胞产生的多药耐药性。结果提示．含有多个核酶的表达系统对MRP1基因的抑制效应优于单核酶的表达系统。因此．该策略可能用于基因治疗肿瘤或其他疾病．     

Cooperation of NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferases reduces menadione cytotoxicity in HEK293 cells

Previous studies have shown that NAD(P)H:quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of menadione (2-methyl-1,4-naphthoquinone, also known as vitamin K3). However, menadiol (2-methyl-1,4-naphthalenediol) formed from menadione by NQO1-mediated reduction continues to be an unstable substance, which undergoes the reformation of menadione with concomitant formation of reactive oxygen species (ROS). Hence, we focused on the roles of phase II enzymes, with particular attention to UDP-glucuronosyltransferases (UGTs), in the detoxification process of menadione. In this study, we established an HEK293 cell line stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with doxycycline (DOX)-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and UGT1A10 (HEK293/NQO1/UGT1A10), and evaluated the role of NQO1 and UGTs against menadione-induced cytotoxicity. Our results differed from those of previous studies. HEK293/NQO1 was the most sensitive cell line to menadione cytotoxicity among cell lines established in this study. These phenomena were also observed in HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells in which the expression of UGT was suppressed by DOX treatment. On the contrary, HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells without DOX treatment were resistant to menadione-induced cytotoxicity. These results demonstrated that NQO1 is not a detoxification enzyme for menadione and that UGT-mediated glucuronidation of menadiol is the most important detoxification process.     

Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells

Fas-associated death domain (FADD) is a common adaptor molecule which plays an important role in transduction of death receptor mediated apoptosis. The FADD provides DED motif for binding to both procaspase-8 and cFLIP molecules which executes death receptor mediated apoptosis. Dysregulated expression of FADD and cFLIP may contribute to inhibition of apoptosis and promote cell survival in cancer. Moreover elevated intracellular level of cFLIP competitively excludes the binding of procaspase-8 to the death effector domain (DED) of FADD at the DISC to block the activation of death receptor signaling required for apoptosis. Increasing evidence shows that defects in FADD protein expression are associated with progression of malignancies and resistance to apoptosis. Therefore, improved expression and function of FADD may provide new paradigms for regulation of cell proliferation and survival in cancer. In the present study, we have examined the potential of FADD in induction of apoptosis by overexpression of FADD in HEK 293T cells and validated further its consequences on the expression of pro and anti-apoptotic proteins besides initiation of death receptor mediated signaling. We have found deficient expression of FADD and elevated expression of cFLIP(L) in HEK 293T cells. Our results demonstrate that over expression of FADD attenuates the expression of anti-apoptotic protein cFLIP and activates the cascade of extrinsic caspases to execution of apoptosis in HEK 293T cells.     

Impact of Cell Type and Epitope Tagging on Heterologous Expression of G Protein-Coupled Receptor: A Systematic Study on Angiotensin Type II Receptor

Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2) receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.     

12(S)-Hydroxyeicosatetraenoic acid induces cAMP production via increasing intracellular calcium concentration

We have found that a 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HETE), induces cAMP production in human normal fibroblast TIG-1 cells. This phenomenon was not observed in other cells tested including human embryonic kidney HEK293 cells. We have speculated that this specific response might be influenced by the kinds of isoform of adenylyl cyclase (AC) present in cells. We found that TIG-1 cells specifically expressed type VIII AC. As type VIII AC is known to be activated by an increase of calcium concentration, we determined the change of intracellular Ca2+ concentration after the addition of 12-HETE. It was elevated not only in TIG-1 cells, but also HEK293 cells, which did not respond to 12-HETE to produce cAMP. The addition of a calcium ionophore elevated the concentration of intracellular cAMP in TIG-1 cells, but it was without effect in HEK293 cells. To show that the expression of this particular isoform of AC is responsible for the positive response to 12-HETE, we transfected this AC isoform into HEK293 cells. The type VIII AC-transfected cells, in contrast to the mock-transfected ones, became very responsive to 12-HETE to produce cAMP. Taken all together the data would strongly suggest that 12-HETE specifically activates type VIII AC via increasing intracellular Ca2+ concentration.     

The P2X7 receptor is a key modulator of aerobic glycolysis

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.     

Three-dimensional constructs induce high cellular activity: Structural stability and the specific production of proteins and cytokines

Naofen has recently been identified from the rat brain/spinal cord cDNA library as a substance reactive against an anti-shigatoxin (Stx)-2 antibody. Naofen mRNA is composed of 4620 nucleotides and encodes 1170 amino acids. Naofen contains four WD-repeat domains in its N-terminus and is ubiquitously distributed in many tissues of the rat. Tumor necrosis factor (TNF)-α enhanced the expression of naofen mRNA in HEK293 cells in a dose-dependent manner. Furthermore, naofen siRNA, which predominantly knocked down the expression of naofen mRNA, significantly reduced both TNF-α-induced caspase-3 activation and apoptosis in HEK293 cells. Overexpression of naofen in HEK293 cells (FLAG-NF) spontaneously induced caspase -3 activation and apoptosis, and showed extremely high susceptibility to TNF-α-induced apoptosis. These results indicated that naofen may function as a novel modulator activating caspase-3, and promoting TNF-α-stimulated apoptosis.     

Protein kinase PKN1 associates with TRAF2 and is involved in TRAF2-NF-kappaB signaling pathway

PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor alpha (TNFalpha) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-kappaB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580-584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-kappaB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-kappaB signaling pathway.     

人P2X7真核表达载体的构建及稳定转染细胞株的建立

目的：构建人P2X7基因的真核表达载体,并通过转染获得稳定表达P2X7分子的HEK293细胞株。方法：以人脑组织P2X7cDNA为模板扩增出P2X7基因,插入到真核表达载体pEGFP-N1中,构建重组质粒pEGFP-N1/P2X7。用X-fect试剂盒将重组质粒转染HEK293细胞,通过G418辅助荧光筛选建立稳定表达P2X7-EGFP细胞株。经流式细胞仪、Western blot和激光共聚焦显微镜检测,了解人P2X7在HEK293细胞中的表达水平及细胞内定位。结果：重组质粒pEGFP-N1/P2X7构建正确,建立了稳定表达人P2X7的HEK293细胞系。Western blot和流式细胞仪检测证实,P2X7在HEK293细胞系中成功表达,激光共聚焦显微镜检测显示P2X7-EGFP定位在细胞膜上。结论：重组载体pEGFP-N1/P2X7构建成功并建立了稳定表达人P2X7的HEK293细胞系,为进一步研究P2X7离子通道结构和功能奠定基础。     

Upregulation of miR-23a～27a～24-2 Cluster Induces Caspase-Dependent and -Independent Apoptosis in Human Embryonic Kidney Cells

miRNAs have emerged as important players in the regulation of gene expression and their deregulation is a common feature in a variety of diseases, especially cancer. Currently, many efforts are focused on studying miRNA expression patterns, as well as miRNA target validation. Here, we show that the over expression of miR-23a∼27a∼24-2 cluster in HEK293T cells induces apoptosis by caspase-dependent as well as caspase-independent pathway as proved by the annexin assay, caspase activation, release of cytochrome-c and AIF (apoptosis inducing factor) from mitochondria. Furthermore, the over expressed cluster modulates the expression of a number of genes involved in apoptosis including FADD (Fas Associated protein with Death Domain). Bioinformatically, FADD is predicted to be the target of hsa-miR-27a and interestingly, FADD protein was found to be up regulated consistent with very less expression of hsa-miR-27a in HEK293T cells. This effect was direct, as hsa-miR-27a negatively regulated the expression of FADD 3′UTR based reporter construct. Moreover, we also showed that over expression of miR-23a∼27a∼24-2 sensitized HEK293T cells to TNF-α cytotoxicity. Taken together, our study demonstrates that enhanced TNF-α induced apoptosis in HEK293T cells by over expression of miR-23a∼27a∼24-2 cluster provides new insights in the development of novel therapeutics for cancer.     

Upregulation of Autophagy-Related Gene-5 (ATG-5) Is Associated with Chemoresistance in Human Gastric Cancer

Autophagy-related gene-5 (ATG-5) is one of the key regulators of autophagic cell death. It has been widely regarded as a protective molecular mechanism for tumor cells during the course of chemotherapy. In the present study, we investigated the expression pattern of ATG-5 and multidrug resistance-associated protein-1 (MRP-1) in 135 gastric cancers (GC) patients who were treated with epirubicin, cisplatin and 5-FU adjuvant chemotherapy (ECF) following surgical resection and explored their potential clinical significance. We found that both ATG-5 (77.78%) and MRP-1 (79.26%) were highly expressed in GC patients. ATG-5 expression was significantly associated with depth of wall invasion, TNM stages and distant metastasis of GC (P<0.05), whereas MRP-1 expression was significantly linked with tumor size, depth of wall invasion, lymph node metastasis, TNM stages and differentiation status (P<0.05). ATG-5 expression was positively correlated with MRP-1 (rp = 0.616, P<0.01). Increased expression of ATG-5 and MPR-1 was significantly correlated with poor overall survival (OS; P<0.01) and disease free survival (DFS; P<0.01) of our GC cohort. Furthermore, we demonstrated that ATG-5 was involved in drug resistant of GC cells, which was mainly through regulating autophagy. Our data suggest that upregulated expression of ATG-5, an important molecular feature of protective autophagy, is associated with chemoresistance in GC. Expression of ATG-5 and MRP-1 may be independent prognostic markers for GC treatment.     

Cisplatin cytotoxicity is enhanced with zoledronic acid in A549 lung cancer cell line: preliminary results of an in vitro study

We tested whether zoledronic acid, a biphosphonate with proposed apoptotic activity, augmented the cytotoxicity of cisplatin and/or gemcitabine in A549 lung cancer cell line. This cell line was subjected to different concentrations of the above chemotherapeutic agents and zoledronic acid. Cytotoxicity was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) assay. Particularly, zoledronic acid in 100 micromolar (microM) concentration augmented the cytotoxicity by cisplatin 1microg/ml from 25% to 70% (Z=3.22, P=0.0072). A significant portion of cells underwent apoptosis with or without zoledronic acid, but more so with the combination treatment as assessed by an Annexin V-FITC apoptosis detection kit. However, 100microM zoledronic acid showed 50% cytotoxicity on its own, but failed to improve cytotoxicity by Gemcitabine. Thus, we show for the first time in a lung cancer cell line that zoledronic acid bears cytotoxic potential on its own and in conjunction with cisplatin. The clinical potential of this finding should be further studied.     

Role of sphingosine kinase 2 in cell migration toward epidermal growth factor

Sphingosine 1-phosphate (S1P), produced by two sphingosine kinase isoenzymes, denoted SphK1 and SphK2, is the ligand for a family of five specific G protein-coupled receptors that regulate cytoskeletal rearrangements and cell motility. Whereas many growth factors stimulate SphK1, much less is known of the regulation of SphK2. Here we report that epidermal growth factor (EGF) stimulated SphK2 in HEK 293 cells. This is the first example of an agonist-dependent regulation of SphK2. Chemotaxis of HEK 293 cells toward EGF was inhibited by N,N-dimethylsphingosine, a competitive inhibitor of both SphKs, implicating S1P generation in this process. Down-regulating expression of SphK1 in HEK 293 cells with a specific siRNA abrogated migration toward EGF, whereas decreasing SphK2 expression had no effect. EGF contributes to the invasiveness of human breast cancer cells, and EGF receptor expression is associated with poor prognosis. EGF also stimulated SphK2 in MDA-MB-453 breast cancer cells. Surprisingly, however, down-regulation of SphK2 in these cells completely eliminated migration toward EGF without affecting fibronectin-induced haptotaxis. Our results suggest that SphK2 plays an important role in migration of MDA-MB-453 cells toward EGF.     

Multidrug Resistance-Associated Protein 1 (MRP1) mediated vincristine resistance: effects of N-acetylcysteine and Buthionine Sulfoximine

Background  

Multidrug resistance mediated by the multidrug resistance-associated protein 1 (MRP1) decreases cellular drug accumulation. The exact mechanism of MRP1 involved multidrug resistance has not been clarified yet, though glutathione (GSH) is likely to have a role for the resistance to occur. N-acetylcysteine (NAC) is a pro-glutathione drug. DL-Buthionine (S,R)-sulfoximine (BSO) is an inhibitor of GSH synthesis. The aim of our study was to investigate the effect of NAC and BSO on MRP1-mediated vincristine resistance in Human Embryonic Kidney (HEK293) and its MRP1 transfected 293MRP cells. Human Embryonic Kidney (HEK293) cells were transfected with a plasmid encoding whole MRP1 gene. Both cells were incubated with vincristine in the presence or absence of NAC and/or BSO. The viability of both cells was determined under different incubation conditions. GSH, Glutathione S-Transferase (GST) and glutathione peroxidase (GPx) levels were measured in the cell extracts obtained from both cells incubated with different drugs.     

Study of expression of desoxyribonucleases in mammalian cells by a protein renaturation method in polyacrylamide gel

Analysis of enzymatic activity in polyacrylamide gel is based on highly effective separation of proteins by SDS-electrophoresis with their subsequent renaturation and detection of enzymatic activity. This method was used to study an expression of DNAases in culturing of cells HEK293, NIH 3T3, U937. We have found that in HEK293 cells the nucleases with molecular weights 47 and 45 kDa were expressed. The localization of DNAases in the cell nuclei was shown as well. Induction of apoptosis in HEK293 cells increase the level of p47 DNAase and causes the expression of novel 50 kDa DNAase. We suggested that those discovered DNAases could take part in apoptotic DNA degradation.