Specially Inhibation Of AQP1 Suppress The Growth Of Hep-2

We use RNAi and acetazolamide to inhibit the expression of AQP1 in hep-2 cells. The different methods have the same result. AQP1 was definitely down-regulate by the drugs, and the tumor cells apoptosised as we proposed. So the drugs for anti-AQP might be a new target for therapeutic of tumors.1. Introduction:When discussing about the therapeutic targets of gene therapy, we could not tell an effective one. Water is one of the most important factors for livings. All the procedures of living actions are performed in water. Tumors are hungrier than the normal tissue. So they demand much more water. The major way of water transduction is simple diffusion. It is well known that a protein named aquaporin has an important role in the water transduction. This study investigated whether the aquaporin in tumor cells express more than the normal tissue and if we down-regulate AQP in cells can induce apoptosis. To detect the AQP in western blot were used Western blot analysis evaluated baseline BCL-XL protein levels in human Hep-2 cell lines BE3 and SK5. Preliminary studies of three 21 nucleotide,double-stranded BCL-XL specific siRNA identified the most potent construct. The cells were then transfected with this construct utilizing oligofectamine, with scrambled siRNA as a control. After 72 hours, protein and transcriptional down regulation were evaluated by Western blot. Flow cytometry was used to determine apoptotic cell death (subG0/G1 phase). Results were compared using the student's t-test.Results have shown that acetazolamide alone can inhibit the invasive potential of cancer cells in vitro and in vivo. Acetazolamide is a kind of sulfanilaminde served as carbonic anhydrase inhibitor. To be interesting, the tissue distribution and even the subcellular localization of AQP1 are similar to that of carbonic anhydrase, suggesting that there are some relation between the two kinds of proteins in their structure and function. It has been reported that acetazolamide has the ability of inhibiting the expression of AQP1 and this can suppress tumor metastasis.To block the AQP gene function we tried two different ways RNAi and acetazolamide. Both methods are effective in down-regulate the gene's expression. And with the down-regulation of AQP, the cells were apoptosis.2. Material and methods2.1 Short-interfering (si)RNA preparation for interferenceThe mRNA sequence to be targeted by 21-nucleotide siRNA duplexes was selected near the NAP domain (5'—3'). This region is transcribed as NAP domain. The sequence was submitted to a BLAST search against the human genome sequence to ensure that only the AQP1 gene of the human genome was targeted. Antisense 5'-3'(AATGACCTGGCTGATGG TGTG-CCTGTCTC) , sense 5-3(AACACACCATCAGCCAGGTCATT- CCTGTCTC),with the help of silencer siRNA Construction Kit(Ambion) , we build the dsRNA.2.2 Cell culturesCell culture.Hep-2,a human laryngeal cancer cell line, was purchased from ATCC, were grown in GIBCO Iscove's Modified Dulbecco's medium (Invitrogen, Grand Island, NY,USA) supplemented with 10% fetal bovine serum,100 U/ml penicillin,and 0.1 mg/ml streptomycin (all from Sigma) in a humidified,5% CO2 incubator at 37℃. Transfections and drug treatmentHep-2 cells were seeded in 6-well plates at 3 *105 cells/well and grown overnight to 50% confluence prior to transfection. All siRNA were transfected with siPORT Amine transfection agent (Ambion Inc. USA) following manufacturer's instructions. For RNAi, lg siRNA expression was used. All transfection experiments were done in triplicate and repeated at least twice with deferent DNA isolates.In order to identify the transfection is effect; we use the siRNA Labeling Kit. (Ambion Inc. USA) Acetazolamide was purchased from Sigma. Three concentrations was selected 1×10-5 mol /L,5×10-5 mol/L,10×10-5mol/L After 24h,48 h, 72h, the cells were harvested for MTT assay, western blot analysis, fluorescence-activated cell sorter(FACS) analysis and trypan blue dye exclusion assay.MTT assay.MTT assay was performed to assess the effect of RNAi and acetazolamide on cell proliferation. Cells (1.5×104 cells/well) were plated in a six-well plate and maintained in RPMI-1640 supplemented with 10% FBS. At 24, 36, and 72 h after seeding, culture medium was removed, cells were treated with 10 ll sterile MTT dye (1 mg/ml, Sigma, USA) for 4 h at 37C, and then 200 ll of DMSO was added and thoroughly mixed for 30 min. Spectrometric absorbance at wavelength of 570nm was measured on a microplate reader (SPECTRA MAX 340, Molecular Devices). Proliferation Index (number of cells in ).Flow cytometry.Flow cytometry. Cell cycle profiles are analyzed by flow cytometry as described previously. Cells were collected and fixed in ice-cold 70% ethanol in phosphate-buffered saline (PBS) and stored at )20 C. After resuspension, 100 ll RNAase I (1mg ml₁) and 100 ll propidium iodide (PI, 400 lg/ml, Sigma, USA) were added and incubated at 37 _C for 30 min. Sample analysis was performed by flow cytometry (Coulter Epics, XL, UK). The cell cycle phase distribution was calculated from the resultant DNA histogram using Multicycle AV software (Phoenix Flow System, San Diego, CA, USA). The apoptotic cells were observed as a subdiploid or'pre-G1'peak. RNA isolation After RNase-free DNase (Ambion) treatment for 15 min at room temperature, RNA was further purified with RNease RNA Isolation Kit (Qiagen, Valencia, CA). Total RNA was reverse transcribed in 20 ll reaction system using Superscript First-Strand Synthesis Kit for RT-PCR (Ambion) under conditions described by the supplier.Assay of transfection rate by Flow cytometric and fluorescence microscopy Southern blot of AQP1Statistical analysis.Data statistical analysis was performed using the SPSS statistics software package (SPSS, Chicago,IL). All results were expressed as means_SD, and p<0:05 was used for significance. 3. ResultsBoth RNAi and acetazolamide down-regulate the AQP1 gene expression in Hep-2 cell lineMorphological changes induced by siRNAs and acetazolamide 4.DiscussionThe decrease in cell volume during apoptosis is a highly conserved phenotype and numerous studies have demonstrated the importance of ion efflux, particularly K+, to this event. While loss of intracellular K+ is concomitant with the acetazolamide, it also occurs measurably before cytochrome C release, caspase-3 activation and DNA degradation. The loss of K+ as a cell dies establishes an osmotic gradient that draws water out of the cell,forcing it to shrink. In the present study, we have presented evidence in apoptotic rat granulosa cells that water follows this gradient primarily through proteinaceous water channels known as AQPs. Inhibition of AQP-mediated water movement not only blocked the acetazolamide, but alsoprevented downstream apoptotic characteristics, suggesting that AQP-mediated water loss is not peripheral to the apoptotic cascade, but an essential component in this step-wise process. The effects of AQP inhibition on the acetazolamide and DNA degradation were neither cell-type nor signal specific and were not mediated by effects on survival pathways. Importantly, overexpression of AQP 1 in Hep-2 cells increased the amount of apoptosis, indicating that the water permeability of the plasma membrane during the acetazolamide may be a rate- limiting step in the early stages of cell death.A reduction in intracellular K+ concentration is essential for activation of apoptotic enzymes, but a loss of ions does not necessarily imply a reduction in concentration, if the loss is matched by an efflux in water and a reduction in intracellular size. We have shown within the same population of cells that the subpopulations that have undergone the acetazolamide have very low water permeability, while the non-apoptotic cells have normal permeability. This change in water permeability during the acetazolamide would thus allow the loss of K+ to be unbalanced from the loss of water and facilitate a decrease in intracellular K+ concentration. We have further shown that AQP 1 in thymocytes is not proteolytically cleavedor removed from the cell membrane following apoptosis, suggesting that this water channel is inactivated by a post-translational, non-degradative mechanism.AQP expression has never been studied in these cells and a deficiency of water channels may be one explanation for the apparent lack of an acetazolamide and ability to die independent of it. All other inducing agents used with these cells, or any other cell types, displayed a strict correlation between the acetazolamide and activation of apoptotic enzymes. Since the acetazolamide, and the subsequent decrease in potassium concentration, is absolutely critical for the activation of apoptoticenzymes, it is reasonable to infer that all cells which express AQPs will utilize one or more of these water channels to mediate water loss during apoptosis. One interesting scenario is cells which do not appear to express AQPs. As more family members become known, a comprehensive analysis will be needed to ensure these cells do not express any water channels.Since the acetazolamide is a nearly universal characteristic during apoptosis, it is also possible that incells, which do not normally express AQPs, one or more of the AQP family members may be upregulatedin response to an apoptotic stimulus. Conversely, they may rely exclusively on simple diffusion of water across their plasma membranes in order to facilitate the acetazolamide. However, based on the rapid and regulated nature of the acetazolamide, simple diffusion through the lipid bilayer seems unlikely.Currently, there is debate in the literature concerning the effect of acetazolamide on K+ channels.Ongoing studies in our laboratory are focused on the mechanisms of inhibition of these water channels following the acetazolamide.The results of this study significantly strengthen the importance of the acetazolamide to apoptosis and suggest for the first time that the water loss occurs through specific proteinaceous water channels. In addition, we have shown that AQP inhibition suppresses the appearance of apoptotic characteristics while overexpression of AQPs enhances the rate of intrinsically induced cell death, suggesting that the water permeability of the plasma membrane may alter the cell's abilityto die. Furthermore, while functional AQPs may be necessary for a cell to shrink and die, theyare also inactivated after the acetazolamide, presumably to allow intracellular K+ to be reduced to levels conducive to activation of the apoptotic program. This inactivation of AQPs after the acetazolamide seems to be accomplished through means other than degradation of these water channels.