With all the extending of GD time, the phosphorylation degree of AKT decreased and disappeared immediately after h in manage cells. On the other hand, from the Grp overexpression group, the phosphorylation level of AKT improved soon after GD for h, then returned to its starting up degree immediately after h and commenced to decrease from h. The expression degree of AKT did not change in management and Grp overexpression cells. Each one of these success showed that Grp overexpression stimulated AKT phosphorylation underneath GD problems. Results of the PIK inhibitor on AKT phosphorylation in Grp overexpression cells AKT is usually phosphorylated in two means: PIK dependent and PIK independent. AKT can be a downstream effector molecule of PIK and is thought to mediate numerous biological actions toward anti apoptotic responses. It had been an intriguing question regardless if Grp activated AKT below GD ailments as a result of PIK. LY, the inhibitor of PIK, was administered h ahead of GD in Grp overexpression cells. Western blot end result showed that, soon after pretreatment with LY, the phosphorylation degree of AKT disappeared in Grp overexpression cells underneath regular disorders compared using the dimethyl sulfoxide pretreated Grp overexpression cells.
This indicated that the activation of AKT was mediated by PIK below usual ailments. Interestingly, following h of GD treatment, the phosphorylation level of AKT during the LY group substantially increased and maintained for h. ROCK inhibitors Quantitative final results indicated that the alteration in the phophorylation degree of AKT was related in DMSO and LY groups below the GD conditions. LY had no result on AKT protein expression underneath the two normal and GD ailments. Each one of these effects illustrated that LY therapy had no results to the activation of AKT by Grp overexpression underneath GDs. This very important finding advised that Grp could modulate AKT activation as a result of one more way. Even though LY did not influence the activation of AKT by Grp overexpression underneath GD circumstances, the number of Bax favourable cells and apoptotic cells within the LY group decreased considerably after GD for h compared to the DMSO group.
Past examine showed that Grp overexpression suppressed apoptosis induced by GD in Pc cells by means of inhibition of Bax conformational peptide synthesis selleck adjust, and present benefits showed that this result was blocked when Grp overexpression cells have been pretreated with PIK inhibitor LY. These information indicated that PIK participated within the suppression of Bax conformational change and apoptosis by Grp overexpression under GDs. The outcomes also suggested that PIK participated within the course of action in which Grp overexpression inhibited Bax conformational modify and subsequent apoptosis by way of other proteins but AKT. We desired to illustrate these unknown occasions. Grp and AKT never physically interact A previous examine showed that AKT can type a complicated with a number of molecular chaperones , this kind of as Hsp and Hsp.
In addition, COX induced BCL expression, doxorubicin resistance, and alterations within the cancer initiating cell phenotype. Products AND Approaches Cell Lines and Servicing The cell line MCF Tet On was obtained from Clontech and maintained at C in RPMI medium supplemented with Tet system authorized fetal bovine serum, units mL penicillin, and g mL streptomycin in a humidified CO environment. The cell line was stably transfected with the plasmids pTREpur COX or pTREpur COX GFP as described previously . Mammosphere Culture Long term mammosphere cultures had been established from trypsindissociated adherent cultures or from uncommon floating cells current in adherent cultures. Mammospheres were cultured in Mammary Epithelium Basal Media supplemented with B , mM L glutamine, units mL penicillin, g mL streptomycin, ng mL bFGF, ng mL EGF, and g mL heparin, as described by Dontu et al The mammospheres had been cultured on minimal attachment cell culture dishes or untreated plastic dishes that are utilised for agar plates. Mammosphere cultures have been passaged every days.
Cells in mammosphere cultures have been collected by centrifugation at , rpm for min at C and then incubated with . trypsin at C for min. Trypsinization was stopped by the addition of mL of MEBM with dietary supplements described over, and centrifuged at , rpm for min at C. The cell pellet was resuspended in mL medium and filtered as a result of a pre wetted m filter to ensure a single cell suspension. Following filtration, the cells have been centrifuged, and the pellet resuspended in L medium. An aliquot order Motesanib was mixed with an equal volume of trypan blue and counted on a hemacytometer to find out the number of cells. Cells were plated in the , to , cells per mL assortment. Western Immunoblotting Expression of BCL protein was detected by Western blot examination. Cell lysates have been ready by getting rid of the media in the cells and washing with sterile phosphate buffered saline . Cells were then lysed by scraping in L of sample buffer per mm dish, containing mM Tris HCl pH sodium dodecyl sulfate, mercaptoethanol, glycerol, and .
bromphenol blue and quickly boiling for min. Cell lysate was centrifuged at rpm for min at room temperature and also the supernatant was stored at C. Samples with equal amounts of protein had been separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis gradient gels with a single lane designated for Bio Rad Dual Shade molecular bodyweight marker then transferred to a . m nitrocellulose membrane. To reduce nonspecific binding, the nitrocellulose Erlosamide membranes were blocked with ECL Advanced Blocking Agent in Tris buffered saline with . Tween in advance of currently being incubated with all the BCL monoclonal antibody clone D . The nitrocellulose membranes have been then incubated with all the secondary antibody in blocking option, washed a variety of times, and produced through the ECL Advance Western blot detection reagents .
Hence our investigation of human intestinal microvasculature endothelial cell will provide a novel method to define the spectrum and treatment of radiation induced gut damage. ROS plays an important purpose in irradiation induced endothelial cells apoptosis, production of ROS by radiation promotes endothelial cell dysfunction and death by which apoptotic endothelial cells drop their interaction using the matrix. Decreased endothelial cells amount in rat’s brain exposed to irradiation has been proven . Radiation induced cellular dysfunction is definitely an inactive injury; cells morphologically are regular but functionally these are defected . Irradiated endotheliumlacks the ability to regulate thrombogenic, inflammatory and coagulation processes months to years soon after irradiation . Substances that raise the SOD expression h following irradiation can substantially cut back the malignant transformation and metastatic probable of irradiated tumor cells . Rosenthal and colleague have shown that orally out there EUK was just about the most energetic mitigator together with the lowest cytotoxicity and highest catalase action .
EUK hts screening , the salenMn complicated alsomitigated radiation induced endothelial cell apoptosis with the highest potency and lowest cytotoxicity . These authors have demonstrated that EUK lowered radiation induced apoptosis by when administered h or much more right after irradiation. They concluded that although it is actually unlikely that SOD mimetics inhibit the radiationinduced DNA damage right, it truly is probable they can lower the prolonged lasting radicals activity and scavenge new radicals extended immediately after irradiation .Mitigating agents, this kind of being a recombinant adenovirus expressing SOD, continues to be shown to mitigate the skin lesions in irradiated mice . In the clinical trial of sufferers getting complete entire body irradiation, an angiotensin converting enzyme inhibitor in conjunction with bone marrow transplantation mitigated the renal failure . These uncovering indicate that absolutely free radical scavengers and anti inflammatory compounds have a important clinical end result when administered soon after irradiation.
Within the current research, we have now demonstrated that irradiation of HIMEC resulted in elevated expression in the professional apoptotic molecules Bax, caspase and inhibition of pro survivalmolecule Bcl expression. Bcl is definitely an anti apoptotic protein Sodium Monofluorophosphate situated from the mitochondrial membrane, which binds and inactivate the pro apoptotic protein Bax, blocks the release of cytochrome c through the mitochondria to your cytosol and prevents caspase activation, which bring about activation of apoptotic pathway . Frequent ways while in the method of programmed cell death would be the release of cytochrome c, greater Bax, and decreased Bcl protein ranges . Within this research, we’ve demonstrated that irradiation resulted in NF?B activation and enhanced the Bax Bcl ratio and caspase activation in HIMEC.
Non precise binding was blocked by pre incubating cells with rat IgG, and appropriately labeled isotype controls had been used routinely to proper for possible nonspecific fluorescence. Listmode information have been collected on a Coulter Epics XL movement cytometer and analyzed using WinList program . EGCG minimizes catenin TCF dependent reporter activity and catenin protein expression in HEK cells Physiologically pertinent concentrations of EGCG were proven previously to inhibit TOPflash reporter action in HEK cells transfected with TCF plus WT catenin . To extend these findings, we transfected HEK cells with TCF plus several catenin mutants, and taken care of the cells with or M EGCG . Concentration dependent inhibition of reporter activity was observed in cells transfected with TCF plus WT DG, DN, DY or SY catenin . The inhibition was most striking in cells transfected with TCF plus catenin, but in all circumstances M EGCG lowered reporter action to a degree comparable with that viewed for empty vector. Endogenous catenin protein was detected at very low ranges inside the total cell lysates of HEK cells handled without any DNA or vector alone .
Catenin amounts had been increased markedly in cells transfected with TCF plus SY catenin , and there was a concentrationdependent reduce in catenin protein expression with , and M EGCG therapy. This reduce in protein expressionwas observed with monoclonal anti catenin antibody, which detects complete catenin, and in addition with anti myc tag antibody, which is unique for that myc tag in transfected catenins. These findings with SY catenin have been recapitulated in HEK cells treated with TH-302 EGCG and WT DG, DN, or DY catenins EGCG decreases nuclear, cytoplasmic and membrane related catenin protein expression, also as membrane associated E cadherin As the catenin protein amounts have been attenuated by EGCG in complete cell lysates, we next investigated the effect of EGCG remedy on catenin expression within the nucleus, cytosol, and membrane related fractions of HEK cells.
As anticipated, in cells transfected with TCF plus SY catenin, cytoplasmic and nuclear catenin protein ranges were strongly improved in contrast with cells transfected with empty vector . There was a concentration dependent lessen in catenin protein expression each while in the cytosol and nucleus of Posaconazole cells taken care of with or M EGCG . Inside the membrane connected fractions, EGCG also attenuated catenin expression ranges, likewise as E cadherin . After normalizing to actin, densitometry analyses indicated that nuclear and membrane connected catenin levels, also as membrane related E cadherin, had been decreased by following therapy with M EGCG, whereas cytoplasmic cateninwas lowered by .
Then, we administered muscimol and baclofen, LY to detect Akt and Ask phosphorylation at min of reperfusion. The cytosolic fraction was detected working with immunoblotting assay. As proven in Fig co applied muscimol and baclofen had a powerful action to Akt activation, Ask phosphorylation, and LY markedly surpassed this result. Rats pretreated with car or LY alone did not have an effect on the Akt and Ask phosphorylation. Furthermore, the protein ranges had been not impacted through the drugs or car. These final results showed that co utilized muscimol and baclofen could activate PI K Akt pathway through phosphorylation of Request serine Co utilized muscimol and baclofen inhibited Inquire downstream MKK and JNK activation within the hippocampal CA region To check the result of co utilized with muscimol and baclofen on Ask downstream target MKK, MKK, two JNK kinases and JNK activation, the experiment was carried out in the next. MKK and MKK had two activated peak at min and day of reperfusion in the hippocampal CA region in our former reports . Consequently, we pick min and day after reperfusion to investigate MKK and MKK activation using immunoblotting assay.
JNK was activated with two peaks occurring at min and days after I R within the hippocampal CA region, the latter activation peak was regarded to account for apoptosis . We selected day soon after reperfusion to Sirolimus investigate JNK activation determined by the consideration that transient brain ischemia leads to cell death of the hippocampal CA pyramidal neurons beginning e days following reperfusion . The study was carried out by immunoprecipitation with p JNKs antibody and after that immunoblotting with JNK antibody. As proven in Fig we discovered that co applied muscimol and baclofen appreciably diminished MKK, MKK and JNK activation at min or day of reperfusion, respectively. It advised that the enhanced Akt activation may perhaps have an effect on Request JNK kinases activation Co applied muscimol and baclofen decreased JNK substrate c Jun and Bcl phosphorylation during the hippocampal CA region We up coming addressed whether or not co applied muscimol and baclofen attenuated the activation of JNK nuclear substrate c Jun and the phosphorylation of non nuclear substrate Bcl in I R.
Nuclear extracts in the CA regions have been subjected to immunoblotting with anti p c Jun antibody or standard anti c Jun antibody, and cytosolic extracts was detected with anti p Bcl antibody MEK Inhibitors immediately after co utilized muscimol and baclofen. Latest reviews had examined that c Jun and Bcl was activated and reached its phosphorylation peak at h and h of reperfusion, respectively . As shown in Fig the drug prevented the elevated c Jun and Bcl phosphorylation observed at h and h immediately after min ischemia.
The docking examination was performed with LigandFit interfaced with Discovery Studio H bondings of the two the aminopyridine moiety with hinge residues M and P as well as the acetyl group with Y contributed to the robust interaction between KRC and c Met KRC inhibits the c Met signaling pathway and proliferation of cancer cells expressing c Met To assess the particular inhibitory impact of KRC on c Metdependent cancer cells, we applied three distinctive cell lines . When the cells were exposed to KRC , KRC particularly inhibited p c Met expression in cancer cells that expressed c Met . c Met continues to be reported to regulate a range of diverse cellular processes this kind of as proliferation and differentiation by modulating the PIK Akt mTOR and Ras Mek signaling pathways . Thus, we determined regardless of whether KRC inhibited the expression of downstream molecules from the PIK Akt mTOR and Ras Mek signaling pathways, as well as p Akt, p mTOR, p pSK, p Raf, p Mek, and p Erk , to elucidate the mechanism accountable for c Met inhibition by KRC . Our outcomes showed that KRC inhibited the expression of p Akt, p mTOR, p pSK, p Raf, p Mek, and p Erk in MKN gastric cancer cells in a dose dependent manner.
We then compared growth costs of MKN , SNU and MKN cells treated with KRC and fluorouracil , a well-known gastric cancer drug, to investigate the inhibitory impact of KRC on c Met dependent cancer cell growth. Interestingly, KRC significantly FTY720 inhibited cell growth compared to FU whilst resulting in only minimal levels of cytotoxicity in Hs usual gastric cells. A lot more importantly, the inhibition of cell growth by KRC was greater in cancer cells expressing c Met than ones that did not . These outcomes showed that KRC might be a possible inhibitor of c Met KRC induces apoptosis and cell cycle arrest in MKN gastric cancer cells Induction of apoptosis by KRC was evaluated by DAPI and TUNEL staining to characterize nuclear morphology. As proven in Fig. A, cells treated with lM KRC presented morphological functions of apoptotic cells, such as bright nuclear condensation and perinuclear apoptotic bodies, when stained with DAPI.
Apoptosis promoted by KRC was confirmed Salinomycin by TUNEL staining, which was applied to detect DNA fragmentation. We also carried out movement cytometric evaluation to watch adjustments of cell cycle profiles induced by KRC . Information from this research uncovered that the KRC treatment increased the amount of cells inside the subG phase, indicating a rise in apoptosis . Moreover, the amounts of Bcl , Bax, and cleaved caspase just after KRC treatment method have been measured by Western blotting. As expected, KRC elevated the expression of cleaved caspase and Bax whereas reducing the expression of Bcl in MKN gastric cancer cells . These findings showed that KRC could induce the apoptosis of MKN gastric cancer cells. Considering apoptosis and growth are linked to cell cycle progression, we next assessed whether KRC promoted cell cycle progression.
They were then dissected underneath a microscope. The salivary gland was excised and transferred into . M phosphate buffer solution, pH and kept within the exact same alternative at C. pairs of horsefly salivary glands were homogenized in . M phosphate buffer answer, pH .andcentrifuged atg for min.The supernatantwas termed as salivary gland extract and was lyophilized Fractionation of horsefly SGE The lyophilized SGE sample was dissolved in ml . M phosphate buffer answer, pH . and after that was utilized to a Sephadex G gel filtration column equilibrated with . M phosphate buffer, pH . in line with our prior strategies. Elution was carried out with the identical buffer, collecting fractions of . ml. The absorbance with the eluate was monitored at nm. Each fraction was subjected to anti angiogenic testing as indicated in experimental protocol. The protein peaks containing examined pharmacological pursuits have been pooled and purified even further by cationic exchange column illustrated in Fig.
FTY720 A Structural examination The amino acid sequences with the N terminus and partial interior amino acid fragments recovered through the trypsin hydrolysis have been determined through the automated Edman degradation on an Utilized Biosystems pulsed liquid phase sequencer, model SDS Polyacrylamide Gel Electrophoresis analysis and protein concentration determination SDS Webpage was carried out underneath decreasing ailments. Protein samples had been loaded onto a polyacrylamide gel. Protein bands had been observed after utilizing a regular Coomassie blue stain. The molecular markers of Escherichia coli b galactosidase , bovine serum albumin , chicken ovalbumin , porcine lactate dehydrogenase , E. coli Rease BSPI , bovine b lactoglobulin and chicken lysozyme were obtained from Fermentas . The protein concentration was determined by a protein assay kit with BSA as being a traditional. PCR based mostly process for large stringency screening of DNA libraries was employed for screening and isolating the clones with some modifications.
Two pairs of primers had been made use of to screen the cDNA sequence, In which the tabRTS will be the unique primer gg tt gt ag gg ta gc during the sense direction constructed as outlined by the peptide sequences established by Edman degradation, tabRTS Tivozanib selleck chemicals will be the specific primer ta cc ct ac aa cc at while in the anti sense direction as well as the Primer II A is described in ??Wise cDNA synthesis??. The DNA polymerase was Benefit polymerase from Clontech . The PCR conditions were: min at C, followed by cycles of s at C, s at C, s at C. DNA sequencing was performed on an Applied Biosystems DNA sequencer, model ABI PRISM Chicken chorioallantoic membrane assay in vivo A chorioallantoic membrane angiogenesis assay was followed since the described technique . Briefly, fertilized domestic chick eggs have been incubated at C inside a humidified incubator for 10 days.
The difference inside the frequencies of Akt activation in between FISH constructive and FISH negative groups was statistically substantial . Lastly, among cases exhibiting disomy, Akt activation was found in instances . As a result, a substantial fraction of tumors devoid of AKT gene obtain also exhibited Akt activation. General, AKT and or AKT gene gains are frequently accompanied by overexpression and activation of Akt, and all FISH optimistic situations revealed particularly higher degree of T Akt overexpression and activation Correlation amongst AKT, AKT, and EGFR gene aberrations EGFR mutation was located in of instances of NSCLC in which AKTs status was characterized . All situations exhibited optimistic p Akt staining, and of those showed intense nuclear staining. Even so, none of those situations exhibited amplification or high level polysomy of chromosome or , while and instances, respectively, of low level polysomy had been observed. For that reason, EGFR mutation is related to activation of Akt and possibly with its nuclear localization, but this occurs in a manner reciprocal to FISH positive ATK gene gain.
Subsequent, EGFR gene gains were found in of instances, such as instances with EGFR amplification and instances of higher level and of low level polysomy of chromosome . Even though amongst these circumstances, Akt was overexpressed in circumstances and activated in instances, none with the circumstances scored as EGFR FISH good showed FISHpositive Tivozanib gene gains of AKT or AKT. In circumstances exhibiting chromosome polysomy, and situations showed low level polysomy of chromosome or , respectively. Therefore, FISH good gene gains in EGFR and AKTs also occurred in a reciprocal manner, while low level polysomy occurs collectively Clinicopathologic analysis We statistically analyzed these outcomes in comparison with the clinicopathologic profiles. IHC expression of p Akt and lymph node metastasis was correlated , suggesting that Akt phosphorylation is actually a feasible predictive factor for metastasis. Having said that, FISH good gene gains of AKT or AKT did not correlate with lymph node metastasis or with other clinicopathologic elements.
In addition, neither of Akt overexpression or activation was correlated with all the tumor size , histologic type , or histologic differentiation . Lastly, IHC positivity, protein levels evaluated by immunoblot, or aberration of AKT or AKT revealed no substantial correlation with survival prices Discussion To date, several oncogenes have already been shown to undergo amplification as a mechanism of cancer development. Those incorporate ERBB in breast cancer, AKT in ovarian cancer, and EGFR in NSCLC . Akt Sinomenine is now identified to become a central node among diverse signaling pathways and plays crucial roles in fundamental physiologic functions and in tumorigenesis . Certainly, you will find quite a few literature reports describing frequent Akt hyperactivation in many different tumors .
Such techniques will let visualization from the D morphology of nanoscale cellular structures, and was used by Huang et al to image microtubules and clathrin coated cellular pits. Cancer diagnostics is more and more reliant upon measurement of various biomarkers at either the genotypic, mRNA or protein degree ideally. There is substantial curiosity within the chance of applying QDs for this goal. Caldwell et al. implemented spectral imaging to measure, in a renal cell carcinoma tissue microarray, typical intensity of QD antibody staining for MDM and actin, demonstrating capability of your procedure to distinguish cancer from typical adjacent tissue. Bostick et al. proposed utilization of QDs for detection of as much as five biomarkers per slide, from which alot more biomarkers could possibly be measured employing a number of slides every stained with five various biomarkers to measure, by QD ISH, nine prognostic genes in AML; unpublished data . Bostick implemented a custom constructed picture evaluation strategy to quantify expression of each biomarker, and also a workflow for your examination, just like that proposed by Byers et al.
and Tholouli et al It will likely be crucial for clinical application that this kind of techniques are robust, standardised, streamlined, quickly, simple to use, and, ideally, automainhibitors; the procedure described by Bostick et al. took seven hours to analyze six biomarkers. Muller et al. created a FISH protocol capable of visualisation of as much as 6 various DNA probes, applying a mixture Quizartinib 950769-58-1 kinase inhibitor of QDs and conventional fluorophores, which, in Pi microscopy has the likelihood of optical resolution down to nm. Many of these applications demand sophisticated image evaluation for image deconvolution, which needs to an extent restricted broad uptake of the multiplex capability of QDs. Tholouli et al Byers et al Sweeney et al and colleagues have extensively explored using QDs for measurement of biomarkers in clinical tissue. In two linked papers Byers et al. and Tholouli et al.
demonstrated multiplex QD ISH in archival clinical tissue samples showing photostability of QDs in excess of a period of months, with each other with preliminary semi quantitative utilization of QD fluorescence intensity to measure FASmRNAexpression in fixedLNCaPcells exhibiting beneficial correlationwith parallel genuine time PCR mRNA measurement. Tofacitinib Tholouli et al. comprehensively examined use of the procedure in EDTA decalcified formalin fixed bone marrow trephine samples, applying strict ISH controls, and demonstrating triplex ISH for XIAP, survivin and Bcl; comparison of expression values obtained by single and triplex ISH showed really good concordance Dwell Cell Imaging There has been substantial interest in utilization of QDs for localisation and monitoring of molecules in residing cells, both in vivo or in vitro, and this discipline continues to broaden at a higher charge than in situ studies.
The significantly less well differentiated human endometrioid cancer AN was obtained from American Style Culture Assortment . Ark, Ishikawa, and AN cells had been grown in RPMI , MEM , and F media, respectively. Each of the media were supplemented with fetal calf serum , g ml streptomycin, units ml penicillin, and mM glutamine. Cells were maintained at C in an environment containing CO and humidity. Oxamflatin and HDAC inhibitor are products of Calbiochem . Antibodies against poly ADP ribose polymerase , Caspase , and caspase were bought from Roche . Rabbit polyclonal antibody for actin was obtained from Santa Cruz Biotechnology . Western blot analysis Ark, Ishikawa, and AN cells have been taken care of with oxamflatin or HDAC Inhibitor as indicated from the figure legends. Cellular proteins were isolated and resolved in SDS Webpage and electro transferred to Immun BlotTM PVDF membrane . The membranes had been blocked for h in PBS buffer containing . Tween and nonfat dried milk. Antibodies against PARP, caspase , and caspase were diluted following the manufacturer’s recommendations.
Main antibody binding was carried out at C overnight with frequent shaking. The anti rabbit or anti mouse antibodies Beta-catenin inhibitor selleck labeled with horseradish peroxidase have been employed at : dilutions. Secondary antibody binding was carried out at space temperature for h. Chemiluminescence detection was carried out with all the ECL plus Western Blotting Detection Strategy . The blots had been re probed with actin antibody and also the final results provided loading controls. Cell growth assay Ark, Ishikawa, and AN cells had been plated at confluence in cmdishes one particular day earlier and counted because the base line level. The cells were treated with Oxamflatin , HDAC I , or DMSO solvent as control. The cell numbers were counted thereafter the moment every day for consecutive days. Floating cells have been washed away and only the living cells have been detached from dishes by trypsin digestion and counted.Development curveswere constructed for individual experimental groups.Average and standard error of every time pointwas calculated depending on three or additional parallel experiments.
Apoptosis assays The Annexin V FITC kit was employed to label apoptotic cells. Cells treated with oxamflatin and HDAC Screening Library selleck I have been washed with cold PBS and diluted in Annexin binding buffer at a concentration of cells ml. cells had been mixed with l of Annexin V FITC stock answer and the binding carried out at room temperature for min inside the dark. The samples were diluted to l and instantly analyzed by movement cytometry for apoptotic cells. For nuclear staining, cells had been washed with cold PBS and fixed with paraformaldehyde, and stained for min with Hoechst dye . The stained cells were washed twice with . triton X , PBS, and observed beneath a fluorescence microscope. Apoptotic cells with condensed or fragmented nuclei have been counted. The results have been presented as percentage of apoptotic cells in total population.