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Nuclear Factor of Activated T cells (NFAT), a family of transcription factors, has been implicated in many cellular processes, including some cancers. For the first time, the present study characterizes the role of NFAT3 in doxorubicin (DOX) mediated apoptosis, migration, and invasion in SNB19 and U87 glioma cells. This study demonstrates specific knockdown of NFAT3 results in a dramatic inhibition of the apoptotic effect, induced by DOX, and favors cell survival. Inhibition of NFAT3 activation by shNFAT3 (shNF3) significantly downregulated TNF-α induction, its receptor TNFR1, caspase 10, caspase 3 and PARP, abrogating DOX-mediated apoptosis in glioma cells. DOX treatment resulted in NFAT3 translocation to the nucleus. Similarly, shNF3 treatment in SNB19 and U87 cells reversed DOX-induced inhibition of cell migration and invasion as determined by wound healing and matrigel invasion assays. Taken together, these results indicate that NFAT3 is a prerequisite for the induction of DOX-mediated apoptosis in glioma cells.
Despite aggressive surgery, radiotherapy, and chemotherapy, malignant brain tumors remain a therapeutic challenge . An inherent resistance to apoptosis may account for the low success rate of glioma treatments [2, 3]. However, because the effector arm of the apoptotic pathway often remains intact, these cells can be induced to undergo apoptosis by direct stimulation of the cell death receptors in the TNFR superfamily .
Nuclear Factor of Activated T cells (NFAT) is a family of transcription factors initially discovered in T cells, which play a crucial role in immunity. More recently, however, the NFAT family has been implicated in many cellular processes including malignancies . Further studies indicate that the NFAT signaling is involved in the regulation of cell growth and development in a wide variety of different tissues and cell types [6, 7]. NFAT3 is one of the five known isoforms of this family of transcription factors, which are known to be involved in activity-dependent gene expression, linking surface receptor stimulation to nuclear events, and ultimately altering the fate of the cell. Unlike NFAT1, -2 or -4, NFAT3 is expressed in non-immune tissues, including heart, brain, and breast [8, 9] where it is thought to influence such varied processes as cardiac hypertrophy, learning and memory, adipocyte differentiation, and breast cancer development [10–15].
NFAT1, -2, -3, and -4 are cytosolic proteins, constitutively expressed in resting cells, and their activation is regulated by calcium and the Ca2+/calmodulin-dependent serine-phosphatase, calcineurin . Upon stimulation, NFATs are dephosphorylated by calcinuerin, and they are then translocated to the nucleus where they bind to consensus DNA sites and control the expression of target genes including: IL-2, IL-4, IL-5, IL-13, IFN-gamma, TNF-α and COX-2 [17–21]. Activation of NFAT is sensitive to calcineurin inhibitors and immunosuppressive agents, such as cyclosporin A and FK506 [16, 22].
DOX is an anti-tumor drug that is being widely used in the treatment of a broad spectrum of cancers [23, 24]. The pro-apoptotic effects of DOX are well established, as it can induce cell death via DNA damage through topoisomerase II inhibition, by inducing expression of the tumor suppressor protein, p53, and by free radical generation through redox reaction [25–27]. DOX has shown a marked cytotoxic effect against malignant glioma cells in vitro . DOX induced up regulation of TNF-α, uPA, IL-8, and MCP-1 in a number of lung cancer cells was reported by Niiya, et al.  (2003). Highly enhanced anti-tumor effect was also reported with systemic administration of TNF-α in combination with DOX for lung tumors [30, 31]. Kalivendi, et al.  documented that DOX activated the transcription factor NFAT, leading to the upregulation of Fas/Fas L-dependent apoptosis through a calcium/calcineurin-signaling pathway in cardiac cells.
So far, there has been no comprehensive study published on the role of DOX in NFAT activation in cancer cells. Given the importance of cell cycle, apoptosis, and angiogenesis phenomena in the development of cell malignancies, it is of considerable interest to understand the mechanisms by which NFAT affects cell growth, differentiation and function. In the present work, we studied the role of NFAT3 in DOX-mediated apoptosis in SNB19 and U87 glioma cell lines. The hypothesis that DOX causes apoptosis in these cells was tested by FACS analysis and DNA fragmentation. For the first time, we have shown the role of the NFAT3 isoform in SNB19 and U87 glioma cell lines in DOX-mediated cellular apoptosis.
pSilencerCMV3.1 (Ambion) was used to construct a vector expressing shRNA for NFAT3 downstream of the cytomegalovirus promoter (Fig. 1A). Exons 4, 5, and 6 were targeted simultaneously by including 21 bases corresponding to each exon. The following was used for the NFAT sequence: aatggatccGCCACTGACCCTACAGATGTTCAAGAGACATCTGTAGGGTCAGTGGCTTCAAGAGATTCAAGAGAGGGTGAGACGGACATCGGGTTCAAGAGACCCGATGTCCGTCTCACCCTTCAAGAGATTCAAGAGACTGGTACTGACTGGCTCCATTCAAGAGATGGAGCCAGTCAGTACCAGaagcttccg. It is 93 bases in length with BamHI and HindIII sites incorporated at the ends, and with a nine base loop region between each 21 base molecule. The oligo was self-annealed in 6X SSC using standard protocols and ligated on to the BamHI and HindIII site of a pSilencerCMV3.1 vector. This yeilded a shRNA expression plasmid for NFAT3, which was designated shNF3. BGH poly-A terminator served as a stop signal for RNA synthesis.
For this study, we used the established human glioblastoma cell lines SNB19 and U87. Cells were grown in Dulbecco's modified Eagle’s medium, supplemented with 1% glutamine, 100 µg/mL streptomycin, 100 units/mL penicillin, and 10% fetal bovine serum (pH 7.2–7.4) in a humidified atmosphere containing 5% CO2 at 37°C. Cells were sub-cultured every three to five days. Cells were grown in 100 mm dishes or six well plate for all treatment conditions and on eight-well chamber slides for immunocytochemistry and tunel assays.
Scrambled vector (pSV) and shNF3 vector were transfected into SNB19 and U87 cells independently by Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) as per the manufacturer’s instructions. Briefly, the cells were cultured in a 100 mm dish to 85–90% confluence. Then, 21 µL of lipofectamine (diluted in 100 µL of serum-free medium) was added dropwise to 7 µg of plasmid DNA (in 100 µL of serum-free medium). This mixture was incubated for 30 min and was then used to transfect each plate in the absence of serum. After six hours, the medium was replaced with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and the cells were incubated for another 12 to 24 hours. DOX (1µM/L) was added to these cells in the absence of serum and incubated for an additional 24 hours. To study the role of TNF-α in DOX induced NFAT3 mediated apoptosis, cells were also treated with TNF-α protein (200 U/ml) for 24 h.
A total of 2×105 of SNB19 and U87 cells were independently cultured in each well of six-well plates to 80% confluence. After transfection with either shNF3 or pSV and subsequent treatment with DOX, the cells were extracted with Trizol reagent (Invitrogen, USA), and the total RNA was isolated as per the manufacturer’s instructions. One µg of total RNA was reverse-transcribed (Superscript II, Invitrogen), and the cDNA was subjected to PCR amplification targeting NFAT3 (forward primer 5’-AGGCCTACAGCCCCAGTG-3’ and reverse primer 5’-CGCCCATTGGAGACATAAAA-3’) and GAPDH (forward primer 5’-AGCCACATCGCTCAGACACC-3’ and reverse primer 5’-GTACTCAGCGGCCAGCATCG-3’). The PCR cycling conditions were: 30 cycles of 95°C for 30 sec, 55°C for 30 sec, 72°C for 45 sec and a final extension of 72°C for 10 min.
SNB19 and U87 cells were washed with ice-cold DPBS 48 hours after treatment and resuspended in 150 µL of radioimmune precipitation assay buffer (20 mmol/L Tris-HCl (pH 7.4), 2.5 mmol/L EDTA, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS, 100 mmol/L NaCl, and 100 mmol/L sodium fluoride) containing 1 mmol/L of sodium vanadate, 10 µg/mL of aprotinin, and 10 µg/mL of leupeptin. Cells were then homogenized using five ultrasonic pulses of one second each. The lysate was centrifuged at 12,000 × g for 10 min at 4°C to remove cellular debris.
Equal amounts of protein were separated on 12% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The membranes were probed overnight with antibodies against NFAT3, phospho-NFAT3, AKT, NFAT4, phospho-AKT, JNK, phospho-JNK, Bcl2, TNF-α, Cyclin E, Cyclin B, Caspase-10, Caspase-3, PARP, and GAPDH (Santa Cruz, CA, USA). The membranes were subsequently washed three times with PBS to remove excess primary antibodies, incubated with appropriate HRP-conjugated secondary antibodies, and then developed according to enhanced chemiluminescence protocol (Amersham, Arlington Heights, IL).
NFAT expression and localization were observed by immunocytochemistry assay according to published standard protocols. Human glioma cells SNB19 and U87 grown in eight-well chamber slides were treated as described previously and were fixed (4% paraformaldehyde, 10 min at room temperature), permeabilized (cold methanol for 2 min), and rehydrated (PBS for 10 min). PBST containing 2% BSA was used for blocking the cells for one hour followed by a two-hour incubation with anti-NFAT3 antibodies (Santa Cruz, CA, USA) at a dilution of 1:300 in PBST containing 2% bovine serum albumin and 1.5% horse serum, followed by a final incubation with Texas Red conjugated secondary antibody (1:1000 in PBS/2% bovine serum albumin, 0.5% tween 20) for one hour. NFAT expression was visualized by fluorescence microscopy (Olympus IX71) and photographed.
SNB19 and U87 cells were cultured in six-well culture plates at a concentration of 2×105 cells/well. After a 24-hour incubation period, cells were transfected with shNF3 vector or pSV. Untreated cells were also cultured under similar conditions and served as the control. Genomic DNA was extracted as per protocol described by Huang, et al. . In brief, the cells were resuspended in 50 µL of PBS, fixed in 1 mL of ice cold 70% ethanol and stored at −20°C overnight. After fixation, the mixture was centrifuged at 6000 rpm at 4°C for 5 min, and the pellet was resuspended in 50 µL of phosphate-citrate buffer (192 parts of 0.2M Na2HPO4 and 8 parts of 0.1M citric acid, pH 7.8) and then left at room temperature for 30 min. After another centrifugation at 10,000 rpm for 5 min, the supernatant was transferred to 0.5 mL Eppendorf tubes and incubated at 37°C with 3 µL of NP40 (0.23%) and 3 µL of RNase (1mg/mL). The solution was further incubated for 30 min after adding 3 µL of Proteinase K (1mg/mL). The DNA content in the mixture was quantified using a spectrophotometer and 10 µg of DNA was electrophoresed on a 2% agarose gel using TAE buffer. DNA fragmentation was visualized under UV light after staining with ethidium bromide.
SNB19 and U87 cells were transfected with either shNF3 or scrambled vector and treated with DOX as described above. After treatment, cells were trypsinized and 2×105 cells were placed into matrigel-coated transwell inserts with an 8-µm pore size. Cells were allowed to migrate through the matrigel for 24 hours. The cells in the upper chamber were removed with a cotton swab. The cells that adhered to the outer surface of the transwell (i.e., the cells which had invaded the matrigel) were fixed, stained using the Hema-3 staining kit, and counted under a light microscope as described previously .
The TUNEL assay was carried out to detect apoptotic cells after the above-described treatments. SNB19 and U87 cells were cultured on eight-well chamber slides at a density of 2×103 per well. After 24 hours, the cells were transfected with either shNFTA3 vector or scrambled vector in serum-free medium. Twenty-four hours later, the cells were treated with DOX and incubated for another 24 hours. The cells were fixed after termination in 10% phosphate-buffered formalin for 15 min. TUNEL staining for detection of apoptotic cells was carried out using the TUNEL Apoptotic Detection kit (Roche Molecular Biochemicals, Indianapolis, IN) as per the manufacturer's instructions. Briefly, the fixed cells were washed three times in PBS (5 min/wash). The cells were then incubated with 0.05% Tween 20 in PBS containing 0.2% bovine serum albumin for 15 min at room temperature. The cells were washed twice in PBS and incubated with 50 µL of terminal deoxynucleotidyl transferase end-labeling cocktail for 60 min at room temperature. The reaction was terminated, and the slides were washed three times in PBS and blocked with blocking buffer for 20 min at room temperature. Then, the slides were incubated with 50 µL of avidin-Texas red in the dark for 30 min at room temperature, washed three times in PBS, and mounted with anti-fading gel mount (Biomeda, Foster City, CA). Slides were allowed to dry in the dark, observed under a fluorescent microscope (Olympus IX71), and photographed.
Induction of apoptosis in response to shRNA-induced downregulation of NFAT3 and DOX individually and in combination was tested using FACS analysis. FACS analysis was performed according to the manufacturer's instructions and 10,000 cells were recorded for each transfection. Cells were treated as described earlier and were trypsinised, washed with 1X PBS, and incubated for 30 min with 1 mL of propidium iodide in the dark. After the incubation period, the cells in propidium iodide were analyzed using a FACS Calibur flow cytometer (BD BioSciences, San Jose, CA) with an excitation wavelength of 488 nm and emission wavelength of 530 nm.
To study cell migration and cell interactions, cells were seeded at a density of 2×106 in a six-well plate and treated as described earlier with shNF3 and DOX. After the transfection and the DOX treatment was complete, a straight scratch was made in individual wells with a 200 µL pipette tip. This point was considered as the zero hour, and the width of the wound was photographed under the microscope. After 18 to 24 hours, the cells were checked for the wound healing and again photographed under microscope. Wound healing was measured by calculating the reduction in the width of the wound after incubation.
SNB19 and U87 cells cultured in 100 mm petri dishes were treated with shNF3 and DOX as described earlier. Harvested cells were washed once with 1X PBS and the cell pellet was resuspended in 300 µL of cytoplasmic extraction buffer (10mM HEPES, pH 7.9; 1.5mM MgCl2, 10mM KCl, 0.5mM DTT, 0.3mM sodium vanadate, 1mM PMSF, 10µg/mL Aprotinin and 10µg/mL leupeptin). This mixture was incubated for 10 min at 4°C and then centrifuged at 6000 rpm at 4°C for 10 min. The supernatant (cytoplasmic extract) was collected and the remaining pellet was suspended in 250 µL of nuclear extraction buffer (20mM HEPES, pH 7.9; 1.5mM MgCl2, 420mM NaCl, 0.5mM DTT, 0.2mM EDTA, 0.3mM Sodium vanadate, 1mM PMSF, 10µg/mL Aprotinin and 10µg/mL leupeptin). The fluid was incubated for 30 min on ice, centrifuged at 13,000 rpm for 5 min at 4°C and the supernatant (nuclear extract) was collected and used for western blot analysis.
We transfected SNB19 and U87 cells with either pSV and/or shNF3. RT-PCR and immunoblot analysis showed significant decrease in NFAT3 expression in shNF3 treated cells compared to controls (Fig. 1C and 1D). GAPDH activity was analyzed at both mRNA and protein levels to serve as a loading control (results confirmed equal loading). Taken together, these results demonstrate the efficient downregulation of NFAT3 with the shNF3 plasmid. To determine the specificity of the shNF3 construct we have immunoblotted the proteins after treatment for other NFAT’s. Figure 1D shows the unaltered NFAT4 expression after the shNF3 treatment.
The mechanism of DOX-induced apoptosis via distinct pathways has been well elucidated in different cancers, including gliomas. To investigate the potential role of NFAT3 in DOX-mediated apoptosis, SNB19 and U87 cells were transfected with the NFAT3 shRNA expression vector or pSV (negative control) and then with DOX. We found that DOX treatment was able to induce apoptosis in glioma cells. Importantly however, suppression of endogenous NFAT3 enhanced cell survival with shRNA alone or shRNA and DOX treatment together as compared to DOX treatment alone (Fig. 2A).
Further studies showed that the role of NFAT3 in DOX-induced apoptosis was mediated by upregulation of its target gene, TNF-α. This conclusion was based on the findings that inhibition of NFAT3 activation by shNF3 dramatically downregulated TNF-α induction, its receptor TNFR1, caspase 10, caspase 3 and PARP, sequentially, and thus led to abrogation of DOX-mediated apoptosis in glioma cells. DOX treatment led to increased expression of the aforementioned molecules whereas shNF3 treatment alone or in combination with DOX showed decreased expression in both cell lines (Fig. 2A), which indicates that NFAT3 is required for DOX-mediated apoptosis. Moreover, caspases 3, 10 and PARP (molecules that mediate the apoptotic mechanism) were cleaved and activated by DOX as shown by western blot analysis of cell lysates (Fig. 2A). Cells treated with shNF3 showed no indication of activated caspases or cleaved PARP. shNF3 inhibited apoptosis was further confirmed by the increased expression of anti-apoptotic molecule, Bcl2. Certain molecules like AIF, p53 and cleaved BID were unchanged with any of the treatments (supplement figure 1). Addition of TNF-α protein increased the expression of caspase-10 and PARP irrespective of the treatments (supplement figure 2).
DOX is known to induce cellular apoptosis by intercalating into the DNA. The results of the TUNEL assay showed increased DNA damage and fragmentation in glioma cells treated with DOX as compared to controls and cells transfected with pSV (Fig. 2B). Knockdown of NFAT3 reduced the DNA damage caused by DOX. We were also able to observe DNA fragmentation after purifying total nuclear DNA, which was electrophoretically separated on an agarose gel. Cells treated with DOX underwent DNA degradation, which indicate apoptosis, whereas shNF3 treatment in combination with DOX showed no degradation of DNA (Fig. 2C).
SNB19 and U87 cells transfected with shNF3 or shNF3 and DOX treatment invaded matrigel-coated filters to a greater extent than did the cells treated with DOX alone (Fig. 3A). The percent of invasive cells after the treatment with shNF3 or shNF3/DOX combination to the lower side of the membrane at 48 hours were 95% (SNB19), 82% (U87) and 90% (SNB19), and 80% (U87) respectively. Only 18% and 15% of the DOX alone treated SNB19 and U87 cells invaded to the lower side of the membrane during the same period of time (Fig. 3B). Respective controls showed 85–100% of cell invasion.
shNF3 treatment in SNB19 and U87 cells also reversed DOX-induced inhibition of cell migration as determined by wound healing assay, wherein a wound was made in a sub-confluent cell monolayer and cells were allowed to migrate into the cell-free area. The distance moved by the cells in treated and untreated wells were compared (Fig. 3C). The percent of wound repaired by cell motility of shNF3 and combination of shNF3 and DOX treatment were 45% (SNB19), 52% (U87) and 35% (SNB19), and 52% (U87) respectively. In contrast, the percent of wound repair in DOX alone treatment were 5% (SNB19) and 10% (U87) (Fig. 3D). These results indicate that after knockdown of NFAT3 and treatment with DOX, the cells were able to divide and survive, suggesting an important role being played by the NFAT3 in DOX-mediated cell migration.
It is known that NFATs in the phosphorylated form reside in cytoplasm and in dephosphorylated form in the nucleus. Immunocytochemistry showed that the shNF3 treatment of cells led to reduction of NFAT3 in the cytosol as well as in nucleus 48 hours after treatment. DOX treatment translocated NFAT3 into the nucleus, and the combination of shNF3 and DOX showed very little or no NFAT3 in cytoplasm or nucleus. In the control cells and the scrambled vector treatment NFAT3 was present only in cytoplasm (Fig. 4). This was also proved by western blot analysis of nuclear and cytoplasmic extracts from these cells. As shown in supplement figure 3, when nuclear extracts were probed with anti NFAT3 antibody, the DOX treatment showed increased expression as compared to the control and no expression with the shNF3 treatment. Similarly the cytoplasmic extracts, when probed with phospho-NFAT3, showed decreased expression of NFAT3 as compared to control and less or no expression in shNF3 treatment. These results indicate that DOX translocates cytosolic NFAT3 into the nucleus and thus leads to apoptosis.
FACS analysis of SNB19 and U87 cells treated with DOX resulted in apoptosis. Percent of cells in sub G0/G1 phase was as high as 61–64% and 66–68% in the cells treated with DOX alone in SNB19 and U87 respectively. Whereas, only 2–13% of cells in sub G0/G1 phase was found in controls and shNF3 with or without DOX treated cells (Fig. 5A). The cell cycle progression into the S phase requires the enzyme cdk2, which is inhibited by p21. In the present study, we observed that the amount of p21 expression was lower after treatment with shNF3 or shNF3 and DOX, than in the treatment with DOX alone. Similarly, increased expression of cyclin E and pcdk2 was observed, which indicates the role of NFAT3 in cell cycle progression and DOX-mediated apoptosis. Expression of cyclin D1 was unchanged (Fig. 5B). Collectively, these results provide direct evidence for the important role of NFAT3 activation in DOX-induced apoptosis by upregulation of TNF-α expression in glioma cells.
Programmed cell death occurs in virtually all cell types and is precisely regulated at the cellular and molecular levels. NFAT proteins have been shown to bind to promoter regions and upregulate the expression of some effectors of apoptosis, such as TNF-α and FasL . Most NFATs perform their function by partnering with other transcription factors and are known for their controversial properties like tumor suppression and pro-apoptotic activity. Yan, et al.  demonstrated that inhibition of COX-2 expression with diminishing endogenous NFAT3 blocked the transformation of Cl41 cells under the treatment of TNF-α. Similarly, Li et al  also documented the role of NFAT3 in regulation of iNOS and in suppression of cell transformation suggesting a tumor suppressor activity of NFAT3. In contrast, tumor development via angiogenesis and apoptotic resistance with the upregulation of COX-2 has also been documented in variety of human cancer cells [36–38]. This could be due to the inability of NFATs to partner with other transcription factors. Studies by Ranger et al.,  revealed that deletion of NFAT1 in cartilage cells leads to the uncontrolled proliferation and exhibit cancer phenotypes both in vitro and in vivo, suggesting a potential tumor suppressor role of NFAT1 in this special cell lineage. NFAT3 was also found to enhance estrogen receptor transcriptional activity in breast cancer cells and play an important role in regulation of breast cancer cell growth [15, 40]. These conflicting results highlight the versatility of NFAT proteins in cell fate determination, depending on their functional modulations under certain condition [6, 41]. Zhang, et al.  demonstrated that NFAT3, as a new kind of cofactor, displays dual transcription modulation modes dependent on tissue types. NFAT3 acted as a transcription activator of estrogen receptors in breast cancer cells and inhibited their transcription activities in all cell types derived from kidney tissue. Recently, NFAT transcriptional activity has been reported to regulate the acetylcholinesterase promoter during ionophore-induced apoptosis in HeLa cells .
In the present study, we demonstrated that treatment of SNB19 and U87 glioma cell lines with shNF3 resulted in downregulation of NFAT3. DOX treatment of these cells resulted in apoptosis in a dose-dependent manner (supplement figure 4). We further investigated the influence of NFAT3 on DOX-mediated apoptosis by NFAT3 downregulation in SNB19 and U87 cells. Western blot analysis showed a decrease in expression of TNF-α, a downstream element of NFAT3, which is known for its roles in immunity and cellular remodeling as well as influencing apoptosis and cell survival . Data from many different studies underlined the importance of TNF-α activation in human cancers. DOX induced TNF-α, uPA, IL-8 and MCP-1 in a number of lung cancer cells with different levels of expression has been reported by. Enhanced anti-tumor effect was also reported with systemic administration of TNF-α in combination with DOX for lung tumors . Cells lacking NFAT1 showed decreased expression of TNF-α . In the present study, decreased expression of TNF-α lowered the expression of TNFR1, caspase 10, caspase 3, and PARP. Similar results were obtained when shNF3 was used in combination of DOX. Moreover, DOX treatment alone increased the above-mentioned molecules. Addition of TNF-α also increased the expression of caspase-10 and PARP. This shows that NFAT3 has a pro-apoptotic role in glioma cells. Thus, we anticipate that NFAT3 represents a putative tumor suppressing mechanism and downregulation of NFAT3 reversed the apoptotic mechanism induced by DOX.
There were certain molecules (e.g., p53, AIF and cleaved BID) that were unchanged with either treatment. p53-mediated apoptosis is common in tumor cells when treated with DOX and is dependent on the Apaf-I/caspase-9 pathway involving cytochrome c release from mitochondria . The p53-mediated activation of calcineurin in lung, renal, colon, and ovarian carcinoma cells and subsequent activation of NFAT to induce apoptosis has been reported as well . However, in the present study, we did not observe any change in the expression of p53 or in cleaved BID, which in turn releases cytochrome c from mitochondria.
Under basal conditions, NFAT3 proteins are present in the cytoplasm in its phosphorylated state and, upon increase in the intracellular calcium, it translocates to nucleus by dephosphorylation at multiple serine residues [48–50]. In the present study, we found that NFAT3 translocated to the nucleus with DOX treatment and that there was little or no NFAT3 in the nucleus of control cells as well as the cells treated with shNF3 alone or in combination of DOX. This could be due to increase in the calcium levels of the cells when treated with DOX. Similar type of results where DOX activated the transcription factor NFAT, leading to the upregulation of Fas/FasL-dependent apoptosis through a calcium/calcineurin-signaling pathway, has been documented in rat cardiac cells . Yaghi and Sims  reported NFAT3 nuclear translocation in smooth muscle cells when treated with PE or 20-HETE.
The NFAT family of proteins plays an important role in regulation of genes that control cell cycle progression, cell development and differentiation, angiogenesis, and possibly tumorigenesis [17, 52–55]. Thus, in the present study the effect of shNF3 on cell cycle progression was studied. We found that DOX treatment led the cells to apoptosis. Interestingly, shNF3 treatment with or without DOX showed only 7–13% of apoptotic cells, suggesting that decreased expression of NFAT3 in the cells will lead to survival and vice-versa. It also suggests the important role played by NFAT3 in DOX-mediated apoptosis. This was proved by the western blot analysis for cell cycle-controlling genes like phospho-cdK2 and cyclin E, which were increased in expression with DOX treatment and decreased with shNF3 alone or with a combination of shNF3 and DOX. These results suggest that NFAT3 is required for DOX to inhibit cell cycle progression and has a central role in cell cycle control. Baksh, et al.  demonstrated that the calcineurin/NFAT1 pathway negatively regulates the expression of cyclin-dependent kinase 4 (CDK4) in Jurkat cells. NFATs need not be always repressors of cell cycle controlling genes. Accumulating evidence suggests that the phosphatase calcineurin plays a major role in the regulation of cell cycle progression by acting during the early stages of G1 phase [57–61]. In addition, it has also been demonstrated that overexpression of NFAT2 in pre-adipocyte 3T3-L1 cells promoted cell cycle progression even under low serum concentrations and induced altered expression of cell cycle-related genes, such as cyclin D1, cyclin D2, pRB, and c-Myc .
Infiltrative and destructive growth patterns of malignant gliomas are characterized by their migratory and invasive properties. Infiltrative growth prevents complete tumor resection and therefore causes significant neurological morbidity and mortality . Jauliac, et al.  reported that two dominant negative NFATs, one deleted in the carboxyl terminus of the sequence necessary for DNA-binding12, and the VIVIT peptide13, which blocks the ability of calcineurin to activate NFAT inhibited carcinoma invasion when transfected into MDA-MB-435. In the present study, we found that downregulation of NFAT3 alone or in combination with DOX increased the migration and invasion when compared to DOX alone treated cells. Impaired migration in DOX treated cells could be due to active apoptosis caused by DOX as seen in Tunel assay. Nevertheless, these data implicate conventional NFATs in carcinoma migration and invasion.
NFAT3-induced, TNF-α-mediated apoptosis by DOX treatment include multiple signal molecules as described earlier. Conflicting role of TNF-α in cell cycle control [30, 44] demands further investigation of whether NFAT3 role in cell cycle control is through TNF- α or mediated by other molecule. These issues are currently being investigated in our laboratory. Since, downregulation of NFAT3 is reversing the DOX induced apoptosis and DOX treatment alone is inducing the apoptosis with the available basal NFAT3, upregulation of NFAT3 may not be required for the cells to undergo apoptosis in the presence of DOX.
In summary, NFAT transcription factors are known for their versatile functions in cell cycle progression, cell development and differentiation. Here, we demonstrate the importance of NFAT3 by siRNA-mediated downregulation of NFAT3, which led to cell survival and subsequent inhibition of DOX mediated apoptosis in SNB19 and U87. Western blot analysis showed the influence of NFAT3 downregulation on apoptotic molecules in both DOX treatment alone and in combination with shNF3. These findings imply that NFAT3 possesses anti-tumorigenic properties and might play an important role in DOX-mediated apoptosis in SNB19 and U87 glioma cell lines.
We thank Shellee Abraham for helping in manuscript preparation, and Diana Meister and Sushma Jasti for manuscript review.
This research was supported by National Cancer Institute Grants CA75557, CA116708, CA138409, (to J.S.R.). The contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH.