Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells

The human astrocytoma cell lines A172 and T98G were obtained from the American Type Culture Collection (Rockville, MD). The U251-MG cell line was graciously donated by Dr. George Perides (Tufts-NEMC). These cell lines were derived from grade IV astrocytomas – glioblastoma multiforme (GBM). Normal human astrocytes (NHA) were obtained from ScienCell (San Diego, CA). A172 and U251-MG cells were cultured in DMEM (ATCC, Rockville, MD) supplemented with 10% FCS. T98G was cultured in MEM (ATCC, Rockville, MD) supplemented with 10% FCS. Normal human astrocytes (NHA) were cultured in astrocyte medium (ScienCell, San Diego, MD).

We designed a double stranded siRNA oligonucleotide against STAT3 (5'-AAC AUC UGC CUA GAU CGG CUA dTdT-3'; 3'-dTdT GUA GAC GGA UCU AGC CGA U-5') and had it synthesized by Dharmacon Research, Inc (Lafayette, CO). Oligofectamine (Invitrogen, Carlsbad, CA) was used as the transfection reagent following manufacturer's directions using 200–600 nmol of siRNA per 10 cm dish. Cells were incubated for 24 hrs in Opti-MEM, at which point appropriate media for the cells supplemented with 30% FCS was added. The cells were then incubated for the amount of time indicated in the Figure Legends.

For Western blot analysis, cells were harvested and lysed with RIPA buffer (0.15 M NaCl, 1% NP40, 0.01 M desoxycholate, 0.1% SDS, 0.05 M Tris-HCl pH 8.0, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/ml each of aprotinin, pepstatin, and leupeptin). For STAT3 analysis, protein samples were electrophoresed on a 10% SDS-PAGE gels for 90 min. at 150 mV and transferred onto Immobilon membranes (Millipore, Bedford, MA) for 60 min at 100 mV in 10% methanol transfer buffer. For caspase-3 analysis the protein samples were separated on a 15% SDS-PAGE and transferred onto Immobilon membranes (Millipore, Bedford, MA) at 20 mV for 20 min and 80 mV for 2 hrs in 20% methanol transfer buffer. The membranes were then probed with each antibody as indicated. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). Anti-phospho-STAT3, anti-STAT3, anti-cleaved Caspase 3, anti-Bcl-xL and anti 42–44 MAP kinase were obtained from Cell Signaling (Beverly, MA). Anti-β-actin antibody was obtained from Sigma (St. Luis, MO).

For cell proliferation assays, cells were incubated for 72 hrs in 96-well plates in quadruplicates. Viable cell number was determined by using MTS assay according to the manufacturer's instructions (CellTiter 96 AQ Non-radioactive proliferation kit, Son Luis, CA). The absorbance was measured at 490 nm with a 96-well plate reader.

Cells were treated with siRNA as described above. After 96 hours, cells were isolated and stained with annexin V-EGFP (BD Biosciences Clontech, Palo Alto, CA). Cells were analyzed using FACS for fluorescence of annexin V positive cells. The fraction of annexin V positive cells in the siRNA treated population was determined using the super-enhanced DMax method of WinList software (Verity Software House, Topsham, ME).

The A172 cell line was treated with STAT3 siRNA as described previously and RNA purified with TRIzol (Invitrogen, Carlsbad, CA). 10 μg of each RNA sample was loaded per well. After transferring onto a nylon membrane (Ambion, Austin, TX), it was probed with a survivin probe (Resgen) or p21 (gift of Sam Lee) labeled with ³²P. Hybridizations were performed with Express Hyb (BD Biosciences Clontech, Palo Alto, CA) and washed following the manufacturers protocol. Blots were exposed to Kodak MS film overnight and developed. Blots were then quantitated using a Molecular Dynamics phosphorimager.

To determine whether STAT3 is expressed and constitutively activated in astrocytoma cell lines, we compared the level of STAT3 expression and tyrosine 705 STAT3 phosphorylation in normal human astrocytes (NHA) to that of three astrocytoma cell lines. Western Blot analysis with anti-STAT3 and anti-phosphotyrosine STAT3 antibodies revealed that STAT3 was overexpressed and overactivated in astrocytoma cells as compared to primary astrocytes (Figure 1). Reprobing the blot with anti-β-actin shows that variability in protein loading could not account for the observed differences in STAT3 expression.

Recent studies show that constitutive activation of STAT3 in variety of tumors directly contributes to their oncogenic potential by inducing proliferation and inhibiting apoptosis [40, 41]. Thus, we sought to determine the effect of inhibiting STAT3 expression in astrocytoma cells.

To inhibit STAT3 expression in astrocytoma cells, we used the RNAi method adapted for mammalian cell culture by Elbashir et al [42]. A number of 21 bp double stranded RNAs to human STAT3 were synthesized and tested for their ability to knockdown STAT3 expression in astrocytomas. Transfection of cells with one of these STAT3 siRNAs (Figure 2a) resulted in a highly significant and reproducible decrease in STAT3 expression levels as judged by Western Blotting (Figure 2b). The degree of knockdown achieved ranged from 75–95% depending upon the experiment and cell line used. STAT3 siRNA inhibited STAT3 expression in normal human astrocytes (NHA) as well as in astrocytoma cell lines (Figure 2b). Mock transfection with oligofectamine or with an siRNA to the green fluorescent protein (GFP) failed to reduce STAT3 expression. The effect of STAT3 siRNA was specific in that it failed to knock down expression of the unrelated proteins β-actin or Map Kinase. STAT3 knockdown by siRNA was found to be time dependent with the maximum effect achieved at 48–72 hrs of siRNA treatment (Figure 2c). This siRNA is human specific and does not efficiently knockdown expression of murine STAT3 (Data not shown).

We noted that upon STAT3 siRNA treatment of some of the astrocytoma lines that cultures were less confluent and some cells became smaller and rounder than control oligofectamine treated cells (Figure 3a). Consistent with this, there were fewer cells in both A172 and U251-MG cultures treated with STAT3 siRNA as compared to control oligofectamine treated cultures 72 hours post transfection (Figure 3b). Transfecting A172 cells a second time with STAT3 siRNA 48 hours after the initial transfection resulted in an even greater reduction in cell number (>90% in A172 and 65% in U251) as compared to oligofectamine controls (data not shown). Viability of normal human astrocytes (NHA) was not significantly affected by STAT3 siRNAi treatment. Another astrocytoma cell line, T98G, was only slightly affected by reductions in STAT3 expression (Figure 3b). Thus, not all astrocytoma cell lines are equally responsive to STAT3 knockdown. The decrease in cell number caused by STAT3 siRNA treatment of astrocytoma cells implies that STAT3 participates in cell cycle progression, cell survival, or both. Staining A172 cells for DNA content after STAT3 siRNA treatment showed an increased proportion of cells in early S phase (data not shown). This could be due to an S phase cell cycle block or to preferential apoptosis of these cells in late S and G2/M.

The cell number decreases observed in conjunction with the morphological observations suggested that astrocytoma cells treated with STAT3 siRNA undergo apoptosis. To confirm that these cells undergo apoptosis in response to STAT3 knockdown, we examined three other indicators of apoptosis in the A172 cell line. First, cultures of astrocytoma cells were stained Hoechst 33258 dye and scored for apoptotic nuclei[43]. A significantly higher percentage of apoptotic nuclei were observed in STAT3 siRNA treated cells than in GFP siRNA or oligofectamine treated cells (Figure 4a). Consistent with this observation, Western blot analysis of astrocytoma cell extracts showed that there is increased cleavage of caspase 3 in STAT3 siRNA treated cells (Figure 4b). Finally, annexin V staining of control and STAT3 siRNA treated cells followed by fluorescence activated cell sorting analysis indicated a 77% increase in the fraction of annexin V positive cells, further confirming that these cells are undergoing apoptosis (Figure 4c)[44]. Together these data indicate that STAT3 regulates an anti-apoptotic program in astrocytomas and that inactivation of STAT3 leads to a rapid induction of apoptosis.

Recent data indicate that constitutive activation of STAT3 induces the expression of a number of anti-apoptotic genes including Bcl-xL, a member of the Bcl-2-family of anti-apoptotic genes [32, 45], and survivin, a member of the IAP, inhibitors of apoptotic proteins family [46]. Moreover, both of these genes are expressed in astrocytomas [47, 48]. In order to determine whether these two genes might be involved in the STAT3 mediated apoptotic block in astrocytoma, Northern and Western blot analyses were performed to examine the expression levels of these two genes. Northern blot analysis showed that STAT3 siRNA reduced expression of survivin by 75% (Figure 5a). Western blot analysis showed that Bcl-xL protein levels were drastically reduced upon treatment with STAT3 siRNA. Thus, these data show that STAT3 regulates the expression of at least two distinct antiapoptotic genes in the A172 cell line.