苏州大学 尹斌 教授
[目的] 检测量子点（quantum dot，QD）对白血病细胞、骨髓细胞、造血干细胞及293T细胞的标记效果。[方法]使用量子点活细胞示踪试剂标记细胞：QD与载体TAT充分混匀，与细胞在37℃条件下孵育1h后镜检；量子点与阳离子型转染试剂TurboFect标记细胞：QD与TurboFect分别先稀释、混匀、室温孵育20min，加到细胞培养板培养，次日观察；QD与脂质体Lipofectamine™ 2000转染细胞：QD与脂质体分别先稀释、室温静置5 min，混匀，室温孵育20 min，滴加到细胞培养板，37℃孵育4-6小时后观察。[结果]采用QD-TAT、QD-TurboFect标记实验，选用细胞系293T、32D-TIP及HL60。标记1h后荧光显微镜下观察：QD单独或QD-TurboFect不能标记各种细胞；QD-TAT标记293T细胞阳性率90%以上， HL60为60%，32D小于10%，阳性细胞胞体呈红色荧光，胞内荧光成簇分布（图1）。次日阳性比例显著减少，荧光变暗。用QD-Lipofectamine2000标记293T与BM细胞5h后，观察发现293T细胞阳性率为40-50%，荧光在细胞内呈点状分布。QD-TAT标记BM细胞效率为20-30%，QD-Lipofectamine2000标记BM的效率小于5%，且QD多聚集在死细胞或细胞碎片（图2-3）。[结论]试剂盒QD-TAT可以成功标记细胞，标记效率与时长因细胞不同而有差别，与QD浓度有一定关系；脂质体可以装载QD标记293T细胞，短时效率较高、荧光较强，随着时间的延长，效率降低、荧光减弱。
图1：QD+TAT标记三种细胞1h后，观察结果，A：32D: 400×，B：293T: 400×，C: HL-60: 200× （QDs+TAT: 10nM）
图2 QD-Lipofectamine2000标记293T与BM细胞5h结果： A:293T-QDs+ Lipo2000 (10 nM)200×, B：293T-QDs+TAT(10nM)400×，C:BM-QDs+TAT (10 nM)400×, D：BM-QDs+TAT(30 nM)400×
图3 QD- Lipofectamine2000标记BM细胞24h结果：A:BM-Lipo2000+QDs(10 nM)400×, B:BM-Lipo2000+QDs(30 nM)400×, C:293T-Lipo2000+GFP(1 µg),100×
二、A small molecule inhibitor of D-cyclin transactivation displays preclinical efficacy in myeloma and leukemia via phosphoinositide 3-kinase pathway【苏州大学毛新良教授课题组】
D-cyclins are universally dysregulated in multiple myeloma and frequently over-expressed in leukemia. To better understand the role and impact of dysregulated D-cyclins in hematological malignancies, we conducted a high throughput screen for inhibitors of cyclin D2 transactivation and identified 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161) that inhibited the expression of cyclins D1, D2, and D3, and arrested cells at the G0/G1 phase. Following D-cyclin suppression, S14161 induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of leukemia, S14161 inhibited tumor growth without evidence of weight loss or gross organ toxicity. Mechanistically, S14161 inhibited the activity of PI3 kinase in intact cells and the activity of PI3 kinases alpha, beta, delta and gamma in a cell-free enzymatic assay. In contrast it did not inhibit the enzymatic activities of other related kinases, including mTOR, DNA-PKcs, and PDK1. Thus, we identified a novel chemical compound that inhibits D-cyclin transactivation via PI3K/AKT signaling pathway. Given the potent anti-leukemia and anti-myeloma activity with minimal toxicity, S14161 could be developed as a novel agent for blood cancer therapy.
Figure 1. S14161 inhibited D-cyclin expression a) The chemical structure of S14161. b) S14161 inhibited cyclin D2 promoter transactivation. NIH3T3 cells were first transfected with pRSV.Luc. and pCCD2.Luc., respectively. Twenty-four hours later, cells were treated with S14161 for 20 hrs followed by luciferase activity assay as described in Material and Methods. c) Myeloma (JJN3, KMS12, LP1, and RPMI-8826) and leukemia (OCI-AML2, K76A, U937, MDAY) cells were treated with 5 µM of S14161 (S1) or vehicle for 24 hrs. After incubation, cells were harvested and total proteins were isolated. Expression of cyclin D1 (CCND1), cyclin D2 (CCND2), cyclin D3 (CCND3), beta-actin and tubulin were measured by immunoblotting. d) KMS11 and K562 cells were treated with increasing concentrations of S1 for 24 hrs. After incubation, cells were harvested and total proteins were isolated. Expression of cyclin D2 (CCND2), cyclin D3 (CCND3), β-actin and tubulin were measured by immunoblotting. e) NIH3T3 cells were transfected with pcDNA3.1-CCND2 (under control of CMV promoter), followed by S1 treatment at indicated concentrations for 24 hrs. Cells were harvested for cyclin D2 expression analysis. Tubulin was used as a loading control. f) LP1 and AML2 cells were treated with 5 µM of S1 for 24 hrs and total mRNA was isolated. Cyclin D2 (from LP1) and D3 (from AML2) expression was measured relative to 18S RNA by real-time RT-PCR. Data represent the mean ± SD percent of D-cyclin expression relative to controls (ΔΔCT normalization) (n =3). g) KMS11 cells were treated with increasing concentrations of S1. Twenty four hours after incubation, cell cycle was measured by PI staining and flow cytometry. Data represented the mean ± SD percent of cells at phases of the cell cycle (n =3). A representative experiment was shown. h) Myeloma cells OPM2 and LP1 were treated with S1 at indicated concentrations for 24 hrs followed by cell lysates and immunoblotting assay against human CDK9 specific antibody. GAPDH was used as a loading control.
Figure 2. S14161 induced cell death and apoptosis in myeloma and leukemia cells and primary patient samples. a) AML patient samples (n =5) and normal hematopoietic cells (PBSC) were treated with increasing concentrations of S14161. Seventy-two hours after incubation, cell growth and viability was measured by the MTS assay. Data represented the mean percentage of viable cells ± SD from experiments performed in triplicate. b) Leukemia (HL60 and U937) and myeloma (JJN3 and KMS11) cells were treated with S1 (5 µM) or vehicle control. Twenty-four hours after treatment, apoptosis was measured by Annexin V staining. A representative experiment is shown. c) Myeloma cells OCI-My5 (Left) was incubated with 5 µM of S1 for the indicated time. After incubation, cells were harvested and total proteins were isolated. Cleavage of PARP and caspase-9 (Casp-9) was measured by immunoblotting. On the right panel, OCI-AML2 cells were treated for 24 hrs at indicated concentrations, followed by evaluation of PARP and caspase-3 (Casp-3). d) Myeloma (OPM2) cells were treated with increasing concentrations of S1 for 24 hrs. After incubation, cells were harvested and total proteins were isolated. Expression of Bim, Bcl2, Mcl1, and loading controls β-actin and GAPDH were measured by immunoblotting. e) Leukemia cells K562 was transfected with cyclin D2 using nanoparticles as vectors (IR, Nanomics Biopharma Inc., China). Twenty-four hours later, cells were harvested for cyclin D2 evaluation (right) or further treated with S14161 for 24 hrs followed by caspase-3 activation analysis using caspase-3 specific antibody. Both pro- and cleaved caspase-3 fragments were detected. GAPDH was used as a loading control.
Figure 3. S14161 delayed the tumor growth in leukemia xenografts. K562 (a, b) and U937 (c, d) leukemia cells were injected s.c. into SCID mice as described in the Materials and Methods. When the tumors were palpable, mice were treated with S14161 (100mg/kg) or vehicle control i.p. for 10 days (n = 10 per group). Tumor growth (a, c)and body weight (b, d) were monitored every other day. Data represented the mean ± SD of a representative experiment. *, p<0.05; ** p<0.001, by the Student’s t-test. e) Tumor samples from U937 xenograft mice models after 10 d treatment of S14161 were excised. Total AKT and cyclin D3 were evaluated by immunoblotting using specific antibodies (left). Relative expression of AKT (middle) and CCND3 (right) was quantitated by densitometric analysis based on the immunoblotting assay result (left). * indicated significant difference (p<0.01) between vehicle control and S14161 treatment.
Figure 4. S14161 inhibited the PI3 kinase signaling pathway. a) Myeloma (H929, OPM2, LP1, and KMS11) and leukemia (OCI-AML2 and THP1) cells were starved overnight, and then treated with S14161 (S1, 100 µM for 2 hr), LY294002 (LY, 100 µM for 30 min) or DMSO (DM, 2 hr) , followed by 100 ng/ml of insulin-like growth factor 1 (IGF1) for 10 min. After incubation, cells were harvested and total proteins were isolated. Expression of AKT and phospho-AKT (p-AKT), and β-actin were measured by immunoblotting. b) KMS11 cells were treated with increasing concentrations of S14161 for 0.5, 1 or 2 hrs, followed by IGF1 stimulation. Cells were then harvested and total proteins were isolated. Expression of AKT, phospho-AKT (p-AKT), and β-actin were measured by immunoblotting. c) JJN3 and K562 cells were treated with S14161 (S1, 2.5 µM) or DMSO control for 24 hr. After incubation, cells were harvested and total proteins were isolated. Expression of AKT and phospho-AKT (p-AKT), and β-actin were measured by immunoblotting. d) Phosphorylated AKT was important for S14161-induced cell death. KMS11 and U266 cells were treated with increasing concentrations of 5 µM of S14161 for 24 hrs followed by apoptosis analysis using Annexin V staining. At the same time, KMS11 and U266 cells were subjected to AKT phosphorylation analysis as described in Materials and Methods. DM, DMSO; I, IGF1; LY, LY294002; S1, S14161.
Figure 5. S14161 inhibited phospho-AKT translocation and accumulation at the cytoplasmic membrane. OPM2 myeloma cells were starved overnight, followed by treatment with S14161 (100 µM for 1 hr), LY294002 (100 µM for 30 min) or DMSO control for 1 hr. Cells were then treated by 100 ng/ml of insulin growth factor 1 (IGF1) for 10 min. Cells were then fixed and stained using antibodies against AKT or phospho-AKT (p-AKT) and DAPI as described in the material and methods. Red indicated AKT or p-AKT, blue indicated nuclei. a) phospho-AKT (arrows) stimulated by IGF; d) total AKT translocated to the plasma membrane after IGF1 treatment; g) Phospho-AKT inhibited by LY294002; j) total AKT located in the nuclei after LY294002 treatment; m) phospho-AKT inhibited by S14161; p) total AKT was restricted to the nuclei after S14161 treatment.
Figure 6. S14161 inhibited the PI3 kinase activity. a) PI3K activity analysis in in vitro cell free system. Increasing concentrations of S14161 were incubated with the PI3 kinase isoforms alpha, beta, gamma, and delta, respectively. Activity of each kinase was determined using the HotSpot technology (Reaction Biology Corp, PA) as described in the Materials and Methods. The results of three independent experiments were presented. b) PI3K activity analysis after S14161 treatment in vivo. OPM2 cells were treated with S14161 as described in Materials and Methods. Cell lysates were prepared and applied for PI3K immunoprecipitation. The PI3K activity was analysed by ELISA as described in Material and Methods.