Introduction: For patients with advanced ovarian cancer there are no effective therapies. The need for alternative treatments with high specificity but with low systemic toxicity can be achieved by targeting key molecular markers associated with cancer cells. Here, we show an innovative proof-of-principle approach for efficient killing of proliferating ovarian cancer cells by inactivating a protein associated with cell proliferation namely, the nuclear Ki-67 protein (pKi-67), using nanotechnology-based photodynamic therapy (PDT). pKi-67 is highly expressed in all proliferating cells and antibodies against this protein are widely used as prognostic tools in tumor diagnosis. Materials and Methods: Anti pKi-67 antibodies were first conjugated to fluorescein isothiocyanate (FITC) and then encapsulated inside liposomes. The liposomes encapsulating antibodies were characterized by dynamic light scattering and transmission electron microscopy. We then evaluated the efficacy of Ki-67 targeting using two in vitro ovarian cancer models - monolayer cultures as well as 3D cultures established in our lab. Cell viability after irradiation with laserlight at 488 nm was assessed using MTT viability assay for monolayer cultures while a Live/Dead assay kit was used for analyzing 3D cultures. Results: After incubation of OVCAR-5 ovarian cancer cells with the liposomal constructs, we used confocal microscopy to image localization of the antibodies to the nucleoli of the cells. Irradiation of these cells with a 488 nm laser led to a significant loss of viability. The efficacy of this approach was further demonstrated in a 3D culture model of OVCAR-5 cells. Incubation of 3D cultures with the liposomal constructs followed by light irradiation led to destruction of their acinar structures over 72 h following treatment. Using two different anti pKi-67 antibodies, where one targets the “active” form of pKi-67 while the other binds to the “inactive” form of the protein; we show that cell killing is specific after inactivation of the “active” Ki-67 protein. Further, the specificity of this approach for pKi-67 positive cells is demonstrated in confluent human lung fibroblasts (MRC-5) where minimal cell death was observed. This is in agreement with the flow cytometry results which show that only small populations of confluent MRC-5 cells express pKi-67. Conclusions: Taken together, our findings suggest that pKi-67 is an attractive therapeutic target in cancer and this approach holds promise as an effective alternative cancer therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C83.
Ineffective erythropoiesis (IE) in β-thalassemia is described as increased expansion of erythroid progenitor cells in combination with accelerated apoptosis and intramedullary hemolysis. However, evidence for this assumption is not particularly strong. In this study we evaluated the kinetics of red blood cell proliferation and survival in thalassemic mice that exhibit levels of anemia consistent with thalassemia intermedia (th3/+) and major (th3/th3), as we described previously. Th3/+ mice show anemia and increased reticulocyte counts (8.9±1.1 g/dL and 17.7±2.6x105/ul compared to 14.9±1.1 g/dL and 2.6±0.4x105/ul in +/+), whereas th3/th3 mice show more severe anemia than th3/+ mice and reduced production of reticulocytes (3.0±1.2 g/dL and 1.8±0.7x105/ul). As soon as two months of age, EPO levels in thalassemic mice are significantly increase over normal mice, 10 times in th3/+ and up to three orders of magnitude in th3/th3 mice. The total number of nucleated erythroid cells (spleen + bone marrow) increased from 3.3±0.9x108 in +/+ to 1.2±0.5x109 (or 3.6 folds) in th3/+ and 1.6±0.6x109 (or 4.8 folds) in th3/th3 mice (N=5 per group), whereas the level of apoptotic cells increased only 2 to 3 folds in percentage in th3/+ and th3/th3, respectively, as observed using the apoptotic Annexin-V and erythroid specific Ter119 markers (9.9±5.0, 14.3±2.5, 24.2±6.7 in BM and 10.6±3.5, 14.8±6.5, 25.4±6.2 in spleen of +/+, th3/+ and th3/th3, respectively; n=3 per genotype). This data was confirmed by TUNEL, cleaved caspase-3/7 and by immunostaining assays. Bilirubin and LDH levels were not different between thalassemic and +/+ mice. Altogether these observations indicated that in thalassemia there is a disproportion between number of proliferating and dying cells with a net increase of erythroid cells. Furthermore, microarray analysis on erythroid cells indicated increased expression of cell cycle promoting genes such as Ki67, Mcm3, Cyclin-A, CDK2 and BclXL (2 to 6 folds compared to +/+ mice, n=5 per genotype). This data was confirmed by Q-PCR, Western blot, immunostaining, clonogenic assay and by analysis of the percentage of erythroid cells in S-phase after in vivo BrdU injection (22, 30 and 44% BrdU+ in +/+, th3/+ and th3/th3, respectively). On the other hand, in th3/th3, which show more apoptosis than th3/+ mice, the cyclin-dependent kinase inhibitor p21 was upregulated both at the RNA (50-folds) and protein level. P53 was also analyzed in th3/th3 mice, showing no expression. In order to investigate the function of p21 in thalassemic erythroid cells, its expression was analyzed on purified erythroid cells isolated from th3/th3 that were transfused (10.4±0.4 g/dL of Hb) or thalassemic mice showing different levels of anemia (6.2±0.2 and 2.1±0.8 g/dL of Hb, respectively; N=3 per each group). We observed that the level of p21 increased with anemia and the severity of the pathology. However, in th3/th3 mice that were injected with BrdU, immunostaining analysis indicated that a large amount of p21+ erythroid cells were also BrdU+. In conclusion, we propose that erythropoiesis in β-thalassemia is characterized by enhanced expression of cell cycle promoting and survival factors that are able to overcome or mitigate p21 cell cycle block and, probably, apoptosis.
Ineffective erythropoiesis (IE) in β-thalassemia has been attributed to erythroid cell death mediated by apoptosis or hemolysis during the maturation process. Historically, ferrokinetic studies in this disease suggested that 60%–80% of erythroid precursors die in the marrow or extramedullary sites. However, several observations have challenged this view. First, the number of apoptotic erythroid cells in patients is low compared to net expansion of the erythroid cell pool. Second, hemolytic markers in β-thalassemic patients are normal or only slightly increased, unless additional pathological conditions appear. Third, our most recent study (Blood, Gardenghi et al, 2007 Jun 1) demonstrated that GI iron absorption in β-thalassemia is increased by the dysregulation of genes such as hepcidin and ferroportin that control iron absorption, resulting in iron levels that exceed the amount required for erythropoiesis. We have undertaken a detailed investigation using cohorts of mice (n>30 per genotype) with β-thalassemia intermedia (th3/+) and major (th3/th3). Using these models, we have previously shown that the severity of anemia (as low as 1 g/dL) inversely correlates with the total number of nucleated erythroid cells (»100 fold compared to wild-type (wt) mice). Cytological analysis has clearly shown that thalassemic spleen specimens were comprised of a homogeneous pre-erythroblastic population. In contrast, the percentage of apoptotic cells and the level of hemolytic markers, such as bilirubin and lactic acid dehydrogenase, slightly increased or were not different compared to wt mice. While not excluding a role for apoptosis, our observations suggest that control of the cell cycle and maturation of erythroid precursors play an important role in IE. We then explored whether the erythroid cell cycle was dysregulated in our model system. We found that erythropoietin (Epo) levels were raised in thalassemic animals by as much as three orders of magnitude. Binding of Epo to its receptor (EpoR), activates antiapoptotic and cell cycle promoting genes, through activation of Jak2 and Stat5. By Western blot we demonstrated up-regulation of EpoR, Stat5 and the antiapoptotic protein BclXL, as well as that of proliferation promoting genes, such as CycA and Cdk2, in purified thalassemic erythroid cells compared to those of wt animals. This data was confirmed by staining both wt and thalassemic liver and spleen sections using the proliferation markers Ki67 and Mcm3, by clonogenic assay and by analysis of the percentage of erythroid cells in S-phase after BrdU injection. In the latter case, we observed 22%, 30% and 44% BrdU+ cells from wt, th3/+ and th3/th3 mice, respectively. In addition, freshly purified thalassemic erythroid cells proliferate faster in vitro than normal cells, a phenomenon blocked by AG490, a Jak2 inhibitor. Significantly, we have been able to reproduce results from our animal studies in humans, comparing normal and thalassemic blood and spleen specimens. In conclusion, we propose that IE in β-thalassemia is likely to be the result of altered cell proliferation and impaired cell differentiation, which in turn limit apoptosis, thereby mimicking tumor-like behavior.
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