A novel and evolutionarily conserved PtdIns(3,4,5)P3-binding domain is necessary for DOCK180 signalling.

The evolutionarily conserved DOCK180 protein has an indispensable role in cell migration by functioning as an exchange factor for Rac GTPase via its DOCK homology region (DHR)-2 domain. We report here that the conserved DHR-1 domain also has an important signalling role. A form of DOCK180 that lacks DHR-1 fails to promote cell migration, although it is capable of inducing Rac GTP-loading. The DHR-1 domain interacts with PtdIns(3,4,5)P(3) in vitro and in vivo, and mediates the DOCK180 signalling complex localization at sites of PtdIns(3,4,5)P(3) accumulation in the cell's leading edge. A form of DOCK180 in which the DHR-1 domain has been replaced by a canonical PtdIns(3,4,5)P(3)-binding pleckstrin homology domain is fully functional at inducing cell elongation and migration, suggesting that the main function of DHR-1 is to bind PtdIns(3,4,5)P(3). These results demonstrate that DOCK180, via its DHR-1 and DHR-2 domains, couples PtdIns(3,4,5)P(3) signalling to Rac GTP-loading, which is essential for directional cell movement.

Phosphorylation of the tumor suppressor p33(ING1b) at Ser-126 influences its protein stability and proliferation of melanoma cells.

ING (inhibitor of growth) tumor suppressors regulate cell-cycle checkpoints, apoptosis, and ultimately tumor suppression. Among the ING family members, p33(ING1b) is the most intensively studied and plays an important role in the cellular stress response to DNA damage. Here we demonstrate that there is basal phosphorylation of p33(ING1b) at Ser-126 in normal physiological conditions and that this phosphorylation is increased on DNA damage. The mutation of Ser-126 to alanine dramatically shortened the half-life of p33(ING1b). Furthermore, we found that both Chk1 and Cdk1 can phosphorylate this residue. Interestingly, while Cdk1 can phosphorylate p33(ING1b) at Ser-126 in nonstress conditions, Chk1 predominantly phosphorylates this residue on DNA damage, which suggests that p33(ING1b) is a downstream target of the ATM/ATR response cascade to genotoxic stress. More importantly, our data indicate that the Ser-126 residue plays a key role in regulating the expression of cyclin B1 and proliferation of melanoma cells.

Formulation and evaluation of mixed polymeric micelles of quercetin for treatment of breast, ovarian, and multidrug resistant cancers.

BACKGROUND: Quercetin (QCT), a naturally occurring flavonoid has a wide array of pharmacological properties such as anticancer, antioxidant and anti-inflammatory activities. QCT has low solubility in water and poor bioavailability, which limited its use as a therapeutic molecule. Polymeric micelles (PMs) is a novel drug delivery system having characteristics like smaller particle size, higher drug loading, sustained drug release, high stability, increased cellular uptake and improved therapeutic potential. In the present study, we have formulated and characterized mixed PMs (MPMs) containing QCT for increasing its anticancer potential. METHODS: The MPMs were prepared by thin film hydration method, and their physicochemical properties were characterized. The in vitro anticancer activity of the MPMs were tested in breast (MCF-7 and MDA-MB-231, epithelial and metastatic cancer cell lines, respectively), and ovarian (SKOV-3 and NCI/ADR, epithelial and multi-drug resistant cell lines, respectively) cancer. RESULTS: The optimal MPM formulations were obtained from Pluronic polymers, P123 and P407 with molar ratio of 7:3 (A16); and P123, P407 and TPGS in the molar ratio of 7:2:1 (A22). The size of the particles before lyophilization (24.83±0.44 nm) and after lyophilisation (37.10±4.23 nm), drug loading (8.75±0.41%), and encapsulation efficiency (87.48±4.15%) for formulation A16 were determined. For formulation A22, the particle size before lyophilization, after lyophilization, drug loading and encapsulation efficiency were 26.37±2.19 nm, 45.88±13.80 nm, 9.01±0.11% and 90.07±1.09%, respectively. The MPMs exhibited sustained release of QCT compared to free QCT as demonstrated from in vitro release experiments. The solubility of QCT was markedly improved compared to pure QCT. The MPMs were highly stable in aqueous media as demonstrated by their low critical micelle concentration. The concentration which inhibited 50% growth (IC50) values of both micellar preparations in all the cancer cell lines were significantly less compared to free QCT. CONCLUSION: Both the MPMs containing QCT could be used for effective delivery to different type of cancer and may be considered for further development.

The role of Bcl-2 family members in the progression of cutaneous melanoma.

The overwhelming problem of cutaneous melanoma is chemoresistance. Subversion of the biochemical changes that lead to chemoresistance intersects the apoptosis pathways. The mitochondrion has been a focal point of this intersection for the development of therapeutic strategies aimed at reducing the progression of melanoma. The Bcl-2 family of apoptotic regulators is arguably the most pivotal component to this mitochondrial response. The shear number of studies conducted on the relationship between melanoma and Bcl-2 members prompted us to evaluate the literature available and discern some rational utility of the data. We have found that there are striking inconsistencies for the expression of Bcl-2 family proteins with melanoma progression, particularly for Bcl-2. Roughly one-third of the data suggests an increase in Bcl-2 expression with advancing melanoma, while another third suggests a decrease. Furthermore, the remaining third found on the whole, a detectable level of Bcl-2 in all tissues of melanocytic origin. These discrepancies are difficult to rectify in light of the apparent success of recent clinical trials utilizing Bcl-2 antisense strategies. The general consensus in the literature is that pro-apoptotic Bax is decreased with melanoma progression while anti-apoptotic Bcl-xL and Mcl-1 appear to increase with progression. We suggest that the biochemical techniques being used for analysis present too great of a heterogeneity, which could be mitigated with more standard procedures and reagents. Finally the utility of 'multi-specific' antisense tactics could be a more effective way of targeting advanced melanoma disease.

Tissue-specific regulation of Fas/APO-1/CD95 expression by p53.

The regulation of Fas/APO-1(CD95), an important member of the tumor necrosis factor (TNF) superfamily involved in membrane-mediated apoptosis, has been a subject of recent research. Ligation of Fas by Fas ligand or an anti-Fas cross-linking antibody triggers receptor trimerization followed by recruitment of FADD to the cytoplasmic domain of the receptor and the activation of the caspase cascade. The tumor suppressor p53 has been shown to upregulate Fas expression under numerous pro-apoptotic stimuli in vitro. Using the p53 knockout mouse model, we demonstrate by Western blot analysis, immunohistochemistry, and semi-quantitative RT-PCR that Fas expression is reduced in spleen and liver from p53-/- mice compared to p53+/+ controls, while similar expression levels were observed in brain, heart, kidney, lung, skin, testis, and thymus between the two groups. While Fas protein was abundant in brain, heart, liver, and spleen, low levels of endogenous expression was observed in other tissues from the p53+/+ and p53-/- mice. These data indicate that p53 regulates Fas expression in a tissue-specific manner.

Tissue-specific regulation of Apaf-1 expression by p53.

The p53 tumor suppressor is a transcription factor that regulates cell cycle and apoptosis in response to a variety of environmental stress conditions. Recent studies indicated that the apoptotic protease activating factor-1 (Apaf-1) is a direct transcriptional target of p53 in DNA damage-induced apoptosis. However, the dependence of Apaf-1 expression on p53 has not been shown in normal physiological conditions. To further investigate their relationship, we first examined Apaf-1 protein levels in seven organs including brain, heart, kidney, liver, lung, spleen, and testis of wild-type (p53+/+) and p53-deficent (p53-/-) mice by Western blot analysis. The heart and spleen of the p53-/- mice showed a reduced Apaf-1 protein expression compared with the p53+/+ controls, while other organs did not show significant difference. The results from Western blot analysis were further confirmed by immunohistochemistry. Semi-quantitative RT-PCR was then employed to determine the expression of Apaf-1 transcripts. Similarly, we detected a lower Apaf-1 mRNA level in the heart and spleen of p53-/- mice compared with the p53+/+ controls, supporting a transcriptional upregulation of Apaf-1 by p53. Taken together, these results demonstrate that p53 regulation of Apaf-1 expression is tissue-specific.

Small molecule-induced mitochondrial disruption directs prostate cancer inhibition via UPR signaling.

We previously identified SMIP004 (N-(4-butyl-2-methyl-phenyl) acetamide) as a novel inducer of cancer-cell selective apoptosis of human prostate cancer cells. SMIP004 decreased the levels of positive cell cycle regulators, upregulated cyclin-dependent kinase inhibitors, and resulted in G1 arrest, inhibition of colony formation in soft agar, and cell death. However, the mechanism of SMIP004-induced cancer cell selective apoptosis remained unknown. Here, we used chemical genomic and proteomic profiling to unravel a SMIP004-induced pro-apoptotic pathway, which initiates with disruption of mitochondrial respiration leading to oxidative stress. This, in turn, activates two pathways, one eliciting cell cycle arrest by rapidly targeting cyclin D1 for proteasomal degradation and driving the transcriptional downregulation of the androgen receptor, and a second pathway that activates pro-apoptotic signaling through MAPK activation downstream of the unfolded protein response (UPR). SMIP004 potently inhibits the growth of prostate and breast cancer xenografts in mice. Our data suggest that SMIP004, by inducing mitochondrial ROS formation, targets specific sensitivities of prostate cancer cells to redox and bioenergetic imbalances that can be exploited in cancer therapy.

Comparative proteomic analysis of a cytosolic fraction from ß3 integrin-deficient cells.

Integrins are heterodimeric transmembrane receptors involved in sensing and transmitting informational cues from the extracellular environment to the cell. This study explored sub-proteome changes in response to elimination of the ß3 integrin using a knockout murine model. Cleavable isotope-coded affinity tagging (cICAT) in combination with sub-cellular fractionation, multiple dimensions of separation and tandem mass spectrometry (MS/MS) were used to characterize differentially expressed proteins among ß3 integrin(-/-) (ß3(-/-)) mouse embryonic fibroblasts and isogenic wild-type (WT) controls. From a cytosolic protein fraction, 48 proteins were identified, in which expression differed by > 1.5-fold. Predominant ontological groups included actin-binding/cytoskeletal proteins and protease/protease inhibitors. Interestingly, ß3 integrin expression was inversely correlated with expression of cathepsin B, a lysosomal cysteine protease, as its expression was greater by over 3.5-fold in the ß3(-/-) cells. This inverse correlation was also observed in stable heterologous cells transfected with ß3 integrin, where the intracellular expression and activity of cathepsin B was lower compared to control cells. Our data suggests that the composition of the cellular proteome is influenced by integrin expression patterns and reveals a strong functional relationship between ß3 integrin and cathepsin B.

A deficiency in the in vivo clearance of apoptotic cells is a feature of the NOD mouse.

Deficiencies in apoptotic cell clearance have been linked to autoimmunity. Here we examined the time-course of peritoneal macrophage phagocytosis of dying cells following the direct injection of apoptotic thymocytes into the peritoneum of NOD mice and BALB/c controls. Macrophages from NOD mice demonstrated a profound defect in the phagocytosis of apoptotic thymocytes as compared to control macrophages. Nonobese diabetic mice also demonstrated a decrease in the clearance of apoptotic cell loads following an apoptotic stimulus to thymocytes (dexamethasone) when compared to BALB/c or NOR controls. Further, NOD mice demonstrated an increase in apoptotic cell load following an apoptotic stimulus to keratinocytes (ultraviolet light, UVB) when compared to control strains. Animals deficient in macrophage phagocytosis of apoptotic debris often manifest an autoimmune phenotype characterized by the production of antinuclear autoantibodies (ANA). We determined whether increased apoptotic cell loads (through repeated exposure to UVB irradiation) could accelerate such autoimmune phenomena in young NOD mice. Following repeated UVB irradiation, NOD mice, but not BALB/c or NOR controls, developed ANA. We propose that abnormalities in apoptotic cell clearance by macrophages predispose NOD mice to autoimmunity.

Regulation of the Mdr1 isoforms in a p53-deficient mouse model.

Both p53 and multidrug transporters play important roles in chemoresistance. A transcriptional dependence of the Mdr1 gene promoter by p53 was first established a decade ago, and despite intense study, the p53-Mdr1 relationship still remains vague in vivo. The general model proposes that wild-type p53 down regulates, while mutant p53 up regulates, the Mdr1 promoter. Given that many studies have utilized cancer cell lines, minimal promoters and non-specific cDNA expression for in vitro experiments, we first sought to confirm the model using dermal fibroblasts isolated from the p53-knockout mice. We show that the gene products of the mouse Mdr1 homologue (Mdr1a and Mdr1b), namely P-glycoprotein (P-gp), appear upregulated at both the protein and mRNA levels in p53(-/-) mFbs compared with p53(+/+) cells. We demonstrate that transient transfection of a mouse p53(WT) expression plasmid into short-term primary p53(-/-) fibroblasts can revert P-gp overexpression. The difference in P-gp levels has functional significance in that p53(-/-) fibroblasts are more resistant to doxorubicin and vincristine treatment and this resistance can be attenuated in the presence of the P-gp inhibitor, verapamil. Furthermore, we demonstrate that in kidney, spleen and testis, P-gp expression is elevated in the absence of p53. In contrast, other organs such as heart, liver, lung, brain, thymus and skeletal muscle, show no difference in expression between p53(+/+) and p53(-/-) mice. Thus, our data shows a tissue-specific regulation of P-gp isoforms by p53 in the context of a p53-null mouse model.

Cancer chemoresistance: the relationship between p53 and multidrug transporters.

Extensive studies indicate that both p53 and multidrug transporters play important roles in chemoresistance. Since the initial reports a decade ago demonstrating a transcriptional dependence of the ABCB1 gene (MDR) promoter by p53, much data have been accumulated. However, despite being the subject of intense study, this p53-MDR relationship remains unclear in human cancers. The data are confounded by variable and contrasting results when considering the in vitro regulation and attempting to draw parallels in tissue specimens. The original model suggested that wild-type p53 downregulates the ABCB1 promoter, whereas mutant p53 increases expression of ABCB1. This review summarizes the data for and against this hypothesis. What emerges from these studies is a complex picture, where data have been obtained in support of this hypothesis, but there are also many circumstances where it is not supported. Taken together, these data suggest that the relationship between p53 and multidrug transporters is conditional. It is dependent on cellular environment, the drug used, and the nature of the p53 mutation.

Rh2, a compound extracted from ginseng, hypersensitizes multidrug-resistant tumor cells to chemotherapy.

Rh2 is a ginsenoside extracted from ginseng that has drawn attention in a few laboratories in Asian countries because of its potential tumor-inhibitory effect. In the present study, we tested Rh2 on many tumor-cell lines for its effects on cell proliferation, induction of apoptosis, and potential interaction with conventional chemotherapy agents. Our results showed that Rh2 inhibited cell growth by G1 arrest at low concentrations and induced apoptosis at high concentrations in a variety of tumor-cell lines, possibly through activation of caspases. The growth arrest and apoptosis may be mediated by 2 separate mechanisms. Apoptosis is not dependent on expression of the wild-type p53 nor the caspase 3. In addition, the apoptosis induced by Rh2 was mediated through glucocorticoid receptors. Most interestingly, Rh2 can act either additively or synergistically with chemotherapy drugs on cancer cells. Particularly, it hypersensitized multidrug-resistant breast cancer cells to paclitaxel. These results suggest that Rh2 possesses strong tumor-inhibiting properties, and potentially can be used in treatments for multidrug-resistant cancers, especially when it is used in combination with conventional chemotherapy agents.