Dibyen Majumdar

Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer

The use of live bacteria in the treatment of cancer has a long and interesting history. We report the use of a purified bacterial redox protein, azurin, that enters human cancer (melanoma UISO-Mel-2) cells and induces apoptosis. The induction of apoptosis occurs readily in melanoma cells harboring a functional tumor suppressor protein p53, but much less efficiently in p53-null mutant melanoma (UISO-Mel-6) cells. A redox-negative mutant form of azurin (M44K/M64E) demonstrates much less cytotoxicity to the UISO-Mel-2 cells than the wild-type protein. Azurin has been shown to be internalized in UISO-Mel-2 cells and is localized predominantly in the cytosol and in the nuclear fraction. In the p53-null UISO-Mel-6 cells, azurin is localized only in the cytosol. Thus, intracellular trafficking of azurin to the nucleus is p53-dependent. Azurin forms a complex with p53, thereby stabilizing it and raising its intracellular level in cytosolic, mitochondrial, and nuclear fractions. Corresponding to an increasing level of p53, an inducer of apoptosis, the level of Bax also increases in mitochondria, allowing significant release of mitochondrial cytochrome c into the cytosol, thus initiating the onset of apoptosis. The M44K/M64E mutant form of azurin, deficient in cytotoxicity, is also deficient in forming a complex with p53 and is less efficient in stabilizing p53 than wild-type azurin. Azurin has been shown to allow regression of human UISO-Mel-2 tumors xenotransplanted in nude mice and may potentially be used in cancer treatment.

Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer

Azurin, a copper-containing redox protein released by the pathogenic bacterium Pseudomonas aeruginosa, is highly cytotoxic to the human breast cancer cell line MCF-7, but is less cytotoxic toward p53-negative (MDA-MB-157) or nonfunctional p53 cell lines like MDD2 and MDA-MB-231. The purpose of this study was to investigate the underlying mechanism of the action of bacterial cupredoxin azurin in the regression of breast cancer and its potential chemotherapeutic efficacy. Azurin enters into the cytosol of MCF-7 cells and travels to the nucleus, enhancing the intracellular levels of p53 and Bax, thereby triggering the release of mitochondrial cytochrome c into the cytosol. This process activates the caspase cascade (including caspase-9 and caspase-7), thereby initiating the apoptotic process. Our results indicate that azurin-induced cell death stimuli are amplified in the presence of p53. In vivo injection of azurin in immunodeficient mice harboring xenografted human breast cancer cells in the mammary fat pad leads to statistically significant regression (85%, P=0.0179, Kruskal–Wallis Test) of the tumor. In conclusion, azurin blocks breast cancer cell proliferation and induces apoptosis through the mitochondrial pathway both in vitro and in vivo, thereby suggesting a potential chemotherapeutic application of this bacterial cupredoxin for the treatment of breast cancer.

Conditional Second-Order Generalized Estimating Equations for Generalized Linear and Nonlinear Mixed-Effects Models

Generalized linear and nonlinear mixed-effects models are used extensively in health care research, including applications in pharmacokinetics, clinical trials, and epidemiology. Because the underlying model may be nonlinear in the random effects, there will generally be no closed-form expression for the marginal likelihood or indeed for the marginal moments. Consequently, estimation is often carried out either using numerical integration techniques or by approximating the marginal likelihood and/or marginal moments using first-order expansion methods. An advantage of the first-order methods is that they do not necessarily require specification of a conditional like-lihood to estimate the regression parameters of interest. However, in many cases they may not take full advantage of the fact that the conditional variance depends on both fixed and random effects. In this article we propose using conditional second-order generalized estimating equations (CGEE2) to estimate both fixed- and random-effects parameters. Under mild regularity conditions, the CGEE2 estimator is shown to be consistent and asymptotically efficient with a rate of convergence depending on both the number of subjects and the number of observations per subject. We compare the CGEE2 estimator against alternative estimators using limited simulation and demonstrate its utility with a numerical example.

A peptide fragment of azurin induces a p53-mediated cell cycle arrest in human breast cancer cells

We report that amino acids 50 to 77 of azurin (p28) preferentially enter the human breast cancer cell lines MCF-7, ZR-75-1, and T47D through a caveolin-mediated pathway. Although p28 enters p53 wild-type MCF-7 and the isogenic p53 dominant-negative MDD2 breast cancer cell lines, p28 only induces a G2-M-phase cell cycle arrest and apoptosis in MCF-7 cells. p28 exerts its antiproliferative activity by reducing proteasomal degradation of p53 through formation of a p28:p53 complex within a hydrophobic DNA-binding domain (amino acids 80-276), increasing p53 levels and DNA-binding activity. Subsequent elevation of the cyclin-dependent kinase inhibitors p21 and p27 reduces cyclin-dependent kinase 2 and cyclin A levels in a time-dependent manner in MCF-7 cells but not in MDD2 cells. These results suggest that p28 and similar peptides that significantly reduce proteasomal degradation of p53 by a MDM2-independent pathway(s) may provide a unique series of cytostatic and cytotoxic (apoptotic) chemotherapeutic agents. [Mol Cancer Ther 2009;8(10):2947–58]

A cell penetrating peptide derived from azurin inhibits angiogenesis and tumor growth by inhibiting phosphorylation of VEGFR-2, FAK and Akt

Amino acids 50–77 (p28) of azurin, a 128 aa cupredoxin isolated from Pseudomonas aeruginosa, is essentially responsible for azurin’s preferential penetration of cancer cells. We now report that p28 also preferentially penetrates human umbilical vein endothelial cells (HUVEC), co-localized with caveolin-1 and VEGFR-2, and inhibits VEGF- and bFGF-induced migration, capillary tube formation and neoangiogenesis in multiple xenograft models. The antiangiogenic effect of p28 in HUVEC is associated with a dose-related non-competitive inhibition of VEGFR-2 kinase activity. However, unlike other antiangiogenic agents that inhibit the VEGFR-2 kinase, p28 decreased the downstream phosphorylation of FAK and Akt that normally precedes cellular repositioning of the cytoskeletal (F-actin), focal adhesion (FAK and paxillin), and cell to cell junction protein PECAM-1, inhibiting HUVEC motility and migration. The decrease in pFAK and pAkt levels suggests that p28 induces a pFAK-mediated loss of HUVEC motility and migration and a parallel Akt-associated reduction in cell matrix attachment and survival. This novel, direct antiangiogenic effect of p28 on endothelial cells may enhance the cell cycle inhibitory and apoptotic properties of this prototype peptide on tumor cell proliferation as it enters a Phase II clinical trial.

A-Optimal Incomplete Block Designs for Control-Test Treatment Comparisons

A-optimal designs for comparing v test treatments with a control in b blocks of size k each are considered. Several series of A-optimal designs are given when the parameters are in the range 2 ≤ k ≤ 8, k ≤ v ≤ 30, v ≤ b ≤ 50. A-optimal designs in blocks of size 2 are extensively studied through a combination of theoretical results and numerical investigations. Tables of approximately A-optimal designs are given when A-optimal designs are not easily available for the case k = 2.

Demonstrating circulation in vasculogenic mimicry patterns of uveal melanoma by confocal indocyanine green angiography.

PurposeVasculogenic mimicry patterns, formed by highly invasive melanoma cells, connect to endothelial cell-lined blood vessels and contain fluid in vitroand in vivo. This study was designed to determine if fluid leaks into vasculogenic mimicry patterns without circulation, or if fluid circulates in and clears from these patterns.MethodsIndocyanine green (ICG) laser scanning confocal angiography (Heidelberg Retinal Angiograph (HRA); Heidelberg Engineering, Heidelberg, Germany) was performed on nine patients with posterior choroidal melanoma in an institutional setting. Blood was drawn before the ICG injection and from the contralateral arm of the ICG injection site and 1 min after the injection. Outcome measures include time to first filling of retinal vessels and vasculogenic mimicry patterns and the time at which no fluorescence could be detected by the HRA instrument. After fluorescence was no longer detected in vessels or patterns, the tubes containing the patients blood was imaged by the Heidelberg HRA.ResultsLooping vasculogenic mimicry patterns were detected focally in five patients within 30 s after injection and were detectable up to 12 min post-injection. Blood drawn before ICG injection did not autofluoresce but ICG-containing blood pooled in the tube continued to fluoresce at 1-month post-injection.ConclusionsVasculogenic mimicry patterns are not part of the endothelial cell-lined vascular system and fluid enters these patterns through leakage. The rapid infusion of ICG into these patterns after injection and the disappearance of fluorescence detectable by the Heidelberg HRA suggest that fluid circulates in these patterns and does not accumulate as a stagnant pool.

Optimal Designs for Comparisons between Two Sets of Treatments.

Abstract : Suppose v treatments are to be compared in b blocks of size k each. Also suppose that the treatments are divided into 2 sets of u and w = v - u treatments. A-optimal designs are obtained for estimating all the differences of two treatments, one from each set. Optimal row-column designs are also obtained. Some new optimal designs for comparing several treatments with a single control are obtained as special cases. Key words include: A-optimal designs, block designs, row-column designs, comparisons between two sets of treatments, control-treatment comparisons, several controls. (Author)

Distinguishing Fibrovascular Septa From Vasculogenic Mimicry Patterns

CONTEXT Molecular analyses indicate that periodic acid-Schiff (PAS)-positive (laminin-rich) patterns in melanomas are generated by invasive tumor cells by vasculogenic mimicry. Some observers, however, consider these patterns to be fibrovascular septa, generated by a stromal host response. OBJECTIVE To delineate differences between vasculogenic mimicry patterns and fibrovascular septa in primary uveal melanomas. DESIGN Frequency distributions, associations with outcome, and thicknesses of trichrome-positive and PAS-positive looping patterns were determined in 234 primary uveal melanomas. Sequential sections of 13 additional primary uveal melanomas that contained PAS-positive/trichrome-negative looping patterns were stained for type I and type IV collagens, laminin, and fibronectin. Real-time quantitative polymerase chain reaction was performed on RNA from cultured uveal melanoma cells for the expression of COL1A1, COL4A2, and fibronectin. RESULTS Trichrome-positive loops were encountered less frequently than PAS-positive loops (10% vs 56%, respectively). Death from metastatic melanoma was strongly associated with PAS-positive (P < .001) but not with trichrome-positive (P = .57) loops. Trichrome-positive loops were significantly thicker than PAS-positive loops (P < .001). The PAS-positive patterns stained positive for laminin, type I and type IV collagens, and fibronectin. Type I collagen was detected within melanoma cells and focally within some PAS-positive patterns. Real-time quantitative polymerase chain reaction revealed 3-fold, 25-fold, and 97-fold increases, respectively, in expression of COL4A2, fibronectin, and COL1A1 by invasive pattern-forming primary melanoma cells compared with poorly invasive non-pattern-forming cells. CONCLUSIONS Fibrovascular septa are rare and prognostically insignificant in uveal melanomas, whereas vasculogenic mimicry patterns are associated with increased mortality. Type I collagen, seen focally in some vasculogenic mimicry patterns, may be synthesized by tumor cells, independent of a host stromal response.

Optimal Step-Type Designs for Comparing Test Treatments with a Control

Abstract The problem of obtaining A-optimal designs for comparing v test treatments with a control in b blocks of size k each is considered. A condition on the parameters (u, b, k) is identified for which optimal step-type designs can be obtained. Families of such designs are given. Methods of searching for highly efficient designs are proposed for situations in which it is difficult to determine an A-optimal design. Under the usual additive homoscedastic model, an A-optimal design minimizes the average variance of the least squares estimators of the control-test treatment comparisons. Majumdar and Notz (1983) gave a method for finding A-optimal designs. Their optimal designs can basically be of two types, using the terminology of Hedayat and Majumdar (1984): rectangular (R), in which every block has the same number of replications of the control, and step (5), in which some blocks contain the control t times and the others t + 1 times. Optimal R-type designs were studied by Hedayat and Majumdar (1985). F...