Photodynamic cell-kill analysis of breast tumor cells with a tamoxifen-pyropheophorbide conjugate

We hypothesized that estrogen receptor (ER) in hormone-sensitive breast cancer cells could be targeted for selective photodynamic killing of tumor cell with antiestrogen-porphyrin conjugates by combining the over-expression of ER in hormone-sensitive breast cancer cells and tumor-retention property of porphyrin photosensitizers. In this study we describe that a tamoxifen (TAM)-pyropheophorbide conjugate that specifically binds to ER alpha, caused selective cell-kill in MCF-7 breast cancer cells upon light exposure. Therefore, it is a potential candidate for ER-targeted photodynamic therapy of cancers (PDT) of tissues and organs that respond to estrogens/antiestrogens.     

Mathematical modeling of ovarian cancer treatments: sequencing of surgery and chemotherapy

Ovarian cancer has long been one of the most common forms of cancer in women. The main treatment for ovarian cancer comprises a combination of surgery and chemotherapy. In an effort to improve treatment strategies, a variety of mathematical models have been developed in the literature. In this paper, we consider a simple mathematical model that incorporates tumor growth as well as the effects of chemotherapeutic and surgical treatments in ovarian cancer. We consider several growth models and combine them with different cell-kill hypotheses. Surgery is assumed to eliminate a fixed fraction of tumor cells instantaneously. We discuss how different models predict the optimal sequencing of chemotherapeutic and surgical treatments. This work has been carried out in the context of ovarian cancer; however, the results may also be useful for other kind of cancers.     

Mathematical model for chemotherapeutic drug efficacy in arresting tumour growth based on the cancer stem cell hypothesis

OBJECTIVE: Cancer stem cells have been identified as the growth root for various malignant tumours and are thought to be responsible for cancer recurrence following treatment. MATERIALS AND METHODS: Here, a predictive mathematical model for the cancer stem cell hypothesis is used to understand tumour responses to chemotherapeutic drugs and judge the efficacy of treatments in arresting tumour growth. The impact of varying drug efficacies on different abnormal cell populations is investigated through the kinetics associated with their decline in response to therapy. RESULTS AND CONCLUSIONS: The model predicts the clinically established 'dandelion phenomenon' and suggests that the best response to chemotherapy occurs when a drug targets abnormal stem cells. We compare continuous and periodic drug infusion. For the latter, we examine the relative importance of the drug cell-kill rate and the mean time between successive therapies, to identify the key attributes for successful treatment.     

Modulation of IMP dehydrogenase activity and guanylate metabolism by tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide)

Tiazofurin, a C-nucleoside, was cytotoxic in hepatoma 3924A cells grown in culture with an LC50 = 7.5 microM. In the culture, a closely linked dose-related response of tumor cell-kill and depletion of GTP pools was observed after tiazofurin treatment. In rats carrying subcutaneously transplanted hepatoma 3924A solid tumors, a single intraperitoneal injection of tiazofurin (200 mg/kg) caused a rapid inhibition of IMP dehydrogenase (EC 1.2.1.14) activity and depleted GDP, GTP, and dGTP pools in the tumor; concurrently, the 5-phosphoribosyl 1-pyrophosphate (PRPP) and IMP pools expanded 8- and 15-fold, respectively. Tiazofurin decreased tumoral IMP dehydrogenase activity and dGTP pools in a dose-dependent manner over a range of 50-200 mg/kg; by contrast, the depletion of GTP and the accumulation of IMP and PRPP pools were near maximum at 50 mg/kg. The increase in PRPP pools may be attributed to an inhibition by IMP of the activity of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8). The IMP dehydrogenase activity and the pools of ribonucleotides returned to the normal range by 24-48 h after the single injection of tiazofurin. However, the markedly depleted dGTP pools remained low for 72 h. Tiazofurin treatment resulted in significant anti-tumor activity in rats inoculated with hepatoma 3924A. The decrease in GTP levels and particularly the sustained depletion in the dGTP pools may explain, in part at least, the chemo-therapeutic action of tiazofurin on hepatoma 3924A. This is the first report showing that a marked therapeutic response was achieved against rapidly growing hepatoma 3924A by treatment with a single anti-metabolite.     

An efficient targeted radiotherapy/gene therapy strategy utilising human telomerase promoters and radioastatine and harnessing radiation-mediated bystander effects

BACKGROUND: Targeted radiotherapy achieves malignant cell-specific concentration of radiation dosage by tumour-affinic molecules conjugated to radioactive atoms. Combining gene therapy with targeted radiotherapy is attractive because the associated cross-fire irradiation of the latter induces biological bystander effects upon neighbouring cells overcoming low gene transfer efficiency. METHODS: We sought to maximise the tumour specificity and efficacy of noradrenaline transporter (NAT) gene transfer combined with treatment using the radiopharmaceutical meta-[(131)I]iodobenzylguanidine ([(131)I]MIBG). Cell-kill was achieved by treatment with the beta-decay particle emitter [(131)I]MIBG or the alpha-particle emitter [(211)At]MABG. We utilised our novel transfected mosaic spheroid model (TMS) to determine whether this treatment strategy could result in sterilisation of spheroids containing only a small proportion of NAT-expressing cells. RESULTS: The concentrations of [(131)I]MIBG and [(211)At]MABG required to reduce to 0.1% the survival of clonogens derived from the TMS composed of 100% of NAT gene-transfected cells were 1.5 and 0.004 MBq/ml (RSV promoter), 8.5 and 0.0075 MBq/ml (hTR promoter), and 9.0 and 0.008 MBq/ml (hTERT promoter), respectively. The concentrations of radiopharmaceutical required to reduce to 0.1% the survival of clonogens derived from 5% RSV/NAT and 5% hTERT/NAT TMS were 14 and 23 MBq/ml, respectively, for treatment with [(131)I]MIBG and 0.018 and 0.028 MBq/ml, respectively, for treatment with [(211)At]MABG. CONCLUSIONS: These results indicate that the telomerase promoters have the capacity to drive the expression of the NAT. The potency of [(211)At]MABG is approximately three orders of magnitude greater than that of [(131)I]MIBG. Spheroids composed of only 5% of cells expressing NAT under the control of the RSV or hTERT promoter were sterilised by radiopharmaceutical treatment. This observation is indicative of bystander cell-kill.     

Steady state kinetics of an enzyme reaction with one substrate and one modifier

This study examines the steady state kinetics of a reaction involving an enzyme, a substrate and a modifier when the reaction follows Michaelis-Menten kinetics. Conditions for Michaelis-Menten kinetics are deduced, and it is shown that an analogue of detailed balance determines the complexity of the rate equations in these cases. A scheme to distinguish many cases of Michaelis-Menten kinetics is presented. It is shown that steady state kinetics are, in general, insufficient to specify the mechanism of a reaction, since different effects of a modifier can give identical steady state kinetic data.     

Oxygen diffusion in a spherical cell with nonlinear oxygen uptake kinetics

The oxygen diffusion in a spherical cell is analyzed in the present work. An oxygen uptake kinetics of the Michaelis-Menten type is employed. The oxygen uptake kinetics predicts the oxygen uptake rates which agree fairly well with the observed data. It has been found that difference between the predicted steady state oxygen tension distribution using the previous simplified oxygen uptake kinetics and that using the present non-linear kinetics is very significant.     

Long tail kinetics in biophysics?

Long tail kinetics describe a variety of data from complex, disordered materials that cannot be described by conventional kinetics. It is suggested that the kinetics of diffusive motion in complex biological media, such as cytoplasm or biomembranes, might also have long tails. The effects of long tail kinetics are investigated for two standard biophysical measurements, fluorescence recovery after photobleaching (FRAP), and dynamic light scattering (DLS). It is shown that long tail kinetic data would yield significantly distorted and misleading results when analyzed assuming conventional kinetics.     

Probing the kinetics of single molecule protein folding

We propose an approach to integrate the theory, simulations, and experiments in protein-folding kinetics. This is realized by measuring the mean and high-order moments of the first-passage time and its associated distribution. The full kinetics is revealed in the current theoretical framework through these measurements. In the experiments, information about the statistical properties of first-passage times can be obtained from the kinetic folding trajectories of single molecule experiments (for example, fluorescence). Theoretical/simulation and experimental approaches can be directly related. We study in particular the temperature-varying kinetics to probe the underlying structure of the folding energy landscape. At high temperatures, exponential kinetics is observed; there are multiple parallel kinetic paths leading to the native state. At intermediate temperatures, nonexponential kinetics appears, revealing the nature of the distribution of local traps on the landscape and, as a result, discrete kinetic paths emerge. At very low temperatures, exponential kinetics is again observed; the dynamics on the underlying landscape is dominated by a single barrier. The ratio between first-passage-time moments is proposed to be a good variable to quantitatively probe these kinetic changes. The temperature-dependent kinetics is consistent with the strange kinetics found in folding dynamics experiments. The potential applications of the current results to single-molecule protein folding are discussed.     

A unified theory of nucleation-rate-limited DNA renaturation kinetics

DNA renaturations under nucleation-rate-limiting conditions on simple DNA such as bacterial and bacteriophage DNA show significant deviation from ideal second-order kinetics when followed by optical density measurements at 260 nm. Ideal second-order kinetics yield linear plots when the data is plotted in the standard reciprocal second-order (RSO) manner. The observed deviations from ideal second-order behavior take the form of steadily downward-curving RSO plots. In this paper, experiments are presented for E. coli and T2 DNA documenting this non-ideal behavior. Since experiments using T4, T5 and B, subtilis DNA yield identical non-ideal behavior, this behavior appears to be a property of DNA renaturation followed by optical density, not a peculiarity of a particular DNA. Identical non-ideal behavior is also seen in kinetics followed by S1 nuclease assay. A theory is developed to explain this deviation from ideal second-order kinetics. The theory also explains why kinetics followed by hydroxyapatite chromatography show nearly ideal second-order kinetics. In contrast to the approach taken by others in developing equations that describe S1 nuclease monitored reactions, our view is that nonideal second-order kinetics are fundamentally due to the reacton of free single strands to yield partially helical duplex species. Later reactions of these species tend to reduce the deviations from non-ideal second-order kinetics.     

Probing the folding and unfolding dynamics of secondary and tertiary structures in a three-helix bundle protein

Fast relaxation kinetics studies of the B-domain of staphylococcal protein A were performed to characterize the folding and unfolding of this small three-helix bundle protein. The relaxation kinetics were initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The kinetics monitored within the amide I' absorbance of the polypeptide backbone exhibit two distinct kinetics phases with nanosecond and microsecond relaxation times. The fast kinetics relaxation time is close to the diffusion limits placed on protein folding reactions. The fast kinetics phase is dominated by the relaxation of the solvated helix (nu = 1632 cm(-1)), which reports on the fast relaxation of the individual helices. The slow kinetics phase follows the cooperative relaxation of the native helical bundle core that is monitored by both solvated (nu = 1632 cm(-1)) and buried helical IR bands (nu = 1652 cm(-1)). The folding rates of the slow kinetics phase calculated over an extended temperature range indicate that the core formation of this protein follows a pathway that is energetically downhill. The unfolding rates are much more strongly temperature-dependent indicating an activated process with a large energy barrier. These results provide significant insight into the primary process of protein folding and suggest that fast formation of helices can drive the folding of helical proteins.     

Product Inhibition in Native-State Proteolysis

The proteolysis kinetics of intact proteins by nonspecific proteases provides valuable information on transient partial unfolding of proteins under native conditions. Native-state proteolysis is an approach to utilize the proteolysis kinetics to assess the energetics of partial unfolding in a quantitative manner. In native-state proteolysis, folded proteins are incubated with nonspecific proteases, and the rate of proteolysis is determined from the disappearance of the intact protein. We report here that proteolysis of intact proteins by nonspecific proteases, thermolysin and subtilisin deviates from first-order kinetics. First-order kinetics has been assumed for the analysis of native-state proteolysis. By analyzing the kinetics of proteolysis with varying concentrations of substrate proteins and also with cleavage products, we found that the deviation from first-order kinetics results from product inhibition. A kinetic model including competitive product inhibition agrees well with the proteolysis time course and allows us to determine the uninhibited rate constant for proteolysis as well as the apparent inhibition constant. Our finding suggests that the likelihood of product inhibition must be considered for quantitative assessment of proteolysis kinetics.     

The kinetic pathway of folding of barnase

To search for folding intermediates, we have examined the folding and unfolding kinetics of wild-type barnase and four representative mutants under a wide range of conditions that span two-state and multi-state kinetics. The choice of mutants and conditions provided in-built controls for artifacts that might distort the interpretation of kinetics, such as the non-linearity of kinetic and equilibrium data with concentration of denaturant. We measured unfolding rate constants over a complete range of denaturant concentration by using by 1H/2H-exchange kinetics under conditions that favour folding, conventional stopped-flow methods at higher denaturant concentrations and continuous flow. Under conditions that favour multi-state kinetics, plots of the rate constants for unfolding against denaturant concentration fitted quantitatively to the equation for three-state kinetics, with a sigmoid component for a change of rate determining step, as did the refolding kinetics. The position of the transition state on the reaction pathway, as measured by solvent exposure (the Tanford beta value) also moved with denaturant concentration, fitting quantitatively to the same equations with a change of rate determining step. The sigmoid behaviour disappeared under conditions that favoured two-state kinetics. Those data combined with direct structural observations and simulation support a minimal reaction pathway for the folding of barnase that involves two detectable folding intermediates. The first intermediate, I(1), is the denatured state under physiological conditions, D(Phys), which has native-like topology, is lower in energy than the random-flight denatured state U and is suggested by molecular dynamics simulation of unfolding to be on-pathway. The second intermediate, I(2), is high energy, and is proven by the change in rate determining step in the unfolding kinetics to be on-pathway. The change in rate determining step in unfolding with structure or environment reflects the change in partitioning of this intermediate to products or starting materials.     

Phosphofructokinase from lycopersicon esculentum fruits-I. Kinetic properties in relation to its substrates

The kinetic properties of phosphofructokinase with regard to its substrates are discussed. Free ATP is inhibitory to the enzyme while the Mg-ATP complex at a concentration up to 5 mM is not. The kinetics with respect to Mg-ATP follow simple Michaelis-Menten kinetics and this pattern is not affected by changes in concentration of the second substrate fructose-6-phosphate (F6P). The kinetics with respect to F6P showed apparent negative co-operative interactions in the presence of saturating levels of Mg²⁺ relative to ATP. In the presence of inhibitory levels of free ATP, the kinetics showed positive co-operative interactions. The relationship between the nature of the kinetics of the enzyme with F6P and the various molecular forms of PFK are discussed.     

Growth and cometabolic reduction kinetics of a uranium- and sulfate-reducing Desulfovibrio/Clostridia mixed culture: Temperature effects

Bioremediation of contaminated soils and aquifers is subject to spatial and temporal temperature changes that can alter the kinetics of key microbial processes. This study quantifies temperature effects on the kinetics of an ethanol-fed sulfate-reducing mixed culture derived from a uranium-contaminated aquifer subject to seasonal temperature fluctuations. The mixed culture contains Desulfovibrio sp. and a Clostridia-like organism. Rates of growth, ethanol utilization, decay, and uranium reduction decreased with decreasing temperature. No significant uranium reduction was observed at 10 degrees C. While both Monod saturation kinetics and pseudo second-order kinetics adequately described the rates of growth and utilization of electron donor (ethanol), model parameters for the pseudo second-order expression had smaller uncertainties. Uranium reduction kinetics were best described by pseudo second-order kinetics modified to include a term for inactivation/death of cells.     

Application of fuzzy-logic models for metabolic control analysis

A priori information or valuable qualitative knowledge can be incorporated explicitly to describe enzyme kinetics making use of fuzzy-logic models. Although restricted to linear relationships, it is shown that fuzzy-logic augmented models are not only able to capture non-linear features of enzyme kinetics but also allow the proper mathematical treatment of metabolic control analysis. The explicit incorporation of valuable qualitative knowledge is crucial, particularly when handling data estimated from in vivo kinetics studies, since this experimental information is scarce and usually contains measurement errors. Therefore, data-driven techniques, such as the one presented in this work, form a serious alternative to established kinetics approaches.     

Protection of apoptotic cell death by protein A

The word Apoptosis or pragrammed cell death is described as the ultimate end of multiple cellular events converging from numerous initiating events to the ultimate death of a cell or organism. Several processes, such as initiation of death signals at the plasma membrane, expression of pro-apoptotic oncoproteins, activation of death proteases, endonucleases etc., that ultimately coalesce to a common irreversible execution phase, lead to cell demise. Counteracting the death signals are cell survival factors. A balance between the cell death and cell survival factors plays a major role in the decision making process as to whether a cell should die or must live. It is, therefore, hypothesized that if the balance can be shifted in favor of cell survival, one might be able to arrest the aging process, save the injured cells or else if the balance is shifted toward cell-kill it might help destroy tumors and other undesirable cells.Protein A (PA) of Staphylococcus aureus has been found to have multifarious biological response modifying properties. It has been shown to possess anti-tumor, anti-toxic, anti-parasitic and antifungal activities. It also acts as a potent immunostimulator. PA can protect bone marrow progenitor cells from zidovudin(AZT)-induced apoptosis and can stimulate immunocyte proliferation, thereby helping to replenish/restore the depleted hematopoietic cell pool. Such ability to replenish hematopoietic cells is a common property of PA observed against a number of toxic drugs/chemicals, such as cyclophosphamide, benzene, aflatoxin, salmonella endotoxin, etc.Interestingly, it was further demonstrated in our laboratory that PA can selectively kill tumor cells without affecting normal cells of the host. A search for the mechanisms of PA action revealed that this bacterial protein could shift the balance between pro- and anti-apoptotic proteins in favor of survival in normal cells, but in favor of cell death in tumor cells at a particular dose level. This unique property of PA suggests that controlled use of such type of Biological Response Modifier might help in controlling both cell growth and death phenomena.     

The complex kinetics of protein folding in wide temperature ranges

The complex protein folding kinetics in wide temperature ranges is studied through diffusive dynamics on the underlying energy landscape. The well-known kinetic chevron rollover behavior is recovered from the mean first passage time, with the U-shape dependence on temperature. The fastest folding temperature T0 is found to be smaller than the folding transition temperature Tf. We found that the fluctuations of the kinetics through the distribution of first passage time show rather universal behavior, from high-temperature exponential Poissonian kinetics to the relatively low-temperature highly non-exponential kinetics. The transition temperature is at Tk and T0 < Tk < Tf. In certain low-temperature regimes, a power law behavior at long time emerges. At very low temperatures (lower than trapping transition temperature T < T0/(4 approximately 6)), the kinetics is an exponential Poissonian process again.     

Hypothesis formulation and testing in aquatic bioassays: a deterministic model approach

The paper examines the significance of toxicant kinetics information obtained from aquatic toxicity bioassays and bioconcentration tests. The data, bioconcentration kinetics and acute mortality versus exposure-duration information for juvenile American flagfish (Jordanella foridae) exposed to 1,4-dichlorobenzene, are interpreted in terms of a one-compartment, first-order kinetics model. The output of the model is used to formulate a testable hypothesis regarding the comparison of toxicant kinetics derived from both bioconcentration test exposures and toxicity bioassays. The model's estimates of the toxicant body burden attained at mortality are compared with theoretical and observed body burdens from literature sources. The use of a simple, deterministic residue-based, one-compartment, first-order kinetics model to evaluate existing data, as well as to formulate hypotheses to direct experimental designs, is examined.     

Kinetics of proton diffusion in the regimes of fast and slow exchange between the membrane surface and the bulk solution

The phenomenological model developed in our recent publications [9,10] is used to investigate the kinetics of proton diffusion from a source to a detector on the membrane surface. In most cases the observed kinetics shows a single diffusional maximum with the exponential ascending front and the power-law descending tail. The kinetics depends on the distance between the source and the detector. If the detector is located inside the proton collecting antenna, the kinetics corresponds to the surface diffusion at the times near the maximum and shortly thereafter, and it turns into the bulk diffusion kinetics at longer times, after the equilibrium is established between the membrane surface and the bulk solution. If the detector is located outside the antenna, the kinetics corresponds to the bulk diffusion at all times where the signal is nonvanishing. What is seen at locations near the antenna radius depends on the exchange regime. In the regime of fast exchange between the surface and the bulk as compared to the bulk diffusion, the kinetics shows a single peak whose location is intermediate between the peaks for the surface and bulk diffusion. In the regime of slow exchange there are two maxima corresponding to the surface and bulk diffusion. In buffered solutions the antenna radius decreases with increasing buffer concentration, which changes the kinetics from the surface to bulk diffusion. The theory is applied to interprete recent experiments on a phospholipid membrane [25]. It is found that (i) the fast exchange regime is operating since only a single maximum is observed; (ii) the shift of the maximum toward longer times with increasing buffer concentration is a manifestation of the transition from the surface to bulk diffusion kinetics. The authors are grateful to Yu. N. Antonenko and A. I. Kotelnikov for helpful discussions. This work was supported by the Russian Foundation for Basic Research (05-03-32104), U.S. Civilian Research and Development Foundation (RUC2-2658-MO-05), U. S. National Science Foundation, and U. S. National Institutes of Health.