Coupled equilibria of a self-associating drug loaded into polymeric nanoparticles.

Doxorubicin (DOX) and other anti-cancer drugs are often formulated using nanoparticles for passive or active targeting and reducing detrimental side effects. Anionic polymers have been shown to effectively facilitate loading of cationic DOX hydrochloride into nanoparticles with high efficiency. One powerful method to study DOX loading into anionic polymeric nanoparticles has been isothermal titration calorimetry (ITC), but the curves are complex and were previously interpreted in a largely qualitative manner only. Here we present detailed quantitative modelling of such ITC data, corroborated by zeta potential measurements and dynamic light scattering. The model takes into account 3 coupled equilibria. First, DOX self-associates in solution to dimers and larger aggregates. This effect is modelled in terms of the stepwise aggregation model. Second, DOX binds with a 1:1 stoichiometry to the carboxylic acids in the polymer at low salt. At about 33% saturation, the nanoparticles collapse in size and the enthalpy of further binding becomes less exothermic. Third, free DOX also stacks onto polymer-bound DOX. This stacking effect is very weak and hardly detected by ITC. It is, however, revealed by a positive zeta potential. The present work demonstrates the power of combining ITC with light scattering and zeta potential measurements for studying the thermodynamics of drug loading into polymeric nanoparticles.

Alterations in plasma lipid profile patterns in head and neck cancer and oral precancerous conditions.

BACKGROUND: The changes in lipid profile have long been associated with cancer because lipids play a key role in maintenance of cell integrity. AIMS: The present study evaluated alterations in plasma lipid profile in untreated head and neck cancer patients as well as patients with oral precancerous conditions (OPC) and its association with habit of tobacco consumption. MATERIAL AND METHODS: This hospital-based case control study included 184 head and neck cancer patients, 153 patients with OPC and 52 controls. Plasma lipids including: (i) Total cholesterol, (ii) LDL cholesterol (LDLC), (iii) HDL cholesterol (HDLC) (iv) VLDL cholesterol (VLDLC) and (v) triglycerides were analysed by spectrophotometric kits. STATISTICAL ANALYSIS USED: Student's t-test was performed to compare mean values of the parameters. RESULTS: A significant decrease in plasma total cholesterol and HDLC was observed in cancer patients (P=0.008 and P=0.000 respectively) as well as in patients with OPC (P=0.014 and P=0.000, respectively) as compared to the controls. The plasma VLDL and triglycerides levels were significantly lower in cancer patients as compared to the patients with OPC (P=0.04) and controls (P=0.059). The tobacco habituates showed lower plasma lipid levels than the non-habituates. Our data strengthen the evidence of an inverse relationship between plasma lipid levels and head and neck malignancies as well as OPC. CONCLUSION: The lower levels of plasma cholesterol and other lipid constituents in patients might be due to their increased utilization by neoplastic cells for new membrane biogenesis. The findings strongly warrant an in-depth study of alterations in plasma lipid profile in head neck cancer patients.

Clinical usefulness of alterations in sialic acid, sialyl transferase and sialoproteins in breast cancer.

Sialic acid, the end moieties of the carbohydrate chains are biologically important and essential for functions of glycoconjugates and are reported to be altered in cancer patients. Two hundred and twenty five breast cancer (BC) patients, 100 patients with benign breast disease (BBD) and 100 healthy females (controls) were enrolled for the study. Eight hundred and twenty four follow-up samples of 225 breast carcinoma patients were also evaluated. The association of sialic acid forms, sialyltransferase and α-2-6 sialoproteins levels with presence and extent as well as prognosis of breast carcinoma was studied. Serum sialic acid forms and sialyltransferase revealed significantly elevated levels among untreated breast cancer patients as compared to the controls, patients with BBD as well as cancer patients in remission. Non-responders showed comparable levels of the markers with those found in breast cancer patients at the time of diagnosis. Higher levels of sialic acid forms at diagnosis were associated with poor prognosis. A positive correlation between serum levels of different forms of sialic acids and extent of malignant disease was observed. The changes in serum proteins with terminal α-2-6 sialic acid correlated well with alterations in the levels of sialic acid forms and sialyltransferase. Malignant tissues showed elevated levels of sialic acid and sialyltransferase as compared to surrounding normal tissues.The results suggested potential utility of these markers in evaluation of clinical outcome.

Evaluation of serum sialic acid, sialyltransferase and sialoproteins in oral cavity cancer.

OBJECTIVE: Altered glycosylation of glycoconjugates is among the important molecular changes that accompany malignant transformation. The aim of our study was to investigate clinical usefulness of circulatory levels of sialic acid, sialoproteins and sialyltransferase for early diagnosis and management of oral cavity cancer (OC) patients. MATERIALS AND METHODS: Blood samples collected from 210 untreated OC patients, 100 patients with oral precancerous conditions (OPC) and 100 healthy males. OC patients were followed after initiation of anticancer treatment and 394 follow-up samples were also collected. Serum sialic acid levels were measured spectrophotometrically. Sialyltransferase activity was analysed using radioassay. Alpha 2-6 sialoproteins were isolated using lectin affinity chromatography. RESULTS: Serum levels of free, protein bound and total sialic acid as well as their ratio with total proteins were significantly elevated in untreated OC patients compared with healthy individuals, patients with OPC as well as complete responders (CR). Levels of the markers were comparable between untreated OC patients and non-responders. We observed positive correlation between serum levels of the markers and extent of malignant disease. Serum sialyltransferase activity showed significant elevations in OC patients compared with the controls (P<0.001), patients with OPC (P<0.05) and CR (P<0.05). Higher sialic acid levels in OC patients at the time of diagnosis showed poor survival. The changes in serum proteins with terminal alpha 2-6 sialic acid correlated well with the alterations in the levels of sialic acid forms and sialyltransferase activity. CONCLUSION: Our results confirmed the elevations in sialic acid and sialyltransferase levels in OC patients and suggested potential utility of these parameters in prognostication and treatment monitoring of this neoplasm. The alterations in these parameters in circulation were in accordance with the changes in alpha 2-6 sialylated proteins.

Serum glutathione-S-transferase and glutathione reductase activity in head and neck cancer patients.

Glutathione, an antioxidant plays an important role in phase-II detoxification of carcinogens. The levels of reduced glutathione are maintained by glutathione-depleting as well as replenishing enzymes such as glutathione-s-transferase (GST) and glutathione reductase (GR), respectively. Pre and post treatment changes in GST and GR activities in head and neck cancer patients were analysed. Serum GST and GR were analysed from untreated head and neck cancer patients (PT) (n=146), controls with habit of tobacco (VHT) (n=25) as well as without (no) habit of tobacco (NHT) (n=25) and patients with oral precancerous conditions (OPC) (n=50). The cancer patients were followed-up after initiation of anticancer therapy. Follow-up blood samples were collected. Serum GST and GR activities were estimated by highly sensitive and specific spectrophotometric methods. Untreated cancer patients showed elevated mean serum GST and GR activities as compared to NHT. Patients with OPC had declined mean GST activity as compared to WHT and untreated cancer patients. Paired t-test revealed that complete responders (CR) showed significantly elevated GST levels and declined GR activities (p < 0.001) as compared to those in PT. No correlation was found between stage of the disease and GST, GR activity. Paired t-test showed significant decreased in GR activity in nonresponders (NR) treated with radiotherapy (p=0.01). The study suggested that analysis of glutathione and glutathione-depleting enzymes can be helpful for treatment monitoring of head and neck cancer patients.

Evaluation of serum alkaline DNase activity in treatment monitoring of head and neck cancer patients.

Our previously published data on breast cancer suggest that serum alkaline DNase, a known circulating tumour marker, can be used for treatment monitoring of cancer patients. Serum alkaline DNase activities were analyzed in 215 untreated head and neck cancer patients. The enzyme activity ranged from 0.17 to 97.97 IKU/l in untreated cancer patients. Responders (n = 314) showed significantly elevated activity of alkaline DNase as compared to untreated cancer patients (p < 0.001). While non-responders (n = 168) showed comparable activity with untreated cancer patients. Serum alkaline DNase activities were significantly elevated in responders as compared to non-responders (p < 0.001). Our clinical studies during follow-up of patients indicated that the variations in serum alkaline DNase activities in individual patients correlate closely with response to therapy. Serum alkaline DNase also appeared to be useful in predicting treatment response in the long-term follow-up of patients. Serum alkaline DNase was systematically examined as a possible indicator for recurrence in patients under complete remission. In conclusion, serum alkaline DNase may be useful as a treatment monitoring in patients with head and neck malignancies.

Distribution of the rubredoxin gene among the Clostridium butyricum species.

With PCR methods, the rubredoxin gene was systematically identified among 11 strains of Clostridium butyricum; this ubiquity means major functions in the metabolism of the Clostridia. The 11 PCR products allowed deduction of a sequence of 26 amino acids corresponding to positions 11-36 of the rubredoxin. They all contained the tyrosines at positions 11 and 13 and the phenylalanine at position 30 characteristic of the rubredoxin, but differed at positions 14-17, 20, 25, 29, and 31, allowing determination of three types of rubredoxins among these 11 strains of C. butyricum.

Evaluation of glycoprotein constituents in head and neck cancer patients undergoing radiotherapy.

BACKGROUND: Alterations in serum levels of several glycoprotein constituents are reported to be useful for treatment monitoring of cancer patients. The purpose of this study was to determine efficacy of sialic acid and seromucoid fraction as treatment monitors for head and neck (H&N) cancer patients undergoing radiotherapy (RT). METHODS: Serum levels of total sialic acid (TSA), lipid-bound sialic acid (LSA), and seromucoid fraction (measured as Mucoid protein [MP] and hexose) were studied in age matched controls and in patients with H&N cancer at diagnosis and during/after completion of RT. The markers were estimated by spectrophotometric methods. RESULTS: Serum levels of sialic acid forms and seromucoid fraction were significantly elevated (p<.001) in untreated H&N cancer patients (n = 32) as compared with controls (n = 50). The marker levels were significantly declined (p<.001) in H&N cancer patients who responded to RT as compared with their levels at diagnosis, whereas the levels were persistently elevated in nonresponders. CONCLUSION: Evaluation of sialic acid forms and seromucoid fraction could be used for monitoring the treatment of H&N cancer patients undergoing RT.

Evaluation of telomerase activation in head and neck cancer.

During replication of the linear chromosomes, telomeres, i.e. the ends of the chromosomes, are not replicated completely by the conventional DNA polymerases. Therefore, normal somatic cells senesce after certain number of cell divisions. Telomerase is a special reverse transcriptase used by most eukaryotes to achieve immortalization. Telomerase activity has been determined in a variety of cancers. However, there are few reports on telomerase activity in head and neck cancer. The etiology of the disease in India is completely different from Western countries. Tobacco consumption is more prevalent in India and the mode of tobacco consumption (e.g. chewing, snuffing, bidi smoking, reverse smoking) is also different. The present study determined telomerase activity in 32 malignant tumour samples of head and neck cancer patients, 11 samples from patients with precancerous/benign lesions and 30 samples of adjacent normal tissues. Telomerase was found to be activated in 80% of the patients with head and neck cancer, 100% of the patients with precancerous/benign lesions and 74% of the adjacent normal tissues. According to the theory of field cancerization, carcinogenic insults (e.g. tobacco) may result into multiple malignant foci. This fact may explain the reason for high telomerase positivity in adjacent normal as well as precancerous/benign tissues. Telomerase activation and the clinical or histopathological characteristics of the head and neck cancer patients were observed to be independent features. This is a preliminary report which has generated a greater interest for in-depth elucidation of the role of telomerase and telomeres in head and neck carcinogenesis in India.

Seromucoid fraction: a useful biomarker for patients with breast cancer.

Seromucoid fraction was measured in terms of mucoid proteins (MP) and hexose content from sera of 1) 47 healthy women, 2) 48 women who had benign breast diseases, 3) 151 untreated patients with breast cancer, and 4) 245 follow-up samples collected from the same breast cancer patients. Mucoid proteins and hexose levels were found to be significantly elevated in untreated patients with breast cancer when compared with the healthy participants (p < 0.001) and patients who had benign breast diseases (p < 0.02 and p < 0.05, respectively). Receiver operating characteristic (ROC) curve analysis revealed potential diagnostic application of both markers for breast cancer. A good correlation was observed between favorable treatment response and decline in serum-marker levels. The markers in patients who did not respond to anticancer therapy remained stable or increased during follow-up. These data indicate that seromucoid fraction can be an useful biochemical marker for breast cancer patients.

Role of sialic acid and alkaline DNase in breast cancer.

Serum levels of sialic acid and alkaline DNase (ADA) were analysed in 495 blood samples collected from 170 breast cancer patients before and during/after anticancer treatment. Fifty-six healthy females were included in the study to define the cutoff values. The markers were analysed by highly sensitive spectrophotometric methods. Statistical evaluation of the data was done using Student's 't' test, paired 't' test and ROC curve analysis. The total sialic acid (TSA), lipid bound sialic acid (LSA) and ADA in sera of untreated breast cancer patients were significantly higher than in controls. ROC curve analysis revealed TSA and LSA to be useful markers for diagnosis of breast cancer. Serum levels of TSA and LSA were significantly decreased in complete responders as compared to their pretreatment values. The pretreatment ADA values showed much individual variation. However, responders showed higher levels of ADA than untreated patients. In nonresponders the values of the biomarkers were comparable with pretreatment levels. The study suggested that TSA and LSA can be helpful in the diagnosis of breast cancer. All three markers can be used for assessment of response to anticancer treatment in breast cancer patients.

Glycoprotein electrophoretic pattern in sera of patients with oral precancerous conditions and upper aerodigestive tract cancer.

Cancer of the upper aerodigestive tract is one of the leading sites of human malignancies in India. Various glycoproteins have been claimed to be specifically associated with cancer. Serum glycoprotein electrophoresis was carried out in sera obtained from 23 healthy individuals [10 without habit of tobacco consumption (NHT) 13 with habit of tobacco consumption (WHT)], 46 patients with oral precancerous conditions (OPC) and 110 untreated patients with upper aerodigestive tract cancer. Eighty-six samples from the cancer patients were also collected after initiation of anticancer therapy. The albumin, alpha, beta and gamma region glycoproteins were quantitated by densitometric scanning after separation by polyacrylamide disc gel (PADG) electrophoresis. Mean values of albumin and alpha region glycoproteins were significantly lower in WHT and patients with OPC as compared to NHT. The gamma region glycoproteins were significantly elevated in WHT, patients with OPC and untreated cancer patients as compared to the NHT. The albumin region glycoproteins were significantly low, whereas, gamma region glycoproteins were significantly elevated in nonresponders as compared to their pretreatment levels. The glycoprotein values in complete responders were comparable with NHT. An extra glycoprotein band was found in the post beta region, in most of the individuals (>50%) with habit of tobacco consumption in all the groups. There was a decrease in the albumin/gamma, alpha/gamma and beta/gamma values in patients with OPC as well as untreated cancer patients as compared to NHT. Albumin/gamma, alpha/gamma and beta/gamma values were lower in nonresponders as compared to their pretreatment value. The results indicate that the alterations in glycoprotein electrophoresis pattern may be useful for early detection of cancer of the upper aerodigestive tract. It may also be helpful in treatment monitoring of cancer patients.

Importance of glycoproteins in human cancer.

Usefulness of cell surface glycoprotein components as markers in early detection of cancer and in monitoring progress during treatment has been evaluated. Total sialic acid (TSA), lipid bound sialic acid (LSA) and seromucoid fractions (SF) have been compared in the sera of healthy human volunteers and patients at different stages of diagnosis and treatment of leukemia, cancer of breast, cervix, and oral cavity. The levels of TSA, LSA and SF are found to be increased in cancer and is proportionate with malignancy. Their levels show decline in patients who respond well to treatment and show increase in patients with recurrence of cancer even before any clinical evidence of recurrence is available. Changes have also been noted in the glycoprotein fractions and their ratios.

Electrophoretic pattern of serum glycoproteins on polyacrylamide disc gel in patients with breast cancer.

Numerous investigators have identified, isolated and characterized serum glycoproteins that are claimed to be specifically associated with malignancy. We have carried out serum glycoprotein electrophoresis on polyacrylamide disc gel in 53 breast cancer patients, at diagnosis as well as during and after therapy. Follow-up samples were divided into complete responders (CR) (n = 138) and nonresponders (NR) (n = 44). Glycoprotein electrophoresis showed multiple bands for each sample which were categorized into four groups: albumin, alpha, beta and gamma. The results revealed a decreasing number of CR and increasing number of NR with elevated (as compared to pretreatment levels) albumin fraction glycoproteins. Gamma region glycoproteins showed the reverse pattern to that of albumin region glycoproteins. The alpha and beta region glycoproteins revealed an increasing number of CR having higher values with increase in follow-up duration. In comparison with their pretreatment values CR showed significantly increased (Paired "t" test) values of albumin, alpha and beta region glycoproteins (p < 0.01, p < 0.001 and p < 0.001, respectively) and decreased gamma region glycoproteins (p < 0.001). The albumin, alpha, beta and gamma region glycoprotein levels were comparable between NR and untreated cancer patients. The variations in albumin, alpha, beta and gamma region glycoproteins correlate with treatment response, which might be useful in the treatment monitoring, and prediction of recurrence in breast cancer patients.

Carbon and electron flow in Clostridium butyricum grown in chemostat culture on glycerol and on glucose.

The metabolism of Clostridium butyricum DSM 5431 was studied in chemostat culture under carbon limitation using either glucose or glycerol. On glycerol, the enzymes glycerol dehydrogenase, diol dehydratase and 1,3-propanediol (1,3-PD) dehydrogenase constitute the branch point that partitions the carbon flux between the competing pathways, i.e. formation of either 1,3-PD or acetate and butyrate. The increasing levels of these enzyme activities with increasing dilution rates (D) explained the constant proportion of glycerol conversion into 1,3-PD. The production of acetate or butyrate constitutes another important branch point and when D increased (i) large amounts of intracellular acetyl-CoA accumulated, (ii) the carbon flux switched from butyric acid to acetic acid, (iii) the specific activity of thiolase was not affected, suggesting this enzyme may be the bottleneck for carbon flux to butyrate biosynthesis providing an explanation for the accumulation of large amounts of intracellular acetyl-CoA, and (iv) high levels of NADH were found in the cell. Oxidation of NADH by 1,3-PD dehydrogenase was linked to the production of 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase. The fact that high intracellular concentrations of NADH were found means that diol dehydratase activity is the rate-limiting step in 1,3-PD formation, avoiding the accumulation of 3-HPA which is a very toxic compound. The specific rate of glucose catabolism (q glucose = 11.1 mmol h-1 g-1) was around four times lower than the specific rate of glycerol catabolism (q glucose = 57.4 mmol h-1 g-1). On glucose-grown cells, reducing equivalents which are released in the glycolytic pathway were reoxidized by the butyric pathway and the low specific formation rate of butyric acid led to an increase in the intracellular level of acetyl-CoA and NADH. Carbon flow was higher on glycerol due to the reoxidation of NADH by both butyric and PD pathways.

Carboxymethylcellulase and Avicelase activities from a cellulolytic Clostridium strain A11.

The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only adhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between "adhered enzyme system" and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the Vmax without significant Km variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose.

Clostridium cellulolyticum Viability and Sporulation under Cellobiose Starvation Conditions.

Depending on the moment of cellobiose starvation, Clostridium cellulolyticum cells behave in different ways. Cells starved during the exponential phase of growth sporulate at 30%, whereas exhaustion of the carbon substrate at the beginning of growth does not provoke cell sporulation. Growth in the presence of excess cellobiose generates 3% spores. The response of C. cellulolyticum to carbon starvation involves changes in proteolytic activities; higher activities (20% protein degradation) corresponded to a higher level of sporulation; lower proteolysis (5%) was observed in cells starved during the beginning of exponential growth, when sporulation was not observed; with an excess of cellobiose, an intermediate value (10%), accompanied by a low level of sporulation, was observed in cells taken at the end of the exponential growth phase. The basal percentage of the protein breakdown in nonstarved culture was 4%. Cells lacking proteolytic activities failed to induce sporulation. High concentrations of cellobiose repressed proteolytic activities and sporulation. The onset of carbon starvation during the growth phase affected the survival response of C. cellulolyticum via the sporulation process and also via cell-cellulose interaction. Cells from the exponential growth phase were more adhesive to filter paper than cells from the stationary growth phase but less than cells from the late stationary growth phase.

Comparison between serum levels of carcinoembryonic antigen, sialic acid and phosphohexose isomerase in lung cancer.

The identification and application of quantifiable tumor markers as adjuncts to clinical care is a story of both success and failure. The present study compared serum levels of carcinoembryonic antigen (CEA) with total sialic acid/total protein (TSA/TP) ratio and phosphohexose isomerase (PHI) in 192 untreated lung cancer patients as well as 80 age and sex matched controls (44 non-smokers and 36 smokers). CEA values were significantly raised (p < 0.001) in smokers as compared to the non-smokers; whereas, TSA/TP and PHI values were comparable between the two groups of the controls. All the biomarkers were significantly elevated (p < 0.001) in untreated lung cancer patients as compared to the controls. Receiver operating characteristic curve analysis revealed higher sensitivities of TSA/TP and PHI as compared to CEA at different specificity levels between 60% and 95%. Mean values of CEA, TSA/TP and PHI were higher in non-responders compared to the responders. The results indicate that TSA/TP and PHI are superior tumor markers than CEA for lung cancer patients.

Assessing benefits of combining biochemical and immunological markers in patients with lung carcinoma.

Establishment of a simple biochemical index for diagnosis of cancer and management of patients with neoplastic diseases remains one of the important goals to be achieved in cancer research. The current investigation determined serum levels of carcinoembryonic antigen (CEA), total sialic acid (TSA) and lipid bound sialic acid (LSA) in 192 untreated lung cancer patients and compared with 80 age- and sex-matched controls (44 non-smokers and 36 smokers). The levels of all the markers were significantly elevated (P < 0.001) in lung cancer patients compared to the controls. CEA and LSA values were significantly raised (P < 0.001 and P < 0.01, respectively) in smokers compared to non-smokers. TSA and LSA values were more sensitive than CEA for diagnosis of lung cancer. Mean values of the three markers were higher in non-responders (n = 5) compared to the responders (n = 10). The results suggest that along with CEA, TSA and LSA might be included as routine parameters in lung cancer patients.

Mercuric reductase activity in the adaptation to cationic mercury, phenyl mercuric acetate and multiple antibiotics of a gram-negative population isolated from an aerobic fixed-bed reactor.

Eighty-eight strains, isolated from an aerobic fixed-bed reactor and identified to the genus level, were examined for resistance to 21 antibiotics, cationic mercury and phenylmercuric acetate. All except three were able to grow on Mueller-Hinton agar plates containing 8 micrograms/ml mercuric chloride, but only 42 exhibited a mercuric reductase and an organomercurial lyase activity. Furthermore, 82 of them were multiply-antibiotic resistant, whereas no positive correlation between this property and cationic mercury volatilization capacity was found. It was concluded that this bacterial community-adapted response to these selective agents, which has been most often shown to be mediated by R plasmids, was the result of two independent phenomena. Moreover, the high percentage of multiple antibiotic and mercury resistance found in this population suggested that simultaneous selections occurred on filters of bacteria which exhibited mucoid colonies and tolerance to these two categories of stress agents.