Regulation of cell cycle transition and induction of apoptosis in HL-60 leukemia cells by lipoic acid: role in cancer prevention and therapy

 

The most common semiquantitative method of evaluation of pulmonary lesions using ¹⁸F-FDG PET is FDG standardized uptake value (SUV). An SUV cutoff of 2.5 or greater has been used to differentiate between benign and malignant nodules. The goal of our study was to investigate the correlation between the size of pulmonary nodules and the SUV for benign as well as for malignant nodules.

Methods

Retrospectively, 173 patients were selected from 420 referrals for evaluation of pulmonary lesions. All patients selected had a positive CT and PET scans and histopathology biopsy. A linear regression equation was fitted to a scatter plot of size and SUV_max for malignant and benign nodules together. A dot diagram was created to calculate the sensitivity, specificity, and accuracy using an SUV_max cutoff of 2.5.

Results

The linear regression equations and (R²)s as well as the trendlines for malignant and benign nodules demonstrated that the slope of the regression line is greater for malignant than for benign nodules. Twenty-eight nodules of group one (≤ 1.0 cm) are plotted in a dot diagram using an SUV_max cutoff of 2.5. The sensitivity, specificity, and accuracy were calculated to be 85%, 36% and 54% respectively. Similarly, sensitivity, specificity, and accuracy were calculated for an SUV_max cutoff of 2.5 and found to be 91%, 47%, and 79% respectively for group 2 (1.1–2.0 cm); 94%, 23%, and 76%, respectively for group 3 (2.1–3.0 cm); and 100%, 17%, and 82%,, respectively for group 4 (> 3.0 cm). The previous results of the dot diagram indicating that the sensitivity and the accuracy of the test using an SUV_max cutoff of 2.5 are increased with an increase in the diameter of pulmonary nodules.

Conclusion

The slope of the regression line is greater for malignant than for benign nodules. Although, the SUV_max cutoff of 2.5 is a useful tool in the evaluation of large pulmonary nodules (> 1.0 cm), it has no or minimal value in the evaluation of small pulmonary nodules (≤ 1.0 cm).     

Functional differences between two major ubiquitin receptors in the proteasome; S5a and hRpn13

It is well known that S5a and hRpn13 are two major ubiquitin (Ub) receptors in the proteasome but little is known about their functional difference in recruiting ubiquitinated substrates. In this study using siRNA-mediated knockdown of S5a or hRpn13, we found that two Ub receptors had different substrate specificity although similar level of accumulation of high molecular weight Ub-conjugates was observed. Interesting enough, depletion of S5a, but not hRpn13, resulted in the Ub-containing aggregates and induced ER chaperones such as Grp78 and Grp94. ERAD substrates such as α-TCR and α1-antitrypsin were also stabilized by the depletion of S5a but not hRpn13. Our results suggest that there is different substrate specificity between S5a and hRpn13 at the level of delivery and S5a may be the major docking site for ERAD substrates.     

Morphological and molecular diversity reveal wide variability among sorghum Maldandi landraces from India

Maldandi is a popular sorghum variety for post-rainy or rabi cultivation in southern and central states of India, which is predominantly used for food purpose. Over time many landraces have been collected from these states which have vernacular connection with Maldandi. Genetic diversity among 82 Maldandi landraces, collected from such geographical regions was evaluated using both morphological (quantitative and qualitative) and SSR markers. In general, both morphological and SSR diversity revealed wide variability among the accessions studied. Euclidean distances based on 17 quantitative traits classified the accessions into two major clusters with two out groups, while the 19 qualitative traits clustered the accessions in one major cluster with six out groups. Sixteen out of 20 (80%) SSR markers detected polymorphism among the accessions with average PIC value of 0.36. Un-weighted neighbor joining clustering grouped the accessions into three clusters with 46, 16 and 17 accessions, respectively throwing three outliers. Average similarity coefficients of 0.62 and 0.34 based on morphological (qualitative) and SSR data indicated presence of wide variability among the Maldandi landraces. The standard check, M 35?C1 (a selection from the original Maldandi) could not be differentiated from EP 98, LG 2, LG 10, IS 4509 and IS 40791 based on qualitative data alone, while EP 54 and IS 33839 were indistinguishable from M 35?C1 solely using SSR markers. Either of the dendrogram threw unique grouping patterns with some identity. Thirteen promising Maldandi accessions selected based on field performance as well as morphological and molecular diversity could be used in the rabi improvement programme. SSR markers combined with morphological traits may effectively be used for designing breeding strategy and management of biodiversity and conservation of Maldandi genetic resources.     

Co-expression of binary and 100 kDa mosquito larvicidal toxins of Bacillus sphaericus in Escherichia coli

The binary (51 and 42 kDa) and 100 kDa mosquito larvicidal toxins of Bacillus sphaericus are expressed at different growth stages of Bacillus. The genes encoding the binary toxin were expressed using T7 expression system of E. coli. In addition, a PCR amplified product containing the coding sequences of the 100 kDa toxin was cloned upstream to the binary toxin genes, and both the toxins were co-expressed in E. coli. Expression studies with these constructs in different E. coli hosts showed that when these two toxins were co-expressed, there was no augmentation of toxicity in comparison to the construct expressing the binary toxin alone. This result apparently indicates that there is no synergism between these two toxins. © Rapid Science Ltd. 1998     

Molecular origin of plasma membrane citrate transporter in human prostate epithelial cells

The prostate is a highly specialized mammalian organ that produces and releases large amounts of citrate. However, the citrate release mechanism is not known. Here, we present the results of molecular cloning of a citrate transporter from human normal prostate epithelial PNT2‐C2 cells shown previously to express such a mechanism. By using rapid amplification of cDNA ends PCR, we determined that the prostatic carrier is an isoform of the mitochondrial transporter SLC25A1 with a different first exon. We confirmed the functionality of the clone by expressing it in human embryonic kidney cells and performing radiotracer experiments and whole‐cell patch‐clamp recordings. By using short interfering RNAs targeting different parts of the sequence, we confirmed that the cloned protein is the main prostatic transporter responsible for citrate release. We also produced a specific antibody and localized the cloned transporter protein to the plasma membrane of the cells. By using the same antibody, we have shown that the cloned transporter is expressed in non‐malignant human tissues.     

“Topological Significance” Analysis of Gene Expression and Proteomic Profiles from Prostate Cancer Cells Reveals Key Mechanisms of Androgen Response

Background

The problem of prostate cancer progression to androgen independence has been extensively studied. Several studies systematically analyzed gene expression profiles in the context of biological networks and pathways, uncovering novel aspects of prostate cancer. Despite significant research efforts, the mechanisms underlying tumor progression are poorly understood. We applied a novel approach to reconstruct system-wide molecular events following stimulation of LNCaP prostate cancer cells with synthetic androgen and to identify potential mechanisms of androgen-independent progression of prostate cancer.

Methodology/Principal Findings

We have performed concurrent measurements of gene expression and protein levels following the treatment using microarrays and iTRAQ proteomics. Sets of up-regulated genes and proteins were analyzed using our novel concept of “topological significance”. This method combines high-throughput molecular data with the global network of protein interactions to identify nodes which occupy significant network positions with respect to differentially expressed genes or proteins. Our analysis identified the network of growth factor regulation of cell cycle as the main response module for androgen treatment in LNCap cells. We show that the majority of signaling nodes in this network occupy significant positions with respect to the observed gene expression and proteomic profiles elicited by androgen stimulus. Our results further indicate that growth factor signaling probably represents a “second phase” response, not directly dependent on the initial androgen stimulus.

Conclusions/Significance

We conclude that in prostate cancer cells the proliferative signals are likely to be transmitted from multiple growth factor receptors by a multitude of signaling pathways converging on several key regulators of cell proliferation such as c-Myc, Cyclin D and CREB1. Moreover, these pathways are not isolated but constitute an interconnected network module containing many alternative routes from inputs to outputs. If the whole network is involved, a precisely formulated combination therapy may be required to fight the tumor growth effectively.     

Lupeol and its ester inhibit alteration of myocardial permeability in cyclophosphamide administered rats

Cyclophosphamide (CP), an alkylating agent widely used in cancer chemotherapy causes cardiac membrane damage. Lupeol, a pentacyclic triterpene, isolated from Crataeva nurvala stem bark and its ester, lupeol linoleate possess a wide range of medicinal properties. The effect of lupeol and its ester was evaluated in CP induced alterations in cardiac electrolytes in rats. Male albino rats of Wistar strain were categorized into 6 groups. Group I served as control. Rats in groups II, V and VI were injected intraperitoneally with a single dose of CP (200 mg/kg body weight) dissolved in saline. CP treated groups V and VI received lupeol and lupeol linoleate (50 mg/kg body weight) respectively, dissolved in olive oil for 10 days by oral gavage. At the end of the experimental period, urinary risk factors, activities of ATPases and electrolytes were measured using standard procedures. CP administered rats showed a significant decrease (P < 0.001) in the activities of ATPases. It was associated with significant alterations (P < 0.001) of electrolytes both in serum and cardiac tissue. The levels of urea, uric acid and creatinine were also significantly (P < 0.001) altered in the serum and urine. Lupeol and its ester showed reversal of the above alterations induced by CP. These findings demonstrate that the supplementation with lupeol and its ester could preserve membrane permeability, highlighting their protective effect against CP induced cardiotoxicity.     

Identity of SMCT1 (SLC5A8) as a neuron-specific Na+-coupled transporter for active uptake of L-lactate and ketone bodies in the brain

SMCT1 is a sodium-coupled (Na(+)-coupled) transporter for l-lactate and short-chain fatty acids. Here, we show that the ketone bodies, beta-d-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, alpha-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na(+)-coupled and electrogenic. The Michaelis constant is 1.4 +/- 0.1 mm for beta-d-hydroxybutyrate, 0.21 +/- 0.04 mm for acetoacetate and 0.21 +/- 0.03 mm for alpha-ketoisocaproate. The Na(+) : substrate stoichiometry is 2 : 1. As l-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co-localizes with MCT2, a neuron-specific Na(+)-independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na(+)-coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of l-lactate and ketone bodies into neurons by a process driven by an electrochemical Na(+) gradient and hence, contribute to the maintenance of the energy status and function of neurons.     

Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy

Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. In this article we describe the system development for the image acquisition for one- and two-photon excitation FRET microscopy. We also describe the precision FRET (PFRET) data analysis algorithm that we developed to remove spectral bleed-through and variation in the fluorophore expression level (or concentration) for the donor and acceptor molecules. The acquired images have been processed using a PFRET algorithm to calculate the energy transfer efficiency and the distance between donor and acceptor molecules. We implemented the software correction to study the organization of the apical endosome in epithelial polarized MDCK cells and dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). For these proteins, the results revealed that the extent of correction affects the conventionally calculated energy transfer efficiency (E) and the distance (r) between donor and acceptor molecules by 38 and 9%, respectively.     

Molecular Mechanism of SLC5A8 Inactivation in Breast Cancer

SLC5A8 is a putative tumor suppressor that is inactivated in more than 10 different types of cancer, but neither the oncogenic signaling responsible for SLC5A8 inactivation nor the functional relevance of SLC5A8 loss to tumor growth has been elucidated. Here, we identify oncogenic HRAS (HRAS^G12V) as a potent mediator of SLC5A8 silencing in human nontransformed normal mammary epithelial cell lines and in mouse mammary tumors through DNMT1. Further, we demonstrate that loss of Slc5a8 increases cancer-initiating stem cell formation and promotes mammary tumorigenesis and lung metastasis in an HRAS-driven murine model of mammary tumors. Mammary-gland-specific overexpression of Slc5a8 (mouse mammary tumor virus-Slc5a8 transgenic mice), as well as induction of endogenous Slc5a8 in mice with inhibitors of DNA methylation, protects against HRAS-driven mammary tumors. Collectively, our results provide the tumor-suppressive role of SLC5A8 and identify the oncogenic HRAS as a mediator of tumor-associated silencing of this tumor suppressor in mammary glands. These findings suggest that pharmacological approaches to reactivate SLC5A8 expression in tumor cells have potential as a novel therapeutic strategy for breast cancer treatment.