Mini-Review on Structure-Reactivity Relationship for Aromatic Molecules: Recent Advances.

Recent advances in quantifying nucleophilic reactivities in chemical reactions and intermolecular interactions of aromatic molecules are reviewed. This survey covers experimental (IR frequency shifts induced by hydrogen bonding) and theoretical (modeling of potential energy surfaces, atomic charges, molecular electrostatic potential) approaches in characterizing chemical reactivity. Recent advances in software developments assisting the evaluation of the reactive sites for electrophilic aromatic substitution are briefly discussed.

SU-E-J-104: Evaluation of Atlas-Based Auto-Segmentation on Daily In-Room CT for Prostate Cancer.

PURPOSE: Anatomy delineation is a major time consuming task to correct for inter-fractional changes in anatomy. Atlas-based auto-segmentation (ABAS) was developed to expedite this process. This study aims to evaluate the performance of ABAS applying to high quality verification CT-imaging acquired using a CT-on-rail system for prostate cancer. METHODS: The prostate, rectum and bladder were manually contoured for seven prostate cancer patients. For each patient, three patient specific atlases were generated consisting of one, four and seven prior image and contour sets. ABAS was applied using these atlases for the last seven daily CT images of each patient. The auto- and manual-contours were compared both geometrically and dosimetrically. The reproducibility of the observation was validated by an experienced radiation oncologist performing the same procedure. The performance of ABAS with patient and non-patient specific atlases were also evaluated on 21 image sets. P<0.05 was considered statistical significant for two-tailed paired student t-test. RESULTS: Contours obtained from ABAS agreed well with the manual ones. With 1-image set atlas, the OI and DSC for the bladder were greater than 96% and 91%, respectively. Both indices were above 81% for rectum and prostate. The consistencies significantly improved by including 4 image sets in the atlas, while a further increase of atlas size to 7 did not show obvious benefits. Dose coverage for the auto- and manual-contours was similar for all organs. Similar results were obtained by the second observer. Compared with non-patient specific atlas, patient specific atlas yielded more accurate contours. The time for ABAS and manual contouring was ∼2 min and ∼20 min per image set, respectively. CONCLUSIONS: With diagnostic quality verification images, ABAS can provide fast and accurate delineations for prostate cancer on a daily basis. The inclusion of more than one CT set in atlas improves the contouring results.

SU-E-J-22: Effect of MLC Leaf Width on MLC Leaf Shifting Algorithm for Concurrent Treatment of Prostate and Pelvic Lymph Nodes.

PURPOSE: Our previous study showed that adjusting selected MLC leaf pairsto follow prostate movement is an effective strategy to account for daily prostate displacement during concurrent treatment with pelvic lymph nodes. MLC leaf width affects the quality of MLC shifting plans for longitudinal prostate motion compensation. This study is to investigate the effect of the MLC leaf width in compensation of the prostate movement. METHODS: Fifty-one daily CT on-rail scans from three patients were available for this study. On these CTs, the prostate, bladder and rectum were manually contoured, and the lymph nodes contours were transferred from the planning CT after rigid bony registration. For each patient, three different IMRT plans were created based on a planning CT using leaf width of 2.5, 5, and 10 mm, respectively. For each CT, the prostate displacement was determined by dual imaging registration and compensated by shifting MLC resulting in a total of 153 MLC shifted plans. RESULTS: Among 51 daily CTs, the average prostate movement along the superior/inferior direction was 1.1±3.7 mm (range: -6 to 6.5 mm). The differences in D99 of the prostate between the dose of the day and dose of the plan were 2.3±3.3%, 1.3±2.0%, and 4.4±5.1% for 2.5, 5, and 10 mm leaf width plans, respectively (p<<0.05). The corresponding differences in D99 of the lymph nodes were 0.7±0.9%, 0.6±0.9%, and 1.4±0.8%. The mean differences in D50 were 0.8%, 1.6%, and 2.7% for the bladder, and 10.0%, 3.9%, and 5.7% for the rectum, respectively. CONCLUSIONS: Using the MLC Shifting method to compensate for prostate movement in the longitudinal direction depends on the MLC leaf width and the magnitude of the prostate motion. The use of leaf width of 5 mm can provide sufficient tumor coverage without significantly affecting doses to the critical structures.

Chemotaxis: moving forward and holding on to the past.

The ability of cells to sense external chemical cues and respond by directionally migrating towards them is a fundamental process called chemotaxis. This phenomenon is essential for many biological responses in the human body, including the invasion of neutrophils to sites of inflammation. Remarkably, many of the molecular mechanisms involved in controlling neutrophils chemotaxis arose millions of years ago in the simple eukaryotic organism Dictyostelium discoideum. Both neutrophils and Dictyostelium use G protein-coupled signaling cascades to mediate chemotactic responses, which are responsible for transducing external cues into highly organized cytoskeletal rearrangements that ultimately lead to directed migration. By using the genetically and biochemically tractable organism Dictyostelium as a model system, it has been possible to decipher many of the signal transduction events that are involved in chemotaxis.

Screening for point mutations in the LDL receptor gene in Bulgarian patients with severe hypercholesterolemia.

Familial hypercholesterolemia (FH) is a common, autosomal dominant disorder of lipid metabolism, caused by defects in the receptor-mediated uptake of LDL (low-density lipoproteins) due to mutations in the LDL receptor gene ( LDLR). Mutations underlying FH in Bulgaria are largely unknown. The aim of the present study was to provide information about the spectrum of point mutations in LDLR in a sample of 45 Bulgarian patients with severe hypercholesterolemia. Exons 3, 4, 6, 8, 9, and 14, previously shown to be mutational hot spots in LDLR, were screened using PCR-single-strand conformation polymorphism (SSCP). Samples with abnormal SSCP patterns were sequenced. Three different, hitherto undescribed point mutations (367T>A, 377T>A, 917C>A) and two previously described mutations (858C>A and 1301C>T) in eight unrelated patients were identified; four of the detected point mutations being missense mutations and one, a nonsense mutation. One of the newly described point mutations (917C>A) is a base substitution at a nucleotide position, at which two other different base substitutions have already been reported. Thus, all three possible base substitutions at this nucleotide position have been detected, making it a hot spot for point mutations causing FH. This is the first such mutational hot spot described in exon 6 of LDLR.

Activation of phospholipase C induces the expression of the multidrug resistance (MDR1) gene through the Raf-MAPK pathway.

Resistance to multiple, unrelated cancer chemotherapeutic drugs can be mediated by P-glycoprotein, the MDR1 gene product. Numerous substances, including chemotherapeutic drugs, heavy metals, growth factors, activated oncogenes, or changes in temperature increase MDR1 gene expression. Because several of these factors regulate cellular function through the activation of phospholipase C (PLC), we postulated that PLC-mediated signaling could be central to regulating the expression of MDR1. Transfection of NIH 3T3 cells with a pMJ30-PLC-gamma 1 expression vector increased the activity of the MDR1 promoter by 2- to 10-fold. PLC-mediated activation required a region between -106 and -99 of the MDR1 promoter. Treatment of cotransfected cells with platelet-derived growth factor further enhanced the activity of the MDR1 promoter. The stimulatory effect of PLC on the MDR1 promoter was increased by cotransfection with constitutively active v-raf and was blocked by the dominant-negative mutant, c-Raf-C4. The activity of mitogen-activated protein kinase (MAPK) was also increased in PLC-gamma 1-transfected cells. Furthermore, PD-98059 and U0126, two MAPK inhibitors, blocked PLC-gamma 1-induced expression of MDR1. The results of Northern blot analysis showed that activation of PLC by heat shock and growth factors increased expression of endogenous MDR1 mRNA in human renal carcinoma cells. These effects were blocked by inhibitors of the PLC-MAPK pathway. In summary, our results indicate for the first time that activation of PLC by a variety of cellular stimuli can regulate the expression of MDR1 and that the transcriptional modulation of MDR1 expression by PLC is mediated by the Raf-MAPK pathway.

Modulation of bcl-2 and cytotoxicity by licochalcone-A, a novel estrogenic flavonoid.

Herbal therapies are commonly used by patients with cancer, despite little understanding about their clinical and biological activity. We recently demonstrated that the herbal combination PC-SPES, which contains licorice root, had potent estrogenic activity in vitro, in animals, and in patients with prostate cancer. Licochalcone-A (LA) is one flavonoid extracted from licorice root with antiparasitic and anti-tumor activity, but the effect on the human estrogen receptor and mechanism of anti-tumor activity is unknown. Recent studies demonstrated that the mechanism of cytotoxic effect by some estrogens may involve modulation of the anti-apoptotic protein bcl-2. In the present study, we determined if LA had estrogenic activity, anti-tumor activity, and modulated the apoptotic protein bcl-2 in human cell lines derived from acute leukemia, breast cancer, and prostate cancer. A yeast growth-based assay under the control of the human estrogen receptor (hER) demonstrated that LA was a phytoestrogen. A cell viability assay demonstrated that LA had anti-tumor activity in all cell lines tested and enhanced the effect of paclitaxel and vinblastine chemotherapy. LA induced apoptosis in MCF-7 and HL-60 cell lines, as demonstrated by cleavage of PARP, the substrate of ICE-like proteases. Immunoblot analysis demonstrated that LA decreased the anti-apoptotic protein bcl-2 and altered the bcl-2/bax ratio in favor of apoptosis. In contrast, the parent compound chalcone or estradiol did not decrease bc1-2 expression. Therefore, these data demonstrate that LA is a phytoestrogen with anti-tumor activity and is capable of modulating bcl-2 protein expression. The modulation of bcl-2 may be dependent on specific structural differences between LA and the parent compound chalcone and independent of LA estrogenicity.

Regulation of expression of the multidrug resistance protein MRP1 by p53 in human prostate cancer cells.

The expression of several drug-resistance genes, including MRP and p53, increases with advancing stage of human prostate cancer. Altered transcription could account for the genotypic alterations associated with prostate cancer progression, and it was recently reported that the promoter of MRP1 is activated in the presence of mutant p53. To determine whether there is a relationship between p53 status and the expression of MRP1, a human, temperature-sensitive p53 mutant (tsp Val(138)) was transfected into LNCaP human prostate cancer cells. In the transfected cell line (LVCaP), the wild-type p53 produced growth arrest at the G1/S interface of the cell cycle, inhibited colony formation, and induced p21(waf1/cip1). Temperature shifting to 38 degrees C (p53 mutant) produced a time-dependent increase in expression of MRP1. This change in MRP1 expression was also seen in isogenic cell lines in which p53 was inactivated by human papilloma virus (HPV)16E6 protein or by a dominant-negative mutant. Functional assays revealed a decrease in drug accumulation and drug sensitivity associated with mutant p53 and increased MRP1 expression. These results provide the first mechanistic link between expression of MRP1 and mutation of p53 in human prostate cancer and support recent clinical associations. Furthermore, these data suggest a mechanism tying accumulation of p53 mutations to the multidrug resistance phenotype seen in this disease.

Treatment of multidrug resistant (MDR1) murine leukemia with P-glycoprotein substrates accelerates the course of the disease.

The prognosis of patients with tumors expressing P-glycoprotein (P-gp), the MDR1 gene product, is generally poor. It is assumed that this is due to decreased tumor responsiveness that results from decreased drug accumulation. We observed that treatment of animals bearing MDR1-transfected leukemic cells with P-gp substrates (i.e., drugs that are transported by P-gp) significantly worsened host survival compared to treatment with vehicle or non-P-gp substrates. This effect was seen with cancer chemotherapeutic agents (paclitaxel and vincristine) and with the MDR modulator, trans-flupenthixol. To determine the mechanism(s) underlying this observation, we studied alterations in pharmacokinetics, pharmacodynamics, and metastasis. We found that the drug-induced acceleration of disease was associated with increased metastases. P-gp(+) cells treated with P-gp substrates demonstrated several pro-metastatic features, including membrane ruffling and invasion through a hepatocyte monolayer. These results suggest that the treatment of MDR tumors with P-gp substrates may produce changes in malignant behavior that could adversely affect therapeutic outcomes.