Resveratrol analog (Z)-3,5,4'-trimethoxystilbene is a potent anti-mitotic drug inhibiting tubulin polymerization

Resveratrol (3,5,4'-trihydroxystilbene) a natural polyphenol present in medicinal plants, grapes and wines, has potent chemopreventive properties on intestinal carcinogenesis. A methylated derivative (Z-3,5,4'-trimethoxystilbene: R3) was synthesized. R3 at 0.3 microM exerted a 80% growth inhibition of human colon cancer Caco-2 cells and arrested growth completely at 0.4 microM (R3 was 100-fold more active than resveratrol). The cis conformation of R3 was also 100-fold more potent than the trans isomer. R3 (0.3 microM) caused cell cycle arrest at the G2/M phase transition. The drug inhibited tubulin polymerization in a dose-dependent manner (IC50=4 microM), and it reduced also by 2-fold ornithine decarboxylase and s-adenosylmethionine decarboxylase activities. This caused the depletion of the polyamines, putrescine and spermidine, which are growth factors for cancer cells. R3 inhibited partially colchicine binding to its binding site on tubulin, indicating that R3 either partially overlaps with colchicine binding or that R3 binds to a specific site of tubulin that is not identical with the colchicine binding site modifying colchicine binding by allosteric influences. The resveratrol derivative (Z)-3,5,4'-trimethoxystilbene (R3) is an interesting anti-mitotic drug that exerts cytotoxic effects by depleting the intracellular pool of polyamines and by altering microtubule polymerization. Such a drug may be useful for the treatment of neoplastic diseases.     

Concomitant Differentiation and Partial Proteasome Inhibition Trigger Apoptosis in Neuroblastoma Cells

Proteasome activity is essential during cAMP-induced terminal differentiation of a murine neuroblastoma cell line (NBP2). However, the mechanisms through which proteasome affects NBP2 differentiation have not been characterized. We hypothesized that proteasome is required to implement the differentiation-mediated effects on cell cycle, and its partial inhibition during differentiation may have adverse consequences. Here we show that partial inhibition of proteasome during cAMP-induced differentiation of NBP2 cells causes apoptosis. Whereas differentiation induced growth arrest at G₁ phase, partial proteasome inhibition during differentiation resulted in the accumulation of cells at G₂M phase. Cell cycle data correlated with the level of cyclin-dependent kinase inhibitors p21WAF and p27Kip1, and cyclin A. While the level of p21 and p27 increased, the level of cyclin A decreased upon differentiation. In contrast, cells treated with proteasome inhibitor in the presence of cAMP-inducing agents showed increased levels of p21 and cyclin A early in the course of differentiation. However, the level of p21 and p27, but not cyclin A, decreased later during concomitant differentiation and partial proteasome inhibition when cells were undergoing apoptosis. Our data suggest that differentiation-mediated growth arrest is dependent on the temporal activity of cell cycle proteins. Partial inhibition of proteasome interferes with differentiation events partly by stabilizing cell cycle proteins and this triggers apoptosis. Thus, differentiating drugs combined with partial proteasome inhibition may impart higher therapeutic efficacy than differentiating agents alone for the treatment of neuroblastoma tumors.     

卵巢癌多细胞球体对紫杉醇耐药及其机制的探讨

背景和目的:多细胞球体(multicellularspheroids,MCS)对传统细胞毒化疗药物的敏感性明显低于单层细胞。本实验旨在探讨卵巢癌MCS耐药的分子机制。方法:以单层细胞为对照,以三维培养方法获得的人卵巢癌A2780MSC和CAOV3MCS为模型,采用台盼蓝拒染法比较紫杉醇对单层细胞和MCS生长的抑制作用,流式细胞仪比较单层细胞和MCS细胞周期的分布和细胞凋亡率;采用流式细胞仪、蛋白质免疫印迹法、激光共聚焦显微镜检测单层细胞及MCS的P-糖蛋白(P-glycoprotein,P-gp)表达及亚细胞分布;采用RT-PCR法检测mdr1mRNA表达水平。结果:(1)不同浓度的紫杉醇(0.2、2.0、10.0、20.0μmol/L)作用后,MCS细胞生长抑制率明显低于单层细胞(PA2780=0.003,PCAOV3=0.015);经20.0μmol/L的紫杉醇作用后,单层细胞的细胞凋亡率明显高于MCS,差异有统计学意义(PA2780=0.034,PCAOV3=0.032)。(2)流式细胞仪、蛋白质免疫印迹法、激光共聚焦显微镜检测提示P-gp在单层细胞中不表达,在MCS中表达明显升高(P均<0.05);RT-PCR证实MCS中有mdr1mRNA表达,而单层细胞中未检出其表达。(3)流式细胞仪检测提示将单层细胞培养成MCS时,G0/G1期细胞比率增加,S期和G2/M期细胞比率降低(P均<0.05)。结论:卵巢癌MCS对紫杉醇化疗耐药性增加,其高表达P-gp,并且与G0/G1期细     

Effect of an omega-3 fatty acid containing lipid emulsion alone and in combination with 5-fluorouracil (5-FU) on growth of the colon cancer cell line Caco-2

Summary. Background: In this study we examined the effects of a fish oil-based lipid emulsion (FO) rich in omega-3 fatty acids, which is used in humans as a component of parenteral nutrition, on the growth of the colon cancer cell line Caco-2. Aim of the study: The aim of the present study was to investigate whether the FO influences growth and chemosensitivity of the colon cancer cell line Caco-2. FO was tested alone and in combination with the anticancer drug 5-fluorouracil (5-FU). Methods: Cell numbers were determined with crystal violet staining, cell cycle distribution was assessed using a flow cytometer and apoptosis was visualized by staining nuclei with diamino-phenylindole hydrochloride. Results: FO inhibited growth of Caco-2 cells in a time and dose dependent manner. FO treatment evoked apoptosis as confirmed by cell morphology. Cell cycle analysis identified an accumulation of cells in the G₂/M phase after incubation with FO. The combined treatment of the cells with FO and 5-FU resulted in a significant enhancement of the growth inhibition seen after exposure to either substance alone. Treatment of the cells with 5-FU specifically blocked the cell cycle in the S phase. The combined treatment of 5-FU with FO showed a further increase in the accumulation of cells in the S phase. Conclusions: In conclusion, FO has a potent antiproliferative effect on Caco-2 cells, at least in part, due to a decrease in the progression of the cell cycle and the induction of apoptosis. The combination of FO with 5-FU results in an additive growth inhibitory effect.     

Neurofibrillary degeneration of the Alzheimer-type: an alternate pathway to neuronal apoptosis?

Neuronal death is a process which may be either physiological or pathological. Apoptosis and necrosis are two of these processes which are particularly studied. However, in neurodegenerative disorders, some neurons escape to these types of death and "agonize" in a process referred to as neurofibrillary degeneration. Neurofibrillary degeneration is characterized by the intraneuronal aggregation of abnormally phosphorylated microtubule-associated Tau proteins. A number of studies have reported a reactivation of the cell cycle in the neurofibrillary degeneration process. This reactivation of the cell cycle is reminiscent of the initiation of apoptosis in post-mitotic cells where G1/S markers including cyclin D1 and cdk4/6, are commonly found. However, in neurons exhibiting neurofibrillary degeneration, both G1/S and G2/M markers are found suggesting that they do not follow the classical apoptosis and an aberrant cell cycle occurs. This aberrant response leading to neurofibrillary degeneration may be triggered by the sequential combination of three partners: the complex Cdk5/p25 induces both apoptosis and the "abnormal mitotic Tau phosphorylation". These mitotic epitopes may allow for the nuclear depletion of Pin1. This latter may be responsible for escaping classical apoptosis in a subset of neurons. Since neurofibrillary degeneration is likely to be a third way to die, molecular mechanisms leading to changes in Tau phosphorylation including activation of kinases such as cdk5 or other regulators such as Pin1 could be important drug targets as they are possibly involved in early stages of neurodegeneration.     

Oral cytology assessment by flow cytometry of DNA adducts, aneuploidy, proliferation and apoptosis shows differences between smokers and non-smokers

Oral cytology and morphometric staining is used to identify malignant keratinocytes in oral premalignant or malignant lesions. To detect and to begin to assess changes in oral keratinocytes exposed to tobacco-derived carcinogens, which are at risk for malignant transformation, a novel method is required. The approach uses oral cytology harvested oral keratinocytes analyzed using flow cytometry (FC) for changes in DNA content, damage, cell cycle and apoptosis. Six smoker and six non-smoker oral keratinocytes were evaluated using flow cytometry in the form of laser scanning cytometry (LSC) and laser microdissection (LMD). Among smokers compared to non-smokers, the method detected and assessed DNA damage from tobacco smoke exposure quantifying an enhanced formation of DNA adducts, such as, 8-hydroxy-2′-deoxyguanine (8-OHdG) which creates oxidation lesions and benzo[a]pyrene(B[a]P), which produces a B[a]P)-N2-dG bulky adduct. Increased DNA content, aneuploidy, percentage of cells in synthesis (S) and G₂+Mitosis (M), and apoptosis were recorded. Tissue and cell controls were used to verify these relationships. Data suggested healthy smokers were at increased risk for malignant transformation of oral keratinocytes because of the changes stated above. Using identical methods, keratinocytes exposed to the tobacco derived carcinogen, B[a]P parallel results obtained from smoke exposure indicating a direct link. Flow cytometric evaluation of oral cytology harvested keratinocytes can be used to measure exposure to tobacco carcinogens, and possibly establish a link to premalignant and malignant transformation before a lesion is noted.     

Lack of effect of isoflavonic phytoestrogen intake on leptin concentrations in premenopausal and postmenopausal women

Objective: To assess the effect of soy isoflavone ingestion on plasma leptin concentrations in premenopausal and postmenopausal women.

Design: Randomized, crossover studies, with blinding of participants and laboratory personnel.

Setting: Procedures involving free-living individuals were carried out at the University of Minnesota General Clinical Research Center.

Patient(s): Fourteen regularly cycling premenopausal women, and 18 postmenopausal women.

Intervention(s): Each premenopausal participant consumed, on a daily basis, each of three soy protein powders containing different levels of isoflavones for three menstrual cycles plus 9 days, with plasma samples collected every other day the last 6 weeks of each diet period. Similarly, each postmenopausal participant consumed each of the three powders for 93 days, with plasma samples collected daily on days 64 to 66 and 92 to 94 of each diet period. The powders, dosed on a per-kilogram body weight basis, provided mean isoflavone intakes of 8, 65, and 130 mg/day, for the control, low-isoflavone, and high-isoflavone diet periods, respectively.

Main Outcome Measure(s): Plasma leptin concentrations.

Result(s): Isoflavone intake had essentially no effect on leptin concentrations in either premenopausal or postmenopausal participants. Concentrations in the premenopausal women were higher during the periovulatory and midluteal phases as compared to the early follicular and midfollicular phases.

Conclusion(s): Despite the well-documented effect of estrogens to enhance leptin production, even high levels of isoflavone consumption do not alter leptin concentrations in women. Further studies are needed to more precisely delineate the nature of estrogenic and/or antiestrogenic effects of isoflavones in humans.     

Influence of menstrual cycle on NK activity

Natural killer (NK) cells are CD3⁻ CD56⁺ and/or CD16⁺ cytotoxic lymphocytes that mediate first-line defense against various types of target cells without prior immunization. To assess the effect of the menstrual cycle and gender on NK activity we evaluated 30 healthy women (mean age 28.1 years, range 21–39) in follicular and luteal phases, 29 postmenopausal women (mean age 58.8 years, range 42–72) and 48 healthy men (mean age 31.6 years, range 21–40). In a flow cytometric test of NK activity, peripheral blood mononuclear effector cells were mixed with K562 targets cells labeled with DiO (3,3′-dioctadecyloxacarbocyanine perchlorate) at effector:target cell ratios of 40, 20, 10 and 5:1. Dead cells were stained with propidium iodide and results were expressed as lytic units per 10⁷ cells. In addition, progesterone levels were determined in the luteal phase of the menstrual cycle of healthy women by a chemiluminescence assay. Our results showed that (1) NK cytotoxicity was higher in the follicular than in the luteal phase of the menstrual cycle (P<0.0001); (2) postmenopausal women and men showed NK activity similar to women in the folicular phase but higher than women in the luteal phase of the menstrual cycle (P<0.05); and (3) there was no correlation between NK activity and levels of progesterone. The data suggest that progesterone does not influence NK activity directly and that other factors may explain the reduction of NK activity in the luteal phase of the menstrual cycle.     

乳腺乳头状瘤病中4种细胞周期调控因子的检测及临床意义

目的　观察不同程度的乳腺乳头状瘤病中 4种细胞周期调控因子蛋白的表达及临床意义 ,为临床上乳腺癌癌前病变的有效诊治提供客观指标。方法　通过免疫组织化学S P法在乳腺轻度、中度、重度乳头状瘤病及导管内癌组各 4 8例患者中检测细胞周期素D1、p16、E2F 1、Rb基因蛋白表达情况。结果　细胞周期素D1、p16、E2F 1、Rb 4种因子表达的阳性率和表达强度分别在 4组间的差异均有显著意义 (χ2 分别为 16 70、2 0 74、4 0 34、4 2 32 ;19 12、2 9 4 7、4 5 0 8、4 6 92 ,均P <0 0 1;在中度和重度乳头状瘤病组间差异也有显著意义 (均P <0 0 5 ) ;E2F 1或Rb在重度乳头状瘤病和导管内癌组间差异有显著意义 (均P <0 0 1) ,但细胞周期素D1或 p16在这两组间差异无显著意义 (均P >0 0 5 ) ;细胞周期素D1及E2F 1表达与由轻至重的乳头状瘤病乃至导管内癌呈正相关 ,相反p16及Rb与之呈负相关 ;Rb阴性或低表达时中度乳头状瘤病发展到重度乳头状瘤病的危险度最大。结论细胞周期素D1等 4种调控因子与乳腺癌的发生发展过程有密切的相关性。E2F 1和Rb可作为重度乳头状瘤病和导管内癌鉴别诊断的参考指标。Rb还有助于筛选重度和中度乳头状瘤病中的高危病例。     

A pilot study on a gene-hormone interaction in female suicide attempts

Abstract. This one-year naturalistic study included all suicide attempters in a catchment area. In the first published set of analyses, an association between menses and suicide attempts was replicated. According to the polymorphism of the serotonin transporter promoter area, the subjects can be classified as S individuals (s/s or s/l) or L individuals (l/l). In the second published set of analyses, L females appeared protected from suicide attempts since they were underrepresented among female (and not male) attempters. This new, unpublished third set of analyses tested for an interaction between the same polymorphism and low hormonal activity (during menses and menopause). In fertile female attempters, the proportion of L women in the menses (41%, 7/17) was significantly higher than expected in the population (15.5 %) and almost significantly higher than in S female attempters (22%,19/87). L females were also overrepresented in postmenopausal attempters. Despite sample size limitations, this gene-hormone interaction needs to be further investigated in female suicide attempters.