Preferential effects of leptin on CD4 T cells in central and peripheral immune system are critically linked to the expression of leptin receptor

Leptin can enhance thymopoiesis and modulate the T-cell immune response. However, it remains controversial whether these effects correlate with the expression of leptin receptor, ObR. We herein addressed this issue by using in vivo animal models and in vitro culture systems. Leptin treatment in both ob/ob mice and normal young mice induced increases of CD4 SP thymocytes in thymus and CD4 T cells in the periphery. Interestingly, expression of the long form ObR was significantly restricted to DN, DP and CD4 SP, but not CD8 SP thymocytes. Moreover, in the reaggregated DP thymocyte cultures with leptin plus TSCs, leptin profoundly induced differentiation of CD4 SP but not CD8 SP thymocytes, suggesting that the effects of leptin on thymocyte differentiation might be closely related to the expression of leptin receptor in developing thymocytes. Surprisingly, ObR expression was markedly higher in peripheral CD4 T cells than that in CD8 T cells. Furthermore, leptin treatment with or without IL-2 and PHA had preferential effects on cell proliferation of CD4 T cells compared to that of CD8 T cells. Collectively, these data provide evidence that the effects of leptin on differentiation and proliferation of CD4 T cells might be closely related to the expression of leptin receptor.     

白花蛇舌草醇提取物对乳腺癌T-47D细胞株生长的抑制作用

目的：观察白花蛇舌草醇提取物对乳腺癌细胞T-47D生长的影响。方法：采用平皿克隆形成实验、软琼脂集落形成实验和BrdUrd（溴脱氧尿苷）掺入实验,观察不同浓度的白花蛇舌草醇提取物（1.0μg/mL,3.0μg/mL,10μg/mL,30μg/mL,100μg/mL）对T-47D细胞锚定依赖性生长和软琼脂集落形成能力及核酸合成的抑制作用。结果：①随着白花蛇舌草醇提取物浓度的升高,对T-47D细胞的生长呈现明显抑制作用;②白花蛇舌草醇提取物可呈浓度依赖性抑制对数生长期T-47D细胞核酸合成。结论：白花蛇舌草醇提取物可能通过影响肿瘤细胞核酸合成而抑制T-47D细胞生长。     

Progestin stimulation of manganese superoxide dismutase and invasive properties in T47D human breast cancer cells

Superoxide dismutase (SOD) occurs in two intracellular forms in mammals, copper–zinc SOD (CuZnSOD), found in the cytoplasm, mitochondria and nucleus, and manganese superoxide dismutase (MnSOD), in mitochondria. Changes in MnSOD expression (as compared to normal cells) have been reported in several forms of cancer, and these changes have been associated with regulation of cell proliferation, cell death, and metastasis. We have found that progestins stimulate MnSOD in T47D human breast cancer cells in a time and physiological concentration-dependent manner, exhibiting specificity for progestins and inhibition by the antiprogestin RU486. Progestin stimulation occurs at the level of mRNA, protein, and enzyme activity. Cycloheximide inhibits stimulation at the mRNA level, suggesting that progestin induction of MnSOD mRNA depends on synthesis of protein. Experiments with the MEK inhibitor UO126 suggest involvement of the MAP kinase signal transduction pathway. Finally, MnSOD-directed siRNA lowers progestin-stimulated MnSOD and inhibits progestin stimulation of migration and invasion, suggesting that up-regulation of MnSOD may be involved in the mechanism of progestin stimulation of invasive properties. To our knowledge, this is the first characterization of progestin stimulation of MnSOD in human breast cancer cells.     

Regulation of leptin gene expression and secretion by steroid hormones.

Previous work has shown that 17 beta-estradiol is the primary ovarian signal regulating body weight and adiposity, although its mechanisms of action remain unclear. We hypothesized that 17 beta-estradiol could enhance leptin levels as a mechanism of its anorectic effects. Administration of 5 microg 17 beta-estradiol subcutaneously (s.c.) for 2 days significantly elevated leptin mRNA levels in adipose tissue as compared to vehicle controls (P < 0.003). A time-course administration of estrogen showed increased mRNA levels in adipose tissue between 6 and 12 h after estrogen injection as compared to vehicle controls (P < 0.03). Corresponding to the increased leptin mRNA levels at 6 and 12 h, elevated plasma leptin levels were observed at 12 h after estrogen administration as compared to controls (P < 0.05). Administration of progesterone (1 mg/rat) after estradiol injection did not enhance the elevated leptin mRNA levels in adipose tissue. Serum leptin levels from cycling rats did not differ significantly between metestrous and proestrous animals. In conclusion, the present studies demonstrate that 17 beta-estradiol can regulate leptin gene expression and secretion in the female rat, thus providing a better understanding of the possible anorectic effect of estrogens.     

Effects of leptin gene expression in mice in vivo by electroporation and hydrodynamics-based gene delivery

In vivo electroporation and hydrodynamics-based gene delivery were utilized to test the effect of leptin gene transfer on food intake, and body and fat weights of mice. Gene transfer of pVRmob by electroporation caused a significant reduction in body weight compared with the control counterpart (p<0.05), although a lesser effect was found in food intake, and the weights of interscapular brown and epididymal fat by electroporation. As might be expected, the hydrodynamics-based transfection method significantly reduced body weight over 1 week post-transfection (p<0.05). Furthermore, epididymal fat was decreased by 50% at 1 week after gene transfer (p<0.001). These results suggest that both electroporation and hydrodynamics-based gene delivery may be effective approaches for systemic delivery of recombinant leptin to the central nervous system, and that the efficiency of gene transfer in hydrodynamics-based gene delivery was markedly higher than that in electroporation at least within the first week after transfection.     

Expression patterns of leptin receptor (OB-R) isoforms and direct in vitro effects of recombinant leptin on OB-R, leptin expression and cytokine secretion by human hematopoietic malignant cells

Several studies have implicated leptin in the pathophysiology of neoplasias. We investigated the direct effect of leptin on malignant hematopoietic tissue that included: primary acute myeloid leukemia (AML) cells, leukemic cell lines and bone marrow biopsies from multiple myeloma (MM) patients. PBMC, T-cells, B-cells and monocytes from healthy subjects served as controls. We defined the patterns of OB-R isoform expression in AML cells and leukemic cell lines in comparison to control cells by RT-PCR. rLeptin upregulated the expression of OB-R and endogenous leptin in AML blasts and certain cell lines but not in control cells. Cytometric Bead Array analysis of pro- and anti-inflammatory cytokines showed that rleptin upregulates IL-6 secretion by AML cells, various cytokines by the leukemic cell lines tested and IL-10 secretion by control PBMC, contributed by monocytes. Western immunoblotting revealed that the effect of rleptin was independent of JAK-2/phospho-JAK-2 protein levels. Finally, MM biopsies stained positive for leptin and, to a lesser extend, OB-R. Immunoreactivity was confined mostly to the nucleus of the myeloma cells. Normal myelocytes, promyelocytes and megakaryocytes stained weakly positive, and erythroid cells were constantly negative. We propose that the leptin/OB-R system is strongly and directly involved in supporting the growth of hematopoietic malignancies.     

Monomeric and oligomeric flavanols are agonists of membrane androgen receptors

The present work reports a new mode of action of the naturally occurring flavanols catechin and epicatechin and their dimers B2 and B5, in the breast cancer T47D cell line, namely, their interaction with membrane androgen receptors. We show that monomeric and dimeric flavanols are complete (B2) or partial displacers of radiolabeled testosterone bound on T47D membranes, with affinities ranging from 1.7 (B5) to 82.2 nM (B2). In addition, they trigger the phosphorylation of the same signaling molecules (FAK, PI3K) as testosterone-BSA, minutes after binding to membrane receptors, leading to actin cytoskeleton polymerization and redistribution, with formation of filopodia and lamellipodia. The PI3K inhibitor wortmannin reverts the effect of polyphenols and testosterone-BSA, providing additional evidence about activation of a similar signaling cascade. Incubation of T47D cells for more than 2 h with polyphenols or testosterone-BSA induces apoptosis, which follows the same time-dependent pattern. We conclude that flavanols (monomers or dimers) are agonists of membrane androgen receptors and could be used as testosterone-protein conjugates for the management of tumors, in which, application of testosterone-BSA induces regression, providing additional data about the mechanism of their antiproliferative action.     

Inhibition of estrone sulfatase by aromatase inhibitor-based estrogen 3-sulfamates

Our rationale is based on the finding that estrone 3-sulfamate (EMATE, 2d), a typical estrone sulfatase (ES) inhibitor, can be hydrolyzed and the pharmacological effect of the free estrogen contributes to the bioactivity of the sulfamate. A number of 3-sulfamoylated derivatives of the good aromatase inhibitors, 2- and 4-halogeno (F, Cl, and Br) estrones and their estradiol analogs as well as 6beta-methyl and phenyl estrones, were synthesized and evaluated as inhibitors of ES in human placental microsomes in comparison with the lead compound EMATE. Among them, 2-chloro- and 2-bromoestrone 3-sulfamates (2b and 2c), along with their estradiol analogs 3b and 3c, were powerful competitive inhibitors with K(i)'s ranging between 4.0 and 11.3 nM (K(i) for EMATE, 73 nM). These four sulfamates as well as the 2-fluoro analogs 2a and 3a inactivated ES in a time-dependent manner more efficiently than EMATE, and 2-halogeno estrone sulfamates 2 also caused a concentration-dependent loss of ES activity. The results may be useful for developing a new class of drugs having a dual function, ES inhibition and aromatase inhibition, for the treatment of breast cancer.     

Inhibition of tumor cell growth by a human B-cell line

An Adenocarcinoma cell line (Breast-M) and an Epstein-Barr virus (EBV)-infected B-cell line (Hairy-BM) were established from breast tumor tissue. The Hairy-BM was CD20⁺, CD25 (Tac)+ and surface immunoglobulin (sIg)+. Hairy-BM suppressed the in vitro proliferation of Breast-M in a time and a dose-dependent manner. The suppression was also found in 5 different human tumor targets showing tumor-Hairy-BM binding, but not; in 2 murine tumor targets showing no significant tumor-Hairy-BM binding. Lytic activity of Hairy-BM was found only against Breast-M.Abbreviations sIg Surface immunoglobulin - CTL Cytotoxic T-cells - NK Natural killer - IL2 Interleukin 2 - LAK Lymphokine activated killer - CSN Culture supernatant - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoolium bromide - PCR Polymerase chain reaction - TIL Tumor-infiltrating lymphocytes - HCL Hairy cell leukemia - TNF Tumor necrosis factor     

Antiproliferative activity of some 1,4-dimethylcarbazoles on cells that express estrogen receptors: part I

Several 9H-carbazole derivatives are used for various pharmacological applications. Many of these compounds demonstrated cytotoxic and anticancer activities. In this work, we have investigated the cytotoxic activity of some substituted carbazoles against cancer cell lines (MCF-7, and ISK). The derivative 2a showed the highest inhibitory activity against both cell lines.     

Regulation of fas ligand expression in breast cancer cells by estrogen: functional differences between estradiol and tamoxifen

During neoplastic growth and metastasis, the immune system responds to the tumor by developing both cellular and humoral immune responses. In spite of this active response, tumor cells escape immune surveillance. We previously showed that FasL expression by breast tumor plays a central role in the induction of apoptosis of infiltrating Fas-immune cells providing the mechanism for tumor immune privilege. In the present study, we showed that FasL in breast tissue is functionally active, and estrogen and tamoxifen regulate its expression. We identified an estrogen recognizing element like-motif in the promoter region of the FasL gene, suggesting direct estrogen effects on FasL expression. This was confirmed by an increase in FasL expression in both RNA and protein levels in hormone sensitive breast cancer cells treated with estradiol. This effect is receptor mediated since tamoxifen blocked the estrogenic effect. Interestingly, tamoxifen also inhibited FasL expression in estrogen-depleted conditions. Moreover, an increase in FasL in breast cancer cells induces apoptosis in Fas bearing T cells and, tamoxifen blocks the induction of apoptosis. These studies provide evidence that tamoxifen inhibits FasL expression, allowing the killing of cancer cells by activated lymphocytes. This partially explains the protective effect of tamoxifen against breast cancer.     

Evaluation of anticancer effects of newly synthesized dihydropyridine derivatives in comparison to verapamil and doxorubicin on T47D parental and resistant cell lines in vitro

Failure of current anticancer drugs mandates screening for new compounds of synthetic or biological origin to be used in cancer therapy. Multidrug resistance (MDR) is one of the main obstacles in the chemotherapy of cancer. Efflux of cytotoxic agents mediated by P-glycoprotein (P-gp or MDR1) is believed to be an important mechanism of multidrug resistance. Therefore, we decided to investigate the antiproliferative effects of seven newly synthesized 1,4-dihydropyridine (DHP) derivatives in comparison to verapamil (VP) and doxorubicin (DOX) on human breast cancer T47D cells and its MDR1 overexpressed and moderately resistant cells (RS cells) using MTT cytotoxicity assay. We also examined the effects of these compounds on cytotoxicity of DOX in these two cell types. The cytotoxicity assays using MTT showed that most of the tested new DHP derivatives and VP at 10 μM concentration had varying levels of toxicity on both T47D and RS cells. The toxicity was mostly in the range of 10–25%. However, the cytotoxicity of these DHP derivatives, similar to VP, was significantly less than DOX when comparing IC₅₀ values. Furthermore, these compounds in general had relatively more cytotoxicity on T47D vs RS cells at 10-μM concentration. Among new DHPs, compounds 7a (3,5-dibenzoyl-4-(2-methylthiazol-4-yl)-1,4-dihydro-2,6-dimethylpyridine) and 7d (3,5-diacetyl-4-[2-(2-chlorophenyl)thiazol-4-yl)]-1,4-dihydro-2,6-dimethylpyridine) showed noticeable potentiation of DOX cytotoxicity (reduction of DOX IC₅₀) compared to DOX alone in both cells, particularly in RS cells. This effect was similar to that of VP, a known prototype of MDR1 reversal agent. In other words, compounds 7a and 7d resensitized RS cells to DOX or reversed their resistance. Results indicate that compound 7d exerts highest effect on RS cells. Therefore, these two newly synthesized DHP derivatives, compounds 7a and 7d, are promising as potential new MDR1 reversal agents and should be further studied on other highly resistant cells due to MDR1 overexpression and with further molecular investigation.     

Evidence that triiodothyronine decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue

Conflicting results have been reported regarding the effect of triiodothyronine (T(3)) on serum leptin and adipose tissue leptin gene expression in human and animals. The aim of the present study was to evaluate the effect of administration of increasing doses of T(3) on serum leptin concentration and on leptin mRNA abundance in white adipose tissue of rats. The results presented in this paper indicate that administration of single different doses of T(3) to euthyroid rats resulted dose dependent increases of serum total T(3) concentrations which are associated with a decrease in white adipose tissue leptin mRNA level. The leptin mRNA level in white adipose tissue was negatively correlated with serum total T(3) concentration (r=-0.8, p<0.001). Like white adipose tissue leptin mRNA level, serum leptin concentration decreased after T(3) administration, and was also negatively correlated with the serum T(3) concentration (r=-0.8, p<0.001). In contrast, administration of T(3) to the same rats led to a significant increase in white adipose tissue expression of the malic enzyme gene (malic enzyme activity and malic enzyme mRNA level), a known target gene for T(3). The results indicate that T(3) exerts a selective inhibitory effect on white adipose tissue leptin gene expression in vivo. A conclusion is that T(3) decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue.     

Inhibition of alpha-globin gene expression by RNAi

RNA interference (RNAi), a process by which target messenger RNA (mRNA) is cleaved by small interfering complementary RNA (siRNA), is widely used for investigations of regulation of gene expression in various cells. In this study, siRNA complementary to 5′ region of exon II of α-globin mRNA was examined for its role in erythroid colony forming cells (ECFCs) isolated from normal peripheral blood donor. On day 6 of cell culture, 1 × 10⁶ ECFCs were transfected with lipofectamine-containing α-globin specific siRNA. After 48 h of transfection, α-globin specific siRNA produced significantly reduction of α-globin mRNA level in a dose-dependent manner, but it did not affect the level of β-globin mRNA. Significantly, decreased numbers of hemoglobinized erythroid cells relative to the control were observed supporting the inhibitory effect of this α-globin mRNA specific siRNA.     

Leptin attenuates gene expression for renal 25-hydroxyvitamin D3-1alpha-hydroxylase in mice via the long form of the leptin receptor

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We previously showed that leptin administration to leptin-deficient obese (ob/ob) mice suppressed mRNA expression and activity of renal 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1). In leptin receptor-deficient (db/db) mice, we presently examined whether leptin affects 1alpha-hydroxylase expression in renal tubules through the active form of the leptin receptor (ObRb). Elevated serum concentrations of calcium and 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in untreated ob/ob mice showed sharp reduction with leptin administration (4 mg/kg, i.p. every 12h for 2 days); no such reduction of elevation occurred in db/db mice. ObRb mRNA was expressed in kidney, brain, fat, lung, and bone in wild-type and ob/ob mice, but not db/db mice. The ob/ob and db/db mice showed large increases in renal 1alpha-hydroxylase mRNA expression and activity. Leptin administration (4 mg/kg) completely abrogated these increases in ob/ob but not db/db mice. Renal 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) mRNA synthesis also was greatly elevated in ob/ob and db/db mice; excesses decreased significantly with leptin administration in ob/ob mice, but increased in db/db mice. Renal tubular cells in primary culture expressed mRNAs including proximal tubules markers (1alpha-hydroxylase and megalin), parathyroid hormone receptor, and vitamin D receptor. Calcitonin receptor mRNA, synthesized mainly in distal tubules, was scant, indicating that most cultured cells were from proximal tubules. Cells did not express ObRb mRNA. Forskolin exposure at 10(-6)M for 3 or 6h significantly increased 1alpha-hydroxylase mRNA. Leptin at 10(-6)M did not change mRNA expression in either presence or absence of forskolin. Accordingly, leptin attenuates renal 1alpha-hydroxylase gene expression through ObRb. Furthermore, leptin appears to act indirectly on renal proximal tubules to regulate 1alpha-hydroxylase gene expression.