Immunomodulation in stable renal transplant recipients with concomitant tacrolimus and sirolimus therapy

BACKGROUND: Long term treatment with immunosuppressive agents results in nephrotoxicity in renal transplant recipients. We explored the effect of combination of Tacrolimus (TAC) and Sirolimus (SRL) on the immune system in renal transplant recipients. METHODS: 10 stable renal transplant recipients were selected to participate in a pharmacokinetic study with a combination of TAC and SRL. Blood was drawn on day zero and 14 days post treatment. Lymphocyte proliferation was quantified by (3)H-thymidine uptake assay (results expressed as counts per minute). The mRNA expression was studied by RT-PCR and serum levels of cytokines were quantified by ELISA and a cytokine bead array system. RESULTS: Lymphocyte proliferative response to PHA (p < 0.05), Con A (p < 0.006) and Anti-CD3 (p <0.005) were significantly decreased in patients who received both TAC and SRL compared to TAC alone. The mRNA expression of proinflammatory cytokines TNF-α (p < 0.05), cyclins G (p < 0.01) and E (p < 05) were decreased, and of TGF-β (p < 0.03) and p21 (p < 0.05) were increased in patients treated with this combination. Circulating levels of IFN-γ (p < 0.04), IL-4 (p < 0.02), and Il-2 (p < 0.03) were significantly inhibited and elevation of TGF-β (p < 0.04) was observed in patients treated with TAC and SRL combination. CONCLUSION: These novel findings demonstrate that addition of SRL to TAC therapy enhances immuno modulation and causes increased immunosuppression providing a rationale for this concomitant therapy

Growth Suppression of Mouse Pituitary Corticotroph Tumor AtT20 Cells by Curcumin: A Model for Treating Cushing's Disease

effectiveness of curcumin to suppress pituitary tumorigenesis. However the molecular mechanism that mediate this effect of curcumin are still unknown.Using the mouse corticotroph tumor cells, AtT20 cells, we report that curcumin had a robust, irreversible inhibitory effect on cell proliferation and clonogenic property. The curcumin-induced growth inhibition was accompanied by decreased NFκB activity. Further, curcumin down-regulated the pro-survival protein Bcl-xL, depolarized the mitochondrial membrane, increased PARP cleavage, which led to apoptotic cell death. Finally, curcumin had a concentration-dependent suppressive effect on ACTH secretion from AtT20 cells.The ability of curcumin to inhibit NFκB and induce apoptosis in pituitary corticotroph tumor cells leads us to propose developing it as a novel therapeutic agent for the treatment of CD

At-Risk and Recent-Onset Type 1 Diabetic Subjects Have Increased Apoptosis in the CD4+CD25+(high) T-Cell Fraction

BACKGROUND: In experimental models, Type 1 diabetes T1D can be prevented by adoptive transfer of CD4+CD25+ FoxP3+ suppressor or regulatory T cells. Recent studies have found a suppression defect of CD4+CD25+(high) T cells in human disease. In this study we measure apoptosis of CD4+CD25+(high) T cells to see if it could contribute to reduced suppressive activity of these cells. METHODS AND FINDINGS: T-cell apoptosis was evaluated in children and adolescent 35 females/40 males subjects comprising recent-onset and long-standing T1D subjects and their first-degree relatives, who are at variable risk to develop T1D. YOPRO1/7AAD and intracellular staining of the active form of caspase 3 were used to evaluate apoptosis. Isolated CD4+CD25+(high) and CD4+CD25− T cells were co-cultured in a suppression assay to assess the function of the former cells. We found that recent-onset T1D subjects show increased apoptosis of CD4+CD25+(high) T cells when compared to both control and long-standing T1D subjects p<0.0001 for both groups. Subjects at high risk for developing T1D 2–3Ab+ve show a similar trend p<0.02 and p<0.01, respectively. On the contrary, in long-standing T1D and T2D subjects, CD4+CD25+(high) T cell apoptosis is at the same level as in control subjects p = NS. Simultaneous intracellular staining of the active form of caspase 3 and FoxP3 confirmed recent-onset FoxP3+ve CD4+CD25+(high) T cells committed to apoptosis at a higher percentage 15.3±2.2 compared to FoxP3+ve CD4+CD25+(high) T cells in control subjects 6.1±1.7 p<0.002. Compared to control subjects, both recent-onset T1D and high at-risk subjects had significantly decreased function of CD4+CD25+(high) T cells p = 0.0007 and p = 0.007, respectively. CONCLUSIONS: There is a higher level of ongoing apoptosis in CD4+CD25+(high) T cells in recent-onset T1D subjects and in subjects at high risk for the disease. This high level of CD4+CD25+(high) T-cell apoptosis could be a contributing factor to markedly decreased suppressive potential of these cells in recent-onset T1D subjects

In Vitro Growth Suppression of Renal Carcinoma Cells by Curcumin

Background: Malignant clear cell renal carcinoma (ccRCC) is an aggressive tumor that is highly resistant to chemotherapy and radiation. Current therapeutic approaches to management of ccRCC have not significantly improved patient survival, therefore novel therapies are needed. The von Hippel-Lindau tumor suppressor gene is frequently mutated in ccRCC resulting in unregulated transcriptional activity of hypoxia-inducible factors (HIF) 1α and 2α. HIF-mediated transcription leads to increased growth factor expression and growth factor receptor (GFR)-mediated signaling. NFκB and STAT3 are phosphorylated in response to GFR activation and modulate gene expression, which promotes cell growth and invasion. Activated NFκB and STAT3 expression is associated with ccRCC pathogenesis. Purpose: The dietary polyphenol curcumin is a well-documented antitumor agent and a known inhibitor of NFκB and STAT3 activation. Given the lack of effective therapies that block ccRCC progression, our objective was to examine whether curcumin could suppress the growth and migration of ccRCC cells, and whether this suppression was mediated via inhibition of NFκB and STAT3 activity. Methods: Human ccRCC cell lines (769-p, 786-o, Caki-1, ACHN and A-498 cells) were exposed to curcumin to assess the impact of curcumin on ccRCC cell viability. To examine the mechanism by which curcumin induced cell death, we used 769-p cells, a highly aggressive human ccRCC cell line that does not express functional von Hippel-Lindau protein. The impact of curcumin on the phosphorylation status and transcriptional activity of NFκB and STAT3, in 769-p cells, was determined. Results: Our results show that in ccRCC cells curcumin decreased cell proliferation and cell viability, abolished clonogenic property, induced apoptosis and blocked cellular migration. The growth suppressive and proapoptotic effects of curcumin were accompanied by decreased phosphorylation and transcriptional activity of NFκB and STAT3. Conclusion: The ability of curcumin to induce apoptosis and inhibit migration of ccRCC cells justifies additional studies that explore the potential of developing curcumin or other NFκB and STAT3 inhibitors as novel therapeutic agents in the management of ccRCC

Curcumin suppresses constitutively activated NFκB activity, down regulates Bcl-_xl and causes mitochondrial membrane depolarization in AtT20 cells.

<p>(<i>A</i>) AtT20 cells were transiently transfected with an NFκB reporter gene, and after 24 hrs treated with either vehicle or the indicated concentrations of curcumin for 4 hrs. Cells were washed and luciferase activity in cell lysates was determined. Normalized luciferase activity was calculated, and data is presented as fold change over control. Each value is the mean ±SEM of 3 separate experiments each performed in triplicates. * indicates significant difference from control, (<i>p<0.05</i>). (<i>B</i>) AtT20 cells were treated with either vehicle or indicated concentrations of curcumin for 24 hrs. Cell lysates were harvested and equal amount of protein was subjected to western blotting with an anti-Bcl-xL Ab. The filter was stripped and reprobed with anti- β tubulin Ab to confirm equal loading. Data shown is from a single experiment, and is a representative of 2 independent experiments yielding similar results. (<i>C</i>) AtT20 cells were treated with either vehicle or indicated concentrations of curcumin for 24 hrs. Cells were washed and labeled with the dual fluorescence mitochondrial specific dye, JC-1, and analyzed by flowcytometry. The dot plots show that in vehicle and 5 µM curcumin treated cells, the % of cells emitting green fluorescence is low, and is indicative of basal apoptosis. However, treatment with 50 µM curcumin, that caused a decrease in Bcl-xL levels, significantly increased the intensity of green fluorescence. The dot plot shown is from a single experiment that is representative of 3 independent experiments. When data from the light scatter plots were quantitated (<i>D</i>) as % of gated cells, our data show that curcumin in a concentration-dependent manner, lead to increased membrane depolarization. Each value is the mean ±SEM of 3 separate experiments. * indicates significant difference from control, (<i>p<0.05</i>).</p

Curcumin suppresses cell proliferation and clonogenic ability of AtT20 cells.

<p>(<i>A</i>) AtT20 cells were treated with the indicated concentrations of curcumin and cell proliferation was determined by MTT assay after 4 days. Data were calculated as % of vehicle control and expressed as mean ±SEM of 3 independent experiments each performed with at least 4 replicates. * indicates significant differences from control, (<i>p<0.05</i>). (<i>B</i>) AtT20 cells were treated with either vehicle or curcumin (5, 10 and 50 µM) for 24 hrs. Cells were trypsinized, washed and re-plated in DMEM medium containing 10% FBS. Cell proliferation was assessed after 4 days. Data were calculated as % of vehicle control and expressed as mean ±SEM of 3 independent experiments each performed with at least 4 replicates. * indicates significant differences from control (<i>p<0.05</i>). (<i>C</i>) AtT20 cells were seeded (1000–3000 cells/well) in a six well plate in complete growth medium containing 10% FBS. Cells were allowed to adhere for 24 hrs, after which medium was replaced with fresh growth medium, containing 10% FBS, together with the indicated concentrations of curcumin. Medium was changed every 3 to 4 days, and colony formation was monitored over a 14–21 day period. Colony formation was detected by crystal violet staining and subsequently photographed. Data shown is from a single experiment, and is a representative of 3 independent experiments yielding similar results. (<i>D</i>) To quantitate colony formation, the number of colonies (each colony consisting of 50 cells or more) in 4 random fields were counted. The average was obtained, and decreases from 0 µM curcumin were calculated and are expressed as % of control. Each value is the mean ±SEM of 3 separate experiments. * indicates significant difference from control, (<i>p<0.05</i>).</p

In Vitro Growth Suppression of Renal Carcinoma Cells by Curcumin

Purpose: Malignant clear cell renal carcinoma (ccRCC) is an aggressive tumor highly resistant to chemotherapy and radiation. Current therapeutic approaches to management of ccRCC have not significantly improved patient survival, therefore novel therapies are needed. Activated NFκB and STAT3 expression is associated with ccRCC pathogenesis. The dietary polyphenol curcumin is a well-documented antitumor agent and a known inhibitor of NFκB and STAT3 activation. Given the lack of effective therapies that block ccRCC progression, our objective was to examine whether curcumin could suppress the growth and migration of ccRCC cells, and whether this suppression was mediated via inhibition of NFκB and STAT3 activity. Methods: Human ccRCC cell lines (769-p, 786-o, Caki-1, ACHN and A-498 cells) were exposed to curcumin to assess the impact of curcumin on ccRCC cell viability. Colony formation assay was used to assess the effect of curcumin on ccRCC cell renewal capability. Effect of curcumin on apoptosis was determined by annexin V binding and mitochondrial membrane depolarization assays. The anti-migratory effect of curcumin on ccRCC cells was assessed using the wound healing assay. Effect of curcumin on NFκB and STAT3 phosphorylation in 769-p cells was determined by western blot analysis. Results: In ccRCC cells, curcumin decreased cell proliferation and cell viability, abolished clonogenic property, induced apoptosis and blocked cellular migration. The growth suppressive and pro-apoptotic effects of curcumin were accompanied by decreased phosphorylation of NFκB and STAT3. Conclusions: The ability of curcumin to induce apoptosis and inhibit migration of ccRCC cells justifies additional mechanistic and preclinical studies that examine the effect of curcumin or other NFκB and STAT3 inhibitors as potential suppressors of ccRCC tumorigenesis