Imatinib inhibits the regrowth of human colon cancer cells after treatment with 5-FU and cooperates with vitamin D analogue PRI-2191 in the downregulation of expression of stemness-related genes in 5-FU refractory cells.

Conventional cytotoxic drugs preferentially eliminate differentiated cancer cells but spare relatively more resistant stem-like cancer cells capable to initiate recurrence. Due to cancer cell plasticity, the stem-like phenotype can be also acquired by cancer cells refractory to treatment with cytotoxic drugs. We investigated whether drugs inhibiting receptor tyrosine kinases could be used to target human colon cancer cells initiating cancer regrowth following conventional cytotoxic treatment. The moderately differentiated cell line HT-29 and poorly differentiated cell line HCT-116 were exposed to 5-fluorouracil (5-FU). Cells that resisted the exposure to 5-FU were subsequently treated with imatinib or sunitinib. Both drugs reduced clonogenicity of 5-FU-refractory cells under normoxic and hypoxic culture conditions. The expression of numerous stemness-related genes was upregulated in cancer cells following the exposure to 5-FU, and remained at a high level in 5-FU-refractory cells undergoing renewal under normoxia, but decreased spontaneously under hypoxia. Imatinib downregulated the expression of stemness-related genes in cells undergoing renewal under normoxia. A combination of imatinib with PRI-2191, an analogue of 1,25-dihydroxyvitamin D3, downregulated stemness-related genes in HCT-116/5-FU cells more efficiently than imatinib alone. A synthetic analogue of 1,25-dihydroxyvitamin D2 (PRI-1906) abolished the effect of imatinib on gene expression in HCT-116/5-FU cells undergoing renewal under normoxia. Sunitinib promoted shift of phenotype of HT-29/5-FU cells undergoing renewal toward stem-like one. It suggests that the phenotype shift toward stemness induced by sequential sunitinib treatment following 5-FU treatment could increase a risk of cancer recurrence. In contrast to sunitinib, imatinib could be used both to interfere with cancer regrowth after conventional chemotherapy and to downregulate the expression of stemness-related genes in residual colon cancer cells capable to initiate cancer recurrence. The findings suggest that imatinib could also be combined with vitamin D analogue PRI-2191 to prevent recurrence more efficiently than imatinib alone and to compensate for vitamin D deficiency resulting from imatinib treatment.

Lenalidomide potentiates CD4+CD25+Treg-related suppression of lymphoma B-cell proliferation.

We have previously found that ex vivo expanded human CD4+CD25+Treg cells suppress proliferation of lymphoma B-cell lines. Here we demonstrate that the immunomodulatory drug lenalidomide potentiates suppression of lymphoma B-cell proliferation by freshly isolated CD4+CD25+Tregs, as well as suppression by Tregs expanded polyclonally in the presence of rapamycin from CD4+CD25+T cells or CD4+CD25+CD127loT cells. The regulation of lymphoma cell proliferation by Tregs pre-expanded with "third-party" allogeneic MoDCs in the presence of rapamycin was also potentiated by lenalidomide. Lenalidomide contributed to the suppression exerted by Tregs despite concomitant downregulation of Treg proliferation. Lenalidomide did not reduce the suppression of conventional T cells by expanded Tregs. The exposure of polyclonally expanded Tregs to lenalidomide did not significantly alter their phenotype. There was no uniform pattern of lenalidomide effect on Treg-mediated regulation of lymphoma B cells freshly isolated from patients. Freshly isolated lymphoma cells activated with multimeric CD40L and IL-4 to support their survival in vitro varied in their sensitivity to lenalidomide, and the regulatory effect of Tregs on such lymphoma cells ranged from suppression to help in individual patients. Lenalidomide potentiated or attenuated Treg effects on the survival of freshly isolated lymphoma cells. A combination of lenalidomide treatment with adoptive transfer of CD4+CD25+Tregs or CD4+CD25+CD127loTregs expanded ex vivo could be used to suppress proliferation of residual lymphoma in select patients with lymphoma responsive to the regulation by Tregs and sensitive to lenalidomide.

The Effect of Analogues of 1α,25-Dihydroxyvitamin D2 on the Regrowth and Gene Expression of Human Colon Cancer Cells Refractory to 5-Fluorouracil.

This study aimed to evaluate the capacity of hypocalcemic analogues of 1α,25-dihydroxyvitamin D2 (1,25D2) and 1α,25-dihydroxyvitamin D3 (1,25D3) to inhibit regrowth and regulate the stemness-related gene expression in colon cancer cells undergoing renewal after exposure to 5-fluorouracil (5-FU). All of the tested analogues of 1,25D2 equally potently decreased the clonogenicity and the proliferative activity of HT-29 cells which survived the exposure to 5-FU, but differently regulated gene expression of these cells during their renewal. 1,25D2 and analogues (PRI-1907 and PRI-1917), as well as 1,25D3 and analogue PRI-2191, decreased the relative expression level of several stemness-related genes, such as NANOG, OCT3/4, PROM1, SOX2, ALDHA1, CXCR4, in HT-29/5-FU cells during their renewal, in comparison to untreated HT-29/5-FU cells. The other 1,25D2 analogues (PRI-1906 and PRI-1916) were not capable of downregulating the expression of these stemness-related genes as the analogues PRI-1907 and PRI-1917 did. All of the tested vitamin D analogues upregulated CDH1, the gene encoding E-cadherin associated with epithelial phenotype. Out of the series of analogues studied, side-chain branched analogues of 1,25D2 (PRI-1907, PRI-1917) and the analogue of 1,25D3 (PRI-2191) might be used to target cancer cells with stem-like phenotypes that survive conventional chemotherapy.

Prenyl Ammonium Salts--New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model.

PURPOSE: Prenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents. METHODS: AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells. RESULTS: All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation--considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination. CONCLUSION: Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.

Human regulatory T cells suppress proliferation of B lymphoma cells.

Activated regulatory T cells (Tregs) suppress proliferation and differentiation of normal B cells. In our study, allogeneic polyclonal CD4 (+) CD25 (+) Tregs and CD4 (+) CD25 (+) CD127(lo)Tregs expanded in vitro in the presence of rapamycin and low dose IL-2 suppressed proliferation of 11 out of 12 established lymphoma B-cell lines. The effect of expanded CD4 (+) CD25 (+) Tregs on survival of freshly isolated lymphoma B cells maintained in culture with soluble multimeric CD40L and IL-4 was variable across lymphoma entities. The survival of freshly isolated follicular lymphoma cells usually decreased in cocultures with CD4 (+) CD25 (+) Tregs. Treg effect on chronic lymphocytic leukemia/small lymphocytic lymphoma cells ranged from suppression to help in individual patients. CD4 (+) CD25 (+) Tregs or CD4 (+) CD25 (+) CD127(lo)Tregs expanded ex vivo with rapamycin could be used to suppress regrowth of residual lymphoma after autologous hematopoietic cell transplantation (HCT), and to counteract both graft-versus-host disease and lymphoma re-growth after allogeneic HCT in select patients with lymphoma susceptible to the regulation by Tregs.

Differential interference of vitamin D analogs PRI-1906, PRI-2191, and PRI-2205 with the renewal of human colon cancer cells refractory to treatment with 5-fluorouracil.

This study was aimed to determine whether hypocalcemic analogs of active forms of vitamins D modulate expression of genes related to stem-like phenotype in colon cancer cell lines HT-29 and HCT-116 undergoing renewal after the treatment with 5-fluorouracil (5-FU). Both lines express vitamin D receptor, but differ in differentiation stage and vitamin D sensitivity. Cells that resisted the 5-FU exposure were treated with synthetic analog of 1,25-dihydroxyvitamin D2 (PRI-1906) and analogs of 1,25-dihydroxyvitamin D3 (PRI-2191 and PRI-2205). Proliferative activity was more profoundly affected by vitamin D analogs in HT-29/5-FU than in HCT-116/5-FU cells. In HT-29/5-FU cells, analogs PRI-1906 and PRI-2191 downregulated the expression of genes related to survival, re-growth, and invasiveness during renewal, while PRI-2205 increased expression of genes related to differentiation only. In HCT-116/5-FU cells, PRI-2191 decreased the expression of stemness- and angiogenesis-related genes, whereas PRI-1906 augmented their expression. The effects in HCT-116/5-FU cells were observed at higher concentrations of the analogs than those used for HT-29/5-FU cells. Out of the series of analogs studied, PRI-2191 might be used to counteract the renewal of both moderately and poorly differentiated cancer cells following conventional treatment.

Promoter DNA methylation and expression levels of HOXA4, HOXA5 and MEIS1 in acute myeloid leukemia.

HOXA genes encode transcription factors, which are crucial for embryogenesis and tissue differentiation and are involved in the early stages of hematopoiesis. Aberrations in HOXA genes and their cofactor MEIS1 are found in human neoplasms, including acute myeloid leukemia (AML). The present study investigated the role of HOXA4, HOXA5 and MEIS1 promoter DNA methylation and mRNA expression in AML. Samples from 78 AML patients and 12 normal bone marrow (BM) samples were included. The levels of promoter DNA methylation were determined using quantitative methylation­specific polymerase chain reaction (PCR; qMSP) and the relative expression levels were measured using reverse transcription quantitative PCR in Ficoll­separated BM mononuclear cells and in fluorescent activated cell sorting­sorted populations of normal hematopoietic progenitors. In total, 38.1 and 28.9% of the patients exhibited high methylation levels of HOXA4 and HOXA5, respectively, compared with the control samples, and MEIS1 methylation was almost absent. An inverse correlation between HOXA4 methylation and expression was identified in a group of patients with a normal karyotype (NK AML). An association between the genes was observed and correlation between the DNA methylation and expression levels of the HOXA gene promoter with the expression of MEIS1 was observed. Patients with favorable chromosomal aberrations revealed a low level of HOXA4 methylation and decreased expression levels of HOXA5 and MEIS1 compared with the NK AML and the adverse cytogenetic risk patients. The NK AML patients with NPM1 mutations exhibited elevated HOXA4 methylation and expression levels of HOXA5 and MEIS1 compared with the NPM1 wild­type patients. Comparison of the undifferentiated BM­derived hematopoietic CD34+CD38low, CD34+CD38+ and CD15+ cells revealed a gradual decrease in the expression levels of these three genes and an increase in HOXA4 promoter methylation. This differentiation­associated variability was not observed in AML, which was classified according to the French­American­British system.

Comparison of promoter DNA methylation and expression levels of genes encoding CCAAT/enhancer binding proteins in AML patients.

CCAAT/enhancer binding proteins (CEBPs) are transcription factors regulating myeloid differentiation. Disturbances of their expression may contribute to leukemogenesis. In this study we compared promoter methylation and expression levels of selected CEBP genes in a group of 78 AML patients, normal bone marrow and hematopoietic precursor cells. CEBPA, CEBPD and CEBPE promoter methylation levels were elevated in 37%, 35.5% and 56.7% of patients. No CEBPZ(DDIT3) methylation was observed. An inverse relationship between CEBPA and CEBPD DNA methylation and expression levels was observed. AML cytogenetic risk groups and patients with particular translocation are characterized by distinct methylation/expression profile of CEBPs encoding genes.