Apoptosis Of Cutaneous T-cell Lymphoma Cells Research Articles

Effects of 9-cis-retinoic acid on the proliferation and apoptosis of cutaneous T-cell lymphoma cells.

The vitamin A derivative 9-cis-retinoic acid (9-cis-RA) has been used for the treatment and prevention of cutaneous T-cell lymphoma (CTCL). However, the precise mechanism by which 9-cis-RA treatment ameliorates CTCL remains elusive. Our research shows that 9-cis-RA inhibits proliferation and induces apoptosis in CTCL cells in a dose-dependent and time-dependent manner. 9-Cis-RA also induced G0/G1 cell cycle arrest by downregulation of cyclin D1. We confirmed that 9-cis-RA significantly decreased phosphorylation of JAK1, STAT3, and STAT5 and downregulated Bcl-xL and cyclin D1, indicating that 9-cis-RA inhibited the activation of JAK/STAT signaling. Meanwhile, 9-cis-RA also activated classical RA-mediated transcription by retinoic acid receptors (RAR) and/or retinoid X receptors (RXR) in a CTCL cell line. Thus, 9-cis-RA may be effective for chemotherapy and may prevent human CTCL by inhibiting proliferation and inducing apoptosis by inhibition of the JAK/STAT pathway and activation of the RAR/RXR pathway.

Vitamin D controls apoptosis and proliferation of cutaneous T‐cell lymphoma cells

Vitamin D controls apoptosis and proliferation of cutaneous T‐cell lymphoma cells

C-CBL E3 Ubiquitin Ligase Is Overexpressed in Cutaneous T-Cell Lymphoma: Its Inhibition Promotes Activation-Induced Cell Death

Mycosis fungoides (MF) and Sezary syndrome (SS) are two major forms of cutaneous T-cell lymphoma (CTCL) characterized by resistance to apoptosis. A central pathway for T-cell apoptosis is activation-induced cell death (AICD) which is triggered through the T-cell receptor (TCR). This results in upregulation of FAS-ligand (FASL) and subsequent apoptosis through the FAS death receptor pathway. It has been known for more than a decade that TCR signaling is defective in CTCL; however, the underlying mechanism has not been apparent. In this report, we show that the E3 ubiquitin ligase, c-CBL, is over-expressed in CTCL and that its knockdown overcomes defective TCR signaling resulting in phosphorylation of PLCg1, calcium influx, ROS generation, up-regulation of FASL and extrinsic pathway apoptosis in CTCL cells expressing adequate FAS. In CTCL cells with suboptimal FAS expression, FAS can be upregulated epigenetically by derepression of the FAS promoter using methotrexate (MTX) which we showed previously has activity as a DNA methylation inhibitor. Using these combined strategies, FAS-low as well as FAS-high CTCL cells can be killed effectively.

NFκB‐dependent Bcl3 regulates survival in cutaneous T‐cell lymphoma (762.1)

Cutaneous T cell lymphoma (CTCL) is characterized by high constitutive activity of NFκB, which promotes cell survival and resistance to apoptosis. Increased expression of the proto‐oncogene Bcl3 belonging to IκB family is associated with the pathogenesis of different types of human cancer, yet, the regulation and function of Bcl3 in CTCL have not been previously studied. Here, we show that human CTCL Hut‐78 and HH cells express high protein levels of Bcl3, which is localized both in the cytoplasm and in the nucleus. NFκB subunits p65 and p50 are highly recruited to the Bcl3 promoter, indicating that Bcl3 expression is regulated by NFκB p65/p50 heterodimers in CTCL cells. Proteasome inhibition by bortezomib (BZ), which induces apoptosis in CTCL cells, inhibits Bcl3 expression. The BZ‐decreased Bcl3 expression is associated with decreased recruitment of NFκB p65 and p50 subunits to the endogenous Bcl3 promoter, but with increased recruitment of NFκB p52. Importantly, suppression of Bcl3 levels by siRNA induces apoptosis and down‐regulates expression of the anti‐apoptotic genes cIAP1 and cIAP2 in CTCL cells. Bcl3 is specifically recruited to the endogenous cIAP1 and cIAP2 promoters, and BZ inhibits this recruitment. These data provide first insights into the regulation and function of Bcl3 in CTCL, and indicate that Bcl3 has an important pro‐survival role in these cells.Grant Funding Source: Support by NIH grants AI085497 and CA173452 to I.V.

SIRT1 is upregulated in cutaneous T-cell lymphoma, and its inhibition induces growth arrest and apoptosis

Silent information regulator type-1 (SIRT1) is the best-studied member of the Sirtuin (Sir2) family of nicotinamide dinucleotide (NAD)-dependent class III histone deacetylases (HDACs), but has not yet been explored in cutaneous T-cell lymphoma (CTCL). We analyzed five CTCL cell lines and lesional tissues using flow cytometry, immunostaining, immunoblotting, cell death, viability, and apoptosis assays, small-molecule inhibitors, and shRNA knockdown. We found strong SIRT1 expression among CTCL lines relative to normal lymphocytes. CTCL cells in lesional tissues also expressed SIRT1 strongly. SIRT1 knockdown resulted in reduced cellular metabolism and proliferation, increased apoptosis, and PARP cleavage products. Tenovin-1, which reversibly inhibits class III HDACs (SIRT1 and SIRT2), reduced SIRT enzymatic activity and SIRT1 expression and led to increased apoptosis. These alterations were accompanied by increased forkhead box O3 (FoxO3) in several cell lines and increased nuclear p53, as well as acetylated p53 in wtp53 MyLa CTCL line. A combination of class I/II and class III HDACIs (vorinostat and tenovin-1) produced significantly greater growth inhibition, cell death via apoptosis, as well as superior p53 promoter upregulation in wtp53 MyLa cells as compared with either agent alone. This occurred in a partially p53-dependent manner, as these effects were blunted by p53 knockdown. Our results indicate that SIRT1 is strongly expressed in CTCL. Its inhibition results in reduced growth and increased apoptosis of CTCL cells. Furthermore, our findings suggest that some CTCL patients, such as those with wtp53, might benefit more from treatment with a combination of different classes of HDACIs than with a single agent.

Abstract B45: The role of combined inhibition of DNA methylation and histone deacetylation in CTCL treatment.

Abstract Introduction: Change in the epigenetic pathways can alter level of gene expressions and trigger neoplastic transformation.1DNA methylation and histone modifications are two main epigenetic mechanisms that regulate gene expression. DNA demethylation results in reexpression of tumor suppressor genes silenced via hypermethylation of their promoter regions and HDACi, approved for cutaneous T cell lymphoma (CTCL) treatment, are shown to synergize with demethylating agents to relieve transcriptional repression2,3. We combined Romidepsin and Azacitidine treatment on CTCL primary cell cultures to investigate cell viability and apoptosis. Methods: Human CTCL cell lines were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), glutamine (2 mM) and streptomycin (100 μg/ml) at 37°C, 5% CO2 and 95% humidity. The CTCL cell cultures were treated with Romidepsin (0.25nM-10uM) and/or Azacitidine (500nM-10uM). After 24 to 72 hrs of combination drug treatment level of apoptosis and necrosis was measured by flow cytometry with Annexin V-FITC detection kit. With the above combination treatment the gene expression levels of cell cycle regulatory genes, HDACs (HDAC1, HDAC2, HDAC3, HDAC6,) and DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) were examined by RT-PCR. The induction of Histone acetylation of H3 and H4 and cell cycle regulatory genes were analyzed by western blotting. Results: The combination treatment showed a noticeable change of cell viability due to an increase in apoptosis and necrosis in a time and dose dependent manner. Furthermore, the RT-PCR results were notable for change in the expression of several of the genes mentioned above, suggesting that the combination of DNA demethylation and histone acetylation has a more selective effect on the induction of apoptosis in CTCL cells. We observed a more pronounced increase cell cycle arrest by induction of cell cycle regulators such as p21 and in acetylation of H3 in the combination treatment than each drug alone suggesting that the this combination treatment, counteracts loss of cell cycle control in CTCL more efficiently. This not only provides a rationale for future clinical trials with combined Romidepsin and Azacitidine treatment but also has initiated us to further explore the possible effects of this combination in HDAC inhibitor resistant CTCL. Conclusion: In the light of our current investigation's outcome, we plan to explore the impact of the cumulative chromatin changes by this treatment combination and try to understand their specific mechanism of cell death induction in cutaneous T cell lymphomas. Further confirmation will be followed by experiments on other CTCL cell cultures and lymphocytes of mycosis fongiodes/Sezary syndrome patients.

Cotreatment with BCL-2 antagonist sensitizes cutaneous T-cell lymphoma to lethal action of HDAC7-Nur77–based mechanism

AbstractPan-histone deacetylase inhibitors, for example, vorinostat and panobinostat (LBH589; Novartis Pharmaceuticals, East Hanover, NJ), have shown clinical efficacy against advanced cutaneous T-cell lymphoma (CTCL). However, the molecular basis of this activity remains unclear. HDAC7, a class IIA histone deacetylase (HDAC), is overexpressed in thymocytes, where it represses expression of the proapoptotic nuclear orphan receptor Nur77. Here, we demonstrate that treatment with panobinostat rapidly inhibits the in vitro and intracellular activity, as well as the mRNA and protein levels of HDAC7, and induces expression and translocation of Nur77 to the mitochondria. There, Nur77 converts death resistance protein Bcl-2 into a killer protein, promoting cell death of cultured and patient-derived human CTCL cells. Treatment with panobinostat improved survival of athymic nude mice implanted with human CTCL cells. Ectopic expression of Nur77 induced apoptosis and sensitized HH cells to panobinostat, whereas combined knockdown of Nur77 and its family member Nor1 was necessary to inhibit panobinostat-induced apoptosis of CTCL cells. Cotreatment with the Bcl-2/Bcl-xL antagonist ABT-737 decreased resistance and synergistically induced apoptosis of human CTCL cells. These findings mechanistically implicate HDAC7 and Nur77 in sensitizing human CTCL cells to panobinostat as well as suggest that cotreatment with an anti–Bcl-2 agent would augment the anti-CTCL activity of panobinostat.

Synergistic interaction of the histone deacetylase inhibitor SAHA with the proteasome inhibitor bortezomib in cutaneous T cell lymphoma

Proteasome inhibitors and histone deacetylase (HDAC) inhibitors are novel targeted therapies being evaluated in clinical trials for cutaneous T-cell lymphoma (CTCL). However, data in regard to tumor biology are limited with these agents. In the present study we analyzed the effects of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the proteasome inhibitor bortezomib on human CTCL cells. Four CTCL cell lines (SeAx, Hut-78, MyLa, and HH) were exposed to bortezomib and/ or SAHA at different concentrations. Cell viability was quantified using the MTT assay. In addition, apoptosis and generation of reactive oxygen species were analyzed. Both agents potently inhibited cell viability and induced apoptosis. After 48 h of incubation, IC50 of bortezomib was noted at 8.3 nm, 7.9 nm, 6.3 nm, and 22.5 nm in SeAx, Hut-78, HH, and MyLa cells, respectively. For SAHA, the IC50 values were at 0.6 microm in SeAx cells, 0.75 microm in Hut-78 cells, 0.9 microm in HH cells, and 4.4 microm in MyLa cells. Importantly, combined treatment resulted in synergistic cytotoxic effects, as indicated by Combination indices values <1 using the median effect method of Chou and Talalay. We furthermore found that combined treatment with both agents lead to a decreased proteasome activity, an upregulation of the cell regulators p21 and p27 and increased expression of phosphorylated p38. In addition, we showed that SAHA reduced the vascular endothelial growth factor production of CTCL cells. Our results demonstrate that bortezomib and SAHA synergistically induce apoptosis in CTCL cells and thus provide a rationale for clinical trials of combined proteasome and histone deacetylase inhibition in the treatment of CTCL.

Arsenic trioxide down-regulates antiapoptotic genes and induces cell death in mycosis fungoides tumors in a mouse model

BackgroundMycosis fungoides (MF) is the most frequent cutaneous T-cell lymphoma (CTCL). Arsenic trioxide (As2O3) has recently been shown to be effective against leukemias, so we studied whether As2O3 induces apoptosis of CTCL cells in vitro. We further investigated if As2O3 is effective in a MF mouse model. Material and methodsAnnexin V/7-amino-actinomycin-D stainings were carried out to investigate if As2O3 induced apoptosis of CTCL cell lines. To study the underlying mechanisms, the effects of As2O3 on various transcription factors and apoptosis regulating proteins were analyzed by western blots, electrophoretic mobility shift assays and transcription factor enzyme-linked immunosorbent assays. The ability of As2O3 to induce tumor regression was investigated in a MF mouse model. ResultsAs2O3-induced apoptosis was paralleled by a reduction of the DNA-binding activities of transcription factors of the NFkB and signal transducer and activator of transcription gene families and reduced expression of the antiapoptotic proteins bcl-1, bcl-xL and mcl-1. Local injections of 200μm As2O3 into tumors caused complete remissions in five of six mice and one partial remission. ConclusionsAs2O3 induced apoptosis of CTCL cells by the down-regulation of transcription factors that stimulate the expression of antiapoptotic genes. Local injection of As2O3 into MF tumor-bearing mice resulted in tumor regression.

Down-regulating constitutive activation of the NF-κB canonical pathway overcomes the resistance of cutaneous T-cell lymphoma to apoptosis

AbstractConstitutive activation of the nuclear factor-kappaB (NF-κB) pathway has been shown to be involved in the resistance of tumor cells to apoptosis in several human malignancies of the hematopoietic lineage. By using electrophoretic mobility shift assay (EMSA) and confocal microscopic analysis, we demonstrate that NF-κB is constitutively activated in cutaneous T-cell lymphoma (CTCL) cell lines HuT-78, MyLa, and SeAx and in peripheral blood lymphocytes (PBLs) from patients with Sézary syndrome (SS) presenting a high ratio of tumor cells, with evidence of p50 and RelA/p65 in DNA-linked complexes. Transfection of SeAx line with a κB/luciferase reporter plasmid showed that translocated NF-κB complexes were functional. Selective inhibition of NF-κB, by transfecting CTCL cell lines with a super-repressor form of IκBα, led to apoptosis. We evidenced down-regulation of NF-κB activation and induction of CTCL cell apoptosis in the presence of proteasome 26S inhibitors ALLN, MG132, and bortezomib. Bortezomib at nanomolar concentrations inhibited constitutive activation of NF-κB and induced apoptosis of CTCL cells, with evidence of an upregulation of Bax expression. These results demonstrate the key role played by NF-κB in the resistance of CTCL to apoptosis and suggest that bortezomib might be useful for the treatment of patients with advanced stages of CTCL refractory to standard antineoplastic chemotherapy.

Primary Cutaneous T-Cell Lymphomas Show a Deletion or Translocation Affecting NAV3, the Human UNC-53 Homologue

Multicolor fluorescent in situ hybridization (FISH) was used to identify acquired chromosomal aberrations in 12 patients with mycosis fungoides or Sézary syndrome, the most common forms of primary cutaneous T-cell lymphoma (CTCL). The most frequently affected chromosome was 12, which showed clonal deletions or translocations with a break point in 12q21 or 12q22 in five of seven consecutive Sézary syndrome patients and a clonal monosomy in the sixth patient. The break point of a balanced translocation t(12;18)(q21;q21.2), mapped in the minimal common region of two deletions, fine mapped to 12q2. By locus-specific FISH, the translocation disrupted one gene, NAV3 (POMFIL1), a human homologue of unc-53 in Caenorhabditis elegans. A missense mutation in the remaining NAV3 allele was found in one of six cases with a deletion or translocation. With locus-specific FISH, NAV3 deletions were found in the skin lesions of four of eight (50%) patients with early mycosis fungoides (stages IA-IIA) and in the skin or lymph node of 11 of 13 (85%) patients with advanced mycosis fungoides or Sézary syndrome. Preliminary functional studies with lentiviral small interfering RNA-based NAV3 silencing in Jurkat cells and in primary lymphocytes showed enhanced interleukin 2 expression (but not CD25 expression). Thus, NAV3 may contribute to the growth, differentiation, and apoptosis of CTCL cells as well as to the skewing from Th1-type to Th2-type phenotype during disease progression. NAV3, a novel putative haploinsufficient tumor suppressor gene, is disrupted in most cases of the commonest types of CTCL and may thus provide a new diagnostic tool.

Retinoids: therapeutic applications and mechanisms of action in cutaneous T-cell lymphoma.

Retinoids, natural and synthetic derivatives of vitamin A, are biological regulators of differentiation, proliferation, apoptosis, and immune response. Retinoic-acid-receptor-selective retinoids (all-trans retinoic acid, 13-cis-retinoic acid, and the synthetic analogs isotretinoin, etretinate and acitretin) have been used for years as monotherapy and/or in combination for treatment of cutaneous T-cell lymphoma (CTCL). Orally administered bexarotene, the first synthetic highly selective retinoid-X-receptor retinoid to be approved by the FDA for CTCL, was shown to be active against the cutaneous manifestations of all stages of CTCL. The topical gel formulation was also effective for early cutaneous manifestations of CTCL or as an adjunct to systemic or phototherapy. Bexarotene treatment induces apoptosis of CTCL cells with down-regulation of its receptors and of survivin, an inhibitor of apoptosis. Identification of new receptor subtype-selective retinoids, combination of various receptor-selective retinoids or other agents, and a new drug delivery system may improve the clinical efficacy of retinoids in the future.