Contributions
Type: Publication Only
Background
Retinoids modulate the activity of a large number of genes through binding to the retinoic acid receptor (RAR/RXR) resulting in a variety of molecular and biochemical consequences. The role of retinoids in different malignancies has been extensively explored. However little is known about their effect on lymphoid malignancies.
Acitretin is a synthetic retinoid which has been shown to induce cell differentiation, and apoptosis in squamous cell carcinoma, where it was shown to induce apoptosis of malignant squamous cells without effecting normal epithelial cells.
Aims
We aimed to investigate the effects of Acitretin on the CLL like cell line MEC-1 and on primary CLL cells.
Methods
Peripheral blood samples were obtained from 28 CLL patients who gave written informed consent (14 treatment naïve, 14 with relapsed disease). The CLL like cell line MEC-1 was obtained and experiments were performed on these and on primary CLL cells, freshly isolated from whole blood using a density centrifugation technique.
Viability assays were performed using the Celltitre96 AquousOne® MTS assay. Migration assays were performed on the Boyden chamber platform where migration of acitretin treated cells toward the chemokine CXCL12 was compared to untreated controls.
Apoptosis was measured using FACS analysis of CLL cells treated with 10microM Acitretin for 24 and 72hrs. Treated cells and vehicle controls were labelled with Annexin 5 and Propidium Iodide to detect early apoptotic cells and dead cells.
Results
Acitretin significantly reduced proliferation of MEC-1 cells at drug concentrations of up to 25µM (28.6% mean reduction, p=0.0002).
Acitretin reduced the proportion of viable CLL cells in 5 out of 7 patients as compared to control in cells from treatment Naïve patients (18% mean reduction, p=0.0001), at drug concentrations up to 20µM.
Furthermore, one clinic patient with early stage CLL was commenced on acitretin for control of squamous cell carcinoma of the skin. His lymphocytosis was found to have resolved 3 weeks into treatment. His lymphocyte count continued to be normal 1 year into follow up.
However, acitretin had no effect on viability of cells from relapsed patients.
Acitretin also had a variable effect on cell migration whereby it reduced the ability of cells to migrate towards the chemokine CXCL12 in 2 out of 5 patient samples and did not appear to effect migration of the cells in 3 patient samples.
Acitretin does not appear to significantly affect the rate of early apoptosis in the primary cells as measured by the proportion of Annexin V/Propidium Iodide labelled cells. Future analysis of additional primay cell lines will further substantiate these results.
Summary
The retinoid derivative acitretin reduced the proliferation rate of MEC-1 cells and the viability of primary CLL cells from treatment Naïve patients in vitro, but had a variable effect on cell migration and did not appear to effect early apoptosis.
Our findings show a potential effect of retinoids in early CLL however the mechanism and clinical significance needs to be further defined.
Keyword(s): Chronic lymphocytic leukemia, Retinoic acid
Session topic: Publication Only
Type: Publication Only
Background
Retinoids modulate the activity of a large number of genes through binding to the retinoic acid receptor (RAR/RXR) resulting in a variety of molecular and biochemical consequences. The role of retinoids in different malignancies has been extensively explored. However little is known about their effect on lymphoid malignancies.
Acitretin is a synthetic retinoid which has been shown to induce cell differentiation, and apoptosis in squamous cell carcinoma, where it was shown to induce apoptosis of malignant squamous cells without effecting normal epithelial cells.
Aims
We aimed to investigate the effects of Acitretin on the CLL like cell line MEC-1 and on primary CLL cells.
Methods
Peripheral blood samples were obtained from 28 CLL patients who gave written informed consent (14 treatment naïve, 14 with relapsed disease). The CLL like cell line MEC-1 was obtained and experiments were performed on these and on primary CLL cells, freshly isolated from whole blood using a density centrifugation technique.
Viability assays were performed using the Celltitre96 AquousOne® MTS assay. Migration assays were performed on the Boyden chamber platform where migration of acitretin treated cells toward the chemokine CXCL12 was compared to untreated controls.
Apoptosis was measured using FACS analysis of CLL cells treated with 10microM Acitretin for 24 and 72hrs. Treated cells and vehicle controls were labelled with Annexin 5 and Propidium Iodide to detect early apoptotic cells and dead cells.
Results
Acitretin significantly reduced proliferation of MEC-1 cells at drug concentrations of up to 25µM (28.6% mean reduction, p=0.0002).
Acitretin reduced the proportion of viable CLL cells in 5 out of 7 patients as compared to control in cells from treatment Naïve patients (18% mean reduction, p=0.0001), at drug concentrations up to 20µM.
Furthermore, one clinic patient with early stage CLL was commenced on acitretin for control of squamous cell carcinoma of the skin. His lymphocytosis was found to have resolved 3 weeks into treatment. His lymphocyte count continued to be normal 1 year into follow up.
However, acitretin had no effect on viability of cells from relapsed patients.
Acitretin also had a variable effect on cell migration whereby it reduced the ability of cells to migrate towards the chemokine CXCL12 in 2 out of 5 patient samples and did not appear to effect migration of the cells in 3 patient samples.
Acitretin does not appear to significantly affect the rate of early apoptosis in the primary cells as measured by the proportion of Annexin V/Propidium Iodide labelled cells. Future analysis of additional primay cell lines will further substantiate these results.
Summary
The retinoid derivative acitretin reduced the proliferation rate of MEC-1 cells and the viability of primary CLL cells from treatment Naïve patients in vitro, but had a variable effect on cell migration and did not appear to effect early apoptosis.
Our findings show a potential effect of retinoids in early CLL however the mechanism and clinical significance needs to be further defined.
Keyword(s): Chronic lymphocytic leukemia, Retinoic acid
Session topic: Publication Only