Review
Novel vitamin D analogues; cytotoxic and anti-proliferative activity against a diffuse large B-cell lymphoma cell line and B-cells from healthy donors

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Highlights

  • We have examined the action of 1,25D3 and vitamin D analogues (VDAs) on malignant and healthy B cells.

  • 1,25D3 and VDAs displayed moderate cytotoxic and pro-apoptotic actions upon DLBCL cells.

  • 1,25D3 and VDAs displayed concentration and time-dependent anti-proliferative actions upon stimulated B-cells from healthy donors.

  • VDAs may offer therapeutic potential for the treatment of DLBCL or conditions benefitted by B-cell depletion.

Abstract

Calcitriol (1,25-dihydroxyvitamin D3, 1,25D3) and vitamin D side-chain modified analogs (VDAs) have gained considerable attention as potential drugs in the treatment of acute myeloid leukemia (AML), yet studies of the impact of 1,25D3 and VDAs upon other haematological malignancies are more limited. To address this gap in knowledge, we have examined the action of 1,25D3 and VDAs on a human cell line (DOHH2, K422) typifying diffuse large B-cell lymphoma (DLBCL) and also peripheral blood B-cells isolated from healthy donors.

1,25D3 and certain VDAs displayed moderate cytotoxic and pro-apoptotic actions upon DLBCL cells. 1,25D3 and VDAs (100 nM) caused the death of approximately 40% DOHH2 cells after 24 h stimulation, similar to their impact on HL-60 cells (acute myeloid leukaemia cell line). In addition, 1,25D3 and VDAs displayed concentration and time-dependent anti-proliferative actions upon stimulated B-cells from healthy donors. The VDAs inhibited proliferation by approximately 30%. Hence VDAs may offer therapeutic potential for the treatment of DLBCL or conditions benefitted by B-cell depletion.

Introduction

Calcitriol (1,25-dihydroxyvitamin D3; 1,25D3) impacts physiological processes in the human body, with perhaps the most notable being the regulation of Ca2+ and phosphate (Pi) transport and bone mineralization [1]. Other actions include control of the growth, differentiation and functional activity of numerous cell types including those of the skin, immune system and pancreas [2].

Numerous studies demonstrate that the active form of vitamin D promotes the differentiation of HL-60 cells (a cell line that typifies acute myeloid leukaemia AML) to a phenotype resembling differentiated monocytes in bone marrow [3], such that majority of cells are now CD14+ cells [4]. Similar results have been obtained for cell lines of biologically related origin, e.g., THP-1 and U-937 cells [5], [6]. In addition to the pro-differentiative effects of 1,25D3, considerable attention has been paid to its cytotoxic and anti-proliferative actions. This has been studied in various mouse models, human cultured breast and colon cancers and also in HL-60 cells, which growth arrest in the G1 phase of cell cycle [7], [8], [9]. It should be noted however that the antitumor activity of 1,25D3 is evident at supra-physiological concentrations, which also promote hypercalcemia and hypercalciuria [10], which are likely to limit therapeutic potential. This has motivated the search for vitamin D analogues (VDAs) with less propensity to increase Ca2+ levels. Some identified compounds retain interaction with the VDR and the anti-proliferative/pro-differentiative capacity but display more limited effect on calcium levels in comparison to 1,25D3 [11].

Lymphoma is a cancer of lymphocytes presenting as either Hodgkin’s (HL) or non-Hodgkin’s lymphoma (NHL); over 90% of all NHL are of B-cell origin. According to the Cancer Research UK statistics over 12,000 people are diagnosed with NHL every year in the UK which makes it the 6th most common cancer in this country and the 11th most common cause of cancer death worldwide. As many as 40% of people diagnosed with this disease are predicted to survive less than 10 years.

It is estimated that 50% of NHL cases in the UK and 25–35% worldwide can be categorised as diffuse large B-cell lymphoma (DLBCL) [12]. DLBCL is an aggressive malignancy of B lymphocytes of germinal centre origin and is characterised by the presence of rapidly proliferating large cells with basophilic cytoplasm and vesicular nuclei [13]. Current treatments for DLBCL include the anti-CD20 monoclonal antibody, rituximab, and cycles of a combination of cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP). Unfortunately, rituximab-CHOP treatment (R-CHOP) can be associated with severe toxicity and only displays failure-free survival of 65% after 3 years [14].

There is little data concerning the therapeutic potential of vitamin D analogues for the treatment of NHL. To address further therapeutic potential from 1,25D3 and VDAs, we investigate in the present study the impact of 1,25D3 and novel VDAs upon DOHH2 and K422, cell lines representative of DLBCL, as well as B-cells from healthy donors.

Section snippets

Vitamin D analogues

1,25D3 and all analogues (PRI-1890, PRI-1906, PRI-2191, PRI-2205; Fig. 1) were synthesized in the Pharmaceutical Research Institute (Warsaw, Poland). The compounds were dissolved in absolute ethanol, the concentration adjusted to 100 μM and the aliquots stored at −20 °C prior to experiments.

Cell culture

HL-60 and DOHH-2 cells were cultured in RPMI-1640 medium (Sigma–Aldrich, UK) supplemented with 1% L-glutamine (Life Technologies), 1% penicillin/streptomycin (Sigma–Aldrich, 10,000 units penicillin and 10 mg

Results and discussion

There are already a few reports describing the impact of vitamin D on B-cells. It is known that normal B-cells express VDR at very low levels though the protein is up-regulated following the stimulation of the cells [15]. Malignant B-cells express VDR and both Hodgkin’s and Non-Hodgkin’s lymphoma cells have relatively high levels of this protein [16]. It has been shown that 1,25D3 inhibits proliferation of anti-CD40, anti-IgM, and IL-21 stimulated B-cells [17]. 1,25D3 has also been shown to

Conclusions

In summary, this study demonstrates that relatively high concentrations of 1,25D3 and vitamin D analogues are cytotoxic to DOHH2 cells (with less pronounced effect in K422 cells) with the extent of killing similar to that observed against HL-60 cells. We have also shown that DOHH2 cells express VDR and that ligand engagement increases expression. From this, we conclude that DOHH2 is a good model to study the effects of 1,25D3 and derived compounds in DLBCL. VDAs also inhibited the proliferation

Acknowledgments

The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013 under REA grant agreement number 315902. PK gratefully acknowledges receipt of a Marie Curie Research Associate post. AK and NMB are partners within the Marie Curie Initial Training Network DECIDE (Decision-making within cells and differentiation entity therapies)

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