Biological evaluation of both enantiomers of fluoro-thalidomide using human myeloma cell line H929 and others

Over the last few years, thalidomide has become one of the most important anti-tumour drugs for the treatment of relapsed-refractory multiple myeloma. However, besides its undesirable teratogenic side effect, its configurational instability critically limits any further therapeutic improvements of this drug. In 1999, we developed fluoro-thalidomide which is a bioisostere of thalidomide, but, in sharp contrast to the latter, it is configurationally stable and readily available in both enantiomeric forms. The biological activity of fluoro-thalidomide however, still remains virtually unstudied, with the exception that fluoro-thalidomide is not teratogenic. Herein, we report the first biological evaluation of fluoro-thalidomide in racemic and in both (R)- and (S)-enantiomerically pure forms against (in vitro) H929 cells of multiple myeloma (MM) using an annexin V assay. We demonstrate that all fluoro-thalidomides inhibited the growth of H929 MM cells without any in-vivo activation. Furthermore, we report that the enantiomeric forms of fluoro-thalidomide display different anti-tumour activities, with the (S)-enantiomer being noticeably more potent. The angiogenesis of fluoro-thalidomides is also investigated and compared to thalidomide. The data obtained in this study paves the way towards novel pharmaceutical research on fluoro-thalidomides.


Introduction
Thalidomide (Fig 1) is a notorious drug, due to the significant socio-scientific impact it had on nearly every sector of the healthcare industry. When it first introduced to the market in Germany on October 1, 1957, it was heralded as a "wonder drug" for a multitude of minor health disorders such as nausea, fatigue, insomnia, coughs, colds and headaches. Four years later, it was banned, leaving a trail of demise and misery for tens of thousands of lives [1,2]. The thalidomide tragedy precipitated a major paradigm shift in pharmacology, leading to the modern concepts of pharmacokinetics and the development of governmental structures responsible for PLOS  desired therapeutic effects [27]. However, the assumption that thalidomide's enantiomers possess different bioactivities, while very probable, cannot be unequivocally established due to its rapid in vivo racemization rate [28]. Consequently, there has been a longstanding significant interest in the syntheses and biological studies of configurationally stable thalidomide analogues [29][30][31]. In this regard, it is interesting to note that a recent study established [32] that thalidomide and its fluoro-derivative, fluoro-thalidomide possess a high magnitude of self-disproportionation of enantiomers (SDE) [33][34][35] when assessed by achiral chromatography. Taking into account that the SDE phenomenon is related to the ability of a chiral compound to form homo/hetero-chiral associations [36][37][38], one can expect that the enantiomers of thalidomide and fluoro-thalidomide might show an explicit preference for the development of homo-or hetero-chiral interactions with chiral biological receptors and, therefore, different bioproperties or bioactivities. While fluoro-thalidomide was first synthesized in 1999 by our group [21], its biological activity is still virtually unstudied. Thus, there are only preliminary reports on its tumour necrosis factor α (TNF-α) suppressive properties [19][20][21] and teratogenicity [39]. It is interesting to note that (S)-fluoro-thalidomide was found to be more potent than (R)-fluoro-thalidomide in inhibiting lipopolysaccharide (LPS)-induced TNF-α production in human blood leucocytes [21]. On the other hand, no significant differences between (S)-fluoro-thalidomide and (R)-fluoro-thalidomide were observed in in vivo biological activity of 5,6-dimethylxanthenone-4-acetic acid-induced TNF-α activity in serum and tumour tissue. Very importantly, in 2011 [39], fluoro-thalidomide was found to be non-teratogenic as the racemic form, i.e., a mixture of (S)-fluoro-thalidomide and (R)-fluoro-thalidomide, reemphasising the importance of studying the biological activity of fluoro-thalidomide and its practical potential. Given that the treatment of multiple myeloma (MM) is one of the most important therapeutic applications of thalidomide the corresponding study of fluoro-thalidomide seems to be of paramount significance. Taking advantage of the recently developed convenient and scalable procedure for the preparation of enantiomerically pure (R)-fluoro-thalidomide and (S)-fluoro-thalidomide [22], we decided to initiate a systematic study of the biological activity of racemic fluoro-thalidomide and its enantiomers (R)-fluoro-thalidomide and (S)-fluoro-thalidomide for multiple myeloma. The angiogenesis of fluoro-thalidomides is also investigated and compared to thalidomide.

Materials
Cytosine β-D-arabinofuranoside was purchased from Sigma-Aldrich, St. Louis, USA. It was used at a concentration of 10 or 20 μg/mL, as a positive control for inducing apoptosis. All other chemicals were of biochemical grade unless otherwise indicated. Thalidomide was prepared according to a reported method but using racemic ornithine [40]. Fluoro-thalidomide, (R)-fluoro-thalidomide and (S)-fluoro-thalidomide were prepared according to a previously published method [22]. They are optically pure, as was confirmed by commonly used spectroscopic and chromatographic techniques. The copies of 19 F NMR, 1 H NMR, and HPLC analyses of the samples may be found in the supporting information.

Assessment of apoptosis by annexin V and propidium iodide staining
To quantify apoptotic cells, an Early Apoptosis Detection kit (MBL, Nagoya, Japan) was used. The substrates thalidomide, fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide or cytosine β-D-arabinofuranoside were added separately to RPMI1640 medium containing each cell type at a concentration of 10 or 20 μg/mL for 24 or 48 h. Cells were centrifuged at 2,000 rpm for 5 min at 4˚C then suspended in 500 μL of binding buffer. Each sample containing treated cells was mixed with 5 μL of fluorescein isothiocyanate (FITC)-conjugated annexin V solution and 5 μL of propidium iodide (PI) solution at room temperature (RT) for 5 min in the dark. Cells were then analyzed by a fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson, Sparks, MD USA).

MTT assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was investigated by evaluating cell viability using an MTT cell proliferation assay kit (Cayman Chemical Co., Ann Arbor, MI USA). H929 cells were incubated with or without thalidomide, fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide or cytosine β-D-arabinofuranoside at a concentration of 20 μg/mL for 24 h. These treated cells were seeded at a density of 1×10 5 cells/well in 100 μL of RPMI1640 medium in 96-well plates (Becton and Dickinson, Franklin Lakes, USA) and cultured at 37˚C for 24 h. 10 μL of MTT reagent was added to each well. Cells were mixed gently, then incubated at 37˚C in a 5% CO 2 incubator. After 3 h incubation, the culture medium was aspirated and 100 μL of crystal-dissolving solution (dilute hydrochloric acid solution with sodium dodecyl sulfate (SDS)) was added to each well and mixed. Then, optical density was measured at 550 nm using a microplate reader (BIO-RAD, Benchmark, Hercules, CA USA).

Morphological observations
H929 cells (1×10 5 cells/mL) were first treated with or without thalidomide, fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide or cytosine β-D-arabinofuranoside for 24 h at a concentration of 20 μg/mL. The cells (1×10 4 cells) were then deposited on glass slide (20 mm 2 ) using a cytospin centrifuge at 750 rpm for 5 min. The glass slides were fixed with Wright's solution (Merck, Darmstadt, Germany) and stained with Giemsa solution (Merck) for observing the morphology under optical microscope (at ×400 magnifications) and the presence or absence of apoptotic bodies.
Detection of Fas or cleaved PARP expression

Tube formation assay
An angiogenesis assay kit (Kurabo) was used following the manufacturer's protocol. HUVECs co-cultured with fibroblasts were cultivated in the presence or absence of various concentration of fluoro-thalidomide, (S)-fluoro-thalidomide, (R)-fluoro-thalidomide, or thalidomide with VEGF (10 ng/mL) at day 1, 4, 7 and 9. The fluoro-thalidomide, (S)-fluoro-thalidomide, (R)-fluoro-thalidomide, or thalidomide was dissolved with dimethyl sulfoxide (DMSO, a final concentration of DMSO was 0.1%). DMSO was added to the control groups. At day 11, cells were fixed with 70% ethanol. The cells were incubated with diluted primary antibody (mouse anti-human CD31, 1: 4000) for 1 h at 37˚C, and with the secondary antibody (goat anti-mouse IgG alkaline phosphatase-conjugated antibody, 1:500) for 1 h at 37˚C. Visualization was achieved using 5-bromo-4-chhloro-3indolyl phosphate/nitro blue tetrazolium. Images were obtained from five different fields (5.5 mm 2 per field) for each well, and tube area, length, joints, and paths were measured using Angiogenesis Image Analyzer Ver.2 (Kurabo) as previously described [41,42].

Statistical analysis
Data were analyzed using Excel software and the Student's t-test was used to assess the statistical significance between treated and untreated samples. Results are expressed as mean ±SD of three independent replicates.

Results and discussion
Multiple myeloma (MM) is one of the common cancers of the blood, characterized by the accumulation of malignant plasma cells in the bone marrow compartment. MM is still an incurable disease with a 5-year survival rate of around 67% [43]. Taking into account that thalidomide monotherapy, or in combination with other drugs [44,45], is one of the most common and effective treatments, we selected MM as the subject of this study.

Assessment of apoptosis by annexin V and PI staining
The annexin V/PI staining method is a commonly used approach to determine if cells are viable or apoptotic/necrotic based on differences in plasma membrane integrity and permeability [46]. To assess the anti-tumour activity of fluoro-thalidomide by apoptosis in H929 cells, we applied the standard annexin V/PI protocol [47] measuring the outcome by a FACS Calibur flow cytometry system. We first compared the effects of fluoro-thalidomide and thalidomide using racemic compounds, thalidomide and fluoro-thalidomide, in vitro on standard annexin apoptosis by flow cytometric analysis (Fig 2A). As expected from the literature [48][49][50][51][52], thalidomide did not induce apoptosis in the in vitro study (also see S1 83.5 or 75.4% respectively. Notably, although the absolute percentage of positive cells increased with a longer incubation time, the relative trend between fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide or cytosine β-D-arabinofuranoside remained constant. Interestingly, was the observation that a difference was observed between the enantiomers (R)-fluoro-thalidomide and (S)-fluoro-thalidomide, where the (S)-enantiomer proved to be more potent to the system. There were several significant observations. Firstly, (S)-configured enantiomer (S)-fluoro-thalidomide was more active than (R)-enantiomer (R)-fluoro-thalidomide. Secondly, the observed differences in biological activities between fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide and cytosine β-D-arabinofuranoside were much more pronounced at a higher (20 μg/mL) concentration than at 10 μg/mL. We next examined apoptosis using other cell lines, in particular Oda (a human IgD-producing cell line), U937 (a human histiocytic lymphoma cell line) and non-cancerous AGLCL (a normal human B cell line). We selected Oda since it produces IgD, and not IgA, in H929 cells.

MTT assay
To further assess toxicity of compounds fluoro-thalidomide, (R)-fluoro-thalidomide and (S)fluoro-thalidomide under study, we conducted an MTT assay measuring, the optical density of the cell samples treated with fluoro-thalidomides fluoro-thalidomide, (R)-fluoro-thalidomide and (S)-fluoro-thalidomide and cytosine β-D-arabinofuranoside at a concentration of 20 μg/mL for 24 h. As can be seen from Fig 3C, optical density decreased by about 53.6% in fluoro-thalidomide, 46.0% for (R)-fluoro-thalidomide, 59.2% for (S)-fluoro-thalidomide and 22.5% in the case of cytosine β-D-arabinofuranoside, relative to untreated cells, which represented 100%. It is important to emphasize that all fluoro-thalidomides were more potent than cytosine β-D-arabinofuranoside, an observation that correlates well with the pharmaceutical potential of these fluoro-derivatives. Furthermore, in this study, we observed a dependency of bio-activity versus stereochemical properties. Thus, enantiomerically pure (S)-fluoro-thalidomide displayed higher potency than its racemic counterpart, i.e., fluoro-thalidomide, while (R)-fluoro-thalidomide was the least potent.

Morphological changes
After H929 cells were incubated with fluoro-thalidomide, (R)-fluoro-thalidomide and (S)fluoro-thalidomide and cytosine β-D-arabinofuranoside at a concentration of 20 μg/mL for 24 h, the morphological features, as presented in Fig 3D. The observed morphological changes indicated that the death of cells was induced by apoptosis, and not merely by toxicity. As can be seen from Fig 3D,   Caspase activity study With these results in hand, we were interested in acquiring more specific data that could point to a possible mechanism of fluoro-thalidomide anti-cancerous activity. To this end, we decided to perform several experiments related to observed apoptosis pathways. It is well known that caspases are key enzymes in the initiation and regulation of cell apoptosis [58]. In particular, among the initiator caspases, caspase-9 initiates the intrinsic apoptotic pathway while caspase-8 is responsible for the extrinsic pathway [59]. Both caspase-9 and caspase-8 activate executioner caspases, such as caspase-3, -6 and -7, leading to the degradation of cellular components and, finally, apoptosis [59,60].  thalidomide was inhibited by the addition of inhibitors of caspase-3, -8 and -9, as well as a caspase-family inhibitor (Fig 4D). Considering these results, one can assume that there might be some degree of similarity between the mechanisms of biological action of fluoro-thalidomide and cytosine β-D-arabinofuranoside. Taking into account the level of activation and inhibition of caspase-3, -8 and -9 in H929 cells, we can conclude that apoptosis initiated by fluoro-thalidomide occurs via a caspase cascade.

PARP or Fas (CD95) expression
PARP is a large family of proteins with 17 members that are critically involved in various cellular processes, including caspase-independent apoptosis [60]. Since PARP is a target of apoptosis-associated caspases, cleavage of PARP strongly suggests caspase activation. The measurements of PARP expression in H929 cells after treatment with fluoro-thalidomides fluorothalidomide, (R)-fluoro-thalidomide and (S)-fluoro-thalidomide and cytosine β-D-arabinofuranoside at a concentration of 20 μg/mL for 24 h are presented in Fig 5A. Anti-cleaved PARP Biological evaluation of both enantiomers of fluoro-thalidomide (Asp214) antibody detected a large fragment (89 kDa) of human PARP1 produced by caspase cleavage. In fluoro-thalidomides fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide or cytosine β-D-arabinofuranoside-treatment experiments, cleaved PARP was detected using anti-cleaved PARP (Asp214) (Fig 5A). Furthermore, the expression of Fas antigen (CD95) [61] was also unmistakably observed ( Fig 5B). However, the level of expression induced by fluoro-thalidomide, (R)-fluoro-thalidomide and (S)-fluoro-thalidomide was relatively weaker than that of cytosine β-D-arabinofuranoside. Nevertheless, the detected PARP expression and Fas (CD95) expression all strongly support that apoptosis observed in the presence of fluoro-thalidomide, (R)-fluoro-thalidomide and (S)-fluoro-thalidomide was induced by a caspase cascade [62].

Bcl-2 expression
The internal apoptosis pathway, due to intracellular stress, involves the permeabilization of the mitochondrial outer membrane by the Bcl-2 family. The Bcl-2 family members comprise three subfamilies, Bcl-2 and its homologues, Bcl-xL and Bcl-w which strongly impede apoptosis in response to cytotoxic stimuli. Among various modes of action of Bcl-2, the phosphorylation of Ser 70 contributes to the inhibition of apoptosis. In other words, the dephosphorylation of Bcl-2 Ser 70 accelerates apoptosis [63,64]. To examine the contribution of Bcl-2 for the apoptosis observation by fluoro-thalidomides, we investigated the expression of Bcl-2 and the phosphorylation of Ser 70 in H929 cells after treatment with fluoro-thalidomide, (R)-fluoro-thalidomide and (S)-fluoro-thalidomide. Cytosine β-D-arabinofuranoside was also examined for comparisons. H929 cells overexpressed Bcl-2, regardless of the treatment. Treatment with thalidomide, fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide or cytosine β-D-arabinofuranoside resulted in 26.0, 40.3, 42.6, 34.3 or 66.1%, respectively of phospho-specific Bcl-2 Ser70, namely activated Bcl-2 expression, after 24 h treatment with 20 μg/mL (Fig 5E). The value was 30.0% without treatment. It should be noted that the phosphorylation of Bcl-2 Ser70 in H929 cells slightly increased after treatment with fluoro-thalidomide, (R)-fluoro-thalidomide or (S)-fluoro-thalidomide instead of dephosphorylation, while the strong apoptosis was observed (Fig 2A). However, the phosphorylation of Bcl-2 Ser70 by the treatment with (S)fluoro-thalidomide was weakest compared to that of fluoro-thalidomide and (R)-fluoro-thalidomide which is good agreement with the fact that (S)-fluoro-thalidomide indicates highest apoptosis observation (Fig 2A). On the other hand, 66.1% of phosphorylation by the treatment with was observed, confirming the activation of on the Bcl-2 signaling pathway [65,66].

Plausible mechanism of the biological activity of fluoro-thalidomides
After considering all of the data discussed above, we propose a plausible mechanism of the biological activity of fluoro-thalidomides. We believe that two pathways are activated. In one pathway, intracellular stress, caused by fluoro-thalidomides, activates caspase-9 by adjusting mitochondrial membrane permeability via the Bcl-2 family. On the other hand, it is the death receptor pathway that is enhanced by fluoro-thalidomides, via both Fas receptor and Fas ligand expression on H929 cells. Even though the contribution of Fas ligand-Fas receptor interactions to the cytotoxic activity of these drugs remains unclear, trimerisation of the Fas receptor leads to subsequent recruitment of caspase-8 [67]. The engaged caspase-8 and caspase-9 cleave and activate further caspases, initiating a caspase cascade, ultimately leading to cell apoptosis.

Fluoro-thalidomide, not thalidomide, promotes VEGF-induced tube formation in HUVECs
To detect a tube formation network, we used a tube formation assay in which HUVECs and fibroblasts were co-cultured. After 11 days of incubation, HUVECs became organized into complex tubular networks exposed to 10 ng/mL of VEGF, and this effect was promoted by fluoro-thalidomide, (S)-fluoro-thalidomide, and (R)-fluoro-thalidomide addition in a concentration dependent manner. However, treatment with thalidomide decreased VEGF-induced tube formation (Fig 6A). To estimate the formation of capillary like structures, we performed a quantitative determination of the tube area, tube length, joints, and paths as indexes. We identified that exposure to 100 μM of fluoro-thalidomide, (S)-fluoro-thalidomide, (R)-fluoro-thalidomide increased HUVEC tube formation. While others have reported that thalidomide decreases HUVEC tube formation [68][69][70] (Fig 6B-6E), interestingly, fluoro-thalidomide did not induce similar effects. While it is widely reported that thalidomide attenuates nitric oxidedriven angiogenesis by interacting with soluble guanylyl cyclase, all fluoro-thalidomides instead induced the activation of angiogenesis. The contrast in these results can be explained by understanding the relationship between bcl-2 and angiogenesis. Previous reports have shown that activation of bcl-2 induces angiogenesis [71]. Based on these facts, and having proven that fluoro-thalidomides cause bcl-2 activation, while thalidomide in contrast de-activates bcl-2, we predict that it is this mechanism which is causing angiogenesis to be either attenuated or activated. This explanation is in good agreement with the data shown in Fig 5E. Previously, others have shown that fluoro-thalidomide is non-teratogenic [39], which due to the direct link between anti-angiogenesis and teratogenic effects is in good agreement with our results.

Conclusion
Our initial study of the anti-tumour activity of fluoro-thalidomides against H929 revealed that a fluorine analogue of thalidomide, i.e., fluoro-thalidomide and its enantiomers are more potent in vitro, compared with the benchmark antitumor drug, cytosine β-D-arabinofuranoside. Moreover, the biological activity of enantiomer (S)-fluoro-thalidomide and fluoro-thalidomide are markedly higher than (R)-fluoro-thalidomide. This work presents the first study of anti-tumor activity of fluoro-thalidomides in racemic, and both (R) and (S)-enantiomerically pure forms. We demonstrate that fluoro-thalidomide, (R)-fluoro-thalidomide and (S)-fluorothalidomide can inhibit the growth of human multiple myeloma cell line H929 and Oda MM cells at a concentration of 20 μg/mL after treatment for 48 h. Furthermore, in sharp contrast to thalidomide, all fluoro-thalidomides do not require any metabolic activation to manifest its strong anti-tumour activity. Moreover, we report the first explicit evidence that the enantiomeric forms of fluoro-thalidomide display different anti-tumor activities in H929 MM cells. In particular, the (S)-enantiomer of fluoro-thalidomide, (S)-fluoro-thalidomide was more potent than its racemate, fluoro-thalidomide or the (R)-configured isomer, (R)-fluoro-thalidomide. (S)-Fluoro-thalidomide was more potent in vitro, when compared with cytosine β-D-arabinofuranoside. It should be noted that (S)-fluoro-thalidomide selectively induced apoptosis of MM cells but were inactive for other cells, including normal human B cells at a concentration of 20 μg/mL after treatment for 48 h. Moreover, all fluoro-thalidomides induce angiogenesis in contrast with the inhibition of angiogenesis by thalidomide. With the reported data of TNF-α suppressive properties and non-teratogenicity of fluoro-thalidomides, the data we disclose in this work strongly suggest that the fluoro-thalidomide would be an attractive therapeutic candidate worthy of systematic evaluation to ascertain its biological activity. Further mechanistic studies including specific ligand-binding study of fluoro-thalidomide is now under investigation.