Cellular uptake of metallated cobalamins

Cellular uptake of vitamin B12-cisplatin conjugates was estimated via detection of their metal constituents (Co, Pt, and Re) by inductively coupled plasma mass spectrometry (ICP-MS). Vitamin B12 (cyano-cob( III )alamin) and aquo-cob( III )alamin [Cbl-OH 2 ] + , which diﬀer in the b -axial ligands (CN (cid:2) and H 2 O, respectively), were included as control samples. The results indicated that B12 derivatives delivered cisplatin to both cellular cytosol and nuclei with an eﬃciency of one third compared to the uptake of free cisplatin cis -[Pt II Cl 2 (NH 3 ) 2 ]. In addition, uptake of charged B12 derivatives including [Cbl-OH 2 ] + , [{Co}-CN-{ cis -PtCl(NH 3 ) 2 }] + , [{Re}-{Co}-CN-{ cis -PtCl(NH 3 ) 2 }] + , and [{Co}-CN-{ trans -Pt(Cyt)(NH 3 ) 2 }] 2+ (Cyt = cytarabin) was high compared to neutral B12, which implied the existence of an additional internalization pathway for charged B12 vitamin analogs. The aﬃnities of the charged B12 derivatives to the B12 transporters HC, IF and TC were similar to that of native vitamin B12.


Introduction
Cyano-cob(III)alamin, also designated vitamin B12, is an essential, water soluble, and non-toxic vitamin even at high doses.Vitamin B12 is involved in the methionine synthesis and the tricarboxylic acid cycle.B12 comprises an octahedral Co III complex with a tetradentate corrin ring, an a-nucleotide loop and a CN À ligand in the b-axial position.The cyanide ligand can be exchanged for other monodentate functions (Fig. 1). 1 Intracellular reduction of the Co III center subsequently forms square pyramidal cob(II)alamin and square planar cob(I)alamin, respectively. 2,3Enzymatic alkylation of cob(I)alamin in the cytosol and mitochondria leads to the formation of two B12-coenzymes with methyl and adenosyl as b-ligands. 4,5B12 is protected from degradation inside living systems through its binding to the proteins haptocorrin (HC, present in saliva/ gastric fluids), intrinsic factor (IF, secreted by parietal cells), and transcobalamin (TC, plasma protein). 6,7Binding of B12 to TC is required for facilitating the uptake of the B12-TC complex via the TC-receptor or CD320 (across most membranes) and megalin (reabsorbed from the urine in kidney cells). 7The B12 specificity follows the order: IF 4 TC c HC. [8][9][10][11] Numerous functional groups on the corrin side chains, b-ligand, and a-nucleotide loop (Fig. 1) are available to coordinate or conjugate drugs and analytical modalities. 12The b-axial ligand is a preferred derivatization site owing to its ease-of-release in cellular environment and its little involvement in interaction with proteins.Nitrogen mustard chlorambucil, 13 colchicine, 14 cisplatin, 15 and insulinomimetic vanadate (V V ) compound 16 are drugs which have been bound to B12 at this site.In addition, fluorophores 17 and radionuclides 18,19 have also been attached to B12 at this position.The ribosehydroxyl group is another convenient derivatization site as it is not involved in protein interaction. 20Fluorophores, 21,22 cytotoxic drugs, 23 insulin, 24 and appetite suppressing peptide hPYY 25 have been coupled to B12 via this position.
Derivatization of B12, however, is not limited to the band ribose-positions.Radioactive 111 In-DTPA and 99m Tc-DTPA were coupled to B12 via the b-side chain. 26,27The { 99m Tc(CO) 3 } moiety was coupled to B12 via ligands coupled to the b-and d-side chains 28 and biotin via either the b-, c-, d-, or e-side chains. 12Increasing structural knowledge of B12 and of its transport proteins is the base for fine-tuning the structures of biologically active B 12 derivatives and mimetics.Several B 12 antagonists were reported, including B12-[c-lactam], 29 and B12 with de novo peptide backbones. 30,31In addition, tissue distribution was interfered by modifying the B12 structure at a critical site (b-side chain) for protein interaction. 32esearch programs have been dedicated to explore the correlation of B12 requirements and DNA synthesis.This, however, is not the only advantage of using B12 as a carrier for anti-cancer drugs.Its water solubility allows convenient administration of the attached insoluble drugs. 15B12 protects protein-peptide drugs 24,25,33 from hydrolysis in the acidic condition of the gastrointestinal track. 34Though the utilization of B12 as drug carrier appeared to be restrained by its cellular loading capacity, 35 a recent study showed that tissue accumulation of B12 increased significantly (up to 350%) upon overdosing of the vitamin. 36n this paper, B12 was applied as a drug carrier for cisplatin and as a carrier of the {fac-Re(CO) 3 } + moiety, which is a model moiety for Tc and Re radiopharmaceuticals. Cisplatin was chosen as it is the most often used platinum based drug in the clinic, its mode of action is binding to DNA in the nucleus, which triggers apoptosis and cell death.Cisplatin is taken up by the cells via passive diffusion or via active transport through the copper transporters (CTR1 and CTR2) and the organic cation transporters (OCTs) and possible also other pathways. 37Once in the cell cisplatin undergoes aquation, chloride exchanged by water, forming positive ions which are more reactive towards the main biological target, DNA. 38We determined the cytosolic and nucleic uptake of cisplatin, vitamin B12, [{Co}-CN-{cis-PtCl(NH 3 ) 2 }] + (1), [{Re}-{Co}-CN-{cis-PtCl(NH 3 ) 2 }] + (2), and [{Co}-CN-{trans-Pt(Cyt)(NH 3 ) 2 }] 2+ (3) (Fig. 2) in human leukemia cells (K562 cells) and non-adherent (EATC)/ adherent (ELA) Ehrlich ascites tumor cells.The uptake was determined by ICP-MS, where the contents of Co, Pt, and Re represent the uptake of B12 and the platinum/rhenium drug, respectively.

Affinity of B12 derivatives towards transport proteins
Affinity to transport proteins of B12 derivatives was measured by Differential Scanning Fluorometry (DSF), where a fluorescent dye interacts with the hydrophobic sites of unfolded proteins and the melting temperature (T m ) of proteins can be inferred from the recorded fluorescence intensity. 41,42Following ligand binding, the protein stability is enhanced and the ligand-bound proteins unfold at higher temperature with respect to the free proteins.B12 derivatives which bind to B12 transport proteins would produce comparable DT m values to those of B12 while DT m values would be much lower if these derivatives do not bind to B12 transport proteins.In Fig. 4, B12 was used as a positive control for the three transport proteins and cobinamide (Cbi) was used as a negative control. 42Despite the chemical modification of the B12 structure, compound 2 retained good affinity to all B12 transport proteins as it has comparable DT m values to those of B12.
To obtain information on the action of these compounds, knowledge on the amount of drug taken up by cells and accumulated in the nucleus with and without B12 is required and is the base for evaluating the efficiency of B12 as a tumor-targeting drug carrier molecule.The metals (Co, Re, Pt) in B12-drugs conjugates can be measured by ICP-MS, i.e., cytosolic and nucleic determination of 59 Co, 195 Pt, and 187 Re reveal the intracellular distribution of these compounds.Fig. 5A shows the cellular cisplatin uptake in K562 cells, determined as the content in the cytosol.The uptake of platinum containing B12 derivatives (1, 2, and 3) is approximately one third compared to the uptake of cisplatin.Similarly it is seen from Fig. 5B that the platinum uptake into cellular nuclei of 1, 2, and 3 was low compared to cisplatin.The low nucleic platinum uptake of 3 was expected as it has a transplatin-based structure. 43,44ore interestingly, substantial extracellular Pt-release from the B12 structures, which would have resulted in comparative cellular uptake to cisplatin, was not observed.These data indicate that Pt is not released in the extracellular media but taken up together with B12.The complexes are protected when conjugated to the B12 structure.Unexpectedly, the cytosolic uptake of B12 was considerably lower than of [Cbl-OH 2 ] + , 1, 2, and 3 (Fig. 5).This indicated that the observed uptake was not mediated by TC as this protein has similar affinities to B12, 2 (Fig. 4), and 3. 42 Therefore TC would have allowed similar accumulation of these compounds in cellular components.
It was previously reported that B12 derivatives are able to enter cells without the support of TC.6][47][48] HeLa cells internalized only 1-2% of free B12 with respect to holo-TC. 45It was later concluded that free B12 supported cell growth ca. 100 to 1000-fold less efficiently than holo-TC. 49However, human skin fibroblasts could internalize free B12 better than holo-TC, up to more than 20% were reported. 46Berliner et al. proposed a free B12-specific receptor on the cell membrane 46 while Hitzig et al. suggested an a2-globulinlike protein in serum as the alternative pathway for cell to internalize free B12 derivatives. 50n this study, we observed higher accumulation of [Cbl-OH 2 ] + as compared to B12.In a previous report, it was found that [Cbl-OH 2 ] + was internalized and retained better than B12 in human skin   fibroblasts culture 51 but the root of this difference was not fully understood.As we also found comparable cellular uptake among compounds 1, 2, 3, and [Cbl-OH 2 ] + , we conclude that the positive charge carried by these compounds allows cells to internalize them better than the neutral B12 derivatives (Fig. 5).Behavioral differences between charged and neutral B12 derivatives were also previously observed. 52It is possible that charged B12 compounds were either distinctly recognized by a B12-specific receptor or they simply passed through the cationic channels on cell membranes.This, however, requires further clarification.In any case, the experimental results highlighted once more the complex multichannel uptake pathways of B12 vitamin analogues including internalization of holo-TC, free B12 derivatives, and charged B12 derivatives.The preferable cellular import of charged B12 compounds should be kept in perspective for future design of pharmaceutical B12 derivatives.
Drugs like cisplatin and cytarabine attached to the b-CN À of B12 are released by reducing enzymes in the cytosol. 15,40,42urthermore, subsequent cytotoxic activities of the released drugs from [{Co}-CN-{cis-PtCl(NH 3 ) 2 }] + (1) and [{Co}-CN-{trans-Pt(Cyt)(NH 3 ) 2 }] 2+ (3) were also demonstrated. 42,52Cisplatin interferes with cell division by binding to DNA.Previous studies demonstrated accumulation of cisplatin in the cytosol and the nucleus in non-adherent EATC and adherent ELA cells. 53It is therefore especially important for compound 1 and 2 to deliver platinum to nucleus for DNA interaction.As evident from Fig. 6, platinum accumulated in the nucleus in EATC and in ELA cells similar to K562 cells.Furthermore, we found that the absolute amount of platinum bound to DNA in cells exposed for 18 hours to 10 mM and 50 mM [{Re}-{Co}-CN-{cis-PtCl(NH 3 ) 2 }] + (2) was 19 and 83 pmol mg À1 DNA (EATC) and 5 and 20 pmol mg À1 DNA (ELA).Hence, cisplatin coupled to B12 derivative did accumulate in the nucleus and was bound to DNA.
The uptake of Pt into cellular components was always higher than Co (Fig. 5 and 6).A correlation between cobalt and platinum uptake for the three compounds 1, 2 and 3 is shown in Fig. 7.The equation for the trend line is y = 1.60X + 4.9.The one outlier point for EATC, cytosol was omitted.The correlation shows that platinum is taken up by a factor of 1.6 higher than cobalt as estimated from the cytosolic and nucleic content.This observation could reflect a more efficient efflux of Co as compared to platinum.

Conclusions
We reported here comparable uptake in cellular nuclei and cytosol of different B12-drug conjugates, including [{Co}-CN-{cis-PtCl(NH 3 ) 2 }] + , [{Re}-{Co}-CN-{cis-PtCl(NH 3 ) 2 }] + , and [{Co}-CN-{trans-Pt(Cyt)-(NH 3 ) 2 }] 2+ .The results underline that these B12 derivatives deliver cispaltin and cisplatin-related drugs to both, cytosol and nuclei, in two different cancer cell lines.It was found that charged B12 derivatives were preferentially taken up into cellular compartments as compared to neutral and native B12.We therefore propose the existence of an additional uptake pathway for charged B12 derivatives in addition to the previously known TC-mediated B12 uptake pathway.Accumulation of released platinum in the nucleus was clearly observed and explains the cytotoxic action of these compounds as previously described. 50

Affinity to transport proteins
Binding to HC/IF and TC was measured by differential scanning fluorimetry (DSF).Details of the method, protein expression and purification was previously described. 42ptake measurement with ICPMS.fractions were separated according to a previously published procedure with certain modifications. 53In details, cells were centrifuged for 1 minute at 700 g and room temperature (adherent ELA cells were trypsinized with 0.25% trypsine/EDTA in PBS before this step).The supernatant was discarded.Pellets were washed (3Â) with 5 ml PBS before chemical lysis. 57Lysis buffer was added.20 ml of the lysate was spared for protein determination (DC Protein Assay, Bio-Rad).Remaining lysate was centrifuged at 600 g, 4 1C for 5 minutes.The pellet was washed (3Â) with 1 ml NaCl/KCl (30 mM/120 mM) solution to isolate nuclei fraction.The supernatant was centrifuged at 5500 g, 4 1C for 15 minutes.Cytosolic fraction was the supernatant part.The nuclei fractions were digested with 100 ml concentrated HNO 3 .
DNA was isolated according to the following procedure.Cells were centrifuged two times for 5 s at 13 000-16 000 g.The precipitate was subsequently lysed with 300 ml lysis solution.100 ml of Protein Precipitation Solution was added and mixed well.After the proteins were precipitated, the supernatant was transferred into isopropanol (300 ml).This mix is centrifuged for 1 minute at 13 000-16 000 g to collect DNA as precipitate.The DNA fractions were washed with 70% ethanol.DNA hydration solution (50 ml) was then added to the dry DNA.The mixture was then incubated in a water bath at 65 1C for 1 hour to dissolve DNA, followed by incubating at room temperature overnight.Both subcellular fractions were diluted with 0.65% HNO 3 /0.1% HCl for ICPMS measurement.

Fig. 7
Fig. 7 Correlation between drug (Pt) and vitamin B12 (Co) in K562, EATC, and ELA cells.Uptake was determined as the nuclear (black symbols) and cytosolic (red symbols) Pt and Co content measured by ICPMS.