Cytotoxic, Antiangiogenic and Antitelomerase Activity of Glucosyl‐ and Acyl‐ Resveratrol Prodrugs and Resveratrol Sulfate Metabolites

Resveratrol (RES) is a natural polyphenol with relevant and varied biological activity. However, its low bioavailability and rapid metabolism to its glucuronate and sulfate conjugates has opened a debate on the mechanisms underlying its bioactivity. RES prodrugs are being developed to overcome these problems. We have synthesized a series of RES prodrugs and RES sulfate metabolites (RES‐S) and evaluated their biological activities. RES glucosylated prodrugs (RES‐Glc) were more cytotoxic in HT‐29 and MCF‐7 cells than RES itself whereas RES‐S showed similar or higher cytotoxicity than RES. VEGF production was decreased by RES‐Glc, and RES‐disulfate (RES‐diS) diminished it even more than RES. Finally, RES‐Glc and RES‐diS inhibited hTERT gene expression to a higher extent than RES. In conclusion, resveratrol prodrugs are promising candidates as anticancer drugs. In addition, RES‐S showed distinct biological activity, thus indicating they are not simply RES reservoirs.


Introduction
Resveratrol (trans-3,5,4'-trihydroxystilbene, RES, 1;S cheme 1) is ap hytoalexin generated in responset oe nvironmental stress or pathogenic attack in grapes, blueberries, peanuts,c ocoa, and plants such as the Japanese knotweed Polygonum cuspidatum.R ES has attracted great attention because of its relevant and varied biological activities, such as its cardiovascular protective properties, [1] its anti-inflammatory activity, [2] and its capacity to extend lifespani nv ariouss pecies. [3] The cancerc hemopreventive and chemotherapeutic potential of RES has been demonstrated in differentm odels of carcinogenesis,b oth in vitro and in vivo. It inhibits proliferation in variousc ancer cell lines, [4] and in animal studies RES was abletoi nterferewith the formation of azoxymethane-induceda berrant cryptf oci in rat colon, [5] reduce mammary tumorf ormation in N-methyl-Nnitrosourea-(NMU)-treated rats, [6] and suppress prostate cancer in SV-40 tagr ats. [7] Extensive in vitro studies have revealed multiple intracellulart argets of resveratrol; this affects cell growth, inflammation,apoptosis, and metastasis. [8] Tumor angiogenesis plays ac ritical role in the development of cancer.R ES and piceid (resveratrol-3-b-glucoside, 2)h ave been shown to inhibit the formation of capillary-like tube formationf rom human umbilical vein endothelial cells. [9] Vascular endothelial growth factor (VEGF) is also crucial for angiogenesis and tumorg rowth. In breast cancer cells, an otable decrease in extracellularV EGF has been associated with apoptosis after resveratrol treatment. [10] Ar elevant observation in 90 % of malignant tumors is the maintenance of telomerel ength (cellular DNA) through the expressiono ft he human telomerase reverse transcriptase (hTERT). This process allows cancer cells to evade the progressives hortening of telomeres and thus avoid apoptosis. Resveratrol showeda ni nhibitory effect on MCF-7 tumor cellsm ainly by the induction of S-phase arrest and apoptosis, with reduced expression of hTERT. [11] However,a si st he case with many polyphenols with putative anticancer properties, the bioavailability of resveratrol is very low. [12] RES is well absorbed but avidly glucuronidated and sulfated, both in the liver and in intestinal epithelial cells. [13] In order to counteract this, high doses of resveratrol have been used in cellular,a nimal, and clinicals tudies. This could be the cause of the toxicityo bserved in certain cases, such as am ultiple myeloma clinical trial with 5gad ay of af ormulation of resveratrol. [14] The low systemic levelso fr esveratrolh ave brought about a debate on the possible contribution of the RES metabolites to the in vivo biological activities attributed to RES. [12,15] It has been proposed that resveratrol metabolites could contribute as aRES reservoir throught heir deconjugation, or that RES metabolites could enter the cell via ABC transporters and interact directly with different intracellular targets. [16] Resveratrol (RES) is an aturalp olyphenol with relevant and varied biological activity.H owever,i ts low bioavailability and rapid metabolism to its glucuronate and sulfate conjugates has openedadebate on the mechanismsu nderlying its bioactivity.R ES prodrugs are being developedt oo vercomet hese problems. We have synthesized as eries of RES prodrugs and RES sulfate metabolites (RES-S) and evaluated their biological activities. RES glucosylated prodrugs (RES-Glc) were more cyto-toxic in HT-29 and MCF-7 cells than RES itself whereas RES-S showeds imilar or higher cytotoxicity than RES. VEGF production was decreased by RES-Glc, and RES-disulfate (RES-diS) diminished it even more than RES. Finally,R ES-Glc and RES-diS inhibited hTERTg ene expression to ah ighere xtent than RES. In conclusion, resveratrol prodrugs are promising candidatesa s anticancer drugs. In addition, RES-S showed distinct biological activity,thus indicating they are not simply RES reservoirs.
Here, we report cytotoxicity,V EGF expression, and telomerase inhibition by five RES prodrugs (3-7)m odified with glucosyl or acyl groups.O ur intention was to determine their potential as cancer chemopreventive or chemotherapeutic agents and if they act directly or as aR ES reservoir.I na ddition, we synthesized three RES sulfate metabolites (8-10)b ya ni mproveds ynthetic route. We tested their biological activities and compared them with RES in order to shed light on the controversy concerning the biological activity of RES metabolites.

Results and Discussion
Synthesis of RES prodrugs and RES sulfate metabolites Diglucosylated RES derivatives, trans-resveratrol-3,5-di-O-b-dglucopyranoside (3)a nd trans-resveratrol-3,4'-di-O-b-d-glucopyranoside (4)w ere synthesized from 1 as previously described. [18] Briefly,m ono-TBDMS (tert-butyldimethylsilyl)-pro-tected resveratrol compounds obtained by random silylation of 1 wereg lycosylated by using 2,3,4,6-tetra-O-benzoyl-d-glucopyranosyl trichloroacetimidatea st he glycosyl donor.O nestep deprotection under aqueous strongb asic conditions yielded the desired products 3 and 4.P iceid acyl derivatives 6 and 7 were synthesized by enzymatic acylation, by using Thermomyces lanuginosus lipase immobilized on granulated silica (LipozymeT LI M) as reportedp reviously. [18] RES sulfate metabolites 8-10 were synthesized by following the strategy of Hoshinoe tal. [19] TBDMS-protected resveratrol derivatives 11-15 were obtained from RES, and each compound was separated by flash columnc hromatography.S ubsequent sulfation of each RES derivativeb yu sing SO 3 . NMe 3 as the sulfating reagenti na cetonitrile under reflux followedb y silyl deprotection resulted in very low yields (10-26 %). The difficulties might have been due to incomplete sulfation and the complicated purification of these highly polar products.T hus, instead, we used microwave-assisted synthesis as reported by Desai and co-workers for highly sulfated organic scaffolds. [20] TBDMS-protected 11-13 were treated with SO 3 . NMe 3 and NEt 3 in acetonitrile and microwavei rradiateda t1 00 8Cf or 20-40 min (Scheme 2). The crude product was purified by LH-20 chromatography,a nd desilylation was carried out with KF in MeOH for 18 h. Final crude purification was carried out by reversed-phase chromatographyt oo btain 8-10 in good yields (73-89 %, calculatedf rom the TBDMS-protected RES derivatives). Mattarei et al. recently reported the synthesis of resveratrol sulfates by as imilar approach:s electivep rotection by silyl ether ands ulfation with chlorosulfonic acid. [21] Cytotoxicity The cytotoxicities of 2-10 weree valuated in vitro against two cancer cell lines:h uman colon adenocarcinoma HT-29 and breast adenocarcinoma MCF-7. In addition, one noncancerous cell line, human embryonick idney cell line (HEK-293), was employed for comparison. [22] RES wasu sed as the positive control. As tandard MTT assay was used (see the Experimental Section), and cytotoxicity values( IC 50 )a re shown in Ta ble 1. In most cases, the values were in the low-to-medium micromolar range. Among the RES derivatives, glucosylated compounds (2, 3,a nd 4) and piceid octanoate 7 showed the highest cytotoxicity for HT-29, with IC 50 values lower than for RES. As imilar trend was observed for MCF-7,w ith the lowest IC 50 values for 2 and 7.
Reported cytotoxicity for 2 in severalc ancer cell lines has a wide IC 50 range, for example 1.5 to 300 mm for MCF-7 cells. [23] IC 50 values for 2 were in the same range as or higher than for 1. [23b, 24] In our case, IC 50 valuesf or 2 were within previously reported values, and lower than for 1 in both cell lines (HT-29 and MCF-7). The differences in IC 50 values for 2 might arise from the different methods used to measure cell viabilityo r from differencesi np urity.
It is important to note that 2-4 werem ore toxic with tumor cells than nontumor ones (an obviously desirable feature). This can be better appreciated by examining the a and b coefficients, which are obtained by dividing the IC 50 values for the noncancerous cell line (HEK-293) by those for at umor cell line (see footnote to Ta ble 1). In general, ah igher value reflects ah igher therapeutic safety margin in the corresponding cell line. Thus, 1 was relativelym ore cytotoxic for the noncancerous cell line than for both cancer cell lines. In contrast, 2 and 3 showedg ood selectivity for HT-29 (a > 5.6 and 4.2, respectively). Zhang et al. [23a] also showedt hat 2 is more potent with cancer cells than non-cancer cells for ar ange of cell lines.
Storniolo et al. reported recently that 2 inhibited Caco-2 cell growth (IC 50 = 25 mm). [25] Because the authors did not observe b-glucosidasea ctivity, they proposed that 2 exhibits antiproliferative effects on intestinal epithelial cells directly and not as ar esveratrolr eservoir.T his could also be the case in our assays with 2 and glycosylatedR ES derivatives 3 and 4.H owever,w e previously observed that the glucosyl or acyl modificationsi n prodrugs 2, 3, 4, 6,a nd 7 retarded their metabolism in Caco-2 cells, although they were metabolized to RES and its conjugates (glucuronates, sulfates, etc.) after 6-24 hincubation. [18] The chemicals tability and metabolism of 1, 2,a nd 7 were evaluateda fter incubation withM CF-7, HT-29, and HEK-293 cells for different times (1, 12, 24, and 72 h; see the Supporting Information). We observed low amounts of 1 (< 3%)a fter short times in the three cell lines, either as ac onsequence of fast metabolism or,m ore probably,b inding to serum proteins such as albumin. [26] Increasing amounts of resveratrol-3-sulfate (8)a nd resveratrol glucuronide were observed, with 8 as the major metabolite. Resveratrol-4'-sulfate (9)w as also observed, but its concentration diminished after 1hof incubation. When 2 and 7 were incubated, we observed similar behaviors in the three cell lines. Their concentrations diminished over time and yielded increasing amountsof8,with 9 as the main metabolite at 1h(decreasing over time). Neither 1 nor resveratrol glucuronide were observed. Therefore, 2 and 7 seem to act as prodrugs thus delivering RES( 1)which is mainly transformed into RES sulfated metabolites in the cells.

VEGF production in HT-29 cells
One of the key factors in angiogenesis is the release of VEGF from cells. We decided to examine whether our RES derivatives and metabolites were able to inhibito ra tl east decrease the activation of VEGF genes in HT-29 tumor cells. We selected glucosylated RES compounds 2 and 3,p iceid octanoate 7,a nd RES sulfate metabolites 8, 9,a nd 10.A gain, RES 1 was used as the positive control.W ed etermined VEGF protein production by ELISA in culture supernatants. Figure 1s hows the results obtained in ELISA measurements after treatmento fH T-29 cells with RES derivatives in DMSO: 2, 3, 7,a nd 10 decreased VEGF secretion in comparison with untreated cells; 3 and 7 were more effective than RES itself. Piceid 2 had been shown previously [9] to inhibit angiogenesis on human umbilical vein endothelial cells at concentrations of 100 to 1000 mm, in accordance with our findings. Twop ermethylated stilbened erivatives [30] and af amily of RES dimers [31] have also been reportedt oi nhibitV EGF production. In the case of the sulfate RESd erivatives, it is surprising that disulfate 10 reduced VEGF secretion whereas monosulfates 8 and 9 did not. It is difficult to imagine that 10 could be ar esveratrol reservoir,asn either 8 nor 9 displayed antiangiogenica ctivity.

Telomerase production
Humant elomerase contains an RNA component (hTER)t hat serves as at emplate for the addition of the repeated nucleotide sequences and ap rotein subunit (hTERT) that catalyzes nucleotide polymerization. Humant elomerase is regulated during development and differentiation, mainly throught ranscriptional control of hTERT,t he expressiono fw hichi sr estricted to cells that exhibit telomerase activity.T his indicates that hTERT is the rate-limiting factor in the enzyme complex. [32] Transcriptional factors c-Myc and Sp1 (amongothers) are implicated in the expression of hTERT,b yu pregulatingh TERT mRNA. [33] Thus, forapreliminarys tudy of the potentiala ntitelomerase activity of RES derivatives, we investigated their ability to inhibit the expression of hTERT and c-Myc mRNA. We selected the same group of RES prodrugs (2, 3,and 7)a nd metabolites (8, 9,a nd 10)a sf or VEGF inhibition.R ES was used again as the positive control.
Treatment of HT-29 cells with RES and the derivatives led to variousd egrees of reduction in the transcription of hTERT and c-Myc mRNA (Figures 2a nd 3). The most active compoundsf or the inhibition of hTERTwere 3 and 10 (both more potent than RES). Particularly interesting is 10,w hichr educed the expression to 39 %(RES: 61 %). Figure 1. Percentage VEGF from HT-29c ells treated with DMSO, 1, 2, 3, 7, 8, 9,a nd 10 (20 mgmL À1 for 1 and 10 mgmL À1 others) related to not treated cells. Data are mean AE SEM(n > 2) relative to control.S tatistical significance was evaluated using one-sample t-tests (***p < 0.001).  For c-Myc inhibition, RES and 3 were the most active (48 % and 66 %r espectively;F igure 3); 2, 8, 9,a nd 10 proved to be inactive. RES and 3 show some correlation between inhibition of c-Myc and hTERT gene expression (61 and 52 %, Figure 3). This indicates that RES and 3 might downregulate the expression of hTERT by reducing the transcription of c-Myc. In contrast, 10 showedt he highest inhibition of hTERTi nt his series but did not downregulate c-Myc. Ad ifferent mechanism seems to be used by 10,t husp ointing out again that this RES metabolite is not just aR ES reservoir but exerts its own biological responses.

Conclusion
RES derivatives 2, 3, 4,a nd 7 displayedh igher antiproliferative activity than RES, and showedl ess toxicity with HEK-293 cells. Moreover,t hey showed an otable decrease in the production of VEGF.T hese derivatives act as RES prodrugs, thus delivering RES more slowly to cancer cells and therefore increasing the biological effect. Therefore, RES prodrugs such as resveratrol-3,5-diglucoside 3 might be promising anticancer drugs. The RES sulfate metabolites (8-10)s howed equalo rh igherc ytotoxicityt han RES for HT-29 and MCF-7 cells together with lower toxicityt owards noncancerous cells. The fact that RES disulfate (10)d ecreased VEGF secretion whereas no activity was observed for the monosulfates (8 and 9)s eems to indicate adistinct activity of 10 rather than just as aRES reservoir.Finally, 10 seemed to decrease hTERT expression by am echanism different to that of resveratrol, as it did not affect c-Myc expression.These results indicate that RES metabolites contribute to the in vivo biological activity observed for RES and do not act only as aR ES reservoir.

Experimental Section
General experimental methods: All commercial chemicals were used without further purification, unless otherwise noted. All reactions were monitored by TLC on precoated Silica-Gel 60 F254 plates, and detected by heating with Mostain (H 2 SO 4 (10 %, 500 mL), (NH 4 ) 6 Mo 7 O 24 ·4 H 2 O( 25 g), Ce(SO 4 ) 2 ·4H 2 O( 1g)). Products were purified by flash chromatography with Merck Silica gel 60 (200-400 mesh). MS analyses were run in the electrospray mode (ESI-MS). NMR spectra were recorded on 300 or 400 MHz spectrometers, at room temperature for solutions in CDCl 3 or D 2 O. Chemical shifts are referred to the solvent signal. Metabolites were purified by Sephadex LH-20 and RP-C18 chromatography.
ELISA analysis: HT-29 cells (~150 000) were placed on six-well dishes, and resveratrol derivatives were added after 24 h. The culture supernatants were collected after 72 h, and secreted VEGF was determined with aH uman VEGF ELISA Kit (Invitrogen/Thermo Fisher Scientific).
RT-qPCR analysis: HT-29 cells were incubated with resveratrol derivative for 48 h. Cells were collected, and total RNA was isolated with an Ambion RNA extraction Kit (Thermo Fisher Scientific). The cDNA was synthesized by MMLV-RT with 1-21 mgo fe xtracted RNA and oligo(dT)15 according to the Maxima cDNA synthesis kit from Thermoscientific. Genes were amplified by use of at hermal cycler and StepOnePlus Ta qMan probes. Ta qMan Gene Expression Master Mix Fast containing the appropriate buffer for the amplification conditions, dNTPs, thermostable DNA polymerase enzyme and apassive reference probe was used. For amplification of each gene primers from Life Te chnologies were used:H s99999903_m1 (bactin), Hs00900055_m1 (VEGFA), Hs00972646_m1 (TERT), and Hs00153408_m1 (MYC).