Yan Liu

Jiaqing Huang

Min Wu

Bi Liu

Qiaofa Lin

Jingjing Wu

Yuhua Ouyang

Xin Guo

Ruyi Huang

Yongmin Zhang
email: yongmin.zhang@upmc.fr

Jianhua Xu

Design, Synthesis of Novel Triptolide-Glucose Conjugates Targeting Glucose Transporter-1 and Their Selective Antitumor Effect

Keywords: triptolide, glucose transporter, triptolide-glucose conjugate, antitumor

Six positional isomers of triptolide-glucose conjugates (TG1α, TG1β, TG2, TG3, TG4 and TG6) were designed and synthesized. These conjugates exhibited better water solubility, and had selective cytotoxicity between the tumor cells with high expression of glucose transport-1 (Glut-1) and non-tumor cells with low expression of Glut-1, in which TG2 formed by triptolide (TPL) and D-glucose C2-OH had the strongest cytotoxicity to tumor cells and lowest toxicity in non-tumor cells, therefore the highest relative therapeutic index, which was 5.7 times that of triptolide and consequent the most powerful selective antitumor activity in vitro. The cytotoxicity of TG2 was highly correlated with Glut-1 function. As a prodrug of triptolide, TG2 could promote RNA Pol II degradation and induce apoptosis as TPL does.

TG2 had a stronger dose-dependent antitumor effect in vivo than TPL and no adverse reaction occurred when its tumor inhibition was higher than 90%, which was associated with its selective distribution in tumor tissues. TG2 could be used as a promising drug candidate for the treatment of solid tumors with high expression of Glut-1, which is worthy of further study.

Introduction

Triptolide (TPL) is a triepoxide diterpene compound isolated from Tripterygium wilfordii which is mainly distributed in southern China, Japan and South Korea [1]. It has multiple pharmacological activities, such as antitumor, anti-inflammatory, anti-rheumatoid, anti-Alzheimer's disease, and immunosuppressive [START_REF] Qiu | Immunosuppressive and anti-inflammatory mechanisms of triptolide, the principal active diterpenoid from the Chinese medicinal herb tripterygium wilfordii Hook[END_REF][START_REF] Tao | The Chinese anti-inflammatory and immunosuppressive herbal remedy Tripterygium wilfordii Hook F[END_REF][4][5]. TPL has shown encouraging antitumor activity against various tumors in vitro and in vivo [START_REF] Gao | Triptolide induces autophagy and apoptosis through ERK activation in human breast cancer MCF-7 cells[END_REF][START_REF] Liu | Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation[END_REF][START_REF] Giri | Pre-clinical evaluation of Minnelide as a therapy for acute myeloid leukemia[END_REF]. In the past decades, the development of TPL and its analogues as immunosuppressive drugs and antitumor drugs has been extensively studied [START_REF] Zhou | 5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide analog mediates immunosuppressive effects in vitro and in vivo[END_REF][START_REF] Xu | Triptolide-targeted delivery methods[END_REF][START_REF] Zhou | Conjugating glucosamine to triptolide to enhance its protective effect against renal ischemia-reperfusion injury and reduce its toxicity[END_REF][12][13].

There is a crucial need to address the safety problems caused by its narrow therapeutic window, poor water solubility, low selectivity and severe multiple organ toxicity (especially gastrointestinal, urogenital and bone marrow toxicity) [START_REF] Du | Novel pH-sensitive nanoformulated docetaxel as a potential therapeutic strategy for the treatment of cholangiocarcinoma[END_REF][START_REF] Zhou | Triptolide-induced oxidative stress involved with Nrf2 contribute to cardiomyocyte apoptosis through mitochondrial dependent pathways[END_REF][START_REF] Xi | Triptolide induces p53-dependent cardiotoxicity through mitochondrial membrane permeabilization in cardiomyocytes-ScienceDirect[END_REF][START_REF] Xi | Triptolide dysregulates glucose uptake via inhibition of IKKβ-NF-κB pathway by p53 activation in cardiomyocytes[END_REF][18]. Therefore, the major strategy to improve the application of triptolide was to selectively deliver it to tumor cells.

There are evidences that the reprogramming of energy metabolism performs a pivotal role in human tumor [19]. Even in sufficient oxygen, tumor cell still tends to produce ATP via glycolysis and convert glucose into intermediate metabolites needed for cell proliferation.

Therefore, cancer cells need more molecules of glucose in comparison with normal cells [START_REF] Pliszka | Glucose Transporters as a Target for Anticancer Therapy[END_REF]. It is well acknowledged that tumor cells are highly dependent on glucose for metabolism, and excessive expression of glucose transporters (Gluts) [START_REF] Calvo | Potential role of sugar transporters in cancer and their relationship with anticancer therapy[END_REF][START_REF] Kunkel | Overexpression of Glut-1 is associated with resistance to radiotherapy and adverse prognosis in squamous cell carcinoma of the oral cavity[END_REF][23]. Among 14 Gluts reported so far, Glut-1 is likely one of the most extensively studied, whose overexpression has been identified in various tumors and correlates well

Results and discussion

Chemistry

Here, we attached triptolide C14-OH to different D-glucose substitution positions via a four-carbon ester bond of succinic anhydride to design and synthesize triptolide-glucose conjugates (TGs) (Fig. 1). The D-glucose exposing a free C1-OH residue was prepared according to the reported method [START_REF] Zhang | Regioselective synthesis of 1I, 1II, 5I, 5II, 6I, 6I, 6II, 6II-2H8-cellobiose[END_REF]. Succinic anhydride was reacted with 1 in the mixture of DCM-DIPEA (10:1) at RT to obtain 2 with carboxyl linker. The esterification of triptolide with 2 was carried out in DCM, in which DMAP was the catalyst and EDCI was the dehydrating agent.

All the benzyl groups in 3 were removed by H2/Pd/C to afford TG1 with a yield of 89% (Scheme 1).

The synthesis of TG2-TG6 was initiated from D-glucose, which was treated with sodium acetate and acetic anhydride in reflux to obtain 15 with all hydroxyl groups protected by acetyl groups. The yield of synthetic intermediate 16 from 15 in one step using BnOH and BF3•OEt2 was only 20%. Therefore, an alternative was performed, 15 was suffered 33% HBr to selectively transform anomeric acetyl group to bromine. The bromide was treated with BnOH in the presence of Ag2CO3 and I2 to give the 16 in 82.8% yield at RT in the dark for 20 h.

The C4 and C6 hydroxyl groups were selectively protected with benzylidene groups to attain 18. Then 18 was treated with dibutyltin oxide, using TBAB and K2CO3 to selectively protect C2-OH or C3-OH with a benzyl group (Scheme S1). Since 4 (two compounds) eluted concurrently during the chromatographic purification process, the two compounds could not be separated and purified. What's more, only one compound could be observed by 1 H NMR, so we used this mixture in the next steps. 5 was directly conjugated with triptolide using EDCI/DMAP in DCM to give 6 with a yield of 62%. 6-2 and 6-3 can be separated and purified by preparative chromatography plate. The HMBC spectrum of 6-2 or 6-3 was shown in Fig. S1 andS2. Hydrogenolysis of 6-2 or 6-3 using Pd/C in MeOH:THF (1:1) generated TG2 or TG3 with yields of 93% and 95%, respectively (Scheme 2). 19 was reduced by NaBH3CN and CH3SiCl in dry DCM with 4Å MS to selectively achieve the C4 free hydroxyl group of 7. Finally, TG4 was prepared following a process similar to that described for the TG1 (Scheme 3). The free hydroxyl group of C6 was obtained by reaction of compound 11 with 0.25 N MeONa at RT for 5 h to give 12. Finally, TG6 was prepared according to a procedure similar to TG1 (Scheme 4). For the synthesis process of all compounds and the characterization of NMR ( 1 H, 13 C) spectra and HRMS of compounds, please refer to the supplementary information. The tests of oil-water partition coefficient (log P) found that the synthesized triptolide glucose conjugates (TGs) were more hydrophilic than their parent compound (Table 1).

Table 1

The oil-water partition coefficient values (log P) of triptolide and TGs.

Selective antitumor activity of TGs

We tested the selective antitumor activity of TGs using MTT assay on between tumor cell lines (SW620, HCT116, HT29, MDA-MB-231, NCI-H460, NCI-H1975) with high expression of Glut-1 (Fig. S3) and non-tumor cell lines (HUVEC, LO2, HaCaT) with low expression of that (Fig. S3). With IC50 value for measuring cytotoxicity, the order of cytotoxicity potency of TGs on the tumor cell lines was as follows: TG2>TG3>TG1β>TG6>TG1α>TG4 (Table 2). Among them, TG2 with the lowest average IC50 on tumor cell lines had the strongest antitumor activity. Meanwhile the order of cytotoxicity potency of TGs on normal cell lines was as follows:

TG6>TG4>TG3>TG1β>TG1α>TG2, suggesting TG2 has the lowest toxicity to non-tumor cells (Table 2). Taking advantage of the relative therapeutic index (RTI, IC50 non-tumor mean/IC50 tumor mean) to measure selective cytotoxicity to tumor cells, the order of TGs selective cytotoxicity on the tumor cell lines was as follows: TG2>TG3>TG1β>TG1α>TG6>TG4>TPL (Table 2). The outcomes indicated that TG2 formed by conjugating D-glucose C2-OH with TPL had the highest selectivity to tumor cells, followed by TG3, TG1β and TG1α. The RTI of TG2, TG3, TG1β or TG1α was 5.7, 2.5, 2.3 and 1.9 times that of TPL, respectively. The results in Table 2 indicated that the RTI of TG2 was 2.5 times that of TG1β namely Glutriptolide 2 reported by He [28]. Furthermore the potency of TG2 on tumor cell lines (Mean IC50 = 1.79 μM) is about 1.64 times that of TG1β (Mean IC50 = 2.94 μM). Our results demonstrated that TG2 not only had the strongest anti-tumor activity but also the highest selectivity to tumor cells, suggesting the D-glucose C2-OH is an ideal site for conjugating with TPL, and the resulting TG2 may be most easily transported into tumor cells through glucose transporters. IC50 values of triptolide and TGs against tumor or non-tumor cell lines and their relative therapeutic index.

RTI: Relative Therapeutic Index = IC50 non tumor mean/IC50 tumor mean

TGs cytotoxicity is associated with Glut-1 function or expression level

Given that TGs were glucose conjugates of triptolide, their selective cytotoxicity on tumor cells may be due to the high expression of Glut-1 in tumor cells. In order to observe the effect of Glut-1 function or expression level on TGs anti-tumor activity in the same genetic background, we treated NCI-H460 cells with TGs combing with Glut-1 inhibitors or Glut-1 siRNA. The Glut-1 expression of Glut-1 silenced NCI-H460 cells was much lower than that of vector-transfected NCI-H460 cells (Fig. S4).

It was shown that in the presence of Glut-1 inhibitors the dose-effect curve of TGs on cell viability moved right accordingly the IC50 values of TGs to NCI-H460 cells were increased in varying degrees (Fig. 2). Similar results were found in Glut-1 silenced NCI-H460 cells, namely the rising IC50 of TGs was found in Glut-1 knockdown NCI-H460 cells (Fig. 3). These data demonstrated that the cytotoxicity of TGs was positively associated with Glut-1 function or expression level, but that of TPL was not. Among all TGs, the increase of IC50 for TG2 was the most significant whether in the presence of Glut-1 inhibitors or Glut-1 knockdowns (Fig. 2H,3H), suggested the cytotoxicity of TG2 was most affected by Glut-1, which was the antitumor activity of TG2 may be more relying on Glut-1. Since TG2 had the strongest selective antitumor activity, therefore we further studied the antineoplastic properties of TG2.

Cell line

Compound (Mean ± SD, μM)

Selective effect of TG2 is highly dependent on Glut-1 of tumor cells

In order to compare the cytotoxic effect of TG2 on tumor cells and non-tumor cells with different Glut-1 expression levels, we utilized both tumor and non-tumor cell lines in different Glut-1 levels produced by knockdown or overexpression to assess the cytotoxicity of TG2 or TPL. It was found that the dose-effect curve of TG2 moved right with Glut-1 knockdown (Fig. 4B) and moved left with Glut-1 overexpression (Fig. 4C,D), which suggested that the cytotoxicity of TG2 to cells increased with the level of Glut-1. However, the degree of this change for tumor cells (NCI-H1975) was greater than that for non-tumor cells (HUVEC) (Fig. 4E), which implied that TG2 may be safety in normal cells even though their Glut-1 expression may be a bit high in a few normal cells in some cases, however its cytotoxicity may not be enhanced proportionately. Glut-1 and GAPDH (loading control). Cell viability was measured by MTT assay. The data represents the mean ± SD of at least three replicate samples. *: p<0.05, compared with the overexpressed vector (OE V) or the knockdown vector (KD V).

TG2 induces RNA pol II degradation and apoptosis in tumor cells

Triptolide-glucose conjugates were able to be hydrolyzed via esterase in cells to release triptolide and intermediates (triptolide succinate) [28]. The main target of TPL for its antitumor activity was RNA Pol II which was degragated by TPL [START_REF] Noel | Triptolide targets super-enhancer networks in pancreatic cancer cells and cancer-associated fibroblasts[END_REF][START_REF] Pan | RNA polymerase -An important molecular target of triptolide in cancer cells[END_REF][START_REF] Tamgue | Triptolide Promotes Senescence of Prostate Cancer Cells Through Histone Methylation and Heterochromatin Formation[END_REF][START_REF] Dienemann | Promoter distortion and opening in the RNA Polymerase II cleft[END_REF][START_REF] Greber | The cryoelectron microscopy structure of human transcription factor IIH[END_REF]. As an inhibitor of RNA polymerase I and II-dependent transcription, TPL mainly resulted in the down-regulation of short-lived mRNA, which associated with its inhibition of proliferation and induction of apoptosis in cancer cells [START_REF] Vispe | Triptolide is an inhibitor of RNA polymerase I and II-dependent transcription leading predominantly to down-regulation of short-lived mRNA[END_REF]. As a prodrug of TPL, TG2 may induce RNA pol II degradation as TPL does. We found that both TG2 and TPL promoted RNA pol II degradation after NCI-H640 cells exposed the drugs for 48 h, however TG2 had a weaker influence on RNA pol II than TPL (Fig. 5).

TG2 has high antitumor effect in vivo

Lung cancer is the most common malignant tumor in human beings. Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers [START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, Ca[END_REF]. The IC50 of TG2 on NSCLC cell line NCI-H1975 was 2.038 μM (Table 2), and NCI-H1975 cells carried EGFR T790M mutation were resistant to the first generation of EGFR-TKI (epidermal growth factor-receptor-tyrosine kinase inhibitor) [START_REF] Tartarone | Clinical approaches to treat patients with non-small cell lung cancer and epidermal growth factor receptor tyrosine kinase inhibitor acquired resistance[END_REF][START_REF] Pao | Rational, biologically based treatment of EGFR-mutant non-small-cell lung cancer[END_REF]. Thereby, we established a nude mouse xenograft model of NCI-H1975 cells to evaluate TG2 antitumor effect in vivo. It was found that NCI-H1975 bearing mice treated with Paclitaxel (PTX) (15 mg/kg, ip, q3d×6), TPL (0.2 mg/kg, ip, qd, d1-d5/week×2), TG2 (1 mg/kg, ip, qd, d1-d5/week×2) and TG2 (2 mg/kg, ip, qd, d1-d5/week×2) resulted in tumor weight inhibition rates by 56.92%, 28.65%, 75.44% and 90.42%, respectively (Fig. 7D). The expression of RNA pol II in tumor tissue was detected by Western blotting, as showed in Fig. S6 that TG2, similar to its parent drug TPL, induced RNA pol II degradation in tumor tissue, which was consistent with its effect in vitro. The antitumor effect of TG2 (1 mg/kg and 2 mg/kg) was significantly stronger than that of PTX (15 mg/kg), a first-line chemotherapy drug for NSCLC and of its parent compound TPL (0.2 mg/kg) (Fig. 7B,C,D). During the experiment period, no adverse reactions such as remarkable weight loss (Fig. 7A) and diarrhea were found in each group of tumor-bearing mice. TG2 showed strong antineoplastic activity against NCI-H1975, proposed that it may have potential clinical value in the treatment of NSCLC with EGFR L858R/T790M mutation.

The high efficient dose of TG2 has lower toxicity in vivo than TPL

It was reported that 0.2 mg/kg of TPL was an effective dose which could produce obvious antitumor activity in vivo [28]. In order to compare the toxicity of TG2 and TPL at their therapeutic dose, the heart, liver, lung and kidney of mice in each group as well as testis tissue that was most sensitive to TPL [START_REF] Xu | Acute and subacute toxicity studies on triptolide and triptolide-loaded polymeric micelles following intravenous administration in rodents[END_REF] were taken for section and H&E stain at the end of the experiment. The results showed that the morphological structures of heart, liver, lung and kidney in TG2 (1 mg/kg and 2 mg/kg) and TPL (0.2 mg/kg) groups had no significant difference compared with the control group (Fig. S7). However, for TPL sensitive testis tissue, irregular spermatogenic tubules, reduced cytoplasmic vacuoles and nuclei, decreased spermatogenic cells, and degeneration of intertubule connective tissue were observed in the TPL group (Fig. 8B). Similar changes were also observed in the TG2 (2 mg/kg) group, however, the pathologic morphological change degree of testis tissue in the TG2 (2 mg/kg) group was less severe than that in the TPL (0.2 mg/kg) group (Fig. 8D), while there was no significant change in testis morphological structure in the TG2 (1 mg/kg) group compared with the control group (Fig. 8A,C). According to above, it was suggested that the effect of TG2 (1 and 2 mg/kg), a high efficient dose, on testis tissue, which was most sensitive to TPL, was notably lower than that of triptolide (0.2 mg/kg), a slight efficient dose in xenograft model of NCI-H1975.

TG2 is stable in serum and selectively distributed in tumor tissues in vivo

Since esterase in plasma is abundant, the ester bond of TG2 could be hydrolyzed by certain esterases and release TPL in plasma, which might cause adverse reaction. Hence we explored the stability of TG2 in serum and the distribution of TG2 in major organs and tumor tissue in vivo. When TG2 was incubated with mouse or human serum at different time points, the content of TPL released from TG2 to be detected in vitro. It was found that TG2 incubated with mouse serum for 8 h, it still existed in its original form, and there was no detectable TPL released. When incubation time extended to 72 h, 66% of TG2 was still present in its original form (Fig. 9A). It was suggested that TG2 was stable at least within 8 h in mice serum. The similar stability of TG2 was found in human serum (Fig. S9). After intravenous injection of TG2 (1 mg/kg) in nude mice, the pharmacokinetic parameters AUC0~3h, MRT0~3h and t1/2 were 83.62 μmol/L*h, 0.56 h and 0.32 h, respectively (Fig. 9B). It revealed that TG2 was almost disappeared in plasma 2 h (went through about 6 folds of t1/2) after intravenous injection. Since the time of TG2 stabilization in serum (8 h) in vitro was considerably longer than that of its existence in plasma (2 h) in vivo.

Therefore, TG2 exists only for a short time in mouse plasma and its resistance from esterase hydrolysis keeps for a relative long time, consequently with little possibility to release TPL in mouse plasma. That is, TG2 was stable in the blood of mice before entering tissues. In order to verify whether TG2 was preferentially distributed in tumor tissues, samples of hearts, livers, lungs, kidneys and tumor were collected from NCI-H1975 tumor-bearing mice after intravenous injection of TG2 or TPL, and the contents of TG2, TPL and TG2 metabolites in these samples was detected by LC-MS. The LC-MS analysis shows that TG2 was hydrolyzed to triptolide and intermediates (triptolide succinate) in tissues. We found that after administration in TG2 (2 mg/kg) group for 1 h (went through about 3 folds of t1/2), the contents of TG2 and its metabolites in tumor tissues were 9.3, 18.8, 15.5 and 7.3 times of those in liver, kidney, lung and heart, respectively, suggesting that TG2 would selectively distribute in tumor tissues. However, there was no selective distribution of triptolide between tumor and organs tissues after administration (Fig. 9C). Fig. 9C showed that even now triptolide succinate was the main hydrolytic product of TG2 in tumor tissues, it could subsequently continue to be hydrolyzed by esterase and ultimately released triptolide to produce sustained antitumor effects.

Conclusions

Taken together, six triptolide-glucose conjugates have been designed and synthesized by using succinic anhydride as a linker between D-glucose C1, C2, C3, C4 and C6-OH and triptolide C14-OH. Among them, TG2 formed by triptolide and D-glucose C2-OH has the strongest selective cytotoxicity to tumor cells. The cytotoxicity of TG2 increased with the high expression of Glut-1, and the effect was more obvious in tumor cells than in non-tumor cells, indicating the cytotoxicity of TG2 to normal cells was lower. As a prodrug of triptolide, TG2 has a similar target with triptolide, both of them can promote RNA Pol II degradation and induce apoptosis. TG2 is stable in serum and selectively distributed in tumor tissues, therefore has a powerful dose-dependent antitumor effect and low toxicity on NCI-H1975 xenografts models.

Experimental

General Procedures of Chemistry

All chemicals were acquired from commercial suppliers as reagent grade quality or better, and used without further purification. All reactions proceed under argon atmosphere and anhydrous conditions unless aqueous reactions. Solvents were used as received or dried over molecular

The reaction was monitored by thin-layer chromatography (TLC) on a pre-coated plate of silica gel 60 F254 (Merck) and detected by ultraviolet light and/or sulfuric acid carbonization. Evaporation of the solvent was performed under reduced pressure below 40 ℃ (water bath). Column chromatography was carried out on silica gel 60 (230-400 mesh, Merck).

1 H NMR and 13 C NMR spectra were measured on Bruker AVANCE DRX 400 spectrometers at 25 ℃. All the measurements were carried out using deuterated solvents. The chemical shifts were referenced relative to trace amounts of the solvent peak, 7.26 ppm ( 1 H) and 77.16 ppm ( 13 C) for CDCl3, 3.31 ppm ( 1 H) and 49.00 ppm ( 13 C) for CD3OD at 25 ℃, and are described in parts per million (ppm). Coupling constants (J) are indicated in Hertz (Hz), with multiplicity mentioned following the usual convention: s, singlet;

quadruplet; dd, doublet of doublets; ddd, doublet of doublet of doublets; dt, doublet of triplets; dq, doublet of quartets; m, multiplet; brs, broad singlet. ESI mass spectra were recorded on an AgilentS3 Technologies 1100 series LC-MSD trap. High-resolution mass spectra (HRMS) were conducted with a Bruker micro TOF spectrometer in electrospray ionization (ESI) mode, using Tuning-Mix as reference.

Synthesis of Succinic Acid Mono-((3R,4S,5R,6R)-3,4,5-tris(benzyloxy)-6-((benzyloxy) methyl) tetrahydro-2H-pyran-2-yl) Ester

(2). Compound 1 was synthesized as previously reported [START_REF] Zhang | Regioselective synthesis of 1I, 1II, 5I, 5II, 6I, 6I, 6II, 6II-2H8-cellobiose[END_REF]. 1 (0.92 g, 1.704 mmol) dissolved in DCM-DIPEA (10:1, 6.6 mL) was treated with succinic anhydride (0.852 g, 8.52 mmol), stirred at RT for 20 h then eliminated the solvent. The residue was dissolved in EtOAc (30 mL), washed with brine, saturated NaHCO3 (aq), dried over Na2SO4, and concentrated. The residue was purified through silica gel column chromatography (CH2Cl2/CH3OH, 50:1) to give compound 2 as a colorless syrup (0.894 g, 82%). The resulting mixture was dissolved in CH2Cl2 (5 mL), washed with water (5 mL), brine (5 mL) and saturated NaHCO3 (aq) (5 mL), dried over Na2SO4, then evaporated to give a residue. The residue was purified by silica gel column chromatography (Cy-EtOAc, 3:1, twice) to give the compounds 3 as a white solid (66.8 mg, 68%). (td, J = 11.9, 5.5 Hz, 1H), 1.06 (s, 3H), 0.96 (d, J = 6.9 Hz, 3H), 0.84 (d, J = 6.9 Hz, 3H). 13 (PVDF) (BioRad, Hercules, CA, USA), and blocked with 5% skimmed milk. The membrane proteins were incubated using primary antibodies for Glut-1 (Abcam, 1:10000), RNA Pol II (Santa Cruz, 1:1000), GAPDH (Abcam, 1:1000), followed through incubation with anti-rabbit IgG-HRP (Abcam, 1:5000), anti-mouse IgG-HRP (Abcam, 1:5000), and enhanced chemiluminescence (GE Healthcare).

Imaging and quantification of protein bands were evaluated with ImageJ software.

Knockdown and overexpression of Glut-1 gene

The lentivirus using for knockdown or overexpression of Glut-1 was purchased from Geenchem (China). The experiment was processed according to the manufacture's protocol. Briefly, cells were infected with the lentivirus containing Glut-1 knockdown or overexpression sequences for 12 h, replaced fresh medium and cultured for 72 h, and then added puromycin to screening successfully transfected cells. The selection process with puromycin was persisted for 14 days with medium replacement every two days. The expression of Glut-1 protein in knockdown or overexpression cells was measured by Western blotting. Experiments were carried out at least three times.

Mitochondrial membrane potential measurement

Mitochondrial membrane potential was detected via TMRE-Mitochondrial Membrane Potential Assay Kit (Abcam). The experiment was processed in accordance with the manufacturer's protocol. Briefly, untreated and treated cells were suspended in 1% BSA DPBS (200 µL), 0.02 µL TMRE (1 mM) were added to each sample and incubated in the dark for 20 min. After discarding the supernatant, the sample was washed with 1% BSA DPBS, then resuspend the sample in 1% BSA DPBS, store at 4 ℃. Subsequently, Samples were attained on a BD FACS Canto II flow cytometer and each sample was acquired 10, 000 events. Cell populations were analyzed using the software FlowJo v10.5.3. Experiments were performed by three independent samples.

ROS level measurement

Cell ROS level was detected by ROS detection kit (Beyotime). The experiment was processed according to the manufacture's protocol.

Briefly, untreated and treated cells were suspended in 1% BSA DPBS (200 µL), 0.2 µL DCFH-DA (10 mM) were added to each sample and incubated in the dark for 30 min. After discarding the supernatant, the sample was wash with 1% BSA DPBS, then resuspend the sample in 1% BSA DPBS, store at 4 ℃. Subsequently, samples were attained on a BD FACS Canto II flow cytometer and each sample was acquired 10, 000 events. Cell populations were analyzed using the software FlowJo v10. were randomized into 3 groups (3 mice for each group). Prior to the experiments, the mice fasted for 12 h with free access to water. The animals were intravenous injection with TG2 (1 mg/kg, 2 mg/kg) and TPL (0.2 mg/kg). After administration for 1 h, the mice were sacrificed and the major organs (heart, liver, lung and kidney) and tumors were collected. Tissue samples were immediately weighted prior to flash freezing with dry ice before storage at -80 ℃ for future analysis. After thawing the tissues, the 1.0 g tissue was added with 2.0 mL normal saline to make homogenate. The homogenate was added methanol to precipitate protein, condensed the supernatant, then reconstituted with methanol. The contents of compounds in different organs or tumor from each group was measured by LC-MS. Three molecular weights were detected at the same time during the determination, 645.3 g/mol (TG2+Na), 483.2 g/mol (intermediate+Na), and 383.2 g/mol (TPL+Na). The LC-MS condition was used for 7.5 min running time at a flow rate of 0.3 mL/min. The eluent was monitored at 220 nm.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 8. All data were analyzed by two-way ANOVA for multiple comparisons. All experiments were performed in triplicate. The values are expressed as the mean ± SD. P<0.05 was considered to be statistically significant differences.

Fig. 1 .Scheme 1 .Scheme 2 .Scheme 3 .Scheme 4 .

 11234 Fig. 1. Structures of C1-C6-substituted positional isomers (1-6) of triptolide-glucose conjugations and parent compound TPL.

Fig. 2 .

 2 Fig. 2. The cytotoxicity of TGs on NCI-H460 cells in the presence of Glut-1 inhibitor. (A-G) TGs cytotoxicity was declined in combination with TGs with Glut-1 inhibitors 10 μM WZB117 or 100 μM Phloretin. (H) The increasing folds of IC50 for TGs in the presence of Glut-1 inhibitors 10 μM WZB117 or 100 μM Phloretin. Cell viability was measured by MTT assay. Data represent the mean ± SD of at least three or more replicates.

Fig. 3 .

 3 Fig. 3. The cytotoxicity of TGs on Glut-1 silenced NCI-H460 cells. (A-G) Glut-1 knockdown declined TGs cytotoxicity in NCI-H460 cells, Glut-1 knockdown with KD1 or KD2, using the vector (KD V) for control. (H) The increasing folds of IC50 for TGs when Glut-1 knockdown. Cell viability was measured by MTT assay. Data represent the mean ± SD of at least three or more replicates.

Fig. 4 .

 4 Fig. 4. The cytotoxicity of TG2 was dependent on Glut-1 expression level. (A) Expression levels of Glut-1 in silenced or overexpressed in NCI-H1975 cells determined by Western blotting. (B) Glut-1 knockdown declined TG2 cytotoxicity but not TPL in NCI-H1975 cells. Cell viability was measured by MTT assay. Data represent the mean ± SD of at least three or more replicates. (C) Glut-1 overexpression enhanced TG2 cytotoxicity but not TPL in NCI-H1975 cells. Cell viability was measured by MTT assay. Data represent the mean ± SD of at least three or more replicates. (D) Glut-1 high overexpression raised TG2 cytotoxicity lightly in HUVEC cells. Expression levels of Glut-1 overexpressed in HUVEC cells determined by Western blotting. Cell viability was measured by MTT assay. Data represent the mean ± SD of at least three or more replicates. (E) Glut-1 level was negatively correlated with the IC50 value of TG2 in NCI-H1975 and HUVEC cells with different levels of Glut-1 expression. Whole cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting against either

  TPL induces tumor cell apoptosis by down-regulating a large number of proteins which play a key role in anti-apoptosis (Bcl-2, Mcl-1, Bcl-xL, XIAP, C-IAP1, C-IAP2)[START_REF] Chance | Hydroperoxide metabolism in mammalian organs[END_REF][START_REF] Gores | Swelling, reductive stress, and cell death during chemical hypoxia in hepatocytes[END_REF][START_REF] Dawson | Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes[END_REF]. The dissipation of mitochondrial membrane potential (MMP) occurred in the initial stage of cell apoptosis. Moreover, excessive ROS production affects mitochondrial permeability and cytochrome C release and results in apoptosis[START_REF] Kim | Mitochondrial permeability transition in the switch from necrotic to apoptotic cell death in ischemic rat hepatocytes[END_REF].We measured MMP using the TMRE probe. Both TG2 and TPL could reduce the relative fluorescence intensity of TMRE and attenuate the accumulation of TMRE (Fig.S5), suggesting MMP dissipation. An increased intensity of DCF fluorescence occurred in NCI-H460 cells following TG2 or TPL treatment, suggesting that ROS accumulation was enhanced (Fig.6A, B), which could result in an apoptotic induction notably in a dose-dependent manner (Fig.6C, D). The outcomes exhibited that TG2 same as TPL could induce the dissipation of MMP, promote the generation of ROS, subsequently result in apoptosis.

Fig. 5 .

 5 Fig. 5. TPL or TG2 promoted RNA pol II degradation. The protein level of RNA Pol II of NCI-H460 cells treated with TG2 or triptolide for 48 h analyzing with Western blotting. Whole cell lysates were resolved by SDS-PAGE and analyzed by Western blotting against either RNA Pol II and GAPDH (loading control).

Fig. 6 .

 6 Fig. 6. TG2 induced ROS accumulation and cell apoptosis. (A) Effect of TG2 on ROS in NCI-H460 cells exposing for 42 h or 48 h. The ROS was measured by DCFH-DA. MFI = Mean Fluorescence Intensity. DCF fluorescence was calculated compared with the control and the results were expressed as the percentage of fluorescence which obtained from the histogram statistics. (B) Effect of TPL on ROS in NCI-H460 cells exposing for 42 h or 48 h. DCF fluorescence was calculated compared with the control and the results were expressed as the percentage of fluorescence which obtained from the histogram statistics. (C) Effect of TG2 on apoptosis of NCI-H460 cells exposing for 48 h. (D) Effect of triptolide on apoptosis of NCI-H460 cells exposing for 48 h. (E) The histogram of apoptosis rate of NCI-H460 cells treated with TG2 or TPL at 48 h. Data represent the mean ± SD of at least three or more replicates. *: p<0.05, **: p<0.01, compared with the control.

Fig. 7 .

 7 Fig. 7. Antitumor effects of TG2 on NCI-H1975 xenograft tumor models in vivo. (A) Body weight curves of the NCI-H1975 tumor-bearing mice in the different groups throughout the 2 week-treatment. (B) Tumor growth curves of the NCI-H1975 tumor-bearing mice in the different groups throughout the treatment. (C) Photographs of the tumors removed from the different groups of tumor-bearing mice after treatment. (D) The effects of TG2 on tumor weight in the different groups of the tumor-bearing mice after the 2 week-treatment. *: p<0.05, **: p<0.01, compared with the control.

Fig. 8 .

 8 Fig. 8. Effect of TG2 and TPL on the morphological structure of testis (H&E stain, ×200). (A) Normal control (saline); (B) TPL (0.2 mg/kg); (C) TG2 (1 mg/kg); (D) TG2 (2 mg/kg).

Fig. 9 .

 9 Fig. 9. TG2 is stable in serum and selectively distributed in tumor tissues in vivo. (A) The stability of TG2 incubated in mice serum at different times. (B) The concentration-time curve of TG2 in plasma following intravenous injection of TG2 (1 mg/kg). The concentration of TG2 (MW: 645.3) in plasma samples was detected by LC-MS. Data represent the mean ± SD, n=3. (C) The tissue distribution of TG2 or TPL in vivo. After intravenous injection TG2 or triptolide (0.2 mg/kg) for 1 h, heart, liver, lung, kidney, and tumor tissues from NCI-H1975 tumor-bearing mice were collected. The concentration of TG2 (MW: 645.3) and TG2 metabolites: triptolide succinate (MW: 483.3) and triptolide (MW: 383.2) in tissue samples of tumor or organs were detected by LC-MS. Data represent the mean ± SD, n=3.

  1 H NMR (400 MHz, Chloroform-d) δ 7.47 -7.21 (m, 18H), 7.15 (dd, J = 6.9, 2.8 Hz, 2H), 5.63 (d, J = 8.0 Hz, 1H), 5.09 (s, 1H), 4.90 (d, J = 10.9 Hz, 1H), 4.86 -4.77 (m, 3H), 4.70 -4.63 (m, 2H), 4.62 (s, 1H), 4.55 (d, J = 10.6 Hz, 1H), 4.52 -4.46 (m, 1H), 3.80 -3.69 (m, 4H), 3.65 -3.54 (m, 2H), 3.50 (d, J = 3.1 Hz, 1H), 3.44 (d, J = 5.4 Hz, 1H), 2.86 -2.56 (m, 5H), 2.32 (dd, J = 18.3, 4.7 Hz, 1H), 2.20 -2.07 (m, 2H), 1.97 -1.82 (m, 2H), 1.64 (s, 1H), 1.55 (dd, J = 12.4, 5.0 Hz, 1H), 1.20

5 . 3 .

 53 Experiments were executed by three independent samples. 4.11. Antitumor effect in vivo and histopathological study Xenografts of NCI-H1975 cells were established by subcutaneous injection into the BALB/c nude mice. Briefly, cells at the logarithmic growth phase (2×10 7 /0.2 mL/mouse) were inoculated into axillary subcutaneous of BALB/c nude mice (male, 5-6 weeks, 18-22 g, purchased from GemPharmatech. Co. Ltd). When the tumor volume reached to 80 mm 3 , the mice were randomized into five groups by tumor size and received various treatments: (a) Normal saline (intraperitoneal injection (ip), qd, d1-d5/week×2); (b) paclitaxel (15 mg/kg, ip, q3d×6); (c) TPL (0.2 mg/kg, ip, qd, d1-d5/week×2); (d) TG2 (1 mg/kg, ip, qd, d1-d5/week×2); (e) TG2 (2 mg/kg, ip, qd, d1-d5/week×2).The body weights of the animals, tumor's long diameter (L) and short diameter (W) were recorded every 3 days, and tumor volume (V) was calculated in accordance to the following formula: V=L×W 2 /2. At the end of the observation, the mice were euthanized and tumors were excised and weighed. The curves of tumor growth was drawn according to tumor volume and days after treatment. The samples of heart, lung, liver, kidney and testis tissue were collected from mice of each group. Samples were fixed with formaldehyde, dehydrated with graded alcohol and xylene, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (H&E). Pathological injury evaluations of heart, lung, liver, kidney and testis were performed under a light microscope (Zeiss, Germany). The tumor weight inhibition (TI) was calculated according to the following formula:

.

4. 12 .

 12 Pharmacokinetic study BALB/c nude mice (male, 6-7 weeks, 22-26 g, purchased from GemPharmatech. Co. Ltd) were administrated by intravenous injection of TG2 (1 mg/kg). After intravenous injection 10, 20, 30, 60, 120 and 180 min, blood samples were collected via orbital venous plexus from 3 mice in each time point, and then centrifuged to gain plasma. The plasma was added methanol to precipitate protein, condensed the supernatant, then reconstituted with methanol. The concentration was determined through HPLC. The eluent was monitored at 220 nm. DAS 3.0 software (Shanghai, China) was used for calculating pharmacokinetic parameters. 4.13. Stability of TG2 in serum 15 μM TG2 was incubated with mice or Human serum at 37 °C for different time (0, 0.5, 1, 8, 12, 18, 60 and 72 h), then stored the sample at -80 °C. The 200 µL methanol was added into the serum samples to precipitate protein, then vortexed and ultrasound for 5 min. The chromatograms were taken by HPLC and detected at 220 nm. All the experiments were in duplicate. 4.14. Distribution of TG2 in major organs and tumor tissue NCI-H1975 tumor-bearing mice were established according to 4.11, when the tumor volume reached approximately 500 mm 3 , the mice

  C NMR (400 MHz, Chloroform-d) δ 173.31, 171.50, 170.69, 160.05, 138.49, 138.35, 138.15, 138.07, 128.54, 128.53, 128.49, 128.06, 127.99, 127.96, 127.95, 127.90, 127.83, 127.80, with BCA protein assay kit (Beyotime). Protein samples were separated by SDS-PAGE and transported onto polyvinylidene difluoride

This work was funded by the National Natural Science Foundation, China (81973364), the Projects of Qihang Fund of Fujian Medical University (2017XQ2017) and Financial supports from the Centre National de la Recherche Scientifique (CNRS) and the Sorbonne Université in France.

ABBREVIATIONS

MMP, mitochondrial membrane potential TMRE, tetramethylrhodamine ethyl ester ROS, reactive oxygen species MTI, mean fluorescence intensity EGFR-TKI, epidermal growth factor-receptor-tyrosine kinase inhibitor NSCLC, non-small cell lung cancer

Funding Sources

Supporting Information

The Supporting Information can be found online at (I). Figures S1-S9, (II). Characterization of Compounds, including NMR Spectra, HRMS Spectra and HPLC Spectra of compounds.

Author Contributions

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.