Supplementary Materials

Materials and methods

1. General tests and measurements

1H and 13C spectra of the emerging Pt(IV) prodrug were recorded in d6-DMSO using a Bruker

AV-400/600 spectrometer. The chemical shifts were relevant to the signals from solvent (1H

NMR: DMSO at δ = 2.50 ppm; 13C NMR: DMSO at δ = 40.45 ppm). ESI-MS was used with an

electrospray interface. ICP-MS was used to determine platinum contents (Optima 5300DV,

PerkinElmer, USA). FACS analysis was applied on a BD Calibur flow cytometer. Confocal

images were mainly captured using Olympus FV3000 confocal microscope.

2. Synthesis of Cx-platin-Cl

To suspend of cis,cis,trans-Pt(NH3)2Cl3OH (101 mg, 0.292 mmol) in DMF (15 mL), CX-4945

(122 mg, 0.351 mmol) and TBTU (113 mg, 0.351 mmol) were mixed. The mixture was

persistently stirring for 10 min at R.T., then TEA (35.6 mg, 0.351 mmol) was mixed. The solution

was kept stirring and heated to 50 under nitrogen atmosphere. Following 36 h reaction, the℃

mixture was condensed and the solvent was taken away in reduced pressure. The remains were

purified on silica gel using column chromatography. The mixed dichloromethane and methanol are

8:1 to give the yellow product (41.5 mg, yield 21.2%). 1H NMR (300 MHz, DMSD-d6): δ ppm

6.38(s, 6H)，7.16 (d, J = 8.1, 1H), 7.49 (t, J = 8.2, 1H), 7.99 (d, J = 8.4, 1H), 8.08 (d, J =8.4, 1H),

8.3 (m, 2H), 8.61 (d, J=5.6, 1H), 8.85 (d, J=8.5, 1H), 9.01 (d, J = 5.6, 1H), 9.7 (s, 1H), 10.2 (s,

1H)；13C NMR (100 MHz, DMSD-d6): δ 116.87, 119.53, 120.52, 121.85, 122.29, 122.60, 124.28,

125.38, 127.58, 128.77, 130.62, 133.28, 135.44, 142.40, 143.48, 147.77, 148.22, 150.41, 173.19；ESI-MS [M-H]-=681.9221

3. Synthesis of Cx-DN604-Cl

To suspend cis,cis,trans-[Pt(NH3)3(3-oxocyclobutane-1,1-dicarboxylate)(Cl)(OH)] (145 mg,

0.292 mmol) in DMF (15 mL), CX-4945 (122 mg, 0.351 mmol) and TBTU (113 mg, 0.351 mmol)

were mixed. The mixture was persistently stirring for 5 min at R.T., then TEA (35.6 mg, 0.351

mmol) was mixed. The solution was kept stirring and heated to 50 under nitrogen atmosphere.℃

Following the reaction of 36 h, the mixture was condensed and the solvent were taken away in

reduced pressure. The remains were purified on silica gel using column chromatography. The

mixed dichloromethane and methanol are 8:1 to give the yellow product (111 mg, yield 50.0%).

1H NMR (300 MHz, DMSD-d6): δ ppm 3.55(s,2H)，3.67(s, 2H)，6.45(s,6H)，7.16 (d, J = 7.6,

1H), 7.46 (t, J = 8.3, 1H), 7.96 (d, J = 8.9, 1H), 8.09 (d, J =8.7, 1H), 8.29 (m, 2H), 8.58 (d, J=5.5,

1H), 8.86 (d, J=8.4, 1H), 9.07 (d, J = 5.6, 1H), 9.7 (s, 1H), 10.2 (s, 1H)；13C NMR (100 MHz,

DMSD-d6): δ 46.18, 46.18, 59.53, 60.52, 116.83, 119.42, 120.44, 121.90, 122.41, 122.56, 124.29,

125.26, 127.50, 128.71, 130.55, 133.28, 142.38, 143.46, 147.80, 148.20, 150.40, 171.68, 176.54,

176.54, 203.71；ESI-MS [M-H]-=768.0402

4. Cell culture

A2780, A2780/cDDP, MCF-7, SGC-7901, HepG2, PANC-1 and non-cancer HUVEC cells

were purchased from Jiangsu KeyGen Biotech Company. The authentication of all cell lines was

confirmed by short tandem repeat (STR) profiling on May 9, 2019. All the cells were cultured and

passed every 1-2 days upon reaching the fourth generation using recommended manufacturer

protocols. For example, A2780/cDDP cancer cell was established from cisplatin-resistant ovarian

carcinoma and cultured in medium of RPMI-1640 supplemented with 10% FBS and additional 1

μM cisplatin. Exponentially growing cultures were sustained at 37 °C with 5% CO2 and 90%

humidity relatively, and grown to 70% confluence before splitting or harvesting.

5. The in vitro cytotoxicity profiles

The cytotoxicity profiles of Cx-platin-Cl and Cx-DN604-Cl in several cancer and non-cancer

cell lines were evaluated with MTT method. The indicated cells were first plated in 96 wells plates

for 12 h, then exposed to the corresponding medium with measured compounds at indicated

concentrations and then incubated for another 72 h at 37 . At the end, the cellular vitality was℃

detected using MTT (Sigma, St. Louis, MO, USA) as previously described [1]. Meanwhile, the

IC50 values were detected using the analysis of nonlinear regression with GraphPad Prism 8.0.

6. The assessment of apoptosis

The apoptotic rates induced by Cx-platin-Cl and Cx-DN604-Cl were detected using Annexin

V-FITC/PI Double-staining Apoptosis Detection kit (KeyGen, Nanjing, China) as described

previously in the manufacturer’s protocol [2].

7. Acridine orange (AO)/ ethidium bromide (EB) dual staining

To confirm indicated necrosis rates in A2780/cDDP cells, AO/EB dual staining assay was

employed to Cx-platin-Cl and Cx-DN604-Cl-treated A2780/cDDP cells as described [2].

8. Detection of activated caspase-3

To quantify activated caspase-3, the cancer cells with a density of 105 grew in 60 mm dishes

and adhered for 12 h, then the cancer cells were pretreated with measured compounds at 15 μM

for 24 h. Then the cells were operated using previous protocol [2]. Relative Fluorescence Unit

(RFU) was used to quantify using Fluorophotometer at an 535 nm emission wavelength after 485

nm excitation.

9. Cloning analyses

The indicated cancer cells were collected and grew at a density of 500/plate. After 2 weeks, 15

μM cisplatin, DN604 and Cx-platin-Cl and Cx-DN604-Cl in medium were added for another 1

week. At the end of 1 week, the cancer cells were eventually fixed in 3% paraformaldehyde and

stained using crystal violet. The cells were counted and the experiment was performed in

triplicate.

10. Luminescence assay

The ATP concentrations were detected using ATP Assay Kit (Beyotime, China). The indicated

cancer cells were seeded and pretreated as mentioned above. Following 6, 12, 24 h treatments, the

mediums were centrifuged at the speed of 500 g for 1 min. The 15 μL supernatant was injected

into the 85 μL luciferin-luciferase reagent for 5 min at R.T.. Then, the luminescence was measured

via using a microplate reader from Bio-Rad Laboratories.

11. ELISA

The intracellular IP3 was detected following the protocols of using inositol 1, 4, 5-

trisphosphate (IP3) Elisa Kit of mouse (Wuhan, China). The indicated cells were collected and

stored at -20℃ for 12 h. After 2 freeze-thaw cycles, the cell lysates were centrifuged at 4 ℃ and

6000 g for 10 min. The supernatant with the volume of 80 μL was incubated for another 2 h at

37℃. While the liquid was discarded, the Biotin-antibody and HRP-avidin with volume of 100 μL

were incubated for 1 h at 37℃. Then, the TMB substrate with 100 μL was prepared and added,

incubated for 30 min at 37 in the darkness. At last, 50 μL stop solution℃ was added and the 450

nm absorbance was detected using a microplate reader from Bio-Rad Laboratories.

12. Cellular uptake

A2780 and A2780/cDDP cancer cells grew in 6-well plates with 105 cells/well and then

incubated with RPMI-1640 supplemented with 10% FBS for 1 days. After that, the indicated cells

were exposed to cisplatin, DN604, Cx-platin-Cl and Cx-DN604-Cl at different concentrations. At

selected time intervals, cells were washed and collected. The protein concentration was measured

with BCA Protein Assay Kit (Thermo Fisher, USA) and the Pt concentration was determined with

inductively coupled plasma-optical emission spectrometer (ICP-MS).

13. Subcellular fractionation

A2780 and A2780/cDDP cells grew in dishes with 106 cells/dish and incubated for 24 h with

RPMI-1640 supplemented with 10% FBS. After 24 h pretreatment with cisplatin, DN604, Cx-

platin-Cl and Cx-DN604-Cl at 15 μM, cells were collected and subcellular fractions were isolated

according to the manufacturer protocol. At last, the total protein concentration was also measured

using a BCA Kit and Pt concentration in each subcellular fraction was quantified with ICP-MS.

14. Pt-DNA adduct

A2780 and A2780/cDDP cells grew in dishes with 106 cells/dish and incubated for 24 h with

the medium of RPMI-1640 supplemented with 10% FBS. Following 24 h pretreatment with

cisplatin, DN604, Cx-platin-Cl and Cx-DN604-Cl at 15 μM, the indicated cells were incubated

with drug-free medium for another 12 h, then collected and DNA was isolated. The DNA

concentration was detected with a NanoDrop 2000 (Thermo Fisher Scientific, USA) and Pt

concentration was determined with an ICP-MS.

15. Intracellular GSH and GSSG assay

A2780 and A2780/cDDP cells were seeded in dishes with 106 cells/dish and incubated for 24

h with 5 mL of RPMI-1640 medium supplemented with 10% FBS. Following 24 h pretreatment

with cisplatin, DN604, Cx-platin-Cl and Cx-DN604-Cl at indicated concentrations, cells were

collected, homogenized and centrifuged. The supernatant was used according to the manufacturer

protocol (Glutathione Fluorometric Assay Kit, BioVision, USA) with a Microplate Reader.

16. Comet Assay

A2780 and A2780/cDDP cells pretreated with cisplatin, DN604, Cx-platin-Cl and Cx-

DN604-Cl at 15 μM for 24 h were mixed with molten LM Agarose as mentioned [2].

17. Immunofluorescence and foci detection

In brief, A2780 and A2780/cDDP cells grew in 35 mm sterile dishes at 37 for 24 h.℃

Following the pretreatment of cisplatin, DN604, Cx-platin-Cl and Cx-DN604-Cl at 15 μM for 24

h, the indicated cancer cells were washed, fixed in 4% paraformaladehyde for 30 min and

permeabilized with PT-5 solution at 4 for 30 min. The cells were blocked with PTB-5 at R.T.℃

for 1 h. The cells were mixed with the polyclonal rabbit antibody of γH2AX (1:500) at 4 for 12℃

h, and then stained with the anti-rabbit secondary antibody which conjugated with Alexa 488

(1:100) and 1 mM DAPI (Invitrogen, USA). The confocal images were captured and recruited in

Adobe Photoshop 8.0.

18. Analysis of SSB repair

SSBs were detected via pretreat cancer cells with 15 μM cisplatin, DN604, Cx-platin-Cl and

Cx-DN604-Cl, respectively, for 0, 15, 30 and 60 min. Then the cells were washed with PBSCMF.

The numbers of SSB were counted as previously described [3].

19. Plasmids

The pEGFP-C1-JWA expression and EGFP-C1-antisense plasmids were described in previous

protocol [4,5]. The expression cassette of JWA siRNA and scrambled shRNA were sub-cloned

into the linearized vector pSEC to generate control and JWA shRNA plasmids, respectively [6],

which was confirmed by DNA sequencing.

20. Luciferase reporter gene assays

The indicated cancer cells incubated in 24-well plates were co-transfected with pGL3-XRCC1

and the JWA or control shRNA plasmid. All the used plasmids were co-transfected with pRL-

SV40. After the treatment with 15 μM measured compounds for 24 min, the lysates were collected

according to the mentioned instruction [7].

21. HCR assay

The Host-cell-reactivation (HCR) assay was carried out to measure DNA repair capacity

(DRC) [8,9]. The LUCconDNA was damaged in vitro by exposure to 15 μM cisplatin, DN604, Cx-

platin-Cl and Cx-DN604-Cl for 1 h at R.T.. After the treatments, the damaged and undamaged

DNA was purified and suspended in TE buffer at 500 mg/ml with pH 7.8. At last, NIH-3T3 cells

were used to be co-transfected with all the LUCconplasmids. The DRC (%) was detected as the

ratio of the damaged plasmid to the undamaged plasmid with the luciferase activity.

22. Molecular Docking

The docking assay was performed with Sybyl-X 2.0. The coordinates for XRCC1 were

extracted from its crystal structure (PDB code 3UKY). The docking studies were performed on the

synthetic Pt(IV) prodrugs. The stereochemical structures of Cx-platin-Cl and Cx-DN604-Cl were

built up. Then the Powell’s method was used to optimize the geometry. After extracted the ligand

naturally, the docking was processed. Automatically, Cx-platin-Cl and Cx-DN604-Cl were docked

into the XRCC1 ATP-binding pocket. The protein was prepared and the automated docking

manner was used presently.

23. Western blot analysis

The Western blot assays were performed as described previously [7]. In brief, the indicated

cell were lysed in 1× lysis buffer (Beyotime Biotechnology, Nanjing, China) for 40 min and

centrifuged for 30 min at 4 ℃, 13,000 g. The concentrations of protein were quantified by Pierce

BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and equal amounts of 30-60 µg protein

was loaded for blots. The primary antibodies used were polyclonal goat anti-JWA (1:1000)

(Imgenex, San Diego, CA, USA); monoclonal mouse anti-APE1 (1:1000) (Abcam, UK) and

monoclonal mouse anti-XRCC1 (1:1000) (Abcam, UK); monoclonal mouse anti-Lig III (1:1000)

(BD Transduction Laboratories, San Diego, CA, USA). Polyclonal rabbit anti-Pro-caspase 3

(1:1000) and polyclonal rabbit anti-caspase 3 (1:1000), monoclonal rabbit anti-PARP (1:1000) and

monoclonal rabbit anti-Cleaved PARP (1:1000) were obtained from Cell Signaling Technology

(Danvers, MA, USA). β-actin (1:5000, Bio-World, USA) was applied for the loading control and

anti-goat, anti-mouse or anti-rabbit IgG HRP conjugated secondary antibodies (1:5000) were

purchased from Bio-World. The corresponding blots were captured by a ChemiDocTM XRS+

system and evaluated using NIH ImageJ software.

24. Animal experiments

The C57BL/6 and nude mice of 8 weeks old male with 18 to 22 g were purchased from

Qinglong Mountain Animal Facility (Nanjing, China). The JWA -/- mice were acquired from the

Jackson Laboratory and the mice were fed based on standard conditions and animal researches

exactly agreed with the protocols of the Ethics Committee of China Pharmaceutical University.

All the experiments are approved by the Instructional Animal Care and Use Committee (IACUC).

Peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained from Red Cross

Blood Centre in Nanjing.

25. Tumor formation following the drug treatments

Tumor xenograft assays were carried out in 8 weeks old mice challenged with 6 × 10 5 ID8

tumor cells. Animal were divided into five groups randomly and administrated with control,

cisplatin, DN604, Cx-platin-Cl and Cx-DN604-Cl at 5 mg/kg/day. Tumor volume was evaluated

every 2 days using the formula below:

V = π × length × width2/6

The ID8 tumors were gathered, weighed and collected in the further study.

26. FACS analysis

The xenografted ID8 tumors were dissected into pieces and digested at 37 for 40 min in 1×℃

HBSS buffer supplemented with 2% FBS, 1 mg/mL dispase and 0.5 mg/mL collagenase I with

further digestion and ammonium chloride at 37 for 5 min; The Spleens derived from ID8℃

tumor-bearing mice were cut and dissociated. The corresponding cells were filtered using a cell

strainer with 70 μm and suspended in 1×HBSS buffer and finally diluted in Ficoll-Pague

(Amershan, USA). The lymphocytes were extracted, stained and analysed with FACS analysis

according to the protocol described previously.

27. Histological and IHC analyses

The key tissues and tumor were fixed and embedded in paraffin, then sectioned for H&E

staining. For IHC assays, the sections with paraffin-embedded were prepared as described

previously [2].

28. In vivo antitumor efficacy

The A2780/cDDP single-cell suspended in PBS was injected into theright oxter of 35 naked

mice subcutaneously which had been divided into five groups randomly. The mice were

administrated via measured compounds i.v. at 5 mg/kg uniformly once a week. The growth of

tumor was monitored and calculated based on the formula below:

Tumor volume (mm3) = 0.5× length × width2

The weight of tumor was assessed as the antitumor effect of measured compounds. Systemic

toxicity was evaluated as mice weight and physical state.

29. Statistical analysis

The results and data were represented as means ± S.D. in at least independent experiments

(n=3). Statistical analyses were executed using Student's t-test. All comparisons compared to

negative controls. *P<0.05 and **P<0.01 indicate significant difference.

Fig. S1. Preparation of Cx-platin-Cl.

Fig. S2. Preparation of Cx-DN604-Cl.

A

B

C

5x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

-ESI Scan (0.670 min) Frag=175.0V wm-052.d

255.21637

315.85923

368.85800

205.14608

681.92212

406.83071138.01083

Counts vs. Mass-to-Charge (m/z)100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500

Fig. S3. Characterization of Cx-platin-Cl. A) 1H NMR spectrum of Cx-platin-Cl. B) 13C NMR

spectrum of Cx-platin-Cl, and C) ESI-MS spectroscopy of Cx-platin-Cl.

A

B

C

5x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4-ESI Scan (0.420 min) Frag=175.0V WM291.d Subtract

768.04021

1099.09192401.95364

Counts vs. Mass-to-Charge (m/z)100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500

Fig. S4. Characterization of Cx-DN604-Cl. A) 1H NMR spectrum of Cx-DN604-Cl. B) 13C

NMR spectrum of Cx-DN604-Cl, and C) ESI-MS spectroscopy of Cx-DN604-Cl.

Fig. S5. The images of full-length western blots were showed. The representative image of each protein is showed and experiments were performed at least three times.

Fig. S6. Emerging Pt(IV) prodrugs mediated an antitumor effect in a A2780/cDDP xenograft

model. A) Tumor growth curve of each mouse from different groups of A2780 and A2780/cDDP

tumor-bearing athymic nude mice during chemotherapy (n = 6). B) The mouse weight in each

group during chemotherapy (n=6). C) The tumor weight in each group at the end of the

experiment. D) Western blot quantifications of pro-caspase-3, Caspase 3, PARP, and cleaved

PARP in A2780/cDDP xenograft tumors following treatment with measured compounds. The

relative density of bands was quantified by Image J. Bar graphs revealing relevant band

densitometry analysis of Western Blot images (n=3). Data represent the mean ± S.D. *P < 0.05,

**P < 0.01 versus control. E) Representative immune-fluorescent and immune-histochemical

images of JWA from six groups; 1000-2000 cancer cells were counted in 10 random fields of each

slide. F) H&E staining of vital organs and tumor as shown. Statistical significance was evaluated

by two-way ANOVA test (*P < 0.05, **P < 0.01 versus control).

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