Emerging JWA-targeted Pt(IV) prodrugs conjugated with CX-4945 to overcome chemo-immune-resistance
Feihong Chen, Sinan Pei, Xing Wang, Qian Zhu, Shaohua Gou*
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, PR China

a r t i c l e i n f o

Article history:
Received 5 October 2019
Accepted 28 October 2019 Available online xxx

Keywords: Pt(IV) prodrug JWA-XRCC1 SSBR
Immunosuppression Cisplatin resistance
a b s t r a c t

Two Pt(IV) prodrugs, Cx-platin-Cl and Cx-DN604-Cl, derived from the conjugation of cisplatin or DN604 with a CK2 inhibitor CX-4945, were constructed to suppress DNA damage repair-related elements. During in vitro biological studies, the Pt(IV) prodrugs had excellent cytotoxicity superior to cisplatin and DN604 to reverse drug resistance. Further mechanistic investigations revealed that the powerful anti- cancer activity of Cx-platin-Cl and Cx-DN604-Cl arisen from its suppression of JWA-XRCC1-mediated single-strand breaks repair. The emerging Pt(IV) prodrugs inhibited the growth of the xenografted tu- mors of C57BL6 and nude mice apart from JWA—/- mice. Between them, Cx-platin-Cl augmented the
infiltration and proliferation of Teff cells, alleviated the recruitment of Treg cells. The results provided
compelling preclinical support that Cx-platin-Cl and Cx-DN604-Cl could reverse chemo-immune resis- tance via decaying JWA-XRCC1-mediated SSBR and immunosuppression, improving the development of emerging Pt(IV) candidate as a potential immunotherapeutic agent for cancer resistant prevention.
© 2019 Elsevier Inc. All rights reserved.

⦁ Introduction

As the most effective anticancer chemotherapeutics in clinic, DNA-targeted agents are diffusely used for treating 50% of malig- nant cancers as the essential components [1,2]. According to sta- tistics, the DNA-targeted agents such as cisplatin, oxaliplatin and carboplatin are approved by FDA, which could exert the anticancer effects via activating severe DNA damage to lead to cell death [3,4]. Our lab proved that with a functional dicarboxylate ligand, the carboplatin analogue DN604 was developed to explore its ability to induce cancer cell apoptosis [5]. However, such drugs commonly generated acquired resistances through activating specific DNA damage repair pathways [6], thus leading to limited clinical appli- cation. Based on that, an effective strategy is to combine platinum- based drugs with various DNA damage response (DDR) suppres- sants to elevated cancer cell sensitivity and combat drug resistance [7]. As we known, casein kinase 2 (CK2) could act as a critical role in both single-strand breaks (SSBs) and double-strand breaks (DSBs) repair [8]. In consequence, DDR pathways could be restrained by CK2 inhibitor CX-4945 processed in the phase II clinical trials [9].

* Corresponding author.
E-mail address: [email protected] (S. Gou).
Thus, the approach of combing Pt(II) drugs with CX-4945 might have the potential advantages to fulfill the task.
JWA, could be activated by heat shock and oxidative stress [10], which might exert various biological functions with separate stress conditions. Recent data revealed that JWA attended to protect cells escaped from distinct DNA strands breaks [11]. As a basic compo- nent of base excision repair (BER)-related complex, JWA could mediate DDR triggered by oxidative stress through increasing XRCC1 expression via MAPK-E2F1 pathway and aparting from XRCC1 degradation by ubiquitin-proteasome, defining the involvement of JWA-XRCC1 in the SSBR and BER processes.
Given the accelerated role of immune checkpoint blockade (ICB) as a therapeutic strategy, the relevance of DNA damage in tumors with the innate immune system has raised concerns, and it is clear that DNA damage repair acts as a key role in drawing attentions to ICB. To some extent, severe DNA damage and subsequent genomic instability are discovered to shape antitumor immune response and thus lead clinic application to immune-directed therapies [12]. The tumor micro-environment (TME) could increase proliferation of effective T cells and suppress populations of regulatory T cells. BER inhibition could positively regulate TME and sensitize cancer cell to DNA-damaging agents [13].
As proof of concept, we have developed a strand of Pt(IV) pro- drugs via conjugating the axial group CX-4945 into Pt(IV) prodrugs

https://doi.org/10.1016/j.bbrc.2019.10.184

0006-291X/© 2019 Elsevier Inc. All rights reserved.

to investigate the effect of anticancer immunotherapy. It has been concluded that emerging Pt(IV) prodrugs could improve anticancer immunotherapy and induce the reversal of chemo-immune resis- tance via suppressing JWA-XRCC1pathways essential for SSBR.

⦁ Results

⦁ Designs and characterizations of Cx-platin-Cl and Cx-DN604-Cl

The designs and characterizations of two Pt(IV) prodrugs are shown in Figs. S1 and S2. Pretreatment of cis,cis,trans- [Pt(NH3)2Cl3OH] and cis,cis,trans-[Pt(NH3)3(3-oxocyclobutane-1,1- dicarboxylate)(Cl)(OH)], respectively, with CX-4945 in the mixture of TBTU and TEA in DMF synthesized Cx-platin-Cl and Cx- DN604-Cl (Fig. 1A), which were characterized by 1H and 13C NMR spectroscopy and ESI-MS (Figs. S3 and S4).

⦁ Cytotoxicity effects of the Pt(IV) prodrugs

The cytotoxicity of the resulting Pt(IV) prodrugs toward cancer and non-cancer cells were determined and the values of IC50 were calculated from a series of doses of the tested compounds for 72 h (Table 1, Fig. 1B). Unlike cisplatin and DN604, Cx-platin-Cl and Cx- DN604-Cl exhibited comparable low IC50 values across the inves- tigated cells. Significantly, Cx-platin-Cl and Cx-DN604-Cl were found to obviously overcome cisplatin-resistance of A2780/cDDP cells via reducing RF from 3.35 to 0.23 and 0.57, respectively. Consistently, Cx-platin-Cl produced the highest population of A2780/cDDP cells in early-stage and late-stage apoptosis (Fig. 1C), which agrees with the findings for the cytotoxicity profiles.
To further verify the cell proliferation inhibition effect of Cx- platin-Cl and Cx-DN604-Cl, colony forming assay was used to compare the capacity of measured compounds. The results in Fig. 1D revealed that fewer colonies on the plates pretreated with two Pt(IV) prodrugs than those treated with cisplatin and DN604. Thus, Cx-platin-Cl and Cx-DN604-Cl were more effective than cisplatin and DN604. The data from AO/EB dual staining assays in Fig. 1E revealed two Pt(IV) prodrugs exhibited higher apoptotic rates than necrotic rate in A2780/cDDP cancer cells, proving that Cx-platin-Cl and Cx-DN604-Cl could trigger a large population of apoptosis in cisplatin-resistant cancer cells. Meanwhile, the cancer cells treated with two Pt(IV) prodrugs exhibited an increased level of activated caspase-3 compared to cisplatin and DN604 (Fig. 1F). The results revealed that Cx-platin-Cl and Cx-DN604-Cl could induce high level of caspase-mediated apoptosis in A2780/cDDP cancer cells.
To explore the role of ATP/IP3 pathway on the Pt(IV) prodrugs- induced apoptosis, we monitored the extracellular concentration of ATP and intracellular levels of IP3. The results in Fig. 1G demonstrated that Cx-platin-Cl and Cx-DN604-Cl could increase the ATP concentration and IP3 levels for 6, 12 and 24 h, respectively.

⦁ The internalization induced by the Pt(IV) prodrugs

The extent of internalization was determined with the treat- ment of 15 mM measured samples in A2780 and A2780/cDDP cancer cells for 3, 6 and 12 h (Fig. 2A) via using ICP-MS, which confirmed that two Pt(IV) prodrugs could enter cancer cells in the time- dependent manners. As a result, the cellular Pt uptake of cisplatin and DN604 in A2780/cDDP cells sharply reduced compared to cisplatin-sensitive A2780 cells, whereas 4- and 3-fold increase of Cx-platin-Cl and Cx-DN604-Cl under the same concentration. Additionally, the results in Fig. 2B captures a snapshot of Pt distri- butions in distinct subcellular fractions of cancer cells pretreated with two Pt(IV) prodrugs. Pt distributions of A2780/cDDP cells
pretreated with the Pt(IV) prodrugs were observed higher than those of A2780 cancer cells, while the opposed data were drawn for the cisplatin-treated groups, as consistent with cellular uptake detection. The fractions in cytoplasm were shown to own the largest contents of Pt and a larger proportion of Pt released from two Pt(IV) prodrugs accumulated in the nucleus in A2780 and A2780/cDDP cells. Therefore, more Pt-DNA adducts were observed in two Pt(IV) prodrugs treated A2780 and A2780/cDDP cancer cells via evaluating the ability of platinum drugs to confirm nuclear Pt- DNA (Fig. 2C). The extent of platinated DNA was detected to be 0.85, 0.47, 7.95 and 4.06 ng/mg for cisplatin, DN604, Cx-platin-Cl and Cx-DN604-Cl in A2780/cDDP cells versus 1.72, 1.24, 7.53 and
3.67 ng/mg for Pt-based drugs in A2780 cells. The significant increased Pt-DNA adduct could be arisen from the avoidance of detoxification-mediated by GSH.
As uptaken into cancer cells, Pt(IV) prodrugs could be reduced to Pt(II) drugs with reductants (GSH and AsA, etc). At the same time, the covalent binding between GSH and the center of Pt(II) drugs competitively could induce cellular detoxification of the Pt(IV) prodrugs. The intracellular oxidized (GSSG) and reduced (GSH) glutathione were detected to explore the ability of the reduction of Pt(IV) prodrugs (Fig. 2D and E). As exposed to cisplatin, the obvious increase of GSH extent was determined in A2780 and A2780/cDDP cancer cells. Conversely, Cx-platin-Cl could restrain GSH growth from 1.46% to 0.42% of A2780 cancer cells and 3.46%e0.72% of A2780/cDDP cancer cells, while 1.13% for Cx-DN604-Cl-treated A2780 cancer cells and 2.82% for Cx-DN604-Cl-treated A2780/cDDP cancer cells. In addition, in contrast to nearly no effect of cisplatin on GSSG level, Cx-platin-Cl and Cx-DN604-Cl could increase their level from 15.46% to 27.2% and 15.35%e24.1% in A2780 cells and 39.46%e47.2% and 37.34%e44.1% in A2780/cDDP cells. The results implied that the intracellular GSH of A2780/cDDP cells was almost 2-fold as high as that of A2780 cancer cells and inhibited by the Pt(IV) prodrugs treatments.
As a DNA cross-linking agent, platinum-based drugs could damage DNA and form Pt-DNA adducts. The comet assay was used to assess the genotoxic effect of the emerging Pt(IV) prodrugs. The results in Fig. 2F reveal that both Pt(IV) prodrugs could produce dramatical prominent tails in contrast to cisplatin and DN604 in tail length and moment. Meanwhile, A2780 and A2780/cDDP cancer cells pretreated with Pt(IV) prodrugs restrained DNA repair pre- vention at 12 h and 14 h (Fig. 2G), which proved that the Pt(IV) prodrugs could apparently trigger DNA damage via ceasing DDR.

⦁ DNA strands breaks induced by the Pt(IV) prodrugs

As a constitutive process in cancer cells, DDR could protect the genome from damage and mutations, which is critical for cell sur- vival. We detected whether Pt(IV) prodrugs could increase DSBs marker, g-H2AX level via g-H2AX foci detection. As shown in Fig. 2HeI, treatment with Pt(IV) prodrugs led to a little increased expression levels of g-H2AX, which indicated that Cx-platin-Cl and Cx-DN604-Cl might cause other DNA strands breaks. The results in Fig. 3A indicated that Cx-platin-Cl could trigger higher number of SSBs compared to cisplatin. As validated in the BER proteins and DNA lesions [14], the HCR assay was carried out in Fig. 3B. A time- dependent repair model was proceeded and the DRC was detected for damaged plasmids, which was reduced to 17.95% and 19.79% for Cx-platin-Cl-treated LUCconplasmid versus undamaged control plasmids in A2780 and A2780/cDDP cancer cells.

⦁ JWA-XRCC1 is required for SSBR pathway involved in the anticancer effect of the Pt(IV) prodrugs

As a basic element of BER complex, JWA could be recruited by

Fig. 1. Emerging Pt(IV) prodrugs enhancement of toxicity to A2780 and A2780/cDDP cancer cells through the augmentation of apoptosis. A) Chemical structures of two Pt(IV) prodrugs. B) In vitro cytotoxicity. C) In vitro apoptosis of A2780 and A2780/cDDP cancer cells pretreated with measured compounds. D) Long-term colony formation assays. E) AO/EB dual staining of A2780/cDDP cancer cells following the treatment of measured compounds at 15 mM for 24 h. The graph shows manual count of apoptotic and necrotic cells in percentage. F) Analysis of caspase-3 activation in cancer cells. G) The extracellular ATP concentration and intracellular IP3 levels induced by measured compounds. Data indicated mean ± SD for n ¼ 3 experiments each performed in triplicate. *P < 0.05, **P < 0.01 compared with NC group.

Table 1
1 2
Cytotoxicity effects of cisplatin, CX-4945, Cx-platin-Cl, Cx-DN604-Cl towards several cancer cell lines with diverse CK2 expression. Cells were treated with the complexes for 72 h and evaluated with IC50 values.
Cell line Cancer Type CK2 expression Cisplatin DN604 IC50 (mM) CX-4945 Cx-platin-Cl Cx-DN604-Cl FI c FI d

A2780 Ovarian High 9.75 ± 7.28 17.26 ± 6.59 26.75 ± 9.75 3.86 ± 0.55 9.03 ± 6.25 1.24 1.23
A2780/cDDP Ovarian High 32.65 ± 2.59 11.49 ± 3.12 18.99 ± 0.64 2.92 ± 0.07 8.48 ± 0.15 11.18 1.35
MCF-7 Breast High 3.51 ± 0.21 9.43 ± 0.32 28.54 ± 6.41 3.67 ± 0.28 7.05 ± 0.73 0.96 1.34
SGC-7901 Stomach Medium 4.86 ± 0.75 29.62 ± 7.76 21.59 ± 7.12 10.73 ± 0.94 18.38 ± 5.15 0.45 1.61
HepG2 Liver Medium 4.40 ± 0.35 23.60 ± 1.97 15.99 ± 0.96 15.50 ± 1.04 34.00 ± 1.78 0.28 0.69
PANC-1 Pancreas Low 12.09 ± 3.05 56.26 ± 10.13 32.15 ± 7.05 15.29 ± 1.79 29.24 ± 7.01 0.79 1.92
HUVEC
RFb Vascular ND 19.06 ± 6.31
3.35 52.98 ± 4.38
1.50 29.23 ± 2.05 27.35 ± 2.77
0.23 35.82 ± 2.03
0.57 0.70 1.48
a Resistant to cisplatin.
b Resistance Factor, IC50(A2780)/IC50 (A2780/cDDP).
c Fold Increase, IC50(Cisplatin)/IC50(Cx-platin-Cl).
d Fold Increase, IC50(DN604)/IC50(Cx- DN604-Cl).

Fig. 2. Cellular uptake of Pt in A2780 and A2780/cDDP cancer cells. A) Pt uptake into A2780 and A2780/cDDP cancer cell after 3, 6, 18 h exposure to measured compounds. B) Subcellular distribution of measured compounds in A2780 and A2780/cDDP cancer cells. C) Pt-DNA adducts in pretreated cancer cells. GSH (D) and GSSG (E) percent of A2780 and A2780/cDDP cancer cells pretreated at Pt concentrations of 0, 1, 4, 16 mM, respectively. F) Comet assay revealing increased chromosomal DNA strand breaks including SBBs, DSBs of DNA cross-links triggered by measured compounds in A2780 and A2780/cDDP cancer cells. G) The level of DNA damage repair secondary to DNA damage induced by the com- pounds. H) Immunofluorescence staining of the determined gH2AX foci ( × 1000). I) gH2AX foci after treatments were counted in 50 individual cells per time points of 8 h and 12 h
in cancer cells. Graph represents average number of foci per cells ±SD. Results are representative of at least n ¼ 3 independent experiments and shown as the mean ± S.D. *P < 0.05,
**P < 0.01 compared with control group.

XRCC1 to DNA damage sites and mediate DNA damage-induced SSBR [15]. The results showed that DRC was declined more than 78% in JWA knockdown cancer cells compared to the untreated
control (Fig. 3C). Conversely, the JWA overexpression in cancer cells remarkably elevated the DRC up to 1.4- and 1.6-fold. The additional SSBR endpoints were investigated to make sure the JWA

Fig. 2. (continued).

requirement in DDR. Stable transfection of JWA knockdown was carried out in cancer cells. Compared to the untreated control, the KD-JWA A2780/cDDP exhibited greater sensitivity to Cx-platin-Cl and Cx-DN604-Cl (Fig. 3D).
In order to understand the mechanism of emerging Pt(IV) pro- drugs at a molecular level, molecular docking studies were
performed to elucidate the binding mode of Pt(IV) prodtugs in the active site of XRCC1. The molecular docking in Fig. 3EeF revealed that Cx-platin-Cl binds to XRCC1 in a hydrophobic pocket, which is surrounded by residues, including Ser105, Glu107, Pro110, Trp142, Ser149, Gln181, Trp184, Asn188, and form hydrogen bonds with Asn146. Moreover, Cx-DN604-Cl binds to XRCC1 in a hydrophobic pocket, which is surrounded by residues, including Ser105, Glu107, Pro110, Trp142, Ser149, Gln181, Trp184, Asn188, and form hydrogen bonds with Leu101, Arg106, Trp184 and Asn188.
As we known, XRCC1 protein levels could be mediated by JWA [10]. In order to elucidate whether JWA plays a part in XRCC1- associated BER complex, the knockdown of JWA in A2780/cDDP cancer cells and vector control were pretreated with 15 mM Cx- platin-Cl for 0.5 h, and cultured in no drug medium to await for DNA rejoining. The level of XRCC1 and its downstream of LigIII were downregulated in the KD-JWA A2780/cDDP, while no change in XRCC1 upstream of APE1 (Fig. 3G). JWA interacted with XRCC1, which could connect with LigIII to PARP-1 to assemble a BER complex [16]. As showed in Fig. 3H, JWA could interact with XRCC1 in control and treated A2780/cDDP cells. The results revealed that JWA might be a key component of BER-related protein complexes suffering severe DNA damage.

⦁ Pt(IV) prodrugs suppressed the growth of JWA-expressing tumor in immunocompetent C57BL6 but not in JWA—/- mice
The mechanisms of severe DNA damage and subsequent genomic instability have obviously shaped the antitumor immune response. To demonstrate the in vivo inhibitory effect of Pt-based drugs on growth of tumor cells, the C57BL6 mice were inoculated with high JWA-expressed ID8 ovarian cancer cells. We observed that Cx-platin-Cl could inhibit tumor growth and decreasing tumor weights and volumes in Fig. 4A and B. To evaluate whether DNA damage-related T-cell-mediated immunity is essential for the antitumor effect of the Pt(IV) prodrugs, we detected the Teff cell and Treg cell within in vivo ID8 tumor xenograft models suffering Cx- platin-Cl and Cx-DN604-Cl treatment. The results showed an obviously increase of tumor-infiltrating and proliferating CD8 Teff cells in ID8 tumors (Fig. 4C). Conversely, the number of infiltration and proliferative Treg cells was decreased in Cx-platin-Cl-treated group (Fig. 4D).
To detect the impact of JWA-XRCC1-SSBR in T-cell-mediated antitumor immunity of Cx-platin-Cl, the JWA—/- mice model was applied to attest whether JWA—/- in tumor or host-derived cells could completely alleviate the antitumor effect of Cx-platin-Cl.
Importantly, we found that Cx-platin-Cl-treatment had no effect on tumor growth suppression in JWA—/- mice inoculated with JWA—/—ID8 tumors (Fig. 4E), suggesting that Cx-platin-Cl exhibited antitumor effect via inhibiting JWA in both the host-derived cells
and inoculated tumor cells.

⦁ Pt(IV) prodrugs increased the number of splenic T cells in ID8 tumor mice model

For spleen dims as a key reservoir for lymphocytes to trigger antitumor immunity, we detected the splenic Teff cell and Treg cell ingredients in ID8 tumor-bearing mice model. The spleens in Cx- platin-Cl-treated group was observed enlarged (Fig. 4F). More- over, H&E staining in Cx-platin-Cl-treated group have no obvious pathological changes (Fig. 4G). FACS analysis revealed that Cx- platin-Cl treatment obviously elevated the percentage of prolifer- ative Teff cells with CD8 and CD4, whereas a remarkably reduction in the percentage of Treg cell in spleen (Fig. 4H). To sum up, the data proved that Cx-platin-Cl treatment could increase the level of splenic Teff cells which could assemble to the tumor sites.

Fig. 3. DNA damage of SSBs and DSBs induced by measured compounds. A) The number of SSB was determined by a neutral comet assay. B) The repair efficiency of A2780 and A2780/cDDP cells on damaged plasmid DNA was detected by HCR assay. C) Knockdown of JWA suppressed DRC of damaged plasmids and overexpression of JWA enhances DRC in A2780 and A2780/cDDP cancer cells. The pGL3 plasmids were transfected as a control for co-transfection efficiency. The renilla luciferase reporter (internal control, Promega) was used to normalize the activity of the LUC reporter. D) JWA knockdown enhances the cell death induced by Cx-platin-Cl. The relative % surviving cells are presented as the means ± S.D. of three independent samples. Molecular modeling of Cx-platin-Cl and Cx-DN604-Cl in complex with XRCC1 NLS peptide: (E) Cx-platin-Cl, (F) Cx-DN604-Cl. G) The expression levels of BER complex components including APE1, XRCC1, LigIII during DNA repair after Cx-platin-Cl treatment in JWA stable knockdown and vector control A2780/

Fig. 4. Cx-platin-Cl treatment enhances the infiltration and accumulation of T cells in ID8 xenograft tumors. A) Tumor weights in immunocompetent mice (n ¼ 6 mice, each). B) Individual tumor growth in immunocompetent mice (n ¼ 6 mice, each). C) Representative percentage of CD8þ effector T cells of total CD45þ cells for vehicle-, cisplatin, Cx-platin-Cl, Cx-DN604-Cl-treated mice (n ¼ 6 mice, each). D) Percentage of CD4þFoxp3þ regulatory T cells of total CD45þ cells for indicated mice. E) Individual tumor growth in JWA—/- mice. F) Gross examination of spleens from tumor-bearing mice that were treated with the indicated compounds (n ¼ 6 mice, each). Spleen weights were detected in tumor-bearing mice. G) H&E staining of spleens as shown. H) Representative dot plots of CD8þ and CD4þ T cells for spleens of tumor-bearing mice that were treated with the indicated compounds (n ¼ 6 mice in three pools, each). Statistical significance was evaluated by two-way ANOVA test (*P < 0.05, **P < 0.01 compared with control group).

cDDP cancer cells. Bar graphs revealing relevant band densitometry analysis of Western Blot images (n ¼ 3). Data represent the mean ± S.D. *P < 0.05, **P < 0.01 versus control. H) The A2780/cDDP cells were pretreated with 15 mM Cx-platin-Cl for 30 min and endogenous protein-protein interaction between JWA and XRCC1 was determined by immuno- precipitation (IP) with JWA or XRCC1 antibodies followed by Western Blot. IgG was used as negative control for IP. Bar graphs revealing relevant band densitometry analysis of
Western Blot images (n ¼ 3). Results are representative of at least n ¼ 3 independent experiments and shown as the mean ± S.D. *P < 0.05, **P < 0.01 versus control.

Table 2
Tumor inhibition rate (TIR) of A2780/cDDP tumor-bearing athymic nude mice following the corresponding treatments.

TIR (%) Cisplatin DN604 Cx-platin-Cl Cx-DN604-Cl A2780/cDDP 26.71 ± 14.92 22.65 ± 12.78 58.28 ± 4.73 36.99 ± 0.92
xenograft model

⦁ Pt(IV) prodrugs inhibited outgrowth of cisplatin resistant ovarian cancer via JWA-XRCC1 SSBR pathway

The antitumor evaluations of the Pt(IV) prodrugs were employed via A2780/cDDP xenograft model. The results in Figs. S4AeC illustrated that tumors inoculated with A2780/cDDP cancer cells increased sharply. Tumor growth in cisplatin-treated group was un-effected. The tumors in Cx-platin-Cl-treated group had developed with no more than 220 mm3, indicating that A2780/ cDDP cancer cell grew at a decelerated rate following the Cx-platin- Cl-treatment. For A2780/cDDP xenograft mice model, 26.71% for cisplatin was small to 58.28% for Cx-platin-Cl and 36.99% for Cx- DN604-Cl, slightly greater than 22.65% for DN604 (Table 2). Addi- tionally, the groups treated with Cx-platin-Cl and Cx-DN604-Cl with the same conditions had nearly no changes in mouse weight, revealing its hypotoxicity and better prognosis for further cancer therapy. The molecule expressions of pro-caspase-3, cas- pase-3, PARP and cleaved PARP in A2780/cDDP xenografted tumors were determined. After mitochondrial dysfunction, caspase 3 could be activated and enhanced cleaved PARP-mediated cellular decomposition [17,18]. As verified in Fig. S4D, two Pt(IV) prodrugs elicited the highest caspase-3 and cleaved PARP in A2780/cDDP xenografted tumor models. Additionally, IFC and IHC analysis demonstrated an obvious decline of JWA expression in Cx-platin- Cl-treated A2780/cDDP xenografted tumors (Fig. S4E). The results of pathological examination (Fig. S4F) yielded no obvious change in the tested vital organs from mice treated with Cx-platin-Cl and Cx- DN604-Cl, while there is important organ damage in the mice treated with cisplatin.

⦁ Discussion

The emerging Pt(IV) prodrugs have been designed as promising agents to combat the drawbacks of Pt(II) agents. Owning kinetic inertness and a low-spin d6 octahedral geometry, Pt(IV) prodrugs were rendered for stable oral administration. Moreover, the intro- duction of axial ligands could improve pharmacological properties, reduce toxic effect and breakdown drug resistance [19]. In present, the cores of Pt(IV) are mainly cisplatin and carboplatin with the bioactive ligands include COX, HDAC and p53 inhibitors [20,21].
DNA-damaging agents act as a critical role in the development of anticancer chemotherapeutics and are world widely used in clinic. DDR is significantly important in maintaining genomic integrity and regulating drug efficacy. Initially, previous studies pointed out that JWA gene is an environmental response gene regulated by a variety of induced differentiation agents and is involved in cell differentiation and apoptosis. JWA is widely acti- vated in cellular responses to stress stimuli or DNA damage and repair processes, thus indicating that JWA may be a target gene functionally. Additionally, Wang [22] confirmed that JWA inhibited the ubiquitination of XRCC1, regulated and stabilized the function of nuclear XRCC1 and participated in BER and SSBR. As we know, the improper DNA damage repair could lead to tumor genome changes which could break the immune balance in TME.
The connection between the host immune system (HIS) and the tumor has been disputed for several decades, the purposes to
awaken the HIS to make cancer cells death showed useful clinical efficacy. Numerous lines of evidence proved that DDR is pivotal in inducing cancer cells sensitivity and response to ICB. A transfer in the regulation of DDR triggered with the exposure to DNA-targeted agents or restrain of DDR pathways can induce an innate immune response mediated by STING [23]. The role of the TME in mediating resistance to DNA-targeted agents has drawn much attention. The HGEOC were used to reveal that fibroblasts could mediate cisplatin resistance via releasing of thiols and counter by T cell-mediated IFNg signaling [24]. Thus, another mechanism while ICB might promote tumor cell apoptosis is via combating stromal cellsemediated protective effects. Given the complicated interac- tion between DNA damage repair and immunosuppression, the suppression of DDR is a promising filed for anti-cancer immunotherapy.
In present, we discovered Cx-platin-Cl and Cx-DN604-Cl struc- turally as potent inhibitors of BER. Compared to cisplatin and DN604, Pt(IV) prodrugs could enhance the extent of Pt uptake and adducts of Pt-DNA, hence suppressing DDR pathways to chemo- immune resistance. More importantly, Cx-platin-Cl and Cx- DN604-Cl present the antitumor activities via inhibiting the JWA- mediated XRCC1 in SSBR thoroughly. Nevertheless, Pt(IV) pro- drugs could be considered as potent inhibitors of JWA-XRCC1 since their treatment causes strong regression in tumor of JWA-intact mice with no effect to suppress tumor growth in JWA deficient mice model.

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Acknowledgements

We are grateful to the National Natural Science Foundation of China, China (21571033 and 81503099) for financial aids to this work. The research was supported by the Zhishan Youth Scholar Program of SEU, China (2242019R40045) and the Fundamental Research Funds for the Central Universities, China (2242017K41024).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.184.

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