Novel CK2-Specific Pt(II) Compound Reverses Cisplatin-Induced Resistance by Inhibiting Cancer Cell Stemness and Suppressing DNA Damage Repair in Non-small Cell Lung Cancer Treatments

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Novel CK2-Speciﬁc Pt(II) Compound Reverses Cisplatin-Induced

Resistance by Inhibiting Cancer Cell Stemness and Suppressing DNA

Damage Repair in Non-small Cell Lung Cancer Treatments

Yuanjiang Wang,^§Xinyi Wang,^§Gang Xu, and Shaohua Gou*

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ABSTRACT: Cancer stem cells (CSCs) have a pivotal impact in drug

resistance, tumor metastasis, and progression of various cancer entities,

including in non-small cell lung cancer (NSCLC). A CK2 inhibitor HY1

was found to show potent CSC inhibitory eﬀects in A549 cells. By taking

advantage of inherent CK2 speciﬁcity and CSC inhibition of HY1, a

Pt(II) agent (HY1-Pt) was developed by conjugation of HY1 with an

active Pt(II) unit to reverse cisplatin-induced resistance in A549/cDDP

cell treatment. In vitro biological studies indicated that HY1-Pt can

target CK2, suppress DNA damage repair, reinforce cellular accumu-

lation of platinum, and reverse resistance apart from eﬀectively inhibiting

CSCs through Wnt/β-catenin signal pathway in A549/cDDP cells.

Signiﬁcantly, HY1-Pt presented an acceptable pharmacokinetic behavior

and exhibited higher tumor growth inhibitory eﬃcacy than cisplatin

either in A549 or A549/cDDP xenograft models with low toxicity.

Overall, HY1-Pt is a promising drug candidate for NSCLC treatment.

CSCs in the quiescent phase can lead to primary and acquired

drug resistance through DNA self-repair, ABC transporters,

and mutation such as point mutation, gene mutation, and gene

ampliﬁcation, responsible for the failure of many cancer

treatments.^10,11Therefore, development of targeted chemo-

therapeutic drugs to abrogate CSCs is a key task in lung cancer

research and clinical application.

Multi-regimen chemotherapy for NSCLC usually involves

the combination of platinum-based drugs like cisplatin and

other drugs with diﬀerent mechanisms of action. According to

the statistics, more than half of chemotherapy regimens

contain platinum-based antitumor drugs.¹²⁻¹⁵However, non-

speciﬁcity and drug resistance in addition to toxicity are still

major inevitable barriers for their clinical use.¹⁶The great

success and drawback of cisplatin have encouraged researchers

to conduct more in-depth and detailed studies on its

mechanism of action, attempting to achieve novel platinum-

based drugs with higher eﬃciency, lower toxicity, and better

reversal of resistance. So far, the mechanism of cisplatin

resistance is recognized as follows: (i) decreased intracellular

1. INTRODUCTION

Lung cancer has become a serious public health issue, and its

morbidity and mortality are increasing year by year, and it has

become the ﬁrst cause of death of malignant tumors in urban

population of China.^1,2Non-small cell lung cancer (NSCLC)

occupies almost 85% of all lung cancers. More serious, nearly

70% of all patients with NSCLC with locally advanced or

metastatic disease need systemic therapy, but they have only a

median survival time of about 18 months in the inoperable

stage.³There are several reasons that NSCLC is diﬃcult to

cure: (i) most of the patients are found to be in the middle or

advanced-stage and those who have received front-line

chemotherapy face fruitless treatments.⁴(ii) Lung cancers

have serious intratumoral heterogeneity. According to

statistics, as many as 63% of NSCLC patients contain more

than one histological subtype.^5,6(iii) The intrinsic and/or

acquired drug resistance from traditional drugs or targeted

tyrosine kinase inhibitors is still a major problem in clinic. So,

new agents are urgently needed to overcome drug resistance.⁷

Lung cancer intratumoral heterogeneity is driven by

subpopulations of tumor cells termed as cancer stem cells

(CSCs).⁸CSCs, a small subset of cancer cells, can diﬀerentiate

into diﬀerent phenotypes with the ability of continuous self-

renewal, diﬀerentiation, and highly tumorigenic subpopula-

tion.⁹Unlike the bulk of tumor cells, CSCs are critically

involved in tumorigenesis, tumor recurrence, cancer invasion,

and metastasis. Moreover, mounting evidence suggests that

Received: January 16, 2021

Published: March 30, 2021

https://doi.org/10.1021/acs.jmedchem.1c00079

J. Med. Chem. 2021, 64, 4163−4178

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Scheme 1. Synthesis of HY1 and HY1-Pt

Figure 1. Inhibitory activity of HY1 on CK2 and its eﬀect on A549 cell stemness. (a) Inhibition rate curves of HY1 and CX-4945 on CK2 enzyme

activity. (b) Western blot analysis on the speciﬁc CK2 phosphorylation targets: phosphorylation of the Akt1 serine 129 and Cdc37 serine 13 in

A549 cells after treatment with CX-4945 and HY1 at 20 μM, respectively, for 24 h. (c) Inhibition rate curves of HY1 and CX-4945 on ALDH1A1

enzyme activity. (d) Percentage of ALDH⁺cells in A549 cells after treatment with CX-4945 and HY1 at 20 μM, respectively, for 24 h. (e)

ALDH1A1 mRNA levels after treatment with CX-4945 and HY1 at 20 μM, respectively, for 24 h in A549 cells. (f) ALDH1A1 protein levels treated

with CX-4945 and HY1 at 20 μM, respectively, for 24 h in A549 cells. Data were obtained via using students’ T-test, n = 3 per group, Error bars =

SD, asterisk indicate statistically signiﬁcant diﬀerences, **P < 0.01, vs control.

platinum accumulation, (ii) detoxiﬁcation of platinum ions by

sulfhydryl molecules, and (iii) increased DNA damage repair

levels.¹⁷⁻¹⁹Besides, there are also other factors that may have

impacts on the mechanism of drug resistance including DNA

hypermethylation, histone modiﬁcation, and chromatin

structure changes.²⁰

Protein kinase CK2 plays a notable role in various essential

and pathological biological processes.²¹⁻²³It has been proven

that CK2 has more than 300 phosphorylated substrates, which

is one of the most pleiotropic proteins in eukaryotic

systems.^24,25CK2 is overexpressed in multiple tumors and

rated as one of the most promising targets for treating many

types of cancer and some life-threatening diseases.²⁶Much

signiﬁcantly, CK2 can play a pivotal regulatory role in DNA

damage repair and cancer stemness by Hedge-hog/Gli1, Wnt/

β-catenin, NF-κB and PI3K, and so forth.²⁷⁻²⁹In addition,

CK2 inhibition also causes an increased uptake of known drugs

in multidrug-resistant cells.³⁰⁻³³

derived a hydrazide compound (HY1) upon the framework of

CX-4945, which was found to exhibit potent capability of

inhibiting cancer cell stemness. By taking the advantage of

inherent CK2 speciﬁcity and CSC inhibition of HY1, a novel

Pt(II) compound was developed by conjugation of HY1 with

an active Pt(II) unit as an antitumor agent, attempting to

enhance antitumor eﬃcacy and overcome cisplatin-induced

resistance by targeting CK2, suppressing DNA damage repair

and inhibiting cancer cell stemness for the ﬁrst time.

2. RESULTS AND DISCUSSION

2.1. Preparation of HY1 and HY1-Pt. CX-4945 and

compound 7 were synthesized in accordance with the

procedures reported previously (Scheme S1).²⁵The synthetic

route of HY1 and HY1-Pt is shown in Scheme 1. Treatment of

compound 7 with hydrazine hydrate in reﬂuxing methanol

aﬀorded HY1 which precipitated from the reaction mixture

upon cooling. Direct condensation between HY1 with DN604,

a known antitumor Pt(II) agent,³⁸resulted in the formation of

Notably, there has been a report that CK2α positively

regulates the population of CD44⁺/CD24⁻and the transcrip-

tional activity of Nothch1, which is concerned in the

maintenance of CSCs.³⁴As CX-4945 is the sole small

molecule CK2 inhibitor in phase I/II clinical trials,³⁵⁻³⁷we

HY1-Pt. The resulting compounds were conﬁrmed by ¹H, ¹³

C

NMR, and HR electrospray ionization-mass spectrometry

(ESI-MS) spectroscopy with purity over 95% determined by

RP-HPLC (see also the Supporting Information).

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Figure 2. Study on the eﬀect of HY1 on the expression of intracellular CD44⁺and the formation of tumorsphere in A549 cells. (a) Statistics of the

experimental results in Figure S1, and the proportion of CD44⁺subsets in A549 cells treated with CX-4945, BBI608, and HY1 at 20 μM,

respectively, for 24 h were statistically analyzed. (b) Morphological image and (c) the number of A549 cells tumorsphere treated with CX-4945,

BBI608, and HY1 at 10 μM, respectively. Error bars = SD (n = 3), **P < 0.01.

Figure 3. Stability of HY1-Pt in Hepes buﬀer at pH 7.4 and 6.0 measured by UV−vis. (a) Stability of HY1-Pt in Hepes buﬀer (pH = 7.4)

containing 2% DMF was recorded via time-dependent UV−vis spectra (scan every 30 min for a total of 24 times) at 37.0 °C. (b) Stability of HY1-

Pt in Hepes buﬀer (pH = 6.0) containing 2% DMF was recorded via time-dependent UV−vis spectra (scan every 10 min for 12 h) at 37.0 °C.

2.2. Inhibitory Activity of HY1 on CK2 and Its Eﬀect

on A549 Cells Stemness. Since HY1 and CX-4945 share the

same scaﬀold of benzo[c][2,6]naphthyridine, we explored

whether HY1 retained the CK2 inhibitory activity of its parent

compound, CX-4945. Thus, the human CK2 assay kit and the

antibodies of the speciﬁc phosphorylation targets of CK2 were

used to evaluate the activity of HY1 with CX-4945 as the

positive control. As illustrated in Figure 1a, HY1 showed dose-

dependent inhibition of CK2 with a higher IC₅₀value than

CX-4945. Besides, HY1 also strongly inhibited the phosphor-

ylation of Akt1 serine 129 and Cdc37 serine 13, two speciﬁc

phosphorylation targets of CK2 (Figure 1b). These results

indicated that HY1 maintained the inhibitory activity of the

parent compound against CK2, even exhibiting slightly

stronger activity than CX-4945.

4945 (IC₅₀= 1.69 0.14 μM). The result indicated that HY1

had strong inhibitory activity on ALDH1A1 enzyme.

ALDH1A1 has been conﬁrmed to play a notable eﬀect in

maintaining the stemness of A549 cells and is expressed

preferentially.⁴¹Therefore, in A549 cells, we hypothesize that

ALDH1A1 has an important eﬀect in facilitating the

toxicological mechanism of HY1. First, the Aldeﬂuor Assay

was selected to determine the proportion of ALDH⁺subsets in

A549 cells. As shown in Figure 1d, the subpopulation of the

ALDH⁺cells were strongly reduced from 13.50 to 2.65% after

treatment with HY1, stronger than that treated with CX-4945

(5.65%). Besides, as shown in Figure 1e, the transcriptional

levels of ALDH1A1 in A549 cells presented a conspicuous

decrease after being treated with HY1 at 20 μM for 24 h.

Meanwhile, the eﬀect of HY1 on the expression of ALDH1A1

in A549 cells was also explored by western blot. As shown in

Figure 1f, as expected, HY1 can also down-regulate the

expression of ALDH1A1 in A549 cells, stronger than CX-

4945.

As CK2α can regulate the population of CD44⁺/CD24⁻

involved in stem cell maintenance,³⁴we studied the inhibitory

eﬀect of HY1 on cancer cell stemness as well. Aldehyde

dehydrogenases (ALDHs) are a superfamily of detoxifying

enzymes that are overexpressed in various cancers.³⁹Increasing

expression of ALDHs, especially ALDH1A1, in a number of

malignancies and CSCs are closely related to poor prognosis,

aggressiveness, and drug resistance in cancer chemotherapy.

Growing lines of evidence prove that ALDH1A1 plays a

signiﬁcant role in tumors and CSCs except for a biomarker of

CSCs and a predictor of the prognosis.⁴⁰As shown in Figure

CD44⁺is a cell surface protein, which can interact with a

variety of growth factors secreted, extracellular matrix,

components, and cytokines by cells present in the tumor

microenvironment.⁴²Therefore, CD44⁺has also been

identiﬁed as a marker for CSCs in a variety of tumors. In

order to study the action of HY1 on cancer cell stemness

further, the expression of CD44⁺in A549 cells treated with

HY1 was determined by ﬂow cytometry using CX-4945 and

BBI608 as positive controls. BBI608, currently in phase II/III

clinical trials, is a STAT3 inhibitor which can potently inhibit

1c, HY1 shows the potent inhibition on ALDH1A1 (IC₅₀

=

0.09 0.01 μM), which is 18.8-fold as much as that of CX-

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Table 1. Cytotoxicity Proﬁles of HY1-Pt and Related Samples against A549 and A549/cDDP Cells

IC₅₀(μM)

b

cell

A549

CX-4945

HY1

HY1-Pt

cisplatin

DN604

DN604/HY1

FI

22.39 2.04

25.08 2.34

18.16 2.21

17.03 1.02

5.10 0.48

4.84 1.38

0.95

4.05 0.30

28.12 1.71

6.94

16.93 1.35

48.56 4.60

2.86

16.76 1.32

18.90 1.65

1.12

0.79

5.81

A549/cDDP

a

RF

a

b

RF (resistant factor) = IC₅₀(A549/cDDP)/IC₅₀(A549). FI (fold increase) = IC₅₀(cisplatin)/IC₅₀(HY1-Pt).

Figure 4. Cellular responses of CX-4945, HY1, cisplatin, and HY1-Pt in A549 and A549/cDDP cells. Statistics of the results of cell cycle arrest in

Figure S3. Cell cycle arrest distributions of (a) A549 and (b) A549/cDDP cells were detected by ﬂow cytometry after treatment with the tested

compounds at 20 μM, respectively, for 24 h. (c) Statistics of the results of apoptosis in Figure S4. A549 and A549/cDDP cells were treated with the

tested compound at 20 μM, respectively, for 24 h, and then, the apoptosis of the cells was detected by ﬂow cytometry. (d) Intracellular

accumulation of platinum in A549 and A549/cDDP cells was detected by ICP-MS after treatment with cisplatin and HY1-Pt at 20 μM,

respectively, for 24 h. (e) Representative images of nuclei from comet assays. A549 and A549/cDDP cells were treated with the tested compounds

at 20 μM, respectively, for 12 h. Error bars = SD (n = 3), *P < 0.05, **P < 0.01.

the expression of the stemness gene, block the formation of

tumorsphere, and kill CSCs isolated from multifarious cancer

types.^43,44As shown in Figure 2a (see also the Supporting

Information, Figure S1), the proportion of CD44⁺subsets in

A549 cells was 61.8%, which is in accordance with other

reports.⁴⁵HY1 showed an extremely strong inhibitory eﬀect on

CD44⁺with an inhibition rate of 93.17%, higher than BBI608

(91.73%) or CX-4945 (79.45%). In addition, the function of

HY1 on stemness phenotypes in A549 cells was also

investigated. The cells were cultured in a three-dimensional

microenvironment without or with CX-4945, BBI608, and

HY1 for 24 h to detect their capability of tumorsphere

formation. After the following 10-day treatment, the number of

multicellular tumorsphere formed was counted. A549 tumor-

sphere cells treated with HY1 displayed reduced sphere sizes

and numbers as compared to those treated with CX-4945,

indicating that the inhibitory eﬀect of HY1 was comparable to

that of BBI608 (Figure 2b,c). Based on the above study, we

assure that HY1 is a bifunctional molecule that inhibits both

CK2 and cancer cell stemness.

at pH 7.40 was studied via RP-HPLC and UV−vis at 37 °C. It

was observed that HY1-Pt maintained its integrity after 12 h

under the condition (Figures 3a and S2a). However, HY1-Pt

was found to disassociate slowly by the hydrazine bond-

breaking to yield HY1 under the tumor acidic microenviron-

ment (pH = 6.0) (Figures 3b and S2b). As the lipid-water

partition coeﬃcient and LLE of a compound directly aﬀects

the absorption in vivo, the log P_o/wvalue of HY1-Pt was

measured as −0.031, and its LLE values were 5.301 and 5.351

in A549 and A549/cDDP cells, respectively, hinting that HY1-

Pt may have good absorption and penetration according to

Lipinski’s rules.⁴⁶

2.4. In Vitro Cytotoxicity. Cytotoxicity of HY1 and HY1-

Pt as well as a physical mixture of equimolar DN604 and HY1

against A549 (sensitive to cisplatin) and A549/cDDP

(resistant to cisplatin) cells was evaluated via MTT assay

using CX-4945, cisplatin, and DN604 as the positive drugs. As

shown in Table 1, cisplatin displayed the most potent

anticancer activity against A549 cells, while presented the

weakest activity against cisplatin-resistant A549/cDDP cells

among the measured compounds. Notably, HY1-Pt had

cytotoxicity nearly as potent as cisplatin in A549 cells, but

2.3. Stability and Physicochemical Properties of HY1-

Pt. Initially, the stability of HY1-Pt in Hepes buﬀer (0.25 M)

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Figure 5. Analysis of the γH2AX expression in A549 and A549/cDDP cells. (a) Expression of γH2AX in two cell lines was analyzed by western blot

after treatment with CX-4945, HY1, cisplatin (cis), and HY1-Pt at 20 μM, respectively, for 24 h. (b) γH2AX foci in two cell lines were determined

via immunoﬂuorescence staining after treatment with CX-4945, HY1, cisplatin, and HY1-Pt at 20 μM, respectively, for 12 h.

Figure 6. Investigation on the inhibition of HY1 on CK2 and DNA damage repair. (a) Western blotting assay on the expression of p-Akt1^S129and

p-Cdc37^S13in either A549 or A549/cDDP cells after treatment with diﬀerent concentrations of HY1-Pt for 24 h. (b) HY1-Pt is docked into the

hydrophobic pocket of CK2α protein (PDB ID: 6isj) utilizing Autodock. Amino acid residues that may be involved in the binding are labeled, and

the yellow dashed indicate the hydrogen bond interaction. (c) The co-localization of MDC1/Aprataxin and the damaged DNA in either A549 or

A549/cDDP cells were detected by immunoﬂuorescence technique after treatment with CX-4945, HY1, cisplatin, and HY1-Pt at 20 μM for 24 h.

the cytotoxicity of HY1-Pt to A549/cDDP cells was 5.81-fold

higher than cisplatin with a RF (resistant factor) value of 0.95.

It was noticed that DN604 and its physical mixture with HY1

showed almost the same cytotoxicity toward A549 cells. Even

though the physical mixture showed 2.57-fold stronger

cytotoxicity than DN604 against A549/cDDP cells, its activity

was 3.90 times weaker than HY1-Pt. Based on the above

results, HY1-Pt as well as its parent compounds CX-4945 and

HY1 was also used to treat diﬀerent cancer cells and human

normal hepatocytes for comparison. As shown in Table S2,

HY1-Pt exhibited more potent activity than HY1 and CX-

4945 against these cancer cells. Signiﬁcantly, HY1-Pt had the

lowest toxicity to LO2 cells among the measured compounds.

These results show that HY1-Pt exhibits strong inhibitory

activity against these cancer cells, especially cisplatin-mediated

drug-resistant cancer cells.

2.5. Cellular Responses. In view of the in vitro anti-

proliferation behavior of HY1-Pt on both A549 and A549/

cDDP cells, its mechanisms of action were investigated. These

two cancer cells were incubated with CX-4945, HY1, cisplatin,

and HY1-Pt at 20 μM for 24 h for studying cell arrest and

apoptosis. As shown in Figure 4a,b (see also the Supporting

Information, Figure S3), in either A549 or A549/cDDP cells,

cisplatin arrests the cell cycle in the S phase, which is in

accordance with other reports.⁴⁷Notably, HY1-Pt arrests the

cell cycle also in the S phase, similar to cisplatin. Meanwhile,

both A549 and A549/cDDP cells are highly sensitive to HY1-

Pt with apoptotic percentages from 66.9 to 85.5%, much more

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Figure 7. Study on the inhibitory eﬀect of the tested compounds on A549/cDDP^CD44brightand its intracellular CD133. (a) Mortality rate of A549/

cDDP^CD44brightcells incubated with CX-4945, HY1, cisplatin, and HY1-Pt at 20 μM, respectively, for 24 h. (b) Expression of CD133 is analyzed in

A549/cDDP^CD44brightcells by ﬂow cytometry. Error bars = SD (n = 3), *P < 0.05, **P < 0.01.

eﬀectively than cisplatin from 46.1 to 19.2% (Figures 4c and

S4). To evaluate the high cytotoxicity of HY1-Pt, the cellular

accumulation of Pt in A549 and A549/cDDP cells were

determined via ICP-MS after being treated with HY1-Pt at 20

μM for 12 h, using CX-4945 as a positive control. Signiﬁcantly,

the Pt accumulation of HY1-Pt in A549 and A549/cDDP cells

is 2.33 and 6.00 times as much as that of cisplatin, respectively

(Figure 4d). As anticipated, the Pt amount of cisplatin in

resistant to cisplatin cells is less than that in sensitive to

cisplatin cells, however, that of HY1-Pt in cisplatin resistant

cells is greater than in cisplatin sensitive cells with an increasing

fold of 1.34. The increased Pt content would promote DNA

damage in cancer cells. As shown in Figure 4e, HY1-Pt

produces longer length tails than cisplatin in these two cancer

cells in a comet assay, while CX-4945 and HY1 display weaker

DNA interactions than two Pt(II) agents. Based on the above,

HY1-Pt has been proved to be capable of eﬀectively promoting

cellular accumulation of platinum, seriously causing DNA

damage and largely inducing programmed cell death, exhibiting

better antitumor activity than cisplatin in the cells either

sensitive or resistant to cisplatin.

2.6. Study on the Expression of γ-H2AX. There are

several kinds of DNA damage, such as base modiﬁcation, in

chain and link-crosslinking, single-strand (SSBs) and double-

strand (DSBs) breaks, among which DSBs are considered to be

the most serious damage to DNA. Since the occurrence of γ-

H2AX is closely related to DSBs in a one-to-one

correspondence, γ-H2AX appears an important indicator to

detect DSBs in cells.^48,49Western blot, ﬂow cytometry, and

immunoﬂuorescence techniques were used to detect the

expression of γ-H2AX in either A549 or A549/cDDP cells.

As illustrated in Figure 5a, HY1-Pt can promote the

phosphorylation of H2AX in two cell lines more than cisplatin,

especially in A549/cDDP cells. Besides, the results showed that

cisplatin caused some DSBs in A549 cells, but such a damage

eﬀect obviously decreased in A549/cDDP cells. In contrast,

HY1-Pt caused DSBs in A549 cells at a higher degree than that

of cisplatin. Assessments of the γH2AX expression by ﬂow

cytometry further veriﬁed that HY1-Pt could lead to severe

DSBs in both A549 and A549/cDDP cells, whose eﬀect was

obviously higher than cisplatin (Figure S5). Furthermore, as

shown in Figure 5b, HY1-Pt also caused more serious DSBs in

A549/cDDP cells with stronger ﬂuorescence intensity than

that in A549 cells.

we explored whether HY1-Pt retained the CK2 inhibitory

activity of HY1.^27,28As illustrated in Figure S6, HY1-Pt

showed dose-dependent inhibition of CK2 enzyme activity,

and its IC₅₀was up to 1.14 0.24 nM. In addition, in either

A549 or A549/cDDP cells, HY1-Pt can inhibit the

phosphorylation of Akt1 serine 129 and Cdc37 serine 13 in

a dose-dependent manner (Figure 6a). The potent inhibition

of HY1-Pt on CK2 prompted us to further explore the binding

mode between HY1-Pt and CK2α protein through molecular

docking. The template of the co-crystal structure of CK2α

complexed with CX-4945 (PDB ID: 6isj) was used. As shown

in Figure 6b, the entire CK2α molecular structure is

completely wrapped in the CK2α active cavity. In addition,

the hydrogen atoms on the amino group act as hydrogen bond

donors form hydrogen bonds with ASP-174 and ASP-155

residues, respectively, while the carbonyl and oxygen atoms in

the ester bond act as hydrogen bond receptors to form

hydrogen bonds with LYS-157 and LYS-48 residues,

respectively. These four hydrogen bonds ﬁrmly rivet HY1-Pt

in the active cavity of CK2α with the lowest binding energy of

−11.21 kcal/mol. These results prove that the hybrid of HY1

into DN604 makes HY1-Pt not only maintain the cytotoxicity

of the active Pt(II) unit but also keep the ability of CX-4945 to

target and inhibit CK2α.

Mediator of DNA damage checkpoint 1 (MDC1) is an

important protein involved in the DSB response pathway.⁵⁰

Using immunoﬂuorescence technique, we checked MDC1 to

explore whether HY1-Pt can inhibit DNA damage repair while

promoting DNA damage. As shown in Figure 6c, HY1-Pt leads

to dramatic reduction in co-localization of MDC1/aprataxin

and the damaged DNA compared with cisplatin in either A549

or A549/cDDP cells, demonstrating that HY1-Pt can suppress

extensive DNA damage repair via inhibiting CK2 compared

with cisplatin.

2.8. Investigation on the Inhibition of A549/

cDDP^CD44brightCells. The expression of CSC surface antigens

plays a key role in invasiveness, promoting cancer recurrence.⁵¹

In solid cancer, CSCs were deﬁned based on CD44 expression

by ﬂow cytometry as CD44^brightpopulation.⁵²In order to

further investigate the inhibitory activity and mechanism of

HY1-Pt on CSCs, CD44^brightpopulation cells in A549/cDDP

were sorted by a ﬂow cytometer. First, using CX-4945, HY1

and cisplatin as positive controls, the inhibition rate of HY1-Pt

to A549/CDDP^CD44brightcells was detected by MTT assay. As

shown in Figure 7a, HY1-Pt maintained a high sensitivity to

A549/CDDP^CD44brightcells with an inhibition rate of 68.36%,

much higher than that of cisplatin (11.47%). In addition, the

2.7. Investigation on the Inhibition of HY1-Pt on

CK2α and DNA Damage Repair. Since CK2 can be used as

a key director in DNA damage repair and cancer cell stemness,

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Figure 8. Study on A549/cDDP^CD44brightcells. (a) qRT-PCR patterns of the ALDH1A1 in A549/cDDP^CD44brightcells treated with cisplatin and

HY1-Pt at 20 μM. (b) Expression of the ALDH1A1 in A549/cDDP^CD44brightcells was explored by western blot after treatment with 10 μM cisplatin

and HY1-Pt at concentrations of 10, 20, and 40 μM, respectively, for 24 h. (c) qRT-PCR patterns of the stem cell genes Nanog, SOX2, and OCT4

in A549/cDDP^CD44brightcells treated with cisplatin and HY1-Pt at 20 μM, respectively. (d) Morphological observation and (e) the number of

A549/cDDP^CD44brightcells tumorsphere treated with cisplatin and HY1-Pt at two doses (5 and 10 μM), respectively. (f) Expression of DKK1, p-

GSK-3β(ser9), and β-catenin in A549/cDDP cells was explored by western blot after treatment with HY1-Pt at concentrations of 10, 20, and 40

μM, respectively, for 24 h. Error bars = SD (n = 3), *P < 0.05, **P < 0.01.

expression of CSC surface antigens CD133⁺in A549/

cDDP^CD44brightcells was 24.37%, which was 7.25 times higher

than that in A549/cDDP cells (Figure S7). Notably, HY1-Pt

also showed dose-dependent inhibition eﬀects, and the

inhibition rate reached 76.78% at 20 μM. In contrast, cisplatin

had hardly signiﬁcant inhibitory activity (Figure 7b).

and OCT4, there is no doubt that HY1-Pt signiﬁcantly down-

regulated the expression of SOX-2 and OCT4 as well, whereas

cisplatin showed the opposite eﬀect. This has a correlation

with the capability of HY1-Pt to inﬂuence stem cells, but

cisplatin preferentially attacks non-stem cells, promoting the

CSC level in the cell population. In addition, the multicellular

sphere formation of A549/cDDP^CD44brightcells after treatment

with cisplatin and HY1-Pt at diﬀerent concentrations,

respectively, was evaluated. Obvious phenotypic changes

happened in these A549/cDDP^CD44bright-derived tumorsphere

in each group. Compared with the control group, the size and

number of A549/cDDP^CD44bright-derived tumorsphere treated

with HY1-Pt were signiﬁcantly reduced, the larger the drug

concentration is, the smaller the volume and the less the

number of tumorsphere are. Although cisplatin has no

signiﬁcant eﬀect on the number and size of tumorsphere, it

may promote the growth of tumorsphere when the

concentration is high (Figure 8d,e). These results hint that

our Pt(II)-based compound may overcome cisplatin resistance

by inhibiting cancer cell stemness.

It is known that stem cells are primarily controlled by Wnt/

β-catenin, Notch, Hedgehog/Gli1, and BMI-1 signal trans-

duction pathway.⁵⁴Therefore, we tried to learn how HY1-Pt

aﬀects Wnt/β-catenin CSCs signal transduction pathway in

A549/cDDP cells via western blot technique. As illustrated in

Figure 8f, the expression of anticancer protein DKK1 was

upregulated with the increasing concentrations of HY1-Pt,

while that of the phosphorylation of GSK-3β(ser9) protein was

signiﬁcantly down-regulated. In addition, HY1-Pt signiﬁcantly

inhibited β-catenin in a dose-dependent manner in A549/

cDDP cells. Therefore, HY1-Pt can inhibit the signaling

pathway of Wnt/β-catenin. It has already been proved that in

The eﬀect of HY1-Pt on ALDH1A1 expression in A549/

cDDP^CD44brightcells was also investigated. As expected, the

transcriptional levels of ALDH1A1 showed a remarkable

decrease in A549/cDDP^CD44brightcells after being treated with

HY1-Pt at 20 μM for 24 h. However, at the same dose,

cisplatin promoted the expression of ALDH1A1 (Figure 8a).

Western blot analysis conﬁrmed this result as well. Besides,

HY1-Pt also showed a dose-dependent inhibition eﬀect on

ALDH1A1 in A549/cDDP^CD44brightcells (Figure 8b). SOX2,

OCT4, and Nanog, essential transcription factors to maintain

the characteristics of stem cells, are currently recognized as the

signature genes of CSCs, which have an important eﬀect in

maintaining self-renewal, proliferation, and pluripotency in

malignant tumors.⁵³To evaluate the impact of HY1-Pt on

cancer cell stemness, we quantitatively analyzed the tran-

scription levels of the signature genes of CSCs in A549/

cDDP^CD44brightcells. As illustrated in Figure 8c, the transcript

levels of the transcription factors in A549/cDDP^CD44brightcells

treated with cisplatin and HY1-Pt were signiﬁcantly diﬀerent.

The transcript levels of Nanog were obviously increased after

being treated with cisplatin, showing its anti-diﬀerentiation

eﬀect on CSCs, which would lead to the enrichment of the

CSC population, in accordance with earlier reports.⁵¹

Importantly, Nanog was down-regulated after being treated

with HY1-Pt, indicating a diversion of the population from

stemness. Due to the synergistic eﬀect of Nanog with SOX-2

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Figure 9. Study on mitochondrial apoptosis pathway. Statistical changes of (a) ROS-positive cells and (b) MMP in A549 and A549/cDDP cell

treatment with CX-4945, HY1, cisplatin, and HY1-Pt at 20 μM for 24 h. (c) Expression levels of Bcl-2, Bax, pro-caspase 3, pro-caspase 9, and

cleaved-PARP in A549 and A549/cDDP cells were explored via western blot after treatment with cisplatin and HY1-Pt at 20 μM for 24 h. Error

bars = SD (n = 3), *P < 0.05, **P < 0.01.

the nucleus, β-catenin binds to TCF/LEF transcription factor

family to activate the promoter of target genes of β-catenin,

thus activating the target genes of Wnt signaling pathway like

CD44, CD24, CD133, ABCG2, ALDH1A1, EpCAM, MMP7,

Vimentin, and Slug. As Wnt1 can increase expression of

ALDH1A1, it promotes the expression of Akt and β-catenin as

well.⁵⁵Phosphorylated Akt has been reported to inactivate

GSK-3β by the phosphorylation of GSK-3β at ser9,

suppressing its capability to degrade β-catenin.^56,57So, it is

suggested that HY1-Pt might regulate the expression of

ALDH1A1 and CD133 via the Akt-GSK-3β(ser9)-Wnt/β-

catenin signaling pathway, thereby inhibiting cancer cell

stemness.

2.9. Study on Mitochondrial Apoptosis Pathways.

Reactive oxygen species (ROS) are inevitable products of cell

metabolism, but accumulation of ROS usually oxidizes and

damages the mitochondria of cells, and metal compounds

induce excess ROS accumulation by disrupting intracellular

redox equilibrium.⁵⁸Mitochondria has emerged as a crucial

coordinator of the cell death signaling network, including

stimulation of caspases and other processes related to cell

death.⁵⁹The mitochondrial membrane potential (MMP)

change in cancer cells is also considered as a key pro-apoptotic

marker for early apoptosis.⁶⁰Therefore, it is interesting to

investigate whether HY1-Pt can induce production of

intracellular ROS, thereby reducing MMP and then inhibiting

the caspase signaling pathway. As shown in Figures 9a and S8,

in A549 and A549/cDDP cells, HY1-Pt can induce the

production of a large amount of intracellular ROS, and the

proportion of ROS-positive cells is 62.4 and 65.7%,

respectively. In contrast, in A549/cDDP cells, the amount of

ROS induced by cisplatin is signiﬁcantly lower than that of

HY1-Pt, the proportion of ROS-positive cells induced by

cisplatin is 2.32-times lower than that of HY1-Pt. Images of

ROS in two cancer cells stained with DCFH-DA (Figure S9)

also indicate that the ﬂuorescence intensity of HY1-Pt is

greater than that of cisplatin, conﬁrming the result obtained by

ﬂow cytometry. In order to investigate the ROS damage to the

mitochondrial membrane by HY1-Pt, we measured the change

of MMP by ﬂuorescent probe JC-1. As shown in Figures 9b

and S10, in both A549 and A549/cDDP cells, HY1-Pt can

seriously induce JC-1 monomer, especially in A549/cDDP

cells, the number of JC-1 monomers induced by HY1-Pt is

3.19-fold as much as that by cisplatin. Meanwhile, more JC-1

monomers were observed in cells treated with HY1-Pt in

ﬂuorescent images (Figure S11). As illustrated in Figure 9c,

HY1-Pt can signiﬁcantly inhibit the caspase signaling pathway,

which is manifested as the decreased expression of pro-caspase-

3 and pro-caspase-9 compared to the control group. Moreover,

signiﬁcant up-regulation of Bax, cleaved-PARP, and down-

regulation of Bcl-2 in A549/cDDP cells were observed after

treatment of HY1-Pt, which were greater than those induced

by cisplatin. Diﬀerent from the dysfunction of cisplatin in

A549/cDDP cells owing to drug resistance, HY1-Pt behaved

in the same way as that in A549 cells as well. These results

imply that as a potent Pt(II) antitumor agent, HY1-Pt can

induce to produce a great deal of ROS, reduce MMP, and

promote the apoptosis of cancer cells through the mitochon-

drial apoptosis pathway.

2.10. Pharmacokinetic Study. As the main hydrolytic

product of HY1-Pt, HY1 generated by the hydrazine bond-

breaking under a tumor acidic condition was ﬁrst studied in

human, rat, and mouse liver microsomes for evaluating its

metabolic stability. As shown in Table 2, the metabolism of

HY1 is not much stable in these three types of liver

microsomes. In addition, the clearance rate (Cl_int) of HY1 in

these liver microsomes is rather fast, especially in the mouse

liver ones.

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Pt with that of cisplatin reported by Tanji Takada and co-

workers.⁶¹At the same dosage (10 mg/kg) by the same

administration (iv), the half-life (t_1/2) of cisplatin in rats is 5.94

h, slightly higher than that of HY1-Pt (5.1 h). However,

cisplatin was found to be diﬃcultly eliminated with a clearance

rate (CL) of 0.06 L·h⁻¹·kg⁻¹, and the residence time is long

(MRT = 7.0 h). In comparison, HY1-Pt has a higher clearance

rate (CL = 3.9 L·h⁻¹·kg⁻¹) and shorter mean residence time

(MRT = 2.7 h) in rats. These data indicated that HY1-Pt had

an acceptable PK behavior superior to cisplatin, which

encouraged us to conduct in vivo tests on its anti-tumor

activity.

Table 2. Liver Microsomal Enzyme Stability of HY1

species

HY1

human

rat

mouse

a

T_1/2(min)

23.88

72.80

37.83

65.66

15.55

351.00

b

Cl_int(mL/min/kg)

a

b

T_1/2is deﬁned as half-life. Cl_intis deﬁned as clearance.

In addition, the pharmacokinetic (PK) analysis of HY1-Pt in

Sprague-Dawley (SD) rats following a single 10 mg/kg dose

administration by intravenous injection (iv) was performed. As

summarized in Table 3, HY1-Pt demonstrated a moderate

2.11. In Vivo Antitumor Activity. Based on the above

study, in vivo antitumor tests of HY1-Pt in A549 xenograft

models were undertaken, in which animals were randomly

divided into ﬁve groups as vehicle, cisplatin (4 mg/kg), and

HY1-Pt at three doses (10, 20, 40 mg/kg). Due to the severe

toxicity of cisplatin, its dose was limited only to the equal

molar amount of platinum in HY1-Pt dosed at 10 mg/kg. As

illustrated in Figure 10, the inhibitory rate (86.92%) of HY1-Pt

at low dose (10 mg/kg) is even higher than that (66.51%) of

cisplatin (4 mg/kg), despite two agents exhibited comparable

in vitro cytotoxicity against A549 cells. It is noticed that HY1-

Pt displayed a dose-dependent inhibitory eﬀect on tumor

growth with a maximum inhibitory rate of 95.03% at 40 mg/

kg.

Table 3. Pharmacokinetic Parameters of HY1-Pt and

Cisplatin in SD Rats

dose (10 mg/kg)

a

parameter

_1/2/h

CL/L·h⁻¹·kg⁻¹

AUC_0‑t/ng h·mL⁻¹

AUC_0‑∞/ng h·mL⁻¹

V_d/L·kg⁻¹

HY1-Pt

cisplatin

T

5.1 0.6

3.9 0.5

2857 374.0

2611 371.0

28.9 7.0

2.7 0.0

5.9 0.7

0.06 0.0

26920 2050

66370 6360

0.6 0.1

MRT_0‑t/h

MRT_0‑∞/h

2.7 0.01

7.0 0.8

a

Data were adopted from ref 61.

To learn whether HY1-Pt can overcome cisplatin resistance

in vivo, we also tested the antitumor activity of HY1-Pt at two

doses (10 and 20 mg/kg) in A549/cDDP xenograft mice with

cisplatin as the positive drug. The mice were administered with

the formulations in the same way as that in the A549 xenograft

half-life and plasma clearance. In addition, HY1-Pt also had a

good apparent volume of distribution, a relatively larger area

under the concentration−time curve, and an acceptable mean

residue time. We also compared the pharmacokinetics of HY1-

Figure 10. In vivo A549 xenograft model study of HY1-Pt and cisplatin. (a) During the observation period, tumor volumes of animals from each

group, including vehicle, cisplatin (4 mg/kg, iv, once a week for 4 weeks) and HY1-Pt (10, 20 and 40 mg/kg, iv, once a week for 4 weeks). (b)

Weights of tumors resected from all groups of mice on the last day. (c) Inﬂuence of diﬀerent samples on body weight changes of the animals. (d)

Images of tumors from ﬁve groups of mice at the end of the experiment. Error bars = SD (n = 5), **P < 0.01, ^##p < 0.01.

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Figure 11. In vivo A549/cDDP xenograft model study of HY1-Pt and cisplatin. (a) During the observation period, tumor volumes of animals in

each group, including vehicle, cisplatin (4 mg/kg, iv, once a week for 4 weeks), and HY1-Pt (10 and 20 mg/kg, iv, once a week for 4 weeks). (b)

Weights of tumors resected from all groups of sacriﬁced mice on the last day. (c) Inﬂuence of diﬀerent samples on body weight changes of the

animals. (d) Images of tumors from ﬁve groups of mice at the end of the experiment. Error bars = SD (n = 5), **P < 0.01, ^##p < 0.01.

model. As shown in Figure 11, cisplatin dosed at 4 mg/kg

displays much weaker activity with an inhibitory rate of 9.09%

owing to the serious resistance as predicted. However, HY1-Pt

dosed at 10 mg/kg with an equivalent molar amount of

platinum in cisplatin is able to eﬀectively inhibit the tumor

growth at a rate of 72.72%. Particularly, HY1-Pt dosed at 20

mg/kg has the highest inhibitory rate of 82.64%, showing the

dose dependence of HY1-Pt treatments.

More signiﬁcantly, there is no conspicuous change in body

weight in all the mice treated with HY1-Pt, revealing that

HY1-Pt has low systemic toxicity and good safety (Figures 10c

and 11c). Furthermore, the H.E. staining of slices from heart,

liver, spleen, lung, and kidney of an animal randomly selected

from each tested group in both A549 and A549/cDDP models

demonstrates that HY1-Pt at the used dosages has almost no

toxic eﬀects on organ tissues compared with cisplatin (Figures

S12 and S13). These results indicate that HY1-Pt not only

eﬀectively inhibits cisplatin-mediated resistance in NSCLC

cells in vitro but also exhibits strong anticancer activity with

low toxicity both in the A549 xenograft model or in the A549/

cDDP xenograft models.

cancer cells with HY1-Pt can dramatically increase the level of

ROS, reduce MMP, and promote apoptosis of cancer cells

through the mitochondrial apoptosis pathway. More signiﬁ-

cantly, HY1-Pt can eﬀectively overcome cisplatin resistance,

exhibiting potent in vivo antitumor activity in both A549 and

A549/cDDP xenograft models with low toxicity. Overall, as the

ﬁrst example of a CSC inhibitor used in conjugation with other

bioactive species in a molecular entity for drug resistance

reversal, HY1-Pt has a potential as a Pt(II)-based drug to treat

with all kinds of solid tumors including NSCLC.

4. EXPERIMENTAL SECTION

4.1. Materials and Instrument. All chemicals in analytical purity

were purchased commercially from InnoChem (Beijing) Technology

Co., Ltd. and Sann Chemical Technology (Shanghai) Co., Ltd. and

used without further puriﬁcation. DN604 and BBI608 were

synthesized according to the method reported in previous

literature.^38,62¹H and ¹³C NMR spectra were recorded on a Bruker

600 MHz spectrometer. High-resolution mass spectroscopy (HR-MS)

was measured by an Agilent 6224 ESI/TOF MS instrument. The

purity of the compounds was detected by a reversed-phase high

performance liquid chromatogram (RP-HPLC) (waters, 2545 system)

on an ODS column (250 × 4.6 mm, 5 μm). Flow cytometry studies

were performed on a BD FACSCalibur ﬂow cytometer. An Olympus

FV1000 confocal microscope was used to record the confocal images.

Besides, all cells used in this work were purchased from Biorn

Lifescience Co., Ltd. Pro-caspase 3, Pro-caspase 9, Bcl-2, Bax,

Cleaved-PARP, phosphor-AKT (ser129), phosphor-Cdc37 (ser13),

ALDH1A1, γ-H2AX, MDC1, DKK1, phospho-GSK-3β (ser9), β-

catenin, and β-actin antibodies were all purchased from Beyotime

Biotechnology.

3. CONCLUSIONS

Our study indicates that HY1-Pt has potent cytotoxicity

activity toward the cancer cells of the tested but with low

toxicity in normal cells. Notably, cisplatin-induced resistant

cancer cells are also sensitive to HY1-Pt as expected. Further

research on the mechanism of action reveals that HY1-Pt can

target CK2 protein, reinforce cellular accumulation of

platinum, inhibit cancer cell stemness, and suppress DNA

damage repair, eﬀectively reversing cisplatin resistance through

the signaling pathway of Wnt/β-catenin by inhibiting

expression of the signature genes of CSCs in A549/cDDP

cancer cells and CK2 overexpression. Besides, treatment of

4.2. Chemistry. 4.2.1. Synthesis and Characterization of

Compound 7 and CX-4945. Compound 7 and CX-4945 were

synthesized in accordance with the procedures reported previously.²⁵

Compound 7: ¹H NMR (600 MHz, DMSO-d₆): δ 10.09 (s, 1H), 9.91

(s, 1H), 8.94 (d, J = 5.6 Hz, 1H), 8.79 (d, J = 8.5 Hz, 1H), 8.77 (d, J =

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5.6 Hz, 1H), 8.34 (t, J = 1.9 Hz, 1H), 8.17 (dd, J = 6.3, 1.4 Hz, 1H),

7.90 (dd, J = 8.4, 1.7 Hz, 1H), 7.42 (t, J = 8.1 Hz, 1H), 7.12 (dd, J =

7.9, 1.4 Hz, 1H), 3.92 (s, 1H) ppm. CX-4945: H NMR (600 MHz,

DMSO-d₆): δ 12.04 (s, 1H), 10.30 (s, 1H), 10.16 (s, 1H),9.08 (d, J =

5.8 Hz, 1H), 9.00−8.97 (m, 1H), 8.94 (d, J = 8.5 Hz, 1H), 8.31−8.28

(m, 2H), 8.12 (d, J = 8.2 Hz, 1H), 8.03 (dd, J = 8.4, 1.5 Hz, 1H), 7.45

(t, J = 8.1 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H) ppm.

(500.00, 100.00, 20.00, 1.00, 0.80 nM) were added to the well as the

“positive control”, with the same solution without the inhibitor as

“blank”. After sealing the plate with a sealing ﬁlm, the setup was

incubated at 37 °C for 30 min. After that, conjugate reagent (50 μL)

was added to each well, excepting blank holes, and incubated at 37 °C

for another 30 min. The color developing reagents A (50 μL) and B

(50 μL) were successively added to each hole, and the mixture was

shaken gently and mixed well, avoiding light for 10 min, and then stop

buﬀer (50 μL) was added to each hole. The reaction mixtures were

incubated at room temperature for 10 min and the value of O.D. was

measured via an enzyme-labeling instrument at a wavelength of 490

nm. The IC₅₀value of each compound represented as the mean SD

was obtained by three parallel experiments.

4.5. Western Blot Tests. A549 and A549/cDDP cells were

diluted into 1 × 10⁵cells/mL mixed liquid with the corresponding

medium and spread in a six-well plate and cultured at 37 °C for 12 h

and then treated with 20 μM of the tested compounds for 24 h. Two

cancer cells were collected, centrifuged, and washed with phosphate

buﬀer saline (PBS) and then lysed in lysis buﬀer (100 mM Tris-Cl,

pH 6.8, 4% (m/v) sodium dodecyl sulfonate, 20% (v/v) glycerol, 200

mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl ﬂuoride, and 1

g/mL aprotinin) and centrifuged at 15000 rpm for 15 min at 4 °C and

collected. The concentrations of total proteins were measured with

BCA protein assay reagents (Imgenex, USA). Protein (20−100 μg)

prepared from the indicated cells was loaded per lane and

electrophoresed in 8 or 10% sodium dodecyl sulfate polyacrylamide

gel electrophoresis and then transferred to poly(vinylidene diﬂuoride)

Hybond-P membrane (GE Healthcare) using a transblot apparatus

(Bio-Rad, USA). The blots were blocked with 5% nonfat milk in

Trisbuﬀered saline with Tween 20 (TBST) buﬀer for 1 h and co-

incubated with primary antibodies (anti-p-AKT(ser129), anti-p-

Cdc37(ser13), anti-ALDH1A1, anti-γ-H2AX, anti-DKK1, anti-p-

GSK3β(ser9), anti-β-catenin, anti-Bcl-2, anti-Bax, anti-pro-caspase-3,

anti-pro-caspase-9, anti-cleaved-PARP, and anti-β-actin and diluted in

TBST overnight at 4 °C. The membrane was washed with TBST and

incubated with secondary antibodies conjugated with peroxidase for 2

h at 37 °C, then washed twice with TBST buﬀer and PBS once, and

visualized with chemiluminescence reagent (Thermo Fischer

Scientiﬁcs Ltd.)

4.6. ALDH1A1 Enzyme Activity Assay. ALDH1A1 kinase

(human) assay/inhibitor screening assay kit and ALDH1A1 enzyme

were purchased from Shanghai Fusheng Industrial Co., Ltd.

(Shanghai, China). The inhibitory activity of CX-4945 and HY1 on

ALDH1A1 enzyme was detected according to manufacturer’s

instruction provided in the ALDH1A1 assay kit. The speciﬁc test

procedure is the same as the CK2 kinase (human) assay/inhibitor

screening assay kit.

4.7. Cell Culture. Six diﬀerent cells were used in this experiment

including A549 (non-small cell lung cancer), A549/cDDP (cisplatin-

resistant lung cancer), PC-3 (prostate cancer), HepG2 (liver cancer),

T24 (bladder cancer), and LO2 (normal live cells). All of them were

originally purchased from the Cell Bank of Shanghai Institute of Cell

Biology. A549, A549/cDDP, and PC-3 cells were cultured with a

complete medium containing 10% fetal bovine serum (FBS) and 90%

Roswell Park Memorial Institute-1640 (RPMI-1640) incomplete

medium at 37 °C in 5% CO₂. HepG-2, T24, and LO2 cells were

cultured at 37 °C in 5% CO₂with Dulbecco’s modiﬁed Eagle medium

supplemented with 10% FBS. The culture medium was also

supplemented with ^D-glucose of 2 g/L, L-glutamine of 0.3 g/L,

sodium bicarbonate of 2 g/L, penicillin of 80 U/mL, and

streptomycin of 0.08 mg/mL, kept in a constant temperature

incubator containing 5% CO₂at 37 °C.

1

4.2.2. Synthesis and Characterization of HY1. To a solution of

compound 7 (1.09 g, 3 mmol) in MeOH (20 mL) was slowly added

hydrazine hydrate (1.5 g, 30 mmol) at room temperature. The

reaction mixture was heated to reﬂux for 72 h and monitored with

thin layer chromatography (TLC). After the substrate was completely

consumed, the reaction liquid was slowly poured into the ice water

mixture to separate out the yellow solid, ﬁltering the precipitate, and

washed with ice methanol (3 × 10 mL) to obtain the crude product of

HY1. The crude product was recrystallized with 1, 4-dioxane (10 mL)

1

to get a bright yellow solid (0.989 g). Yield: 90.8%. H NMR (600

MHz, DMSO-d₆): δ 10.14 (s, 1H), 10.08 (s, 1H), 9.63 (s, 1H), 8.95

(d, J = 6.0 Hz, 1H), 8.80 (d, J = 12.0 Hz, 1H), 8.55 (d, J = 6.0 Hz,

1H), 8.33 (t, J = 1.8 Hz, 1H), 8.21 (d, J = 1.6 Hz, 1H), 8.07 (d, J = 6.0

Hz, 1H), 7.91 (dd, J = 12.0, 1.6 Hz, 1H), 7.42 (t, J = 6.0 Hz, 1H),

7.12 (dd, J = 8.0, 1.4 Hz, 1H), 4.63 (s, 2H) ppm. ¹³C NMR (150

MHz, DMSO-d₆): δ 165.77, 150.47, 148.13, 147.69, 143.76, 142.40,

134.73, 133.35, 130.52, 127.52, 126.26, 124.17, 123.00, 122.83,

122.60, 121.47, 120.57, 119.56, 116.79 ppm. HR-MS (m/z): (ESI)

calcd for C₁₉H₁₄ClN₅O [M + H]⁺, 364.0965; found, 364.0967.

4.2.3. Synthesis and Characterization of HY1-Pt. HY1 (0.181 g,

0.5 mmol), DN604 (0.193 g, 0.5 mmol), and 3−5 drops of acetic acid

were mixed in methanol (20 mL). The mixture was stirred and heated

at 60 °C for 48−72 h in the dark. The reaction was monitored by

TLC. After the reaction ﬁnished, the resulting deposits were ﬁltered

and washed with methanol (3 × 10 mL) to obtain the crude product

HY1-Pt. The crude product was recrystallized with dimethylforma-

mide (DMF)/1, 4-dioxane (1:4, v/v) to get a bright yellow solid

(0.365 g). Yield: 93.4%. ¹H NMR (600 MHz, DMSO-d₆): δ 11.15 (s,

1H), 10.19 (s, 1H), 9.67 (s, 1H), 8.99 (d, J = 4.2 Hz, 1H), 8.86 (d, J =

7.7 Hz, 1H), 8.58 (d, J = 3.8 Hz, 1H), 8.26 (s, 1H), 8.23 (s, 1H), 8.14

(d, J = 7.2 Hz, 1H), 7.93 (d, J = 7.3 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H),

7.14 (d, J = 6.9 Hz, 1H), 4.26 (s, 6H), 3.74 (s, 4H) ppm. ¹³C NMR

(150 MHz, DMSO-d₆): δ 176.45, 176.23, 163.28, 159.72, 150.55,

148.18, 147.80, 143.63, 142.33, 135.12, 133.28, 130.62, 127.50,

126.91, 124.23, 123.84, 122.80, 122.69, 121.73, 120.64, 119.68,

116.83, 56.40, 49.60, 46.96 ppm. HR-MS (m/z): (ESI) calcd for

C₂₅H₂₂ClN₇O₅Pt [M + H]⁺, 731.1091; found, 731.1353.

4.3. Purity of Final Compounds. The purity of all ﬁnal

compounds was determined by RP-HPLC (waters e2695, US)

equipped with an ODS column (250 × 4.6 mm, 5 μm) and a 2489

UV/vis detector. Detailed steps: CX-4945, HY1, and BBI608 were

dissolved in chromatographic pure methanol and ﬁltered by organic

syringe ﬁlters of 0.45 μM aperture. The purity of these compounds

was investigated at 25 °C as the column temperature, using

acetonitrile/water (55:45, v/v) as the mobile phase at the ﬂow rate

of 0.8 mL/min. The purity of CX-4945, HY1, and BBI608 was 99.43,

95.58, and 99.70%, respectively, analyzed by the peak area percentage

method. HY1-Pt was dissolved with a small amount of dimethyl

sulfoxide (DMSO), diluted with methanol, and ﬁltered with an

organic syringe ﬁlter of 0.45 μM aperture. The purity of HY1-Pt was

investigated at 25 °C as the column temperature, using methanol/

water (60:40, v/v) as the mobile phase at the ﬂow rate of 1.0 mL/

min. The purity of HY1-Pt was 97.40%, analyzed by the peak area

percentage method.

4.4. CK2 Activity Assay. CK2 kinase (human) assay/inhibitor

screening assay kit and CK2 enzyme were purchased from Shanghai

Fusheng Industrial Co., Ltd. (Shanghai, China). The inhibitory

activity of CX-4945, HY1, and HY1-Pt on CK2 enzyme was detected

according to manufacturer’s instruction provided in the CK2 assay kit.

Brieﬂy, the CK2 reaction solution was added into each well of 96-well

plates after thawing, and ﬁve diﬀerent concentrations of HY1-Pt

(500.00, 100.00, 20.00, 1.00, 0.80 nM) were added to each well as the

“test inhibitor” and ﬁve diﬀerent concentrations of CX-4945 and HY1

4.8. Detection of ALDH Activity. For the evaluation of the

expression levels of ALDH1A1 in A549 cells exposed to CX-4945 (20

μM) and HY1 (20 μM), ALDEFLUOR (Stem cell Technologies,

Vancouver BC, CA) staining was measured using the FITC channel of

ﬂuorescent activated cell sorting according to the manufacturer’s

instruction.

4.9. Real-Time Quantitative PCR. Total RNA was isolated by

using TRIZOL from A549 and A549/cDDP^CD44brightcells treated for

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24 h with equitoxic concentration of tested compounds corresponding

to IC₅₀,_72h. RNA was reverse-transcribed with Luna universal one-step

RT-qPCR (GeneAmp 9600, PerkinElmer), and the resulting cDNA

was subsequently submitted to thermal cycling on Quantitative Real-

time PCR (ABI step one plus, ABI). The thermal proﬁle was as

follows: reverse transcription for 10 min at 55 °C, initial denaturation

for 1 min at 95 °C, followed by 39 thermal cycles of denaturation for

10 s at 95 °C, and extension for 30 s at 60 °C. Primer sequences are

listed in Table S2. All runs included melting curve analysis and

template-free negative technical controls to conﬁrm speciﬁc single-

product ampliﬁcation. β-actin mRNA was used as an internal control.

The relative expression of mRNA is represented as fold increase

(2^−ΔΔCt).⁶³

4.10. Spheroid Formation Assay. Brieﬂy, the A549 and A549/

cCDDP^CD44brightcells were plated on Ultra-Low Attachment Surface

24 well-plates (3473, Corning) in serum-free 1640 medium

supplemented with B27 (1:50, 17504044, Invitrogen), epidermal

growth factor (EGF) (20 ng/mL, AF-100-15, Peprotech), and β-

ﬁbroblast growth factor (β-FGF) (10 ng/mL, 100−18B, Peprotech)

at a density of 800 cells per well. When the number of cells in a

spheroid were more than 10, it was considered to have formed a clone

sphere. After treatment with diﬀerent drug formulations for 10 days.

The number and sizes of tumorsphere were measured on day 10.

4.11. Assessment of CD44 Expression. A549 cells were seeded

on 6-well plates at the density of 1 × 10⁵cells per well. After

incubating the samples for 12 h, the cells were treated with CX-4945

(20 μM), BBI608 (20 μM), and HY1 (20 μM), respectively, for 24 h.

Then, the cells were stained with FITC-conjugated anti-CD44

according to the manufacturer’s respective instructions. CD44

expressions were assessed by ﬂow cytometry. Calculation of inhibition

rate was based on ﬂuorescence intensity.

4.12. Stability of HY1-Pt. HY1-Pt was dissolved in pH = 6.0 and

7.4 Hepes buﬀer (0.25 M) containing 2% DMF and incubated at 37

°C in the dark, respectively. The solution behavior of HY1-Pt was

detected and recorded by UV−vis (Shimadzu UV-2600) with

wavelength ranging from 225 to 450 nm and scanned every 30 min

for a total of 24 times. In addition, the stability of HY1-Pt was also

detected by RP-HPLC (waters, 2545 system) on an ODS column

(250 × 4.6 mm, 5 mm) at diﬀerent time points with an eluent of

methanol/water (60:40, v/v) at a ﬂow rate of 1 mL/min.

4.13. Measurement of Water/Octanol Partition Coeﬃcient

(log P_o/w). The lipophilicity (log P_O/W) of HY1-Pt was measured

using the shake-ﬂask method in the 1-octanol/distilled water system.

Octanol was saturated with a distilled water by shaking the two-phase

mixture overnight. A portion of 5 mL of pre-saturated octanol

containing HY1-Pt was incubated with 5 mL of distilled water in a 15

mL tube. The tube was shaken at room temperature in the dark for 24

h. Centrifugation was carried out at 2500 rpm for 15 min to separate

diﬀerent phases, and the platinum concentrations in these two phases

were determined by ICP-MS. The ﬁnal result was expressed as a mean

of three independent experiments. The log P_O/Wwas calculated using

the following equation

4.15. Cell Cycle Test. A549 and A549/cDDP cells were diluted

into 1 × 10⁵cells/mL mixed liquid with the corresponding medium

and spread in a six-well plate and cultured at 37 °C for 12 h. Two

cancer cells were incubated with 20 μM of the tested compounds for

24 h. The cells were collected into diﬀerent centrifugal tubes, added

about 500 μL of 70% ice ethanol, and placed at −20 °C for overnight.

Centrifugation was carried out at 1500 rpm for 5 min to remove the

supernatant, and then 1 mg/mL PI staining solution (containing 10%

RnaseA) was added to the cells and stored in the dark for 30 min. The

sample was measured via ﬂow cytometry (FAC Scan, Becton

Dickenson), and the data was analyzed with FlowJo software

(TreeStar, Inc.).

4.16. Apoptosis Assessment. Detection of apoptosis of A549

and A549/cDDP induced by CX-4945, HY1, cisplatin, and HY1-Pt

was made by the Annexin V-FITC apoptosis detection kit according

to the manufacturer’s protocol. The A549 and A549/cDDP cells in

logarithmic growth phase were collected and suspended with the

corresponding culture medium after centrifugation and plated on a

six-well plate with a cell concentration of 1 × 10⁵cells per well. After

the cells have completely adhered to the wall, the original culture

medium was replaced with a fresh medium and treated with 20 μM of

the tested compounds, respectively, for 24 h at 37 °C. Then, the cells

were incubated with Annexin V-FITC and then PI for 10 min in the

dark at room temperature; cells were analyzed by ﬂow cytometry

(FAC Scan, Becton Dickenson) and the apoptosis value was obtained

using Cell-Quest software (BD Biosciences).

4.17. Cellular Accumulation of Platinum. A549 and A549/

cDDP cells with a concentration of 1 × 10⁵cells/mL were seeded in

6-well plates until reached about 80% conﬂuence and then incubated

with cisplatin (20 μM) and HY1-Pt (20 μM), respectively, at 37 °C.

The cells were harvested and stored in a diﬀerent centrifuge after 12 h

co-incubation, washed with cold PBS (3 × 3 mL), and digested with

200 mL 65% HNO₃to obtain a completely uniform solution. After

diluted with pure water, intracellular platinum content was detected

via ICP-MS.

4.18. Comet Assay. A549 and A549/cDDP cells were incubated

with 20 μM of the tested compounds for 12 h, combined with molten

LM Agarose (Trevigen) at a ratio of 1:10 (v/v), and pipetted onto

Comet Slide (Trevigen) immediately. In the dark, slides were

incubated at 4 °C for 10 min, and then the cells were immersed in

prechilled Lysis buﬀer and incubated at 4 °C for 60 min. In the dark,

the DNA was immersed in the alkaline solution of pH > 13 (200 mM

NaOH, 1 mM EDTA) for 40 min. Single cell electrophoresis was

carried out at 25 V voltage for 25 min via alkaline electrophoresis

solution (200 mM NaOH, 1 mM EDTA). The slides were rinsed with

Tris-HCl solution of 0.4 mM of pH = 7.5 three times, stained with PI

for 30 min in the dark, and visualized by microscopy.

4.19. Immunoﬂuorescence Test. Brieﬂy, A549 and A549/cDDP

cells were grown to conﬂuence in sterile watchglasses for 24 h at 37

°C and then treated with 20 μM of CX-4945, HY1, cisplatin, and

HY1-Pt for 24 h. All cells were washed with PBS, ﬁxed in 4%

paraformaladehyde/PBS for 30 min, and permeabilized with PT-5

solution [0.3% Triton X-100 in PBS] for 30 min at 4 °C. The cells

were blocked by incubating with PTB-5 [0.5% BSA in PBS] for 1 h at

room temperature. Dishes were then incubated with the primary

antibody (1:500) overnight at 4 °C. Cells were stained with the

secondary antibody conjugated with Alexa 488 (1:100, Invitrogen,

Carlsbad, USA). Microscopy was performed on a Leica TCS NT

confocal scanner equipped with an ArKr-Laser on the Leica DM IRBE

inverted microscope (lens: HCX PlanApo 63x oil/NA1.32). Confocal

images were displayed as maximum projections and assembled in

Adobe Photoshop 7.0.

4.20. Molecular Docking. Molecular docking of HY1-Pt to

CK2α (PDB ID: 6isj) was carried out by AutoDock, in which the

Iterated Local Search Globule Optimizer was applied as optimization

algorithm.⁶⁴Ligand HY1-Pt was prepared in ChemBioDraw Ultra

14.0 and minimized with the ChemBio3D Ultra 14.0. Before the

docking process, the nature ligand and water molecules were removed

from the crystal structure. Subsequently, all hydrogen atoms were

added to each protein and ligand to be docked, and each coordinate

log P = log[([[C]_initial− [C]_final])/[C]_final

]

o/w

4.14. Cytotoxicity Measurement. MTT assay was used to

evaluate the cytotoxicity of all compounds synthesized in this work.

Diﬀerent cells were grown on 96-well plates at a cell density of 1 ×

10⁵cells/mL and incubated at an atmosphere of 5% CO₂and 95% air

at 37 °C for 12 h. After the cells were completely attached, the

complete medium was replaced, and the samples were diluted into

ﬁve concentrations and added to the wells in turn. Each concentration

was set up with three identical experimental groups to ensure that the

volume of each well was 100 μL. The MTT (3-(4,5)-

dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide) solution

with the concentration of 5 mg/mL was added to each well after

72 h. Next, the culture medium was removed and about 100 μL of

DMSO was added to each well to dissolve completely after 3−4 h.

The O.D. value was read at 490 nm enzyme labeling instrument. The

IC₅₀values of each group can be calculated by SPSS software.

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ﬁle of protein and ligand was generated as PDBQT ﬁle using

AutoDockTools-1.5.6. A grid box for the binding site covered the

catalytic site of the protein or 50 Å in the three dimensions for the

allosteric binding site. The coordinate of the binding site is X =

33.303, Y = 6.104, Z = 14.131. The box had 0.375 Å grid spacing and

centered at the geometric center of the protein. When using Autodock

software to simulate the binding mode between HY1-Pt and CK2α

protein, the force ﬁeld parameters added to Pt were as follows

“atom_par Pt 2.75 0.080 12.000−0.00110 0.0 0.0 0 -1 -1 1 # non H-

bonding”. In each docking experiment, the best binding mode was

selected according to the binding aﬃnity calculated by the scoring

function in AutoDock. Docking results were analyzed and visualized

with PyMOL 2.4.

S47 LC-MS/MS analysis. Positive control samples were prepared as

described above, except for the test compound that was replaced with

the known P450 substrate (1 μM Midazolam).

4.26. Pharmacokinetic Assay. The pharmacokinetic proﬁle of

HY1-Pt was investigated in 12 h fasted male SD rat following a single

dose given intravenous injection (iv). HY1-Pt was dissolved in DMF:

Tween 80: 5% glucose injection = 10:2:88. Three male SD rats

weighing about 250−270 g were treated with HY1-Pt (10 mg/kg).

Blood samples (0.3 mL) of each rat were collected from the rat’s

posterior orbital vein at 0.0833, 0.25, 0.50, 0.75, 1, 2, 3, 4, 6, 8, 12, and

24 h following iv dosing. Samples were centrifuged at 4000 rpm for 10

min, and the plasma was collected and stored at −40 °C until analysis.

Samples were analyzed by LC-MS/MS technique. The data of blood

concentration (c)−time (T) measured after iv administration were

processed by Das 2.0 software. The appropriate mathematical model

was selected to ﬁt, and the pharmacokinetic parameters were

calculated.

Preparation of standard curves: 100 μL of blank plasma was added

to 10 μL of the standard solution of HY1-Pt at diﬀerent

concentrations (30, 60, 150, 300, 600, 1500, 3000, 6000, 15000,

and 30000 ng/mL), respectively, to prepare plasma samples

equivalent to ropivacaine with plasma mass concentrations of 3, 6,

15, 30, 60, 150, 300, 600, 1500, and 3000 ng/mL. The samples with

diﬀerent concentrations (5 μL) were analyzed by the LC-MS/MS

technique, the ratio Y of the peak area of each component to the

internal standard peak area was used to perform linear regression

operation on the plasma mass concentration X, and the regression

equation obtained was the standard curve. The regression equation of

HY1-Pt is Y = 0.6962X − 0.0137 (r = 0.9998), and the minimum

quantitative limit all is 3 ng/mL.

4.27. In Vivo Antitumor Eﬃcacy of HY1-Pt. (a) A549 xenograft

model: 5 × 10⁷A549 cells in 0.1 mL of sterile PBS were injected

subcutaneously into the ﬂank of ﬁve-week-old, weighing about 16−18

g, male BALB/c athymic nude mice; they were all purchased from

Shanghai Sipo-Bikai laboratory animal co. Ltd. (China). When the

tumors reached a volume of 100−120 mm³in all mice, 25 mice were

randomly divided into ﬁve groups with ﬁve mice per group and

treated via intravenous injection through the tail vein with 0.1 mL 5%

glucose injection (once a week for 4 weeks), cisplatin (4 mg/kg, once

a week for 4 weeks), and HY1-Pt (10, 20 and 40 mg/kg, once a week

for 4 weeks). Cisplatin was dissolved in normal saline, HY1-Pt was

dissolved in DMF: Tween 80: 5% glucose injection = 10:2:88. Tumor

volumes and body weights were recorded twice for a week. The

calculation formula of tumor volume (TV, mm³) is TV = 0.5 × length

× width². Growth curves were plotted using the average tumor

volume within each experimental group at the set time points via

origin 9.0. 25 mice were sacriﬁced after the last treatment; then tumor

weight was evaluated as the antitumor activity of the corresponding

groups, and the heart, liver, spleen, lung, kidney, and tumor tissues

were excised for H.E. staining. The body weight and physical state of

the mice were measured simultaneously as an indicator of systemic

toxicity.

4.21. A549/cDDP^CD44brightCell Sorting. In order to enrich the

cells by CSCs, A549/cDDP cells were stained for its CSC-marker

CD44 with FITC-conjugated antibodies and subsequently sorted with

magnetic anti-FITC microbeads. The eﬀectivity of sorting was further

checked by ﬂow cytometry (Figure S6a,b). CD44-birght A549/cDDP

(A549/cDDP^CD44bright) cells were selected for further experiments as

CSC-like population. The A549/cDDP cells were sorted by using the

magnetic separation method based on anti-ﬂuorophore microbeads

from Miltenyi Biotec, Gladbach, Germany. Brieﬂy, A549/cDDP cells

were stained with CD44-FITC for 10 min at 4 °C; then, the cells were

labeled with anti-FITC microbeads for 15 min at the same

temperature. Cells were magnetically sorted by using the magnetic

separation method based on anti-ﬂuorophore microbeads (MS

columns).

4.22. Assessment of CD133 Expression. The A549/

cDDP^CD44brightcells were seeded on 6-well plates at the density of 1

× 10⁵cells per well. After incubating the samples for 12 h, the cells

were treated with cisplatin (10 μM) and diﬀerent concentration of

HY1-Pt, respectively, for 24 h. Then, the cells were stained with PE-

conjugated anti-CD133 according to the manufacturer’s respective

instructions. CD133 expressions were assessed by ﬂow cytometry.

Calculation of the inhibition rate was based on ﬂuorescence intensity.

4.23. ROS Measurement. The A549 and A549DDP cells were

diluted into 1 × 10⁵cells/mL mixed liquid with the corresponding

medium and spread in a six-well plate and cultured at 37 °C for 12 h.

Two cancer cells were treated with various compounds: vehicle

(DMSO), CX-4945 (20 μM), HY1 (20 μM), cisplatin (20 μM), and

HY1-Pt (20 μM) for 24 h, respectively. The cells were washed two

times with PBS, and 2,7-dichlorodi-hydroﬂuorescein diacetate

(DCFH-DA) (Molecular Probe, Beyotime, Haimen, China) was

used as a probe to measure the level of ROS according to the diﬀerent

ﬂuorescence intensity by Confocal photography at the same voltage.

Besides, after diﬀerent groups of cells were washed twice with PBS,

the production of ROS was also detected by ﬂow cytometry.

4.24. Mitochondrial Membrane Potential Measurement.

The A549 and A549DDP cells were diluted into 1 × 10⁵cells/mL

mixed liquid with the corresponding medium and spread in a six-well

plate and cultured at 37 °C for 12 h and then treated with 20 μM of

the tested compounds for 24 h. In the dark, the medium was replaced

followed by staining with a JC-1 ﬂuorescent probe for 30 min at 37

°C, and then the cells were washed twice with PBS, and the eﬀects of

various compounds on MMP of tumor cells were analyzed with ﬂow

cytometer and confocal photography.

(b) A549/cDDP xenograft model: the procedure was the same as

that of the A549 xenograft model except that A549/cDDP cells were

used and HY1-Pt was given in two groups at doses of 10 and 20 mg/

kg, respectively.

4.28. Statistical Analysis. The data shown in this manuscript

4.25. Metabolic Stability. The metabolic stability of HY1 in

incubation with human, rat, and mouse liver microsomes was

determined through the commercially available service provided by

Shanghai ChemPartner Co., Ltd. (Shanghai, China). Liver micro-

somal incubations were conducted in triplicate. Incubation mixtures

(0.5 mg/mL microsome protein, pH 7.4 of 100 mM phosphate buﬀer,

1 μM compounds in DMSO) were ﬁrst shaken for pre-incubation at

37 °C. The reaction was initiated by adding NAPDH to obtain a

concentration of 1 mM NAPDH in the mixture. For metabolic

stability studies, aliquots of the incubation sample mixture were

collected at 0, 5, 15, 30, and 45 min. After collection of samples, the

reaction was terminated with DMSO containing the internal standard

(200 ng/mL Tolbutamide). The mixture was then centrifuged to

remove the protein, and the supernatant was subsequently applied to

were expressed as means

SD from at least three independent

experiments, each in triplicate samples for individual treatment or

dosage. Statistical analyses were performed using an unpaired, two-

tailed Student’s t-test, and signiﬁcance of diﬀerence is indicated as *P

< 0.05 and **P < 0.01.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00079.

■

sı

The synthetic route of CX-4945; the proportion of

CD44⁺subsets induced by CX-4945, BBI608, and HY1;

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this work. Nanjing Biorn Life Science Co. Ltd is acknowledged

for the in vivo tests.

RP-HPLC analysis on the stability of HY1-Pt; ﬂow

cytometry analysis on the cell cycles, apoptosis, and the

expression of γH2AX in A549 and A549/cDDP cells

induced by HY1-Pt and related sample; the inhibitory

rate curves of HY1-Pt on CK2 enzyme activity; ﬂow

cytometry analysis on the expression of CD133⁺in

A549/cDDP and A549/cDDP^CD44brightcells induced by

cisplatin and HY1-Pt; the production of ROS, changes

of JC-1 and MMP in A549 and A549/cDDP cells

induced by the HY1-Pt and related sample were

detected by the ﬂow cytometer and confocal laser

scanning microscope; H.E. staining of tissues in the nude

mice xenograft model of A549 and A549/cDDP; tables

of the stability of HY1 in incubation with human, rat,

and mouse liver microsomes; tables of the data of the

plasma concentrations at diﬀerent time points after

intravenous injection of HY1-Pt in rats; tables of the

inhibitory eﬀects of measured samples on the A549

cancer cell xenograft mice model; tables of the inhibitory

eﬀects of measured samples on the A549/cDDP cancer

ABBREVIATIONS

■

AKT, protein kinase; ALDH1A1, aldehyde dehydrogenase

1A1; A549, human NSCLC cell; A459/cDDP, resistant to

cisplatin A549 cells; Bax, Bcl-2 associated X protein; Bcl-2, B-

cell lymphoma-2 protein; cDDP, cisplatin; CK2, casein kinase

2; Clk2, cell division cycle-like kinase 2; CSC, cancer stem cell;

DCHF-DA, 2,7-dichlorodi-hydroﬂuorescein diacetate; DCM,

dichloromethane; DMEM, Dulbecco’s modiﬁed Eagle me-

dium; EGF, epidermal growth factor; β-FGF, β-ﬁbroblast

growth factor; FITC, ﬂuorescein isothiocyanate isomer; GSK-

3β, glycogen synthase kinase-3β; HepG2, human liver

hepatocellular cancer cell; HR-MS, high resolution mass

spectrometry; ICP−MS, inductively coupled plasma−mass

spectrometry; IC₅₀, the half maximal inhibitory concentration;

LO2, human normal hepatocytes cell; MMP, mitochondrial

membrane potential; MMP-7, matrix metalloproteinase 7;

NMR, nuclear magnetic resonance; PARP, poly ADP-ribose

polymerase; PBS, phosphate buﬀer saline; PC-3, human

prostate cancer cell; PI, propidium iodide; qRT-PCR,

quantitative real-time PCR; RF, resistant factor; ROS, reactive

oxygen species; γH2AX, phosphorylation of H2AX; RP-HPLC,

reversed-phase high performance liquid chromatography;

TMS, tetramethylsilane; T24, human bladder cancer cell;

UV−vis, ultraviolet and visible spectrophotometry

1

cell xenograft mice model; and ﬁgures of H NMR, ¹³C

NMR, HR-MS spectra, and RP-HPLC chromatograms

(PDF)

Molecular formula strings (CSV)

Atomic coordinates of HY1-Pt (PDB)

AUTHOR INFORMATION

■

Corresponding Author

REFERENCES

■

Shaohua Gou − Jiangsu Province Hi-Tech Key Laboratory for

Biomedical Research and Pharmaceutical Research Center

and School of Chemistry and Chemical Engineering,

Southeast University, Nanjing 211189, PR China;

orcid.org/0000-0003-0284-5480; Email: 2219265800@

qq.com

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Yuanjiang Wang − Jiangsu Province Hi-Tech Key Laboratory

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Xinyi Wang − Jiangsu Province Hi-Tech Key Laboratory for

Biomedical Research and Pharmaceutical Research Center

and School of Chemistry and Chemical Engineering,

Southeast University, Nanjing 211189, PR China

Gang Xu − Jiangsu Province Hi-Tech Key Laboratory for

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00079

Author Contributions

^§Y.W. and X.W. contribute equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We would like to thank National Natural Science Foundation

of China (Project no. 22077015), Fundamental Research

Funds for the Central Universities (Project 2242020K30059),

and Graduate Research and Innovation Fund of Jiangsu

Province, China (Grant KYCX19_0102) for ﬁnancial aid to

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Novel CK2-Specific Pt(II) Compound Reverses Cisplatin-Induced Resistance by Inhibiting Cancer Cell Stemness and Suppressing DNA Damage Repair in Non-small Cell Lung Cancer Treatments

DOI: 10.1021/acs.jmedchem.1c00079

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