1
Te-Chung Hsia 1,2 , Ju-Hwa Lin 3 , Shu-Chun Hsu 3 , Nou-Ying Tang 1 , Hsu-Feng Lu 4 ,
Shin-Hwar Wu 5 , Jaung-Geng Lin 1,* and Jing-Gung Chung 3,6,*
1 School of Chinese Medicine, 3 Department of Biological Science and Technology,
China Medical University, Taichung 404, Taiwan,
2
Department of Internal Medicine,
China Medical University Hospital, Taichung 404, Taiwan, 4 Department of Clinical
Pathology, Cheng Hsin General Hospital, Taipei 112, Taiwan,
5
Division of Critical
Care Medicine, Department of Medicine, Changhua Christian Hospital, Changhua
500, Taiwan,
6
Department of Biotechnology, Asia University, Wufeng, Taichung 413,
Taiwan
Running title: Cantharidin affects DNA damage and DNA repair protein expressions in NCI-H460 cells
Correspondence to : Prof. Jing-Gung Chung, Department of Biological Science and
Technology, China Medical University, No 91, Hsueh-Shih Road, Taichung 404,
Taiwan. Tel.: +886-4-2205-3366 ext 2161. Fax: +886-4-2205-3764.
E-mail: jgchung@mail.cmu.edu.tw
Prof. Jaung-Geng Lin, School of Chinese Medicine, China Medical University, No 91,
Hsueh-Shih Road, Taichung 40402, Taiwan. Tel: +886-4-22053366 ext 3113. Fax:
+886-4-2205 3764.
E-mail: jglin@mail.cmu.edu.tw

2
Abstract.
Cantharidin is one of the major compounds from mylabris and it has cytotoxic effects in many different types of human cancer cells. Previously, we found that cantharidin induced cell death through cell cycle arrest and apoptosis induction in human lung cancer NCI-H460 cells. However, cantharidin-affected DNA damage, repair and associated protein levels in NCI-H460 cells have not been examined. In this study, we determined if cantharidin induced DNA damage and condensation and altered levels of proteins in NCI-H460 cells in vitro . Incubation of NCI-H460 cells with 0, 2.5, 5, 10 and 15 μM of cantharidin caused a longer DNA migration smear
(Comet tail cantharidin also increased DNA condensation. These effects were dose-dependent. Cantharidin (5, 10 and 15 μM) treatment of NCI-H460 cells reduced protein levels of ataxia telangiectasia mutated (ATM), breast cancer 1, early onset
(BRCA-1), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O 6 -methylguanine-DNA methyltransferase (MGMT) and mediator of DNA damage checkpoint protein 1 (MDC1). Protein translocation of p-p53, p-H2A.X (S140) and MDC1 from cytoplasm to nucleus was induced by cantharidin in
NCI-H460 cells. Taken together, the present study showed that cantharidin caused
DNA damage and inhibited levels of DNA repair associated proteins. These effects may contribute to cantharidin-induced cell death in vitro .
Key words : Cantharidin, DNA damage, Comet assay, DNA repair, human lung cancer
NCI-H460 cells
Introduction
Cancer is a major cause of death worldwide. Lung cancer has both a high
prevalence and mortality cancers (Bostancioglu et al., 2013). In Taiwan, lung cancer
is associated with 36.8 deaths per 100,000 annually, and it is the leading cause of

3 death in males and females based on 2010 reports from the Department of Health,
R.O.C. (Taiwan). Numerous pharmacological approaches have been used for treating lung cancer but the outcomes are unsatisfactory. Efficacy is not optimal and side effects are common. New compounds from natural plants or live organisms including insects have been gaining attention. In the Chinese population, one of the animal-derived Chinese medicines is the dried body of mylabris ( Mylabris phalerata
Pallas). It has been used to treat malign sores and to relieve blood stasis (Wang et al.,
2000). Cantharidin, one of the compounds extracted from mylabris (Hundt et al., 1990;
Wang et al., 2000), inhibits cAMP phosphodiesterase activity (Zhang and Chen, 1985),
acts as a strong protein phosphatase, and it is a phosphatase 2A inhibitor (Graziano et
al., 1988; Li et al., 1993). It was reported that cantharidin may induce cystitis through
secondary necrosis and COX 2 over-expression in a human bladder carcinoma cell
line (Huan et al., 2006). Cystitis was caused by cantharidin administration in rats acting through c-Fos and COX-2 over-expression (Huan et al., 2012). Cantharidin is a
topical vesicant and keratolytic agent and it is available in Canada (Wong et al., 2012).
Recently, it was reported that the treatment of molluscum contagiosum (MC) by
cantharidin can experienced minimal side effects in clinical patients (Coloe Dosal et al., 2012).
Cantharidin induced apoptosis in human cancer cell lines such as colon cancer
cells (Huang et al., 2011; Liu et al., 2012), bladder cancer cells (Kuo et al., 2010),
pancreatic cancer cells (Li et al., 2010) and in multiple myeloma cells (Sagawa et al.,
2008). Recently, it was reported that cantharidin repressed MCF-7 cell adhesion to platelets through down-regulation of α2 integrin (Shou et al., 2013). Cantharidin can
cause cytotoxicity and cell death in different human cancer cell lines. There are no published studies on effects of cantharidin-induced DNA damage and DNA repair genes and proteins in cancer cells. Therefore, we investigated the effects of

4 cantharidin on DNA damage and DNA repair associated genes and proteins in
NCI-H460 human lung cancer cells. Cantharidin induced DNA damage and changed
DNA associated genes and protein levels in NCI-H460 cells.
Materials and methods
Chemicals and reagents. Cantharidin, dimethyl sulfoxide (DMSO), propidium iodide
(PI) and Trypsin-EDTA were purchased from Sigma Chemical Co. (St. Louis, MO,
USA). Minimum essential medium (MEM), fetal bovine serum (FBS), L-glutamine and penicillin-streptomycin were purchased from GIBCO
®
/Invitrogen Life
Technologies (Carlsbad, California, USA). Primary antibody (anti-ATM, -ATR,
-BRCA-1, -14-3-3σ, -DNA-PK and –MGMT) were obtained from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA, USA).
Cell culture. The NCI-H460 human lung cancer cell line was purchased from the Food
Industry Research and Development Institute (Hsinchu, Taiwan). Cells were maintained in MEM medium with 2 mM L-glutamine, 10% FBS, 100 Units/ml penicillin and 100 μg/ml streptomycin in a 75 cm 2 tissue culture flasks and grown at
37°C under a humidified 5% CO
2 atmosphere.
Cell viability assay. Cell viability was determined by counting viable cells using trypan
blue exclusions, and flow cytometry as described previously (Lu et al., 2009).
NCI-H460 cells (2x10
5
cells/well) in 12-well plates were incubated with 0, 5, 10, 15 and 20 μM of cantharidin for 24 h. Cells were stained with PI (5 μg/ml) and analyzed by flow cytometry (Becton-Dickinson, San Jose, CA, USA) or counted using a
microscope as previously described (Ji et al., 2012; Lu et al., 2009).

5
Comet assay for examining the DNA damage. NCI-H460 cells (2x10 5 cells/well) were placed into 12-well plates for 24 h then were treated with 0, 2.5, 5, 10 and 15 μM of cantharidin, vehicle (1 μl DMSO) and 0.1% of H
2
O
2
(positive control) for 24 h or exposed to different concentration of cantharidin for 48 h in DMEM medium. At the end of incubation, cells were harvested and DNA damage determined using the Comet
assay as described previously (Ji et al., 2012; Lu et al., 2009).
4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining for DNA damage and condensation. NCI-H460 cells (2×10 5 cells/well) were maintained in 6-well plates for
24 h and then were treated with 0, 1, 2.5, 5, 10 and 30 μM of cantharidin for 24 h.
Cells were then DAPI stained as described previously (Chiang et al., 2013; Chiu et al.,
2013). At the end of staining, the cells were examined and photographed using a
fluorescence microscope at 200x.
Western blotting assay for protein levels of associated with DNA damage and repair.
NCI-H460 cells (2x10
5
cells/well) were placed into 12-well plates for 24 h then were treated with 0, 5, 10 and 15 μM of cantharidin for 48 h. The total protein was determined by a Bio-Rad assay Kit. The protein levels of p53, p-p53, p-H2A.X,
MGMT, 14-3-3σ , ATM, BRCA-1, DNA-PK and MDC1 were examined by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western
blotting as described previously (Chiang et al., 2013; Chiu et al., 2013).
Protein translocation determined by confocal laser microscopy. NCI-H460 cells (5×
10
4
cells/well) were maintained on 4-well chamber slides and were incubated with 0,
10 and 15 μM of cantharidin for 24 h and then were fixed in 4% formaldehyde in
PBS for 15 min followed by addition of 0.3% Triton-X 100 in PBS for 15 min to

6 permeabilize the cells. For immunostaining, non-specific binding sites were blocked
by using 2% BSA as described previously (Wu et al., 2011; Yang et al., 2010). Green
fluorescence anti-p-p53, anti-p-H2A.X and anti-MDC1 were used to individually stain cells overnight. Next day, all samples were washed twice with PBS followed by staining with FITC-conjugated goat anti-mouse IgG (secondary antibody) and then all samples were stained using the PI (red fluorescence) to do nuclein examination.
All samples were washing twice with PBS then were examined and photo-micrographed under a Leica TCS SP2 Confocal Spectral Microscope as
described previously (Wu et al., 2011; Yang et al., 2010).
Statistical analysis. Data are presented as the means

S.D. Comparisons between cantharidin-treated and control groups were performed by Student’s t -test. Statistical significant was determined at the * p < 0.05 and *** p < 0.001 levels
Results
Cantharidin induced cytotoxic effects in NCI-H460 cells. To determine effects of cantharidin on NCI-H460 cells, cells were incubated with 0, 5, 10 and 15 μM of cantharidin for 24 and 48 h. The percentage of viable cells was determined using flow cytometry. Figure 1 shows that cantharidin decreased the percentage of viable cells, and these effects were dose-dependent manner.
Cantharidin-induced DNA damage in NCI-H460 cells. In order to examined whether or not cantharidin induced cell death through the induction of DNA damage in
NCI-H460 cells, The Comet assay was used to determine if cantharidin damaged
DNA. Cantharidin induced DNA damage in NCI-H460 cells in a dose-dependent manner (Fig. 2A and B). It can be seen that higher concentrations of cantharidin

7 caused longer DNA migration smears (Comet tail) (Fig. 2A).
Cantharidin-induced DNA damage and condensation in NCI-H460 cells. Figures 1 and 2 showed that cantharidin caused cell death and DNA damage as quantified by flow cytometry and the Comet assay, respectively. To further examine toxic effects of cantharidin, DNA condensation was analyzed. Cantharidin induced DNA condensation in NCI-H460 cells and these effects were dose-dependent (Fig. 3A &
B).
Effects of cantharidin on levels of proteins associated with DNA damage and repair in
NCI-H460 cells. Based on the results from the Figures 2 and 3 indicated that cantharidin induced DNA damage in NCI-H460 cells, in order to further investigate whether or not cantharidin affected DNA damage and repair associated protein expressions, NCI-H460 cells were treated with 0, 5, 10 and 15 μM of cantharidin for
48 h. Then all samples were harvested for Western blotting and results were showed in
Figure 4. Cantharidin treatment for 48 h reduced protein levels of p53, MGMT,
14-3-3σ, ATM, BRCA-1, DNA-PK and MDC1 (Fig. 4).
Cantharidin promotes translocation of specific proteins associated with DNA damage and repair in NCI-H460 cells. Based on the results from Western blotting (Fig. 4) indicated that cantharidin inhibited the protein levels of p-p53, p-H2A.X and MDC1 in NCI-H460 cells, we further investigated whether or now involved the translocation of those protein thus, we used confocal laser microscope and results showed in Figure
5A, B and C, that cantharidin stimulated translocation of the p-p53 (Fig. 5A), p-H2A.X (Fig. 5B) and MDC1 (Fig. 5C), in NCI-H460 cells when compared to control groups. These observation indicated cantharidin induced DNA damage may

8 also involved these proteins in NCI-H460 cells.
Discussion
Based on the viability examinations indicated that cantharidin induced cell death in a concentration-dependent manner (Fig. 1), furthermore we find that a concentration
-dependent increase in DNA damage was observed in NCI-H460 cells, which was assayed by Comet assay and DAPI staining (Fig. 2 and 3). Comet assay (single cell gel electrophoresis) showed that a significant increase in the tail moment of the comets of NCI-H460 cells, the longer of comet tail mean that the higher DNA damage
(Fig. 2). DAPI staining indicated that increased the dose of cantharidin led to increased DNA condensation (Fig. 3). Numerous studies have recognized that Comet assay is a significant and sensitive technique for measuring the DNA damage by using
culture cells after exposed to chemical agents (Ashby et al., 1995; Donatus et al., 1990;
Pool-Zobel et al., 1994). Furthermore, numerous studies also showed that during the
process of excision repair of DNA, which can be used comet assay to measure the
trend-break formation (Olive et al., 1990; Tice et al., 1990). There are several reports
on cantharidin induced apoptosis and cell death in cancer cells (Huang et al., 2011;
Kuo et al., 2010; Li et al., 2010; Liu et al., 2012; Sagawa et al., 2008). Previously we
reported that cantharidin increased ROS production and decreased the level of
mitochondrial membrane potential (ΔΨm) in colo 205 cells (Huang et al., 2011). In
the present study we found that cantharidin induced DNA damage in NCI-H460 cells which may be involve production of ROS. Additional studies are needed to establish how cantharidin interacts with DNA of cancer cells.
DNA repair play an essential role for the survival of both normal and cancer cells.
It is well documented that DNA damage of cells can be reduced or repair based on the
DNA repair systems in cells after exposure to chemical agents by eliminating DNA

9
lesions or adding new DNA bases (Olive et al., 1990; Tice et al., 1990). In the present
study, we examined whether cantharidin affects levels of proteins associated with
DNA damage and repair. Proteins whose levels were reduced were ATM, BRCA-1,
14-3-3σ, DNA-PK, MGMT, p-p53, p-H2AX and MDC1.
It is well known that agent induced DNA damage in cells that will led to the
increased the expression of p53 (Bishayee et al., 2013; De Luca et al., 2013), herein,
we found that cantharidin promoted the expression of p-p53 which were examined by using Western blotting (Fig. 4A) and were further confirmed by confocal laser microscopy (Fig.5A). Western blotting results showed that cantharidin inhibited the expressions of ATM in NCI-H460 cells. It was reported that in cells after occurs of double-stranded DNA breaks that can activate two master checkpoint kinases (ATM
and ATR) (Choi et al., 2009; Shiotani and Zou, 2009) in order to maintain genomic
integrity. Cantharidin increased levels of p-H2A.X in NCI-H460 cells. H2A.X has been reported to play a critical role in the efficient accumulation of DNA repair
factors at the break site (Bassing et al., 2002; Celeste et al., 2003; Celeste et al., 2002).
We found that cantharidin reduced levels of BRCA-1 in NCI-H460 cells,
BRCA-1 is a tumor suppressor and plays an important role in DNA repair, cell cycle checkpoint control and maintenance of genomic stability in breast and ovarian cancer
(Venkitaraman, 2002). In women with breast cancer, BRCA-1 promoter methylation was found to be positively associated with increased mortality (Xu et al., 2009). Our
results also showed that cantharidin reduced protein levels of 14-3-3σ in NCI-H460 cells. In breast cancer patients, 14-3-3σ overexpression is a novel molecular marker of disease recurrence and 14-3-3σ has been suggested to be an effective therapeutic
target (Neal et al., 2009).
DNA-dependent protein kinase (DNA-PK) is one of the key proteins involved in
DNA damage repair (Dejmek et al., 2009). Cantharidin reduced proteins levels of

10
DNA-PK (Fig. 4B) in NCI-H460 cells. Recently, it was reported that sub-nuclear induction of complex DNA damage can elicit the pan-nuclear activation of ATM and
DNA–PK (Meyer et al., 2013).
Cantharidin inhibited protein levels of O
6
-methylguanine DNA methyltransferase
(MGMT). Increased levels of MGMT activity are associated with poor response to
temozolomide drugs (Friedman et al., 1998; Hegi et al., 2005). MGMT reduces cytotoxicity of therapeutic or environmental alkylating agents (Jesien-Lewandowicz et al., 2009). It was reported that mice overexpressing human MGMT were resistant
to methylating agent induced tumor formation (Dumenco et al., 1993; Zaidi et al.,
1995). In conclusion, cantharidin induces DNA damage in NCI-H460 cells and
reduces levels of proteins associated with DNA repair including ATM, MGMT,
BRCA-1, 14-3-3σ, DNA-PK and MDC1 (Fig. 6).
Acknowledgement
This work was supported by grant CMU-Asia from China Medical University,
Taichung, Taiwan.
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Figure legends
Figure 1.
Cantharidin affected the percentage of viable human lung cancer NCI-H460 cells. The NCI-H460 cells at the density of 2x10 5 cells/well were maintained in
12-well plates then were incubated with cantharidin (0, 5, 10, 15 and 20 μM) for 24 and 48 h. Then all samples were stained with PI (5 μg/ml) and analyzed by flow cytometry (Becton-Dickinson, San Jose, CA, USA) as previously described. * P <0.05 and *** P <0.001 were considered significant.
Figure 2.
Cantharidin-induced DNA damage in NCI-H460 cells was examined by
Comet assay. The NCI-H460 cells at the density of 2x10
5
cells/well were maintained in 12-well plat then were incubated with 0, 2.5, 5, 10 and 15 μM of cantharidin for 24 h and DNA damage was determined by Comet assay as described in Materials and
Methods. A: representative picture of Comet assay for dose-dependent effects; B:
Comet length (folds of control). The 0.1% H
2
O
2
was acted as positive control.
Arrow showing the comet tail (DNA damage). * P <0.05 and *** P <0.001 were considered significant.
Figure 3.
4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining for DNA damage and condensation in NCI-H460 cells. The 2×10 5 cells/well of NCI-H460 cells

16 were maintained in 6-well plates for 24 h and were treated with 0, 1, 2.5, 5, 10 and 30
μM of cantharidin for 24 h. Then cells in each treatment were DAPI stained as described previously. At the end of staining, the cells were examined and photographed using a fluorescence microscope at 200x. * P <0.05 and *** P <0.001 were considered significant.
Figure 4.
Western blotting assay for protein levels of associated with DNA damage and repair in NCI-H460 cells. The NCI-H460 cells at the density of 2x10
5
cells/well were maintained in 12-well plat then were incubated with 0, 5, 10 and 15 μM of cantharidin for 24 h. The total proteins from each treatment were collected and determined by Bio-Rad assay Kit. The amounts of proteins from each treatment were measured by sodium dodecylsulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting as in Materials and methods.
Figure 5. Confocal laser system microscopy was used for examining protein translocation in NCI-H460 cells. NCI-H460 cells (5× 10
4
cells/well) were kept on
4-well chamber slides and were incubated with 0, 10 and 15 μM of cantharidin for 24 h and then were fixed in 4% formaldehyde in PBS for 15 min followed by using
0.3% Triton-X 100 in PBS for 1 h then followed by immunostaining as described in
Materials and methods. A:p-p53; B: p-H2A.X; C:MDC1 examined and photo-micrographed under a Leica TCS SP2 Confocal Spectral Microscope.
Figure 6. The possible flow chart for cantharidin-induced DNA damage and repair aossociated proteins in human lung cancer NCI-H460 cells.

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