Anti-parasitic Drug Ivermectin Exhibits Potent Anticancer Activity Against Gemcitabine-resistant Cholangiocarcinoma In Vitro

Abstract. Background/aim: The antiparasitic drug,

ivermectin (IVM), exerts anticancer activities in diverse

cancer types. However, its anticancer activity against

cholangiocarcinoma (CCA), especially the drug-resistant

phenotype, has not yet been explored. Materials and

Methods: IVM was tested for its anticancer activity against

gemcitabine-sensitive (KKU214) and gemcitabine-resistant

(KKU214

GemR

) CCA cell lines in vitro using the

sulforhodamine B and clonogenic assays as well as cell-

cycle analysis. Results: IVM treatment inhibited cell

proliferation and colony formation of both KKU214 and

KKU214

GemR

in a dose- and time-dependent manner.

KKU214

GemR

cells were more sensitive than KKU214 to

IVM treatment. IVM treatment caused S-phase cell-cycle

arrest and also cell death as indicated by an increase of sub-

population in KKU214

GemR

cells treated with IVM for

48 h. Conclusion: IVM exerts anti-CCA activities and

gemcitabine-resistant KKU214

GemR

cells are more sensitive

to IVM treatment. Thus, IVM might be useful as an

alternative treatment for CCA, especially in patients who do

not respond to gemcitabine.

Chemotherapeutic drugs have been developed and used as

first-line treatment of several cancers. Although satisfactory

outcomes are usually achieved at the beginning of treatment,

the efficacy any drug tends to decline over time. Cancer cells

can adapt to the toxic effects of chemotherapy and eventually

become tolerant of the drug (1, 2). Drug resistance is the

major impediment to effective cancer therapy and has been

observed in several cancers including cholangiocarcinoma

(CCA), a bile-duct cancer originating from bile-duct

epithelial cells. Although surgical resection can be effective

for CCA treatment, approximately two-thirds of CCA

patients are inoperable (3). Hence, chemotherapy is a

treatment of choice and has been used to control disease

progression and improve survival and quality of life for

unresectable CCA patients (4). Gemcitabine (2’, 2’-

difluorodeoxycytidine) is a deoxycytidine analogue which

has been widely used for treatment of CCA (3). However,

several clinical studies have indicated that response rates and

overall survival rates are relatively low (4), likely due to

acquired drug resistance. Therefore, it is necessary to

identify other agents or drugs that can be used against drug-

resistant CCA.

Ivermectin (IVM) is a derivative of avermectin and was

discovered in 1970. IVM is one of the most effective anti-

parasitic drugs and is widely used in human and veterinary

medicine (5). Apart from its anti-parasitic activity, IVM has

been shown to exert anti-cancer activities including

inhibition of proliferation as well as induction of apoptosis

and autophagy (6-8). The anticancer activities of IVM have

been observed both in vitro and in vivo in a number of solid

tumors such as colon cancer (9), glioblastoma (10), ovarian

cancer (11) and in hematological malignancies such as

leukemia (12). IVM has also been shown to reverse the drug

resistance of cancer cells (13-15). Therefore, IVM is a

promising anticancer agent and could possibly be used

against drug-resistant cancers.

It is not known whether IVM can exert anticancer activity

against CCA, and especially against drug-resistant CCA.

Herein, we explored the anti-CCA activity of IVM and

4837

Correspondence to: Prof. Somchai Pinlaor, Ph.D., Department of

Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen

40002, Thailand. Tel: +66 43348387, Fax: +66 43 202475, e-mail:

psomec@kku.ac.th

Key Words: Ivermectin, cholangiocarcinoma, drug resistance,

gemcitabine, KKU214

GemR

ANTICANCER RESEARCH 39: 4837-4843 (2019)

doi:10.21873/anticanres.13669

Anti-parasitic Drug Ivermectin Exhibits

Potent Anticancer Activity Against

Gemcitabine-resistant Cholangiocarcinoma In Vitro

KITTI INTUYOD

1,2

, CHARIYA HAHNVAJANAWONG

, PORNTIP PINLAOR

2,4

and SOMCHAI PINLAOR

1,2

Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand;

Cholangiocarcinoma Research Institute, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand;

Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand;

Centre for Research and Development in Medical Diagnostic Laboratory,

Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand

demonstrated that IVM inhibits CCA cell growth and

clonogenicity in dose- and time-dependent manner in both

gemcitabine-sensitive and gemcitabine-resistant CCA cell

lines in vitro. Interestingly, the anticancer properties of IVM

were more prominent in gemcitabine-resistant CCA cells.

These results suggest that IVM is a promising agent for

treatment of CCA.

Materials and Methods

Materials. Dulbecco’s Modified Eagle’s Medium (DMEM), sterile

phosphate-buffered saline (PBS), fetal bovine serum (FBS) and

penicillin-streptomycin were purchased from Gibco (Grand Island,

NY, USA). Trypsin-EDTA solution and flat-bottomed 96-well plates

were obtained from Corning (Corning, NY, USA). FxCycle

PI/RNase staining solution was obtained from Invitrogen (Thermo

Fisher Scientific Inc., Waltham, MA, USA). Ivermectin (IVM),

dimethyl sulfoxide (DMSO), trichloroacetic acid (TCA) and

sulforhodamine B (SRB) were purchased from Sigma Aldrich (St.

Louis, MO, USA). Gemcitabine (Gemzar) was purchased from Eli

Lilly (Indianapolis, IN, USA).

Human CCA cell lines. Gemcitabine-sensitive CCA KKU214 (16)

and gemcitabine-resistant KKU214 (KKU214

GemR

) cell lines (17)

were established from Thai CCA patients as described previously.

Both cell lines were maintained in DMEM supplemented with

10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at

37˚C with 10% CO

in a humidified incubator. KKU214

GemR

was

maintained in the presence of gemcitabine. Before use in an

experiment, this cell line was cultured in a drug-free medium for

one passage.

Assessment of cell proliferation. CCA cells seeded in flat-bottomed

96-well plates (2,000 cells/well) were treated with either DMSO

(control) or different concentrations of IVM dissolved in DMSO for

24, 48 and 72 h. Thereafter, the cells were fixed with cold 40% TCA

for 1 h in a refrigerator and washed 3 times with running tap water.

Then, the fixed cells were stained with 0.4% (w/v) SRB solution in

1% acetic acid for 1 h at room temperature. Excess SRB solution

was removed by washing with 1% acetic acid and SRB dye was

dissolved with 10 mM Tris buffer pH 10.5. Absorbance at 492 nM

was measured using an ELISA reader (Tecan group ltd., Männedorf,

Switzerland). Absorbance obtained from DMSO-treated cells was

used as control.

Clonogenic assay. Approximately 1,000 cells per well of CCA cell

lines were grown in 6-well plates and treated with different

concentrations of either gemcitabine or IVM. The culture medium

was changed every 2 days and cells were grown for approximately

2 weeks. The cells were then fixed with 4% paraformaldehyde,

stained with 0.5% crystal violet and dissolved with 33% acetic acid.

The absorbance at 620 nM was measured using an ELISA reader

(Tecan group ltd.).

Cell cycle analysis. One million CCA cells treated with either

DMSO (control) or 4 μM IVM for 48 h were washed with ice-cold

PBS and fixed with 70% ethanol overnight at –20˚C. After washing

twice with ice-cold PBS, 500 μl of FxCycle PI/RNase staining

solution were added and the cells were incubated at room

temperature in the dark for 30 min. Finally, stained cells were

detected using a BD FACSCanto II flow cytometer (BD biosciences,

San Jose, CA, USA) and data analyzed using BD FACSDiva

software (BD biosciences).

Statistical analysis. Data are expressed as mean±SD. Student’s t-test

was used to test differences between experimental groups. A value

of p<0.05 was considered statistically significant. Non-linear

regression analysis was carried out to calculate IC

. All statistical

analyses were performed using Graphpad Prism 7.0 for Mac

(GraphPad Software, Inc., CA, USA).

Results

IVM treatment suppressed proliferation of CCA cells and

displayed potent growth inhibitory activity against

gemcitabine-resistant CCA cells. Both gemcitabine-

sensitive (KKU214) and gemcitabine-resistant

(KKU214

GemR

) CCA cell lines (17) were treated with

different concentrations of IVM for up to 72 h. The SRB

assay showed that cell proliferation in both cell lines

ANTICANCER RESEARCH 39: 4837-4843 (2019)

4838

Figure 1. IVM treatment inhibited cell proliferation of KKU214 and KKU214

GemR

CCA cell lines. The anti-proliferative activity of IVM was

evaluated using the SRB assay. Both KKU214 and KKU214

emR

CCA cell lines were treated with different concentrations of IVM for (A) 24 h, (B)

48 h and (C) 72 h. DMSO-treated cells were used as controls. The experiment was performed in triplicate. IVM: Ivermectin.

decreased in dose- and time-dependent manner (Figure 1).

Potent cell proliferation inhibition (more than 75%) was

achieved in both CCA cell lines treated with 32 μM of IVM

for 48 h or 72 h (Figure 1B and C). Interestingly,

KKU214

GemR

cells, which are extremely resistant to

gemcitabine, were more sensitive to IVM treatment than

the parental KKU214 cell line. This was particularly

apparent at concentrations of IVM of 4 μM or higher, at all

time points. Non-linear regression analysis showed that the

concentrations of IVM at 48 h and 72 h for KKU214

cells were 11.41 μM and 7.27 μM, respectively.

Corresponding values for KKU214

GemR

cells were 4.05 μM

and 3.15 μM, respectively (Figure 2). These findings

indicated that IVM exhibited anti-CCA potential, especially

for gemcitabine-resistant CCA cells.

IVM treatment inhibited clonogenicity of CCA cells. A

clonogenic assay was employed to test the ability of IVM to

inhibit unlimited division and colony formation of CCA cell

lines (18). In line with the SRB assay, IVM treatment

significantly inhibited colony formation of KKU214 cells in

a dose-dependent manner compared to untreated controls

(p<0.0001), but was less effective than a similar dosage of

gemcitabine (Figure 3A-C). In contrast, gemcitabine

treatment was ineffective against the KKU214

GemR

CCA cell

line (Figure 3A and D), whereas IVM treatment significantly

suppressed colony formation of KKU214

GemR

cells

compared to untreated controls (p<0.0001, Figure 3B and

D). Once again, the inhibitory effect of IVM on colony

formation was more potent in KKU214

GemR

cells compared

to KKU214 cells. These results suggest that IVM exerts its

Intuyod et al: Anticancer Activity of Ivermectin Against Cholangiocarcinoma Cells

4839

Figure 2. Gemcitabine-resistant KKU214

GemR

cells were more sensitive to IVM treatment than gemcitabine-sensitive KKU214. Non-linear regression

analysis was performed to calculate the half-maximal inhibitory concentration 50 (IC

) of IVM at 48 h and 72 h for (A, B) KKU214 and (C, D)

KKU214

GemR

CCA cell lines. Data generated from the SRB assay were used for calculation.

anti-CCA activity partly through inhibition of unlimited

division and colony formation.

IVM treatment induced cell-cycle arrest of gemcitabine-

resistant KKU214

GemR

. Since the SRB assay demonstrated

the growth inhibition properties of IVM against CCA cell

lines, we then examined whether induction of cell-cycle

arrest contributed to this phenomenon. Flow cytometry

demonstrated that the cell-cycle profiles of the IVM-treated

KKU214 cells did not differ from those of controls (Figure

4A-C). However, treatment with 4 μM of IVM for 48 h

significantly induced cell-cycle arrest at the S phase in

KKU214

GemR

cells compared to controls (p<0.01, Figure

4D-F). Furthermore, the population of sub-G

of IVM-

treated KKU214

GemR

cells, an indicator of apoptotic cell

death (19), was significantly higher compared to controls

(2.93±0.83 vs. 0.67±0.15, p<0.05, Figure 4D-F). Therefore,

in addition to inhibition of growth and colony formation,

IVM treatment can induce apoptotic cell death of

KKU214

GemR

cells.

Discussion

Resistance to conventional chemotherapy has been the most

important obstacle for effective treatment of many cancer

types including CCA. In addition to the increasing chance of

relapse, the development of cancer drug resistance could

possibly make treatment nearly impossible (20). A number

of therapeutic approaches to circumvent this problem have

been proposed such as targeted therapy (21), nanomedicine

ANTICANCER RESEARCH 39: 4837-4843 (2019)

4840

Figure 3. IVM treatment inhibited colony formation of KKU214 and KKU214

GemR

CCA cell lines. The effect of (A) gemcitabine treatment and (B)

IVM treatment on colony formation of both CCA cell lines was investigated using a clonogenic assay. Relative differences in colony formation of

GemR

CCA cell lines treated with either gemcitabine or IVM compared to controls was investigated by measuring

absorbance at optical density (OD) 620 nM. The experiment was performed in triplicate. * and **** indicate a significant difference at p<0.05

and p<0.0001, respectively. N.S.: No significant difference.

(22), drug combination therapy using drugs with different

actions (23), etc. However, the efficacy of these approaches

for treatment of drug-resistant cancer has been questioned.

Herein, we demonstrated that the anti-parasitic drug IVM

exerts anticancer activity against CCA cells. Importantly,

IVM also exerted a potent anticancer activity against

gemcitabine-resistant CCA cells.

Due to its safety and high efficacy, IVM has been used for

treating both endo- and ectoparasites of animals and humans

for decades (24). IVM is also known to act against several

cancer types. A number of mechanisms have been found to

be associated with the anticancer activity of IVM. However,

an antiproliferative function has been widely reported (8). We

found that IVM treatment effectively inhibited cell

proliferation and colony formation of both gemcitabine-

sensitive and gemcitabine resistant CCA cell lines. Our

findings are in general agreement with a previous report

investigating colon cancer (9). Notably, non-linear regression

analysis found that the IC

of IVM against KKU214 cells

was higher than that in KKU214

GemR

cells. Increased

expression levels of chloride channels (12), which are usually

observed in drug resistant cancer cells (25-27), might be

responsible for the difference in susceptibility to IVM

between these cell lines. Furthermore, there might be a

difference in transcriptomic profile between gemcitabine-

sensitive and gemcitabine-resistant CCA cell lines (28). Cell-

cycle analysis found that IVM treatment induced cell death

of KKU214

GemR

cells as demonstrated by the significant

increase in the sub-G

population. The induction of death

of KKU214

GemR

cells by IVM may be mediated by the

induction of mitochondrial dysfunction and oxidative DNA

damage as demonstrated in renal cancer cells (29) and

glioblastoma (10). However, significant cell-cycle arrest was

not observed in the KKU214 CCA cell line treated with 4 μM

of IVM for 48 h, consistent with the cell proliferation assay

in which treatment with 4 μM of IVM did not cause a

significant decrease in cell-proliferation rate compared to

controls. These results indicate that drug resistant cells are

more susceptible to IVM-induced cytostasis and cell death.

However, the precise mechanism by which IVM exerts

anticancer activity against CCA cells, especially drug resistant

cancer cells, remains to be elucidated.

Conclusion

IVM treatment exhibited anti-CCA potential as shown by

inhibition of cell growth and colony formation of CCA cell

lines. The anticancer activities of IVM were more potent in

gemcitabine-resistant KKU214

GemR

cells. Furthermore, IVM

treatment also induced S-phase and sub-G

cell-cycle

Intuyod et al: Anticancer Activity of Ivermectin Against Cholangiocarcinoma Cells

4841

Figure 4. IVM treatment caused S-phase and sub-G

cell-cycle arrest of KKU214

GemR

. Cell-cycle analysis of (A-C) KKU214 and (D-F)

KKU214

GemR

CCA cell lines after treatment with either DMSO (controls) or 4 μM of IVM for 48 h, was performed by flow cytometry. Cell-cycle

distribution was visualized using BD FACSDiva software. The experiment was performed in triplicate. * and ** indicate a significant difference at

p<0.05 and p<0.01, respectively. N.S.: No significant difference.

arrest in gemcitabine-resistant KKU214

emR

cells. Our

results highlight the therapeutic value of IVM: this drug

could be used for treatment of CCA, especially in patients

ho do not respond to gemcitabine treatment.

Conflicts of Interest

The Authors have no conflicts of interest to declare regarding this

study.

Author’s Contributions

KI, CH, PP, and SP designed the experiments. KI performed the

experiments. KI, CH, PP, and SP performed the data analysis. KI

and SP drafted the manuscript. All Authors approved the final

version of manuscript for publication.

Acknowledgements

KI thanks the scholarship under the post-doctoral training program

from research affairs and graduate school, Khon Kaen University,

Thailand (Grant no. 60163) and also Khon Kaen University research

fund (Grant no. KKU61004405). The Authors thank Prof. David

Blair for critical reading and English editing of the manuscript.

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Received August 23, 2019

Revised August 28, 2019

Accepted August 30, 2019

Intuyod et al: Anticancer Activity of Ivermectin Against Cholangiocarcinoma Cells

4843