Cepharanthine exerts antitumor activity on cholangiocarcinoma by inhibiting NF‐κB (2024)

Abstract

Cholangiocarcinoma (CCA) is a major cause of cancer deaths in northeast Thailand. It is aggressive, highly metastatic, and responds poorly to traditional chemotherapy. We demonstrated the potential for Cepharanthine (CEP), a biscoclaurine alkaloid extracted from Stephania cepharantha, to treat CCA. CEP significantly inhibited growth of human CCA cell lines in a dose‐ and time‐dependent manner, regardless of the histologic type of tumor origin. Increasing cell apoptosis via caspase‐3 and capase‐9 activation was demonstrated in CEP‐treated cells. We found that CEP controlled the growth of CCA cells through nuclear factor‐kappa B (NF‐κB) inactivation by inhibiting nuclear translocation. CEP treatment effectively reduced tumor size in CCA‐inoculated mice without serious side effects. CEP also increased cell apoptosis in primary histocultures of CCA patients’ tissues; this was demonstrated by immunohistochemistry using TUNEL staining. Our results suggest that CEP possesses therapeutic potential against human CCA. (Cancer Sci 2010)

Cholangiocarcinoma (CCA) is an aggressive and lethal cancer arising from biliary epithelia within either the intrahepatic or extrahepatic biliary tracts. This cancer is rare worldwide but it is the most common liver cancer in northeast Thailand. Several conditions associated with chronic inflammation have been identified as risk factors for CCA. Infection with the liver fluke Opisthorchis viverrini is the most common risk factor for CCA in Thailand and in Southeast Asia; whereas Clonochis sinensis infection is the general risk for CCA in East Asia. For non‐liver fluke‐associated CCA, primary sclerosing cholangitis is the predisposing factor in Western countries.⁽¹⁾ According to our research, there were no significant differences in the clinical features and biological behaviors of these three different etiologic CCAs; however, differences in the molecular signature of CCAs from different etiologic CCAs were recognized. A comparison of gene expression profiles for mass‐forming CCA (from Thai Opisthorchis viverrini‐related CCA) and Japanese (non‐liver fluke‐related CCA) revealed that the liver fluke‐related CCA exhibited increased expression of genes involved in xenobiotic metabolism, whereas those of non‐fluke‐related CCA showed enhanced genes related to growth factor signaling.⁽²⁾

Early detection of CCA is difficult since there are no specific symptoms during the early stages of tumor development. Consequently, the majority of CCA patients present with advanced incurable disease so these people are not good candidates for curative surgery. Even in those who have undergone complete surgical resection, recurrence is common and the 5‐year survival rate unfavorable.^{(3, 4)} Novel treatment strategies directed against this malignancy are, therefore, urgently needed.

Cepharanthin (CEP), a biscoclaurine alkaloid extracted from the roots of Stephania cepharantha Hayata, is widely used in Japan for the treatment of various acute and chronic diseases without any serious side effects.⁽⁵⁾ It is known to have anti‐inflammatory, anti‐allergic, and immunomodulatory activities and hence is used for chemoprevention and treatment of many diseases. Regarding inflammation and shock, CEP affects antitumor activity,⁽⁶⁾ cell cycle arrest,⁽⁷⁾ anti‐proliferative, and pro‐apoptotic actions,^{(8, 9)} as well as anti‐tumor invasion⁽¹⁰⁾ in many cancer cells. The potent enhancement of CEP on the sensitivity and restoration of anticancer drugs in cancer cells is also reported.⁽⁷⁾

As corroboration of the anti‐inflammatory and antitumor activity of CEP, we planned to demonstrate the antitumor activity of CEP in human CCA cell lines, in a CCA‐inoculated mouse model and in primary cultures of human CCA tissues, and to clarify the mechanism by which CEP acts on nuclear factor‐kappa B (NF‐κB) activity. To our knowledge, our findings provide the first evidence of the use of CEP to modulate the growth of CCA.

Materials and Methods

Cell lines.  Human CCA cell lines were cultured in DMEM supplemented with 10% fetal calf serum, 1%l‐glutamine, and 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C and 5% CO₂.

Cell viability test.  MTT assays were applied to test cell viability. In brief, 3 × 10³ cells per well were seeded in a 96‐well dish and incubated with or without various concentration of CEP for 24, 48, and 72 h at 37°C, 5% CO₂. Subsequently, 10 μL MTT (0.5 mg/mL final concentration) was added to each well. After 4 h of additional incubation, 100 μL of 0.01 N HCl in isopropanol was added to dissolve the crystals. Absorption at 570 nm for each sample was determined with an auto‐matic ELISA plate reader (Multiskan; Thermo Electron, Vantaa, Finland).

DNA fragmentation assay.  Briefly, 10⁶ cells were lysed in 100 μL of 10 mM Tris–HCl buffer (pH 7.4) containing 10 mM EDTA and 0.5% Triton X‐100. After centrifugation for 5 min at 20 000g, the supernatant was treated with 2 μL of 10 mg/mL RNase‐A and 2 μL of 10 mg/mL proteinase K. Subsequently, 20 μL of 5 M NaCl and 120 μL absolute isopropanol were added to the samples and kept at −20°C for 12 h. After centrifugation for 15 min at 20 000g, the pellets were dissolved in 20 μL of TE buffer (10 mM Tris–HCl and 1 mM EDTA) and loaded onto a 1.5% agarose gel in 1 × TBE buffer (89 mM Tris Base, 89 mM boric acid, and 2 mM EDTA) and electrophoresed at 100 volt for 30 min and stained with ethidium bromide.

Western blot analysis.  Protein of whole cell or nuclear lysates were separated by 10% SDS‐polyacrylamide gel electrophoresis⁽¹¹⁾ and blotted onto a PVDF membrane (GE Healthcare, Tokyo, Japan). Detection was performed using the Enhanced Chemiluminescence Western Blotting Detection System (ECL; GE Healthcare Bio‐Science, Buckinghamshire, UK). Primary antibodies used were: anti‐p65 (F‐6), anti‐p50 (NLS), anti‐p52 (C‐5), anti‐IκBα (C‐21), anti‐phospho (Ser32)‐IκBα (B‐9), anti‐IKKαβ (H‐470), anti‐phospho (Thr23)‐IKKαβ, anti‐Actin (C‐2), and anti‐Histone H1(N‐16) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase‐3 (9662), and caspase‐9 (9502) (Cell Signaling Technology, Danvers, MA, USA). Quantification of the western blots was performed using NIH Image software (NIH, Bethesda, MD, USA). Relative density was evaluated and normalized with actin or Histone H1.

Nuclear extraction.  Cells were washed with PBS, lyzed in 1 mL hypotonic buffer (10 mM HEPES‐KOH [pH 7.9], 1.5 mM MgCl₂, 10 mM KCl, 0.1% NP‐40, 0.5 mM DTT, and 0.5 mM PMSF) and incubated on ice for 15 min. Nuclei fraction was collected by centrifugation at 200g for 1 min, lysed with 100 μL of nuclear lysis buffer (50 mM HEPES‐KOH [pH 7.9], 0.1 mM EDTA and 10% glycerol, 420 mM KCl, 5 mM MgCl₂, 0.1 mM DTT, 0.5 mM PMSF, and 2 μg/mL Aprotinin), and incubated on ice for 30 min. Nuclear lysate was obtained by centrifugation at 18 000g for 10 min.

Electrophoretic mobility shift assay.  An electrophoretic mobility shift assay (EMSA) was performed using a 2nd generation DIG Gel Shift Kit (Roche Diagnostics, Mannheim, Germany). Double‐stranded oligonucleotide probes containing the mouse immunoglobulin kappa (Igκ) light‐chain NF‐κB consensus site were purchased from Promega (Madison, WI, USA). The oligonucleotide was 3′ end‐labeled with a digoxigenin‐11‐ddUTP. The nuclear extract (10 μg protein) was incubated with 1 μg of poly [d(I‐C)], 0.1 μg of poly‐L‐lysine, and DIG‐labeled oligonucleotide in binding buffer (20 mM HEPES [pH 7.6], 1 mM EDTA, 10 mM (NH₄)₂SO₄, 0.2% Tween 20, and 30 mM KCl) for 15 min at 25°C. After the incubation, 5 × loading buffer (0.25 × TBE and 60% glycerol) was added, and the samples separated on 5% acrylamide gel in 0.5 × TBE buffer (89 mM Tris Base, 89 mM boric acid, and 2 mM EDTA). The oligonucleotide was electroblotted onto a positively charged nylon membrane and immunodetected using anti‐digoxigenin‐alkaline phosphatase. For analysis of the specific binding to κB sequence, a 200 × excess of the non‐biotin‐tagged NF‐κB oligonucleotides was added to the sample as a competitor.

In vivo assay.  Eight‐ to 10‐week‐old male NOD/Scid/Jak3 deficient (NOJ) mice⁽¹²⁾ were housed and monitored in the animal research facility according to the institutional guidelines. All experimental protocols were approved by the Institutional Animal Care and Use Committee, Kumamoto University. Mice were subcutaneously injected with 4 × 10⁶ cells of KKU‐M213 and KKU‐M214 at both flank sides, and were intraperitoneally injected with PBS or CEP (10 mg/kg/day) on the next day and every day for 17 days thereafter. On day 18, serum was collected from each animal and the tumors removed, weighted, and fixed in 4% paraformladehyde overnight for immunohistochemistry study.

Terminal deoxynucleotide transferase‐mediated dUTP nick‐end labeling (TUNEL) assay.  Histologic analysis of DNA fragmentation was used to identify apoptotic cells in the paraffin sections of the HDRA. In situ TUNEL was done using the DeadEnd Colorimetric TUNEL System (Promega). TUNEL‐positive cells were quantified in at least four high‐power fields (×40) of randomly selected tissue sections.

Statistical analysis.  The results were presented as a mean ± SD for at least three separate experiments. Statistical significance was determined using the Student’s t‐test and P < 0.05 was required for statistical significance.

Results

Cepharanthin inhibits growth of CCA cells by induction of apoptosis.  The effects of CEP on the growth of CCA cells were observed in seven human CCA cell lines established from primary tumors of liver fluke‐associated CCA patients with different histological types; namely, KKU‐M055 and KKU‐100 (poorly differentiated CCA), KKU‐M139 (adeno‐squamous CCA), KKU‐M156 (moderately differentiated CCA), KKU‐M214, KKU‐OCA17 (well‐differentiated CCA), and KKU‐M213 (mixed papillary and non‐papillary CCA). As shown in Figure1(a), CEP inhibited the growth of all CCA cell lines examined. The effective doses were varied; however, CEP at 20 μg/mL effectively suppressed growth of all cell lines to <40% of the control. CEP significantly inhibited the growth of KKU‐M213 and KKU‐M214 (P < 0.001) in a dose‐ and time‐dependent manner (Fig.1b,c).

Discussion

Cepharanthine (CEP) – alkaloids from the root extract of Stephania cepharantha Hayata – has been widely used in Japan with a variety of biological and pharmacological effects for acute and chronic diseases including cancer.⁽¹⁴⁾ Recently, it was demonstrated that CEP acts via inhibition of NF‐κB activation.^{(15, 16)} We demonstrated that biscoclaurine alkaloid CEP effectively limits growth of CCA in vitro and in vivo via inactivation of NF‐κB action. CEP exhibited potent pro‐apoptotic effects on CCA cells resulting in induction of cell death and reduction of tumor growth in NOD/Scid/Jak‐3‐deficient mice without any observed side effects. Moreover, CEP effectively reduced tumor growth of patient tissues, as determined by histoculture drug response assay.

CEP inhibited growth of CCA cell lines of KKU‐M213 and KKU‐M214 at concentrations ranging between 2.5 and 20 μg/mL (4.12–32.96 μM), which are clinically achievable,^{(17, 18)} suggesting the clinical efficacy of CEP for CCA. Several mechanisms for CEP‐mediated inhibition of tumor cell survival have been proposed.⁽¹⁴⁾ Our results clearly showed that CEP suppressed tumor growth by inducing cell apoptosis in CCA cells as evidenced by DNA ladder formation, a hallmark feature of apoptosis. The apoptosis‐inducing effects of CEP have been demonstrated in a variety of cancer cells.^{(9, 19, 20)} Production of reactive oxygen species and induction of p27 were evident in CEP‐treated neutrophils⁽²¹⁾ and squamous cell carcinoma,⁽¹⁶⁾ respectively.

In our study, CEP‐induced apoptosis in CCA cells is partly via activations of caspase‐3 and caspase‐9, both enzymes known to play a key role in apoptosis regardless of the stimuli.⁽²²⁾ CEP at high doses can induce excessive production of oxygen reactive species in cancer cells⁽²⁰⁾ and consequently acts directly on the mitochondrial membrane, resulting in the release of cytochrome C and activating caspase‐9 and caspase‐3.⁽⁹⁾

Nuclear factor‐kappa B (NF‐κB) is an extensively anti‐apoptotic transcription factor whose DNA binding is potently and rapidly induced by tumor nectrosis factor‐α (TNF‐α), infrared radiation, and other stimuli, especially chemotherapeutic drugs in almost all cells. Constitutive NF‐κB activation has been observed in a wide variety of cancers including those of the prostate,⁽²³⁾ colon,⁽²⁴⁾ lung,⁽²⁵⁾ and CCA.⁽²⁶⁾ It is also associated with a resistance to apoptosis, since many of its target genes coded for anti‐apoptotic molecules such as cIAPs, c‐FLIP, A20, and BclXL.⁽²⁷⁾ Many findings indicate that the blockade of NF‐κB signaling pathways produces a new and effective strategy for anticancer therapy.^{(27, 28)}

Because of the pivotal role that NF‐κB plays in promoting tumor cell proliferation, survival, migration, and angiogenesis, it is a likely target for experimental and/or pharmacological intervention for the treatment of cancer. CEP has been shown to exert its effect via inhibition of NF‐κB activation. In general, the IκB form a complex with NF‐κB which prevents the translocation of NF‐κB from the cytoplasm into the nucleus. The specific phosphorylation of IκBα by an IKK complex causes degradation of IκB and allows the translocation of NF‐κB to the nucleus. It has been shown that treatment of primary effusion lymphoma cells with CEP abrogated NF‐κB activity by blocking the phosphorylation of p65.⁽¹⁵⁾

In our study, CEP clearly reduced the nuclear translocations of NF‐κB (p50, p52, and p65) and, as a result, abolished NF‐κB–DNA complex formations, as shown by the EMSA assay. The mechanism by which CEP inactivates NF‐κB activity is not well understood; however, inactivation of NF‐κB by inhibiting nuclear translocation seems to be the general mechanism of CEP regardless to cancer origin. CEP effectively reduced growth of human primary effusion lymphoma cell lines, in vitro and in vivo, via decreasing protein expression of p65 and inhibition of nuclear translocation.⁽¹⁵⁾ In addition to the present study, decreases in the expression of NF‐κB p50, p65 and inhibition of NF‐κB nuclear translocation by caffeic acid phenethyl ester also effectively suppressed growth of extrahepatic CCA‐derived cell lines.⁽²⁹⁾ From these perspective, activation of the NF‐κB pathway may play a central role in oncogenesis of CCA and drugs or chemicals targeting inhibition of NF‐κB action may efficiently inhibit growth of CCA.

CEP, a biscoclaurine alcakloid, is known to possess various biological and pharmacological activities.⁽¹⁴⁾ Apart from the anti‐inflammatory function via NF‐kB inhibition, CEP also exerts various activities which suppress tumor growth, such as: (i) inhibiting the activity of P‐glycoprotein;^{(30, 31)} (ii) inducing cell cycle arrest by down‐regulation of cyclin E and up‐regulation of p27;⁽³²⁾ (iii) inducing apoptosis effects through the activation of caspase‐9 and caspase‐3; and (iv) regulating the expression of Bcl‐2 and Bax protein.^{(8, 33)} From this perspective, utilizing CEP as an antitumor agent in CCA should have more of an advantage than merely using it is an anti‐inflammatory agent.

In our study, the effect of CEP on suppression of tumor growth was demonstrated in CCA‐inoculated mice without any side effects. Similar effects and the safety of CEP have been reported for studies on leukemia cells,⁽³⁴⁾ hepatocellular carcinoma,⁽³⁰⁾ and primary effusion lymphoma.⁽¹⁵⁾ The effect of CEP on patients’ tissues determined by the histoculture drug response assay was first demonstrated in our study. The number of apoptotic cells as indicated by positive TUNEL staining was significantly higher in tissues cultured in the presence of CEP in a dose‐dependent manner. This three‐dimensional, native state, histoculture assay simulates the tumor architecture in the body and may reflect the CEP‐responsiveness of tumors if CEP is given as a supplement to CCA patients.

CEP effectively inhibited growth of CCA cell lines regardless of histologic type. As shown in this study, growth of seven CCA cell lines established from intrahepatic CCA with different histologic types was significantly suppressed by CEP treatment. A similar effect was also observed when human tissues from different histological types of CCA were treated with CEP. These results imply that CEP may efficiently inhibit growth of CCA regardless of the type of tumor origin.

In this study, we used a relatively high amount of CEP (10–20 μg/mL for in vitro study and 10 mg/kg for in vivo study). As for the mice study, as high as 10–100 mg/kg of CEP has been effectively used in several studies without obvious adverse effects.^{(14, 15)} Moreover, a dose of 40–100 mg/body/day of CEP has been used in several clinical applications such as idiopathic thrombocytopenic purpura (ITP) and in cancer patients without any side effects.^{(17, 35)} These findings suggest that CEP is an efficient alternative anticancer agent and that higher dose of CEP can be safely used for cancer patients.

In conclusion, we demonstrated that CEP exhibits antitumor activity against human CCA cells by enhancing apoptosis through the inhibition of NF‐κB activation. The antitumor activity of CEP was shown in CCA cell lines, as demonstrated in CCA‐inoculated mice and confirmed in the primary histoculture of patients’ tissues. For the first time, it has been demonstrated that CEP abrogates NF‐κB activity by inhibiting NF‐κB activation, leading to the inhibition of NF‐κB nuclear translocation and DNA‐binding activity. Since CEP exhibits diverse pharmacological effects, there may be other pathways apart from NF‐κB pathway undergirding the action of CEP. In view of the fact that CEP is a safe drug, which has been used in Japan for several decades⁽¹⁴⁾ and effectively suppresses the growth of CCA cells in culture, patient tissues, and tumor‐inoculated mice without obvious side effects, we propose that CEP be approved as a novel drug of choice for the treatment of CCA patients.

Disclosure Statement

The authors have no conflict of interest.

Acknowledgments

This project was supported by the Research Team Strengthening Grant, National Genetic Engineering and Biotechnology Center, National Science and Technology Development Agency, Thailand, and the Royal Golden Jubilee – PhD Program for W. Seubwai and S. Wongkham (PHD/0034/2549). We thank Mr. Bryan Roderick Hamman and Mrs. Janice Loewen Hamman for assistance with the English‐language presentation of the manuscript.

References