Background
Cholangiocarcinoma (CCA) is one of the important public health problems in Southeast Asia, particularly Thailand. It is an uncommon adenocarcinoma which arises from the epithelial cells of bile ducts anywhere along the intrahepatic and extra hepatic biliary tree excluding the papilla of Vater and the gall bladder [
1]. The highest prevalence of CCA in Northeast of Thailand (with age-standardized incidence rate of 33.4
per 100,000 in males and 12.3
per 100,000 in females) has been associated to the consumption of improperly cooked and preserved cyprinoid fish species which contains the liver fluke,
Opisthorchis viverrini [
2‐
5]. In other Asian countries like China
, Korea and Japan,
Clonorchis sinensis is the main risk factor for CCA [
6].
The major challenge for CCA control and treatment is the lack of early diagnosis and resistance of this type of cancer to most chemotherapeutics as well as radiotherapy [
7]. At present, surgical resection of detectable tumors and adjunctive therapy with chemotherapeutic agents including gemcitabine,
cis-platin and
5- fluorouracil (5-FU) leads to an improvement in the 5-year survival rate, despite low clinical response rate and extremely high recurrence rate [
7]. Discovery and development of effective alternative chemotherapeutics for CCA is therefore the first priority needed to be focused.
Plants have formed the basis of traditional medicine systems which have been used for thousands of years and the use of plant-based systems continues to play an essential role in health care. It is estimated that approximately 80% of the population in developing countries rely on traditional medicines for their primary health care [
8,
9]. In China, traditional herbal preparations account for 30 to 50% of the medicines consumed [
10]. In industrialized countries on the other hand, adaptation of traditional medicine often termed complementary or alternative medicine, also play an important role in the health care system of about 20% of the population [
10]. Plants afford a rich repository of remedies with diverse chemical structures and bioactivities against several health disorders including cancers. Several modern anticancer drugs
, i.e.
, vinblastine, vincristine, etoposide, teniposide, paclitaxel, vinorelbine, docetaxel, topotecan and irinotecan have been developed from plant sources [
11,
12]. Thailand is a country which is rich in a wide range of tropical habitats and remarkable biodiversity. Traditional medicines are used for treatment of various infections and chronic diseases including cancers [
13]. Candidate medicinal plants or herbal formulations commonly used in Thai traditional medicine were screened for their anti-CCA activities [
11,
14,
15]. Among these, ethanolic extract of the leaves of
Kaempferia galanga Linn. was shown to exhibit promising in vitro cytotoxic activity against CCA [
14]. The highly aromatic rhizome of this plant is valued in Southeast Asian countries as a spice to flavor rice and also in folk medicine. Indigenous medicinal practitioners use the rhizome extract of
Kaempferia galanga Linn. for various medical purposes including treatment of scariasis, bacterial infections, cancers, cardiotonic and CNS stimulant. In addition, it is also applied externally for abdominal pain in women and for rheumatism [
16‐
18]. Nevertheless, there has been little evidence or report on the in vivo toxicity and anti-CCA activity of this plant
. The objective of the study was to confirm the anti-CCA potential as well as toxicity of the crude ethanolic extract of
K. galangal Linn. (rhizome) both in vitro and in animal models.
Discussion
Qualitative analysis of the marker EPMC in the test extract was made between sample retention time with retention times of the standard. From the high content of volatile oil in the rhizome of
K. galangal Linn., the marker compound EPMC was detected using HPLC with a major peak area of about 94.09%. As one of the phenolic compounds, the chemical marker EPMC, in
K. galangal can be considered to have higher solubility in organic solvents including dichloromethane and ethanol as compared with that of water [
28]. Apart from CCA, this compound has been shown to inhibit proliferation of human hepatocellular liver carcinoma (Hep G2 cell line) in a dose-dependent manner and annexin-fluorescein isothiocyanate and propidium iodide staining showed an increased early apoptotic population in human hepatocellular carcinoma cells [
29]. Variation of the content of EPMC from the volatile oil of
K. galanga Linn. was reported from various studies as 31.77% [
18] and 80% [
30]. This variation could be due to different extraction and analytical procedures. In addition, phytochemicals are mostly minor plant constituents whose concentration varies considerably according to seasonal and agronomic factors, the variety, age, and part of the plant examined [
31].
Results of cytotoxicity test showed moderate activity of both the
K. galanga Linn. rhizome extract and its bioactive compound EPMC against CL-6 cell lines with median IC
50 of 64.2 and 49.9 μg/ml, respectively. Their potency of cytotoxicity and selectivity on CCA cells was similar, but was about 1.5 to 2-fold of 5-FU (107.1 μg/ml). It was noted however for the relatively low potency of the extract (about 50%) compared with that reported during the initial screening of the leaf extract against the same CL6 cell line (mean ± SD IC
50 = 37.36 ± 3.98 μg/ml, SI = 2.9) [
14]. The IC
50 of the plant extract and 5-FU appears to be too high above recommended threshold for any compound or extract to be regarded as anticancer agent. CCA is the cancer that is highly resistant to anticancer drugs and this multidrug resistant nature might explain the low sensitivity of this type of cancer to chemotherapeutics including those obtained from medicinal plant sources [
4,
32]. There is a general agreement between a particular tumor type and its corresponding clinical cancer with respect to their response to a given drug or set of drugs [
33]. Since the tumor cell line (CL-6)was derived from human CCA tumor, the patient from whom this tumor line was obtained might have received 5-FU based chemotherapy. This may explain the relatively low sensitivity of the cells to 5-FU compared with the extract. With regard to cytotoxic activity against other cancer cells, the ethanolic extract of
K. galanga Linn. rhizomes has been reported to exhibit potent activity against SW 620 (human colorectal adenocarcinoma cell line (IC
50 = 6.13 ± 0.52 μg/ml), DU145 (human prostate cancer cell line (IC
50 = 10.51 ± 0.34 μg/ml), PA1 (human ovarian teratocarcinoma cell line (IC
50 = 10.53 ± 0.22 μg/ml), and B16F10 (murine melanoma cell line (IC
50 = 12.63 ± 1.24 μg/ml) [
34]. The discrepancy in cytotoxic activities of the extract against various cancer cells could be associated with variations in geographic origins of the herb or extraction methods, and in particular, difference in sensitivity of the cancer cells.
Results from the acute toxicity test with
K. galangal Linn. extract indicated virtually no toxicity with respect to mortality and morbidity (body weight changes, internal organ weights, and signs of abnormalities of the internal organs at gross and microscopic levels) at the highest dose level of 5000 mg/kg body weight (single oral dose). In the subacute toxicity test (daily doses for 30 days), relatively low toxicity was observed up to the dose of 1000 mg/kg body weight. At higher dose levels (3000 and 5000 mg/kg body weight), deaths of mice and behavioral signs of lethargy and piloerection were observed. Nevertheless, significant differences in body weight changes, organ weights, post-mortem gross organ lesions and histopathological changes were not observed between different dose levels and control. The effect of the extract on mortality of mice was dose-independent manner and only observed during the first two weeks. Comparatively higher frequency of death incidence was observed in mice receiving the extract at the dose of 3000 compared with 5000 mg/kg body weight (4/10 vs. 3/10 mice). This difference was minor and could be explained by variability in the response of the animals as well as the complication of the gavage methodology [
35]. The observation of death during the first two weeks of the subacute toxicity period suggests immediate toxic effect of the extract which could lead to severe adverse effects such as organ function loss leading to death [
36]. In a study conducted in mice with the dichloromethane extract of
K. galanga Linn. relatively lower dose level (100 mg/kg body weight) and EPMC (120 and 160 mg/kg body weight) for 28 consecutive days, no significant signs of morbidity was observed [
30]. In rats, no toxicity was observed when treated with the extract at the oral dose of up to 100 mg/kg body weight [
37].
With regard to the toxicity of the extract on hematological and biochemical profiles, most of values except hematocrit, MCHC, BUN and AST, were not significantly different between the extract treated and control groups. This was in agreement with that previously reported in rats in a subacute toxicity test of
K. galanga Linn. rhizome extract at the oral dose of up to 1000 mg/kg body weight [
38]. A significant increase in hematocrit (3000 mg/kg body weight) and a decrease in MCHC (all the 3 dose levels) were observed in male mice compared with control. In addition, a significant increase in BUN and AST was found in male and female mice receiving the extract at the dose levels of 5000 and 1000 mg/kg body weight, respectively. The observed variability in toxicity in male and female mice could be due to normal variation among animal groups. The changes in some of the laboratory parameters however, remained within the normal ranges reported in mice, suggesting that such variations were not associated with the
K. galangal extract [
27,
38].
The results from the current toxicity study indicate that oral administration of
K. galanga Linn. extract up to 1000 mg/kg body weight was well tolerated and this dose level was therefore considered as the maximum tolerated dose level for further evaluation of anti-CCA activity of the extract in CCA-xenografted nude mouse model. The nude mouse/human tumor xenograft system provides a useful model for cancer therapy studies involving human neoplasms. Moreover, the system lends itself to the development of screening protocols for the identification of potential anticancer drugs which would be clinically effective against a given type of cancer [
24,
33]. Despite the fact that not all human tumors can be successfully xenografted, the histology and biochemical properties of the tumors that do grow in nude mice closely resemble those of the original tumor specimens [
25]. The rhizome extract of
K. galanga Linn. at 1000 (high) and 500 (medium) mg/kg body weight and 5-FU showed significant anti-CCA activity in CL6-xenografted nude mice based on TV progression, TGI and inhibitory on lung metastasis compared with the control group. The anti-CCA activity of the extract was clearly seen at the high dose level of 1000 mg/kg body weight (TGI 58.41%, median survival time 62 days, proportion of mice without lung metastasis 33.3%). Although the extract and 5-FU did not arrest tumor growth or progression during the observation period (Fig.
3), the rate of tumor growth was considerably slow particularly for the high dose extract and 5-FU treated groups. The rapid increase in TV progression observed in all groups might be due to multidrug resistance nature of the CCA tumor [
32]. A significant prolongation of the mean survival time of CCA-xenografted nude mice treated with 5-FU compared with untreated control (55 ± 0.87 days vs 40.0 ± 0.57 days) was also reported in our previous study [
15]. Metastasis is a major cause of treatment failures and death in many cancers including CCA. Macrometastasis examination at autopsy in this study revealed lung metastasis of CL-6 tumor in all mice receiving low dose extract and untreated control mice in the current study. The high metastatic rates observed in these groups could be associated with the higher respective tumor burdens in these groups. In addition, delay in the autopsy time due to prolongation of the survival time observed in most animals led to metastatic spread of the tumor to the lungs.
Acknowledgements
The authors would like to thank and acknowledge Associate Professor Adisak Wongkajornsilp, Department of Pharmacology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Thailand for his kind support of the cholangiocarcinoma cell line (CL-6).