Anticancer activities against cholangiocarcinoma, toxicity and pharmacological activities of Thai medicinal plants in animal models

Cholangiocarcinoma (CCA) is a devastating cancer with increasing worldwide incidence and mortality rate. It is an important public health problem in several parts of Southeast Asia, particularly in the northeastern region of Thailand. The major cause of CCA in Thailand is the consumption of improperly cooked and fermented fresh water cyprinoids fish called "Pla-ra" or "Pla-som", which contains Opisthorchis viverrini (OV) and nitrosamine. Lack of effective diagnostic tool and chemotherapeutics are the major constraints for controlling this type of cancer. Chemotherapy of CCA is largely ineffective and clinical efficacy of the standard treatment with 5-fluorouracil (5-FU) is low. Furthermore, the resistance of CCA to chemotherapy and radiotherapy is a major problem [1]. The discovery and development of chemotherapeutics that are effective for the treatment and control of CCA is urgently needed.

Throughout history, natural products have afforded a rich repository of remedies with diverse chemical structures and bioactivities against several heath disorders including cancer. It is estimated that 122 drugs from 94 plant species have been discovered through ethnobotanical leads [2]. Additionally, the use of herbs as complementary and alternative medicine has increased dramatically in the last 20-25 years. According to the World Health Organization (WHO), traditional medicines are relied upon by 65-80% of the world's population for their primary health care needs [2]. In a previous study [3], we screened a total of 28 plants and 5 herbal formulations used in Thai traditional medicine for the treatment of various ailments for their cytotoxic activities against human CCA cell line CL-6. Ethanolic extracts of 5 plants (rhizomes of Zingiber officinale Roscoe and Atractylodes lancea thung. DC, fruits of Piper chaba Hunt., flowers of Mesua ferrea, and leaves of Kaemferia galangal) and 1 herbal formulation (Pra-Sa-Prao-Yhai) showed promising activities against CL-6 cell line with IC₅₀ (concentration that inhibits cell growth by 50%) values of less than 50 μg/ml.

In our effort to search for new herbal medicines with promising activity against CCA, we further investigated the in vivo anti-CCA, including the pharmacological activities (motor coordination, antihypertension, analgesia, anti-inflammation, anti-ulcer, and anti-pyrexia) of the ethanolic extracts of rhizomes of Zingiber officinal Roscoe (ZO), Atractylodes lancea thung. DC (AL), fruits of Piper chaba Hunt. (PC), and Pra-Sa-Prao-Yhai formulation (PPF) in CCA-xenograft nude mouse model. Due to the potential therapeutic interest for the treatment of cancer, the anti-CCA and pharmacological activities of curcumin (CUR), the phenolic compound isolated from rhizome of Curcuma longa Linn. was also investigated. ZO, commonly known as ginger, is a familiar condiment for various foods and beverages and is used in folk medicine in Asia and in tropical areas for various purposes such as relief for colds, fevers, and digestive problems and as treatment for nausea and vomiting as well as for arthritis. Some pungent constituents present in ZO possess potent antioxidant and anti-inflammatory activities and some of them exhibit cancer preventive activity in experimental carcinogenesis [4]. AL, the dried rhizome of A. lancea thung. DC called "Cang Zhu" in China or "Khod-Kha-Mao" in Thailand, has traditionally been used as a stomachic in China and Japan [5]. PC, the fruit of P. chaba Hunt. commonly called "Dee Plee", has been used in Thai medicine as an anti-flatulent, expectorant, carminative, antitussive, antifungal, uterus contracting agent, sedative-hypnotic, appetizer, counter-irritant, and is also useful in asthma, bronchitis, fever, and inflammation [6]. PPF is a Thai traditional medicine used for the treatment of fever in children [7]. This remedy consists of a mixture of various parts of 18 medicinal plants as described in Table 1.

The present study demonstrated promising anticancer activity against CCA in nude mouse xenograft model of the ethanolic extract of AL at all dose levels, as well as the extracts of ZO, PPF, and CUR compound at the highest dose level. PC produced no significant anti-CCA activity. Results from the acute and subacute toxicity tests both in mice and rats indicated safety profiles of all test materials in a broad range of dose levels. No significant toxicity, except stomach irritation and general CNS depressant signs (reduced alertness and locomotion, and diminished response to touch and balance), was observed. Stomach irritation occurred in all animals immediately after feeding them with a high dose of the test materials. Investigation of pharmacological activities of the test materials revealed promising anti-inflammatory (ZO, PPF, and AL), analgesic (CUR and PPF), antipyretic (CUR and AL), antihypertensive (ZO and AL), and anti-ulcer (CUR, ZO, and AL) activities.

The ethanolic extract of AL was shown to possess the most potent anti-CCA activity similar to 5-FU with regards to the reduction of tumor mass, prolongation of survival time, and inhibition of lung metastasis. All dose levels significantly reduced tumor size (by 97.3%), prolonged survival time (by 208.5%), and inhibited lung metastasis (by 95% of total lung mass) compared with the untreated control. To the best of our knowledge, the present study is the first study that demonstrated the anticancer activity of AL. Interestingly, AL extract exhibited prominent inhibitory effect on lung metastasis. Metastasis is one of the major problems in the treatment of several cancer types. In severe stage CCA, metastatic tumors in the lungs are CCA cancers which developed in the lung tissues by spreading from the liver origin through the bloodstream or lymphatic system to the lungs. Histopathological examination at autopsy revealed lung metastasis in all mice following xenografting with CL-6. Significant reduction of lung metastasis to only 5% of the total lung mass was observed in the xenografted mice treated with AL (Figure 4) whereas metastasis of more than 90% of the total lung mass was found in the untreated mice. The anti-metastatic action of AL corresponded well with its observed antihypertensive effects (reduction of systolic and diastolic blood pressure, heart rate, and mean arterial pressure). Furthermore, AL also exhibited anti-ulcer (all dose levels), anti-inflammatory (high dose), and antipyretic (high dose) activities. The observed significant anti-ulcer activity (96-98%) of AL confirms its use to improve stomach damage partly through anti-ulcer effects [8]. This anti-ulcer activity was more potent than the reference drug omeprazole given at a dose of 20 mg/kg body weight. The compound 2-[(2'E)-3',7'-dimethyl-2',6'-octadienyl]-4-methoxy-6-methylphenol isolated from the rhizome of AL showed strong inhibitory effects on 5-lipoxygenase (5-LOX) and cyclooxygenase-1 (COX-1) [9], the two key enzymes responsible for the metabolism of arachidonic acid, either to prostaglandins and thromboxanes or to leukotrienes, which play a central role in the regulation of different physiological processes, but also cause pain, inflammation, and hypersensitivity.

Significant anti-CCA activity of rhizome extract of ZO was also observed in the xenograft mouse model. High dose ZO significantly reduced tumor volume (by 35.8%), and all dose levels significantly prolonged survival time (by 161.5%) compared with the control group. Inhibition of lung metastasis by about half was seen at high dose. Furthermore, the extract possessed anti-inflammatory at all dose levels. In our previous study, the anti-CCA activity of ZO was demonstrated in OV/dimethylnitrosamine induced-CCA hamster model [10]. The extract significantly prolonged survival time and survival rate of the cancerous animals. The anticancer properties of ZO have been attributed to the presence of certain pungent vallinoids, viz. [6]-gingerol and [6]-paradol, as well as some other constituents like shogaols and zingerone [11]. A number of mechanisms that may be involved in the chemopreventive activity of ZO and its components have been reported from the laboratory studies in various experimental models [11]. Various studies have shown that a wide range of ZO constituents inhibit production of nitric oxide, inflammatory cytokines and enzymes prostaglandin synthase, and arachidonate-5-LOX in a dose-dependent manner. The latter in turn, inhibits the synthesis of leukotrienes from both COX-1 and COX-2 and LOX, respectively [12]. ZO has also demonstrated a significant reduction of inflammation in animals compared with conventional drugs [12]. A significant anti-ulcer activity of the ethanolic extract of ZO was also observed. In a previous study [13], the gastroprotective effect of 50% ethanolic extract of ZO was assessed in rats in the ethanol and acetic acid-induced ulcer models at different doses (500 mg/kg and 1.5-5 g/kg body weight, respectively). The extract showed a dose-dependent inhibition of the ulcer index in ethanol and acetic acid-induced ulcers. It prevented the oxidative damage of the gastric mucosa by blocking lipid peroxidation and by a significant decrease in superoxide dismutase and increase in catalase activity.

The anti-CCA activity of PPF extract was moderate; nevertheless, it exhibited diverse pharmacological activities. High dose PPF significantly reduced tumor volume and prolonged survival time compared with the untreated group. Inhibition of lung metastasis occurred on an average 60% of the lung mass. The anticancer activity of PPF is likely to be due to the activities of various components in the formulation. Recently, we demonstrated the cytotoxic activity against CL-6, a CCA cell line, of the two components of PPF: Mimusops elengi Linn. (flower) (IC₅₀ = 48.53 μg/ml) and Kaempferia galangal ("Proh-hom" in Thai; leaf) (IC₅₀ = 37.36 μg/ml) [3]. In addition, previous in vitro and in vivo studies reported the anticancer potential of Nigella saliva (commonly known as black seed or black cumin) [14], Angelica sinensis (Oliv.) Diels [15], Anethum graveolens Linn. [16], Foeniculum vulgare Mill. [17], Angelica dahurica Benth. [18], Mammea siamensis Kosterm. [19], Myristica fragrans Houtt. [20], and Syzygium aromaticum Linn. (cloves) [21]. The bioactive compound derived from N. sativa oil is thymoquinone, which was shown to exhibit anti-tumor activities, including anti-proliferative and pro-apoptotic effects on cell lines derived from breast, colon, ovary, larynx, lung, myeloblastic leukemia, and osteosarcoma [22‐26]. Mechanistically, thymoquinone reportedly induced apoptosis in tumor cells by suppressing NF-κB, Akt activation and extracellular signal-regulated kinase signaling pathways, and also inhibits tumor angiogenesis [27]. Recently, the serine/threonine Polo-like kinases (Plk) which are over-expressed in many types of human cancers have been identified as targets for thymoquinone. Cytotoxic activity towards several cell lines in vitro including apoptotic activity was also shown with a novel polysaccharide isolated from a rhizome of A. sinensis (Oliv.) Diels named APS-1 d [15]. Among the coumarins isolated from the bark of M. siamensis Kosterm, theraphin C showed the strongest inhibitory activity on cell proliferation in DLD-1 (colon cancer), MCF-7 (breast adenocarcinoma), HeLa (human cervical cancer), and NCI-H460 (human lung cancer) cell lines [19].

With regards to the pharmacological activities of PPF, anti-inflammatory and CNS suppression effects were observed at all doses, whereas reduction of blood pressure and analgesic effects were shown at high dose and antipyretic effect was shown at the medium dose level of 2,000 mg/kg body weight. Promising antipyretic, anti-hypertensive, anti-inflammatory, and analgesic activities of various components of PPF have previously been shown. For example, the seed of N. sativa was shown to exhibit antihypertensive property [14]. The anti-inflammatory action of nutmeg has been shown to be due to the myristicin that it contains [28]. Anti-inflammatory effect of thymoquinone from the seed of N. sativa was shown to be due to the inhibitory effect on eicosanoid generation, namely thromboxane B2 and leukotrienes B4, by inhibiting both COX and LOX enzymes, respectively [29]. The ethanol fractions of A. sinensis (Oliv.) Diels and A. dahurica were shown to exert an anti-inflammatory effect through the suppression of NF-κB-dependent activity [30]. Flavonoids and stilbenoids isolated from the stem wood of D. loureiri were shown to exhibit COX-1 and COX-2 inhibitory activity [31]. The anti-inflammatory mechanisms of byakangelicin, the active component of A. dahurica Benth, involve the inhibition of tumour necrosis factor-α, histamine release, and of PGE2 through decreased COX-2, besides its potent antioxidant effects [32]. The analgesic activity of PPF observed in the present study was found to be centrally acting. The antinociceptive effects of N. sativa oil and the active compound thymoquinone were shown to be through indirect activation of the superspinal mu₁ and kappa opioid receptors [33].

Recent studies have substantiated and provided scientific evidence regarding the prophylactic and therapeutic potential of CUR as anticancer in several types of cancer including colon, lung, breast, liver, and prostate. Interestingly, the anticancer activity of CUR against CCA in the hamster model was reported recently [34]. The compound exhibited an anticarcinogenic potential via increasing the survival of hamsters, suppression of the various events involved in multiple steps of carcinogenesis such as transcription factor, NF-κB, AP-1, and STAT-3, and ability to suppress pro-inflammatory pathways on COX-2 and iNOS. In the present study, the CUR compound was shown to possess moderate anti-CCA activity. High dose significantly reduced tumor volume (by 40.5%) and inhibited lung metastasis (by 60%). All doses significantly prolonged survival time (by 160.8%) compared with the control group. Undisputed scientific evidence suggests that CUR suppresses all three stages of carcinogenesis: initiation, promotion, and progression [34]. Due to a vast number of biological targets with virtually no side effects, CUR has achieved the potential therapeutic interest to cure immune related, metabolic diseases, and cancer. The intricate mechanism of action of CUR involves various biological targets viz transcription factors: NF-AT, AP-1, signal transducers and activator of transcription (STAT), p53 and kinases: mitogen-activated protein kinases, cytokines release, and the receptors found on different immune cell type. These actions of CUR greatly affect the innate and adaptive arms of immunity especially in the pathological conditions [34]. The peripheral analgesic and anti-ulcer effects were shown in this study following all dose levels of CUR. Surprisingly, no significant anti-inflammatory activity was observed. The analgesic activity of CUR is consistent with the previous findings of suppression of nociception in both tail flick and acetic acid-induced writhing tests following intraperitoneal injection of CUR extract [35]. Tuorkey and Karolin (2009) [36] demonstrated the anti-ulcer activity of curcumin that was displayed by attenuating the different ulcerative effectors including gastric acid hyper-secretion, total peroxides, myeloperoxidase (MPO) activity, IL-6, and apoptotic incidence.

The results suggest that plants used in Thai traditional medicine for the treatment of various ailments may provide reservoirs of promising candidate chemotherapeutics for the treatment of CCA. In addition, results from pharmacological activity tests rationalize the ethnomedical use of this plant in a number of ailments. These plants have multiple pharmacological actions on various human physiological systems that would support the treatment of chronic disease like cancer. Moreover, the use of herbal medicines is safe compared with synthetic drugs. Further studies are required to determine the molecular mechanisms and active ingredients of all test materials particularly AL, which are responsible for their anti-CCA activities.