Background
Many cancer patients use therapies promoted as viable alternatives to conventional cancer treatment with questionable outcomes. Such unproven modalities can be potentially harmful. Furthermore, an even greater proportion of cancer patients use complementary therapies such as herbs and supplements along with conventional cancer treatment such as chemotherapy and radiation therapy. Some of these may have been proven to be adjunctive approaches that control symptoms and enhance quality of life. There is much controversy as to whether these natural health products should be taken during conventional cancer treatments and both sides of the divide provide valid arguments. More importantly, the drug-herb interaction effects of such complementary therapies with chemotherapy agents are often not studied during clinical trials or even receive post-marketing surveillance [
1,
2]. Cancer development and progression is usually not driven by single cells. The tumor microenvironment drives the drug resistance and tumor survival [
3‐
5]. It is hard to believe any single agent may effectively suppress cancer development and progression. Researchers have been actively targeting the Mother Nature to explore any potential regimen for cancer. Despite dietary or plant-isolated compounds [
6‐
12] exhibiting a potent anticancer effect, thorough scientific investigation should be conducted in order to validate their effects on cancer treatment.
Clinacanthus nutans, or Sabah Snake Grass (SSG) as it is locally known in Malaysia, is a plant with indigenous origins in Southeast Asia, although its actual origin is unknown. It was originally isolated from Sabah, West Malaysia and hence, it is named after the location. The genus
Clinacanthus consists of two species,
C. nutans Lindau and
C. siamensis Brem, with both belonging to the family Acanthaceae.
C. nutans, a small shrub, is often cultivated and has long been used in Thailand as a traditional medicine for the treatment of skin lesions resulting in a
C. nutans preparation for the relief of minor skin inflammation [
13,
14]. Among cancer patients in Malaysia, SSG has been known to cure the latter stages of liver cancer; however, its consumption is advised to be carried out only following conventional treatments of chemotherapy and radiotherapy due to possible adverse effects that could arise. To the best of our knowledge, this claim has no scientific evidence to support it, and is made purely due to the cautioning of concomitant use of chemotherapy agents with other unproven agents. Several isolated studies have therefore investigated the claims. It was suggested the methanolic extracts of
C. nutans had effects on human lung cancer (NCI-H23), cervical cancer (HeLa), liver cancer (HepG2), leukemia (K-562, Raji), neuroblastoma (IMR32), gastric cancer (SNU-1) and colon cancer (LS-174 T) cells. However the most active extract, chloroform extracts exhibited only a very low potency (IC
50 = 47.31–47.70 μg/mL) against cancer cells [
15]. The criteria established by the American National Cancer Institute for a crude extract to be considered as a potential cytotoxicity agent, it would achieve an IC
50 less than 30 μg/mL when tested against a cell line. In another study, however,
C. nutans methanolic extracts showed no significant cytotoxicity until at the highest concentrations tested under normoxic conditions [
16]. Furthermore,
C. nutans extracts tested against cyclophosphamide against COR-L23 cancer cell line with and without microsomal incubation did not show a significant (
p > 0.05) reduction in IC
50 values [
17]. Thus, it is rather challenging for clinicians to recommend or to discourage the use of
C. nutans in achieving the desired therapeutic outcomes. The need to standardise the experimental procedures, including using the standardised extracts, and to use a standardised in vitro anticancer procedure, is of the utmost importance to mitigating the anticancer potential of
C. nutans.
In our previous study, we have prepared the standardised polar and non-polar fractions of
C. nutans leaves and stem. These extracts were found to exhibit anti-inflammatory properties through inhibiting Toll-like Receptor 4 (TLR-4) activation and nitric oxide production, one of the key inflammatory mediators. The total phenolic contents and total flavonoid contents were correlated with its anti-inflammatory potency. The polar leaf extracts were also found to inhibit the hallmark inflammatory mediators, such as p65, p38, pERK, pJNK and pIRF3. More importantly, we have established that these standardised bioactive extracts of
C. nutans had no cytotoxicity on human embryonic kidney cells and macrophages [
18]. In this study, we aimed to expand our knowledge by investigating the anticancer effects of these standardised
C. nutans leaves and stem in human cancer cells. Since most patients are likely also to take both chemotherapy agents and
C. nutans concomitantly
, we also investigated the interaction between chemotherapy agents and
C. nutans. The current investigation was also designed to determine the possible cell death behind the interaction between
C. nutans extracts and gemcitabine in pancreatic cancer cells.
Discussion
In recent years, the use of complementary and alternative medicine among cancer patients has been raising. This exponential increase could be attributed to the enhanced awareness of the general public to social media who are marketing the benefits of complementary medicine and the overall misgivings towards conventional cancer treatments. Furthermore, patients consume complementary and alternative medicine because they believe it will improve their quality of life. Some patients may even think complementary and alternative medicine can prolong life and promote cancer remission. These common complementary and alternative medicines include herbs, vitamins, minerals, homeopathy, naturopathy and specialized diets [
33‐
36]. Numerous studies reported that 48–88% cancer patients used complementary and alternative medicine as part of their cancer therapy [
37‐
39]. Despite the popularity of complementary and alternative medicine, there is limited research evaluating the scientific efficacy of complementary and alternative medicine in cancer treatment as well as its interaction with conventional cancer treatment. In addition, compared to patients receiving conventional cancer treatment, patients who chose complementary and alternative medicine also showed higher refusal rates of surgery, chemotherapy, radiotherapy, and hormone therapy. These factors lower the 5-year overall survival rates, inadvertently leading to greater mortality risks [
36]. Hence, the need for conclusive evidence of the evidence of complementary and alternative medicine in cancer treatment is needed.
Based on folk medicine,
C. nutans, or Sabah Snake Grass is one of the popular herbs and perceived to have anti-cancer effects [
13‐
15]. To the best of our knowledge, only several isolated studies have thus far investigated the claims. Our previous study [
18] suggested
C. nutans extracts exhibit potential immunomodulator effects. Evidence suggests pancreatic cancer may susceptible to immunotherapies or immunodulators [
40]. It is the interest of Asians to thoroughly investigate the role of
C. nutans in cancer. The extracts were found to induce low anti-proliferative effect (IC
50 = 47.31–47.70 μg/mL) against in vitro lung, cervical, liver, leukemia, gastric and cancer cells [
15]. Our study has also shown that most of LP, LN, SP and SN extracts have very weak (IC
50 > 30 μg/mL) anti-cancer effects when tested against 4 breast cancer cells, 4 colorectal cancer cells, 4 lung cancer cells, 4 endometrial cancer cells, 4 nasopharyngeal cancer cells and 3 pancreatic cancer cells, suggesting
C. nutans may have no or very week anti-proliferative effect in cancer cells. The most potent
C. nutans extracts amongst all tested extracts was SN extracts, with IC
50 around 30.91–39.12 μg/mL on SW1990, AsPC1 and BxPC3. There were extensive phytochemistry studies, isolating many compounds from
C. nutans, trying to investigate the anti-cancer effects of these isolated compounds. These flavonoids and terpenoids were also commonly isolated from various other plants [
41,
42].
Previous study [
42] published extensive metabolite profiling of
C. nutans extracts. The research team identified betulin, lupeol and beta-sitosterol (Sigma Aldrich, USA) in stem extracts, which were not identified from other
C.nutans extracts. Isovitexin, vitexin, rutin, chlorogenic acid and gallic acid (Sigma Aldrich, USA) were found in other extracts but not in SN extracts. In the attempt to correlate the bioactivity of these compounds to the observed anticancer effects of SN extracts, we purchased the pure compounds and tested it individually in our in vitro cytotoxicity assay. However, none of these compounds exhibited a promising anti-cancer effect against human pancreatic cancer cells (Additional file
1: Table S4). The observed anticancer effects may be due to novel compounds or a combinatory of compounds. To date, none of the isolated compounds or extracts have reached the patient as a promising anti-cancer agent. It is very unlikely a single isolated compound or a single extract from
C. nutans alone may render a promising anticancer effects. Thus, it is not recommended to consume these extracts as a total replacement to conventional cancer chemotherapy.
According to the locals, many of the cancer patients were also taking the
C. nutans extracts while receiving the chemotherapy and some of them claimed they have a ‘better recovery’. We were interested to find out whether the anti-cancer effects exhibited by chemotherapy may be enhanced or abolished in the present of extracts
C. nutans. Thus, we then proceeded to investigate the effects of SN extracts (the most potent extracts) together with gemcitabine (the first line chemotherapy for pancreatic cancer patients) in both squamous pancreatic cancer cells (SW1990 and BxPC3) as well as progenitor pancreatic cancer cells (AsPC1). To our surprise, SN extracts strongly enhanced gemcitabine sensitivity in the squamous subtype of pancreatic cancer cells (SW1990 and BxPC3) but not the progenitor pancreatic cancer cells (AsPC1), and the favourable DRI trend obtained might also be exploited as added benefit of combining SN with gemcitabine in squamous pancreatic cancer cells. To date, there is no report of synergistic interaction between SN extracts when combined with gemcitabine in any cancer treatment study. A healthy individual will have no squamous cells in the pancreas. As compared to progenitor cells carcinoma, squamous cell carcinoma of the pancreas is associated with poor prognosis, more aggressive with higher metastasis rate and no targeted therapy [
43,
44]. The major difference between squamous and progenitor carcinoma would be the transcription factors in endodermal development and differentiation. TGF-β and MYC were usually overexpressed among the squamous carcinoma, but not progenitor carcinoma [
44]. In view of the upregulation of TGF-β among squamous carcinoma, gemcitabine treatment may down-regulate TGF-β in pancreatic tumor and resulting a better treatment outcomes achieved among the squamous carcinoma. The underlying mechanism behind gemcitabine inhibitory effect on TGF-β could be driven by its modulatory effect on STAT3 phosphorylation [
45] Alternative explanation to the observed stronger synergism against squamous carcinoma may be attributed to MYC inhibitor in SN extracts (eg: fisetin [
46]) may downregulate MYC to enhance the cytotoxicity effect of gemcitabine. Kim N et al. elucidated fisetin enhanced cytotoxicity effect of gemcitabine in squamous carcinoma via inhibition of MYC signalling [
46]. Both possible explanations may require further phytochemistry studies and molecular mechanistic studies to identify the underlying mechanism. Nevertheless, the combination of SN extracts and gemcitabine may have more promising outcomes against pancreatic ductal carcinoma especially the squamous carcinoma. Current research has been revolving around discovering ways to target the molecular subtype as a roadmap for precision medicine in pancreatic cancer [
47].
Our findings may encourage exploration of the role of nutraceuticals (SN extracts) in combining with pharmaceuticals (gemcitabine) for patients with squamous pancreatic adenocarcinoma. There have been several plant extracts found in the past which has synergistic interactions when used in combination with gemcitabine. Yu J and coworkers reported that the extract of Pao Pereira enhanced the anti-tumor effect of gemcitabine in mice bearing pancreatic tumors with reduced metastatic lesions in peritoneum [
48]. The same research team also reported that
Rauwolfia vomitoria extract can potentiate gemcitabine’s anti-tumor effects, via reduction in tumor burden and prevented metastatic potential in an orthotopic pancreatic cancer mouse model [
49]. Alternative study by Archana Pandita and coworkers suggested that gemcitabine may synergise with betulinic acid and thymoquinone, both of which are common dietary compounds, by inhibiting the proliferation of pancreatic cancer cells, inducing apoptosis and down-regulating pyruvate kinase M2 isoforms [
50]. Irofulven (a compound from sesquiterpene mushroom metabolites) [
51], cucurbitaicin B (Cucurbitacae family) were some of the pure compounds isolated from plants that can enhance the anti-tumor effects of gemcitabine [
52]. Not forgetting that, in the presence of SN extracts, we can reduce the dose of gemcitabine by 2.38–5.28 fold and maintain the effects of gemcitabine in pancreatic cancer cells. By reducing the dose of gemcitabine, we can potentially reduce the toxicities associated by gemcitabine [
53‐
55]. Nanotechnology in formulation, such as niosomes or liposomes, may be able to co-deliver SN extract and gemcitabine together [
56]. These studies supported that SN extracts could be used to potentiate a chemotherapy, and are worth further investigation.
It is known that bcl-2, cIAP-2, livin, survivin and XIAP are the anti-apoptotic markers that prevent cancer cells undergoing apoptosis [
20,
22]. Thus, by suppressing these anti-apoptotic proteins, the combination of SN extracts and gemcitabine, may induce inhibition in cell proliferation and apoptosis. Particularly, high expression of these anti-apoptotic proteins is associated with higher risk of chemoresistance and poorer prognosis among cancer patients [
57,
58]. Our data clearly shown that the downregulation of antiapoptotic proteins, particularly bcl-2, cIAP-2, and XIAP by the combination of SN extracts and gemcitabine could be probably enhancing the pro-apoptotic proteins, such as bax levels leading to apoptotic cell death. TLR-4, the innate immune receptor, is related to immune evasion and carcinogenesis [
59]. A study shown TLR-4 were expressed in AsPC1, BxPC3, and SW1990 cells [
60]. Surprisingly, even though the previous study [
18] suggested that SN extracts can reduce TLR-4 activities, the level of TLR-4 in cancer cells was not affected by SN extracts or in combination with gemcitabine. These results are in favour of our hypothesis that SN extracts synergised the anticancer effects of gemcitabine via upregulation of pro-apoptotic proteins, and downregulating the antiapoptotic proteins, independent of TLR-4 expression. Since TLR-4 is an upstream innate immune receptor, extracts that do not inhibit TLR-4 expression may have less implication for the immune system of the host. It is important to maintain the immune system of the host, in view of the fact that most cancer patients may suffer immunosuppression due to the non-selective cytotoxicity of the chemotherapies.
The crux of the study is to provide evidence-based data to support or refute the traditional practices of C. nutans in cancer. Hence, we emphasised on phenotypic investigation, rather than detailed mechanistic investigation because the phenotypic observation or therapeutic response has yet to be established. To the best our knowledge, our results is the first to confidently suggest that C. nutans extracts should not be used alone or as a replacement to chemotherapy since the extracts were not potent in all the 23 human cancer cells. Since the study was only conducted only in vitro, future study should emphasis on its investigating the synergistic effects on pancreatic tumor animal model with detailed mechanistic study. The mechanistic study may further explore correlation of pro- and anti-apoptotic markers with hallmarks of cancer inflammatory markers such as TLR-4, p38, p65, pERK and pJNK. The animal study would also provide evidence whether the combination of SN extracts and gemcitabine may alter the tumor microenvironment and tumor-immune system interaction. Also, it would be useful to determine the active metabolites from SN extracts. Our findings suggested the commonly isolated compounds (such as vitexin and isovitexin) had no anti-cancer effects. It is strongly believe that other active metabolites in SN extracts have yet to be uncovered. Detailed chromatography studies would be of useful to isolate and to characterise the active metabolites. With the known active metabolites isolated and characterised, it would be clearer to determine the pharmacodynamic and pharmacokinetics interaction between the active metabolites and gemcitabine. It may provide new evidence for future study to investigate the potential of these metabolites in enhance chemosensitivity and to reverse chemoresistance in human pancreatic cancers.
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