Anticancer Properties of Phytochemicals Present in Medicinal Plants of North America

Cancer is one of the most severe health problems in both developing and developed countries, worldwide. Among the most common (lung, stomach, colorectal, liver, breast) types of cancers, lung cancer has continued to be the most common cancer diagnosed in men and breast cancer is the most common cancer diagnosed in women. An estimated 12.7 million people were diagnosed with cancer across the world in 2008, and 7.6 million people died from the cancer during the same year [1]. Lung cancer, breast cancer, colorectal cancer and stomach cancer accounted for two-fifths of the total cases of cancers diagnosed worldwide [1]. More than 70% of all cancer deaths occurred in lowand middle-income countries. Deaths due to cancer are projected to continuously increase and it has been estimated that there will be 11.5 million deaths in the year 2030 [1] and 27 million new cancer cases and 17.5 million cancer deaths are projected to occur in the world by 2050 [2]. According to Canadian cancer statistics, issued by the Canadian Cancer Society, it is estimated that 186,400 new cases of cancer (excluding 81,300 non-melanoma skin cancers) and 75,700 deaths from cancer will occur in Canada in 2012 [1]. The lowest number of incidences and mortality rate is recorded in British Columbia. Both incidence and mortality rates are higher in Atlantic Canada and Quebec [3].


Pathophysiology of cancer
Cancer is a population of abnormal cells which divide without control, with the ability to invade other tissues. Cancer and some of the other chronic diseases share common pathogenesis mechanisms, such as DNA damage, oxidative stress, and chronic inflammation [10]. It is understood that both environmental factors and chemical carcinogens play a key role in the initiation and progression of cancer. Among the major environmental factors are asbestos, polluted air near industrial emission sources, exposure to secondary tobacco smoke, indoor air pollution such as radon, drinking water containing arsenic, chlorination by-products, and other pollutants [11]. Chemicals with carcinogenic activity can be classified as DNA reactive (e.g.: nitrogen mustards, chlorambucil, epoxides, aliphatic halides, aromatic amines), epigenetic (e.g.: chlordane, pentachlorophenol, hormones, cyclosporin, purine analogs), dichlorodiphenyltrichloroethane, phenobarbital, minerals (e.g.: asbestos), metals (e.g.: arsenic, beryllium, cadmium) and unclassified carcinogens (e.g.: acrylamide, acrylonitrile, dioxane) [12]. DNA-reactive carcinogens act in the target cells of tissue(s) of their carcinogenicity to form DNA adducts that are the basis for neoplastic transformation [12]. Epigenetic carcinogens lack chemical reactivity and hence, do not form DNA adducts. These carcinogens are produced in the target cells of tissue(s) of their carcinogenicity. Effects of epigenetic carcinogens indirectly lead to neoplastic transformation or enhance the development of tumors from cryptogenically transformed cells [12].
Carcinogenesis is a multi-step process consisting of tumor initiation, promotion and progression [13]. Cancer initiation can be blocked by activating protective mechanisms, either in the extracellular environment or intracellular environment by modifying trans-membrane transport, modulating metabolism, blocking reactive oxygen and nitrogen species, maintaining DNA structure, modulating DNA metabolism and repair, and controlling gene expression [10]. Tumor promotion is the second stage of carcinogenesis and is followed by tumor progression. Both stages can be suppressed by inhibiting genotoxic effects, favoring antioxidant and anti-inflammatory activity, inhibiting proteases and cell proliferation, inducing cell differentiation, modulating apoptosis and signal transduction pathways and protecting intercellular communications [10]. In addition, tumor progression can also be inhibited by affecting the hormonal status and the immune system in various ways and by inhibiting tumor angiogenesis [10].

In vitro anticancer activity of phytochemicals and extracts of medicinal plants
Cultured cancer-derived cell lines with comparison to normal healthy cell lines are commonly used to assess the anticancer properties of isolated phytochemicals and extracts of medicinal plants ( Table 2). The anticancer properties of ethanolic extract of leaves, pulp and seeds of, Annona glabra (L.), commonly known as pond apple, were shown, using human drug-sensitive leukemia (CEM) and its multidrug-resistant-derived (CEM/VLB) cell lines [52]. The most potent anticancer activity was shown in the seed extract of A. glabra [52]. Both dried rhizome hexane extract and dried fruit hexane extract, partitioned from total methanol extract, of Aralia nudicaulis (L.) caused death of cancer cell lines such as human colon cancer cell (WiDr), human leukemia cell (Molt) and human cervix cancer cell (HeLa) at a lower concentration, than that of required for the death of normal cells [53]. Eupatoriopicrin, a sesquiterpene lactone isolated from Eupatorium cannabinum (L.) (Bonesets), indicated anticancer properties on FIO 26 (fibrosarcoma) cells with an IC 50 =1.5 µg/ml [22]. Methanolic extracts of Hypericum perforatum (L.) (St. Johns wort) possessed strong antiproliferative activity in the human prostate cell line (PC-3) and the major constituents, hyperforin and hypericin, synergistically contributed to the reduction of the PC-3 cells proliferation [24]. Maslinic acid, (Figure 2(a)) a triterpene from Olea europaea (L.) (Olive), has shown to be significantly inhibitory in cell proliferation of the human colorectal adenocarcinoma cell line (HT29) in a dose dependent manner [33]. The major components in the extract were identified to be oleuropein, hydroxytyrosol, hydroxytyrosol acetate, luteolin-7-O-glucoside, luteolin-4'-O-glucoside and luteolin [34] ( Figure 2). All these phytochemicals inhibited the proliferation of cancer and endothelial cells with IC 50 , at the low micromolar range [37]. Methanolic leaf extract of Plantago lanceolata (L.) (Ribwort plantain) inhibited the growth of three different cell lines; human renal adenocarcinoma (TK-10), the human breast adenocarcinoma (MCF-7) and the human melanoma (UACC-62) cell lines and the MCF-7 was totally inhibited [54]. Further, the ethanolic extract of P. lanceolata (L.), produced by maceration with ethanol : water, showed significant antiproliferative activity on cervix epithelioid carcinoma (HeLa), breast adenocarcinoma (MCF-7), colon adenocarcinoma (HT-29) and human fetal lung carcinoma (MRC-5) [38]. Several chemical constituents ( Figure  3) from Silybum marianum (Milkthistle) have been isolated and their cytotoxic and anticancer potential has been investigated, in vitro, using both cancer and normal healthy cell lines. Silymarin, isolated from seeds of S. marianum, is a mixture of series of flavolignans, major constituents being: silybin A and B, (also known as silibinin), isosilybin A and B, silychristin, and silydianin [55,56].  Silybin possessed a dose-dependent growth inhibitory effect on parental ovarian cancer cells (OVCA 433), drug-resistant ovarian cancer cells (A2780 WT) and doxorubicin (DOX)-resistant breast cancer cells (MCF-7) [55]. Both L and D diastereoisomers of silybin inhibited A2780 WT cell growth at low IC 50 reported with L-diastereoisomer [55]. Furthermore, silybin potentiated the effect of Cisplatin (CDDP, a platinum analog; cis-diamminedichloroplatinum [II]) in inhibiting A2780 WT and CDDP-resistant cell growth. Cisplatin is an inorganic metal complex which acts as an alkylating agent [57]. Similar results recorded with doxorubicin (DOX) on MCF-7 DOX-resistant cells when silybin associated with doxorubicin. Doxorubicin ((7S,9S)-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyace tyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione) is an anthracycline antibiotic isolated from Streptomyces peucetius var caesius [57]. The effect of silybin-CDDP and silybin-DOX combinations resulted in a synergistic action, as assessed by the Berembaum isobole method [55]. Silymarin demonstrated to have marked inhibition of cell proliferation with almost 50% inhibition in a time dependent manner on the human breast cancer cell line (MDA-MB 468), at 25 µg/mI concentration, after five days of treatment. Its potential anticancer activity was dose dependent and showed a complete inhibition of cancer cells at 50 and 75 µg/mI concentrations at the beginning of Day 2 of exposure [56]. Induction of apoptotic cell death of human prostate cancer (DU145) treated with silibinin is shown to be due to activation of caspase 9 and caspase 3 enzymes [58].

Evidence from animal studies for anticancer activity of North American medicinal plants
Anticancer and antiproliferative potential of some North American medicinal plants has also been studied in animal studies (Table 3). In vivo antitumor activities of Achyranthes aspera (L.) (Devil's Horsewhip) on athymic mice, with are subcutaneous xenograft, harboring human pancreatic tumor were demonstrated, using the leaf extract. The leaf extract significantly reduced both tumor weight and volume in mice treated with leaf extract intraperitonealy [14]. Intravenous administration of 40 mg/kg body weight eupatoriopicrin, a sesquiterpene lactone present in E. cannabinum, significantly delayed the growth of tumor in Lewis lung tumour-bearing syngeneic C57B1 female mice [22]. A 70% inhibition of tumor growth in PC-3 cells, orthopedically implanted into the dorsal prostatic lobe in athymic nude mice, was observed, upon their receiving 15 mg/kg intraperitoneal H. perforatum methanolic extract [24]. Lantadene A is a pentacyclic triterpenoid, isolated from the weed, Lantana camara (L) [59]. Feeding of female Swiss albino mice (LACCA) with a dose of 50 mg/kg body weight of Lantadene A twice a week for 20 weeks, showed potential chemopreventive activity. This chemopreventive activity could be linked to the expression of transcriptional factors and a significant decrease in the mRNA expression of AP-1 and c-fos), NF-kB (p-65) and p53 was observed in Lantadene A treated mice skin tumors [59]. Silibinin decreased tumor multiplicity by 71% (P < 0.01) in wild type mice, but did not show any such considerable effect in iNOS -/-mice upon oral feeding of 742 mg/kg body weight silibinin for 5 days per week for 18 weeks [60]. Lesser effects of silibinin in iNOS -/-mice suggested that most of its chemopreventive and angiopreventive effects were through its inhibition of iNOS expression in lung tumors [60]. Treatment of a purified diet, containing 0.5% to 1.0% silibinin on a transgenic adenocarcinoma of are mouse prostate (TRAMP) model, decreased the weight of the tumor in both the prostate and seminal vesicle, when compared with control mice [61].
Treatment of silibinin significantly decreased tumor angiogenesis and proliferation and also there was increased apoptosis in prostate tumor tissue samples in the TRAMP model [61]. The protective effect of silibinin was also demonstrated in mouse skin with tumors caused by acute and chronic UVB-exposure-caused mitogenic and survival signaling and associated biological responses [62]. Mice were treated with silibinin, either topically (9 mg in 200 ml acetone/mouse) or orally (1% of diet), and both administrations strongly inhibited UVB-induced skin tumorigenesis in a long-term study [62]. Thymine dimers are formed in DNA, immediately after UVB irradiation, and are considered as an early and important biomarker for UVB induced DNA damage [62]. A noticeable, 71% reduction (P < 0.001) of thymine positive cells was obtained in the mice treated with 1% (w/w) silibinin before the UVB exposure, compared with the UVB alone group [62]. Oral feeding of 200 mg/kg of silibinin for 5 days per week, for 33 days, significantly inhibited human non-small-cell lung cancer cells (NSCLC A549) tumor xenograft growth in nude mice, in a time-dependent manner [63]. This accounted for 58% (P = 0.003) reduction in tumor weight per mouse and intraperitoneal administration of 4 mg/kg doxorubicin, once a week for four weeks, showed 61% (P = 0.005) reduction in tumor weight. However, interestingly, in silibinin-doxorubicin combination, 76% (P = 0.002, versus control) decrease in tumor weight per mouse was observed, that which was significantly different from either treatment alone, showing enhanced efficacy [63].

Mode of action of selected phytochemicals of North American medicinal plants
Apoptosis (programmed cell death) is the principal mechanism through which unwanted or damaged cells are safely eliminated from the body. This programmed cell death is mediated via either an extrinsic apoptotic pathway or an intrinsic apoptotic pathway [65]. These two apoptosis signaling pathways differ in the origin of their apoptosis signal, but converge upon a common pathway [66].
The extrinsic pathway is initiated by the stimulation of the cell surface 'death receptor' due to the binding of death ligand and the intrinsic pathway is also known as the mitochondrial pathway in which an intracellular apoptotic signal initiates the process [68]. Various natural extracts, obtained from medicinal plants grown in North America, have been found to induce apoptosis pathways at different levels ( Figure 4 and Table 04). Leaf extract of Achyranthes aspera activated caspase-3 and induced caspase-3 mRNA in tumor cells. It also decreased Akt-1 transcription, as well its phosphorylation. Suppression of pAkt-1 and a corresponding activation of caspase 3 by the leaf extract, induced apoptosis of tumor cells [14]. It was also found that maslinic acid, isolated from O. europaea, inhibited considerably the expression of Bcl-2 (B-cell lymphoma 2), whilst increasing that of Bax. Maslinic acid stimulated the release of mitochondrial cytochrome-c and activated caspase-9 and caspase-3 [33]. These results showed the activation of the mitochondrial apoptotic pathway, in response to the treatment Using Old Solutions to New Problems -Natural Drug Discovery in the 21st Century of HT29 colon-cancer cells with maslinic acid. The major flavonoid present in P. lanceolata, luteolin-7-O-β-glucoside, as well as aglycon luteolin, acted as potent poisons for DNA topoisomerase I on cancer cell lines [54]. Silibinin (major bioactive component from S. marianum) markedly activated the DNA-PK-p53 pathway for apoptosis, in response to UVBinduced DNA damage [69]. DNA-PK pull-down assay showed that silibinin pre-treatment strongly increased binding of DNA protein kinase with p53 [69].   Akt (Protein kinase B); Bcl-2 (Protein kinase B); Bax (Bcl-2-associated X protein); Topo-1 (Topoisomerase 1); p53 (tumor protein 53); Ser15 (Serine 15); NF-kB (nuclear factor kappalight-chain-enhancer of activated B cells); AR (Androgen Receptor)

2.
The mechanism of Maslinic acid (MSA) (isolated from Olea europaea) is regulated via Bcl-2 inhibition and Bax induction, producing mitochondrial disruption, cytochrome-c release, leading finally to the activation of caspases 9 and caspase 3.

3.
Luteolin-7-O-β-glucoside and its aglycon, luteolin (major bio-active constituents of Plantago lanceolata) showed DNA topoisomerase I poison activities and Topoisomerase mediated DNA damage might be the possible mechanism which induce apoptosis.

4.
Silibinin (SBN) (extracted from Silybum marianum) pretreatment enhance DNA-PK (DNA Protein kinase) associated kinase activity as well as the physical interaction of p53 with DNA-PK and it preferentially activates the DNA-PK-p53 pathway for apoptosis.

Mode of action References
Achyranthes aspera Inhibition of microtubule assembly 70 Table 4. Mode of action of anticancer activity of phytochemicals present in selected North American medicinal plants

Conclusion
Currently, natural products, especially plant secondary metabolites such as isoprenoids, phenolics and alkaloids, have been demonstrated to be the leading providers of novel anticancer agents. Thiese important groups of phytochemicals represent a vast majority of chemical groups, including alkaloids, flavonoids, flavonols, flavanols, terpenes and terpenoids, phenols, flavonolignans and steroids. Potential anticancer properties of these phytochemicals have been shown by both cell culture (in vitro methods) and animal (in vivo methods) studies. However, in vitro and in vivo findings should be strengthened by valid human clinical trial data before introducing to the medicine cabinet as natural therapeutics or drugs.