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Table of Contents

    Topic review

    Natural Products against Colorectal Cancer

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    Submitted by: Mohammad Basyuni

    Definition

    Cancer is a deadly disease that has become a burden to everyone. There were 9.8 million cancer deaths reported in 2018. Colorectal cancer (CRC) is the third most common type of cancer globally, with 1.80 million cases, and it ranks second with the highest mortality in the world, i.e., 862,000 deaths.

    1. Introduction

    Cancer is a deadly disease that has become a burden to everyone. There were 9.8 million cancer deaths reported in 2018. Colorectal cancer (CRC) is the third most common type of cancer globally, with 1.80 million cases, and it ranks second with the highest mortality in the world, i.e., 862,000 deaths [1]. The increase in CRC in developing countries is possibly due to an increase in the aging population, modern living habits, dietary habits, and an increase in risk factors for CRC, which include genetic diseases, smoking, alcohol, and lack of exercise. The percentage of CRC deaths in Indonesia in 2014 was 10% of the 103,000 CRC mortality rates in men and 8.5% of the 92,000 in women [2][3]. In Indonesia, CRC is an interesting case. For instance, the median age of colorectal cancer patients is younger than the western population. This means that productive young people are affected more, thus posing a heavy economic burden on their families [4][5][6].

    Cancer treatment with chemotherapy agents is still an option, but the multi-drug resistance (MDR) mechanism has resulted in reduced efficacy of chemotherapy drugs [7]. The chemoprevention agents referred to here generally have the role of inhibiting tumor growth through cell cycle arrest mechanisms [8], stimulating apoptosis, or inhibiting the expression of proteins that play a role in MDR [9]. Various efforts are needed to develop new treatment methods for more effective therapy and prevention of degenerative diseases [10]. Alternative options such as the use of medicinal plants in the treatment of degenerative diseases can decrease any side effects [11]. Usually, lower effects may affect low efficacy, so there may be a trade-off. Therefore, there is a need to develop targeted therapy that uses super toxic plant-derived toxins as warheads that are to be conjugated to monoclonal antibodies targeting the CRC-specific antigens [12].

    One of the strategies in the discovery and development of drugs for the prevention of degenerative diseases is by exploring natural products, especially plants, that have the potential to be sources of antioxidants. Plants are known to have an important role in drug discovery [13]. Natural products are secondary metabolites produced by plants, animals, and microorganisms in response to external stimulation such as changes in nutrition, infection, and competition [14].

    2. Analysis on Results

    The PRISMA flow diagram of the publication screening is presented in Figure 1. Initially, 465 publications were identified through the systematic literature search string described in Table 1. Following the relevance of the initial papers to the defined research questions for this study, the literature screening, which consisted of title, abstract, and full-text screenings, resulted in 38 final papers for further data extraction. Overall, the screening processes only included 10% of the initially identified papers in Scopus, PubMed, and Google Scholar publication databases. The literature search results from major scholar databases such as Scopus as well as Google Scholar suggested the approximate publications that were recorded out of publications with duplicates (Figure 1). The cut-off date of the literature search was 13 February 2021.
    Figure 1. The PRISMA flow diagram showing the selection of eligible studies.
    Table 1. Search string composition adopted from defined PICO with desired focus on studies from Indonesia in both English and Bahasa Indonesia.
    Language Geographical Location Population Search Terms Intervention Search Terms
    English Indonesia Colorectal cancer* OR “colorectal cancer*” Secondary metabolite* OR natural product*
    Bahasa Indonesia Indonesia “Kanker kolon” atau kanker usus besar* Produk bahan alam* atau metabolit sekunder*
    As presented in Table 2, the research publication on colorectal cancer based on natural products derived from Indonesian plants has increased since 2008. The trend of publications seems to have increased significantly, starting in 2016, and it was the highest in 2018. Afterward, the positive trend continued until 2020, with the most publications in the form of journal articles.
    Table 2. The trend of publication on natural products in colorectal cancer in Indonesia: (a) year vs the number of publications; (b) types of publications reported.
    Year Number of Publication (a)
    1990 1
    2007 1
    2008 2
    2012 2
    2013 1
    2015 2
    2016 4
    2017 5
    2018 8
    2019 7
    2020 5
    Types of Publication Publication Reported (b)
    Book/Book Chapter 1
    Journal Article 35
    Thesis 2
    Table 3 exhibits that the distribution of natural products having the potential activity against colorectal cancer cells in Indonesia is dominated by terpenoids, followed by phytosterols, phenolics, alkaloids, and polyisoprenoids. The type of cell line that is most often utilized in the cytotoxic activity analysis of natural products against colorectal cancer cells is the WiDr cell line (followed by HT-29 cells and HCT-116 cells). WiDr cells are human colorectal cancer cells isolated from the colon of a 78-year-old woman, a derivative of other colorectal cancer cells, namely HT-29 cells [15]. This data led to the MTT in vitro assay as a general method that was used to analyze the cytotoxic activity of a natural product against colorectal cancer cells, followed by other in vitro and in vivo methods. Commonly known anticancer methods include the MTT assay method, apoptosis, enzymatic inhibition by the in vitro method, and the use of mice and rats in the in vivo method. Azoxymethane, a metabolite of dimethyhydrazine (DMH), has been used extensively by many investigators to induce colon tumors and to study the effects of nutritional factors and chemopreventive agents in colon carcinogenesis. Azoxymethane (AOM) induces colon cancer in experimental animals; in a mechanism that is mediated by glutathione (GSH) depletion and impairing the total antioxidant capacity in colonic cells of rats [16][17]. In anticancer studies, researchers prefer the in vitro method, as can be seen from the larger research ratio than the in vivo method from results screened in the Scopus, Pubmed, and Google Scholar databases. A more complete explanation of the distribution of the types of methods, objects used, and natural products analyzed from 38 articles is summarized in Table 4.
    Table 3. Systematic review results summarized from 38 publications: (a) types of natural product; (b) the number of used cell lines; (c) colorectal cancer cytotoxicity analysis method.
    Types of Natural Product Number of Natural Product Reported (a)
    Phenolic 8
    Phytosterol 9
    Carotenoid 1
    Terpenoid 17
    Alkaloid 8
    Flavonoid 5
    Peptide 3
    Polyketide 2
    Polyisoprenoid 5
    Carbolyc acid 1
    Fatty acid 5
    Glycoside 2
    Aromatic compound 1
    Types of Cell Lines Number of Used Cell Lines (b)
    HCT-15 3
    Colo205 1
    HT-29 5
    CaCo-2 2
    HCT-116 6
    SW-480 1
    CRC 2
    Colo320DM 1
    WiDr 16
    ADC 1
    AOM CRC Rat Model 2
    Colorectal Cancer Cytotoxicity Analysis Method Number of Used Method (c)
    MTT in vitro assay 22
    In vivo 3
    Others in vitro 8
    Table 4. Systematic review results summarized from 38 publications.
    Types Method Colorectal Cancer Cytotoxic Analysis Method Types of Object/CRC Cell Lines Types of Natural Products The Concentration of the Tested Samples IC50 Value / % Cell Viability / % Inhibition Cytotoxicity Categorize [18] Mechanism of Actions
    In vivo Colonic lesions induced by azoxymethane (AOM) Rats Non-nutritive compounds in fruits, vegetables, and fruits [19] Control of cell proliferation in ACFs and/or normal-appearing crypts of rats exposed to AOM [19].
        Rats Ethanol extract of Phaleria macrocarpa fruits (mostly flavonoids contains) [20] The crude ethanolic extract of P. macrocarpa had high antioxidant activity and it modulated the oxidative stress as proved by the up-regulation of glutathione-s-transferase and superoxide dismutase [20].
        Rats Water extract of Premna oblongifolia Merr. Leaves (polyphenolic compound) [21] Natural dietary fiber and antioxidant sources (as found in fruits, vegetables, and plant extracts) may exhibit a protective effect against CRC [21].
      Xenograft model nude mice (carrying HCT-15 cells) [22] Mice Lissoclibadins (polysulfur aromatic alkaloids) from Ascidian lissoclinum [22] Lissoclibadin 1 suppressed tumor growth in nude mice. Lissoclibadin 1 induced cell death via apoptosis due to the mitochondrial cytochrome dependent activation (intrinsic pathway) of the caspase-9 and caspase-3 cascade pathway [22].
    In vitro MTT assay [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43]; MTS assay [44][45][46]; WST assay [22]; SRB assay [47]; apoptosis with double staining method [30][41][43][48]; cell cycle analysis [49] gene expression analysis [32][33]; mitochondrial membrane potential ѱm (MMP), cytochrome c release analysis and NFkB translocation [45]; caspase activity and inhibitor assays [45]; protein extraction, protein array and western blotting analyses [45]; computational molecular docking and statistical analyses [41][46] HT-29 [20][45][46][50]; HCT-15 [22][23][39]; Colo205 [44]; WiDr [24][26][27][28][30][32][33][34][35][36][37][38][40][41][49]; HCT-116 [25][29][31][42][43][47]; CaCo-2 [45] Catechin 7-O-apiofuranoside and didesmethyl tocotrienol [20] 12.5–200 µg/mL 68% (inhibition) Not classified The fraction of P. macrocarpa exhibited the highest activity as anti-proliferative against HT-29 cells. The compounds had antioxidant activity leading to a cytoprotective effect. The mechanisms of this chemoprevention included up-regulation of Bax and proliferation-promoting proteins (PCNA) [20].
          Lissoclibadins (polysulfur aromatic alkaloids) from Ascidian lissoclinum [22] 5 μM 4.0 μM Significant / strong Lissoclibadin 1 exerted the most potent cytotoxic effects and mainly promoted apoptosis through an intrinsic pathway with the activation of a caspase-dependent pathway in HCT-15 cells [22].
          Leptoclinidamide and (−)-leptoclinidamine from Leptoclinides dubuis [23] 0 to 27 μM The compounds not displayed activity against cell line Not classified Not active against cancer cell line [20][22].
          Curacyclin A and B from the latex of Jatropha curcas L. [44] 1–1000 µg/mL The compounds did not have any effect on cell line No cytotoxicity
          The ethanol extracts from 95 ascidians collected at North Sulawesi, Indonesia; shermilamine B and kuanoniamine D [39] 0 to 27 μM 6.7 µM (shermilamine B) and 4.1 μM (kuanoniamine D) Significant / strong Shermilamine B and kuanoniamine D was classified in the pyridoacridine alkaloids, which have been known to exhibit various bioactivities such as cytotoxicity, inhibition of topoisomerase II, anti-HIV activity, Ca2+ releasing activity, and intercalation with DNA [39].
          Crude ethyl acetate extract of endophytic fungi isolated from Annona muricata leaves; alkaloid compounds [24] 25; 50; 100; 200; 400 µg/mL 20.80 µg/mL Moderate Alkaloid compounds in endophytic fungal extract of isolate Sir-SM2 had a high cytotoxic effect on the colon cancer cell and the lowest toxicity to normal cells compared with other fungal extracts. The compounds have an alkylating activity that can cause breakage and damage of DNA strands, leading to the cancer cells death [24].
          Fungi derived from the marine sponge Neopetrosia chaliniformis [34], Acanthostrongylophora ingens [40], Aspergillus nomius NC06 [43] 100 ppm [34] 70.31% (cell viability) [34] Not classified Marine-derived fungus NC06 from sponge N. chaliniformis AR-01 showed the most selective cytotoxicity against the WiDr cell line compared to the Vero cell line [34].
          100 µg/mL [40] 12.88% (cell viability) [40] Strong cytotoxicity (≤ 50%) Not presented
          100; 10; 1; 0.1 µg/mL [43] 5.28 µg/mL [44] Significant / strong Not presented
          Polyisoprenoids (polyprenol and dolichol) from Nypa fruticans, Rhizophora mucronata, Ceriops tagal, Avicennia alba, Avicennia marina and Avicennia lanata leaves [27][28][35][36][37][49] 15.625; 31.25; 62.50; 125; 250; 500 μg/mL [37] 276 µg/mL (C. tagal) and 278 µg/mL (R. mucronata) [37] No cytotoxicity Polyisoprenoids induced apoptosis in the early-apoptosis phase and caused cell cycle arrest in the G0-G1 stage while decreasing the expression of Bcl-2 and cyclin-D1. In addition, the polyisoprenoid had a SI value for classification as highly selective and enables the suppression of COX-2 expression in WiDr cells [35][36][37].
          15.625; 31.25; 62.525; 125; 250; 500 µg/mL [27] 180.2 µg/mL (N. fruticans) [27] Low
          15.625; 31.25; 62.525; 125; 250; 500 μg/mL [28] 180.186 μg/mL (N. fruticans) [28] Low
          500; 250; 125; 62.5; 31.25 µg/mL [35] 154.987 µg/mL (A. marina) and 305.928 µg/mL (A. lanata) [35] Low and no cytotoxicity
          500; 250; 125; 62.5; 31.25; 15.625 µg/mL [36] 173.775 μg/mL (A. alba) [36] Low
          Ethyl acetate extract from Trichoderma reesei strain TV221 (EAFTrR) associated with marine sponge: Stylissa flabelliformis [37] 2000, 1000, 500; 400; 300; 250; 200; 150; 125; 100; 75; 62,5; 50; 25 µg/mL 88.88 µg/mL Low The extract has the potential of having anti-cancer genes through the capability to spur apoptosis. The mechanism of inhibition of cancer cell growth may go by cell cycle arrest, cell cycle delay, or apoptotic mechanism [38].
          Alpinumisoflavone from Erythrina poeppigiana [41] 100.0; 50.0; 25.0; 12.5; 6.25; 3.25 μg/mL 5.63 µg/mL Significant / strong Alpinumisoflavone is a flavonoid that has a pyran ring as pyranisoflavonoid. The presence of hydroxyl group in A-ring in position 5 increase the cytotoxic activity of flavonoids. The presence of hydroxyl group in B-ring in positions 4’ is shown to increase the cytotoxicity of flavonoids [41].
          Dichloromethane extract of Canna indica rhizomes [26] 2000, 1500, 1000, 750; 500; 250; 125 ppm 361.83 ppm No cytotoxicity The extract contained a compound that could induce apoptotic activity and cell cycle in the WiDr cells [26].
          Nine lichen species from six different locations in East Java, Indonesia [30] 1024, 512; 256; 128; 64; 32 μg/mL 324 μg/mL No cytotoxicity Not presented
          Arcangelisia flava L. Merr chloroform extract [31] 50; 100; 200; 300; 400 µg/mL 121.637 µg/mL Low The chloroform extract of A. Flava was capable to trigger apoptosis in the WiDr cells [32].
          Piper crocatum Ruiz & Pav ethanol extract [33] 1; 10; 100; 500; 1000, 2000, 4000 µg/mL 727 μg/mL No cytotoxicity The ethanol extract of P. crocatum had an activity to induce apoptosis and suppress COX-2 protein expression in WiDr cells [33].
          Peptides from Platycephalus fuscus [46] 0.005 mg protein/mL 91.04% (inhibition) Not classified The further cell-based study is essential to observe the mechanistic pathways and structure or function relationship of peptides in stimulating apoptosis [46].
          Cycloart-24-ene-26-ol-3-one from Aglaia exima leaves [45] 0.39–200 μM 2.4 µM (HT-29); 5.6 µM (CaCo-2) Significant / strong It is bound to tumor necrosis factor-receptor 1 (TNF-R1) leading to the initiation of caspase-8 and, through the activation of Bid, in the activation of caspase-9. This activity causes a reduction in mitochondrial membrane potential (MMP) and the release of cytochrome-C. The activation of caspase-8 and -9 both acts to commit the cancer cells to apoptosis through downstream caspase-3/7 activation, PARP cleavage and the lack of NFkB translocation into the nucleus [45].
          Seaweeds (extracted in four kind of organic solvents): Gracilaria verrucose [51]; Ulva luctuca and Eucheuma cottonii [29]; Eucheuma Sp. [42] 200; 100; 50; 25; 12.5; 6.25; 3.125; 1.5625 μg/mL [51] 43.9 μg/mL (G. verrucose) [51] Moderate Not presented
          51.2; 25.6; 12.8; 6.4; 3.2; 1.6; 0.8; 0.4 µg/mL [29] 69.3 μg/mL (U. luctuca) and 21.4 μg/mL (E. cottonii) [29] Low and moderate
          51.2; 25.6; 12.8; 6.4; 3.2; 1.6; 0.8; 0.4 μg/mL [42] 16.82 μg/mL (Eucheuma Sp.) [42] Significant / strong
          2-O-β-glucopyranosil cucurbitacin D, isolated from the ethyl acetate soluble fraction of Benalu batu (Begonia sp.) [25] 6.25; 12.5; 25; 50 μg/mL 0.002 μg/mL and 6.88% (cell viability) Significant / strong The presence of cucurbitacin type triterpenoid could be a marker compound for Begonia plant species. It exhibited potent cytotoxic activity against HCT-116 via apoptosis induction with a significant percentage of early and late apoptosis [25].
          Chloroform fraction of Garcinia mangostana fruits hulls [47] 0.01–100 μM 15.8 µM Moderate The chloroform fraction contained bioactive compounds that induced significant antiproliferative and cytotoxic potentials via induction of apoptosis and cell cycle arrest at G0/G1-phase, necrosis, and apoptosis in HCT-116 cells [47].
          Polygonumins A from Polygonum minus [31] 100; 50; 25; 12.5; 6.25; 3.13 µg/mL 3.24 µg/mL Significant / strong The sugar moiety, a sucrose unit, was recognized to be critical to the topoisomerase inhibition activity as antitumor drugs [31].
          Gyrinops versteegii (Gilg.) Domke leaves extract (chloroform and ethanol solvents). The most abundant compounds detected in both extracts were fatty acids, namely palmitic acid, stearic acid, and pentadecanoic acid [51] Not determined Not presented (the first reported study on metabolite profiling of G. versteegii leaves extract, the result supported further study on G. versteegii as the anticancer-resource plant)
          (S)-2-hydroxy-3-(octanoyloxy)propyl tetracosanoate, (S)-3-(((S)-11-acetoxy octadecanoyl)oxy)propane-1,2-diyl diacetate, docosanedioic acid, 2,5-dimethylnonadecane, lupeol, stigmasterol, b-sitosterol, heptadecanoic acid, hexanedioic acid, 1,6-bis[(2R)-ethylhexyl] ester, and 1,3-di-O-[2′,2′-di- (p-phenylene)] were isolated from the leaves of Garcinia daedalanthera Pierre, collected from Indonesia [52] Not displayed 19.2 μM (lupeol) Moderate Not presented
          Fulvoplumierin; allamcin; allamandin; 2,5-dimethoxy-p-benzoquinone; plumericine; and lignan liriodendrin (from bark or Plumeria rubra) [53] Not displayed 0.1 µg/mL (plumericine); 0.3 µg/mL (allamcin and allamandin); 1.3 µg/mL (fulvoplumierin); 1.4 µg/mL (2,5-dimethoxy-p-benzoquinone); 16 µg/mL (liriodendrin) Significant / strong Not presented

    3. Discussion and Future Directions

    Cancer is the second leading cause of death in the world after cardiovascular disease. Meanwhile, colorectal cancer ranks in the top three in the number of causes of death and is ranked as the second most common cancer type in men and third in women [54][55]. A comprehensive therapy development to treat colorectal cancer is needed to reduce patient mortality. This therapy development is expected to be capable of overcoming the resistance of conventional chemotherapy agents that already exist today. Resistance of WiDr cells to 5-fluorouracil (5-FU)—an antimetabolite chemotherapeutic agent—is mediated by an increased expression of thymidylate synthetase enzyme which is the main inhibitory target of 5-FU [56][57]. WiDr is also one of the cells that have low sensitivity to treatment with 5-FU. WiDr cells are widely applicable in Indonesia due to proper carcinogenesis and tumorigenicity studies and anti-tumor testing on potency disclosure of bioactive compounds from natural products for further development. Besides, WiDr cells are identical toHCT-15 and HT-29 cell lines because they are derived from the same patient and most likely from the same tumor [57]. Overall, WiDr cells are suitable to be used as models in screening the novel compound as a co-chemotherapeutic agent with 5-FU. Combination therapy (co-chemotherapy) of 5-FU with a chemo-preventive agent is an alternative to overcome resistance, increase efficacy, and reduce adverse effects. These facts that lead the research and development of natural products become important for the future directions of medicinal plants in colorectal cancer therapy.

    Plants are one of the largest sources of natural products that are used to discover and develop novel chemotherapeutic agents. In particular, several discovered novel compounds from plants have unique mechanisms of action, greater efficacy, or lower adverse effects compared to the conventional chemotherapy drugs currently in use. Bioactive compounds from medicinal plants in Indonesia have also been studied to exhibit anticancer activities in the colorectal, namely terpenoids, phytosterols, alkaloids, phenolics, flavonoids, and polyisoprenoids ( the basic structure for one of several derivatives from those bioactive compounds is presented in Figure 2 ).

    Figure 2. The basic structure for one of the various derivatives from bioactive compounds of terpenoids, phytosterols, alkaloids, phenolics, flavonoids, and polyisoprenoids.

    In the current paper, we conducted a systematic review that provided predictions for several secondary metabolites sourced from natural products to be utilized as an alternative treatment against colorectal cancer in Indonesia. They are also prospective candidates for future co-chemotherapy agents in safety, quality, standardization, and efficaciousness. This finding emphasized the potential of several natural products as anticancer agents against colorectal cancer cells.

    Leading the discovery is the main component of today’s early pharmaceutical research. The exploration of novel drugs for colorectal cancer therapy that has a fast progression can be a drug repurposing strategy to bypass preclinical steps that usually require laborious and resource-intensive work. In addition, it is also considered that the development of existing agents in the future can be more easily utilized by the community. For those purposes, the exploration of Indonesia’s frequently used natural resources biodiversity is the best choice. In the past, drug development is based on trial and error, so it was costly and time-consuming. Today, molecular modeling, with the aid of computer hardware and software (computational method), has reduced the risk and the process of discovery is more effective in cost and time [58].

    4. Conclusions

    In summary, this entry revealed bioactive compounds from natural products of Indonesian plants that have been researched and have potential as anticancer agents that are most commonly experienced in men and women including colorectal cancer. The general method used for the analysis of the cytotoxic activity of colorectal cancer cells is the in vitro method using the MTT assay, the most widely used cell line is WiDr. The most studied bioactive compounds that have any activity against colorectal cancer are terpenoids, phytosterols, phenolics, alkaloids, and polyisoprenoids, but other natural products may have the potential to be developed from this study.

    The entry is from 10.3390/molecules26164984

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