Biocompounds—dietary sources and mechanisms of action involved in modulation of apoptosis.
A big challenge for a successful colon cancer treatment is the lack of eradication of the entire tumour cell population and consequent development of chemoresistance. Control of cell number from tissues and elimination of cells predisposed to malignant transformation, having an aberrant cell cycle or presenting DNA mutations, might be performed by a cellular ‘suicide’ mechanism — the programmed cell death, or apoptosis. Coordinated activation and execution of multiple subprograms are needed, added by a good knowledge of the basic components of the death machinery, besides their interaction to regulate apoptosis in a coordinated manner. Triggering apoptosis in target cells is a key mechanism by which chemotherapy promotes cell killing. Many anti‐cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. New therapeutic approaches in cancer induce tumour cells to undergo apoptosis and break the cancer cell resistance to apoptosis commands. Administrations of natural compounds that prevent induction, inhibit or delay the progression of cancer, or induce inhibition or reversal of carcinogenesis at a premalignant stage represent chemoprevention strategies. Several natural compounds have been shown to be promising based on their anti‐cancer effects and low toxicity; alternative approaches might be taken into account to obtain a stronger anti‐tumour response when lower concentrations of anti‐cancer drugs are used, and to diminish the undesirable side‐effects.
- colon cancer
- tumour evasion
- bioactive compounds
- combined therapy
Cancer is a disease of cells that is thought to evolve along a multi‐step process: the transformation of normal cells, tumour progression and advanced metastasis, that involve a complex series of events such as genetic alterations, aberrant progression of the cell cycle, resistance to growth inhibition, proliferation without dependence on growth factors, replication without limit, evasion of apoptosis, induction of angiogenesis and modification of cell adhesion . For an accurate prediction, prevention, early detection and development of anti‐cancer drugs, it is essential to identify the stages of development and use basic information . The lack of eradication of the entire tumour cell population and the consequent development of chemoresistance represent main obstacles to a successful treatment in many malignancies, including colon cancer [3, 4]. The control of cell number from tissues and elimination of those predisposed to malignant transformation, having an aberrant cell cycle or presenting DNA mutations, might be performed by a cellular “suicide” mechanism, the programmed cell death or apoptosis [5, 6]. Elucidating the mechanisms of programmed cell death process seems to be of great importance for carcinogenesis, tumour evasion, and to have practical implications for anti‐cancer therapy since many anti‐cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction [7, 8].
Several therapeutic agents used in colon cancer treatment, e.g. fluoropyrimidines, cisplatin, oxaliplatin, irinotecan have been shown to induce resistance in cancer cell killing, and their number are rapidly increasing, possibly through the modulation of survival cell components, such as proliferative or anti‐apoptotic proteins [9, 10]. Triggering apoptosis in target cells represents a key mechanism by which chemotherapy promotes cell killing. Continuing efforts are made for discovering new molecular target‐based molecules , and new therapeutic approaches in colon cancer involve restored cellular mechanisms responsible for the induction of apoptosis in tumour cells [12–15].
A main strategy in colon cancer treatment might be the combined multi‐drug chemotherapy, the reason being the potential additive or synergistic tumour cytotoxicity produced . The focus on finding new therapeutic strategies has recently shifted to natural products. Various plants and their bioactive compounds have been shown to have anti‐carcinogenic and anti‐proliferative effects towards the colon cancer cells. Studies have also reported positive correlation between the antioxidant activity of plants and their anti‐proliferative effects, suggesting the potential action of antioxidants in inhibiting cancer cell growth. For example, the flavonoids display a wide range of biological activities, including anti‐inflammatory and cytoprotective activities, and several are known to act as anti‐cancer reagents .
The administration of synthetic or natural compounds that prevent induction of cancer, inhibit or delay its progression, or reverse carcinogenesis at a premalignant stage could represent useful strategies because of their potential clinical application in combined treatments with anti‐cancer drugs . By combining natural compounds with anti‐cancer drugs, it might be obtained an increase of cancer treatment effects, specifically in highly invasive colon cancer cells, while in non‐tumour cells the use of natural compounds could reduce the cytotoxic side effects .
2. Biology of colon cells: normal versus carcinogenic
Colorectal cancer (CRC) is the third most common malignancy worldwide, being frequently diagnosed in advanced stages. Recent data added to the molecular explanations of growth dysregulation, metastasis formation, extension of life span, and loss of maintenance of genomic and epigenetic integrity in cancer suggest models for their causal connection. The mechanisms of growth control, senescence, and anchorage dependence are linked at the molecular level .
The adult colon epithelium contains three cell types that arise from a multipotent stem cell: absorptive epithelial, enteroendocrine and Goblet cells. Colonic epithelial cells are configured in deep invaginations into the wall of the colon named crypts: from stem cells located at the base of the crypt, they arise and migrate to the luminal surface of the crypt where they are shed. Stem cells divide asymmetrically: the “old” DNA is retained in the stem cell population, and the new synthesised DNA is donated to daughter cells that migrate up the crypt and are ultimately shed. Stem cells are particularly vulnerable to developing mutations that might evolve into a malignant clone. Therefore, the cells located at the base of crypts, presumably stem cells, are highly prone to apoptosis, able to counteract dangerous mutations . The result of the imbalance between cell proliferation and apoptosis determines colorectal tumour growth. Relatively undifferentiated tumours with higher proliferative potential are often more aggressive than well‐differentiated ones . The molecular mechanisms of cell division and apoptosis are similar in normal and tumour cells, but in tumour cells, these mechanisms are aberrantly regulated. Four cellular functions are inadequate regulated in tumour cells: (1) control of cell proliferation is inefficient; (2) genetic and chromosomal structure is destabilized; (3) cellular differentiation program is frequently altered; (4) the control of apoptosis is disturbed .
Multiple sequential genetic changes are needed to occur in order to ensure colorectal cancer evolution. During progression of normal epithelial to carcinoma cell in colorectal cancer, TP53, KRAS, BRAF and PIK3CA gene alterations play important roles. Gene alterations cause disruption of signalling pathways in which they are involved, accompanied by increased proliferative potential and decreased apoptosis of cells . Along with genetic mutations, colon carcinogenesis is accompanied by epigenetic changes that lead to altered expression of key genes. Three major epigenetic regulatory mechanisms are described: (a) DNA methylation, (b) the covalent modifications of histones and (c) non‐coding RNA interference .
3. Programmed cell death in normal versus carcinogenic colon cells
Apoptosis represents a cellular “suicide” mechanism which allows control of the number of cells from tissues and removal of cells that present DNA mutations or have an aberrant cell cycle, predisposed to malignant transformation . Thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion, and therefore for anti‐cancer therapy like restoration of cellular mechanisms responsible for the induction of apoptosis in tumour cells [23, 24]. Abnormalities in apoptotic function contribute to both pathogenesis of colorectal cancer, and its resistance to chemotherapeutics and radiotherapy .
3.1. Apoptosis pathways
Apoptosis is an active, specialized form of cell death with distinct biochemical and genetic pathways that play a critical role in normal tissue homeostasis and development. Under stress, such as precancerous lesions, the mechanisms involved in repairing DNA damage are activated and potentially harmful cells are removed, and carcinogenesis is blocked . Lack of regulation of the apoptosis pathways may promote tumorigenesis and induce resistance to treatment in cancer cells .
The apoptotic process displays morphological features of the cells: cellular shrinkage with nuclear chromatin condensation and nuclear fragmentation, membrane blebbing, and cell‐self‐fragmentation into apoptotic bodies. Apoptosis is initiated by two basic signalling pathways:
3.2. Evasion mechanisms of apoptosis in colon cancer
Apoptosis is subverted during tumorigenesis through the systematic loss of regulatory control mechanisms, ultimately resulting in the generation of a malignant phenotype and resistance to chemotherapy and radiation therapy. Several potential mechanisms and factors involved were taken into account to explain the defects in apoptotic signalling and the increased activation of anti‐apoptotic pathways that were observed in colon cancer cells:
(a) Disrupted balance between pro‐ and anti‐apoptotic proteins
Many proteins exert pro‐ or anti‐apoptotic activity in cells, and the ratio between them plays an important role in the regulation of cell death. Over‐ or under‐expressed genes were also found to contribute to carcinogenesis by reducing apoptosis in cancer cells. Key regulatory proteins of apoptotic machinery, such as Bcl‐2 (including Bcl‐xl and Bax) and IAP family, undergo changes in expression during the transition from adenoma to carcinoma, and therefore, they were used as prognostic biomarkers . Pro‐ and anti‐apoptotic mediators can regulate mitochondrial outer membrane permeability and release of cytochrome
When there is a disruption in the balance of anti‐apoptotic and pro‐apoptotic members of the
Overexpression of the anti‐apoptotic Bcl‐2 family member Bcl‐XL predicts poor prognosis in patients with colonic adenocarcinomas, conferring a multidrug resistance phenotype [46, 47]. Bcl‐w, another anti‐apoptotic Bcl‐2 family protein, plays a general role in the progression from adenoma to adenocarcinoma in the colorectal epithelium; it is frequently expressed in colorectal adenocarcinomas at significant higher levels in TNM stage III tumours, positive correlated with node involvement . In primary colorectal adenocarcinomas, elevated levels of expression for Bcl‐xL and Bcl‐w were reported to be associated with reduced expression of Bax . Regarding the pro‐apoptotic members of Bcl‐2 family like Bcl‐10, Bax, Bak, Bid, Bad, Bim, Bik, and Blk, increasing evidences suggest the involvement of Bak and Bax in the release of cytochrome
One of the best known tumour suppressor proteins is
(b) Reduced caspase activity
During apoptosis process, the caspases implicated are either
(c) Impaired death receptor signalling
Several receptors and ligands that modulate the programmed cell death were described: TNF receptor superfamily, Fas/Fas‐L, CD27, death receptors and ligands, receptors phosphatases. Signalling via death receptors could be impaired in human cancers via downregulation of receptor surface expression as part of an adaptive stress response. Death receptors and their ligands are key players in the extrinsic pathway of apoptosis. The extrinsic signalling pathway leading to apoptosis involves transmembrane death receptors that are members of the tumour necrosis factor (TNF) receptor gene superfamily . Several abnormalities in the death signalling pathways that can lead to evasion of the extrinsic pathway of apoptosis have been identified: the downregulation of receptor expression, the impairment of its function, as well as a reduced level in the death signals, all of which contribute to impaired signalling and a reduction of apoptosis. Reduced membrane expression of death receptors and abnormal expression of decoy receptors have also been reported to play a role in the evasion of the death signalling pathways in various cancers [12, 70] (Figure 1).
(d) Altered redox status in apoptosis induction
The oxidative stress process is characterized by an increased generation of reactive oxygen species (ROS) accompanied by a dysfunction of the antioxidant systems which exist in every cell, dependent on the metabolic state of the cell [71, 72]. The increased metabolic activity, mitochondrial dysfunction, peroxisome activity, oncogene activity, increased activity of oxidases, cyclooxygenases, lipoxigenases could be responsible for the generation and release of reactive oxygen species in tumour cells [73–75]. Low levels of ROS may influence processes like angiogenesis, cell proliferation and survival, while intermediate levels of ROS cause transient or permanent cell‐cycle arrest and induce cell differentiation. When ROS production does not irreversibly alter cell viability, they can act as primary messengers, modulating several intracellular signalling cascades that lead to cancer progression . High levels of ROS induce cell apoptosis or necrosis by causing an alteration of membrane permeability, a genetic instability, oxidative modifications that lead to less active enzymes or proteins more susceptible to proteolytic degradation . Furthermore, ROS plays a crucial role in regulating expression of genes associated with cancer cell proliferation, angiogenesis, invasion and metastasis by activating transcription factors such as NF‐κB, activator protein‐1 (AP‐1) and hypoxia inducible factor‐1 (HIF‐1α) .
Excessive production of ROS in tumour cells induces apoptosis or necrosis, and acts as an important inhibitor of cancer cell proliferation. Fas ligand mediates the induction of ROS, essential for the initiation of apoptotic signalling cascade and activation of the intrinsic apoptotic machinery by disruption of mitochondrial membrane integrity  (Figure 1). The transformed cells use ROS signals to drive proliferation to tumour progression. Tumour cells present an increased basal oxidative stress, making them vulnerable to chemotherapeutic agents that further augment ROS generation or weaken antioxidant defences of the cell . Human colorectal tumours have increased levels of different markers of oxidative stress, such as ROS, nitric oxide (NO), lipid peroxides, glutathione peroxidase (GPx), catalase (CAT), and decreased cytosine DNA methylation [81–83].
ROS‐sensitive signalling pathways are persistently elevated in many types of cancers, including colon cancer . Reactive oxygen species can act as second messengers in cellular signalling. For example, hydrogen peroxide (H2O2) regulates protein activity through reversible oxidation of its targets, including protein tyrosine phosphatases, protein tyrosine kinases, receptor tyrosine kinases and transcription factors [85, 86]. The mitogen‐activated protein (MAP) kinase/Erk cascade, phosphoinositide‐3‐kinase (PI3K)/Akt‐regulated signalling cascades, as well as the IκB kinase (IKK)/nuclear factor κ‐B (NF‐κB)‐activating pathways are regulated by ROS. The extracellular signal‐regulated kinase pathway (ERK) mediates signal transduction involved in cell proliferation, differentiation, and migration . Activation of ERK in tumour cells by biocompounds (e.g. resveratrol, quercetin) results in anti‐proliferative effects, such as apoptosis, senescence, or autophagy [88–91]. Then, ERK can activate apoptotic enzymes or phosphorylate transcription factors that regulate the expression of pro‐apoptotic genes . Cell death in tumour cells treated with resveratrol and quercetin was accompanied by increased ROS levels and p53 expression, decreased Bcl‐2 expression, depolarization of the mitochondrial membrane, cleaved caspase‐3, and DNA fragmentation . Elevated levels of ROS triggered by treatment with biocompounds might inhibit dual‐specificity phosphatases (DUSPs) that dephosphorylate and inactivate MAPKs, leading to ERK activation and promoting cancer cell death. Therefore, biocompounds might induce apoptosis in colon cancer cells via activation of the MEK/ERK pathway .
Mitochondrial release of H2O2 and NO upon apoptotic signals leads to the activation of
In addition, ROS play an important role in the regulation of IKK/NF‐κB pathway. NF‐κB is a redox‐regulated sensor for oxidative stress that is activated by low doses of hydrogen peroxide. The activation of NF‐κB is mediated through the NF‐κB‐inducing kinase (NIK) and IκB kinase (IKK) complexes. Degradation of IκB translocates NF‐κB to the nucleus, where it acts as a transcription factor to induce the expression of anti‐apoptotic and anti‐inflammatory genes . Peroxisome proliferator‐activated receptor‐gamma (PPARγ) has been shown to exert an inhibitory effect on cell growth in most cell types. The expression of PPARγ was significantly increased in tumour tissues from human colon cancer, and the occurrence of apoptosis induced by PPARγ ligands was sequentially accompanied by reduced levels of NF‐κB and Bcl‐2. PPARγ‐Bcl‐2 feedback loop might control the life–death continuum in colonic cells, while a deficiency in generation of PPARγ ligands could precede the development of human colon cancer .
4. Bioactive compounds and colon cancer
Recent studies focused on the discovery of new chemotherapeutic agents among natural products since many plants and their bioactive compounds displayed anti‐carcinogenic and anti‐proliferative effects towards colon cancer cells . Positive correlations between antioxidant activities of plants and their anti‐proliferative effects, suggesting the potential action of antioxidants in inhibiting cancer cell growth, were also reported . Among them, over 5000 flavonoids were found in vegetables and fruits, wines, seeds, nuts, grains and teas, herbs, and represent a class of plant secondary metabolites, known for their antioxidant properties . The position of hydroxyl groups and other features in the chemical structure of flavonoids are important for their antioxidant and free radical scavenging activities . The dietary compounds could interfere with specific stages of the carcinogenic process, inhibiting cell proliferation and inducing apoptosis in different types of cancer cells . In addition, they might affect the expression of several detoxifying enzymes and their ability to modulate protein‐signalling cascades .
4.1. Dietary sources and functional features
Since the 1950s, despite extensive clinical trials, mortality from colon cancer is a major public health problem in developed countries as a result of high consumption of animal fat or red meat and low intake of fibres or vegetables . Protective factors include physical activity and increased intakes of dietary fibre, fish, nuts, dairy products, fruits and vegetables, while other factors, including weight and obesity, waist circumference, smoking, alcohol consumption, and red and processed meat intakes increase the risk of colorectal cancer [104, 105]. Using simple lifestyle modifications, changing the diet might substantially reduce the risk of colorectal cancer and could complement screening, so that CRC could be preventable in 90% of cases . Over the last decade, different drugs and nutritional elements have been studied in preclinical as well as clinical trials and proved to have potential benefit in the field of CRC prevention . Chemoprevention, the use of drugs or other agents to inhibit the development or progression of malignant changes in cells represents an alternative approach to reduce the mortality from colorectal cancer as well as other cancers .
|Biocompounds||Source||Mechanisms of action||Refs.|
|Grapes and red wine,|
mulberries, peanuts, seeds
Activation of MEK/ERK
Increase of ROS
and p53 levels
|Soybeans, fava beans,|
Inhibition of PTK
Inhibition of AKT
|Vegetables (capers, radish|
leaves, dill, cilantro,
fennel, red onion,
radicchio, kale), fruits
(cranberry, black plums,
blueberry, apples), seeds,
nuts, tea, red wine
|Bcl‐2, EGFR downregulation|
Cyclin D1, survivin inhibition
Inhibition of Wnt/beta‐catenin
Increase of ROS and p53
levels Activation of
|Turmeric, curry, mustard||NF‐κB inhibition|
Modulation of MAPK pathway
Downregulation of survivin and
|Parsley, celery, dandelion,|
coffee, chamomile tea
|Modulation of survival and|
death effectors (PI3K, AKT,
ERK, STAT3, JNK,
|Green tea, white|
tea, black tea
|Modulation of ROS production|
Inhibition of growth
signalling (EGF, VEGF, IGF‐I)
Inhibition of MAPK and
p21 pathways Downregulation
|Milk thistle seeds||Bcl‐2 downregulation|
Decrease of cyclin D1
and c‐myc expression
Upregulation of death
receptors DR4, DR5
and tomatoes (skin)
|Losses in mitochondrial|
Intracellular ROS production
Sustained ERK activation
|Pomegranate||Downregulation of Bcl2‐XL|
Caspase‐3 and caspase‐9
inhibition Suppression of AKT
|Broccoli, Brussels sprouts,|
cabbage, cauliflower, kale,
collards, kohlrabi, mustard,
turnip, radish, arugula,
|Upregulation of Bax, p21|
G2/M cell‐cycle arrest
|Disruption in mitochondrial|
Activation of caspase‐3,
caspase‐8 and caspase‐9
Inactivation of PI3K/Akt
pathway Bax upregulation;
Increase of ROS production
|Tomatoes, red carrots,|
|Bax and FasL upregulation;|
Bcl‐2 and Bcl‐XL
Downregulation of Akt,
Different natural compounds display a wide range of biological activities, including anti‐inflammatory and cytoprotective activities, and several are known to act as anti‐cancer reagents. Curcumin from turmeric, genistein from soybean, tea polyphenols like epigallocatechin gallate from green tea, resveratrol from grapes, sulforaphane from broccoli, isothiocyanates from cruciferous vegetables, silymarin from milk thistle, diallyl sulphide from garlic, lycopene from tomato, rosmarinic acid from rosemary, apigenin from parsley, gingerol from ginger and quercetin (Table 1) have high antioxidant activities, and demonstrated anti‐proliferative effects against various cancer cell lines .
5. Bioactive compounds and their role in modulation of apoptosis in colon cancer
Results of clinical trials revealed that colon cancer can be successfully treated by chemotherapy, if the tumour selective detection can be substantial increased. In this regard, there is an increasing demand for biomarkers for risk assessment, early detection, prognosis and surrogate end points. This will be possible by the introduction of new drugs with more precise mechanisms of action, such as those acting specifically upon well‐known aberrant pathways (e.g. apoptosis, cell signalling) . New drugs can initiate or modulate the apoptosis cascade acting on caspases, Fas, Bax, Bid, APC or molecules which promote colon cancer cell survival (p53 mutants, Bcl‐2 or COX‐2) .
The implementation of new treatment options (and the management of metastatic colon cancer) must take into account the role of apoptosis in colon tumorigenesis with a highlight on the mechanisms leading to chemotherapeutic resistance as well as immune system evasion . From this point of view, apoptosis can be considered as a potential target for cancer treatment at various stages of tumour progression, while chemoprevention as well as the apoptotic mechanisms could be utilized in the prevention and management of tumorigenesis [35, 135].
5.1. Mechanisms and targets involved
5.2. Epigenetic mechanisms related to apoptosis, influenced by natural compounds
Several papers pointed the role of various natural compounds that target the
HDAC activity is also inhibited by
6. Combined therapy of colon cancer: current strategies and future directions
Conventional therapeutic approaches, including chemotherapy, radiotherapy and surgery, are limited for the treatment of advanced colon cancer, in prevention of the disease recurrence, and are associated with a high risk of complications, highlighting the need to develop new therapeutic strategies. The majority of CRC patients receive chemotherapy using multiple agents that are currently approved for the treatment in the appropriate setting, but many patients have tumours intrinsically resistant to them. However, it is a complex process to select the optimal chemotherapy for each patient, and the difference between theory and practice is still a problem. That's why new concepts and modern technologies are promoted by precision medicine in order to achieve a personalized treatment for cancer patients.
6.1. RTCA: useful tool for screening biocompounds and drugs
Contrast data are available on the anti‐cancer effects of biocompounds in colon cancer, whether they could influence the effects of oncolytic drugs against the cell growth and apoptosis of human colon cancer cells, or which might be the proper concentrations of the compounds with cytotoxic or cytostatic potential. Real‐time impedance data obtained by the xCELLigence System (ACEA Biosciences) might be used to generate compound‐specific profiles which are dependent on the biological mechanisms of action of each compound used. The actual kinetic response of the cells within an assay prior or subsequent to certain manipulations provides important information regarding the biological status of the cell, such as cell growth, cell arrest, morphological changes and apoptosis . Changes in a cell status, such as cell morphology, cell adhesion or cell viability lead to a change in cell index (CI), which is a quantitative measure of cell number present in a well. For example, the cytotoxicity versus proliferative capacity of genistein, resveratrol or 5‐fluorouracil was assessed in LoVo colon cancer cell line in order to modulate the chemosensitivity of colon cancer cells to drug treatment, and overcome the chemo‐resistance. The entire length of the assay was presented, allowing informed decisions regarding the timing of certain manipulations or treatments, choice of the proper concentrations for further end‐point assays, such as flow‐cytometry techniques or molecular biology approaches [186, 187].
6.2. Modulation of apoptosis by combined therapy
Many anti‐cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. By combining natural compounds with anti‐cancer drugs, an increase of the effects might be obtained, specifically in highly invasive cancer cells, while in non‐tumoral cells the natural compounds could reduce the cytotoxic side effects  (Figure 2).
5‐Fluorouracil (5‐FU) that blocks the thymidylate synthase (TS), which is essential for DNA synthesis;
Capecitabine that blocks thymidylate synthase (orally administered prodrug converted to 5‐FU)
Oxaliplatin that inhibits DNA replication and transcription by forming inter‐ and intra‐strand DNA adducts/cross‐links;
Irinotecan that inhibits topoisomerase I, an enzyme that facilitates the uncoiling and recoiling of DNA during replication;
Bevacizumab, a monoclonal antibody, which binds to vascular endothelial growth factor (VEGF) ligand;
Cetuximab, a monoclonal antibody to epidermal growth factor receptor (EGFR) (chimeric), that blocks the ligand‐binding site;
Panitumumab, a monoclonal antibody to EGFR (fully humanized), that blocks the ligand‐binding site .
Several anti‐cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction [23, 189]. The pattern and extent of the cell damage induced by chemotherapeutics, like fluoropyrimidines, in human cancer cells have been suggested to depend also on the pathways downstream from drug‐target interactions that once triggered will initiate programmed cell death (apoptosis) [190, 191]. 5‐Fluorouracil (5‐FU) is one of the widely used chemotherapeutic drugs targeting various cancers, but its chemoresistance remains as a major obstacle in clinical settings. Several groups reported the induction of apoptosis by 5‐fluorouracil (5‐FU) in HT29  or LoVo human colon cancer cell lines [186, 187]. The long exposure of colon cells to 5‐FU treatments influences both pro‐ and anti‐apoptotic molecules like P53 and Bax, or Bcl‐2 and Bcl‐XL . Several studies showed that 5‐FU inhibits DNA proliferation in colon cancer cells by inhibiting the enzyme thymidylate synthase, leading to apoptosis, a mechanism of active cell death characterized by rapid loss of plasma membrane integrity, DNA fragmentation and altered expression of numerous genes [46, 52, 104] (Figure 2).
The biocompounds extracted from botanicals may be used as chemopreventive and therapeutic agents for various human cancers, inclusive colon cancer . The active biocompounds might induce cancer‐selective cell death by increasing production of reactive oxygen species. The cancer cells have increased levels of ROS accompanied by a highly active antioxidant defense system; therefore, the tumour cells are unable to recover from additional oxidative stress and die. It is accepted that mitochondria‐derived ROS play a critical role in their pro‐death and chemopreventive responses. The natural biocompounds inhibit mitochondrial electron transport chains causing ROS production, thus triggering apoptotic cell death . By combining flavonoids with anti‐cancer drugs, it might be obtained an increase of the desired effects, specifically in highly invasive cancer cells, while in non‐tumour cells the cytotoxic side effects could be reduced . In vitro, studies showed that LoVo colon cancer cells were markedly sensitized to apoptosis by both 5‐FU and genistein compared to the 5‐FU treatment alone. When time of incubation was increased, treatments with GST and/or 5‐FU had much stronger effects on the induction of apoptosis in LoVo cells, evaluated by using annexin‐V/FITC and PI double staining, followed by flow‐cytometry analysis . Similar studies demonstrated the additive effect of GST to anti‐cancer drug treatment, and in reversing the multi‐drug resistance [13, 14].
Experimental assays showed that resveratrol (RSV) induced higher levels of early and late apoptosis compared to untreated or 5‐FU‐treated LoVo cells. When treatments were prolonged to 72 h, stronger effects were observed both for RSV alone and combined treatments with 5‐FU . Flow‐cytometry analyses showed that treatments with 25 μM 5‐FU or 50 μM RSV slightly increased the expression of the pro‐apoptotic molecules p53 and Bax. The combined treatments of 50 μM RSV and 25 μM 5‐FU induced a higher increase of p53 expression compared to the non‐treated cells. Also the increase of Bax expression was much higher for the combined treatments compared to non‐treated cells or treated cells with 5‐FU alone. Both RSV and 5‐FU treatments seemed to decrease Bcl‐2 expression, but the effect was stronger for the combined treatments. Combined treatments induced a higher increase of pro‐apoptotic antigen expression, both for P53 and Bax, compared to 5‐FU treatment .
Therefore, addition of flavonoids and other natural compounds might be an alternative approach in order to obtain the same or a stronger anti‐tumour response, enhance the chemosensitivity of tumours to anti‐cancer drugs, or diminish the undesirable side effects by using lower concentrations [194, 195].
A big challenge for a successful treatment of colon cancer is the lack of eradication of the entire tumour cell population and the consequent development of chemoresistance. Since many anti‐cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction, elucidation of the mechanisms that govern the programmed cell death process seems to be of great importance for carcinogenesis, tumour evasion, and to have practical implications for anti‐cancer therapy. Many therapeutic drugs used in cancer treatment proved to induce resistance in cancer cell killing, and their number are rapidly increasing, possibly through the modulation of survival cell components such as proliferative or anti‐apoptotic proteins. Contrast data are available on the anti‐cancer effects of natural compounds in colon cancer, whether they could influence the effects of oncolytic drugs against the growth and apoptosis of human colon cancer cells, or which might be the proper concentrations of compounds with cytotoxic or cytostatic potential. From a large number of natural compounds investigated, several have been shown to be promising, based on their anti‐cancer effects related to apoptosis. A newly arising field involves therapeutic approaches in cancer in order to induce tumour cells to undergo apoptosis and break the cancer cell resistance to apoptosis commands. Therefore, manipulation of the mechanisms of programmed cell death process could be of outstanding importance for malignant transformation, and alternative approaches might be used to obtain a stronger anti‐tumour response, and/or diminish the undesirable side effects by using lower concentrations of anti‐cancer drugs. Thus, new concepts and modern technologies are promoted by precision medicine in order to achieve a personalized treatment for cancer patients.