Open access peer-reviewed chapter

Modulation of Apoptosis in Colon Cancer Cells by Bioactive Compounds

By Marinela Bostan, Mirela Mihaila, Camelia Hotnog, Coralia Bleotu, Gabriela Anton, Viviana Roman and Lorelei Irina Brasoveanu

Submitted: September 28th 2015Reviewed: March 30th 2016Published: September 7th 2016

DOI: 10.5772/63382

Downloaded: 1614

Abstract

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.

Keywords

  • colon cancer
  • apoptosis
  • tumour evasion
  • bioactive compounds
  • combined therapy

1. Introduction

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 [1]. 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 [2]. 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 [11], and new therapeutic approaches in colon cancer involve restored cellular mechanisms responsible for the induction of apoptosis in tumour cells [1215].

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 [1]. 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 [16].

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 [17]. 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 [18].

Advertisement

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 [2].

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 [19]. 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 [2]. 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 [20].

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 [21]. 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 [22].

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 [5]. 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 [19].

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 [25]. Lack of regulation of the apoptosis pathways may promote tumorigenesis and induce resistance to treatment in cancer cells [19].

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: the extrinsic pathway, initiated by external stimuli and via activation of death receptors on the cell surface, such as tumour necrosis factor‐α (TNF‐α), Fas (CD95/APO1) and TNF‐related apoptosis‐inducing ligand (TRAIL) receptors, and the intrinsic(or mitochondrial) pathway,activated by intracellular stimuli and characterized by mitochondrial outer membrane permeabilization and release of mitochondrial cytochrome c(cyt‐c) [26]. There is an overlap between the two apoptotic pathways: the extrinsic pathway usually also activates the intrinsic pathway, and both pathways result in the recruitment and activation of cysteine‐aspartic acid proteases (caspases) [27, 28]. Upon receiving specific signals instructing the cells to undergo apoptosis, the caspase family of proteins is typically activated and cleaves key cellular components required for normal cellular function, including structural proteins in the cytoskeleton and nuclear proteins such as DNA repair enzymes [29]. Caspases can directly signal apoptosis or use mitochondria as an intermediate and additional point of regulation in apoptosis signalling [30].

(a) the mitochondrial pathway(the intrinsic pathway) is activated by a wide variety of cytotoxic drugs, DNA damage, growth factor deprivation, oxidative stress, Ca2+ overload and oncogene activation [29, 30]. It is regulated by formation of the mitochondrial permeability transition pore (MPTP), composed by Bcl‐2 family members and voltage‐dependent anion channels on the outer mitochondrial membrane [31], leading to mitochondrial outer membrane permeabilization (MOMP). The drop of mitochondrial membrane potential initiates the osmotic swelling of the matrix by water influx and release of cytochrome cfrom mitochondrial intermembrane space into the cytoplasm. Cytochrome cthen associates with apoptotic protease‐activating factor 1 (APAF‐1) and caspase‐9 forming apoptosome complex. The activation of caspase‐9 and/or caspase‐8 leads to caspase‐3 cleavage, endonuclease activation, and ultimately nuclear DNA fragmentation, which is the hallmark of apoptosis [31, 32]. B cell leukaemia/lymphoma 2 (Bcl‐2) family proteins are central regulators of the intrinsic pathway, which either suppress or promote changes in mitochondrial membrane permeability required for the release of cyt‐c and other apoptogenic proteins [33].

(b) the extrinsic pathwaystarts with the stimulation of specific death receptors upon binding of their ligands, like tumour necrosis factor (TNF), tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and CD95 (Fas or APO1) [34]. Death receptors are transmembrane proteins with a death domain in their cytosolic region; the ligands binding causes oligomerization of these receptors, exposing their death domains (DD) in their cytosolic tail, which rapidly bind to Fas‐Associated Death Domain (FADD). Several of the DD‐containing TNF‐family receptors use caspase activation as a signalling mechanism, including TNFR1/CD120a, Fas/APO1/CD95, DR3/Apo2/Weasle, DR4/TrailR1, DR5/TrailR2, and DR6 [35, 36]. Binding of these receptors at the cell surface results in the recruitment of several intracellular proteins, including some procaspases, to the cytosolic domains of these receptors, forming a “death‐inducing signalling complex” (DISC) that triggers caspase‐8 activation [37, 38]. In the case of TNFR1, after the ligand binds to TNFR1, the cytosolic region of the receptor does not bind FADD, but TRADD adaptor, as well as several other signalling proteins, some of them being involved in the activation of NF‐κB transcription factor. The initial complex is then released from the receptor, TRADD binds to FADD in the cytosol, and caspase‐8 is recruited. The downstream signalling depends on additional interactions with proteins like FLICE‐like inhibitory proteins (c‐FLIP), forming a complex that contains heterodimers of caspase‐8 and c‐FLIP that will inhibit apoptosis. However, if NF‐κB activity is blocked or disrupted, or c‐FLIP expression is inhibited, caspase‐8 is activated and cell undergoes apoptosis [30, 39]. Between the death receptor pathway and the mitochondrial pathway of apoptosis, there is an overlap. Caspase‐8 has another substrate in the cell, BID, a BH3‐only protein: when caspase‐8 cleaves BID, the protein is translocated to the mitochondria to promote MOMP and to initiate mitochondria‐dependent apoptosis [40].

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 [7]. Pro‐ and anti‐apoptotic mediators can regulate mitochondrial outer membrane permeability and release of cytochrome cfrom the mitochondria into the cytoplasm [41] (Figure 1).

When there is a disruption in the balance of anti‐apoptotic and pro‐apoptotic members of the Bcl‐2 family, the result is a dysregulation of apoptosis in the affected cells. This can be due to the overexpression of one or more anti‐apoptotic proteins, or the underexpression of one or more pro‐apoptotic proteins, or both [42, 43]. In colorectal cancer, the dysregulated expression of Bcl‐2 family members may be associated with disease outcomes. Bcl‐2 expression is restricted to the basal epithelial cells in normal and hyperplastic mucosa, but in dysplastic polyps and carcinomas, it is extended to the parabasal and superficial regions. Bcl‐2 expression is increased in hyperplastic polyps and markedly increased in almost all adenomas, while carcinomas show weaker Bcl‐2 expression, indicating the decrease of apoptosis during progression from adenoma to carcinoma [44, 45].

Figure 1.

Role of biocompounds in modulation of intrinsic and extrinsic pathways of apoptosis.

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 [42]. In primary colorectal adenocarcinomas, elevated levels of expression for Bcl‐xL and Bcl‐w were reported to be associated with reduced expression of Bax [42]. 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 c, based on phosphorylation of both Bak and Bax that facilitate their homo‐oligomerization and subsequently the localization in mitochondria [29, 48]. Mutations in both Bax and Bak genes confer cells the resistance to apoptosis [49, 50]. In colon cancer, Bax gene is frequently mutated in hereditary non‐polyposis colorectal cancers [51] and microsatellite mutator phenotype [52, 53]. Decreased Bax expression was correlated with poor prognosis and progression towards metastasis [54].

One of the best known tumour suppressor proteins is p53, encoded by the tumour suppressor gene TP53 located on the short arm of chromosome 17 (17p13.1). The oncogenic property is due to a p53 mutations [55, 56], and half of all colorectal cancer cases show mutations in TP53 gene that were correlated with adenoma‐to‐carcinoma transitions and aggressive subsets of colorectal cancer [57, 58]. TP53 is a tumour suppressor gene in the mitochondrial apoptotic pathway, and one of the key regulators of cell‐cycle control and apoptosis. Tumour cells presenting p53 mutations are defective in the induction of apoptosis. Its expression is downregulated by survivin and Bcl‐2 [59]. The molecular mechanisms that are employed by p53 to induce cell death in the context of suppressing cancer progression include the transcriptional regulation of pro‐apoptotic PUMA expression, the generation of oxidative free radicals within mitochondrial components, the reduction of COX‐2/PGE2 synthesis, and the induction of death receptor 5 [60, 61] (Figure 1).

Inhibitors of apoptosis (IAPs family)suppress apoptosis through inhibition of effector caspases [62]. The expression of inhibitor of apoptosis proteins (IAPs) is dysregulated in colorectal cancer. The anti‐apoptotic regulators belonging to the IAP family, including XIAP, cIAP, and survivin, bind to caspase‐3 and caspase‐9 and thereby inhibit c aspase activity (Figure 1). Moreover, XIAP‐associated factor 1 (XAF1) negatively regulates the anti‐apoptotic function of XIAP. Molecules like c‐FLIP, XIAP, cIAP2, and survivin have increased expression levels in colon cancer patients, and this has been correlated with disease progression and poor survival [47, 63, 64].

(b) Reduced caspase activity

During apoptosis process, the caspases implicated are either initiator caspases(e.g. caspase‐2, caspase‐8, caspase‐9 and caspase‐10) which are primarily responsible for the initiation of the apoptotic pathway, or effector caspases(caspase‐3, caspase‐6 and caspase‐7), which are responsible in the actual cleavage of cellular components during apoptosis [65]. In the initiation and execution of apoptosis, caspases remain important players; therefore, low levels of caspases or impairment in caspase function may lead to a decrease in apoptosis and carcinogenesis [66, 67]. More than one caspase can be downregulated, contributing to colon cancer cell growth and development. Studies on differential expression by cDNA array showed a downregulation of both capase‐8 and caspase‐10, phenomenon that influences the pathogenesis of carcinomas [68].

(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 [69]. 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 [7375]. 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 [76]. 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 [77]. 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) [78].

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 [79] (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 [80]. 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 [8183].

ROS‐sensitive signalling pathways are persistently elevated in many types of cancers, including colon cancer [84]. 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 [87]. Activation of ERK in tumour cells by biocompounds (e.g. resveratrol, quercetin) results in anti‐proliferative effects, such as apoptosis, senescence, or autophagy [8891]. Then, ERK can activate apoptotic enzymes or phosphorylate transcription factors that regulate the expression of pro‐apoptotic genes [92]. 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 [93]. 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 [94].

Mitochondrial release of H2O2 and NO upon apoptotic signals leads to the activation of c‐Jun N‐terminal kinases (JNKs). In response to ROS, JNKs catalyze the phosphorylation and downregulation of anti‐apoptotic proteins such as Bcl‐2 and Bcl‐XL. JNK influences the composition of the Bax/Bcl‐2 complex by increasing the expression of Bax, leading to the formation of Bax homodimers and dissipation of mitochondrial membrane integrity [95, 96]. In response to the increased generation of ROS, the MAPK family member p38MAP is also implicated in apoptotic signalling [97].

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 [98]. 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 [99].

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 [13]. 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 [13]. 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 [100]. The position of hydroxyl groups and other features in the chemical structure of flavonoids are important for their antioxidant and free radical scavenging activities [70]. The dietary compounds could interfere with specific stages of the carcinogenic process, inhibiting cell proliferation and inducing apoptosis in different types of cancer cells [101]. In addition, they might affect the expression of several detoxifying enzymes and their ability to modulate protein‐signalling cascades [102].

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 [103]. 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 [106]. 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 [107]. 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 [108].

BiocompoundsSourceMechanisms of actionRefs.
ResveratrolGrapes and red wine,
mulberries, peanuts, seeds
Caspase activation
NF‐κB inhibition
FasL induction
Activation of MEK/ERK
pathway Bcl‐2
downregulation
Increase of ROS
and p53 levels
[9294,
111, 137,
166, 167]
GenisteinSoybeans, fava beans,
lupin, coffee
NF‐κB inhibition
Caspase activation
Inhibition of PTK
Inhibition of AKT
pathway mdm2
downregulation
[113, 114]
QuercetinVegetables (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
signalling pathway
Increase of ROS and p53
levels Activation of
MEK/ERK pathway
[9194,
164]
CurcuminTurmeric, curry, mustardNF‐κB inhibition
ROS induction
Modulation of MAPK pathway
Downregulation of survivin and
IGF‐1
expression
[141143,
146, 147]
ApigeninParsley, celery, dandelion,
coffee, chamomile tea
Modulation of survival and
death effectors (PI3K, AKT,
ERK, STAT3, JNK,
Mcl‐1)
[119, 168]
Epigallocatechingallate(EGCG)Green tea, white
tea, black tea
Modulation of ROS production
NF‐κB inhibition
Inhibition of growth
factor‐dependent
signalling (EGF, VEGF, IGF‐I)
Inhibition of MAPK and
p21 pathways Downregulation
of survivin
[148, 149,
151155,
157, 176]
SilibininMilk thistle seedsBcl‐2 downregulation
Bax upregulation
Decrease of cyclin D1
and c‐myc expression
Upregulation of death
receptors DR4, DR5
[17, 127,
136, 138,
139]
NaringeninGrapefruits, oranges
and tomatoes (skin)
Losses in mitochondrial
membrane potential
Caspase‐3 activation
Intracellular ROS production
Sustained ERK activation
[129, 163]
Pomegranate juicePomegranateDownregulation of Bcl2‐XL
Caspase‐3 and caspase‐9
activation NF‐κB
inhibition Suppression of AKT
pathway
[131, 132,
158, 160]
SulforaphaneBroccoli, Brussels sprouts,
cabbage, cauliflower, kale,
collards, kohlrabi, mustard,
turnip, radish, arugula,
watercress
Upregulation of Bax, p21
G2/M cell‐cycle arrest
[121, 122,
177, 178]
Ellagic acidStrawberries, walnuts,
pecans
Disruption in mitochondrial
membrane potential
Activation of caspase‐3,
caspase‐8 and caspase‐9
Inactivation of PI3K/Akt
pathway Bax upregulation;
Bcl‐2 downregulation
Increase of ROS production
[169, 170]
LycopeneTomatoes, red carrots,
watermelons, papayas
Bax and FasL upregulation;
Bcl‐2 and Bcl‐XL
downregulation
Downregulation of Akt,
NF‐κB
[171, 172]

Table 1.

Biocompounds—dietary sources and mechanisms of action involved in modulation of apoptosis.

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 [13].

Resveratrol(RSV, trans‐3,4′,5‐trihydroxystilbene), a naturally occurring polyphenol phytoalexin, is abundant in a wide variety of plants and their products, including grapes and red wine, mulberries, peanuts, seeds, and has anti‐inflammatory, antioxidant, anti‐neoplastic, anti‐carcinogenic, anti‐tumorigenic, cardioprotective, neuroprotective, anti‐aging and antiviral effects [102, 109]. Resveratrol exhibited anti‐colon cancer properties by inhibiting cell proliferation, inducing apoptosis, decreasing angiogenesis, and causing cell‐cycle arrest [110, 111].

Genistein(GST, 4′,5,7‐trihydroxyisoflavone) is a natural compound found in lupin, fava beans, soybeans, coffee and occurs in Asian diet, rich in soy products [112]. It is a strong topoisomerase inhibitor, similarly to etoposide and doxorubicin. It has a wide spectrum of activity, expressed in protecting cells from malignant transformation, reducing proliferation of tumour cells and stimulating apoptosis [113, 114].

Quercetin(QCT, 3,3′,4′,5,7‐pentahydroxyflavone) is an important dietary flavonoid, which presents in different vegetables (e.g. capers, radish leaves, dill, cilantro, fennel, red onion, radicchio, kale), fruits (cranberry, black plums, blueberry, apples), seeds, nuts, tea and red wine. It is involved in suppression of tumour‐related processes, including oxidative stress, proliferation and metastasis. QCT has also received greater attention as pro‐apoptotic flavonoid with a specific and almost exclusive activity on tumour cell lines rather than normal, non‐transformed cells [115]. The anti‐tumour effect found in SW480 colon cancer cell line was related to the inhibition of Cyclin D (1) and survivin expression, as well as Wnt/beta‐catenin signalling pathway [116].

Curcumin(CRM, 1,7‐bis‐(4‐hydroxy‐3‐methoxyphenyl)‐1,6‐heptadiene‐3,5‐dione) is a diarylheptanoid and the principal curcuminoid of turmeric, extracted from Curcuma longa; it possesses anti‐inflammatory and antioxidant properties, and has a strong inhibitory effect on cell proliferation in the HT‐29 and HCT‐15 human colon cancer cell lines [117, 118].

Apigenin(APG, 4′,5,7‐trihydroxyflavone) is one of the most common flavonoids widely distributed in fruits and vegetables, such as parsley, celery, dandelion coffee and chamomile tea. However, apigenin only showed a modest anti‐tumour activity towards cancer cells. New strategies are needed to enhance apigenin's anti‐tumour efficacy [119].

Epigallocatechin 3‐O‐gallate (EGCG)[(2R,3R)‐5,7‐dihydroxy‐2‐(3,4,5‐trihydroxyphenyl)‐3,4‐dihydro‐2H‐1‐benzopyran‐3‐yl 3,4,5‐trihydroxybenzoate] is the most abundant catechin in tea, a polyphenol found in high quantities in the dried leaves of white tea, green tea and, in smaller content in black tea [120]. EGCG was found to exert profound anti‐inflammatory, antioxidant, anti‐infective, anti‐cancer, anti‐angiogenic, and chemopreventive effects [120].

Sulforaphane(1‐Isothiocyanato‐4‐methylsulfinylbutane) is found in cruciferous vegetables such as broccoli, Brussels sprouts, cabbage, cauliflower, bok choy, kale, collards, Chinese broccoli, broccoli raab, kohlrabi, mustard, turnip, radish, arugula and watercress. Young sprouts of broccoli and cauliflower are particularly rich in glucoraphanin. It is produced when the enzyme myrosinase transforms glucoraphanin, a glucosinolate, into sulforaphane upon damage to the plant which allows the two compounds to mix and react [121]. Sulforaphane (SFN) is a naturally occurring chemopreventive agent, inducing the cell‐cycle arrest and apoptosis in colon cancer. However, little is known about the differential effects of SFN on colon cancer and normal cells [122].

Lycopeneis a bright carotenoid pigment and phytochemical found in tomatoes, red carrots, watermelons and papayas. Although lycopene is chemically a carotene, it has no vitamin A activity. Also foods that are not red may contain lycopene, such as asparagus or parsley. Lycopene exhibited potential anti‐carcinogenic activity, and the consumption of tomatoes was associated with reduced risk of several types of human cancer, including colon cancer [123, 124].

Glucobrassicinis a type of glucosinolate found in cabbages, broccoli, mustards, horseradish and woad. The main hydrolysis product after glucobrassicin is degraded by myrosinase is indole‐3‐carbinol, which was found to have apoptosis‐inducing effects in a concentration‐ and time‐dependent manner in human colon cancer cells [125, 126].

Silibininis the major active constituent of silymarin, a standardized extract of the milk thistle seeds, that contains a mixture of flavonolignans and was shown to induce apoptosis in colon cancer cells [127, 128].

Naringenin(5,7‐dihydroxy‐2‐(4‐hydroxyphenyl)chroman‐4‐one) is a flavanone, considered to have a bioactive effect on human health as antioxidant, free radical scavenger, anti‐inflammatory, carbohydrate metabolism promoter and immune system modulator. It can be found in grapefruits, oranges and tomatoes (skin) [129].

Pomegranatejuice obtained from Punica granatum is rich in polyphenol compounds such as gallo, ellagitannin and flavonoid classes. It possesses therapeutic activity such as anti‐atherogenic, anti‐parasitic, anti‐microbial, antioxidant, anti‐carcinogenic and anti‐inflammatory effects [130]; in preclinical animal studies, oral consumption of pomegranate extract inhibited the growth of lung, skin, colon and prostate tumours [131]. It was shown that pomegranate juice derivatives promote apoptosis of colon cancer cells by inducing the intrinsic pathway, but no effect was shown on the extrinsic pathway [132].

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) [133]. 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) [134].

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 [69]. 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

Grape seed extractthat consists in a mixture of polyphenols was able to decrease cyclin D1, and c‐myc expression, preventing cycle cell disruption, reduce expression of iNOS and COX‐2 decreasing oxidative cellular stress [136]. On the other hand, in vitrostudies performed on CRC cell lines showed that grape seed extract induces apoptosis via activation of caspase‐3, caspase‐8, and caspase‐9, and also generation of ROS. It is worth mentioning that proapoptotic activity of grape seed extract has no effect in normal colonocytes [107].

Resveratrol has anti‐CRC effects by inhibiting tumour initiation and progression by affecting caspase activation, NF‐κB inhibition and FasL induction. Resveratrol could suppress inflammatory responses through decreasing nitric oxide levels and inhibiting the phosphorylation of the IκB enzyme complex, thus suppressing the activation of NF‐κB dependent mechanisms [111, 137]. It was also described to interfere with mitochondrial functions by inhibiting mitochondrial ATP synthesis through its binding to F1‐ATPase. In addition, resveratrol can antagonize anti‐apoptotic proteins that prevent the induction of apoptosis in cancer cells. Resveratrol induces p53‐independent upregulation of p21, p21‐triggered cell‐cycle arrest and subsequently cell‐cycle‐dependent depletion of the anti‐apoptotic protein survivin, thereby sensitizing cancer cells to TRAIL‐induced apoptosis. Moreover, it suppresses expression levels of additional anti‐apoptotic proteins (e.g. Bcl‐xL and MCL‐1). The anti‐tumour activities of resveratrol are also due to its ability to interfere with the phosphatidylinositol‐3 kinase (PI‐3K)/AKT and the MAPK pathways [13] (Table 1).

Silibinin:Studies in vitroand in vivohave shown a chemopreventive role in CRC by interfering in proliferation, signalling pathway and inflammation processes [17]. It was also found to cause decrease of cyclin D1 and c‐myc expression [127]. Moreover, silibinin modulates the expression of anti‐apoptotic proteins (Bcl‐2, Mcl‐1, X‐linked inhibitor of apoptosis protein, and survivin) [136]. Silibinin can induce apoptosis by downregulation of the anti‐apoptotic protein Bcl‐2 and upregulation of the pro‐apoptotic protein Bax, inverting the Bcl‐2/Bax ratio. Sibilin also promotes apoptosis by upregulating transcription of the death receptors DR4 and DR5 [138], inhibits TNF‐α activation of NF‐κB, decreases expression of COX‐2 and iNOS [139]. Silibinin decreased the expression of IL‐1, TNF‐alpha and their downstream target MMP7, all of them being upregulated during colon carcinogenesis [127, 138]. In a study evaluating silibinin pharmacodynamics, Hoh et al. [140] showed that silibinin is not toxic to normal colonic epithelium (Table 1).

Curcuminis effective in apoptosis triggering, inhibiting DNA mutations, cancer cell proliferation, metastasis and inflammation. Curcumin induces the production of ROS in concentration that leads to p21 protein upregulation, and consequently inhibiting cancer cell growth [141]. Moreover, curcumin interferes with the protein kinase (MAPK) pathway, which in turn decreases production of TNF‐α and COX‐2 as well as downregulates the expression of NF‐κB and IL‐6 [142, 143]. The downregulation of NF‐κB levels has also effect on expression level of c‐myc, cyclin D1 and Bcl‐2 genes, and finally modulates the cell cycle [144]. It promotes cancer cell apoptosis by inducing expression of proapoptotic proteins (Bax, Bim, Bak, Noxa) and inhibiting expression of anti‐apoptotic proteins (Bcl‐2, Bcl‐xl) [143]. Curcumin prevents the formation of metastases by decreasing vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 expression [144]. In recent in vitro study in CRC cells, Patel et al. [145] have shown that curcumin inhibits the receptor expression of HER2 and insulin‐like growth factor 1 (IGF‐1) which is well known to create resistance to 5‐fluorouracil and oxaliplatin. Curcumin downregulates the expression of survivin and IGF‐1 by activating the expression of p53 and reducing TNF‐α levels, leading to activation of apoptotic signal [146, 147] (Table 1).

Epigallocatechin 3‐Gallate Epigallocatechin 3‐gallate (EGCG)has a strong antioxidant activity preventing ROS formation, blocking cancer cell proliferation and metastasis formation by down‐regulating the expression of growth factors (epidermal growth factor, IGF‐1, VEGF) [148]. EGCG blocks the cell cycle through the modulation of both MAPK and p21 pathways [149]. Furthermore, by upregulation of p53, EGCG induces apoptosis in CRC cells [150]. EGCG promotes cell growth arrest and induces apoptosis by affecting regulatory proteins of the cell cycle and inhibition of NFκB [151153]. Some reports point out that the ROS‐related effects may contribute to the anti‐proliferative and pro‐apoptotic activity of EGCG [154]. The effects are associated with modulation of reactive oxygen species (ROS) production. Although EGCG has a dual function of antioxidant and pro‐oxidant, EGCG‐mediated modulation of ROS production is reported to be responsible for its anti‐cancer effects. The EGCG‐mediated inhibition of NF‐κB signalling is also associated with inhibition of migration, angiogenesis and cell viability [155]. Furthermore, it inhibits growth factor‐dependent signalling (e.g. EGF, VEGF and IGF‐I), the MAPK pathway, proteasome‐dependent degradation and expression of COX‐2. EGCG seems to directly interact with and modulate the character of membrane lipid rafts, which explains the ability to alter signalling processes of growth factor receptors. Furthermore, EGCG inhibits telomerase, topoisomerase II and DNA methyltransferase 1, thereby affecting the functions of chromatin [153, 156]. EGCG has a protective effect against NO‐induced apoptosis in HDPC by scavenging ROS and modulating the Bcl‐2 family [157] (Table 1).

Pomegranatemay inhibit cancer cell proliferation and apoptosis through the modulation of cellular transcription factors and signalling proteins. In previous studies, pomegranate juice and its derivate inhibited proliferation and induced apoptosis in colon cancer cells, significantly suppressed TNF‐α‐induced COX‐2 protein expression. It also reduced phosphorylation of the p65 subunit and binding to NF‐κB, and abolished TNF‐α induced AKT activation, playing an important role in the modulation of cell signalling in colon cancer cells [158]. The pomegranate juice exhibited a dose‐ and time‐dependent decrease in cell proliferation, inducing cell‐cycle arrest in the G0/G1 and G2/M stages of the cell cycle, followed by apoptosis [159]. In the same regard, Larrosa et al. [160] have shown that induction of apoptosis was due to the downregulation of Bcl2‐XL protein as well as activation of caspase‐3 and caspase‐9, but not caspase‐8 (Table 1).

Citrus flavonoids:Volatile oil of Citrus aurantifolia, showed 78% growth inhibition of human colon cancer cells, induced the characteristic pattern of DNA fragmentation, via caspase‐3 dependent pathway along with modulation of apoptosis‐related protein expression [161].

Orange(Satsuma mandarin) juice contains a lot of flavonoids, which are potential chemoprotective compounds, with the capacity to suppress the expression of several cytokines (TNF‐α, IL‐1β, IL‐6) as well as inflammatory enzymes (COX‐2 and iNOS). In human colon cancer cell lines, the mechanism that induced the inhibition of their growth acted by blocking the cell cycle in G0/G1 phase and reducing levels of cyclins (A, D1 and E) [162].

Naringenin:Treatment with naringenin derivates resulted in significant apoptosis‐inducing effects concomitant with losses in mitochondrial membrane potential, caspase activation, intracellular ROS production and sustained ERK activation [129]. In human colon adenocarcinomas, it induced activation of p38/MAPK, leading to the pro‐apoptotic caspase‐3 activation and poly (ADP‐ribose) polymerase cleavage [163] (Table 1).

Quercetinplays a role in inhibiting tumorigenesis in colon cells through antioxidant, anti‐inflammatory, anti‐proliferative and pro‐apoptotic mechanisms. Quercetin downregulated Bcl‐2 through the inhibition of NF‐κB and inhibited phosphorylation of EGFR suppressing downstream signalling in colon carcinoma cells. It augments TRAIL‐induced apoptotic death and inhibits cyclin D1, survivin expression and Wnt/beta‐catenin signalling pathway [91, 164] (Table 1).

Apigenin:Studies have shown that apigenin induces cell‐cycle arrest and causes apoptosis in different cancer cells including colon cancer through modulation of various survival and death effectors, such as PI3K, AKT, ERK, STAT3, JNK and Mcl‐1 [119] (Table 1).

Genisteininhibits the activation of NF‐κB and Akt signalling pathways, both of which are known to maintain a homeostatic balance between cell survival and apoptosis; antagonizes estrogen‐ and androgen‐mediated signalling pathways in the processes of carcinogenesis [114]. It has antioxidant properties, being a potent inhibitor of angiogenesis and metastasis. Genistein is a known inhibitor of protein‐tyrosine kinase (PTK), which may attenuate the growth of cancer cells by inhibiting PTK‐mediated signalling mechanisms. Genistein also inhibits topoisomerase I and II, 5α‐reductase and protein histidine kinase, all of which may contribute to the anti‐proliferative or pro‐apoptotic effects [113]. It also down‐regulates mdm2 at both transcriptional and post‐translational levels [165] (Table 1).

5.2. Epigenetic mechanisms related to apoptosis, influenced by natural compounds

Several papers pointed the role of various natural compounds that target the epigenetic mechanismsin order to modulate the biologic activities, including apoptosis [173]. Epigenetic control mechanisms are reversible and natural compounds that target them may contribute to the development of new and attractive therapeutic strategies. A review of some natural compounds that target apoptosis through epigenetic mechanisms is presented below.

EGCGhas a role in inhibiting histone deacetylases (HDACs) [174]. Site‐specific acetylation of histones is essential to switch between permissive and repressive chromatin structures. Chromatin remodelling allows the regulation of gene expression, and it takes place under the action of enzymes responsible for acetylation (histone acetyl transferase, HAT) and deacetylation (histone deacetyl transferase, HDAC) [175]. An increased HDAC activity is characteristic to many cancers and is associated with alterations of several cellular mechanisms, including apoptosis. Therefore, by targeting HDACs, it is possible to modulate several cell processes like cell cycle, cell differentiation and apoptosis. Combined treatments of EGCG and sodium butyrate (NaB), in physiologically achievable concentrations, were shown to promote apoptosis and induce cell‐cycle arrest in RKO, HCT‐116 and HT‐29 colon cell lines [176]. Both EGCG and NaB are epigenetic regulators, and treatment with these two compounds reduced both the expression of survivin (which has anti‐apoptotic activity), as well as the expression of enzymes involved in epigenetic regulation (HDAC and DNA methyltransferase- DNMT) [176].

HDAC activity is also inhibited by sulforaphane(SFN) and induces an increased histone acetylation, mainly at p21 and Bax 2 promoters, events that lead to G2/M cell‐cycle arrest and apoptosis [177]. Oral administration of SFN in mice increased p21 expression via HDAC inhibition, while in APC‐knock‐out mice the same treatment leaded to specific increase of acetylation in H3 and H4 with decrease of intestinal polips [178]. Moreover, SFN was found to inhibit DNMTs (enzymes responsible for DNA methylation) in CaCo‐2 colon cancer cell line [179]. Another epigenetic mechanism used by SFN to exert its activity is through miRNAs: in NCM460 and NCM356 normal colon epithelial cells SFN upregulated 15 miRNAs, among which several were involved in apoptosis (miR‐9, miR‐135b) [180].

Resveratrolactivates type III HDAC inhibitors, sirtuin 1 (SIRT1) and p300 [181] which in turn negatively modulates the expression of survivin. On the other hand, in colon cancer cells, resveratrol inhibits the cell growth and induces apoptosis through miRNAs [182, 183]. Combined treatment of quercetin and resveratrol induced apoptosis in colorectal cancer cells through downregulation of miR‐27a [184].

Advertisement

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 [185]. 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 [115] (Figure 2).

Figure 2.

Available colon cancer therapeutic agents with disparate mechanisms of action.

A wide variety of currently available cancer therapeutical agents (Figure 2), with disparate mechanisms of actions, lead to the same mode of cell death [188]:

  1. 5‐Fluorouracil (5‐FU) that blocks the thymidylate synthase (TS), which is essential for DNA synthesis;

  2. Capecitabine that blocks thymidylate synthase (orally administered prodrug converted to 5‐FU)

  3. Oxaliplatin that inhibits DNA replication and transcription by forming inter‐ and intra‐strand DNA adducts/cross‐links;

  4. Irinotecan that inhibits topoisomerase I, an enzyme that facilitates the uncoiling and recoiling of DNA during replication;

  5. Bevacizumab, a monoclonal antibody, which binds to vascular endothelial growth factor (VEGF) ligand;

  6. Cetuximab, a monoclonal antibody to epidermal growth factor receptor (EGFR) (chimeric), that blocks the ligand‐binding site;

  7. Panitumumab, a monoclonal antibody to EGFR (fully humanized), that blocks the ligand‐binding site [188].

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 [192] 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 [193]. 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 [94]. 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 [80]. 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 [186]. 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 [186]. 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 [187]. 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 [187].

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].

7. Conclusions

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.

© 2016 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite and reference

Link to this chapter Copy to clipboard

Cite this chapter Copy to clipboard

Marinela Bostan, Mirela Mihaila, Camelia Hotnog, Coralia Bleotu, Gabriela Anton, Viviana Roman and Lorelei Irina Brasoveanu (September 7th 2016). Modulation of Apoptosis in Colon Cancer Cells by Bioactive Compounds, Colorectal Cancer - From Pathogenesis to Treatment, Luis Rodrigo, IntechOpen, DOI: 10.5772/63382. Available from:

chapter statistics

1614total chapter downloads

1Crossref citations

More statistics for editors and authors

Login to your personal dashboard for more detailed statistics on your publications.

Access personal reporting

Related Content

This Book

Next chapter

BRAF Mutation in Colorectal Cancer

By Louisa Lo, Timothy Price, Joanne Young and Amanda Townsend

Related Book

First chapter

Acute Biliary Pancreatitis

By Mehmet Ilhan and Halil Alıs

We are IntechOpen, the world's leading publisher of Open Access books. Built by scientists, for scientists. Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals. We share our knowledge and peer-reveiwed research papers with libraries, scientific and engineering societies, and also work with corporate R&D departments and government entities.

More About Us