Abstract

Present review article describing effects of garlic components on induction of apoptosis, cell cycle check points and inhibition of cancer cell proliferation. Garlic derived organic compounds hold unique therapeutic potential and induces apoptotic signaling pathways; do cell cycle arrest, and show cytotoxic and anti-invasive activity to a variety of cancer cells. Regular dietary intake of garlic cut down neoplastic growth, cancer cell proliferation, and induces programmed cell death. Garlic components act as tumor suppressor agents and prevent aberrant cell expansion by slowing down the cell cycle, or by inducing apoptosis. These apoptosis inducers can act on various apoptosis-related proteins to promote apoptotic cell death. Garlic based nanoformulations can target oncogenic mutations which disrupt apoptosis. These can inhibit cellular changes leading to tumor initiation, progression of metastasis. Garlic derivatives can control DNA damage thereby slow down oncogenic changes and induce caspase activity which halts or stalls mutation in p53 gene and thereby stops growth of lung, colon, and breast cancer. More often, new combinations of garlic components may stop cancer progression as well as multistage carcinogenesis. No doubt garlic components can be used in designing best possible molecular targets of cancer cells and that could be recommended for clinical management of different types of cancer.

1. Introduction

Allium sativum, commonly known as lahsun in Hindi (Garlic in English) belongs to family Alliaceae and plant order liliales ¹ Plant has been used for centuries as a nutraceutical, prophylactic and therapeutic medicinal agent. Garlic is a perennial, erect, bulbous herb indigenous to Asia but it is commercially grown in many parts of the world. It is a main singular and combined foodstuff which is used in Traditional Medicine of India. Allium sativum L. is an important aromatic plant and has multiple uses ². It is a rich source of organo-sulfur compounds, which are thought to be responsible for its flavor and aroma, as well as its potential health benefits ³. It is also used for both culinary and medicinal purposes by many cultures for centuries ⁴. Garlic is a multi-component pioneer food contains good nutraceuticals which are highly protective to lung cancer and increase the life expectancy in cancer patients ^{4, 5, 6}. Dietary consumption of green garlic bulbs reduce all types of cancer risks ⁷. It stops invasion and progression of cervical cancer ⁸. Homemade garlic ailments and preparations are used as herbal drug for complementary therapy of cancer. Garlic oil inhibits proliferation of AsPC-1, PANC-1, and Mia PaCa-2 cells while garlic tea shows strong therapeutic potential against lung and colorectal cancer ⁹. Both black and green garlic extracts (BGE) check proliferation of lung ¹⁰ and colorectal cancer and inhibits other types of carcinoma ¹¹. It contains S-allylcysteine (SAC), one of the major water-soluble compounds significantly inhibit oncogenesis ¹² while, S-Allylmercaptocysteine (CySSA) is known to exhibit anti-cancer effects ¹³. Alliin, isolated from garlic (Allium sativum) prevents LPS-induced inflammation in 3T3-L1 adipocytes. Similarly, DATS prevents tumor progression and promotes apoptosis in ectopic glioblastoma xenografts in SCID mice via HDAC inhibition ¹⁴ (Table 1). Cyclic sulfoxides garlicnins B2, B3, B4, C2, and C3 isolated from Allium sativum are toxic to cancer cells ¹⁵. Conjugates of daidzein-alliinase are used as a targeted pro-drug enzyme system against ovarian carcinoma ¹⁶.

Both allicin and allyl-mixed disulfides with proteins and small thiol molecules show good anticancer activity ³ and are considered as safe botanicals ¹⁷ (Table 1). These compounds induce of apoptosis in tumor cells and show multiple therapeutic potential ¹⁸ against different types of cancers ¹⁹. Alliin, prevents LPS-induced inflammation in 3T3-L1 adipocytes) ²⁰ while thiacremonone shows anticancer activity and does down regulation of peroxiredoxin ²¹. Black garlic extract is strong inhibitor of lung cancer cell proliferation and radio sensitization ¹⁰ (Table 1). Thiacremonone (2, 4-dihydroxy-2, 5-dimethyl-thiophene-3-one is sulfur compound from garlic ²¹ shows inhibition of NF-kappaB and cancer cell growth with IC(50) values about 100 microg/mL in colon cancer cells) ²². Garlic, silver bullets are used for carcinoma surveillance in upper endoscopy for Barrett's esophagus ²³ (Table 1).

Induction of apoptosis depends on the redox-state of the cell, with anti-oxidants being able to prevent sulfide-induced apoptosis ²⁴. Both growth arrest and induction of apoptosis is associated with a considerable reduction of the level of cdc25C and p53 gene mutations and its expression in cells. Garlic derived polysulfides induce apoptosis ²⁴ and can control cancer cell proliferation, angiogenesis and metastasis at physiological dose. Garlic components may participate in regulation of cellular differentiation and cell death much similar to oncogenes and tumor suppressor genes or induce their activity that play an important role in this process by regulating either cellular proliferation. There is a possibility that garlic may restore all three different categories of cancer associated genes. First category of proto-oncogenes and their oncogenic counterparts encodes proteins that induce cellular proliferation. Some of these proteins function as growth factors or growth factor receptors in normal cells, and its expression is carefully regulated. Garlic possesses apoptosis inducers which act on several apoptosis-related proteins to promote apoptotic cell death. Even its components also bind to most of the growth factor receptors and normalize any type of aberration related to gene function. A second category of genes called tumor suppressor genes or anti-oncogenes encodes proteins that inhibit excessive cell proliferation. Inactivation of these genes results in unregulated proliferation. Garlic antioxidants and enzymatic inhibitors check unregulated cellular proliferation by making interference in metabolic pathways which synthesize cancer growth promoting factors.

A defective or inefficient apoptosis is a characteristic feature of cancer cells that needs induction but garlic derived polysulfides lead to induction of apoptosis ²⁴. These may induce apoptosis through p53-dependent and independent pathways, which facilitate cytochrome c release from mitochondria. Hence, there should be more appropriate and authentic information is needed on target specificity of cancer agents which induce apoptosis. Though from biochemical researches cytotoxic potential of garlic components is established but target specificity explanation is still lacking. Therefore, all intriguing possibilities that are responsible for defects in apoptotic pathways which contribute to treatment failure can be resolved by making appropriate combinations of garlic components for cancer control. It also needs a thorough understanding about apoptotic signaling pathways to unravel novel drug targets and finding proper therapeutic solutions. Further, these should be compared for better cancer therapeutics with effect of certain oncogenes such as E1A and c-myc. Among all efficacies new induction of apoptotic signaling pathways in cancer cells be needed to resist apoptotic insults, with strong efforts to target the mitochondrion for restoring efficient cell death signaling in cancer cells. Hence, there is an immense need to make conceptual framework that can link cancer genetics with cancer therapy. Present review article describes use of garlic components for inducing apoptosis via small molecule drug treatments, its receptor ligation, and cytotoxic action. Further, nanoformulations which could target oncogenic mutations, disrupt apoptosis, reduce the chances of tumor initiation, progression and metastasis should prepare. These nanoformulations may induce apoptosis by making changes in mitochondrial membrane potential, phosphatidylserine membrane localization, DNA content, and antigen cleavage. Hence, best possible drug targets must explore to stop cancer progression and producing selective pressure to override apoptosis so then multistage carcinogenesis could be finished by opting new combinations of garlic components. Garlic based herbal products and targeted molecules can be recommended for clinical management of various cancer types.

2. Anticancer Activity

CASP3 protein is an important member of the cysteine-aspartic acid protease (caspase) family ²⁵. Caspases exist as inactive proenzymes in the cells which undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. This protein cleaves and activates caspases 6 and 7; and the protein itself is processed and activated by caspases 8, 9, and 10. It is the predominant caspase involved in the cleavage of amyloid-beta 4A precursor protein, which is associated with neuronal death in Alzheimer's disease. After alternative splicing this gene results in two transcript variants that encode the same protein ²⁶ Caspase-3 is activated in the apoptotic cell both by extrinsic (death ligand) and intrinsic (mitochondrial) pathways ²⁷. Garlic induces caspase activity which halts or stalls mutation in p53 gene and thereby stops growth of lung, colon, and breast cancer. It supports non-apoptotic roles of many effectors to the apoptotic signaling pathways. Similarly, diallyl trisulfide-induce apoptosis of bladder cancer cells which is caspase-dependent and regulated by PI3K/Akt and JNK pathways ²⁸. DADS also induce phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane and activate caspase-3 activity. Like caspase 3, caspase 2, plays important role in cell cycle regulation, DNA repair, and tumor suppression ²⁹. Garlic components act upon two pathways of target cell apopotosis stimulated by CTLs and through Fas pathway in which ligation of trimeric Fas units by CTL borne Fas ligands leads to the association of death domains of Fas with FADD. These components regulate a series of reactions that could lead to apoptosis of target cell. Garlic components may induce CTL functions and activating caspase cascade that results in the apoptotic death of the cell death by invoking mitochondrially mediated death pathways.

Normally in healthy cell three important genes reside i.e. proto-oncogenes, oncogenes, and a third category of cancer associated genes which regulate programmed cell death. These genes encode proteins that either block or induce apoptosis; bcl-2 is an anti-apoptosis gene. DATS-induce apoptosis and does down-regulation of Bcl-2, Akt and cyclin D1 protein levels, and up-regulation of Bax, Fas, p53 and cyclin B protein levels in Capan-2 cells ³⁰ (Table 1). More specifically carcinogens and viruses might alter the regulated function of these genes and converting them into potent cancer causing genes. In such condition garlic derived components mainly sulfur compounds, saponins and vitamins can evoke tumor suppressor gene and stop conversion of proto-oncogenes in to oncogenes. All this is possible due to genetic rearrangements of proto-oncogenes and mutations. Obviously, conversion of proto-oncogenes into oncogenes involves mutation that may result in production of qualitatively different gene products that may harm normal physiological metabolism of a normal cell. Further, viral integration into the host genome serves to convert a proto-oncogene into a transforming oncogene. Retroviruses have shown to integrate within the c-myc proto-oncogene. Such transformed or metastatic cells can be depleted by programmed cell death only thorough apoptosis induction pathways. That is possible by garlic component dosage as DADS modulates the cellular levels of Bax, Bcl-2, Bcl-xL, and Bcl-w in a dose-dependent manner, and induce apoptosis in the MCF-7 breast-cancer cell line through interfering with cell-cycle growth phases in a way that increases the sub-G(0) population and substantially halts DNA synthesis ²⁸.

Apoptosis is followed by two types of death pathways, namely, the extrinsic pathway and the mitochondria-mediated pathway. Both are linked and molecules used in one pathway can influence the other ³¹. Apoptosis imparts cell shrinkage, blebbing of plasma membrane, affect maintenance of organelle integrity, condensation and fragmentation of DNA, followed by ordered removal of phagocytes ³². Apoptosis is a suicidal self managed operation that kills cancer cells and maintain minimal damage to surrounding tissues. Apoptosis is regulated by the Bcl-2 family of proteins i.e. Bcl-2, Bcl-XL, and Mcl-1 which are anti-apoptotic proteins that possess all the four domains (BH1-4). These proteins regulate apoptotic events and share homology in any of the four common Bcl-2 homology (BH) domains ³³. The second category of Bcl-2 family of proteins contains BH domains 1, 2 and 3. These proteins would include Bax, Ki-67 and Bak. Bax is a pro-apoptotic protein that resides in the cytosol under physiological conditions. The third group of Bcl-2 family of proteins is the BH3-only proteins. The BH3-only family members include Bim, Bad, Bmf, Noxa and Puma. They act by neutralizing the anti-apoptotic proteins ³³. Apoptosis is induced in experimental systems through a variety of methods but two known methods i.e. biological induction and chemical induction are well used. In biological induction activation of Fas or TNF receptors by their respective ligands, or by cross-linking with an agonist antibody, induces apoptosis of Fas- or TNF receptor-bearing cells. In chemical apoptosis inducers act on several apoptosis-related proteins to promote apoptotic cell death ³⁰.

Few important derivatives from garlic such as allicin, ajoene, DAS, DADS, DATS, and SAMC, have been found to induce apoptosis and cell cycle arrest in various cancer cell lines grown in culture. There are many more possibilities that different natural products may choose different pathway to maintain apoptosis in cancer cells. Garlic oil induce programmed cell death, cell cycle arrest, and show pro-apoptosis effects in AsPC-1 cells ³⁴ and human pancreatic carcinoma cells ³⁴. Depending on the agent selected and the concentrations used, apoptotic events can be detected between 8–72 h post-treatment. S-allylcysteine is a potential therapeutic compound isolated from garlic derivative that suppresses proliferation and induces apoptosis in human ovarian cancer cells in vitro ³⁵. S-allylcysteine is also used to enhance apoptosis in oral cancer (Table 1) and human breast cancer cells through ROS-mediated activation of JNK and AP-1 ³⁶. Allicin inhibits cell growth and induces apoptosis in U87MG human glioblastoma cells through an ERK-dependent pathway ³⁷. It induces p53-mediated autophagy in Hep G2 human liver cancer cells ^{4, 38} and induces apoptosis in EL-4 cells in vitro by activation of expression of caspase-3 and -12 and up-regulation of the ratio of Bax/Bcl-2 ³⁹. It also induces apoptosis in gastric cancer cells through activation of both extrinsic and intrinsic pathways ⁴⁰. Allicin purified from fresh garlic cloves induces apoptosis in colon cancer cells via Nrf2 ⁴¹. while diallylpolysulfides induce growth arrest and apoptosis in cancer cells (Busch et al., 2010) ²⁴. Allicin induce autophagic cell death in human HCC Hep G2 (p53(wild type) cells, it may also induce apoptotic cell death through caspase-dependent and caspase-independent pathways by overproduction of reactive oxygen species (ROS) in human HCC Hep 3B (p53(mutation)) cells. Moreover, in cell death mechanism p53 knocked down Hep G2, and silenced the p53 gene using siRNA-mediated silencing. However, allicin treatment induces apoptotic cell death in p53 knocked down Hep G2 cells similar to that of Hep 3B cells ⁴. Allyl sulfides inhibit cell growth of skin cancer cells through induction of DNA damage mediated G2/M arrest and apoptosis ³⁹.

In normal cells growth occurs at regular interval through well controlled phases of mitotic cell division. Apoptosis is highly important event because it controls cell number and support healthy cells to live and operate to kill old cells or defective cells. Diallyl tetrasulfide induce mitotic arrest to apoptosis ⁴². It also induces apoptosis in human primary colorectal cancer cells) ⁴³ and AGS gastric cancer cell line ⁴⁴. Diallyl trisulfide sensitizes human melanoma cells to TRAIL-induced cell death by promoting endoplasmic reticulum-mediated apoptosis) ⁴⁵. It induces apoptosis and inhibits proliferation of A549 cells in vitro and in vivo ⁴⁶. Similarly, di-allyl disulfide plays important role of in NF-κB mediated transient G2-M phase arrest and apoptosis in human leukemic cell-lines ⁴⁷. Allyl sulfides inhibit cell growth of skin cancer cells through induction of DNA damage mediated G2/M arrest and apoptosis) ³⁸ (Wang et al., 2010). Similarly, diallyl trisulfide ³⁰ and S-alkenylmercaptocysteine (CySSR) with sodium selenite induce apoptosis in pancreatic cells ¹³. DATS-induced apoptosis is related with induction of pro-apoptotic Bax protein and p53 protein expression that was found upregulated and translocation to nucleus in MCF-7 cells ⁴⁸. DATS inhibits phosphatidylinositol 3'-kinase/Akt activation that, in turn, results in modulation of Bcl-2 family proteins, leading to enhanced apoptosis of T24 cells) ³⁹. This is the possible mechanism by which allyl sulfides suppress neoplastic cell proliferation ⁴⁹. S-benzyl-cysteine mediate cell cycle arrest and apoptosis involving activation of mitochondrial-dependent caspase cascade through the p53 pathway in human gastric cancer SGC-7901 cells ⁵⁰ (Table 1). Thiacremonone inhibits lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decrease of xIAP, cIAP and Bcl2 expression and inhibition of glutathione peroxidase activity of of PRDX6 through interaction ) ²¹.

Histone deacetylase inhibitors (HDIs) interfere with the function of histone deacetylase and are as possible treatments for cancers ^{51, 52}. Sulfur compounds from garlic showed modulation of acetylation ⁵³. These are cytostatic agents that inhibit the proliferation of tumor cells in culture in vitro while in vivo induce cell cycle arrest, cell differentiation and/or apoptosis. Histone deacetylase inhibitors exert their anti-tumor effects via the induction of expression changes of oncogenes or tumour suppressor genes, through modulating the acetylation/deactylation of histones and/or non-histone proteins such as transcription factors ⁵⁵. Histone deacetylation inhibitors (HDACi) also suppress cancer growth and induce apoptosis in cancer cells. Similarly, DADS shows similar properties as HDACi in MCF-7 cells as it lowers the removal of an acetyl group from an acetylated substrate and induces histone-4 (H4) hyper-acetylation that may be responsible for the induction of apoptosis in breast cancer cells ⁵⁶. In addition, di-allyl disulfide causes inhibition of histone deacetylation may also an important check point ⁵⁶. Histone deacetylase inhibition induces the accumulation of hyperacetylated nucleosome core histones in most regions of chromatin but affects the expression of only a small subset of genes, leading to transcriptional activation of some genes, but repression of an equal or larger number of other genes. Garlic compounds inhibit histone deacetylase activity and induce histone hyperacetylation in vitro as well as in vivo ⁵⁶. Thus both histone acetylation and deacetylation play important roles in the modulation of chromatin topology and the regulation of gene transcription ⁵⁶ and are responsible for the induction of apoptosis in breast cancer cells ⁵⁶. DADS is able to increase PGC1α expression in a ROS-dependent manner and to induce mitochondrial biogenesis at early stage of treatment that precede cell cycle arrest and apoptosis outcome ⁵⁷. DADS also elicits the increase of PGC1α within nuclear compartment, the decrease of PGC1α non-active acetylated form and does induction of nuclear-encoded mitochondrial genes such as TFAM and TFBM1) ⁵⁷. There are clear evidences about involvement of histone acetylation in modulation of gene expression by diallyl disulfide and allyl mercaptan ⁵⁷.

Cell cycle checkpoints are control mechanisms in eukaryotic cells which ensure proper division of the cell. Each checkpoint serves as a potential halting point along the cell cycle, during which the conditions of the cell are assessed, with progression through the various phases of the cell cycle occurring when favorable conditions are met. There are three known checkpoints: the G1 checkpoint, also known as the restriction or start checkpoint; the G2/M checkpoint; and the metaphase checkpoint, also known as the spindle checkpoint. The cell cycle checkpoints play an important role in the control system by sensing defects that occur during essential processes such as DNA replication or chromosome segregation, and inducing a cell cycle arrest in response until the defects are repaired ⁵⁸. Cell cycle in eukaryotes is a complex process, which is maintained by a network of regulatory proteins, or cell cycle control system. This system monitors and dictates the progression of the cell through the cell cycle ⁵⁹. Cell cycle checkpoints are maintained through the regulation of the activities of a family of protein kinases known as the cyclin-dependent kinases (CDKs), which bind to different classes of regulator proteins known as cyclins, with specific cyclin-CDK complexes being formed and activated at different phases of the cell cycle. Those complexes, in turn, activate different downstream targets to promote or prevent cell cycle progression ⁶⁰.

In normal cells, the cell cycle is tightly regulated to ensure faithful DNA replication and chromosomal segregation prior to cell division. Following DNA damage, the cell cycle can be transiently arrested to allow for DNA repair or activation of pathways leading to cell death (apoptosis). But in normal cells DNA damage is self repaired and cell tries to maintain cell cycle normal. There are certain cell cycle check points such as late G2 check point controls progression of cell cycle from G2 to M phase. Here, active Cdk1 (cdc2) for formation of complex, cyclin B1 is required. In next step for regulation of the cdc-B1 complex inhibitory phosphatases are needed at a pair of amino acids in the roofs of the active site by Wee1 (Figure 1 and Figure 2). After dephosporylation of these sites by the phosphatase Cdc25C increases Cdk activity. At this stage DNA damage activates Chk1 which inactivates Cdc25C through phosphorylation of cdc25C. This results in the phosphorylation and activity of cdc2-B1 and G2-M arrest (Figure 1 and Figure 2). Thus, G1/S is an important checkpoint that controls progression of cells through the restriction point (R) into the DNA synthesis S-phase (Figure 1 & Figure 2). During G1 stage, the tumor suppressor Rb binds and inhibits transcription factor E2F. More specifically, diallyl trisulfide-induced G2/M phase cell cycle arrest in DU145 cells is associated with delayed nuclear translocation of cyclin-dependent kinase 1 ^{61, 47} while synthetic polysulfane derivatives induce cell cycle arrest and apoptotic cell death in human hematopoietic cancer cells ⁶² (Table 1). DAS also induce G0/G1 cell cycle arrest ⁶³ and apoptosis in HeLa cells through caspase- and mitochondria and p53 pathways. It significantly inhibits the growth and induces apoptosis of human cervical cancer HeLa cells in vitro ⁶⁴. DAS-induced G0/G1 phase arrest is mediated through the increased expression of p21, p27, and p53 with a simultaneous decrease in CDK2, CDK6, and CHK2 expression. DAS exposed Ca Ski cells undergo apoptosis, with significant morphological changes and DNA condensation. It leads to alter the ratio of Bax/Bcl-2, and induce mitochondrial dysfunction, that results in release of cytochrome c for causing apoptosis in Ca Ski cells. Due to these properties DAS is considered as potential chemotherapeutic agent for the treatment of cervical cancer ⁶⁵. S-Allylmercaptocysteine does cell cycle arrest at the G2/M and sub-G1 interphases ¹³.

Diallyl disulphide, also found in Garlic oil, causes a redistribution of cell-cycle growth phases, induces apoptosis, and enhances butyrate-induced apoptosis in colorectal adenocarcinoma cells (HT-29)) ⁵⁶. Garlic sulfur components may activate phosphorylation of Rb by cyclin-bound cyclin dependent kinases (CDK) in late G1 and induce dissociation of Rb protein and permits E2F-mediated transcription of S-phase-promoting genes ⁶⁶. INK4 and Kip/Cip family inhibitors control CDK activity and prevent entry into S-phase after. DNA damage activates response pathways through ATM/ ATR and Chk1/2 kinases to block CDK activity ⁶⁷, leading to cell cycle arrest and DNA repair or cell death ⁵⁸ (Figure 1 and Figure 2). DATS suppress the viability of cultured human pancreatic cancer cells (Capan-2) and it increases the proportion of cells in the G2/M phase and induced apoptotic cell death. DATS enhance the expression of Fas, p21, p53 and cyclin B1, but down regulate the expression of Akt, cyclin D1, MDM2 and Bcl-2) ³⁰. DATS induces cell cycle inhibition and elevated levels of cyclin B1 and p21, and reduced levels of cyclin D1 in Capan-2 cells and H6C7 cells) ³⁰. DATS reduce mitosis in tumors, decrease HDAC activity, increase acetylation of H3 and H4, and inhibit cell cycle progression. It decrease pro-tumor markers (e.g., survivin, Bcl-2, c-Myc, mTOR, EGFR, VEGF) and promote apoptotic factors (e.g., bax, mcalpian, active caspase-3), and induced DNA fragmentation. There also occurs an increase in p21Waf1 expression, which is correlated with increased p53 expression and MDM2 degradation following DATS treatment ¹⁴. DATS also enhance the expression of Fas, p21, p53 and cyclin B1, but down regulated the expression of Akt, cyclin D1, MDM2 and Bcl-2) ¹⁴. DATS induce cell cycle inhibition and elevate levels of cyclin B1 and p21, and reduce levels of cyclin D1 in Capan-2 cells and H6C7 cells ³⁰. DATS causes cytotoxicity which is mediated by the generation of ROS and subsequent activation of the ASK1-JNK-Bim signal transduction pathway in human breast carcinoma MDA-MB-231 cells ⁴⁴. S-benzyl-cystein also-mediate cell cycle arrest and operate apoptosis that involves activation of mitochondrial-dependent caspase cascade through the p53 pathway in human gastric cancer SGC-7901 cells ⁵⁰. Tumor cells lacking p53 may continue to replicate damaged DNA and do not undergo apoptosis. Finally, cell cycle stalls in the cells with the damaged DNA.

DAS treatment of cultured cell also invokes changes typical of apoptosis such as morphological changes, DNA damage and fragmentation, dysfunction of mitochondria. It also induce cytochrome c release and increase expression of pro-caspase-3 and -9. DAS also promote the release of AIF and Endo G from mitochondria in HeLa cells ⁶⁴. In fact G2/M checkpoint prevents cells containing damaged DNA from entering mitosis (M). Activated CDK1 (cdc2) bound to cyclin B promotes entry into M-phase. Wee1 and Myt1 kinases and cdc25 phosphatase competitively regulate CDK1 activity; Wee1 and Myt1 inhibit CDK1 and prevent entry into M-phase, while cdc25 removes inhibitory phosphates. DNA damage activates multiple kinases activity ⁶⁸ mainly phosphorylate kinases Chk1/2 and tumor suppressor protein p53. Chk1/2 kinases stimulate Wee1 activity and inhibit cdc25C, preventing entry into M-phase ⁶⁹. Phosphorylation of p53 promotes dissociation between p53 and MDM2 and allows binding of the transcription factor to DNA (Figure 1 and Figure 2). SFN Sulforaphane (SFN) and NaHS activated p38 mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinase (JNK) ⁷⁰. Pre-treatment of PC-3 cells with methemoglobin decreased SFN-stimulated MAPK activities. DATS inhibits phosphatidylinositol 3'-kinase/Akt activation that, in turn, results in modulation of Bcl-2 family proteins, leading to enhanced apoptosis of T24 cells ) ³⁸. Crushed garlic inhibit the proliferation of tumour cells by inducing G(2)/M cell cycle arrest and apoptosis) ⁷¹. Similarly, ajoene also inhibit WHCO1 cell growth, inducing G(2)/M cell cycle arrest and apoptosis by caspase-3 activation. Here, vinyl group serves to enhance the anti-proliferation activity manifold ⁷¹.

DADS inhibit the activation and nuclear translocation of p65, p50, and c-Rel subunits of nuclear factor (NF)-B and other transcription factors, such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response element-binding protein, in B16F-10 melanoma cells ⁷². In also does down regulation of pro-inflammatory cytokine production and gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). DADS induces caspase-dependent apoptosis through a mitochondria-mediated intrinsic pathway in B16F-10 melanoma cells by activating p53 and caspase-3 gene expression and suppressing pro-inflammatory cytokines and NF-B-mediated Bcl-2 activation ⁷². DADS treatment of colonic adenocarcinoma cells (HT-29) initiates a cascade of molecular events characteristic of apoptosis. These include a decrease in cellular proliferation, translocation of phosphatidylserine to the plasma-membrane outer-layer, activation of caspase-3 and -9, genomic DNA fragmentation, and G(2)/M phase cell-cycle arrest ⁵⁶. Short-chain fatty acids (SCFAs), particularly butyrate (abundantly produced in the gut by bacterial fermentation of dietary polysaccharides), enhance colonic cell integrity but, in contrast, inhibit colonic cancer cell growth ⁵⁶. Combining DADS with butyrate augmented the apoptotic effect of butyrate on HT-29 cells. More likely, anti-cancerous properties of DADS are of greater benefit when supplied with other favorable dietary factors (short chain fatty acids/polysaccharides) that likewise reduce colonic tumor development ⁵⁶.

DATS-mediated G2/M phase cell cycle arrest was found to be independent of reduced complex formation between cdk1 and cyclin B1, but it is correlated with delayed nuclear translocation of cdk1. DATS-mediated G2/M phase cell cycle arrest in DU145 cells results in differential kinetics of nuclear localization of cdk1 and cyclin B1 ⁷³. DAS treatment increased the accumulation of sub-G1 DNA which also increases the proportion of cells in the G2/M phase in a dose-dependent manner. In addition, DAS-induced apoptosis shows a decrease in the level of Bcl-2 expression and an increase in the level of Bax expression, and cytochrome c was remarkably released from mitochondrial into the cytosol by DAS. Furthermore, caspase-9 and caspase-3 were activated by DAS, and DAS cleaved PARP. DAS decreases cell proliferation and induce apoptosis via mitochondrial signaling pathway in ATC cells ⁷⁴. Here, both p38MAPK and caspase-3 are seems to be involved in the process of DADS-induced apoptosis in human HepG2 cells and interact with each other ⁷⁵. Diallyl trisulfide inhibits phorbol ester-induced tumor promotion, activation of AP-1, and expression of COX-2 in mouse skin by blocking JNK and Akt signaling ⁷⁶.

DADS treatment causes significant transcriptional induction of p21 in early hours of treatment, which is due to increased nuclear translocation of NF-κB and its specific binding to p21 promoter. It also induce transient G2/M phase arrest, which in later hours is lost leading to apoptosis via intrinsic mitochondria-mediated pathway through generation of reactive oxygen species followed by changes in mitochondrial membrane potential ⁴⁷. DADS induces reversible G2/M arrest through NF-κB mediated pathway in human leukemic cell lines, like U937, K562, and Jurkat, lacking wild type p53. However, failure of G2/M arrest make the system incapable of the damage repair system that leads to apoptosis ⁴⁷. Similarly, BGE and radiotherapy combination significantly induce Lewis cells' apoptosis in G2/M stage which decreases the expression of bcl-2, and up-regulated the expression of bax ¹⁰. BGE sensitize the lung cancer Lewis cells to ionizing irradiation. This effect might be probably caused by changing the cell cycles and affecting expressions of bax and bcl-2 ¹⁰.

Spindle check points ⁷⁷ are important to repair DNA damage and cancer therapy ⁷⁸ (Figure 1 and Figure 2). These checkpoints ensure proper chromatid attachment prior to progression from metaphase to anaphase. Similarly, both SCF and APC/C protein complexes play prominent roles, with APC-cdc20 initiating the entry into anaphase by promoting ubiquitin-mediated degradation of multiple substrates, including cyclin B ⁷⁹. It indicates that regulatory proteins have important role in repairing of DNA level defects and occurrence of mutation in these proteins induce stressed in cancer prone cells. It is true that proteins like SCF and APC/C ubiquitin ligases also control cell growth ⁸⁰. There occurs a mutual regulation between the spindle check point and APC/C ⁸¹. Organosulfur compounds modulate the activity of several metabolizing enzymes that activate (cytochrome P450s) or detoxify (glutathione S-transferases) carcinogens and inhibit the formation of DNA in different tissues ¹⁹.

3. Inhibition of Production of Reactive Oxygen Species

Reactive oxygen species (ROS) are formed as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis ⁸². These are also formed due to environmental stress mainly due to heat exposure, ionizing radiation ⁸³, pollutants, tobacco, smoke, drugs and other agents. Their levels get increased dramatically if no antioxidant is used in the diet ⁸². ROS reactive intermediates cause significant damage to cell structure and function. ROS are also formed intracellularly through multiple mechanisms and effect NADPH oxidase (NOX) complexes found in cell membranes, mitochondria, peroxisomes, and endoplasmic reticulum ^{84, 85}. Cancer cells show extremely high ROS levels that shows weaker antioxidant defense system. Further, cellular ROS stress level elevated due to over drug dosage and cut down tolerability threshold, a induce cell death. ⁸⁶ (Kong et al., 2006). On the other hand, normal cells show lower cellular stress and show very high capacity to fight ROS and its related changes ⁸⁷. Garlic chemical constituents showed very multiple therapeutic efficacy and are proved useful for preventing diseases associated with reactive oxygen species ⁸⁸. Aged garlic extract scavenges superoxide radicals ⁹¹ and induce apoptosis in cancer cells ^{89, 90}. Diallyl trisulfide does inhibition of cell proliferation and migration in cancer cells and acts as chemopreventive drug ⁹¹. The DATS-induced apoptosis in MDA-MB-231, MCF-7, and BRI-JM04 cells was found to be associated with reactive oxygen species (ROS) production ⁹¹. However, over expression of Cu, Zn-superoxide dismutase (Cu,Zn-SOD) as well as Mn-SOD conferred significant protection against DATS-induced ROS production and apoptotic cell death in MDA-MB-231 and MCF-7 cells. The DATS treatment caused ROS generation, but not activation of Bax or Bak, in MCF-10A cells. Furthermore, the DATS-mediated inhibition of cell migration was significantly attenuated by Cu,Zn-SOD and Mn-SOD over expression in association with changes in levels of proteins involved in epithelial-mesenchymal transition. The DATS-mediated induction of heme oxygenase-1 was partially attenuated by overexpression of Mn-SOD. There seems a critical role for ROS in anticancer effects of DATS ⁹¹. DATS also increases reactive oxygen species (ROS) production in primary colorectal cancer cells ⁴³. Diallyl tetrasulfide acts independently of reactive oxygen species and tubulin represents one of its major cellular targets ⁴² (Kelkel et al., 2012). It induces production of reactive oxygen species (ROS) in normal cells similar to cancer cells in a time and dose dependent manner ⁹². This is the main reason that both garlic and its derivatives are used as conventional drug in many countries for clinical treatment of cancer ⁹³.

Garlic derivatives such as ajoene, induces apoptosis in human promyeloleukemic cells, accompanied by generation of reactive oxygen species and activation of nuclear factor kappaB) ⁹⁴ and show antiproliferative effects) ⁹⁵. Allicin a sulfur compound breaks into diallyl disulfide (DADS) (66%), diallyl sulfide (DAS) (14%), diallyl trisulfide (9%), and sulfur dioxide ⁹⁶. Allicin easily reacts with amino acids and proteins, creating a -SH group. It binds to protein and fatty acids in the plasma membrane, and is trapped before absorption. It circulate in the blood stream ⁹⁷ but did not detect in the blood sample after the ingesting raw garlic or pure allicin ⁹⁴. Allicin is metabolized into allyl methyl sulfoxide (AMS) and released into the breath after dietary intake ⁹⁷. Allicin shows antitumoral activity in murine lymphoma L5178Y) ⁹⁸ while DATS-induced apoptosis of human CNE2 cells ⁷⁵ (Table 1). Allicin acts as an antioxidant in the blood and remove out all reactive oxygen species generated due to metabolic stress.

Garlic-derived compound S-allylmercaptocysteine (SAMC) is associated with microtubule depolymerization and c-Jun NH(2)-terminal kinase 1 activation ⁹⁹. These components also show induction of apoptosis in breast cancer cell lines ¹⁰⁰, and did attenuation of cell migration and induction of cell death in rat sarcoma cells ¹⁰¹. The garlic-derived organosulfur component ajoene decreases basal cell carcinoma tumor size by inducing apoptosis ¹⁰². Z-ajoene, a natural compound purified from garlic shows antimitotic and microtubule-interaction properties ¹⁰³. A protein fraction from aged garlic extract enhances cytotoxicity and proliferation of human lymphocytes mediated by interleukin-2 and concanavalin A ¹⁰⁴. More specifically, garlic preparations were found active against human tumor cell proliferation ¹⁰⁵ and show antitumor ¹⁰⁶ and anti-cancer effects ¹⁰⁷. Allium vegetables in the daily diet play important role in the prevention of cancer ¹⁰⁸ (Table 1) (Figure 5b).

4. Control of DNA Damage

There are so many extracellular agents such as ionizing radiation, UV light, environmental chemicals and alkyltaing agents which cause DNA damage in cells that finally induce carcinogenesis. Many potential therapies are used to target carcinogenesis through a series of alternatives including plant-derived pharmaceutical agents. A large number of these herbal remedies are particularly derived from garlic and are used to treat cancer patients mainly for inhibition of cancer cell proliferation and growth. DATS inhibits cell growth of human melanoma A375 cells and basal cell carcinoma (BCC) cells by increasing the levels of intracellular reactive oxygen species (ROS) and DNA damage. It also induce G2/M arrest, endoplasmic reticulum (ER) stress, and mitochondria-mediated apoptosis, including the caspase-dependent and -independent pathways ¹⁰⁹. Similarly, DADS was found to be effective in inhibiting BaP-induced cell proliferation, cell cycle transitions, reactive oxygen species, and DNA damage in a normal cell line. It also inhibits environmentally induced breast cancer initiation ¹¹⁰. Diallyl polysulfides play potential role as chemopreventive and therapeutic agents in cancer treatment due to their selective antiproliferative effects.

Some herbal medicines including garlic show potentially beneficial effects against cancer progression and ameliorate chemotherapy-induced toxicities ¹¹¹. As most of the anticancer drugs undergo Phase I and/or II metabolism and are substrates of P-glycoprotein, breast cancer resistance protein, multidrug resistance associated proteins, and/or other transporters. Therefore, induction and inhibition of these enzymes seems to be essential and mechanism of action of transporters is important for herb-anticancer drug interactions and action. However, endogenous mechanisms causing DNA damage such as spontaneous chemical hydrolysis, intracellular interaction with reactive oxygen groups and errors in replication and recombination can be restored by identifying correct and appropriate bio-molecules from garlic in form of a formulation (Figure 4). In addition chemical carcinogen induced changes such as depurination, de-amination, and reactive oxygen species including superoxide anion (an ion and a free radical) formation cab ne controlled by highly permeable oxygen bearing molecules from garlic. These compounds conjugated with certain cofactors and vitamins can finish reactive oxygen species formed by the effect of ionizing radiation. Both garlic oil and sulfur rich compounds can minimize the formation of free radicals due to oxidant reactions with proteins and other biomolecules. Diallyl sulfide (DAS), one of the main active constituents of garlic, causes growth inhibition of cancer cells in vitro and promotes immune responses in vivo ⁶⁴.

Moreover, garlic based drugs or crude extracts prepared green garlic cloves induce apoptosis through the generation of reactive oxygen species in Hep3B human hepatocarcinoma cells ¹¹². These also target DNA repair mechanisms, and do restoration of a series of metabolic and signaling pathways. Temperature- and pressure-treated green garlic does inhibition of ENNG-induced pyloric stomach and small intestinal carcinogenesis in mice ¹¹³. DNA damage also occurs due to mistakes in DNA replication or recombination cause strand breaks to be left in DNA. An incorrect proof reading also results in incorporation of mismatched bases. When damage occurs due to effect of carcinogens it remain repairable but long term natural genetic changes cannot be reverted. Possible potential anti-cancer agents from garlic such as diallyl trisulfide can check these events in eukaryotic cells due by imposing external stress on “checkpoints” of cell cycle. Drug may target effectively on proper timing of cellular events that can stop cell cycle or delay it. Thus, passage of anticarcinogenic effect through a checkpoint from one cell cycle phase will also require coordinated set of proteins that monitor cell growth and DNA integrity. Using natural products/molecules in a state of deficiency of transcription and translation of cell cycle proteins may activate DNA synthesis and protein translation. Garlic products can stop uncontrolled cell division or propagation of damaged DNA and save cells from genomic instability that give rise tumorigenesis (Figure 3). Diallyl trisulfide does transcriptional repression and inhibition of nuclear translocation of androgen receptor in human prostate cancer cells ¹¹⁴. It also inhibits estrogen receptor-α activity in human breast cancer cells ¹¹⁵ (Table 1). Thus garlic based herbal medicines have shown potentially beneficial effects on cancer progression and may ameliorate chemotherapy-induced toxicities (Figure 4).

DNA damage resulted in an impaired cellular response due to hypersensitivity generated by carcinogenic agents. If this DNA damage remains defective for longer period it results in mutations that lead to altered protein functions and generate impaired cellular responses. Normal cells react to DNA damage by stalling progress through the cell cycle at a check point until the damage has been repaired or triggering apoptosis if the damage remains un-reparable. All this is initiated by ATM protein briefly which sense DNA damage and relays the signal to the p53 protein which is synthesized under regulation of p53 gene that is known as the guardian of the genome ¹¹⁶. It plays a critical role in intrinsic tumor suppression via two mechanisms either through cell cycle arrest or by induction of apoptosis. A variety of triggers such as DNA damage, oncogene activation, and telomere erosion can lead to the activation of p53. An increase in p53 transcription inhibits cell cycling. Possibly p53 may be knocked out by mutation or by the action of an inhibitor such as the MDM2 gene product which binds p53 and targets for its degradation. S-Allylmercaptocysteine (CySSA) from garlic increases in phospho-p53, Bax and Bad levels which indicate that apoptosis occurs via the mitochondrial pathway ¹³ (Figure 4). SAMC-induced apoptosis is associated with the Id-1 pathway and that the inactivation of Id-1 enhances the ability of SAMC to inhibit the survival, invasion and migration of bladder cancer cells ¹¹⁷.

DADS induces apoptosis in the MCF-7 breast-cancer cell line through interfering with cell-cycle growth phases in a way that increases the sub-G(0) population and substantially halts DNA synthesis ⁵⁶. It also induces phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane and activates caspase-3 ⁵⁶. It also modulates the cellular levels of Bax, Bcl-2, Bcl-xL, and Bcl-w in a dose-dependent manner, and show involvement of Bcl-2 family proteins in apoptosis ⁵⁶. Diallyl trisulfide (DATS) induces apoptosis and inhibits the growth of many cancer cell lines. Mainly it induces apoptotic cell death in human primary colorectal cancer cells through a mitochondria-dependent caspase cascade signaling pathway through a significant decrease of the anti-apoptotic Bcl-2 that resulted in up-regulation of the ratio of Bax/Bcl-2 and the activity of caspase-3, -8, and -9 ^{44, 46}. It also increases in the levels of cytochrome c, Apaf-1, AIF and caspase-3 and caspase-9 activity ⁴⁴. Eventually, DATS induce the apoptosis and inhibit the cancer cell proliferation in a concentration- and time-dependent manner ⁴⁶. Exposure to DATS additionally induced endogenous endoplasmic reticulum stress markers and intracellular Ca2⁺ mobilization, upregulation of Bip/GRP78 and CHOP/GADD153, and activation of caspase-4 ⁴⁶. It also makes an increase in reactive oxygen species (ROS) production in primary colorectal cancer cells but decrease in the level of ΔΨm was associated with an increase in the Bax/Bcl-2 ratio which led to activation of caspase-9 and -3. No doubt, DATS can offer protection against chemically-induced neoplasia as well as oncogene-driven spontaneous cancer development in experimental animals. DATS do alteration in carcinogen-metabolizing enzymes, operate cell cycle arrest, and impose induction of apoptotic cell death, suppression of oncogenic signal transduction pathways, and inhibition of neoangiogenesis ¹¹⁸. DATS did not affect the activity of sulfurtransferases and lowered sulfane sulfur level in HepG2 cells. But is possible that sulfane sulfur containing DATS may be bioreduced in cancer cells to hydroperthiol that leads to H(2)O(2) generation, thereby influencing transmission of signals regulating cell proliferation and apoptosis ¹¹⁹. Similarly, allicin also inhibit the cell viability of U87MG human glioma cells in a dose- and time-dependent manner. Allicin-induced inhibition of cell viability was due to apoptosis of cells. The mechanisms of apoptosis were found to involve the mitochondrial pathway of Bcl-2/Bax, the MAPK/ERK signaling pathway and antioxidant enzyme systems ³⁷ (Figure 4).

DATS also exerts chemopreventive potential via ER stress and the mitochondrial pathway in BCC cells ⁴¹. DAS, DADS, and DATS induce down regulation expression of PI3K, Ras, MEKK3, MKK7, ERK1/2, JNK1/2, and p38 and then lead to the inhibition of MMP-2, -7, and -9. DAS, DADS, and DATS inhibited NF-κB and COX-2 for leading to the inhibition of cell proliferation ¹²⁰. DADS inhibit the activities of matrix metalloprotease (MMP)-2 and -9 in AGS cells in dose-dependent manner. It decreases expression of their mRNA and proteins and repressed the levels of claudin proteins (claudin-2, -3, and -4 ¹²¹. In addition, both diallyl tri- and tetrasulfide are reported as strong inducers of an early mitotic arrest and subsequent apoptosis ⁴². Diallyl tetrasulfide acts independently of reactive oxygen species and tubulin represents one of its major cellular targets. Tubulin depolymerization prevents the formation of normal spindle microtubules, thereby leading to G2/M arrest. Moreover, c-jun N-terminal kinase, which is activated early in response to diallyl tetrasulfide treatment, mediates multisite phosphorylation and subsequent proteolysis of the anti-apoptotic protein B-cell lymphoma 2 ⁴² (Figure 4).

SAC suppress the proliferation of PC-3 cells and led to cell cycle arrest at the G0/G1 phases, as well as inducing cell apoptosis which was accompanied by the decreased expression of Bcl-2 and increased expression of Bax and caspase 8 ¹²². This chemopreventive effect of SAC may be associated with the suppression of carcinogenesis factors such as N-methylpurine DNA glycosylase and OPN. SAC also significantly suppresses the phosphorylation of Akt, mammalian target of rapamycin, inhibitor of κBα and extracellular signal-regulated kinase 1/2 in tumour tissues ¹²². But SAC-mediated suppression of cyclin D1 protein was associated with an augmented expression of the cell-cycle inhibitor p16(Ink4). SAC also inhibit expression of cyclo-oxygenase-2, vimentin and NF-κB p65 (RelA) ¹²³. In addition, both SFN Sulforaphane (SFN) and NaHS activate p38 mitogen-activate protein kinases (MAPK) and c-Jun N-terminal kinase (JNK) ¹²⁴. Thus, pre-treatment of PC-3 cells with methemoglobin decreases SFN-stimulated MAPK activities. Suppression of both p38 MAPK and JNK reversed H(2)S- or SFN-reduced viability of PC-3 cells. H(2)S mediates the inhibitory effect of SFN on the proliferation of PC-3 cells. Therefore, H(2)S-releasing diet or drug might be beneficial in the treatment of prostate cancer ¹²⁴. SAC also hinder the migration and invasion of MHCC97L cells corresponding with up-regulation of E-cadherin and down-regulation of VEGF. It also significantly induced apoptosis and necrosis of MHCC97L cells through suppressing Bcl-xL and Bcl-2 as well as activating caspase-3 and caspase-9) ^{124, 125}. In addition, SAC could significantly induce the S phase arrest of MHCC97L cells together with down-regulation of cdc25c, cdc2 and cyclin B1 ¹²⁵. Similarly, phytoalexins show specific targets in cancer cells that include signal transduction pathways, transcription factors, cell cycle checkpoints, intrinsic and extrinsic apoptotic pathways, cell invasion and matrix metalloproteinase, nuclear receptors, and the phase II detoxification pathway ¹²⁶. Thiacremonone inhibits tumor growth accompanied with the reduction of PRDX6 expression and glutathione peroxidase activity, but its shows an increase in expression of cleaved caspase-3, -8, -9, Bax, p21 and p53) ²¹. Garlic extract induce cytotoxic and apoptotic effects in HL-60 cells that is mediated by oxidative stress ¹²⁷, phosphatidylserine externalization, caspase-3 activation, and nucleosomal DNA fragmentation and formation of MDA, a by-product of lipid peroxidation and biomarker of oxidative stress ¹²⁷ (Figure 4).

5. Inhibition of Carcinogenesis

Diallyl trisulfide inhibits benzo(a)pyrene-induced precancerous carcinogenesis in MCF-10A cells ¹²⁸ and induce apoptosis in human basal cell carcinoma cells via endoplasmic reticulum stress and the mitochondrial pathway ³⁹. Diallyl sulfide, diallyl disulfide and diallyl trisulfide affect drug resistant gene expression in colo 205 human colon cancer cells in vitro and in vivo ¹²⁰ (Table 1). Diallyl trisulfide (DATS) is used to prevent growth of pancreatic ³¹ and prostate cancer cells PC-3) ¹²⁹, and HCT116 cells ¹⁴³. Diallyl trisulfide (DATS) inhibits mouse colon tumor in mouse CT-26 cells allograft model in vivo) ⁶⁴. Diallyl disulfide inhibits the proliferation of HT-29 human colon cancer cells by inducing differentially expressed genes ¹³⁰. Diallyl sulfide inhibited murine WEHI-3 leukemia cells in BALB/c mice in vitro and in vivo ¹³¹ and induces molecular mechanisms which target cancer chemoprevention ¹³². It also induces Ca2+ mobilization in human colon cancer cell line SW480 ¹³³ (Table 1).

Fresh garlic extract induces growth arrest and morphological differentiation of MCF7 Breast cancer Cells ¹³⁴. Sulfide compounds found in dietary garlic supplements ¹³⁵ increase C reactive protein concentration ¹³⁶ and enhance therapeutic potential against infectious agents ¹³⁷. These effect cell proliferation, caspase 3 activities, thiol levels and anaerobic sulfur metabolism ¹¹⁹ and modulate peroxisome proliferation activated receptor gamma co-activator 1 alpha in human hepatoblastoma HepG2 cells ⁵⁷. Garlic oil shows protective effects on hepatocarcinoma induced by N-nitrosodiethylamine in rats ¹³⁸. Similarly, S-allylcysteine (SAC), a garlic derivative suppresses proliferation and metastasis of hepatocellular carcinoma ¹²⁸ and induces cell cycle arrest and apoptosis in androgen-independent human prostate cancer cells ¹²². Oxidative species and S-glutathionyl conjugates aids in the apoptosis induction by allyl thiosulfate ¹³⁹.

The aged garlic extract (AGE) contains S-allyl-L-cysteine (SAC) which is a potential therapeutic agents for Alzheimer's disease ¹⁴⁰. It is a major water- soluble compound found in aged garlic exerts cytotoxic activity by involving activation of mitochondrial-dependent apoptosis through p53 and Bax/Bcl-2 pathways in human gastric cancer SGC-7901 cells ⁴⁴. It inhibits tumor progression and the epithelial-mesenchymal transition in mouse xenograft model of oral cancer ¹²³. It also shows ameliorating effects against Aβ-induced neurotoxicity and cognitive impairment ¹⁴¹. It induces inhibition of gastric cancer cell growth in vitro and in vivo ³⁹. Similarly, S-benzyl-cysteine (SBC) a structural analog of S-allylcysteine (SAC), mediates cell cycle arrest and apoptosis that involves activation of mitochondrial-dependent caspase cascade through the p53 pathway in human gastric cancer SGC-7901 cells ⁴². S-allylmercaptocysteine (SAMC) shows potent therapeutic effect on human cancer cells by causing apoptosis ^{10, 142}. Hydrogen sulfide mediates the anti-survival effect of sulforaphane on human prostate cancer cells ¹²⁴. Garlic-derived allyl sulfides cause inhibition of skin cancer progression ³⁹ (Wang et al., 2012) and show modifying effect on colon carcinogenesis in C57BL/6J-ApcMin/⁺ mice ¹⁴³.

However, intake of fruits and vegetable reduce the risk of gastric cancer ^{144, 145} and restore health and reduces the chances of occurrence of physiological diseases ¹⁴⁶. Both garlic and garlic-derived organic sulfur compounds are used for controlling breast cancer ^{147, 148}. Besides, sulfur compounds garlic also contains saponins, flavonoids vitamins, minerals, hence daily consumption of garlic vegetables daily can lower down the risk of hematologic malignancies and other lifestyle diseases ^{149, 150, 151}. Garlic dietary supplements also contain multivitamins and folic acid which show strong anticancer effects in breast cancer survivors ¹⁵². These lower down oxidative stress and induce apoptotic mechanisms in acute promyelocytic leukemia ^{127, 170}. Green garlic shows cardioprotective effects ¹⁵³ (Alkreathy et al., 2012) and is used as a complementary and alternative medicines for treatment of breast cancer ¹⁵⁴. It shows potential beneficial effects in oncohematology ¹⁵⁵ (Table 1).

Consumption of s-allylcysteine inhibits the growth of human non-small-cell lung carcinoma in a mouse xenograft model ¹⁵⁶. Similarly, S-allylmercaptocysteine effectively inhibits the proliferation of colorectal cancer cells under in vitro and in vivo conditions ¹⁵⁷. S-allylmercapto-L-cysteine shows anticancer activity in implanted tumor of human gastric cancer cell ⁴⁴ (Table 1). S-allyl cysteine attenuates oxidative stress associated cognitive impairment and neurodegeneration in mouse model of streptozotocin-induced experimental dementia of Alzheimer's type ¹⁵⁸. There occurs a relationship between lipophilicity and inhibitory activity against cancer cell growth of nine kinds of alk(en)yl trisulfides with different side chains ¹⁵⁹ (Table 1). Garlic oil suppressed the hematological disorders induced by chemotherapy and radiotherapy in tumor-bearing mice ¹⁶⁰. Thiacremonone, a sulfur compound isolated from garlic, attenuates lipid accumulation partially mediated via AMPK activation in 3T3-L1 adipocytes ¹²⁴. Artesunate combined with allicin shows synergistic anticancer effect in osteosarcoma cell line in vitro and in vivo ¹⁶¹. Moreover, garlic components showed antioxidant activity ¹⁷² inhibit fibrosarcoma tumor growth in BALB/c mice positive ¹⁶³.

Oil-soluble compounds such as diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS) and ajoene are more effective than water-soluble compounds in protection against cancer. DADS, a major organosulfur compound derived from garlic, decrease carcinogen-induced cancers in experimental animals and inhibit the proliferation of various types of cancer cells. Its mechanisms of action include: the activation of metabolizing enzymes that detoxify carcinogens; suppression of the formation of DNA adducts; antioxidant effects; regulation of cell-cycle arrest; induction of apoptosis and differentiation; histone modification; and inhibition of angiogenesis and invasion ¹⁶⁴. No doubt DAC inhibitory effects of garlic organosulfur compounds might play a role in primary cancer prevention at doses achievable by human diet. ¹⁶⁵. However, the decrease of the peripheral total white blood cells (WBCs) count induced by CTX/radiation was significantly suppressed by GO co-treatment. No doubt, GO consumption may benefit for the cancer patients receiving chemotherapy or radiotherapy ¹⁶⁰.

Diallyl disulfide shows anti-invasive activity through tightening of tight junctions and display inhibition of matrix metalloproteinase activitiy in LNCaP prostate cancer cells ⁷⁴. Diallyl trisulfide does inhibition of cell proliferation and migration in cancer cells and acts as chemopreventive drug ⁹¹. Similarly, natural tetrasulfides also show antiproliferative effect of in human breast cancer cells that is mediated through the inhibition of the cell division cycle 25 phosphatases ¹⁶⁶. Garlic reduces the risk of colorectal polyps ¹⁶⁷ and accelerates red blood cell turnover and splenic erythropoietic gene expression in mice ¹⁶⁸. Boiled garlic does inhibition of 1, 2-dimethylhydrazine-induced mucin-depleted foci and O⁶-methylguanine DNA adducts in the rat colorectum ¹⁶⁹ while crude garlic extract and its fractions showed cytotoxic effect ¹⁷⁰ on malignant and nonmalignant cell lines ¹⁷¹. Garlic provides great protection against physiological threats ¹⁷² and lower downs risk of gastric cancer ^{10, 173}. DAS also does growth inhibition and induce apoptosis in anaplastic thyroid cancer cells by mitochondrial signaling pathway ⁷⁴ while allyl sulfur compounds causes cellular detoxification of carcinogens and is used in cancer therapy ¹⁷⁴. Garlic phytochemicals counteract the cardiotoxic side effects of cancer chemotherapy ¹⁷⁵ (Table 1). SAMC inhibit the survival, invasion and migration of bladder cancer cells ¹¹⁷.

Garlic derived natural products showed chemopreventive effects and successfully prevent colorectal, ¹¹ and prostate cancer ¹⁷⁶. These also show onco-cardiological prevention ¹⁷⁷ reduce the risk of invasive cervical) ⁷⁵ and ovarian carcinoma ¹⁶. Diallyl trisulfide induces Bcl-2 and caspase-3-dependent apoptosis via downregulation of Akt phosphorylation in human T24 bladder cancer cells) ³⁹ (Wang et al., 2010). Garlic powder supplemented diet shows chemopreventive effects in diethylnitrosamine-induced rat hepatocarcinogenesis) ¹⁷⁸ (Figure 5a). It also shows chemoprotection against cyclophosphamide toxicity in mice ¹⁷⁹ and protects against adriamycin induced alterations in mouse red blood cells (Thabrew et al., 2000) ¹⁸⁰. Garlic inhibits methylcholanthrene-induced carcinogenesis in the uterine cervix of mice ¹⁸¹ and shows prevention of 4-nitroquinoline 1-oxide-induced rat tongue ¹⁸² and hamster cheek pouch carcinogenesis. It shows protective effects against bromobenzene toxicity to precision cut rat liver slices ^{183, 184}. Diallyl disulfide shows prevention of chemically induced skin tumor development ¹⁸⁵. Organosulfur compounds from garlic and onions protect from benzo[a]pyrene-induced neoplasia and glutathione S-transferase activity in the mouse ¹⁸⁶ (Table 1). Onion and garlic oils and extracts inhibit tumor promotion in experimental animals ^{187, 188}. Diallyl sulfide, a flavor component of garlic (Allium sativum), inhibits dimethylhydrazine-induced colon cancer ¹⁸⁹.

6. Conclusion

Organosulfur compound isolated from garlic have been shown to have anticancer activity both in vitro and in vivo. These are also used as pharmaceuticals for treatment of acute and chronic diseases and are thought to be powerful instrument in maintaining health as medicine and nutraceutical. Garlic components do restoration of cell death pathways via the targeting of mitochondrial proteins. These can normalize the central pathway which can frequently impair cancer cells and contributes to the development of resistance to conventional chemotherapy. Garlic derived molecules can be formulated and designed to target oxidants or pro-oxidants and to activate pro-apoptotic proteins as well as to block these proteins. Garlic based herbal products and targeted molecules can find good solutions for clinical management of various cancer types. Today garlic based herbal medicines are attracting public health authorities, pharmaceutical industries because of its larger use in prevention and treatment of so many diseases and disorders. Garlic derived dietary supplements are highly demanded by nutritionists, physicians, food technologists and food chemists. No doubt garlic and its derived herbal products are providing optimal health and quality of life.

Conflict of Interests

Author has no conflict of interests. The author alone is responsible for the content and writing of the paper.

Acknowledgements

Author is thankful to Prof. Ashok Kumar, Vice Chancellor, D D U Gorakhpur University, Gorakhpur for his kind support.

Abbreviation

DAS=diallyl sulfide

DADS= diallyl disulfide

DATS= diallyl trisulfide

SBC=S-benzyl-cysteine

SAMC =S-allylmercaptocysteine

SAC =S-allylcysteine (SAC)

ROS =reactive oxygen species

BSO =buthionine sulfoximine

CySS=S-alkenylmercaptocysteine

caspase=cysteine-aspartic acid protease.

References