Molecular Targets of Isothiocyanates in Cancer: Recent Advances

Abstract

Cancer is a multistep process resulting in uncontrolled cell division. It results from aberrant signaling pathways that lead to uninhibited cell division and growth. Various recent epidemiological studies have indicated that consumption of cruciferous vegetables such as garden cress, broccoli, etc., reduces the risk of cancer. Isothiocyanates (ITC) have been identified as major active constituents of cruciferous vegetables. ITCs occur in plants as glucosinolate and can readily be derived by hydrolysis. Numerous mechanistic studies have demonstrated the anti-cancer effects of ITCs in various cancer types. ITCs suppress tumor growth by generating reactive oxygen species or by inducing cycle arrest leading to apoptosis. Based on the exciting outcomes of pre-clinical studies, few ITCs have advanced to the clinical phase. Available data from pre-clinical as well as available clinical studies suggests ITCs to be one of the promising anti-cancer agents available from natural sources. This is an up-to-date exhaustive review on the preventive and therapeutic effects of ITCs in cancer.

1. Introduction

Cancer is the leading cause of deaths worldwide, accounting for 7.6 million deaths according to recent statistics. The number of deaths due to cancer is projected to increase to 13.1 million in 2030. These figures implicate marginal efficacy of present standard available therapies to cancer patients, implying the urgent need to identify new strategies/agents that can be included in cancer preventive or therapeutic regimen.

Historical evidence purports nature being a prodigious source of many drugs and drug leads for various ailments, including cancer [1]. Several epidemiological studies have been published over the past few decades that indicate a strong correlation between intake of fruits & vegetables and reduced risk of cancer [2–4]. Basic benefits of using bioactive dietary agents are low cost, well known applications in traditional medicinal system, accessibility and minimal or no toxicity.

Epidemiological and case-control studies continue to support the notion that consumption of cruciferous vegetables reduces the risk of developing various types of cancers such as pancreatic, prostate, ovarian and breast [5–11]. Isothiocyanates (ITCs) occur in cruciferous vegetables as glucosinolates and are converted to ITCs by the action of the enzyme myrosinase. ITCs from these vegetables are also released by cutting or chewing or by intestinal micro flora present in humans [12] (Figure 1). ITCs have been shown to have substantial chemopreventive activity against various human malignancies [13, 14]. Some of the widely studied ITCs that have potent anti-cancer effects are Allyl isothiocyanate (AITC), Benzylisothiocyanate (BITC), Phenethylisothiocyanate (PEITC) and Sulforaphane (SFN). Unless stated, most of the studies mentioned in this article used 95–98% pure ITCs for evaluating anti-cancer effects. This exhaustive review highlights the specificity of ITCs against various targets in cancer.

Figure 1.

Mechanism of cellular uptake of ITCs

2. Chemoprevention by ITCs

An individual's susceptibility to cancer is determined by numerous factors including maintenance of a critical balance between phase I and phase II enzymes. Phase I primarily consists of cytochrome P450 enzymes, which play an important role in metabolizing the xenobiotics and carcinogens. However, in this process, several chemicals or pro-carcinogens are activated or converted into highly reactive electrophilic metabolites. The generated electrophiles can disturb the genomic stability by causing DNA damage. Chemopreventive effects of ITCs are exerted by inhibition of the bio-activation of carcinogens by phase I drug metabolizing enzymes [15–18]. The mechanistic studies by Morse et. al. and others indicate that administration of ITCs prevents the tumor promoting effects of various chemical carcinogens in different animal models [19–21]. PEITC has been shown to possess significant chemopreventive properties against tobacco-induced carcinogens in rodent models of lung and esophageal cancers [22, 23]. AITC also inhibits NNK (a tobacco derived carcinogen) induced tumors in rats [24]. Similar to other ITCs, AITC induces phase II detoxifying enzymes quinone reductase and glutathione S-transferase in the animal tissues [25]. Cytochrome P450E1 and N-dimethylnitrosoamine demethylase (NDMAd) are major enzymes that cause bio-activation of tobacco specific nitrosoamines. These enzymes can be inhibited by glutathione conjugates of ITCs [26]. ITCs also inhibit various isoforms of CYP450 directly; for example BITC suppresses cytochrome P450 2E1 while sulforaphane inhibits cytochrome P450 1A2 [27, 28]. Sulforaphane also has been shown to inhibit steroid and xenobiotic receptor (SXR), a nuclear hormone receptor that regulates expression of CYP3A4 [29]. Zhou et. al. showed specific antagonism by sulforaphane to inhibit drug clearance due to SXR-induced activity of CYP3A4.

Phase II enzymes like glutathione-S-transferase (GST), NADPH quinine oxidoreductase and UDP-glucuronosyltransferases play an important role in detoxifying carcinogens as well as xenobiotics. ITCs are known to induce phase II enzymes, which further explain the cancer chemo-preventive activity of ITCs [28, 30–34]. GST catalyzes the conjugation of glutathione with electrophilic compounds making them more water soluble and facilitating their removal from the body [35, 36]. It is well known that ITC-GSH conjugate is exported out by MRPs [37]. As a result of continuous conjugation and efflux of the conjugate, intracellular GSH level drops significantly within 3h of ITC treatment. This time also coincides with the induction of GST and MAPK [38]. Due to non-availability of GSH, ITCs bind with other vital cellular proteins causing their thiocarbamoylation [37]. Although being electrophilic, no studies have reported direct binding of ITCs to cellular DNA [39]. In addition PEITC has been shown to de-methylate the promoter region of GSTP1 to induce the expression of GSTP1 [40]. ITCs also induce GSTs which scavenges ROS [41]. The action of phase II enzymes is primarily regulated by the antioxidant or electrophile response element (ARE/EpRE). The latter can be activated by the transcription factors such as the basic leucine zipper (bZIP) Nrf2, which heterodimerizes with Maf G/K to exhibit its effects. ITCs induce the Nrf2 transcription factor to activate ARE, which in-turn translates into the activation of mitogen activated protein kinase (MAPK) ERK/JNK, PI3K and PKC [41–44]. SFN induces epigenetic modifications by inhibition of HDAC 1, 4, 5 and 7. In addition, SFN induced de-methylation at the promoter region of Nrf2 causes enhanced expression of Nrf2 in the TRAMP mice model for prostate cancer [45]. SFN's chemopreventive effects mainly depend on induction of phase II enzymes through the activation of antioxidant response elements like Keap1/Nrf2 [31, 46, 47]. SFN mediated induction of Nrf2 was found to be through the activation of heme oxygenase 1 and inhibition of p38 in hepatoma cells [42]. Furthermore, several studies have shown induction of thioredoxin reductase as well its substrate thioredoxin by SFN in various cancer cell lines [48–50]. Inhibition of key survival pathway such as NF-kB and AP-1 by ITCs also contributes to the chemopreventive effects of ITCs [44].

ITCs thus modulate phase I and II enzymes to reduce the bio-activation of carcinogens as well as enhanced detoxification. This dual mechanism leads to reduced binding of carcinogens with the DNA and hence less mutagenic or carcinogenic effects.

These studies suggest existence of mutually distinct mechanisms of chemo-preventive and chemo-therapeutic effects of ITCs. Specific targets have been identified that mediate chemotherapeutics effects of different ITCs against human cancers [30, 41, 44, 51–53]. These targets might vary with the structural variations amongst ITCs as well as the nature and origin of cancer. Several studies demonstrate that ITCs modulate cancer cell signaling by acting on multiple targets to suppress growth and progression of cancer cells [41, 53].

3. Uptake of ITC by Cancer Cells

The uptake of anti-cancer agents is an important limiting factor for efficacy. Most of the ITCs can be taken up by the cells through passive diffusion. The cellular uptake of ITCs correlates with the induction of phase II detoxifying enzymes important for chemo-preventive activity. It was observed that the intracellular concentrations of ITCs can reach up to 100–200 folds higher than the extracellular concentrations. For example when hepatoma cells were incubated with 100μM SFN for about 30 minutes, the intracellular concentrations reached about 6.4mM [38]. The magnification of intracellular concentration was due to the formation of dithiocarbamates, as ITCs rapidly conjugate with thiols, particularly GSH. Uptake of ITCs in cancer cells was GSH dependent. The uptake was reduced if GSH concentration was increased. The ITC-GSH conjugate being the substrate of MRPs is transported out of the cells. This mechanism of uptake and cellular accumulation can be vital in designing the dose regimens of these ITCs. The dose will require the adjustment for high accumulation as well as to compensate for the rapid export through transport proteins likes MRPs [54]. The shuttling of ITC-GSH causes prompt depletion of intracellular GSH, resulting in the perturbation of cellular redox homeostasis. This could be one plausible mechanism of reactive oxygen species (ROS) generation by ITCs.

4. Chemotherapeutic Targets

A. Benzylisothiocyanate (BITC)

BITC occurs in cruciferous vegetables like cabbage, mustard, watercress, cauliflower and horseradish that constitute a significant proportion of our daily diet. Accumulating evidence suggests the anti-cancer effects of BITC through suppression of initiation, growth and metastasis of human cancers in various mouse models [55–61]. BITC induces apoptosis selectively in cancer cells through multiple mechanisms [55, 60, 62]. Major anti-cancer effects of BITC are due to the generation of reactive oxygen species. BITC causes cell cycle arrest as well as disruption of mitochondrial membrane potential to initiate mitochondrial pathway of apoptosis [57, 63]. Studies from our laboratory have demonstrated the anti-cancer effect of BITC against pancreatic tumor growth via inhibition of key molecules overexpressed in cancer such as AKT, STAT3, HDAC and NF-kB (Table 1) [7, 55, 56, 59]. The targets of BITC can be divided as per their role against cancer promoting mechanisms.

Table 1.

Chemo preventive effects of ITCs

Compounds	Efficacy	Organ	Mechanism		Dose/Duration	Cell line	References
BITC	Anti-angiogenesis	Pancreas	↓VEGF, MMP-2, HIF-a	In vitro	5, 10,20 μM/24 h	BxPC-3,PanC-1	[77]
			↑RhoB
			↓STAT-3 phosphorylation (Tyr-705), HIF-a, VEGFR-2, VEGF, MMP-2, CD31, RhoC.	In vivo	12 μmol/40 days
BITC	Anti-angiogenesis	Breast	↓CD31, VEGF, VEGFR	In vitro	2.5, 5, 7.5μuM/24 & 48h	MDA-MB-231	[78]
				In vivo	6 & 9 μmol/14days
BITC	Apoptosis	Pancreas	↓PI3K, AKT, PDK1, mTOR, FOXO1, FOXO3a	In vitro	5, 10, 20 μM/24h	BxPC-3,PanC-1	[55]
			↑Bim, p27, and p21	In vivo	12 μmol/45 days
BITC	Apoptosis	Breast	↓mTOR, FOXO1	In vitro	2.5, 5μM/6 & 9h	MDA-MB-231, MCF-7, MDA-MB-468, BT-474, BRI-JM04	[68]
BITC	Apoptosis	Pancreas	↓NF-kappaB, cyclin D1, ↑HDAC1, and HDAC3	In vitro	10 μM/24h	BxPC-3, Capan-2	[56]
BITC	Apoptosis	Pancreas	↑ERK, JNK, P38, ROS	In vitro	10 μM/24h	Capan-2, MiaPaCa-2	[57]
BITC	Apoptosis	pancreas	↑ATR (Ser-428), Chk2 (Thr-68), Cdc25C (Ser-216), Cdk-1 (Tyr-15)and induction of p21 Waf1/Cip1	In vitro	2.5, 5, 10 μM/24, 48 h	BxPC-3, PanC-1	[58]
			↓NF-kappa B
BITC	Apoptosis	pancreas	↓STAT-3, Mcl-1, Bcl-2	In vitro	5–40 μM/24 h	BxPC-3, AsPC-1, Capan-2, MiaPaCa-2, Panc-1	[59]
				In vivo	60 μmol/wk/6 weeks
BITC	Apoptosis, Cell cycle arrest	Brain	↑ROS	In vitro	10, 20μM/24h	U87MG	[82]
BITC	Apoptosis	pancreas	↑H2A.x, p21, Chk2	In vivo	10 μmol/L/24 h	Capan-2	[60]
			↓CyclinB1, Cdc2, Cdc25C
BITC	Apoptosis, Cell cycle arrest	pancreas	↓Cdk1, cyclin B1, Cdc25B, NF-kappaB	In vitro	5, 10 μM/24 h	BxPC-3	[61]
			↑IkappaBa
BITC	Apoptosis	Ovary	↓Bcl-2, ERK1/2 and Akt	In vitro	10 μM/48 h	SKOV-3, KLE, SW954, SW756, HL60	[62]
			↑caspase-3, −9, Bax, JNK1/2 and p38
BITC	Apoptosis, cell cycle arrest	Bone	↓cyclin A, cyclin B1, ↑Chkl, p53, caspase-9 and −3, ROS	In vitro	7.5 μM/0, 12, 18, and 24 h	U-2 OS	[79]
BITC	Anti-metastasis	Lung	↓MMP-2, Twist, betacatenin, Akt and NFkappaB	In vitro	5 μM/24 h	L9981	[80]
			↑ROS
BITC	Anti-tumorigenesis	Leukocyte	↓hydrogen peroxide, ROS	In vitro	1, 10, 100 μM/24 h	HL-60	[81]
				In vivo	81 or 810 nmol/20 weeks
PEITC	Apoptosis	Breast	↓HER2, EGFR, STAT-3, BCL-XL, XIAP, ROS	In vitro	10 μM/24 h	MDA-MB-231, MCF-7	[95]
			↑Bax, Bim, HER2	In vivo	12μmol/33 days
			↓HER2, EGFR, STAT-3
PEITC	Apoptosis	Prostate	↓Bcl-2, XIAP	In vitro	1 μM/24 h	PC-3, DU145	[97]
			↑Bax, Bak	In vivo	9 μmol/38 days
PEITC	Apoptosis	Leukemia	↑ROS, NO	In vitro	10 μM/ 1–6 h	HL-60	[98]
			↓GSH
PEITC	Apoptosis	Breast	↑ROS, caspases 9 and 3	In vitro	20 μM/6 h	MDA-MB-231, MCF-7	[99]
			↓GSH, Bax
PEITC	Apoptosis	T-cell	↑JNK	In vitro	20μM/24h	Jurkat	[101]
PEITC	Apoptosis	Lung	↑JNK, p38, Erk1/2, AP-1	In vivo	15 micromol/g/140 days		[102]
PEITC	Apoptosis	Colon	↓SOS-1, PKC, ERK1/2, Rho A, MMP-2 and -9, GRB2, NF-κB, iNOS, COX-2	In vitro	2.5 μM/24, 48 h	HT29	[103]
PEITC	Anti-metastasis	Liver	↓MMP-2,MMP-9	In vitro	0.1–5 μM/24 h	SK-Hep1	[104]
			↑TIMP1,2
PEITC	Anti-angiogenesis	Prostate	↑VEGF, VEGF receptor 2, Akt, EGF, G-CSF	In vitro	4 μM/24 h	HUVEC, PC-3	[105]
PEITC	Anti-angiogenesis	Breast	↓HIF1alpha, CAIX, GLUT1, BNIP3, VEGF-A, 4E-BP1	In vitro	16 μM/24h	MCF7	[106]
PEITC	Apoptosis, cell cycle arrest	Prostate	↑p21WAF-1/Cip-1 and p27Kip1	In vivo	8 μmol/g/9 weeks	BALB/c male mice	[122]
			↓cyclins D and E, Rb
PEITC	Apoptosis, cell cycle arrest	Oral squamous cell	↑p53, p21, pl7, Bax, Bid	In vitro	5 μM/72 h	HSC-3	[116]
			↓cyclin E, CDK2, Bcl-2
PEITC	Apoptosis	multiple myeloma	↑PARP, caspases-3 and -9, c-jun, HSP27	In vitro	2.5, 5 and 10 μM/12, 24, 48 h	MM. 1S	[51]
			↓Mcl-1, X-IAP, c-IAP and survivin
PEITC	Apoptosis	Breast	↑p57 Kip2, p53, BRCA2, IL-2, and ATF-2	In vitro	3 μM/48 h	MCF-7	[124]
PEITC	Apoptosis, Cell cycle arrest	Prostate	↑p53, WEE1, caspase-3, -8, -9	In vitro	20 μM/24,48 h	DU 145	[117]
			↓CDC25C
PEITC	Cell cycle arrest	Lung	↓cell growth	In vitro	3, 6, 9 μM/24, 48, 72 h	A549, H1299	[125]
PEITC	Apoptosis	Ovary	↓EGFR, AKT, p-GSK	In vitro	40 μM/24 h	SKOV-3, OVCAR-3 , TOV-21G	[109]
			↓Tumor growth	In vivo		12 μmol/42 days
PEITC	Apoptosis	Oral cancer	↑caspase-3,-8, BID, DR5	In vitro	10 μM/48 h	HN22	[113]
PEITC	Apoptosis	Cervical cancer	↑DR4, DR5, caspase-3, PARP	In vitro	5 μM/48 h	HEp-2, KB	[114]
			↓ERK1/2
PEITC	Apoptosis	Breast	↑caspases 7 and 9, PARP	In vitro	3–30 μM/24h	MCF-7	[118]
			↓Bcl-2, XIAP
PEITC	Apoptosis	Breast	↓ER-α36, ERK 1/2	In vitro	10 μM/48 h	MCF7, H3396, MDA-MB-231,SK-BR-3	[110]
SFN	Apoptosis	Liver	↑TrxR1	In vitro	12 μM/24 h	HepG2	[49]
SFN	Apoptosis	Breast	↑TrxR1	In vitro	3, 6 μM/24 h	MCF-7	[50]
SFN	Apoptosis	Prostate	↑Ac-histone H4, Bax, P21, ↓HDAC	In vitro	15 μM/24 h	BPH-1, LnCaP, PC-3	[133]
SFN	Apoptosis, Cell cycle arrest	Bladder	↑p27	In vitro	5–20 μM	T24	[147]
SFN	Apoptosis, Cell cycle arrest	Colon	↑cyclins A and B1, bax, cytochrome c	In vitro	15 μM/24 h	HT29	[148]
SFN	Apoptosis, Cell cycle arrest	lymphoblastic leukemia	↑caspases 3, 8, and 9, p21	In vitro	7.5 μM/24 h	LCL, Nalm-6, Jurkat, KOPTK1	[149]
			↓Cdc2/Cyclin B1, AKT, mTOR
SFN	Apoptosis	Bladder	↓tumor volume	In vivo	12 mg/kg/5 weeks	athymic mice	[135]
			↑caspase 3 and cytochromec
SFN	Apoptosis	Prostate	↓p65, VEGF, cylcin D1, and Bcl-X	In vitro	20 and 30 μM/24 h	PC-3	[136]
SFN	Cell cycle arrest	Colon	↑ERK,JNK,p38,p21	In vitro	6.25, 12.5, 25, 50 and 100 μM/−24h	HT-29	[150]
			↓cyclin D1
SFN	Apoptosis	Colon	↑ERKl/2 and Akt	In vitro	20, 30 μM/24 h	Caco-2	[151]
SFN	Apoptosis	Breast	↑caspase-8, caspase-3, cytochrome c	In vitro	0, 5, 15, or 25 μmol/L for 48 h	MDA-MB-231,MDA-MB-468, MCF-7, T47D	[137]
			↓Bcl-2, HDAC, ERalpha, EGFR, EGFR2
SFN	Apoptosis	Prostate	↓IAP, cIAP1, cIAP2, XIAP, p65	In vitro	20 μM/ 24 h	LNCaP, PC3	[143]
			↑Apaf-1, E2F1
SFN	Anti-angiogenesis, anti-metastasis	Pancreas stem cell	↓Nanog, Oct-4, VEGF, PDGFRα, Zeb-1	In vivo	0–20 mg/kg/6 weeks	NOD/SCID/IL2Rgamma mice	[144]
SFN	Apoptosis	prostate	↑ROS, Fas, caspase-8, Bid	In vitro	40μM/4h	PC-3	[145]
SFN	Anti-metastasis	Oral carcinoma	↓MMP-1, -2	In vitro	1μM/24h	YD8, YD10B, YD15	[152]
SFN	Apoptosis	Breast	↓tumor growth	In vitro	25 or 50 mg/kg/24 days	KPL-1 athymic mice	[153]
				In vivo
SFN	Anti-metastasis	Bladder	↓ZEB1, Snail	In vitro	0–20μM/24h	T24	[154]
			↑E-cadherin
AITC	Apoptosis, Cell cycle arrest	Prostate	↓Bcl-2, cyclinBl, Cdc25B, Cdc25C	In vivo	10μmol/26 days	Nude mice	[168]
			↑BID
AITC	Anti-proliferation	Colon	↓mitosis	In vitro	12 μM/24 h	HT-29	[164]
AITC	Apoptosis	Prostate	↑AP-1, ERK1/2, JNK1/2, Elk-1 and c-Jun	In vitro	50 μM/24 h	PC-3	[165]
AITC	Apoptosis, Cell cycle arrest	Bladder	↓ α- and β-tubulin	In vitro	30 μM/24 h	UM-UC-3	[167]
			↑cytochrome c, caspase-9,-3, Bcl-2, JNK
AITC	Anti-metastasis	Liver	↓MMP-2/-9	In vitro	0.1–5 μM/24 h	SK-Hep 1	[169]

A.1. Cell Proliferation and Growth

PI3K/AKT pathway has been shown to be activated in about 59% of the pancreatic tumors, and it also promotes cell division in other cancer forms [64, 65]. PI3K stimulation results in the phosphorylation of AKT at Thr-308 and Ser-473 through PDK1 activation [66]. Studies by Boreddy et. al. have shown that BITC inhibits PI3K/AKT signaling. BITC prevents the phosphorylation of AKT at both Thr-308 and Ser-473 along with suppression of PI3K (Tyr-458), PDK1 (Ser-241), mTOR (Ser-2448) [55]. The inhibition of mTOR signaling by BITC was also observed in human prostate cancer cells [67]. These studies showed that BITC had negligible effect on normal human pancreatic ductal epithelial (HPDE-6) cells, suggesting the specificity of BITC towards cancer cells [55]. These results also showed up-regulation of pro-apoptotic proteins like Bim, p21 and p27 due to nuclear accumulation of Forkhead Box Protein 1 (FOXO1). Inhibition of phosphorylation of FOXO1 (Ser-256) and Forkhead Box Protein 3a (FOXO3a) by BITC was due to the de-phosphorylation of AKT in pancreatic cancer cells [55]. Interestingly, BITC also reduced acetylation of FOXO proteins by reducing the level of CREB-binding protein (CBP) protein [55]. FOXO1 suppression was also shown to be responsible for BITC initiated cell-death in breast cancer cells [68].

NF-kB is a transcription factor that regulates cellular inflammation, immunity and proliferation [69, 70]. Batra et. al. showed that BITC-mediated downregulation of HDAC1 and HDAC3 expression was associated with the acetylation of NF-kB in pancreatic cancer cells [56]. BITC treatment significantly suppressed the phosphorylation of NF-kB at Ser-276 and Ser-536 in BxPC-3 and Capan-2 cells in a dose and time dependent manner [56, 61]. BITC reduced NF-kB protein expression in BxPC-3 cells but not in Capan-2 cells, indicating that BITC acts differentially on different cell lines [56]. The Capan-2 cells have wild type p53, whereas BxPC-3 cells harbor mutated p53, hence the role of p53 in BITC mediated down-regulation of NF-kB expression cannot be ruled out and remains to be explored further. The mechanistic studies revealed that neither IkB phosphorylation nor expression levels were altered by BITC, whereas IKK expression was down-regulated. Hence, down-regulation of IKK by BITC treatment could be the reason for inhibition of NF-kB phosphorylation (Ser-536) [56, 61].

Signal Transducer and Activator of Transcription 3 (STAT3) is hyper-activated in significant number of malignancies like breast cancer, pancreatic cancer, gastric cancer and head & neck cancer as well as in cancer stem cells where it enhances tumor aggressiveness and progression [71–73]. Sahu and Srivastava have shown that BITC suppresses the phosphorylation (Tyr-405 & Ser-727) and expression of STAT3 in pancreatic cancer cells lines such as BxPC-3, PanC-1, Capan-2 and MIA PaCa-2 [59]. The role of STAT3 in the anti-cancer effects of BITC was confirmed by STAT3α overexpression or through activation by Interleukin-6 (IL-6), which abrogates the effects of BITC (Table 1) [59].

A.2.Angiogenesis

The growing tumors are nourished through processes such as angiogenesis and neovascularization. Angiogenesis is mainly promoted by hypoxia inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) [74]. STAT-3 has been shown to be a positive regulator of VEGF and HIF-1α [75, 76]. Boreddy et al. demonstrated that BITC inhibits angiogenesis in chicken chorioallantoic membrane (CAM) and rat aortic ring assay [77]. This clearly indicates the anti-angiogenic potential of BITC. BITC-mediated suppression breast cancer xengrafts was associated with inhibition of critical angiogenic factors like CD31 and VEGF [78]. Furthermore, BITC down-regulated the expression of HIF1-α, VEGFR-2, MMP-2, Rho A, Rho C and RAC1, 2 and 3 in pancreatic, but the inhibition of VEGF, HIF-1α and MMP-2 was not observed in STAT3 overexpressing BxPC-3 cells [77]. This undoubtedly suggests that inhibition of tumor growth and angiogenesis by BITC correlates with STAT3 inhibition.

A.3.Mitochondrial Cell-death

Generation of reactive oxygen species (ROS) is an important mechanism to induce cell death, specifically in cancer cells. As shown by us and others, BITC significantly induced ROS generation in pancreatic cancer cells and glioma as well as other cancer models [57, 79–82]. ROS generation leads to disruption of mitochondrial membrane potential and release of pro-apoptotic molecules resulting in activation of caspase-mediated cell death [63, 81, 83]. Furthermore, BITC-mediated down-regulation of MCL-1 in human leukemia cells was also found to be correlated with the mitochondrial pathway of apoptosis [84]

A.4.Cell-cycle Arrest

ROS induced by BITC also DNA damage and G2/M cell cycle arrest as detected through increased phosphorylation of H2A.X (Ser-139) and ChK2 (Thr-68) [57, 60, 61]. Antioxidants block the effects of BITC confirming the role of ROS in cell cycle arrest [57]. BITC treatment increased the phosphorylation of the MAP kinases, such as ERK (Thr202/Thy204), JNK (Thr183/Tyr185) as well as p38 (Thr180/Tyr182) in a dose-dependent fashion [57, 62]. It was later found that BITC-induced cell cycle arrest was executed only through ERK, while the other MAP kinases were playing role in the induction of apoptosis [57].

A.5.Invasion and Metastasis

Metastasis is initiated by key regulators like matrix-metalloproteinases, Twist and β-catenin. A study showed that BITC treatment inhibited cell migration and invasion in lung cancer cells. This was accompanied with reduced expression of MMP-2, Twist and β-catenin [80]. Another study showed that oral administration of 5 and 10mg/kg BITC suppressed the expression of MMP-2& 9 in the sera and lungs of mice injected with 4T1 breast cancer cells [85]. BITC also inhibits the process of epithtelial to mesenchymal transition through FOXQ1 suppression in breast cancer cells, leading to reduced metastatic potential [86]. The data available for anti-metastatic effects of BITC is insufficient to prove the anti-metastatic efficacy. Hence, additional elaborate studies are required to establish the role of BITC in metastasis.

A. 6. In vivo Studies

Our in vivo studies indicated that BITC is well tolerated at a dose of 12 μmol/day (72mg/kg) in mice. Interestingly, in vivo tumor growth was markedly arrested by BITC treatment in athymic nude mice as compared to controls [59]. These results showed that after 6 weeks of 12 μmol/day BITC treatment by oral gavage, average tumor volume in BITC-treated mice was about 48% less as compared to the control group [59]. LC-MS analysis showed that after 46 days of BITC (12 μmol/day) treatment, mean concentration of 6.5±0.1 μmol/L (39mg/L) (n=10) & 7.5±0.3 μmol/g (45mg/g) (n=10) BITC was observed in the plasma and tumors of treated mice respectively [55]. These results suggest a reasonable bioavailability of BITC and also that the therapeutic concentration could be achieved in vivo by oral administration. No untoward side effect or change in body weight was observed, the suggesting that 12μmol/day BITC was relatively safe. Furthermore, suppression of in vivo angiogenesis by 12 μmol/day (72mg/kg) treated mice was observed by reduction of hemoglobin content by 76% in matrigel plugs implanted in the mice as well by 61% in the excised tumor xenografts, as compared to respective controls [77]. These results signify the potential anti-tumor and anti-angiogenic effects of BITC. The molecular targets of BITC have been described in detail in Table 1. Interestingly, dietary BITC also suppressed the growth of cancer stem cell in MMTV-neu breast cancer transgenic mice model along with inhibition of major stem cell markers like Oct4, SOX-2 and Nanog [87]. In contrast, the activation of NOTCH2 signaling by BITC was found to impede the therapeutic benefits of BITC [88]. A recent study from our group showed that the absorption and bioavailability of BITC can be enhanced by making the nanoemulsion of BITC [89].

A. 7. Toxicity studies

No major evidence of BITC side effect exists for the doses that are commonly used for anti-cancer studies. A study has shown that oral administration of BITC (0, 50, 100 and 200mg/kg) for 4 weeks caused reduction in body weight and reduced food consumption only at highest doses [90]. In addition, the study revealed that BITC treatment caused increase in serum cholesterol and decrease in triglycerides, accompanied with renal dysfunction. Furthermore, in this study BITC treatment reduced the weight of almost all the organs except the adrenals, where the weight was increased. Some transitory hematological changes like reduced hemoglobin and lymphocyte count with increased platelets, eosinophils and neutrophils were observed in BITC treated rats. It is pertinent to note that no significant signs of toxicity were observed at the dose of 50mg/kg. Although these changes were observed at higher doses (100 and 200mg/kg) of BITC, no mortality was reported [90]. The high doses like 100–200mg/kg BITC, which were associated with some side effects, are unlikely to be used for anti-tumor effects. So far the therapeutic doses of BITC, which suppresses in vivo tumor growth are much lower and not associated with any side effects and hence can be considered relatively safe.

B. Phenethylisothiocyanate (PEITC)

PEITC is another isothiocyanate that occurs conjugated with glucosinolate in many cruciferous plants. PEITC is abundantly present in plants such as watercress, garden cress and in some non-cruciferous plants like turnips and radishes [30, 91]. Watercress is the most prolific source of PEITC, which can release approximately 2–6 mg PEITC/ounce (0.07 to 0.21mg of PEITC/g) in humans [91, 92].

The effective concentrations of PEITC vary from 0.12μM to 14μM [93, 94]. Like BITC, PEITC also induces ROS generation selectively in cancer cells [95, 96]. Mechanistic studies have shown that PEITC disrupts mitochondrial electron transport chain (ETC) by inhibiting Complex I and III activity and reduces oxygen consumption rate in prostate cancer cells [97, 98]. Furthermore, PEITC is known to inhibit ROS-detoxifying mechanisms to enhance ROS-mediated cytotoxicity [96–98]. This was further proven in cells with varying levels of anti-ROS mechanisms that showed differential sensitivity towards PEITC [99, 100].

Two general mechanisms that have been identified for the anti-cancer activity of PEITC include cell cycle arrest and apoptosis induction [44, 101, 102]. Few studies also suggest anti-angiogenic and anti-metastatic effects of PEITC by mechanisms similar to BITC [80, 103–106]. PEITC has been shown to act on about 30 different targets present in cancer cells [107]. Mi et al. have shown that PEITC alters the function of critical amino acids of proteins and peptides through covalent interactions [107].

B.1.Cell Proliferation and Growth

Studies suggest that PEITC has multiple targets like AKT, EGFR and HER2 in cancer cells, which promote anti-apoptotic mechanisms in cancer cells. As discussed earlier, AKT (Protein kinase B) is frequently overexpressed in cancers and regulated by oncogenes like EGFR and HER2 [108]. Our studies demonstrated that PEITC inhibits EGFR and HER2 in ovarian and breast cancer cells [95, 109]. PEITC caused significant inhibition of activated EGFR (Tyr1068) to suppress the growth of ovarian cancer cells. Furthermore, PEITC reduced the phosphorylation of AKT and mTOR expression [109]. In this study PEITC also disrupted the complex of Raptor and Rictor with mTORC1 and mTORC2 [109]. In another study we observed inhibition of HER2 and AKT in breast cancer cells. These observations suggest that PEITC inhibits AKT activation by suppressing EGFR and HER2 expressions to suppress anti-apoptotic signaling in cancer cells (Table 1). Furthermore, PEITC also inhibits HDACs, the major epigenetic regulators resulting in the inhibition of androgen receptor in prostate cancer cells [40].

B.2.Angiogenesis

Similar to BITC, PEITC also inhibits vascular endothelial growth factor (VEGF), a major promoter of angiogenesis. Xiao and Singh showed suppression of VEGF by PEITC, which was later shown to be mediated through suppression of HIF1α [105, 110–112]. Based on the evidence provided in these studies, it can be suggested that PEITC inhibits angiogenesis mainly by inhibiting VEGF.

B.3.Mitochondrial Cell-death

Accumulating evidence from several studies showed induction of apoptosis signaling by PEITC. PEITC has been shown to activate death receptors and Fas-mediated extrinsic apoptotic pathway in oral and cervical cancer cells [113–115]. PEITC treatment also resulted in the activation of intrinsic pathway of apoptosis. PEITC modulates mitochondrial proteins like BCL2, BID and BAX, causing the release of cytochrome c into cytosol to induce intrinsic apoptosis pathway [94, 116–119]. However, the release of cytochrome c by PEITC treatment into cytosol to induce apoptosis was contradicted by a study conducted by Wu et. al. [120]. Further in-depth studies are thus required to delineate the exact mechanism of PEITC.

B.4.Cell-cycle Arrest

PEITC as well as its N-acetyl cysteine conjugate causes activation of Retinoblastoma (Rb) protein in prostate cancer cells, leading to attenuation of cell cycle progression [39, 121]. Furthermore, a G0/G1 phase cell cycle arrest by PEITC was associated with activation of p53 in oral squamous carcinoma cells, in multiple myeloma, osteogenic sarcoma and breast cancer cells and G2/M cell cycle arrest in prostate cancer cells [51, 52, 116, 117, 122, 123]. Interestingly, lung carcinoma cells expressing mutated p53 were shown to be more sensitive to PEITC as compared to cells with wild type p53 expression [107, 124].

B.5.Invasion and Metastasis

PEITC inhibits cancer cell invasion by inhibiting matrix metalloproteinases (MMP) and suppresses activity of ERK and NF-kB to inhibit metastasis [103, 104]. We recently demonstrated in vivo anti-metastatic potential of PEITC using a unique mouse model of breast cancer metastasis [94]. This model utilizes MDA-MB-231-Luc2 brain-seeking breast cancer cells that lodge in the brain from blood circulation when injected into the left ventricle of mouse heart. These cells later grow to form metastatic tumors in brain. Oral administration of 10 μmol PEITC (65mg/kg) for 10 days significantly prevented the seeding of breast cancer cells into the brain in this model. We also observed that PEITC administration suppressed the growth of metastasized tumor in the brain and enhanced the survival of mice bearing tumors in the brain [94]. This was the first evidence of in vivo anti-metastatic effects of PEITC in breast cancer model, but further studies are required to establish similar efficacy in other cancer forms. The molecular targets of PEITC have been described in detail in Table 1.

B.6. In vivo Studies

PEITC mediated inhibition of anti-apoptotic pathways was observed in the preclinical mouse model studies [125]. PEITC has a dose dependent bioavailability of about 70 – 110% by oral administration, which is a probable reason for in vivo efficacy [126]. Treatment of brain metastatic breast cancer has always been a problem due to the presence of blood brain barrier. Organ distribution study has revealed a fair availability of PEITC in brain suggesting better chances of PEITC to cross blood brain barrier [127]. This could be the reason for the anti-metastatic effects of PEITC [94].These studies indicate a high anti-tumor efficacy of PEITC in all organs including brain by oral administration. Orally administered PEITC causes significant inhibition of major oncogenic pathways like EGFR, HER2 and AKT in various in vivo cancer models leading to tumor growth suppression [94, 95, 109, 128]. These results clearly re-enforce potential for in vivo efficacy of PEITC.

B.7.Toxicity Studies

In addition to the beneficial effects, it is also essential to evaluate the probable side effects of PEITC. It was observed that i.p. administration of 80 and 160mg/kg PEITC caused increase in body weight of mice but reduction in the weights of liver and spleen [129]. Interestingly, preventive effects of PEITC were observed on acetaminophen induced hepatotoxicity and mortality [130]. These mutually contradicting observations make it important to establish a well-defined toxicity profile of PEITC using appropriate controls and population size.

B.8.Clinical Studies

Three clinical studies are currently under progress to test anti-cancer effects in humans. A phase I lung cancer study with PEITC conducted at MD Anderson Cancer Center was recently completed; however, the findings have not yet been published. Another phase I clinical study at the same institution has been planned to test the anti-leukemic effects of PEITC. Notably, a recent phase I clinical trial (NCI CN-55120) reported that 10μM PEITC can be achieved in the plasma after intake of 200 mg PEITC orally in human volunteers [131]. A phase II trial is also under progress in lung cancer patients at the Masonic Cancer Center, University of Minnesota in collaboration with the National Cancer Institute. The outcomes of these studies will provide data on the efficacy and toxicity of PEITC in humans.

C. Sulforaphane (SFN)

SFN is an isothiocyanate mainly present in broccoli and Brussels sprouts. Studies have shown that SFN is highly effective in blocking carcinogenesis. SFN inhibits HDAC activity to promote cell cycle arrest and apoptosis in Nrf2−/− cells suggesting Nrf2 independent mechanism of SFN [132, 133].

C.1.Cell proliferation and Growth

SFN acts on certain molecular targets like survivin and NF-kB that are vital for cancer cell survival [134, 135]. SFN induces apoptosis in breast cancer cells by the inhibition of Estrogen receptor (ER), EGFR1 and HER2, which are particularly important for the growth of breast cancer [136]. Recently SFN was shown to cause DNA damage through enhanced acetylation of DNA repair proteins. This effect was shown to be specific for cancer cells as there were no epigenetic changes or DNA damage observed in non-cancer cells [137]. Interestingly, based on the methylation of DNA and Cyclin D2 by SFN, a clinical trial (NCT01265953) has also been initiated at Portland, VA Medical Center [138]. Studies suggest significant epigenetic changes induced by SFN in various cancer models.

C.2.Angiogenesis

Very few studies have reported the anti-angiogenic effects of SFN. The suppression of VEGF and MMP-2 has been shown by SFN treatment [139, 140]. Another study indicated that VEGF suppression was mediated through inhibition of FOXO1/AKT pathway [141]. However, no further evidence exists for the anti-angiogenic effects of SFN. Due to the lack of sufficient evidence, anti-angiogenic activity cannot be considered as a critical mechanism of SFN.

C.3.Mitochondrial Cell-death

Another important mechanism of action of SFN was inactivation of inhibitors of apoptosis proteins (IAPs) [142]. SFN-mediated IAP inhibition was associated with BCL-2 inhibition suggesting activation of intrinsic apoptosis pathway [143]. SFN also causes generation of mitochondrial ROS in cancer cells that further leads to release of cytochrome c into cytosol augmenting cell apoptosis [144]. Interestingly, a ROS independent activation of MEK/ERK pathway was shown to lead to caspase dependent apoptosis in neuroblastoma cells [145].

C.4.Cell-cycle Arrest

SFN was shown to induce p27-mediated G0/G1 phase cell cycle arrest [146]. In addition SFN causes irreversible cell cycle arrest in G2/M phase followed by caspase-mediated apoptosis [147]. Recent studies have shown that SFN induces G2/M arrest through the activation of p21 (CIP1/WAF1) and inhibition of Cdc2/Cyclin B1 complex independent of p53 [148]. This study showed that apoptosis following G2/M arrest was induced by caspase and PARP activation in leukemia cells [148]. Specific activation of MAP kinases like ERK, JNK and p38 in response to SFN treatment was shown to be involved in inducing cell cycle arrest [149, 150].

C.5.Invasion and Metastasis

SFN exhibits potent anti-metastatic effects by suppressing cell migration and invasion. Jee et. al. observed that the anti-cell migratory effect of SFN was associated with MMP suppression [151]. Recently EMT was shown to be an important mechanism of SFN to inhibit cell migration and metastasis in different cancer types [143, 151-153]. Li et. al. have shown that SFN modulates Sonic hedgehog pathway to suppress self-renewal capacity of the pancreatic cancer stem cells and reduce EMT characteristics [143]. Significant suppression of SNAIL and ZEB-1 marked by the re-expression of E-cadherin was observed by SFN treatment that lead to reversal of EMT [153]. EMT prevention by SFN was also associated with induction of miR-200c and re-expression of the estrogen receptor [154]. The details of molecular targets of SFN have been described in Table 1.

C.6.In vivo Studies

Kanematsu et. al. demonstrated the in vivo efficacy of SFN against tumor growth and metastasis in breast cancer [152]. Pharmacokinetic studies show good bioavailability of SFN after oral administration. A concentration of 20μM in plasma was achieved after oral administration of 50 μmol SFN/rat (35 mg/kg) [155]. In a human study it was shown that after consumption of 200 μmol SFN (35.5mg), about 2 pmol/mg (0.355 ng/mg) SFN was detected in the breast tissue suggesting its availability at the tumor site [156]. The cumulative concentration of SFN in the small intestine was shown to be sufficient to inhibit tumor growth in the colonic tissue [157]. These studies clearly indicate bioavailability and favorable pharmacokinetic profile of SFN which can be instrumental for future development of SFN as an anti-cancer agent.

C.7.Toxicity Studies

Along with the anti-cancer activity of SFN, it is important to study its toxicity to assess to benefit to risk ratio. An increase in hepatoxicity indicators AST, ALT and LDH in plasma was observed with SFN (1.6mg/mouse/day (64mg/kg) for 14 weeks) administration in mouse bearing Benzo(a)pyrene [B(a)P] (100mg/kg b.wt.) induced lung cancer [158]. Interestingly, opposite findings were reported in another study. The rats were pre-treated with 3mg/kg SFN by intra-peritoneal injection. One hour later an intestinal ischemia/reperfusion surgery was performed to induce toxicity. It was observed that SFN administration increased the SOD levels along with reduction of myeloperoxidase, ALT and AST levels in serum [159]. Both the studies used significantly different concentrations of SFN, which can explain the opposite observations. However, due to the lack of confirmatory evidence, overall no conclusion can be drawn about the toxicity of SFN.

D. Allylisothiocyanate (AITC)

AITC is an aliphatic isothiocyanate derived from sinigrin and is excreted as NAC conjugates in the urine [160]. A recent study demonstrated a short term reversible DNA damage when AITC was provided in the diet [161]. Cancer cells in general are more susceptible to DNA damage leading to cell death. This explains the enhanced sensitivity of cancer cells towards AITC. The cytotoxic effects of AITC were shown to be specific to cancer cells [162]. Smith et. al. demonstrated apoptosis induction by AITC in colorectal cancer cells [163].

D.1.Cell Proliferation and Growth

AITC targets specific signaling molecules to suppress cancer cell growth. ERK and JNK signaling were involved in the activation of AP-1 by AITC to suppress cancer cell growth [164, 165].

D.2.Mitochondrial Cell-death

Geng et. al. observed that AITC resulted in the phosphorylation of BCL-2 to induce apoptosis, whereas mutated BCL-2 abrogated the cytotoxic effects of AITC [166].

D.3.Cell-cycle Arrest

Srivastava et. al. demonstrated the in vivo efficacy of AITC in prostate cancer [167]. This study indicated that cell growth arrest in G2/M phase by AITC was associated with the inhibition of cyclin B1, cell division cycle (Cdc)25B and Cdc25C.

D.4.Invasion and Metastasis

The anti-metastatic effects of AITC have been demonstrated through suppression of cell migration and invasion. It was observed that AITC inhibits MMP2/9 to exhibit anti-metastatic effects in hepatoma cells [168]. Furthermore, AITC exerts anti-angiogenic effects to suppress tumor growth by down-regulating angiogenic factors like nitric oxide and tumor necrosis factor α (TNFα) (Table 1).

D.5.In vivo Studies

AITC was shown to inhibit tumor and ascites formation from Ehrlich ascites tumor cells in mice. This study also revealed enhanced survival of ascites-bearing mice with AITC treatment [169]. Furthermore, i.p administration of 25μg AITC/animal (1 mg/kg) in mice inhibited tumor-directed capillary formation suggesting inhibition of angiogenesis. AITC treatment also reduced serum nitric oxide and TNFα levels indicating reduction in inflammatory markers by AITC [170]. These studies suggest a good in vivo efficacy of AITC. Nonetheless, more studies are required to confirm the in vivo activity against contemporary targets in cancer.

D.6.Toxicity Studies

Pre-clinical studies have demonstrated some toxicity induced by AITC. Significant hematological changes were observed with AITC treatment. Subcutaneous administration of 20 mg/kg AITC reduced WBC counts by 25% along with marked reduction of lymphocytes and monocytes. In addition, increase in neutrophil and corticosteroid levels were observed indicating stress induced by AITC. The AITC treatment caused reduction in thymus weights while increasing the weights of adrenals [171]. These observations suggest significant effect of AITC on blood profile and organ weights. Interestingly in another study, i.p administration of 25μg AITC/animal every day for 5 consecutive days showed reduced WBC count at the 9^th day after starting the treatment [172]. Perhaps the differences between these observations could be due to different doses and the time points of analysis after AITC administration. Another study showed increased AST levels at high doses of AITC (100–150 mg/kg), but no change was observed at lower dose (50 mg/kg) suggesting dose dependent toxicity induced by AITC [173]. Interestingly, oral administration of AITC resulted in bladder toxicity in rats. This was found due to free AITC cleaved from urinary metabolites [174]. Taken together studies suggest that AITC exhibits toxic side effects, cautioning its use. Further in-depth studies are required to establish the toxicity profile of AITC so that the dose for anti-cancer effects can be titrated effectively.

5. Potential for Combination Therapy

Cancer cells contain multiple aberrant signaling pathways which lead to drug resistance and therapy failure in many patients. Combination therapy is known to kill cancer cells more effectively through diverse mechanisms simultaneously. ITCs exhibit a diverse range of cellular targets for anti-cancer effect. This property of ITCs makes them highly desirable for combinatorial therapeutic approaches. Several combination strategies have been tested in pre-clinical studies by combining ITCs amongst themselves or with conventional or new anti-cancer therapies (Table 2) [58, 175-182].

Table 2.

Effects of combinations of ITC with other anti-cancer agents

BITC	Concentrations and treatment time of ITCs	Combination with other agents	Concentrations	Cancer Type	Targets affected by combination treatment	Effect of combined treatment	References
BITC	5μM (18h)	TRAIL	10ng/ml (6h)	Pancreatic	↑ Caspase cleavage, ↓XIAP, ↑ BID cleavage, ↑ PARP cleavage	Synergistic	[192]
	20μM (1h)	Cisplatin	15, 30, 45μM (48h)	Lung	↓ β-Tubulin	Sensitization	[193].
	10μM (3days)	SFN	10μM (3days)	Pancreatic	↓ STAT3	Synergistic	[202]
	9, 12μmol(10 week)	PEITC (10 week)	12μmol	Lung	↓ Chemically induced tumorigenesis	Synergistic	[203]
	2.5μM (24h)	Radiation	5Gy (24–48h)	Pancreatic	↑ Caspase cleavage, ↑ G2/M cell cycle Arrest, ↑ ATR, ↑ Chk2, ↑ Cdc25c, ↑ Cdk-1, ↑ p21Wafl/Cip1	Synergistic	[58]
PEITC	5, 10μM (8, 24h)	Cisplatin	5, 10μM (8, 24h)	Cervical	↑ ERK, ↑ JNK, ↑ p38, ↑ MAPK	Synergistic	[181]
	20μM (1h)	Cisplatin	15, 30, 45μM (48h)	Lung	↓ β-Tubulin	Sensitization	[193]
	0.1, 0.5, 2.5, 5μM (24h)	Adriyamycin	20, 25μM (24h)	Cervical	↓ pkc, ↓ Telomerase	Synergistic	[195]
	0.1, 0.5, 2.5, 5μM (24h)	Etoposide	20, 25μM (24h)	Cervical	↓ pkc, ↓ Telomerase	Synergistic	[195]
	5μM (48h)	Paclitaxel	10nM (48h)	Breast	↑ G2/M cell cycle Arrest	Synergistic	[196]
	5μM (24h), 25mg/kg (50 days)	Platinum agents	40 μM (24h), 5mg/kg (50 days)	Lung	↓ GSH mediated export, ↑ ROS, ↑ DNA damage	Sensitization	[177]
	5μM (24h)	Metformin	8mM (24h)	Ovarian (Cisplatin resistant)	↑ ROS	Synergistic	[178]
	2.5μM (6h)	Vorinostat	2μM (18h)	Leukemia	↑ ROS	Synergistic	[179]
	2μM (24h)	Docetaxel	1nM (24h)	Prostate	↑ XIAP, ↓ BCL-2	Synergistic	[97]
	0.025% in diet (10 and 16 weeks)	Curcumin	1% in diet (10 and 16 weeks)	Prostate	↓ PDK-1/AKT	Synergistic	[208]
	2.5μmol (28 days)	Curcumin	3μmol (28 days)	Prostate	↓ AKT	Synergistic	[209]
	10μM (24h)	Curcumin	25μM (24h)	Prostate	↓ EGFR, ↓ AKT, ↓ NFkB	Synergistic	[210]
	2μM (24h)	SFN	0.4μM (24h)	Leukemia	↓ iNOS, ↓ COX-2, ↓ prostaglandin E2 ↓ tumor necrosis factor (TNF), ↓ interleukin-1 (IL-1)	Synergistic	[206]
	0.008% in diet (17 weeks)	d-limonene, Indole-3carbinol	0.63%, 0.18% in diet (17 weeks)	Lung	↓ Chemically induced tumorigenesis	Synergistic	[183]
SFN	20μM (24, 48h)	Radiation	2Gy (24, 48h)	Osteosarcoma	↑ Caspase cleavage, ↓ ERK, ↓ AKT, ↓ G2/M cell cycle arrest	Synergistic	[176]
	10μM (0, 12, 24, 48, 72h)	Radiation	4Gy (0, 12, 24, 48, 72h)	Head and Neck Cancer	↓ AKT, ↓ MCL-1	Synergistic	[184]
	3μM (24h)	Arsenic trioxide	1μM (24h)	Multiple Myeloma	↑ ER stress, ↑ HSP90, ↑ PERK, ↑ eIF2α, ↑ Unfolded protein response, ↑ ROS	Synergistic	[185]
	20–30μM (16h)	5-FU	45–60μM (16h)	Salivary gland adenoid cystic carcinoma	↓ NFkB	Synergistic	[182]
	25μM (24h)	Adriyamycin	0.6–10μM (24h)	Adriyamycin resistant mouse fibroblasta with mutant p53	↑p53	Sensitization	[194]
	1–20μM (6, 24h)	Oxaliplatin	100nM – 10μM (6, 24h)	Colorectal Cancer	↑ DNA fragmentation	Synergistic	[197]
	10μM (72h) 3mg/kg (3 days)	Sorafenib	20μM (48h) 60mg/kg (3 days)	Pancreatic cancer stem cells	↓ALDH1, ↓ NFkB	Synergistic	[180]
	25μM (24h)	Resveratrol	25μM (24h)	Glioma cells	↓ LDH, ↓ AKT, ↑ Caspase 3 cleavage	Synergistic	[186]
	20μM (48, 72h)	Diindolylmethane	20μM (48, 72h)	Colon	↑ G2/M cell cycle arrest	Antagonistic (At low concentrations) Synergistic (At higher concentrations)	[201]
		Quercetin		Pancreatic cancer stem cells	↓ BCL-2, ↓ XIAP, ↑ Caspase 3 cleavage	Synergistic	[187]
	10, 20μM (16h)	Quercetin	25, 50μM (16h)	Melanoma	↓ MMP-9	Synergistic	[188]
	10μM (6days)	Epigallocatechin gallate	20μM (6days)	Ovarian	↓ hTERT, ↓ BCL-2	Synergistic	[213]
	25μM (24h) 45mg/kg (3, 12h)	Epigallocatechin gallate	20, 100μM (24h) 100mg/kg (3, 12h)	Prostate	↓ Nrf2, ↓ AP-1	Synergistic	[212]

Radiation therapy is an important intervention for majority of cancers. Radiation has been shown to activate some important cancer cell survival signaling molecules like AKT, ERK and MCL-1 which lead to reduced efficacy. Our studies have shown that when BITC was combined with radiation therapy, a 2.8 fold increase in apoptosis and cleavage of caspase-3 was achieved in pancreatic cancer cells [58]. In addition to increased apoptosis, inhibition of NF-kB and activation of p38 was also observed with the combination of BITC and radiation therapy [58]. The combination of BITC or SFN with the radiation therapy caused increased G2/M cell cycle arrest [58, 175]. Combination of SFN with radiation therapy also showed inhibition of activation of critical molecules like AKT, ERK and MCL-1 along with induction of endoplasmic reticulum stress, explaining its efficacy [175, 183-187].

TNF-related apoptosis-inducing ligand (TRAIL) is a potential chemotherapeutic agent. Interestingly, TRAIL death receptors are highly expressed on cancer cells but not on normal cells making the cancer cells more susceptible to TRAIL-induced apoptosis as compared to normal cells [188, 189]. However, resistance to TRAIL is reported in many cancer cells [189, 190]. Our studies showed that BITC sensitized pancreatic cancer cells to TRAIL-induced apoptosis by activating both intrinsic and extrinsic pathway [191].

Accumulating evidence shows that combination of ITCs with conventional chemotherapeutics improves the efficacy against resistant cancer cells. Studies suggest synergistic activity of ITCs with common anti-cancer agents like cisplatin, adriamycin, etoposide, paclitaxel, metformin, vorinostat and docetaxel [176, 181, 192-194]. Both BITC and PEITC increased the apoptotic effects of cisplatin through depletion of beta-tubulin, but the combination did not affect DNA platination [180, 192]. Furthermore, reversal of the resistance to cisplatin was observed with PEITC, which was mediated by depletion of cellular GSH [176]. The combination of Metformin and PEITC also showed high efficacy in cisplatin resistant cancer cells [177]. PEITC and SFN caused inhibition of anti-apoptotic proteins like protein kinase C (α, β, ε and ς) and telomerase, while increasing pro-apoptotic protein kinase Cб to enhance the apoptosis caused by adriamycin and etoposide [194]. Also, the combination of adriamycin with SFN-induced sensitivity in resistant cancer cells by the effect of adriyamycin independent of p53 [193]. An HDAC inhibitor, vorinostat induced ROS to increase resistance in cancer cells. PEITC treatment suppressed the cytoprotective antioxidant response through depletion of cellular ROS, to reverse the resistance in leukemia cells [178]. The efficacy of taxanes was also enhanced by PEITC in different forms of cancer [97, 195]. The combination of SFN with oxaliplatin caused increased DNA fragmentation, suggesting synergism through oxaliplatin dependent mechanism [196]. NF-kB is a known target of SFN [197]. NF-kB inhibition by SFN mediated synergism with sorafenib and 5-fluorouracil (5-FU) to inhibit pancreatic cancer stem cell survival and salivary gland adenoid cystic carcinoma respectively [179, 181]. These observations suggest that ITCs can utilize the mechanisms of action of conventional agents or can induce independent effects to exhibit synergism.

Although most of the combinations exhibited synergistic effects in cancer cells, a combination of 5-FU with SFN showed antagonistic activity in the normal cells by modulating G2/M cell cycle phase [198]. This suggests that ITCs protect normal cells from the toxic effects of conventional therapeutic agents. Another study showed that the combination of cisplatin with ITCs was selectively effective in cancer cells [180]. Although the mechanism of selectivity remains to be elucidated, these observations clearly suggest an urgent need for clinical testing of the combination therapies of ITCs with conventional anti-cancer chemotherapeutics.

ITCs have been shown to offer synergism amongst themselves and other anti-cancer compounds. 3, 3'-diindolylmethane (DIM) is an important constituent of cruciferous vegetables and exhibits anti-cancer effects [199]. DIM synergizes with SFN leading to enhanced cell cycle arrest in colon cancer cells [200]. Amongst other ITCs, combination of BITC with SFN or PEITC was more effective in preventing pancreatic and lung cancer than the individual treatment [201, 202]. Curcumin is a well-known dietary agent with remarkable anti-cancer activity [203, 204]. The combination of curcumin with ITCs caused significant reduction in the levels of inflammatory markers. These observations advocate the possible synergistic or additive effect of curcumin in combination with ITCs [205, 206]. Several other studies re-enforce the enhanced anti-cancer effect of PEITC with curcumin through inhibition of pro-survival pathways like AKT, EGFR and NF-kB [207–209]. Epigallocatechin gallate (EGG), a green tea agent, has significant anti-cancer potential [210]. The chemo-preventive effects of the combination of SFN with EGG were successfully shown in transgenic model of prostate cancer through the induction of Nrf2 and AP-1 in Nrf2-deficient mice [211]. Furthermore, the combined treatment of SFN with EGG enhanced apoptosis in paclitaxel-resistant cancer cells by inhibiting hTERT and BCL-2 expression, showing therapeutic anti-cancer potential [212]. Taken together, it is clear from the above the studies that ITCs can be used for combination therapeutics in cancer treatment, especially for the resistant cancers. The combinations of ITCs with various anti-cancer agents and their prime mechanism of action have been summarized in Table 2.

6. Conclusion

Current epidemiological studies have certain limitations, such as differential exposure of the populations leading to misclassification, improper controls and possibility of recall bias. Hence, better designed studies are required to establish the role of ITCs as neutraceuticals for cancer prevention and treatment. Furthermore, better designed studies along with detailed mechanistic studies can provide us with an opportunity to use ITCs as the lead for synthesis of more potent and safe drugs through chemical modifications. It is important to note that some studies were done using extracts of ITCs from the vegetables. Few studies have shown that ITCs are susceptible to hydrolytic degradation at high temperatures and basic conditions [213, 214]. Thus the observations made by extracts of ITCs could be questionable especially if the extraction procedure was not appropriate or standard. These observations require further confirmation using pure forms of ITCs.

Recent studies have revealed many novel cancer targets. Specifically targeting these can enhance the efficacy of new as well as conventional therapies. Hence, it is important to test the efficacy of ITCs against new targets. Current preclinical evidence presented in the review provides an insight into potential anti-cancer mechanisms of action of the ITCs as well as their selectivity towards the cancer cells. Some clinical studies have been initiated already for some ITCs. Nonetheless, further detailed studies are required to establish the safety and efficacy profiles of these agents based on which they can be streamlined for further human studies. Based on the current data, it is evident that ITCs possess highly potential anti-cancer activity, but further detailed toxicity and clinical studies are required to warrant their future clinical benefits.

Figure 2.

Chemopreventive effects of ITCs

Figure 3.

Chemotherapeutic targets of ITCs

List of Abbreviations

ITC

Isothiocyanate

BITC

Benzylisothiocyanate

AITC

Allyl isothiocyanate

SFN

Sulforaphane

PEITC

Phenethyl isothiocyanate

GST

Glutathione-S-transferase

VEGF

Vascular Endothelial Growth Factor

STAT3

Signal Transducer and Activator of Transcription 3

IL-6

Interleukin 6

FOXO

Forkhead Box Protein

MAPK

Mitogen-activated Protein Kinase

Rb

Retinoblastoma protein

ROS

Reactive Oxygen Species

EMT

Epithelial to mesenchymal transition

EGG

Epigallocatechin gallate

SXR

Steroid and Xenobiotic receptor

CYP

Cytochrome P450

ER

Estrogen receptor

EGFR

Epidermal growth factor receptor