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This version of the book chapter is a pre-print and the final version is available in the book
"Curcumin: Synthesis, Emerging Role in Pain Management and Health Implications, " edited by
Daniel Loïc Pouliquen, pages 347-378 ; Nova Science Publishers, ISBN: 978-1-63321-319-7.
Chapter
CURCUMIN ANALOGUES FOR THE TREATMENT
OF BREAST CANCER REVISITED: THE
NANOTECHNOLOGY APPROACH
Vignesh Sundararajan1, Rhonda J. Rosengren1
and Khaled Greish1,2*
1Department of Pharmacology and Toxicology,
University of Otago, Dunedin, New Zealand
2Department of Oncology, Faculty of Medicine,
Suez Canal University, Egypt
ABSTRACT
Curcumin, the principal active component of turmeric, has anti-cancer and anti-
inflammatory properties and is effective in animal models of cancers such as prostate and
colon. Curcumin has been widely studied as an anti-cancer drug for breast cancer, and
also recently for one of the subtypes known as triple negative breast cancer (TNBC),
which lack the hormonal receptors. However, limited bioavailability, water insolubility,
and rapid metabolism are the main factors that hinder the development of curcumin as an
anti-cancer drug. Curcumin has been encapsulated in liposomes, micelles, solid lipid
nanoparticles, and conjugated with silica nanoparticles to improve its efficacy. Another
approach to overcome the problems with curcumin is the development of synthetic
curcumin analogues, which are more potent than curcumin in TNBC cell lines. Yet, some
of the curcumin analogues synthesised have the same problems associated with curcumin
when administered in animal models of TNBC. The same technology used for curcumin
encapsulation has been used to improve the pharmacokinetics of novel curcumin
analogues. Thus, this book chapter focusses on using nanotechnology as an approach to
improve the efficacy of curcumin and its analogues for use as a potential adjuvant drug
therapy for breast cancer.
Keywords: Breast cancer, curcumin, nanocurcumin, curcumin analogues, nanoformulation of
curcumin analogues, clinical trials
Vignesh Sundararajan, Rhonda J. Rosengren and Khaled Greish
2
INTRODUCTION
The use of active compounds from plants is based on the medicinal system of
predominantly the South-East Asian countries [1]. Curcumin or diferuloylmethane, a phenolic
compound extracted from the roots of Curcuma longa, has been used widely as a colouring
agent, food additive and in Indian traditional medicine [2]. It possesses antioxidant, anti-
inflammatory, and anti-cancer properties [2]. The constituents of curcumin available
commercially are 77% curcumin, 18% demethoxycurcumin, and 5% bisdemethoxycurcumin
[2]. Curcumin has been used as an anti-inflammatory agent due to its ability to block nuclear
factor-kappaB (NFκB) activation [3]. In tumour cells, it inhibits various proteins that control
proliferation, invasion and angiogenesis [3]. Because of its anti-inflammatory properties,
curcumin has been used against neurodegenerative, cardiovascular, pulmonary, metabolic,
and autoimmune diseases [3].
In various cancer models, curcumin elicits anti-inflammatory action by inhibiting
cyclooxygenase-2, lipoxygenase, and inducible nitric oxygenase [4]. At the molecular level,
curcumin interacts with a wide variety of target molecules. Some of the target molecules are
enzymes such as haem oxygenase-I [5], Ca(2+)-ATPase [6], and Na,K-ATPase [7],
telomerase, glutathione S-transferase (GST) [8], transcription factors such as NFB [8],
notch-1 [8], signal transducers and activators of transcription (STAT) [8], and β-catenin[8],
protein kinases such as mitogen-activated protein kinase (MAPK) [4], janus kinase (JAK) [8],
and protein kinase A (PKA) [8], receptors such as oestrogen receptor-α (ER-α) [8], human
epidermal growth factor receptor 2 (Her2) [8], and epidermal growth factor receptor (EGFR)
[8] and anti-apoptotic proteins such as bcl-2 [4]. Curcumin also inhibits chemical-induced
carcinogenesis at the initiation and progression stages [9] and the bioactivation of
environmental carcinogens such as benzo[a]pyrene by acting as a competitive inhibitor of
cytochrome P450/A1 [10]. Furthermore, by inducing haem oxygenase-1 via the activation of
the transcription factor Nrf2, curcumin protects the body from various forms of stress [5].
I. CURCUMIN AS AN ANTI-CANCER AGENT FOR BREAST CANCER
Breast cancer is estimated to be the most frequently diagnosed cancer in women,
contributing to 16% of all female cancers and is also one of the major causes of death
worldwide [11]. The most common form of breast cancer is invasive ductal carcinoma,
contributing to 85% of all breast cancer cases [11]. The conventional prognostic markers
predicting the overall survival include tumour size, tumour grade, and axillary lymph node
status [12]. Some of the newer markers include DNA ploidy, S-phase fraction (SPF), p53,
Her2, and EGFR [12]. Sub-classification of invasive ductal carcinoma is based on the
immunostaining of tumour tissues for ER, progesterone receptor (PR), and Her2 [13].
Tumours lacking the expression of these receptors are referred to as ‘triple negative breast
cancer’ (TNBC) [13]. As breast cancer is a heterogeneous disease, a more descriptive
classification based on gene- expression profiling of tumours is used to classify into five
major groups: luminal A, luminal B, normal breast-like, Her2 overexpression, and basal-
like [14].
Curcumin Analogues for the Treatment of Breast Cancer Revisited
3
Table 1. Summary of breast cancer subtypes and their characteristics.
Breast cancer subtype Characteristics
Luminal A ER (+), with high levels of ER expression. These tumours have a
better prognosis.
Luminal B ER (+). Comparatively low levels of ER expression and
overexpression of Her2. Increased expression of proliferation genes
such as CCNB1, MKI67 and MYBL2. Compared with luminal A
tumours, these have a poor prognosis.
Her2 overexpression ER (-), PR (-), and Her2 (+). Comparatively better survival rates
because of the ability to target them using herceptin.
Normal breast-like ER (-). Characterised by their resemblance to normal breast tissue
with elevated expression of genes associated with adipose cells, other
non-epithelial cells and low expression of genes associated with
luminal epithelial cells.
Basal-like (Triple
Negative cancer)
Lack ER, PR, and Her2. Very aggressive, difficult to treat, and
associated with poor prognosis. Characterised by the expression of
genes associated with basal epithelial cells.
The term triple negative breast cancer is frequently used for the basal-like tumours as 80-
90% of the triple negative tumours have the characteristics of basal-like type [14]. Table 1
summarises the breast cancer subtypes and their characteristics.
The triple negative tumours are difficult to treat [15] and are characterised by relapse
within three years with poor survival rates, especially in the first three years of post-
recurrence [15, 16]. The standard treatment for patients with TNBC is cytotoxic
chemotherapy [17]. The most common immunohistochemical and gene expression changes
in TNBC include p53 and BRCA1 mutations, overexpression of EGFR, caveolin-1, P-
cadherin, basal cytokeratins (CK5, CK17), and Ki67 [18]. Because the triple negative
tumours express neither ER, PR nor Her2, they are highly resistant to some of the drugs
used to treat breast cancer [19].
Though TNBC accounts for a minority of the breast cancers, they have high mortality
rates due to their aggressive nature and lack of targeted therapies [23].
Compared to other types of cancers, TNBC has a younger mean age of diagnosis [24].
Patients diagnosed with TNBC are more likely to have a larger tumour size and a grade III
tumour [24]. The majority of TNBC patients have distant recurrence and the median survival
rate is 4.2 years [24]. TNBC is more frequently seen in women of African and Hispanic
ancestry. Visceral metastasis is more commonly seen with TNBC, with the lungs and brain
being the most common sites of metastasis, and bone metastasis is seen only rarely [25-27].
Several studies show that curcumin has potent anti-cancer activity against different types
of breast cancer cell lines and in animal models [28-34].
1) Anti-Cancer Effect of Curcumin in Breast Cancer Cell Lines
Several mechanisms exist for the anti-cancer effect elicited by curcumin in breast cancer
cells. Cell cycle progression is a tightly regulated process controlled by protein kinases,
Vignesh Sundararajan, Rhonda J. Rosengren and Khaled Greish
4
consisting of cyclins and cyclin-dependent kinases (Cdks). Cyclin D, along with Cdk4 and
Cdk6 regulate the passage of cells through various phases of the cell cycle. Cyclin D1 is often
aberrantly up-regulated in breast cancer cells. Treatment of MCF-7 breast cancer cells with 10
µM of curcumin resulted in apoptosis of cells at the G2 phase of cell cycle. Interestingly, the
study didn’t find cell cycle arrest and there was no alteration in cyclin D1 expression levels
even after 48 h of treatment [35]. However, in ER (-) MDA-MB-231 cells, curcumin elicited
cell cycle arrest at the G2/M phase in a concentration-dependent manner [36].
Loss of p27, a Cdk inhibitor that can predict the prognosis in breast cancer, is often
associated with a poor prognosis. In the MDA-MB-231/Her2 cell line which overexpresses
Her2 and Skp2, a p27 degrading protein, curcumin treatment at 30 µM elicited cell cycle
arrest at G1 phase by down-regulating the expression of Her2 by 91%, Skp2 by 82%, cyclin E
by 80%, Cdk2 by 48%, and Cdk4 by 57%, respectively. At the same time, it increased the
expression of p27, a Cdk inhibitor by 417%. However, at 50 µM, curcumin elicited apoptosis
by up-regulating the expression of the pro-apoptotic protein Bax by 45%, cleaved caspase-3
by 213% and cleaved PARP by 7145% [37]. Curcumin can also induce apoptosis in breast
cancer cells via the mitochondrial-mediated pathway [38]. This often involves the loss of
mitochondrial membrane potential, an increase in mitochondrial membrane permeability
leading to opening of the transition pore, release of cytochrome c from mitochondria which
causes caspase-3 activation and PARP cleavage [39].
Studies examining the mechanism of action of curcumin demonstrated that it acted by
affecting various intracellular pathways. Treatment of MDA-MB-468 breast cancer cells with
curcumin (40 µM) resulted in the inhibition of EGF- stimulated phosphorylation of EGFR
and downstream targets ERK1/2, JNK and Akt [40]. NFκB is a transcription factor that is
involved in lobuloalveolar development of the mammary gland and is also frequently
activated in breast cancer. Its activation leads to cell proliferation, angiogenesis, and
metastasis [41]. MDA-MB-231 cells when treated with curcumin at 5 µg/mL resulted in a
significant decrease in NFκB activation at 48 h [42]. Activation of NFκB has also been linked
to development of resistance towards various chemotherapeutic agents. For example,
curcumin inhibited NFκB activation in taxol-treated cells, thus sensitising it to the effects of
taxol [43]. Another signalling pathway that is commonly activated in breast cancer is the
Wnt/β-catenin pathway. Curcumin at 20 µM reduced the expression of several components of
the pathway such as β-catenin, dishevelled, cyclin D1, and slug in both MCF-7 and MDA-
MB-231 breast cancer cells [44].
Matrix metalloproteinase (MMP) are endopeptidases that facilitate metastasis and
invasion by the destruction of the basement membrane. MMP-2 and MMP-9 are often up-
regulated in breast cancer patients [45-47]. Curcumin at 40 µM decreased the expression of
MMP-2, MMP-9 and up-regulated the expression of tissue inhibitor of metalloproteinases
(TIMP) genes, thus inhibiting invasion and metastasis [47]. MDA-MB-231/Her2 cells treated
with curcumin at concentrations of 30, 40, and 50 µM resulted in inhibition of cell migration,
colony formation and reduced expression of MMP-2 and MMP-9 [37].
Curcumin has also been studied as a drug for TNBC. Curcumin modulated BRCA1, and
caused apoptosis in TNBC cell line. Curcumin elicited DNA damage and promoted
phosphorylation, cytoplasmic retention of BRCA1 protein. Curcumin inhibited NFκB, and
phosphorylation of p65 NFκB. It also prevented migration and anchorage-independent
growth of TNBC cells [17].
Curcumin Analogues for the Treatment of Breast Cancer Revisited
5
2) Chemosensitisation of Multidrug Resistant Breast Cancer Cells by
Curcumin
Development of multidrug resistance (MDR) to chemotherapeutic agents is a major
challenge in cancer treatment. The resistance is conferred by ATP-dependent efflux of the
drug due to the overexpression of ATP-binding cassette (ABC) transporters, such as ABCB1
(P-glycoprotein), ABCC1 (MRP1), and ABCG2 (breast cancer resistance protein,
mitoxantrone resistance-associated protein) [48-50]. Chearwae et al. studied the ability of
curcumin, demethoxycurcumin, and bisdemethoxycurcumin to modulate the function of the
breast cancer resistance protein ABCG2 in MCF-7 FLV1000 and MCF-7 AdVp3000 cells
overexpressing the wild-type 482R and the mutant 482T ABCG2 transporter, respectively.
The amino acid at position 482 of ABCG2 plays an important role for substrate and inhibitor
specificity, and mutations at position 482 can alter the specificity. Curcuminoids (5 µM)
inhibited the efflux of ABCG2 substrates mitoxantrone and pheophorbide-a, and the most
potent effect was observed with curcumin.
(A)
(B)
Figure 1. Schematic representation of chemosensitisation of breast cancer cells by curcumin. (A)
Treatment with anti-cancer drug alone leads to ATP-mediated efflux of the drug, leading to treatment
failure. (B) Treatment with anti-cancer drug and curcumin inhibits the efflux of anti-cancer drug, thus
sensitising the cells to the drug.
Vignesh Sundararajan, Rhonda J. Rosengren and Khaled Greish
6
Treatment of MCF-7 AdVp3000 cells with non-toxic concentrations (10 µM) of curcumin
mixture and curcumin, demethoxycurcumin, and bisdemethoxycurcumin resulted in
sensitisation to mitoxantrone, topotecan, SN-38, and doxorubicin. Overall, curcumin was the
most potent modulator of ABCG2 [51]. Since curcumin undergoes extensive metabolism and
tetrahydrocurcumin (THC) is the final metabolite, more stable, and easily absorbed in vivo,
the same study was performed using THC. THC at 50 µM inhibited the efflux of ABCG2
substrates mitoxantrone and pheophorbide-a in 482R-ABCG2 overexpressing MCF-7
FLV1000 cells. THC also increased the accumulation and inhibited the efflux of [3H] -
vinblastine in a concentration-dependent manner in drug-resistant MCF-7 MDR cells.
Moreover, THC increased the mitoxantrone sensitivity in a concentration-dependent manner
in MCF-7 AdVp3000 cells, but not in drug-sensitive MCF-7 cells [52]. Thus, curcumin and
THC both exhibited a significant inhibitory action on the efflux of ABCG2 and ABCC1, and
sensitised the drug-resistant cells to anti-cancer agents (Figure 1).
3) Anti-Cancer Effect of Curcumin in Animal Models of Breast Cancer
Curcumin also possesses anti-cancer activity in animal models of breast cancer as
reported by various studies. A study by Masuelli et al. showed that curcumin delayed the
growth of mammary tumours in BALB-neuT mice. These transgenic mice develop aggressive
tumours at 16 weeks of age. The mice were dosed with curcumin, corn oil and water.
Administration of curcumin significantly increased the tumour-free time period and also
decreased the rate of tumour multiplicity. Mice dosed at 6 weeks of age with water had
palpable tumours by week 22 and those dosed with corn oil had palpable tumours by week
23. All the mice in curcumin-treated group exhibited tumours by week 27. At week 27, the
tumour multiplicity was 5.4, 5.9, and 2.8 for corn oil, water and curcumin-treated mice,
respectively. At 16 weeks of age when these mice developed invasive breast carcinoma, water
and corn oil-treated mice had palpable tumours by 23 and 22 weeks. However, in curcumin-
treated mice, palpable tumours developed in all mice only by week 25. The tumour
multiplicity at that stage was 5, 6.2, and 3.2 for corn oil, water and curcumin-treated mice.
Curcumin was also safe with no abnormal clinical parameters reported [31]. In another study,
curcumin was effective in animal models of Her2 overexpressing (BT-474) tumours.
Administration of curcumin intraperitoneally at a dose of 45 mg/kg twice a week, for 4
successive weeks resulted in a decrease in tumour volume. The study investigated the effects
of curcumin, herceptin, and a combination of both on tumour volume. When compared to the
control group, treatment with curcumin resulted in an approximately 77% decrease in tumour
volume, while a combination of curcumin and herceptin resulted in the smallest tumour
volume (88% decrease). In another set of experiments, the effects of taxol, taxol and
herceptin, taxol and curcumin, and a combination of curcumin, herceptin, and taxol on
tumour size were investigated. Mice treated with taxol and curcumin showed a 24% decrease
in tumour volume, compared to taxol-alone treated group. However, the smallest tumour
volume was observed in the group dosed with a combination of taxol, curcumin, and
herceptin (46% decrease). Interestingly, the taxol and curcumin-treated group had a similar
anti-tumour activity compared to the taxol and herceptin-treated group [32]. Carroll et al.
tested the hypothesis that curcumin could be effective against dimethylbenzanthracene
(DMBA)- induced mammary tumours, accelerated by medroxyprogesterone acetate (MPA).
Curcumin Analogues for the Treatment of Breast Cancer Revisited
7
Female Sprague-Dawley rats were gavaged a single dose of DMBA (20 mg/rat) to induce
mammary tumours on day 0. The animals were dosed intraperitoneally with curcumin (200
mg/kg) from day 26-50 and on day 30, animals were implanted with 25 mg MPA pellets. The
curcumin-treated rats exhibited a reduced incidence of MPA-driven tumours. On day 42, the
group dosed with MPA alone had a tumour incidence of 21%, whereas those treated with
MPA+curcumin had a tumour incidence of 6%. At the end of the experiment on day 52, the
curcumin+MPA-treated rats showed a 50% reduction in tumour multiplicity and a 34%
reduction in the levels of MPA-induced vascular endothelial growth factor (VEGF) [33].
Curcumin also inhibited metastasis in female athymic nude mice grafted with a highly
metastatic human breast cancer cell line MDA-MB-435LVB into the mammary fat pad. The
tumours were removed when they reached a size of 10 mm and from day 5 after tumour
removal, the animals fed with a diet mixed with 2% w/w curcumin had decreased lung
metastasis [43]. Similar results were found in another study with immunodeficient mice
injected intercardially with MDA-MB-231 cells. The animals were fed with either 1% casein
(control) or 1% curcumin. 68% and 17% of the curcumin-treated and control group showed
significantly decreased lung metastases [34].
II. PROBLEMS ASSOCIATED WITH CURCUMIN’S
PHARMACOKINETICS AND BIOAVAILABILITY
Though curcumin has shown great potential as an anti-cancer agent in several cancer
models, inherent problems associated with it such as poor absorption, rapid metabolism, and
water insolubility have hindered its successful development as a chemotherapeutic agent.
1) Pharmacokinetics
An ex-vivo study using everted rat intestinal sacs incubated with curcumin (50-750 µg)
showed that the compound had poor intestinal absorption. Approximately 30-80% of
curcumin was lost from the mucosal side, with only less than 3 % being found in the tissue.
The serosal fluid didn’t show any presence of curcumin. The authors also reported the
transformation of the compound during intestinal absorption [53]. The fate of curcumin in rats
was studied using Sprague-Dawley rats. Rats of both sexes were orally gavaged with
curcumin (1 g/kg) dissolved in arachis oil. Three days after the oral dosage, excretion via the
faecal route accounted for 65-85% of loss. The maximum elimination was observed at 48 h.
Curcumin was detectable in plasma 3 h after injection, but was beyond the limit of detection
at 6 h. However, when injected intravenously, curcumin was cleared rapidly and was beyond
the limit of detection within an hour. These results show that curcumin has a very low
absorption and is also rapidly metabolised [54]. Another study also showed that curcumin
administered orally at a dose of 2 g/kg to rats produced a maximum serum concentration at
0.83 h; serum levels of curcumin were also low in humans when administered a dose of 2 g,
even at 1 h after dosing. Supplementing curcumin with piperine to inhibit glucuronidation
resulted in a significant improvement in bioavailability and absorption [55]. The
pharmacokinetic properties of curcumin when given orally vs intraperitoneally (i.p.) in mice
Vignesh Sundararajan, Rhonda J. Rosengren and Khaled Greish
8
produced a completely contrasting result. With oral administration of 1 g/kg, the maximum
concentration in plasma (0.22 µg/ml) was detected at 1 h, and was below the limit of
detection within 6 h (5 ng/ml). However, when dosed intraperitoneally, the concentration was
much higher in plasma at 15 min (2.25 µg/ml), which rapidly declined within an hour [56].
Tissue distribution studies with curcumin in orally gavaged rats show that 0.015% of the
administered curcumin deposited in the liver, kidneys, and body fat after 3 h and small
intestine had the maximum accumulation. Liver perfusion studies also demonstrated that 49
% of the curcumin was excreted as glucuronide conjugates, and also as conjugated sulphate
[54, 57]. Studies investigating curcumin conjugation show that the enzyme responsible for
such reactions are mainly located in liver, kidney and intestinal mucosa [58]. Intravenous
injection of curcumin in rats and liver perfusion experiments show the accumulation of the
compound in liver and kidney, with the liver being the major site of metabolism [54]. Some
groups have used radioactive curcumin to trace the metabolism and elimination of curcumin
after dosing. For this, mice were dosed with [14C] curcumin (100 mg/kg) intraperitoneally and
the radioactivity in different organs was measured. Intestinal mucosa had the highest
radioactivity, followed by kidney, liver and plasma. Negligible amounts were found in brain,
heart, lungs and muscle. After 4 h, the peak values reduced to 20-33% of the initial values
[59].
2) Bioavailability
Another factor that contributes to the low bioavailability of curcumin is its clearance
from the body. Shoba et al. studied the pharmacokinetics of curcumin in rats and human
volunteers. Rats orally administered with curcumin 2 g/kg had an absorption half-life of 0.31
± 0.07 h and an elimination half-life of 1.7 ± 0.5 h. The same dose in humans was below the
limit of detection in serum [55]. Yang et al. studied the oral bioavailability of curcumin in rat
using tandem mass spectrometry. Oral administration of curcumin (500 mg/kg) resulted in an
elimination half-life of 28.1 ± 5.6 min, whereas i.v injection of curcumin (10 mg/kg) resulted
in an elimination half-life of 44.5 ± 7.5 min [60].
Aqueous solubility of curcumin is also a major factor contributing to its low
bioavailability and reduced pharmacological benefits. Curcumin is hydrophobic and water
insoluble, but soluble in organic solvents such as methanol [61]. Curcumin undergoes
hydrolytic degradative reactions in aqueous solutions. At pH below 7, it is stable but aqueous
solubility is poor. Without a stabilising agent, curcumin is very unstable in aqueous solutions
at pH >7. Under alkaline conditions, the hydrolytic degradation products formed are feruloyl
methane, ferulic acid and vanillin. In organic solvents, curcumin also undergoes
photodegradation. [62, 63]. In order to improve the water solubility and stability of curcumin,
cyclodextrin complexes of curcumin were prepared and investigated by Tønnesen et al. At pH
5, the water solubility of curcumin was increased by a factor of 104. The complex formation
also resulted in an increase in hydrolytic stability under alkaline conditions, but a decrease in
photostability was observed at the same time, compared to curcumin in organic solvents [63].
In another study, curcumin rapidly degraded after 6 h incubation in 0.01 M, pH 7.4 solution
of PBS. Only 6% of the initial curcumin remained intact. This was attributed to the
conversion of curcumin into its glucuronide and sulphate conjugates [64]. Wang et al. also
observed in their study that 90% of curcumin degraded when added to 0.1 M phosphate buffer
Curcumin Analogues for the Treatment of Breast Cancer Revisited
9
(pH 7.2). It was also highly unstable after incubation at 37°C in serum-free media for 1 h.
However, the stability was better in 10% foetal calf serum and in human blood, where more
than 50% of curcumin still remained intact after 8 h [65]. Moreover, curcumin forms
aggregates in aqueous media owing to its poor solubility [66].
All these factors greatly affect the efficacy and targeted delivery of curcumin in animal
models and clinical studies. Owing to its low bioavailability and rapid elimination from the
systemic circulation, high doses of curcumin are required to achieve accumulation in tumours
and significant results in clinical trials. Hence, nanoparticle formulation of curcumin is
currently being tested.
III. NANOTECHNOLOGY FOR IMPROVING CURCUMIN’S
PHARMACOKINETICS
Owing to the poor bioavailability and rapid metabolism of curcumin, nanotechnology has
been used to improve the efficacy of curcumin. Some of the commonly used nanotechnology
approaches include nanoparticles, liposomes, micelles, and phospholipid complexes. All these
novel approaches improved the absorption and biodistribution. The nano forms are more
effective than curcumin because of their physiochemical properties [67]. Nanocurcumin has
several advantages such as better solubilisation, tumour-specific accumulation, high stability,
reduced clearance from the body, and controlled release (Figure 2). These features make it a
better candidate than curcumin [68].
A study in rats intravenously administered with curcumin and nanocurcumin showed that
nanocurcumin had greater tissue distribution in different organs due to increased half-life and
mean residence time. Curcumin was mainly found in liver and kidneys, where it was
metabolised and eliminated from the body. This significantly decreased the systemic
circulation of curcumin. However, nanocurcumin was found mainly in the spleen and lungs,
and very little in the metabolising organs and heart. Both curcumin and nanocurcumin crossed
the blood-brain barrier, but the nanoformulation had better biodistribution in brain compared
to curcumin. Overall, the authors concluded that the nanosized formulation resulted in a better
biodistribution pattern [69].
Figure 2. Schematic representation of the factors involved in tumour suppression by nanocurcumin.
Vignesh Sundararajan, Rhonda J. Rosengren and Khaled Greish
10
Another advantage of nanocurcumin is that it provides controlled release of drug. It also
demonstrated better uptake and efficacy in breast cancer cells. This led to overall improvements
in the retention, efficacy, and bioavailability of curcumin [70]. Nanocurcumin was also more
soluble than curcumin, as curcumin-loaded PLGA nanospheres were completely soluble in
aqueous media without any aggregate formation [66]. Also, using inert hydrophilic polymers such
as poly(ethylene glycol) and poly(vinyl alcohol) can help in overcoming opsonisation and non-
specific accumulation of nanoparticles in organs [71]. Nanocurcumin is a superior drug carrier
with a loading capacity up to 25 wt/wt% and encapsulation efficiency of 70-90%. Thus,
controlled release of curcumin from nanoparticles dramatically improves the accumulation of
curcumin in tumours [68]. In animal models, curcumin nanoparticles greatly enhanced the
accumulation of curcumin in tumour tissues, resulting in increased number of apoptotic cells and
decreased cell proliferation [72].
1) The EPR Effect
The concept of using nanomedicine to deliver drugs selectively to the tumour tissue is
based on the phenomenon of ‘enhanced permeability and retention’ (EPR) effect [73], first
described by Maeda et al. in 1985 while working towards improving the pharmacological
properties of the anti-cancer drug neocarzinostatin by conjugating it to styrene-co-maleic acid
(SMA) [74]. This phenomenon makes use of the abnormal vasculature of the tumour tissues.
Tumour tissues are characterised by irregularly shaped, dilated, highly permeable, and leaky
blood vessels with large pores [73, 75]. The endothelial cells of the tumour blood vessels
have wide fenestrations, lack a smooth muscle layer, and functional receptors for angiotensin
II (AT-II) [76]. They also have defective lymphatic drainage in that the macromolecules are
not cleared effectively from the interstitial space [76]. Vascular mediators such as bradykinin,
nitric oxide (NO), and prostaglandins also contribute to the enhanced permeability of the
tumour tissues [77]. The submicron size of the nanoparticle also helps in its selective
accumulation in tumour tissue. The nanoconstructs must range in size from approximately 10
to 100 nm to utilise the EPR effect. Those smaller than 10 nm are cleared by the kidneys and
those ranging from approximately 100-200 nm are cleared by the reticulo-endothelial system
(RES) [78]. The molecular weight of the drug plays an important role in its ability to
accumulate in tumour tissues. Nanoparticles larger than 40 kDa have a longer circulation
time, and a reduced clearance rate from the body [76]. Neutrally-charged molecules circulate
in the blood longer than the negatively and positively-charged ones, which are eliminated
rapidly by the phagocytic cells of the liver [79]. Surface modification using polyethylene
glycol (PEG) and its derivatives reduces the negative charge and prevents rapid clearance of
the drug by RES [79, 80]. Taken together, all these factors help in selective accumulation of
nanodrugs in tumour tissues.
2) In vivo Effects of Nanocurcumin in Animal Models of Breast Cancer
Gupta et al. studied the effect of silk-fibroin (SF) derived curcumin nanoparticles in
MDA-MB-453 breast cancer cells, which have a high expression of Her2. SF nanoparticles
with 10% SF coating and less than 100 nm in size had better uptake, efficacy, and release rate
Curcumin Analogues for the Treatment of Breast Cancer Revisited
11
[70]. Curcumin, when encapsulated in poly (lactic-co-glycolide) (PLGA), a biodegradable
polymer showed a six-fold increase in uptake in triple negative MDA-MB-231 breast cancer
cells. The nanocurcumin was superior to free curcumin with an IC50 value of 9.1 µM in
MDA-MB-231 cells. The nano form elicited concentration-dependent anti-proliferative
effects and inhibited colony formation with just 40% release of the drug from the polymer. It
also caused an 8-fold increase in the number of apoptotic cells [81]. In another approach,
curcumin was conjugated with oligo (ethylene glycol) to form Cur-OEG nanoparticles. It
elicited apoptosis in several cancer cell lines and reduced tumour volume in a MDA-MB-468
xenograft model. The nanoparticle also did not cause acute or chronic toxicity when dosed
with 100 or 250 mg/kg [82]. In an in vivo study using MDA-MB-231 xenografts, a single
dose of PLGA-curcumin nanoparticles injected subcutaneously in mice demonstrated
sustained release of the drug, with much higher accumulations in metastatic sites such as
lungs and brain. The nanoparticle also reduced the tumour size by 49%, compared to empty
nanoparticle-treated mice, by down-regulating the expression of markers of angiogenesis,
metastasis and proliferation such as VEGF, MMP-9, Ki-67, and cyclin D1. The nanoparticle
treated tumours also had much smaller and poorly developed CD31 positive microvessels.
Interestingly, in the same study, repeated systemic administration of curcumin (4.4 mg,
intraperitoneally) did not result in inhibition of tumour growth [83].
Recently, magnetic nanoparticles have gained significant interest because of the
numerous unique properties they possess such as ultra-fine size and superparamagnetic
properties. These properties allow their modification for specific purposes such as drug
delivery, magnetic resonance imaging (MRI), and immunoassays. Curcumin encapsulated in
multi-layered polymer coated nanoparticle (F127250-CUR) demonstrated enhanced cellular
uptake, sustained release, and efficacy. F127250-Cur had an IC50 value of 11.9 µM compared
to free curcumin which had an IC50 value of 18.8 µM in MDA-MB-231 breast cancer cells. It
elicited a significant decrease in the colony densities at 4 µM in MDA-MB-231 cells after 10
days of treatment. F127250-Cur was also haemocompatible, thus offering a non-toxic, yet
effective drug delivery system [84]. Chun et al. studied the effect of NanoCurc, curcumin
encapsulated in a polymer composed of N-isopropylacrylamide, vinylpyrrolidone and acrylic
acid in female Sprague–Dawley rats injected with N-methyl-N-nitrosourea (MNU) to induce
breast cancer. Two different experiments were performed. In the first study, MNU-rats were
administered either NanoCurc dissolved in phosphate-buffered saline or an empty
nanoparticle in PBS. The NanoCurc delivered approximately 168 µg of curcumin per teat.
Two intraductal (i.duc) injections were given, the first on day 14 post-MNU exposure and the
next 4 weeks after the first injection. Rats were then observed for 34 weeks post-MNU
exposure for tumour incidence. While the rats treated with empty nanoparticles had a tumour
incidence of 22%, the NanoCurc-treated rats had a tumour incidence of 8%. In the second
study, the rats were randomised into four groups and were administered either oral free
curcumin (200 mg/kg), i.duc free curcumin, i.duc NanoCurc (168 µg of curcumin) or i.duc
empty polymer. Three treatments were given. The first one was at day 14 post-MNU
exposure. The other two treatments were at 7 and 14 days following the first injection. The
rats were observed for 24 weeks post-MNU exposure for the presence of palpable tumours.
Among the treatment groups, the i.duc NanoCurc rats had the smallest mean tumour size
(1672.7 mm3). This was associated with a decrease in NFB (approximately 84% decrease),
and proliferation marker Ki67 (approximately 93% decrease). Interestingly, the groups treated
intraductally with both free curcumin and NanoCurc had a similar decrease in the expression
Vignesh Sundararajan, Rhonda J. Rosengren and Khaled Greish
12
of Ki-67. The main mechanism behind the action of NanoCurc was via regulating cell
proliferation, which was evident by the decrease in the mitosis marker phospho-histone H3
[85].
3) Targeted Drug Delivery by Passive or Active Processes
Drugs can be delivered selectively to tumour tissue either by passive or active processes.
Passive targeting refers to the accumulation of drug into the tumour interstitium through the
highly permeable, leaky tumour capillaries either by passive diffusion or convection, based on
the EPR effect [86]. Convection is the movement of particles by fluid based on the
equilibrium between the hydrostatic and osmotic pressures [75]. Active targeting refers to the
conjugation of a targeting moiety to the nanoparticle, and its receptor must be highly
expressed on the surface of the cancer cell. After binding to the receptor, the nanoparticle is
internalised via receptor-mediated endocytosis. The low pH of the endosome then facilitates
in the release of drug [87]. Some of the commonly used ligands for targeted drug delivery
include lectins, transferrin, folate, lipoproteins, and opsonins [87, 88].
Polymeric micelles have also been used as a drug carrier because of their ability to
exploit the highly permeable and disorganised tumour vasculature via the EPR effect. This
allows micelles greater in size than 40 kDa to accumulate preferentially in tumour tissues [89,
90]. In a recent study, Liu et al. studied the efficacy of curcumin- loaded polymeric micelles
(Cur-M) via passive targeting, as a drug for breast cancer using the highly metastatic 4T1
mouse mammary tumour cell line in BALB/c mice. Cur-M had an encapsulation efficiency of
99% and showed a sustained release rate. Both free curcumin and Cur-M demonstrated
concentration-dependent cytotoxicity in 4T1 cell line, but Cur-M produced a statistically
significant cytotoxicity at a curcumin concentration of 20 µg/mL. The Cur-M-treated mice
(30 mg/kg) when compared to control, showed a 76% reduction in tumour burden, a
significantly higher (45%) median survival, and a 79% reduction in tumour nodules in the
lungs. Moreover, tumour tissues from Cur-M-treated mice had more apoptotic cells (16%)
compared to control (2%), a 73% smaller CD31 mean microvessel density, and decrease in
proliferation of cells evident by a weak Ki-67 immunoreactivity (25% for Cur-M, 68% for
control) [72].
By using the principle of active targeting, Mulik et al. studied the effect of transferrin-
mediated solid lipid nanoparticles containing curcumin (Tf-C-SLN) against MCF-7 breast
cancer cells. The nanoparticles showed increased cellular uptake, stability, cytotoxicity and a
slower release rate. The cytotoxicity of Tf-C-SLN was compared with curcumin solubilised
surfactant solution (CSSS) and curcumin-loaded SLN (C-SLN). All the three formulations
demonstrated concentration-dependent anti-proliferative activity; however Tf-C-SLN was
more superior. At concentrations of 3 and 9 µM of Tf-C-SLN, the cell viability was
42.4±1.78%, 14.5±0.7%, respectively. The treatment with 27 µM resulted in a significant
difference between CSSS and Tf-C-SLN. Even at the highest concentration of 81 µM, Tf-C-
SLN was more superior. Compared to other formulations, Tf-C-SLN at concentrations above
3 µM induced higher reactive oxygen species (ROS) generation, with significant increase
after 12 h. Further experiments to study the mechanism behind the cytotoxicity of Tf-C-SLN
demonstrated apoptotic induction which was confirmed by a concentration-dependent
Curcumin Analogues for the Treatment of Breast Cancer Revisited
13
increase in the number of apoptotic cells, higher percentage of DNA content in subG1 phase,
and higher percentage of cells with mitochondrial membrane potential loss [91].
Liposomal formulation of curcumin, which is a passive mode of drug delivery, has also
been studied as an alternative approach to increase the solubility and efficacy of curcumin.
Specifically, liposomes and cyclodextrins have been studied more extensively as a drug
carrier; however, some of their undesirable properties have led to a combinational approach to
encapsulate drugs. For example, cyclodextrins could be haemolytic, rapidly removed by the
kidneys and cause local toxicity. The drug to lipid mass ratio also affects the efficiency of
liposomes in encapsulating hydrophobic drugs. A novel approach involves use of drugs-in-
cyclodextrins-in liposomes (DCL) [92]. A curcumin loaded DCL (HPγCD- curcumin
liposome) was cytotoxic against MCF-7 breast cancer cells with an IC50 value of 11.5 µM ±
1.1. The study compared the cytotoxicity of HPγCD- curcumin liposome against conventional
curcumin liposome and DMSO-curcumin. Though the conventional curcumin liposome had a
lower IC50 value (10.2 µM ± 0.8) among all the three formulations, its encapsulation level was
only 0.8 mg/ml, compared to 1.3 mg/ml for HPγCD- curcumin liposome. Thus, HPγCD-
curcumin liposome enabled higher drug delivery [93].
IV. CURCUMIN ANALOGUES
An alternative approach to overcome the problems associated with curcumin is the
synthesis of curcumin analogues by modifying the chemical structure of curcumin.
Chemically, curcumin is a bis-α, β-unsaturated β-diketone which acts as a linker for two o-
methoxy phenols attached to two terminal positions. The molecule exhibits keto-enol
tautomerism at physiological pH, but the bis-keto form predominates in acidic and aqueous
neutral solutions [94, 95]. It has been reported that the instability of curcumin is due to the
enolic – OH moiety and the presence of the β-diketone moiety is not a definite prerequisite
for curcumin’s anti-cancer activities as curcumin analogues without this show anti-
proliferative activity [94]. By modifying the phenolic ring as well as the β-diketone moiety, a
wide range of curcumin analogues have been synthesised that are more potent than curcumin
[96]. Analogues having the cyclohexanone ring are referred to as the first-generation
analogues. The cyclohexanone containing derivative 2, 6-bis ((3- methoxy-4-hydroxyphenyl)
methylene)-cyclohexanone (BMHPC) had an IC50 value of 5.0 µM and elicited cytotoxicity,
anti-angiogenic properties against MDA-MB-231 and murine endothelial cells [97] (Figure
3).
Figure 3. Chemical structure of BMHPC [97].
Vignesh Sundararajan, Rhonda J. Rosengren and Khaled Greish
14
Among all the analogues, the fluorinated compound EF24 (3, 5-bis-(2-
fluorobenzylidene)-piperidin-4-one, acetic acid salt) demonstrated the most potent
cytotoxicity in MDA-MB-231 cells with an IC50 value of 0.8 µM ±0.4. EF24 at 100 mg/kg
also elicited tumour regression by 45% in a xenograft model of MDA-MB-231 cells at a dose
lower that the maximum tolerated dose (MTD) of 200 mg/kg i.v. without any toxicity [97]
(Figure 4). EF24 at 10 µM inhibited cell proliferation and induced growth arrest in G2/M
phase of the cell cycle in ER (-) breast cancer cells. EF24 caused apoptosis by caspase-3
activation, phosphatidylserine externalisation and by depolarisation of the mitochondrial
membrane potential [98]. Studies focussing on the mechanism of action of EF24 in MDA-
MB-231 cells showed that the analogue elicited its anti-proliferative activity by down-
regulating the expression of the pro-angiogenic transcription factor hypoxia inducible
transcription factor (HIF-1) post-transcriptionally in a VHL-dependent, but proteasome-
independent mechanism [99]. EF24 also demonstrated anti-angiogenic action when
conjugated with coagulation factor VIIa (fVIIa) to target tissue factor (TF), which is
overexpressed in tumour vascular endothelial cells. The conjugate induced apoptosis in
MDA-MB-231 and HUVEC cells. Intravenous injections of the conjugate containing 50 µM
of EF24 significantly reduced tumour burden in a MDA-MB-231 xenograft model [100].
By exchanging the β-diketone moiety for an αβ-unsaturated ketone, Lin et al. studied the
effect of curcumin analogues FLLL11 and FLLL12 in different ER (+) and ER (-) breast
cancer cells (Figure 5). Compared to curcumin, FLLL11 and FLLL12 had much lower IC50
values: 0.3-5.7 µM and 0.3-3.8 µM, respectively. At a concentration of 10 µM, the analogues
down-regulated the expression of Akt, STAT3, and Her2. They were also more effective than
curcumin in inhibiting cell migration, colony formation, and inducing apoptosis. Moreover,
these analogues together with doxorubicin elicited a more pronounced inhibition of cell
viability, compared to doxorubicin or the analogues alone. MDA-MB-231 cells were treated
with doxorubicin at different concentrations and cell viability was determined 72 h after
treatment. The viable cells were approximately 55% for 200 nM and 37% for 400 nM.
Similarly, the viable cells were approximately 37% with 5 μM of FLLL11. When the drugs
were combined, synergism was observed with all the three different combinations of
doxorubicin and FLLL11. The most potent effect was observed for the concentration 400 nM:
5 μM (doxorubicin: FLLL11), with approximately 18% of the cells being viable. A similar
study was performed with doxorubicin and FLLL12. Approximately, 38% and 35% of MDA-
MB-231 cells were viable after treatment with 400 nM and 5 μM of doxorubicin and
FLLL12, respectively. Synergism was observed with all the three different combinations of
doxorubicin and FLLL12. Specifically, treatment at concentrations 100 nM: 5 μM and 400
nM : 5 μM resulted in approximately less than 10% viable cells 72 h after treatment [101].
Figure 4. Chemical structure of EF24 [99].
Curcumin Analogues for the Treatment of Breast Cancer Revisited
15
Figure 5. Chemical structure of FLLL11 and FLLL12 [101].
Figure 6. Chemical structure of GO-Y030 [103].
In another study, more than 50 curcumin analogues were synthesised and screened by
Ohori et al. Several curcumin analogues displayed enhanced efficacy and decreased the levels
of oncoproteins at a much lower concentration than curcumin. These analogues were
symmetrical 1, 5-diarylpentadienone with an alkoxy substitution at positions 3 and 5 of the
aromatic rings. During their study, the authors found that addition of a methyl group to the p-
hydroxy group relative to the β- unsaturated ketone moiety caused enhanced cytotoxicity and
the presence of a 5-carbon tether was more efficacious than a 7-carbon tether [102]. GO-
Y030, one of the potent compounds from the aforementioned screening had an IC50 value of
1.2 µM compared to 19.3 µM for curcumin in MDA-MB-231 cells. At concentrations as low
as 2.5 µM, GO-Y030 induced apoptosis by PARP cleavage and inhibited STAT3
phosphorylation at 5 µM. Also, 1 µM treatment of MDA-MB-231 cells with the analogue
decreased colony formation by 95%, compared to control [103] (Figure 6).
By replacing the diketone moieties of natural curcumin analogues demethoxycurcumin
and bisdemethoxycurcumin with hydrazine derivatives, Shim et al. synthesised a novel
synthetic curcumin analogue called hydrazinocurcumin (HC) [104]. HC had IC50 values of
3.37 and 2.56 µM in MDA-MB-231 and MCF-7 cells, respectively, and elicited a
concentration-dependent inhibition of cell viability 72 h after treatment. HC inhibited colony
formation by 95% compared to control at 5 µM and elicited cell cycle arrest at the G1 phase
at 10 µM, in both the cell lines. Concentration-dependent anti-apoptotic action was also seen
48 h post treatment, with 10 µM treatment causing 14% and 26% cells to undergo apoptosis
in MDA-MB-231 and MCF-7 cells, respectively. HC at 10 and 20 µM treatment for 24 h in
both the cell lines decreased STAT3 phosphorylation and inhibited its downstream targets
such as MMP-9, MMP-2, Mcl-1, cyclin D1, c-Myc, Bcl-xl, survivin, and VEGF. Moreover,
HC was more potent than curcumin at reducing the migration and invasion in both the cell
lines [105] (Figure 7).
Vignesh Sundararajan, Rhonda J. Rosengren and Khaled Greish
16
Figure 7. Chemical structure of HC [104].
Figure 8. Chemical structure of Compound 15H [106].
Figure 9. Chemical structure of compound 23 [107].
Figure 10. Chemical structure of compound 18 [108].
By introducing benzimidazole groups into the feruloyl scaffold, 16 curcumin analogues
were synthesised and tested for cytotoxicity, among which the compound 15H had an IC50
value of 1.9 µM against ER (+) breast cancer cell line MCF-7 [106] (Figure 8).
Fuchs et al. synthesised a series of heptadiendione analogues (compounds 1-13) and
pentadienone analogues (compounds 13-24). Among all the analogues, compound 23 was the
most potent with an IC50 value of 0.4 and 0.6 µM in MCF-7 and MDA-MB-231 cells,
respectively [107] (Figure 9).
Curcumin Analogues for the Treatment of Breast Cancer Revisited
17
Mono ketone analogues with a piperidone ring also displayed potent cytotoxicity when
compared to curcumin. Specifically, compound 18 with N-ethylpiperidone ring was the most
potent with IC50 values ranging from 2.6-5.5 µM in different breast cancer cells [108] (Figure
10).
Another novel curcumin analogue, 5-bis (4-hydroxy-3-methoxybenzylidene)-N-methyl-
4-piperidone (PAC) was synthesised by removal of methylene, carbonyl groups and
introducing an N-methyl-4-piperidone into the curcumin structure (Figure 11). It was 5 times
more potent than curcumin in apoptotic induction and was 10 times more potent in ER (-)
breast cancer cells than ER (+) cancer cells. Treatment of MDA-MB-231 cells with 10 µM of
PAC caused cell-cycle arrest at G2/M phase. PAC mediated its effect by decreasing the
expression of NFκB, survivin, cyclin D1, Bcl-2 and up-regulating the expression of p21 in
vitro and in vivo. In xenograft models of MDA-MB-231, PAC at 100 mg/kg/day significantly
reduced tumour size and triggered apoptosis. Moreover, PAC significantly increased
bioavailability (5-fold) an hour after injection, and solubility (27-fold), when compared to
curcumin [109].
Studies have also been conducted to test the efficacy of curcumin analogues on 7, 12-
dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis in mice.
Dibenzoylmethane (DBM) is a chemical analogue of curcumin and lacks the phenolic
hydroxyl groups on the aromatic rings (Figure 12). Female Sencar mice were either dosed
with DMBA (1 mg per mouse, once weekly for 5 weeks) and AIN-76A diet (control) or 1%
DBM in AIN-76A diet, beginning two weeks prior to the first dose of DMBA and continuing
until they were sacrificed. The mice were sacrificed at 20 weeks after the last dose of DMBA
treatment. Mice dosed with 1% DBM in their diet showed decreased mammary tumour
multiplicity, tumour incidence, and increased latency time. Three weeks after the last dose of
DMBA treatment, the first tumours appeared in the control group, whereas in the mice fed
with 1% dietary DBM, the first tumours appeared 18 weeks after the cessation of DMBA
treatment. Tumour incidence was also reduced by 97-100%. Dietary DBM treatment also
resulted in inhibition of the proliferation rate of the mammary gland epithelial cells by 53%,
and formation of DMBA-DNA adducts in mammary glands by 72%. Mechanistic studies
showed that dietary DBM lowered serum oestradiol levels, increased the levels of hepatic
cytochrome P450 enzymes, and increased hepatic hydroxylation and glucuronidation of
oestradiol. In vitro studies showed that DBM competed with oestradiol for oestrogen receptor
sites and elicited concentration-dependent inhibition of DMBA hydroxylation [110, 111].
Figure 11. Chemical structure of PAC [109].
Vignesh Sundararajan, Rhonda J. Rosengren and Khaled Greish
18
Figure 12. Chemical structure of DBM [110].
Figure 13. Chemical structure of RL90 and RL91 [114].
Figure 14. Chemical structure of RL66 and RL71 [115, 116].
Figure 15. Chemical structure of B02 and B33 [117].
Synthesis of second-generation curcumin analogues involves the modification of the
central structure of the curcumin moiety by the introduction of heterocyclic rings. 18
curcumin analogues were synthesised, some of which elicited potent cytotoxicity in ER (-)
breast cancer cell lines, MDA-MB-231 and SKBr3, compared to curcumin [112]. Mechanistic
studies of two analogues, 2, 6-bis (pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2, 6-
bis (pyridin-4-ylmethylene)-cyclohexanone (RL91) (Figure 13) showed their ability to
modulate the expression of cell signalling proteins such as NFB, Akt, EGFR, β-catenin, and
Her2. Treatment with these analogues also resulted in the activation of stress kinases by
phosphorylation of JNK1/2 and p38 MAPK. In MDA-MB-231 cells, 3 µM of RL90 or 2.5
µM of RL91 caused cell cycle arrest in G2/M phase. Compared to control, the number of
apoptotic cells increased by 164% and 406% with RL90 and RL91, respectively [113].
Curcumin Analogues for the Treatment of Breast Cancer Revisited
19
More potent analogues were obtained by modification of the curcumin scaffold to include
N-methylpiperidone, tropinone or cyclopentanone core groups. In MDA-MB-231 cells,
3,5-bis (pyridine-4-yl)-1-methylpiperidin-4-one (RL66) and 3,5-bis (3,4,5-
trimethoxybenzylidene)-1 methylpiperidin-4-one (RL71) (Figure 14) had IC50 values of 0.8
and 0.3 µM, respectively [112]. In SKBr3 cells, RL71 at 1 µM induced cell cycle arrest in
G2/M phase, time-dependent anti-apoptotic effect with 35% cells undergoing apoptosis after
48 h, and increased the expression of cleaved caspase-3 and p27. RL71 also down- regulated
the expression of Her2 phosphorylation with complete inhibition after 12h. In MDA-MB-231
and MDA-MB-468 cells, RL71 (1 µM) significantly down-regulated the expression of Akt
phosphorylation and transiently increased the stress kinases JNK1/2 and p38 MAPK. RL71
also elicited anti-angiogenic properties by inhibiting HUVEC cell migration (46% compared
to control) and their ability to form endothelial tubes. RL71 at 8.5 mg/kg was also orally
bioavailable and produced a peak plasma concentration 5 min after oral dosing [115]. RL66
also had a similar mechanism of action in ER (-) breast cancer cell lines. Treatment of SKBr3
cells with 2 µM of RL66 resulted in cell cycle arrest, apoptosis, decrease in Her2
phosphorylation, and increase in the expression of p27, caspase-3 48 h post treatment. RL66
at 2 µM down-regulated the expression of Akt phosphorylation and transiently increased the
stress kinases JNK1/2 and p38 MAPK. RL66 inhibited HUVEC cell migration by 46% and
endothelial tube formation. Importantly, RL66 at 8.5 mg/kg by oral dosing daily, for 10
weeks, suppressed tumour growth in a MDA-MB-468 xenograft model by 48% and decreased
the microvessel density in the tumours by 57%, when compared to control. Thus, RL66
demonstrated potent anti-cancer activity in vitro and in vivo [116].
A series of mono-carbonyl curcumin analogues have been synthesised by deleting the β-
ketone moiety, considered responsible for the limitations of curcumin. The synthesis of these
compounds involved different 5-carbon linkers such as cyclopentanone, acetone, and
cyclohexanone. The analogues exhibited an increased stability and pharmacokinetic profile.
Oral dosing of male Sprague-Dawley rats with 500 mg/kg of compound B02 and B33 (Figure
15) resulted in an increase in the plasma concentration to 0.82 µg/ml and 4.1 µg/ml,
respectively, whereas curcumin had a plasma concentration of 0.091 µg/ml. There was also a
decrease in the plasma clearance of the compound B02 (125.4 L/kg/h) and B33 (38.98
L/kg/h), compared to curcumin (835.2 L/kg/h). The half-life of B02 was twice that of
curcumin. Furthermore, there was an increase in the cytotoxicity of these analogues towards
tumour cell lines by the replacement of benzene ring with a hetero aromatic ring [117].
V. NANOTECHNOLOGY TO IMPROVE CURCUMIN
ANALOGUE’S PHARMACOKINETICS
Though the majority of curcumin analogues synthesised are more potent and stable than
the parent compound in breast cancer cell lines, the same problem with curcumin was seen
when the analogues were tested in xenograft models. For example, the most potent curcumin
analogue, RL71, showed limited in vivo activity due to limited solubility, bio-distribution,
and stability [118]. Other laboratories are also now focusing on nanoparticle formulation of
curcumin analogues to further improve the efficacy of the drug and achieve tumour specific
targeting [119, 120].
Vignesh Sundararajan, Rhonda J. Rosengren and Khaled Greish
20
To improve the efficacy of RL71, SMA micelles were used as a drug carrier because of
their amphiphilic nature and ability to improve the pharmacokinetics of the drug. Different
loadings of SMA-RL71 (5, 10, and 15%) were prepared and their physiochemical
characteristics were assessed. The cytotoxicity of SMA-RL71 was also compared with free
RL71 in different TNBC cell lines. The micelles had a near neutral zeta potential and 15%
loading had a slower release rate, and higher cellular uptake. In TNBC cell lines, 15% SMA-
RL71 was more stable than free RL71 and elicited a higher cytotoxicity with an IC50 value of
0.54 µM in MDA-MB-231 cells. The 15% SMA-RL71 was also cytotoxic in a tumour
spheroid model. Overall, 15% SMA-RL71 showed characteristics favourable for preclinical
studies in xenograft models [118].
Hydrazinocurcumin, a curcumin analogue described in earlier sections, was also
encapsulated in a nanoparticle (HC-NP) and tested for its effect on the tumour
microenvironment in breast cancer cell lines and animal models. The local tumour
microenvironment can recruit and program tumour associated macrophages (TAMs) to
transform into a tumour initiating phenotype called M2 having the following expressions: IL-
10 (high), IL-12 (low), and TGF-β (high). STAT3 signalling plays a crucial role in
malignancy through crosstalk between tumour cells and TAMs. Co-culture of 4T1 mouse
breast cancer cells with RAW264.7 cells resulted in a transformation from M1 to M2
phenotype. The RAW264.7 cells referred in this step as ‘educated’ (E-RAW264.7) were re-
educated (RE-E- RAW264.7) when treated with 18 µM of HC-NP, resulting in the
transformation of M2 phenotype to M1 phenotype macrophages with the following
characteristics: IL-10 (low), IL-12 (high), and TGF-β (low). Cell migration and invasion were
affected by a decrease in the expression of pSTAT3, MMP-9, MMP-2, and VEGF. The
treatment also resulted in a decrease in the number of cells in the S-phase (38%) compared to
control (54%). Treatment of BALB/c female mice with 1 mM of Legumain-targeting-HC-
NPs (Leg-HC-NPs) 10 days after tumour induction, at 3 day intervals within 15 days resulted
in a significant decrease in tumour weight by approximately 71% and an increase in survival
by more than 2 months. Immunohistochemical analysis showed that the percentage of Ki67-
positive cells in the Leg-HC-NPs group was approximately 18, compared to 68 for control,
while the percentage of STAT3-positive cells was approximately 15, compared to 50 for
control. Furthermore, there was a 70% decrease in CD31 positive microvessel density in the
Leg-HC-NPs group compared to control. Moreover, Leg-HCNPs-treated mice had a 3-fold
decrease in pulmonary metastasis when compared to control [119].
VI. CLINICAL TRIALS
There have been a limited number of clinical trials examining curcumin in breast cancer
patients. However, the studies conducted so far in healthy volunteers and breast cancer
patients show that curcumin has minimal toxicity and is well tolerated. Specifically, Lao et al.
investigated the maximum tolerated dose, and pharmacology and toxicology of curcumin in
healthy volunteers. Dose escalation ranged from 500 mg up to 12,000 mg. Only seven of the
twenty-four volunteers exhibited grade I toxicity with symptoms such as diarrhoea, headache,
nausea, rash, and yellow stools. Curcumin was undetectable in serum until 8,000 mg.
Curcumin was detected in serum of two volunteers administered with 10,000 and 12,000 mg.
Curcumin Analogues for the Treatment of Breast Cancer Revisited
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The study concluded that a single oral dose of 12,000 mg was well tolerated and warranted
further investigations [121]. Another phase I clinical trial in 25 patients with high-risk or pre-
malignant lesions demonstrated that curcumin was safe up to 8,000 mg/day orally for 3
months. Peak serum concentration of curcumin was detectable 1-2 h after oral intake of
curcumin. Moreover, histological improvement of pre-cancerous lesions was seen after
administration of curcumin for 3 months [122]. Curcumin was also investigated as a
combinational therapy along with docetaxel in patients with advanced and metastatic breast
cancer. In the phase I clinical trial with 14 patients, docetaxel was administered as a 1 h-
perfusion, 100 mg/m2 every 3 weeks for six cycles. Curcumin was administered orally from
500 mg/day, followed by dose escalation for seven consecutive days for six cycles. The dose
limiting toxicities were diarrhoea and neutropenia. The combinational therapy was as safe
compared to docetaxel treatment alone, with no reported haematological toxicities or febrile
aplasia. Furthermore, the combinational therapy elicited anti-tumour activity leading to
improvements in biological and clinical responses. Biological response included changes in
the level of tumour markers CEA, CA15.3 and anti-angiogenic marker VEGF after six cycles
of treatment in eight patients with measurable lesions. Although no significant decrease was
observed in the levels of CA15.3, a significant decrease in CEA levels was observed from the
third cycle (approximately 30% decrease from the baseline).
Table 2. Summary of completed, active and yet to be completed
clinical trials with curcumin in breast cancer patients
Investigation Status Result Reference
Pilot study of curcumin for
women with obesity and high
risk for breast cancer using a
nanoemulsion formulation.
Recruiting - [126]
Phase II study of curcumin vs
placebo for chemotherapy-
treated breast cancer patients
undergoing radiotherapy to
determine if curcumin reduces
NFκB DNA binding in such
patients.
Not yet
open for
recruitment
- [127]
Curcumin for the treatment of
radiation-induced dermatitis in
breast cancer patients.
Completed 6 g of oral curcumin daily
during radiotherapy reduced the
severity of radiation-induced
dermatitis in breast cancer
patients.
[124]
Phase I dose escalation clinical
trial of docetaxel and curcumin
for advanced or metastatic
breast cancer.
Completed Curcumin at 6,000 mg/day for
seven consecutive days every 3
week, along with a standard
dose of docetaxel elicited anti-
tumour activity with no major
toxicities.
[123]
Vignesh Sundararajan, Rhonda J. Rosengren and Khaled Greish
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The anti-angiogenic effect of the combinational treatment was investigated by measuring the
levels of VEGF, which was significantly decreased after three cycles of combinational
treatment (30% decrease below the baseline and 21% decrease after the sixth cycle). Clinical
responses included partial response after six cycles in three patients and no residual tumour in
a patient with stable disease after six cycles of treatment. Other responses included stable
pulmonary lesions accompanied by decrease in tumour markers. From the clinical trial,
curcumin was recommended at 6,000 mg/day for seven consecutive days every 3 weeks,
along with a standard dose of docetaxel [123].
Curcumin has also been investigated as a drug to treat radiation-induced skin damage in
breast cancer patients [124]. Radiotherapy (RT) is commonly used in the management of
breast cancer and a major side effect is radiation-induced inflammatory reactions in the skin
[125]. In a randomised, double-blind, placebo-controlled clinical trial with 30 non-
inflammatory or carcinoma in situ breast cancer patients undergoing RT without
chemotherapy , a dose of 2 g of curcumin orally three times a day was investigated. RT was
given for four to seven weeks and patients were administered either curcumin capsules or
placebo capsules containing dicalcium phosphate. Oral curcumin at 6 g/day significantly
reduced the extent of radiation dermatitis and moist desquamation. It was well tolerated
without any major clinical events being reported [124]. Table 2 summarises the list of active,
completed and yet to be completed clinical trials with curcumin for breast cancer.
CONCLUSION
There is no doubt that curcumin has potent anti-cancer activity in in vitro as well as
animal models of breast cancer. However, drawbacks associated with curcumin such as poor
bioavailability and rapid metabolism hinder its development as a potential anti-cancer drug.
Extensive studies with curcumin have provided key information about its structure to
chemically modify the parent compound in order to produce more potent curcumin analogues.
Along with this, advances in drug delivery systems have enabled the delivery of curcumin
and its analogues selectively to tumour tissues using nanotechnology. This has helped
overcome the inherent problems associated with curcumin. It is expected that in the near
future, either curcumin in a nano form or curcumin analogues in either a free or nano form
will emerge as potential adjuvant drug candidates for treating aggressive and metastatic breast
cancer.
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