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Introduction
Mustard gas, the first used drug for cancer chemo-therapy, was used as a chemical warfare agent during World War I and
was reported to cause severe toxic effects, one being the suppression of bone marrow leading to low white blood cell
counts[1]. Studies thereafter with other forms of mustards confirmed the antineoplastic action of nitrogen mustard and thus
launched the new era of cancer
chemotherapy[1]. The majority of chemotherapeutic drugs can be broadly
classified[2] into: alkylating agents (nitrogen mustards, ethyleneimines, methylmelamines, methylhydra-zine derivatives, alkyl sulfonates,
nitrosoureas, triazenes, and platinum compounds); antimetabolites (folic acid, pyrimidine, and purine analogs); natural
products (vinca alkaloids, taxanes, epipodophyllotoxins,
camptothecins, anthracycline antibiotics, and anthracenedione); hormones and antagonists (adrenocortical
suppressants, adrenocorticosteroids, progestins, estrogens,
anti-estrogens, androgens, anti-androgens,
gonadotropin-releasing hormone analogs, and aromatase inhibitors);
biological response modifiers; differentiating agents;
proteasome inhibitors; substituted urea; tyrosine kinase
inhibitors; and monoclonal antibodies. This review intends
to focus on the combination studies (both in vitro
and in vivo) of silibinin, an established cancer chemo-preventive
agent with the 2 classes of antineoplastic agents: the
platinum compounds belonging to the class of alkylating agents
and the anthracycline antibiotics.
Alkylating agents
The chemotherapeutic and cytotoxic effects of
alkylating agents are directly related to the formation of carbonium
ion intermediates or related transition complexes that
alkylate DNA[3,4]. The potential of these drugs to induce cell
death via interference with DNA integrity in fast
proliferating tissues forms the basis for their therapeutic and toxic
properties[3,4]. Platinum complexes do not alkylate DNA;
however, they covalently bind to nucleophilic sites on DNA
and share many pharmacological attributes to the alkylating
agents and are thus included in this
group[3,4]. In 1965, the platinum coordination complexes were first identified as
potential antiproliferative agents based on a study which
showed that a current delivered between platinum electrodes
resulted in the inhibition of Escheria coli
proliferation[3]. The inhibition of bacterial replication was later related to the
formation of inorganic platinum-containing compounds in
the presence of ammonium and chloride
ions[3]. After this discovery, many platinum-containing compounds were
synthesized and tested against different experimental tumor
systems. Cisplatin and other members of the group, like
carboplatin and oxaliplatin, have shown great clinical
efficacy[5]. The platinum agents have broad antineoplastic
activity against epithelial malignancies and have been used
for the treatment of head and neck, bladder, esophagus, lung,
colon, testicular, and ovarian
cancers[4]. The platinum complexes inhibit telomerase (a specialized DNA polymerase
expressed in malignant cells that mediates telomere extension
at the 3' ends of telomere DNA) activity, react with DNA to
form both intrastrand and interstrand cross-links, and the
DNA adducts so formed inhibit DNA replication and
transcription and lead to breaks and
miscoding[4]. These
adducts are recognized by p53 and other checkpoint
proteins and result in the induction of
apoptosis[5]. Malignant cells with mutant or absent p53 fail to arrest cell cycle
progression and do not undergo apoptosis and are thus
resistant to these drugs[5]. Other mechanisms that contribute to
the resistance of the tumor to the platinum drugs are a
reduction in drug accumulation in cancer cells due to barriers across
the cell membrane, drug inactivation, alterations in drug
target, and faster removal of the DNA
adducts[4,5]. The cisplatin adducts are removed by nucleotide exchange repair,
homologous recombinant repair, and translesion synthesis,
which serve as cellular defense mechanisms against the toxic
effects of the platinum drugs and thus contribute to tumor
resistance to cisplatin[5].
Mismatch repair (MMR) increases the cytotoxicity of the
cisplatin in tumor cells by the binding of the MMR complex
to DNA adducts; dysfunction of this type of DNA repair
also results in increased resistance of tumor cells to this
drug[5]. The DNA adducts of cisplatin have been also shown
to inhibit the anti-apoptotic function of NF-κB by inhibiting
the binding affinity of NF-κB to the specific DNA
elements[5]. Thus, NF-κB activation/inhibition is involved in the
resistance/sensitivity of tumor cells to cisplatin. Carboplatin
shows cross-resistance with cisplatin and forms similar types
of DNA adducts, but with different sequence
preferences[5].
The adverse reactions of cisplatin include renal toxicity,
gastrointestinal toxicity, peripheral neuropathy, and
ototoxicity[3]. Carboplatin is less toxic to kidneys and the nervous
system; however, hematological adverse effects are more
common[3].
Anthracyclines
The anthracycline antibiotics, another class of
chemotherapeutic agents, are derived from the
fungus Streptococcus
peucetius[6,7]. They have a tetracyclic ring structure
attached to the sugar, daunosamine[6,7]. Cytotoxic agents in
this class have quinone and hydroquinone moieties on
adjacent rings that permit the exchange of
electrons[6,7]. These compounds intercalate with DNA and directly affect
transcription and replication[6,7]. These drugs form a tripartite
complex with DNA and topoisomerase II (an
ATP-dependent enzyme that binds to DNA and produces double-strand
breaks at the 3' phosphate backbone, allowing strand
passage and the uncoiling of supercoiled DNA, after which it
religates the DNA strands). The formation of the tripartite
complex with anthracyclines inhibits the religation of the
broken DNA strands and leads to
apoptosis[2,7]. Thus, the defect in DNA double-strand break repair mechanisms
results in cell damage causing cell cycle arrest and apoptotic
death; however, an overexpression of transcription-linked
DNA repair contributes to resistance to these
drugs[7].
In addition to be topoisomerase II poison, the
chemotherapy drugs in this class also possess potential for
generating free radicals (by interaction with iron), thereby causing
irreversible cardiomyopathy[8], which is related to the total
dose of the drug. Doxorubicin also produces formaldehyde
in iron-mediated free radical reactions; formaldehyde and
doxorubicin then react to form a conjugate which
intercalates with DNA by a process referred to as "virtual cross
linking"[7]. Treatment with doxorubicin also results in
malondialdehyde formation and DNA intercalation-oxopropenylation, which results in DNA
damage[7]. Doxorubicin also activates p53_DNA binding which causes the
induction of Cip1/p21 and results in the
G1 arrest of p53-proficient
cells[7]. The overexpression of p21 is also
associated with resistance to doxorubicin as it is associated with
increased G1 arrest that facilitates DNA repair before the cells
undergo DNA replication[7]. The p53-deficient cells, not
arrested at the G1 phase, progress through to the S phase
where the expression of α-isoform of topoisomerase II is
increased during DNA synthesis; these cells become
sensitive to doxorubicin[7].
Another mechanism of resistance to doxorubicin involves
the role of proteasome. Doxorubicin is transported to the
nucleus via the complex formation with proteasome in the
cytoplasm, thus in cells with increased nuclear
sequestration of proteasome, there are reduced levels of transported
doxorubicin and these cells show resistance to
doxorubicin[7]. Doxorubicin causes the activation of
NF-κB in cancer cells, which in turn inhibits apoptosis induced by doxorubicin;
cells with increased activity of NF-κB are thus resistant to
doxorubicin[7].
The clinical use of the drugs in this class is thus limited
due to the development of resistance in tumor cells, toxicity
to normal tissues, and adverse cardiac
effects[7,8]. However, it is important to note that daunorubicin and idarubicin are
effective in patients with acute leukemias, whereas
doxorubicin and epirubicin have shown clinical efficacy against
solid tumors in their clinical
manifestations[7,8]. The structurally similar agent mitoxantrone (anthracenedione) is used
in high-dose chemotherapy in advanced
hormone-independent prostate cancer and acute myeloid leukemia. It causes
less cardiac toxicity than the anthracyclines, and its toxicity
includes myelosuppression and
mucositis[9,10].
Anticancer effects via the modulation of cell
cycle checkpoints
In order to understand how the anticancer agents
eventually inhibit tumor cell growth or how a particular chemo
combination would ultimately result in a synergistic effect,
an understanding of cell cycle regulation is essential. The
cell cycle is divided into 2 basic parts: mitosis and interphase.
Mitosis (nuclear division) corresponds to the separation of
daughter chromosomes and ends with cell division (cytokinesis). Interphase involves both cell growth and DNA
synthesis in an orderly manner in preparation for cell
division[11,12].
While the cell grows at a steady rate throughout
inter-phase, DNA is synthesized only during a portion of
interphase[11,12]. Cytokinesis is followed by the
G1 phase that corresponds to the interval between mitosis and the
initiation of DNA replication[11,12]. During the
G1 phase, the cell is metabolically active and continuously grows, but does not
synthesize DNA. G1 is followed by DNA synthesis (S) phase
followed by an interval following the termination of DNA
synthesis (G2) phase, during which cell growth continues
and proteins are synthesized in preparation for mitosis and
then the mitotic (M) phase[11,12]. Different cellular processes,
such as cell growth, DNA replication, and mitosis, are
coordinated during cell cycle progression by a series of
checkpoints and feedback controls that regulate progression
through various phases of the cell cycle and prevent entry
into the next phase of the cell cycle until the events of the
preceding phase have been completed[13].
There is a major cell cycle regulatory point that controls
progression from G1 to S, and having passed this regulatory
point, the cells are committed to entering the S phase and
undergoing 1 cell division cycle even in the absence of
further growth factor stimulation[14]. If appropriate growth
factors are not available in G1, progression through the cell
cycle stops at this point and the arrested cells then enter a
quiescent stage of the cell cycle called
G0, in which they are metabolically active but cease growth, have a reduced rate
of protein synthesis, and can remain for a long period of time
without proliferating until they receive external
signals[13,14].
While the proliferation of most cells is regulated in
G1, some cell cycles are controlled in
G2, which prevents the initiation of mitosis until DNA replication is
completed[14,15]. Progression is also arrested at the
G2 checkpoint in response to DNA damage, which allows time for the damage to be
repaired before being passed on to daughter
cells[14,15]. DNA damage not only arrests the cell cycle in
G2, but also arrests cell cycle progression in
G1, which may allow repair of the damage to take place before the cell enters the S phase where
the damaged DNA would be
replicated[13_16]. Arrest at the
G1 checkpoint is mediated by p53, which is rapidly induced in
response to damaged DNA. It is important to emphasize
here that mutations in the p53 gene are most common in
human cancers[16]. Another important checkpoint occurs
toward the end of mitosis, which monitors the alignment of
chromosomes on the mitotic spindle and ensures that the
chromosomes do not separate until a complete complement
of chromosomes has been organized for the distribution of
the daughter cells[12,15].
Cell cycle progression is regulated by the activity of
cyclin-dependant kinases (Cdk) in association with
their regulatory subunits known as cyclins as well as their negative
regulators, which are named Cdk
inhibitors[13]. The activation of
different Cdk is specific to the distinct phases of the
cell cycle; for example, the association of Cdk4/Cdk6 with
D-type cyclins controls the early and
mid-G1 phase progres-sion, whereas
Cdk2_cyclin E regulates late
G1 and G1_S transition, Cdk2_cyclin
A for S-phase progression, and cell division cycle (Cdc)2 (also known as Cdk1)_cyclin B1
for G2_M phase transition (Figure
1)[13]. The activity of Cdk during cell cycle progression is regulated by different
molecular mechanisms, where it first involves the
association of Cdk with their cyclin partners, the formation of
specific Cdk/cyclin complexes being controlled by cyclin
synthesis and degradation[13]. To further elaborate on that as an
example (Figure 1), cyclin B1 is a regulatory subunit required
for the catalytic activity of the Cdc2 protein kinase. Cyclin
B1 synthesis begins in the S phase, it then accumulates and
forms a complex with Cdc2 throughout S and
G2 during which Cdc2 is phosphorylated at 3 regulatory
positions[13_16]. Phosphorylation at threonine-161 by Cdk-activating kinase (CAK)
is required for Cdc2 kinase activity. The inhibitory
phosphorylation of Cdc2 at tyrosine-15 and threonine-14 is
catalyzed by protein kinase Wee1, which inhibits Cdc2 activity
and leads to the accumulation of inactive Cdc2/cyclin B
complexes throughout the S and G2
phases[13_16]. The dephosphorylation of Cdc2 at threonine-14 and tyrosine-15 by
protein phosphatase Cdc25C causes the activation of the
Cdc2/cyclin B complex and results in the transition from the
G2 phase to the M
phase[13_16]. Cell cycle arrest at this
checkpoint is mediated by Chk2 and/or Chk1 kinase, activated by
ataxia telangiectasia mutated (ATM) and ATM and Rad3
related (ATR), respectively, in response to DNA damage or
incomplete replication[15]. Chk1 and Chk2 phosphorylate
protein phosphatase Cdc25C, and thus prevent Cdc25C from
dephosphorylating and activating
Cdc2[15]. In the absence of such activation, however, the progression to mitosis is
blocked and the cell remains arrested in
G2[15]. Once activated, the Cdc2 protein phosphorylates a number of target
proteins that initiate the events of the M
phase[13_16]. The Cdc2 activity also triggers the degradation of cyclin B, which in
turn inactivates Cdc2, leading the cell to exit from mitosis
and undergo cytokinesis[13_16].
Apart from the regulation of their activity by
phosphoryla-tion, the activity of Cdk could also be controlled by the
binding of Cdk inhibitors to Cdk/cyclin complexes (Figure
1)[17]. Members of the Cip/Kip family regulate the progression
through the G1 and S phases by inhibiting the activity of the
complexes of Cdk2/4/6 with cyclins A, D, and E, and the
members of the Ink4 family of Cdk inhibitors are specific for
complexes of Cdk4 and 6 with cyclin D. The Ink4 inhibitors
thus regulate progression only through the G1
phase[13,17]. Furthermore, retinoblastoma (Rb), a tumor suppressor gene
whose inactivation leads to tumor development, is a key
substrate protein of the Cdk4, 6/cyclin D complexes and is
frequently mutated in a variety of human
tumors[18]. Studies have revealed that the activity of Rb is regulated by changes
in its phosphorylation status where it becomes
hyperphos-phorylated by Cdk4, 6/cyclin D complexes as cells pass
through the restriction point in G1 (Figure
1)[18]. In its hypo-phosphorylated form, Rb binds to the members of the E2F
family of the transcription factors and thus acts as a
repressor where the Rb/E2F complex suppresses the transcription
of E2F-regulated genes to drive cell cycle progression to the
next phase[18]. The phosphorylation of Rb by the Cdk4,
6/cyclin D complexes results in its dissociation from E2F; E2F
then causes the activation of genes required for cell cycle
progression[18]. E2F induces cyclin E and then Cdk2/cyclin
E maintains Rb phosphorylation and this creates a positive
feedback loop that is responsible for irreversible
G1_S transition[17,18]. Cyclin A/B-dependent Cdk essential for S phase
progression are subsequently activated and maintain the
hyperphosphorylated form of Rb[17,18]. Upon withdrawal of
mitogens, cyclin D synthesis stops and Cip/Kip proteins are
available to bind to cyclin E/A_Cdk2, which blocks their
catalytic activity and results in cell cycle
arrest[17,18]. At this stage, in response to DNA damage, p53 stimulates the
expression of the Cip1/p21 which inhibits several Cdk/cyclin
complexes and thus inhibits Rb phosphorylation and directly
inhibits DNA replication thereby stopping S phase
progression[15,16,18].
Taking all these basic cell biology concepts together, it
is reasonable to speculate that the efficacy of any anticancer
agent would greatly depend upon its ability to target cell
cycle checkpoints, either by the modulation of the cell cycle
regulatory molecules (by direct or indirect action) thereby
resulting in cell cycle arrest as a primary/ secondary effect or
by targeting the damaged cells towards the end of their
mitotic phase. The inhibition of cell growth is mediated
either by arresting the cells in the G1 or S phase, bypassing
the G2_M arrest via the G2_M checkpoint abrogation that
would drive the cells to apoptosis, or by enhancing the
G2_M checkpoint arrest[19]. The anticancer effects of drugs
favor both the contrasting hypothesis:
G2_M checkpoint abrogation or
G2_M cell cycle
arrest[20,21]. The mechanisms involved in both
G2_M checkpoint abrogation or
G2_M cell cycle arrest might be totally contrasting and conflicting,
however, the endpoint of both these effects has been
increased cytotoxicity[19].
Combined chemotherapy: a synergistic approach with "reduced toxicity"
Most chemotherapeutic drugs effectively target fast
dividing cells causing their damage and are thus also
referred to as "cytotoxic
drugs"[22]. Consistent with this, the
fast growing normal cells, such as those involved in hair
growth and the mucosal lining of intestinal epithelium, are
also effected leading to alopecia and intestinal complications.
In addition, the chemotherapeutic drugs cause a wide
variety of side_effects, like nausea, vomiting, anemia,
malnutri-tion, immunosuppression, myelosuppression, hemorrhage,
cardiotoxicity, hepatotoxicity, nephrotoxicity, ototoxicity, and
non-specific neurocognitive
problems[22].
The above listed toxic manifestations clearly suggest that
the dosage of the drug is a critical factor; if the dose is too
low, it will not be effective against the tumor, while at high
doses the toxicity signs will be effective. Accordingly, the
dosing schemes have been generated wherein the dose is
adjusted according to the patient's body surface area and
blood volume[22]. Additionally, a number of strategies are
being used in the administration of chemotherapeutic drugs,
which include[22]: (1) combined modality chemotherapy that
involves the use of drugs with other cancer treatments, like
surgery and radiation therapy; (2) neoadjuvant chemotherapy
or preoperative treatment where preceding surgery or
radiation therapy, chemotherapeutic drugs are given to reduce
the tumor size and make the local therapies more effective;
(3) adjuvant chemotherapy or postoperative treatment, which
involves continued drug intake even after surgery to reduce
the risk of recurrence; (4) palliative chemotherapy that
involves drug dosing only for decreasing the tumor load; it
does not have any curative effects, but is expected to
increase life expectancy slightly; and (5) combination
chemotherapy, which involves treating a patient with a
number of different drugs simultaneously.
In designing the specific regimens of combination
chemotherapy for clinical use, a number of factors need to be
considered; drugs should be most effective in combination,
that is, their molecular/biochemical interactions should
result in a synergistic response and thus it is more effective
to combine drugs that do not share common mechanisms of
activity and resistance and do not overlap in their major
side-effects. In an effort to develop effective strategies that
increase the therapeutic potential of anticancer drugs with
less systemic toxicity, more strategies are being directed
towards the investigation of dietary supplements and other
phytotherapeutic agents (known for their high anticancer
efficacy and low toxicity to normal tissues) for their
synergistic efficacy in combination with anticancer
drugs[23,24].
One such dietary agent is silibinin, which is a
polyphenolic flavonoid isolated from the seeds of milk thistle
(Silybum marianum). Both silibinin and its cruder form, known as
silymarin, have clinically established hepatoprotective
activity and have been used as dietary supplements against
liver toxicity for over 3 decades[25]. Silibinin has shown
promising chemopreventive and anticancer effects in various
in vitro and in vivo
studies[26_29]. Studies conducted by us and
others have shown that silibinin and silymarin have
anticancer activity against breast, skin, androgen-dependent
and-independent prostate, cervical, bladder, hepatocellular,
colon, ovarian, and lung cancer cells in culture and several
in vivo animal cancer model
systems[26]. The administration of these compounds to various laboratory animals has not
shown any toxic or adverse effects in various acute,
sub-chronic and chronic tests; there is no known
LD50 for silymarin and silibinin in these
animals[30,31]. This is also supported by
our in vivo studies in animal models where we have used
high doses of silibinin, up to 2 g/kg per d by oral gavage or
1%(w/w) in the diet, and observed no apparent signs of toxicity[27,29].
Combination studies of silibinin with chemo-therapeutic agents
Several in vitro and in vivo combination studies of
silibinin and chemotherapeutic drugs were carried out to
analyze the effects of such a combination on growth
inhibition, cell cycle regulation, and apoptosis. As
summarized below, the 3 most important cancer systems, namely
prostate, breast, and lung, have been investigated in
detail-ed efficacy and mechanistic studies employing silibinin
combination with chemotherapy agents (Figure 2).
Silibinin and chemo combination in prostate cancer
Studies by our group have shown that silibinin possesses
strong anticancer efficacy against both androgen
dependent and independent prostate carcinoma (PCa) cells in cell
culture, where it inhibited cell growth and induced cell cycle
arrest in LNCaP, PC-3, and DU145
cells[32_34]. Mechanistic studies conducted by us have also shown that the
anticancer efficacy of silibinin against PCa cells is associated with
the induction of differentiation morphology, a reduction in
the prostate specific antigen level, the induction of cell cycle
arrest accompanied by an increase in Cdk inhibitors, the
inhibition of Cdk kinase activity, a decrease in the
phosphorylation of Rb and Rb-related proteins, and their increased
interaction with the E2F family of transcription
factors[28]. We have also observed that silibinin inhibits the
in vivo growth of DU145 xenografts in athymic nude mice mediated in part
by an induction in the insulin-like growth factor-binding
protein-3 level, the inhibition of proliferation and angiogenesis,
and an induction in apoptosis[35]. Together these
in vitro and in vivo studies clearly indicate that silibinin has strong
efficacy against PCa, and therefore, our next goal was to
assess whether synergistic action against PCa growth also
exists when silibinin is used in combination with
chemotherapeutic drugs.
Our chemo combination studies (Figure 2) showed that
silibinin sensitizes the hormone refractory DU145 prostate
carcinoma cells to cisplatin- and carboplatin-induced cell
growth inhibition and apoptotic
death[36]. The in vitro
effects of silibinin and the platinum compounds, cisplatin (2
µg/mL) and carboplatin (20 µg/mL), on DU145 cells
were
assessed either alone or in combination. The findings of
these studies revealed that the 48% cell growth inhibition
observed with cisplatin increased to 50%_100%
(P<0.05_0.001) when used in combination with 50_100 µmol/L silibinin.
Similarly, the growth inhibition by carboplatin increased from
68% to 80%_90% (P<0.005_0.001) when used in
combination with 50_100 µmol/L silibinin. The combination also
resulted in a stronger G2_M arrest accompanied by a
decrease in the expression levels of Cdc2, cyclin B1, and
Cdc25C, which as earlier described, are essential for the
transition from the G2 phase to the M phase of the cell cycle.
Furthermore, compared to the effect of the single agent alone,
there was a moderate increase in the Chk1 and Wee1 protein
levels.
A similar chemo combination of silibinin and
doxorubicin (Figure 2) also showed a strong synergism in the
growth inhibitory effects in DU145 cells (combination
index [CI]: 0.23_0.58). In this study, a dose-dependant
effect of silibinin (10_100 µmol/L) and doxorubicin (10_100
nmol/L) on growth inhibition was determined followed by a
combination of different doses of silibinin and doxorubicin,
which resulted in a stronger synergistic growth inhibition[37]. Among the combinations studied, a combination of
100 µmol/L silibinin and 25 nmol/L doxorubicin was most
effective and showed an increase in growth inhibition of up
to 80% compared to 43% and 53% when either of the agents
were used alone. Further studies with this combination
revealed that the synergistic effect was more profound when
the cells were pretreated with either of the agents for 24 h
followed by cotreatment with the other agent for another
24 h. Both cotreatments and simultaneous treatments showed
an induction of G2_M arrest. Thus, regardless of the
treatment regimen, the combination of silibinin and doxorubicin
caused a strong G2_M arrest compared to
G1 arrest by silibinin or a moderate
G2_M arrest by doxorubicin alone. This
G2_M arrest was also accompanied by a decrease in the expression
levels of Cdc25C, Cdc2, and cyclin B1, together with a
decrease in Cdc2 kinase activity and a moderate increase in
Chk1/2 levels. There was also a ~3-fold increase in apoptosis
when the 2 drugs were used in combination. Similar
combination studies were also carried out in LNCaP cells, an
androgen-dependant PCa cell line[37]. However, the
synergistic effect was more profound in DU145 cells (the advanced
prostate cancer cells) compared to the androgen-dependant
LNCaP cells (Figure 2). Together, these studies showed that
silibinin strongly synergizes the therapeutic effect of
doxorubicin in advanced human prostate carcinoma DU145 cells
via an induction of G2_M arrest and apoptotic cell death.
Combination studies of silibinin with docetaxel and
mitoxantrone were also carried in PC-3, DU145, and LNCaP
prostate cancer cell lines (Figure
2)[38]. A combination of silibinin (10_40 µmol/L) and docetaxel (2.5_5 nmol/L)
exhibited little or no synergy in growth inhibitory and apoptotic
effects in PC-3, DU145, and LNCaP cells, however, silibinin
was able to overcome the relative resistance of PC-3 cells to
mitoxantrone (25_200 nmol/L)-induced apoptosis. In this
study, the combination of silibinin and mitoxantrone was
also able to exhibit a synergistic action for apoptosis
induction in DU145 and LNCaP cells and caused a synergistic
decrease in the cell viability of all 3 cell lines.
Recently, we conducted a phase I clinical trial with silibinin
in prostate cancer patients, in which 13 g of oral
silybin_phytosome daily in 3 divided doses was administered to
patients with advanced prostate cancer; each course was 4
weeks in duration[26]. An important observation in this study
was that 100 µmol/L plasma level of free silibinin was achieved
in the patients. This observation is highly significant with
regards to the achievement of the physiological levels of a
dose that was found to be highly effective in various
in vitro studies including chemo combinations as discussed earlier.
Silibinin and chemo combination in breast cancer
We also assessed the synergistic effect of silibinin with
chemotherapy agents in estrogen-dependant MCF-7 and
-independent MDA_MB468 human breast carcinoma cell lines
(Figure 2)[39]. In these cell lines, extensive dose- and
time-dependant studies were initially carried out with the single
agents followed by several combinations (10_100 µmol/L) of
silibinin with doxorubicin (10_75 nmol/L), cisplatin (0.2_2
µg/mL), and carboplatin (2_20 µg/mL) to assess their
synergistic/additive or antagonistic effect towards cell growth
inhibition and apoptotic death. Combinations of these 3 drugs
with silibinin showed strong synergistic effects on cell
growth inhibition in both the cell lines, however, the
strongest effect was due to a combination of 75 nmol/L
doxorubicin and 100 µmol/L silibinin (CI: 0.35 for MCF-7 cells and CI:
0.45 for MDA_MB468 cells). A combination of silibinin and
doxorubicin also showed strong apoptotic death in both the
cell lines. The silibinin and carboplatin combinations
increased the apoptotic effect in MCF-7 cells only, while the
combination with cisplatin did not show any additional
apoptotic effect in any of these cell lines.
Silibinin and chemo combination in lung cancer
Studies conducted by our group have also shown that
silibinin strongly inhibits growth and induces apoptotic
death in both human small cell lung carcinoma (SCLC) and
non-small cell lung carcinoma (NSCLC) cells together with
an alteration in cell cycle
checkpoints[40]. Silibinin caused an increase in the
G0_G1 population in SCLC SHP77 cells and an
increase in the S phase in NSCLC A549 cells at lower
treatment times followed by an increase in the
G0_G1 phase at 72 h. Further studies in A549 cells showed that a combination
of 25 nmol/L doxorubicin and 60 mmol/L silibinin increases
cell growth inhibition to 85%, which was significantly higher
than that caused by either agent alone (Figure
2)[41]. The combination also resulted in a
~37-fold increase in apoptosis, suggesting a synergistic effect on apoptotic cell death as
well. In a follow up of these cell culture studies, we have
also shown that oral feeding of silibinin (200 mg/kg body
weight, 5 d/week for 33d) inhibits tumor growth, cell
proliferation and angiogenesis and induces apoptosis in A549
tumor xenografts in nude mice. There was also a synergistic
effect when silibinin was used in combination with
doxorubicin (4 mg/kg body weight, intraperitoneally once a week
on d 1, 8, 15, and 22; a total of 4 doses). Furthermore, there
was also a reduction in doxorubicin-induced systemic
toxicity in mice when the drugs were used in
combination[41]. Other studies have also shown that there is an activation of
NF-κB by doxorubicin, which was possibly responsible for
the resistance of A549 cells to this
drug[42,43]. Based on the results of our earlier studies where silibinin sensitized DU145
cells for TNFα-induced apoptosis by inhibiting
NF-κB activation via an inhibition of IKKα kinase activity, which led to
the increase in the cytoplasmic IκBα level and ultimately
inhibited the nuclear translocation of the p65 and p50
subunits of NF-κB[44], we assumed that silibinin would also
reverse the NF-κB-based chemoresistance of A549 cells to
doxorubicin. We confirmed this assumption when the
combination treatment of cells with doxorubicin and silibinin was
able to decrease the activation of NF-κB by doxorubicin
together with the retention of p65 and p50 in the cytosol by
silibinin, which in turn significantly reduced the
doxorubicin-induced resistance in A549 cells (Figure
2)[41]. The chemo combination with silibinin thus enhanced the therapeutic
efficacy of doxorubicin towards the inhibition of lung tumor
growth.
Conclusion
In the unfortunate scenario where the most effective
cytotoxic drugs face limited clinical applications due to their
high toxicity and increased chemoresistance, combination
therapy/prevention is gaining increased attention as an
effective alternative to increase therapeutic efficacy and
minimizes the systemic toxicity of these chemotherapeutic agents.
In this regard, the present review summarizes the effects of
the combination of silibinin and chemotherapeutic drugs on
growth inhibition, cell cycle regulation, and apoptosis
induction in prostate, breast, and lung cancer cells. Together,
the results indicate a synergistic effect of silibinin on growth
inhibition, the reversal of chemoresistance, apoptosis
induction, and a strong increase in G2_M checkpoint arrest
when given in combination with these drugs. These results
are highly significant with respect to the combined
chemotherapy approach where the criteria for combination are that
the response has to be synergistic and that the drugs should
not share common mechanisms of resistance and not
overlap in their major side-effects. Accordingly, we emphasize
that silibinin is an ideal candidate to be used in clinical
combination with the chemotherapeutic drugs which would
increase their therapeutic efficacy and at the same time reduce
the adverse effects associated with their high doses.
How-ever, more extensive studies in this direction are needed in
the near future, not only with silibinin, but also several
additional non-toxic and naturally-occurring cancer
chemopreventive agents to justify their potential clinical
application in combination chemotherapy.
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