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Cervical cancer is one of the most common cancers among women worldwide. Current standards of care for cervical cancer includes surgery, radiation, and chemotherapy. Conventional chemotherapy fails to elicit therapeutic responses and causes severe systemic toxicity. Thus, developing a natural product based, safe treatment modality would be a highly viable option. Curcumin (CUR) is a well-known natural compound, which exhibits excellent anti-cancer potential by regulating many proliferative, oncogenic, and chemo-resistance associated genes/proteins. However, due to rapid degradation and poor bioavailability, its translational and clinical use has been limited. To improve these clinically relevant parameters, we report a poly(lactic-co-glycolic acid) based curcumin nanoparticle formulation (Nano-CUR). This study demonstrates that in comparison to free CUR, Nano-CUR effectively inhibits cell growth, induces apoptosis, and arrests the cell cycle in cervical cancer cell lines. Nano-CUR treatment modulated entities such as miRNAs, transcription factors, and proteins associated with carcinogenesis. Moreover, Nano-CUR effectively reduced the tumor burden in a pre-clinical orthotopic mouse model of cervical cancer by decreasing oncogenic miRNA-21, suppressing nuclear β-catenin, and abrogating expression of E6/E7 HPV oncoproteins including smoking compound benzo[a]pyrene (BaP) induced E6/E7 and IL-6 expression. These superior pre-clinical data suggest that Nano-CUR may be an effective therapeutic modality for cervical cancer.
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Scientific RepoRts | 6:20051 | DOI: 10.1038/srep20051
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Curcumin Nanoformulation for
Cervical Cancer Treatment
Mohd S. Zaman
1
, Neeraj Chauhan
1
, Murali M. Yallapu
1
, Rishi K. Gara
1
, Diane M. Maher
2
,
Sonam Kumari
1
, Mohammed Sikander
1
, Sheema Khan
1
, Nadeem Zafar
3
, Meena Jaggi
1
&
Subhash C. Chauhan
1,3
Cervical cancer is one of the most common cancers among women worldwide. Current standards of care
for cervical cancer includes surgery, radiation, and chemotherapy. Conventional chemotherapy fails
to elicit therapeutic responses and causes severe systemic toxicity. Thus, developing a natural product
based, safe treatment modality would be a highly viable option. Curcumin (CUR) is a well-known
natural compound, which exhibits excellent anti-cancer potential by regulating many proliferative,
oncogenic, and chemo-resistance associated genes/proteins. However, due to rapid degradation
and poor bioavailability, its translational and clinical use has been limited. To improve these clinically
relevant parameters, we report a poly(lactic-co-glycolic acid) based curcumin nanoparticle formulation
(Nano-CUR). This study demonstrates that in comparison to free CUR, Nano-CUR eectively inhibits cell
growth, induces apoptosis, and arrests the cell cycle in cervical cancer cell lines. Nano-CUR treatment
modulated entities such as miRNAs, transcription factors, and proteins associated with carcinogenesis.
Moreover, Nano-CUR eectively reduced the tumor burden in a pre-clinical orthotopic mouse model
of cervical cancer by decreasing oncogenic miRNA-21, suppressing nuclear β-catenin, and abrogating
expression of E6/E7 HPV oncoproteins including smoking compound benzo[a]pyrene (BaP) induced
E6/E7 and IL-6 expression. These superior pre-clinical data suggest that Nano-CUR may be an eective
therapeutic modality for cervical cancer.
Cervical cancer is one of the most common and deadly cancers among women worldwide and is associated with
persistent Human Papillomavirus (HPV) infection
1
. Only a small subset of women with chronic HPV infection
progresses to develop the disease
2
. Additional factors are needed to acquire an immortal phenotype and to fur-
ther advance towards malignant and invasive phenotypes
3,4
. In addition to HPV infection, cigarette smoking and
smoke carcinogen (benzo[a]pyrene, BaP), are known risk factors associated with cervical cancer
2,5
. Viral mor-
phogenesis is increased subsequent to BaP treatment of cells infected with the high-risk HPVs, 31, 16 and 18 in
organotypic ra cultures derived from a cervical intraepithelial neoplasia type I cell line
6
. Moreover, micro RNAs
(miRNAs), small noncoding RNAs that regulate the expression of protein-coding genes, also play an impor-
tant role in the development of carcinogenesis. Resistance to chemo/radio-therapies with prolonged treatment,
resulting in an invasive form of cancer, requires the development of novel therapeutic modalities to conquer
chemo-resistance and improve the overall life expectancy of patients.
Curcumin (CUR) is a natural polyphenol compound that is derived from the rhizome of the medicinal plant
Curcuma longa Linn. It has been widely used in traditional Indian medicine for its ecacy against inammation,
respiratory diseases and other disorders
7,8
. Due to its anti-inammatory and anti-carcinogenic qualities, it has
also been extensively studied in the eld of cancer therapeutics. CUR has shown dose-dependent chemopreven-
tive and chemotherapeutic eects in a number of studies and pre-clinical trials
9,10
. Curcumin exhibits cytotoxic
eects in cervical cancer cells in a concentration-dependent and time-dependent manner and its activity was
found to be higher in HPV infected cells
11
. Curcumin has been proven to downregulate HPV18 transcription by
selectively inhibiting AP-1 activity, which reverses the expression dynamics of c-fos and fra-1 in cervical cancer
cells
11
. Superior inhibitory action of curcumin against cervical cancer cells
12–14
was due to the inhibition of tel-
omerase activity, Ras, and ERK signaling pathways, cyclin D1, COX-2 and iNOS activity, and the mitochondrial
pathway. Interestingly, curcumin acted upon multiple targets and due to pretreatment was in turn able to revert
1
Department of Pharmaceutical Sciences and Center for Cancer Research, University of Tennessee Health Science
Center, Memphis, Tennessee, 38163, USA.
2
Cancer Biology Research Center, Sanford Research, Sioux Falls,
South Dakota, 57104, USA.
3
Department of Pathology, University of Tennessee at Memphis, Memphis, TN, USA.
Correspondence and requests for materials should be addressed to S.C.C. (email: schauha1@uthsc.edu)
Received: 04 November 2015
Accepted: 23 December 2015
Published: 03 February 2016
OPEN
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Scientific RepoRts | 6:20051 | DOI: 10.1038/srep20051
the proliferative eects of cervical cancer cells. A recent proteomic study suggests that curcumin induces signif-
icant changes in tumor-related proteins that are associated with cell metabolism, cell cycle, and carcinogenicity
in HeLa cells
15
. Additionally, curcumin acts as a sensitizer for chemotherapy and radiation in cervical cancer
therapy. We reported a detailed cellular suppressive mechanistic role of curcumin in a three-dimensional cervi-
cal cancer ra culture system
5
. Our study demonstrated that curcumin inhibited cell motility, induced apopto-
sis, decreased the expression of HPV oncoproteins, and restored tumor suppressor proteins. At present, about
55 clinical trials are listed on clinicaltrial.gov related to curcumin and cancer therapy (as of December 2, 2015),
which suggest its translational and clinical potential. Furthermore, it has demonstrated no toxicity to healthy
organs at higher doses such as 8 g/day in clinical trials
16
. However, it suers from very low systemic bioavailability,
poor pharmacokinetics, poor absorption ability, high metabolic rate, inactivity of metabolic products, together
with rapid elimination and clearance from the body
17–19
. Some studies have shown that a trace amount of CUR
was detected in the serum of humans when 4–12 g/day of CUR was administered
17,20
. Although curcumin has
inspired considerable interest for its extensive physiological activities, its poor bioavailability restricts its clinical
translation.
Nanoparticle technology provides an eective way to deliver anti-cancer drug(s) into tumors
21
. To circumvent
curcumins inherent issues, our lab has developed a curcumin nanoparticle formulation (Nano-CUR), (Fig.1A,B)
based on poly(lactic-co-glycolic acid) (PLGA), an FDA approved polymer. is formulation has shown to be
Figure 1. Nano-CUR improves cellular accumulation of curcumin in cervical cancer cells. (A) Structural
components of Nano-CUR formulation. (B) Representative transmission electron microscopy image of Nano-
CUR particles. (C) Fluorescent images of live cells showing increased uptake of CUR/Nano-CUR with increase
in dose. Original magnication 200X. (D) Samples were analyzed by ow cytometry for cellular uptake. Fold
change in mean uorescence normalized to the respective vehicle controls. Error bars show SEM; N = 3, average
of 3 independent experiments, done in triplicate; *p < 0.05.
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eective for improved therapeutic eects in metastatic ovarian and breast cancer cells
22
. e objective of this
study was to implement this ecient Nano-CUR formulation to achieve improved anti-cancerous eects on cer-
vical cancer. Our results indicated that Nano-CUR eectively inhibited growth of cervical cancer cells (Caski
and SiHa), induced apoptosis, and arrested cell cycle in the G1-S transition phase. Moreover, Nano-CUR formu-
lation eectively reduced the tumor burden in NOD SCID gamma (NSG) orthotopic xenogra mouse model.
Additionally, Nano-CUR treatment caused a marked decrease in the levels of miRNA-21 (an onco-miRNA asso-
ciated with chemo-resistance), in in vitro and in vivo models
23,24
, and enhanced the expression of miRNA-214 (a
tumor suppressor)
25,26
, when compared to free CUR, besides decreasing the levels of IL-6 (Interleukin-6) cytokine
expression which was found to be enhanced with BaP treatment. Altogether, these results suggest that Nano-CUR
provides a benecial approach for a rational strategy to widen the chemo-preventive and therapeutic modality for
the overall management of cervical cancer.
Results
Internalization of Nano-CUR in cervical cancer cells. e ecient internalization of drug mole-
cules contributes to their improved anti-cancer activity. To examine this phenomenon, we selected two HPV
infected cervical cancer cell lines (Caski and SiHa) as our in vitro model. SiHa cells have 1–2 copies of integrated
HPV 16
27
. Caski cells are a robust model of HPV because they contain about 600 copies of integrated HPV 16
28
.
Fluorescence microscopy and ow cytometry techniques were used to examine the internalization patterns of
CUR and Nano-CUR. e internalization was found to increase with an increase in the concentration of CUR
and Nano-CUR in Caski and SiHa cervical cancer cells (Fig.1C,D). Fluorescence microscopy analysis revealed
that cells incubated with free CUR exhibited presence of CUR in the periphery and cytoplasm (Fig.1C). Whereas,
in the case of Nano-CUR, internalization was more ecient by endocytosis process due to smaller size, and there-
fore, greater accumulation of CUR was observed on the cell membrane and cytoplasm (Fig.1C). is could be
due to the result of greater interaction of Nano-CUR with the cell surface. is eect might aid internalization of
the drug. A similar improved uptake of Nano-CUR was observed in the ow cytometry analyses (Fig.1D).
Nano-CUR inhibits growth of cervical cancer cells. To assess the anti-cancer potential of CUR and
Nano-CUR in cervical cancer, Caski and SiHa cells were treated with 10, 20, and 25 μ M CUR or Nano-CUR
for 48 hrs. Both cell lines showed an inhibitory eect of CUR/Nano-CUR on cellular proliferation, especially at
higher concentrations (20 and 25 μ M) (Fig.2A). To determine the long-term eect of CUR/Nano-CUR on the
growth of cervical cancer cells, we performed a colony formation assay (Fig.2B,C). Nano-CUR was found to be
more eective as compared to free CUR in reducing the clonogenic ability of cervical cancer cells (Fig.2B,C).
Nano-CUR inhibits cell cycle and induces apoptosis. To elaborate functional eects of CUR and
Nano-CUR on cervical cancer, Caski and SiHa cells were treated with varied concentrations of CUR, Nano-CUR
and their respective vehicle controls (DMSO and PLGA NPs) for 24 hrs as described in Methods. Aer 24 hrs, cells
were trypsinized and processed for ow cytometry analyses for cell cycle and apoptosis. CUR and Nano-CUR
were found to arrest Caski and SiHa cells in the G1-S transition phase (Fig.3A). e eect was more pronounced
at higher concentrations. Nano-CUR was found to be more eective in comparison to free CUR in inducing
apoptosis in Caski and SiHa cells (Fig.3B).
Nano-CUR eectively inhibits tumor growth in NOD SCID gamma (NSG) orthotopic xeno-
graft mouse model. An orthotopic mouse model was developed and used to assess the ecacy of CUR
and Nano-CUR to inhibit tumor growth in NSG mice. For that, Caski cells were injected directly into the cervix
of NSG mice. Following tumor development (~200 mm
3
), the mice were administered with intra-tumoral injec-
tions of CUR, Nano-CUR and their respective vehicle controls (PBST and PLGA NPs, respectively). Figure4A
illustrates the mice treated with various treatment groups. e tumor volumes were measured and presented on
the days shown in Fig.4B,C. At the termination of the experiment, mice were euthanized, interestingly CUR and
Nano-CUR treatment (Fig.4B,C) demonstrated inhibition of tumor growth over their respective control groups.
However, Nano-CUR treatment was found to be more eective than all other treatment groups. Average tumor
volume in all groups are as follows: Nano-CUR (637 ± 68 mm
3
) < CUR (816 ± 94 mm
3
) < PLGA NPs (control
NPs; 1042 ± 166 mm
3
) < PBST (1115 ± 184 mm
3
) (Fig.4B,C).
Nano-CUR eectively represses the expression of HPV oncoproteins in tumor tissues. HPV
oncoproteins, E6/E7, initiate the dysregulation of cellular proliferation and apoptotic mechanisms by targeting
p53 and retinoblastoma (Rb) tumor suppressor proteins, respectively
29
. is leads to oncogenesis and the devel-
opment of cervical carcinoma. Also, higher expression of Ki67, a commonly used marker for cell proliferation, is
associated with high-grade cervical intraepithelial neoplasia (CIN)
30
. Immunohistochemistry done on the tissue
slides made from the orthotopically generated mice tumors treated with CUR/Nano-CUR, and their respective
controls, revealed that Nano-CUR was more eective in suppressing E6/E7 and Ki67 expression levels in mice
tumors when compared with free CUR (Fig.4D).
Nano-CUR efficiently modulated BaP induced expression of miRNAs in cervical cancer
cells. As mentioned earlier, in addition to HPV infection, cigarette smoking and smoke carcinogens, such as
benzo[a]pyrene (BaP), are known risk factors for cervical cancer. Our prior work has shown that BaP increased
the level of HPV oncoproteins, E6/E7
5
. us, Caski cells were treated with BaP alone and in combination with
CUR and Nano-CUR. BaP was found to increase the expression of oncogenic miRNA-21 and decrease the expres-
sion of the tumor suppressor miRNA-214 which was abrogated by CUR/Nano-CUR treatment. is indicates
that CUR and Nano-CUR treatments can mitigate the oncogenic eects of BaP by decreasing the expression of
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miRNA-21 and increasing the expression of the tumor suppressor miRNA-214. Nano-CUR treatment however,
was found to be more eective than free CUR in inducing these eects (Fig.5A,B).
Nano-CUR inhibits BaP induced IL-6 expression in cervical cancer cells. Previous studies have
shown that the IL-6 induces the expression of miRNA-21 in cancer cells
31
. us, we sought to investigate the eect
of BaP on IL-6 in Caski cells. BaP was found to increase the IL-6 expression as compared to its vehicle control.
CUR and Nano-CUR were observed to decrease the oncogenic eects of BaP by decreasing the expression of
IL-6. Again, Nano-CUR was more eective than free CUR at similar concentrations in decreasing IL-6 expression
(Fig.5C).
Restoration of anti-survival pathways aected by BaP through Nano-CUR. miRNA-21 is asso-
ciated with several cancers and involved in chemoresistance and metastasis. It is also responsible for activat-
ing a number of pro-survival pathways
31,32
. To determine mechanisms that are involved in enhanced expression
of miRNA-21, we investigated the modulations of several transcription factors and proteins that are related to
miRNA-21. Western blot studies indicated that BaP treatment induced activation of transcription factors STAT-3
Figure 2. CUR/Nano-CUR inhibit proliferation and clonogenic potential of cervical cancer cells.
(A) Proliferation was determined using MTS method with Caski and SiHa cell lines. Results were normalized
to control wells treated with appropriate amounts of vehicle, DMSO for CUR and PLGA for Nano-CUR.
Error bars show SEM; N = 3, average of 3 independent experiments, done in replicates of 6; *p < 0.05.
(B,C) Clonogenic potential was performed with Caski and SiHa cells. (B) Representative colony formation
images of cervical cancer cells upon treatment. (C) Inhibitory eect of treatments measured by colony counting.
Graphs represent average of three independent experiments, each replicated three times; *p < 0.05.
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and STAT-5 (i.e., increased the phosphorylated form of STAT-3 and STAT-5), and NF-kB (Fig.5D). BaP was
also responsible for decreasing the expression of the anti-survival phosphatase PTEN, a direct target of miRNA-
21
33,34
, in addition to increasing the inactive form of PTEN, phospho-PTEN (p-PTEN)
35,36
. CUR and Nano-CUR
were observed to mitigate the oncogenic eects of BaP by reducing the expression of phosphorylated forms of
STAT-3 and STAT-5, and decreasing NF-κB. Moreover, they also increased the expression of PTEN and reduced
the inactivated form of PTEN, p-PTEN (Fig.5D). CUR and Nano-CUR were also instrumental in decreasing the
cytoplasmic amount of β -catenin, which is a direct target of miRNA-214
32,37
.
In vivo assessment of miRNA-21 and its direct target, PTEN, in mice tissues treated with CUR/
Nano-CUR. To examine the in vivo eects of CUR and Nano-CUR on miRNA expression, we investigated
the expression of miRNA-21 in mice tumors treated with CUR, Nano-CUR and their respective vehicle controls.
In situ hybridization of miRNA-21 in mice tumor tissues showed that CUR and Nano-CUR eectively reduced
miRNA-21 expression as compared to their respective controls. Additionally, immunohistochemical analysis
showed that CUR and Nano-CUR signicantly enhanced the expression levels of PTEN, which is a direct target
for miRNA-21 and a well-known tumor suppressor (Fig.5E).
Nano-CUR reduces BaP and IL-6 enhanced migration of cervical cancer cells. Previous studies
have shown that BaP enhances cell motility through dierent mechanisms in cancer cells
38–40
. BaP alone was
observed to increase cell migration in Caski and SiHa cells. IL-6 further enhanced the motility of the cells in
combination with BaP. CUR and Nano-CUR were found to eectively reduce BaP and IL-6 mediated enhanced
cellular motility of these cells (Fig.6A).
Figure 3. Eect of CUR and Nano-CUR on cell cycle and apoptosis in cervical cancer cells. (A) CUR/Nano-
CUR treatment arrests Caski and SiHa cells at the G1-S transition. Cervical cancer cells were treated with CUR/
Nano-CUR for 24 hrs. Samples were analyzed by ow cytometry for cell cycle analysis. Error show SEM; N = 3,
average of 3 independent experiments, done in triplicate. (B) CUR/Nano-CUR treatment induces apoptosis in
Caski cervical cancer cell line. Flow cytometry was done to detect the early apoptosis (Annexin V +ve) or late
apoptosis (Annexin V +ve and 7AAD +ve).
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Nano-CUR suppresses BaP induced nuclear translocation of β-catenin. β -catenin is a key medi-
ator of Wnt signaling, and its deregulation, resulting in its nuclear accumulation, which in turn can cause cancer
progression and metastasis
41
. In normal cells, β -catenin is anchored at the plasma membrane with E-cadherin,
and is maintained at low levels in the cytoplasm by an APC/axin-dependent degradation complex. Mutations
occurring in β -catenin itself or its degradation complex proteins result in the inability to degrade β -catenin,
which leads to the increase of protein levels of β -catenin that accumulate in the nucleus
42–44
. us it binds to the
members of LEF-1/TCF family of transcription factors, subsequently resulting in stimulation of gene expres-
sion and production of proteins involved in cell transformation
45,46
. erefore, persistent nuclear localization
of β -catenin is the key initiating and driving event for transformation of normal (non-tumor) cell to tumor cell.
Treatment with BaP resulted in an increase in the nuclear translocation of β -catenin in comparison to control as
evidenced through confocal microscopy (Fig.6B). Nano-CUR treatment was found to be more eective than free
Figure 4. Nano-CUR signicantly reduces tumor growth and oncogenic HPV E6/E7 and Ki67 proteins in
cervical cancer orthotopic mouse model. Tumor formation in female nude mice by injecting 4 million Caski
cells. (AC) Nano-CUR was able to reduce the tumor burden by a considerable amount as compared to free
CUR. Error bars show SEM; N = 6; *p < 0.05. (D) Immunohistochemical (IHC) analysis showed that CUR
and Nano-CUR were eective in suppressing the expression of HPV oncogenic proteins E6 and E7 and cell
proliferation marker Ki67 in mice. Original magnication 400X.
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CUR, especially at higher concentrations, in reducing nuclear β -catenin accumulation and enhancing β -catenin
translocation to the plasma membrane.
Discussion
Cervical cancer is the third most common cancer in women worldwide
47
. Invasive cervical cancer and the
precursor lesions are the result of persistent infection by oncogenic HPVs, predominantly HPV types 16, 18,
31, etc.
48
. However, HPV infection only is not enough to immortalize and transform the epithelial cells of the
host. Additional co-factors are needed to acquire an immortal phenotype and to further develop into a blatant
Figure 5. Eect of BaP, CUR and Nano-CUR on miRNAs, cytokine IL-6 expression and related pathways
in Caski cells. BaP was found to: (A) increase the expression of oncogenic miRNA-21, and (B) decrease
the expression of tumor suppressor miRNA-214. Nano-CUR was found to be more eective than free CUR
in mitigating the oncogenic eects of BaP, by decreasing miRNA-21 and increasing miRNA-214. (C) BaP
increased the expression of oncogenic IL-6, which was decreased eectively by CUR and Nano-CUR. (D) CUR
and Nano-CUR restore anti-survival pathways aected by BaP by suppressing oncogenic transcription factors
and restoring tumor suppressor PTEN. Control-DMSO + PLGA; BaP-Benzo(a)pyrene; CUR; Nano-CUR.
miRNA expression was assessed through qRT-PCR using Taqman primers and IL-6 expression was analyzed
using a cytokine assay kit. Western blotting was done using commercially available antibodies. e experiments
were done in triplicate. Error bars show SEM; N = 3; all concentrations are in μ M; *p < 0.05. (E) ISH (In situ
hybridization) of miRNA-21 and IHC of PTEN in mice tumor tissues treated with CUR and Nano-CUR.
Blue marks correspond to miRNA-21 and brown marks correspond to PTEN expression. ISH and IHC were
conducted according to the manufacturer’s protocol for FFPE tissues of control and treated orthotopic mice to
detect the expression of miRNA-21 and PTEN. Original magnication 200X.
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malignant and invasive phenotype
3,4
. In addition to HPV infection, cigarette smoking is a known risk factor with
smoke carcinogens such as benzo[a]pyrene (BaP)
2,5
. BaP levels have been found to be elevated in the cervical
mucus of women who smoke
49
. BaP has been shown to stimulate high levels of the viral oncoproteins E6 and E7,
besides enhancing virion synthesis in cell lines prolically infected with HPV
5
. Furthermore, mixtures containing
BaP, such as cigarette smoke condensate, have been shown to induce remarkable microRNA alterations in rodent
lungs and in in vitro human bronchial models
50,51
.
Conventional cytotoxic chemotherapy/radiation therapy in patients with recurrent and metastatic cervical can-
cer is restricted due to the eventual development of drug resistance and systemic toxicity. erefore, it is imperative
to implement biologically safe and eective natural compounds as anti-cancer and chemopreventive drugs that
have already been widely used in humans and have fewer systemic side eects. One such promising molecule is cur-
cumin, a natural polyphenol compound found in turmeric. is molecule is widely studied in the literature ( > 8,350
peer-reviewed articles, http://www.ncbi.nlm.nih.gov/pubmed/?term = curcumin, search resulted on December 02,
2015) and has been used in more than 125 clinical trials (https://clinicaltrials.gov/ct2/results?term = c urcumin&-
Search = Search, search resulted on December 02, 2015). is molecule has a number of pharmacological activities,
including anti-oxidant and anti-mutagen properties, and can inhibit the development of carcinogenesis. In our pre-
vious report, we demonstrated the anti-cancer eects and reversion of BaP-induced oncoproteins with curcumin
treatment in cervical cancer cells. Based on curcumins excellent pleiotropic properties, its clinical implication that
includes cancer treatments has increased signicantly in recent years. However, its inherent bioavailability and rapid
degradation characteristics restrict its use in clinical settings. erefore, our current study utilized a novel strategy to
treat cervical cancer with a curcumin nanoformulation, i.e., Nano-CUR (Fig.1A,B).
Figure 6. Eect of CUR and Nano-CUR on the suppression of BaP and IL-6 induced cellular migration.
(A) CUR and Nano-CUR were able to suppress BaP and IL-6 induced cellular migration in cervical cancer cells.
(B) Inhibition of enhanced nuclear translocation of β -catenin by BaP through CUR and Nano-CUR. For this
study, 80,000 Caski cells were seeded overnight in 4-well chamber slides, then treated with BaP alone and in
combination with CUR/Nano-CUR for 24 hrs. Subsequently, they were xed and processed for immunostaining
using anti β -catenin Ab. Confocal images are shown for nuclear staining (Blue for DAPI) and β -catenin (red for
Cy3), using a Nikon confocal microscope. Original magnication 200X.
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Our data, for the rst time, demonstrated signicant accumulation of curcumin in cervical cancer cells
when we use the Nano-CUR formulation (Fig.1C,D). is indicates that curcumin nanoformulation uptake is
favored by superior endocytosis due to its smaller size. A similar enhanced cellular uptake was observed even
in other types of cancer cells
22
. Such internalization leads to pronounced eects due to the drugs. As expected,
we observed Nano-CUR to be more eective in reducing cell viability and clonogenicity of cervical cancer cells
(Fig.2). Furthermore, the superior functional eects of Nano-CUR was conrmed through ow cytometry by
performing cell cycle and apoptosis assays. Nano-CUR was observed to be more eective in arresting cells in
G1-S transition as compared to free CUR at higher concentrations (Fig.3A). Additionally, Nano-CUR was found
to be more eective in comparison to free CUR in inducing apoptosis in cervical cancer cells, wherein it induced
63–88% late apoptosis at higher concentrations (Fig.3B).
To further verify the improved therapeutic ecacy of the Nano-CUR formulation, we generated an ortho-
topic mouse model of cervical cancer (Caski cells) using NSG mice. Nano-CUR treatment was more eective
to inhibit the tumor growth over all other groups (Fig.4B,C). is demonstrates that curcumin showed better
therapeutic eects in its nanoformulation form (Fig.4). Previous studies from our lab
5
have shown that curcumin
decreases the expression of the oncogenic HPV protein E7 in in vitro models. is led us to conrm the status of
HPV oncogenic proteins E6 and E7 in NSG mice tumor tissues that were collected aer the treatment. CUR and
Nano-CUR treatment eectively suppressed the expression of E6 and E7 oncoproteins. Nano-CUR, however, was
more ecient than free CUR in decreasing the expression levels of Ki67, a well-known marker for cell prolifera-
tion (Fig.4D).
Furthermore, the anti-oncogenic eects of CUR and Nano-CUR were measured against smoke carcinogen,
BaP. e cancer causing eects of BaP, such as increasing the expression of oncogenic entities miRNA-21 and IL-6
and decreasing the expression of the tumor suppressor miRNA-214, were mitigated by CUR and Nano-CUR.
ese eects were even more signicant with Nano-CUR treatment (Fig.5A–C). CUR/Nano-CUR were also
eective in restoring the anti-survival pathways aected by BaP treatment through decreasing the expression
of oncogenic transcription factors such as p-STAT3/5, NF-κ B and β -catenin and enhancing the expression of
the tumor suppressor phosphatase PTEN, besides suppressing its inactive form p-PTEN (Fig.5D). Polycyclic
aromatic hydrocarbons like BaP or its derivatives are known to increase the expression of pro-inammatory
cytokines such as IL-8 or IL-6
52,53
in various organs and cancers. IL-6 mediated activation of transcription factor
STAT3 (to its activated form p-STAT3) is a common oncogenic process and is one of the mechanisms through
which chronic inammation contributes to cancer development and progression
31
. Previous studies have demon-
strated that an upstream enhancer that contains two STAT3 binding sites controls the gene encoding miRNA-21
and miRNA-21 expression is dependent on binding of the activated form of STAT3 (p-STAT3) to its promoter
regions
31
. Moreover, these studies also show that miRNA-21 gene transcription is controlled by IL-6 and entails
STAT3. e overexpression of miRNA-21 has been observed in several cancers and all of those cancers have
constitutively activated STAT3 (p-STAT3)
53,54
. Furthermore, NF-κ B has also been shown to increase miRNA-21
expression in a number of studies
55,56
. All of this corroborates our nding in cervical cancer that BaP treatment
leads to an increase in miRNA-21 expression. Additionally, CUR and Nano-CUR restore the expression of the
tumor suppressor phosphatase PTEN, a direct target of miRNA-21 (Fig.5A,D). We also validated the expression
levels of miRNA-21 and PTEN in mice tumors generated through the establishment of the orthotopic model of
cervical cancer. In situ hybridization of the tissue slides showed that CUR and Nano-CUR were eective in abro-
gating the expression of miRNA-21 and thus increased expression of PTEN (Fig.5E). CUR and Nano-CUR were
also able to suppress IL-6 (Fig.5C) and BaP-induced migration of cervical cancer cells (Fig.6A). IL-6 is known
to enhance the cellular migratory properties of cancer cells
57,58
. Polycyclic aromatic hydrocarbons (PAHs) such
as BaP are also known to enhance cellular migration in several types of cells
39,56,58
. It would be interesting to see
if BaP alone or in combination with pro-inammatory cytokines such as IL-6 can lead to enhanced inamma-
tion which is a precursor of oncogenesis
59
. Additionally, Nano-CUR was found to be more eective than free
curcumin in suppressing nuclear translocation of β -catenin and restoring membrane bound β -catenin in Caski
cervical cancer cells (Fig.6B). Nuclear localization of β -catenin is the key initiating and driving event for transfor-
mation of normal (non-tumor) cell to tumor cell (as discussed in Results). Moreover, β -catenin is a direct target of
miRNA-214, a tumor suppressor miRNA in cervical cancer (as mentioned earlier in Results).
In conclusion, this study clearly is an advancement of an earlier report of curcumin nanoformulation tested
against cancer cells
60
. Our ndings show that PLGA based Nano-CUR signicantly inhibits growth of cervical
cancer cells and regulates the expression of miRNAs and various oncogenic and tumor suppressor proteins asso-
ciated with cervical cancer (Fig.7). In vivo experiments show that Nano-CUR is ecacious in reducing the tumor
burden. erefore, Nano-CUR may be a novel chemo-preventive and therapeutic modality for the management
of cervical cancer.
Materials and Methods
Cell Culture. Caski and SiHa cervical cancer cell lines were purchased from American Type Culture
Collection (Manassas, VA). ese cell lines were expanded and low passage frozen aliquots were stored in liquid
nitrogen. For conducting experiments, cells were thawed and grown for < 6 months. SiHa cells were maintained
in Dulbeccos Modied Eagles medium (DMEM) containing 4.5 g/L of glucose, 10 nM of nonessential amino
acids, 100 nM of sodium pyruvate, 1x antibiotic/antimycotic (Sigma, St. Louis MO), and 10% heat-inactivated
FBS (Atlanta Biologicals, Lawrenceville, GA). Caski cells were grown in Roswell Park Memorial Institute (RPMI)
medium containing 10% heat-inactivated FBS and 1x antibiotic/antomycotic. All cells were incubated in a
5% CO
2
incubator at 37 °C.
Preparation of Nano-CUR formulation. e PLGA based CUR nanoformulation was synthesized as
described previously
22
. Briey, 90 mg of PLGA solution in 10 mL of acetone was mixed with 10 mg of CUR for
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5 min. is solution was added dropwise to 20 mL of aqueous solution containing 1% wt/vol PVA and 10 mg of
poly(l-lysine), over a period of 10 min on a magnetic stirrer at 800 rpm. e resulting suspension of Nano-CUR
particles was stirred at room temperature for ~24 hrs to evaporate the acetone solvent completely. Larger aggre-
gates and un-bound polymers were removed by centrifugation at 5000 rpm in an Eppendorf Centrifuge 5810 R
(Eppendorf AG, Hamburg, Germany) for 10 min. Nano-CUR particles were recovered by ultracentrifugation at
30,000 rpm using a Rotor 30.50 in an Avanti J-30I Centrifuge (Beckman Coulter, Fullerton, CA, USA). Particles
were washed twice and then freeze-dried using the Labconco Freeze Dry System ( 48 °C, 133 × 10
–3
mBar;
Labconco, Kansas City, MO). Control PLGA nanoparticles were similarly prepared by dissolving polymer in
organic solvent without CUR, with the rest of the method being the same.
Cellular uptake. To determine the uptake and internalization ecacy of Nano-CUR, we utilized uores-
cent microscopy and ow cytometry. For qualitative assessment, Caski and SiHa cells (2 × 10
5
cells/well in 2 mL
media) were plated in 6-well plates and allowed to attach overnight. Cells were then treated with 5, 10, and 20 μ M
CUR or Nano-CUR and their vehicle controls (DMSO and PLGA NPs, respectively) for 6 hrs. Aer 6 hrs, cells
were washed with PBS twice and replaced with phenol-red free medium. Live adherent cells were examined under
a uorescent microscope (Nikon Eclipse Ti Microscope, Melville, NY) and images were captured at 200X. For
quantitative measurement, cells (2 × 10
5
cells per well in 2 mL media) were plated in 6-well plates and allowed
to attach overnight. Cells were then treated with 5, 10, and 20 μ M CUR or Nano-CUR and their vehicle controls
(DMSO and PLGA-NPs, respectively) for 6 hrs. Aer 6 hrs, cells were washed with PBS, trypsinized, and collected
Figure 7. Schematic representation of CUR/Nano-CURs eect on the suppression of E6 and E7 HPV
proteins and IL-6 pathways to reduce the tumor burden in an orthotopic cervical cancer mouse model.
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Scientific RepoRts | 6:20051 | DOI: 10.1038/srep20051
in 2 mL media. Further, these cells were centrifuged, washed with PBS twice, and collected in 2 mL phenol-red
free medium for ow cytometry analysis. About 10,000 cells in medium were injected into an Accuri C6 Flow
Cytometer (BD Biosciences, San Jose, CA, USA) to determine the uorescence levels in the FL1 channel (488
excitation, Blue laser, 530 ± 15 nm, FITC/GFP). FL1 channel measures the green uorescence generated by the
curcumin or Nano-CUR.
Cell proliferation assay. e eect of CUR and Nano-CUR on cell growth was determined by cell prolif-
eration assay using CellTiter96 Aqueous One Solution (MTS) reagent (Promega, Madison, WI). Briey, cervical
cancer cells (5000 cells/well) were plated in 100 μ L DMEM/RPMI medium in 96-well tissue culture plates and
incubated overnight. CUR was dissolved in DMSO and diluted in cell culture media. e cells were treated with
varying concentrations of CUR/Nano-CUR or equivalent amounts of vehicle controls (DMSO/PLGA) for 48 hrs.
Cell proliferation was determined by adding 20 μ L of MTS reagent, incubating for 2 hrs at 37 °C and measuring
the absorption at 490 nm using a SPECTRA Max Plus Plate Reader (Molecular Devices, Sunnyvale, CA). e
percent proliferation in CUR/Nano-CUR treated cells was determined by normalizing the cells with no treatment
(considered as 100%).
Colony forming assay. For this assay, cervical cancer cells were seeded at 500 cells per well in 2 mL media
in 6-well plate and allowed for 24 hrs to attach. Aer that, cells were treated with concentrations of 2.5, 5, and
10 μ M CUR/Nano-CUR for an additional 12–14 days. e cells were washed, xed in cold methanol, and stained
with hematoxylin. Visible colonies (~50 cells) were counted manually and reported as the ratio of the number of
colonies in treated cells divided by the number of colonies in the vehicle (DMSO/PLGA) control.
Cell cycle analysis. Cell cycle arrest was analyzed by the Telford method. Caski and SiHa cervical cancer
cells (1 × 10
6
) were plated in a 100 mm dish and allowed to attach overnight. e following day, cells were exposed
to either 10 or 25 μ M CUR, Nano-CUR or equivalent amounts of controls for 24 hrs, trypsinized, washed, xed
with 70% ethanol, stored at 4 °C for an hr, stained with propidium iodide (Sigma-Aldrich, St. Louis, MO; 50 μ g in
1 mL Telford reagent) in the dark for 4 hrs at 4 °C and analyzed by an Accuri C6 Flow Cytometer in FL2 channel.
Annexin V-7AAD staining. Detection of apoptosis was determined by using Annexin V-7AAD staining.
Caski and SiHa cervical cancer cells (1 × 10
6
) were plated in a 100 mm dish and allowed to adhere overnight. e
next day, cells were treated with 10, 20 and 25 μ M CUR, Nano-CUR or equivalent amounts of controls for 24 hrs.
At the end of indicated time, both adherent and oating cells were collected and stained with Annexin V and
7-AAD (BD Biosciences) at 5 μ L of each/100 μ L of cell suspension. Cells were incubated for 20 min in the dark at
room temperature and analyzed with an Accuri C6 Flow Cytometer in FL2 and FL3 channels.
Orthotopic model of cervical cancer in NSG mice. Six-week-old female NSG mice (Cancer Research
Animal Core, UTHSC, Memphis, TN) were used to generate an orthotopic model of cervical cancer. e mice
were maintained in a pathogen-free environment and all procedures were carried out as approved by the UTHSC
Institutional Animal Care and Use Committee (UTHSC-IACUC). All the procedures and methods were car-
ried out in “accordance” with the approved guidelines of UTHSC-IACUC. Briey, Caski cells (4 × 10
6
cells/per
mouse) were dispersed in 100 μ L 1X PBS and 100 μ L Matrigel (BD Biosciences) and injected directly into the
cervix of the mice. e animals were periodically monitored for tumor development and the tumor volume was
measured from day 5 aer injection using a digital Vernier caliper. When the tumor volume reached ~200 mm
3
,
CUR, Nano-CUR (100 g/mice) and their respective vehicle controls (PBST and PLGA NPs, respectively) were
injected intra-tumorally. e tumor volume was calculated using the ellipsoid volume formula: tumor volume
(mm
3
) = π/6 × L × W × H, wherein L is length, W is width, and H is height. e tumor was regularly monitored
and allowed to grow until the tumor burden reached a maximum volume of 1100 mm
3
. At the time of sacrice,
the mice tumors were removed, xed in formalin, embedded in paran, and sliced into 5 micron sections for
further processing and analysis.
Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR). Caski
cervical cancer cells were seeded in a 6-well plate, allowed to attach, and treated with varied concentrations of
CUR/Nano-CUR and their vehicle controls (DMSO/PLGA) and BaP in their respective combinations for
4 days. Total RNA was extracted from treated cells using TRIzol reagent (Invitrogen, Life Technologies, Grand
Island, NY). e integrity of the RNA was measured with an RNA 6000 Nano Assay kit and 2100 Bioanalyzer
(Agilent Technologies, Santa Clara, CA). For miRNA detection, 100 ng total RNA was reverse transcribed into
cDNA using specic primers designed for miRNA analysis (Applied Biosystems, Foster City, CA). e miRNA
expression levels were determined by qRT-PCR using the Taqman PCR master mixture (no AmpErase UNG) and
specic primers designed for detection of mature miRNAs (Applied Biosystems). e expression of miRNA was
normalized with the expression of endogenous control, U6snRNA.
IL-6 measurement. Caski cervical cancer cells were treated using modified culture medium (RPMI-
1640 + 2% FBS). Briey, aer treatment with BaP alone or in combination (BaP and CUR/Nano-CUR) for 48 hrs,
culture medium was centrifuged at 10,000 g for 10 min at 4 °C. OD was measured at 450 nm. IL-6 (eBioscience,
San Diego, CA, USA) measurement was performed as per manufacturer’s instructions. Quantication of IL-6 was
performed by standard curve prepared by recombinant human IL-6 (provided with the kit) and normalized with
live cells. All experiments were performed in triplicate and repeated at least three times.
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Migration assay. Cell migration assay was performed in Cornings 96-well HTS transwell as per manu-
facturer’s instructions with minor modications. Briey, Caski cells (50,000 cells/well) were seeded in upper
chambers of the plate containing serum-free culture medium. Cells were further treated with BaP alone, or in
combination (BaP and CUR/Nano-CUR) for 18 hrs. Cells were allowed to migrate from upper chamber (5% FBS)
towards lower chamber which has 10% FBS. Cells in the upper chamber were completely removed by cotton swab.
Cells were xed with 4% paraformaldehyde prepared in PBS for 30 min. ese cells were stained with Giemsa
stain and migrated cells were observed by using a light microscope. Migrated cells were counted in six random
elds of view and the experiments were performed in triplicate.
Western blotting. Actively growing Caski cervical cancer cells were used for Western blot analysis. Briey,
Caski cells were washed with ice-cold phosphate buer saline (PBS) and lysed in 2X radioimmunoprecipitation
assay (RIPA) buer aer the respective treatments. Protein content was analyzed using Nanodrop 2000 (ermo
Scientic, Wilmington, DE), and equivalent amount of protein samples were electrophoretically separated on
4–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, and transferred to a pol-
yvinylidene diuoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA). Following blocking with 5%
bovine serum albumin (BSA; 5 mL for one hour), the membranes were probed overnight at 4 °C for various pro-
teins using specic primary antibodies (all from Cell Signaling Technologies, Danvers, MA). e western blots
were incubated with HRP-labeled secondary antibody and the protein bands were developed using Lumi-Light
Plus chemiluminescent reagent (Roche, Indianapolis, IN).
Immunouorescence and Confocal Microscopy. Immunouorescence staining was performed to
determine the eect of BaP on the nuclear translocation of β -catenin. 80,000 Caski cells were seeded overnight in
4-well chamber slides (Nalgene Nunc Intl.., Rochester, NY), then treated with BaP alone and in combination with
CUR/Nano-CUR for 24 hrs. Subsequently, they were xed and processed for immunostaining. Briey, cells were
xed in 2% para-formaldehyde, permeabilized, blocked and then incubated with primary antibody, anti β -catenin
Ab (Cell Signaling Technologies). Following washing, cells were incubated with Cy3 labeled secondary antibody
(Jackson Immunoresearch Labs, Westgrove, PA). Aer further washing, the cover slips were mounted on glass
slides using aqueous anti-fade medium (Vector Laboratories, Burlingame, CA). Slides were examined under a
laser confocal microscope (Nikon Corporation).
In situ hybridization for miRNA-21. In situ hybridization was conducted according to the manufacturer’s
protocol for FFPE tissues of control and treated orthotopic mice to detect the expression of miRNA-21 using a
Biochain kit (Biochain, San Francisco, CA). DIG-labeled LNA oligonucleotides (Exiqon, Woburn, MA) were used
for overnight hybridization at 50 °C. e staining was carried out as per Exiqon user manual instructions. Briey,
aer deparanization, specimens were xed in 4% para-formaldehyde in DEPC-PBS for 20 min and subjected to
digestion using 2X standard saline citrate and 0.1% Triton-X for the next 25 min. Slides were pre-hybridized with
pre-hybridization solution for 4 hrs at 48 °C followed by hybridization of the slides with hybridization buer and
probe (Digoxigenin labeled) at 45 °C overnight. Aer stringent washing of slides with various grades of standard
saline citrate, the slides were blocked using 1X blocking solution provided in the kit. e tissues were subse-
quently incubated overnight with the AP-conjugated anti-digoxigenin antibody. e slides were washed twice
for 5 min with 1X Alkaline Phosphatase buer. e nal visualization was carried out with NBT/BCIP overnight
followed by nuclear fast red counterstaining. e slides were mounted, imaged and analyzed under ScanScope
®
XT/ XT2 system (Aperio, Vista, CA). All the reagents used for the assay were provided in the kit from Biochain.
Immunohistochemical analysis of cervical orthotopic tumors. IHC analysis for HPV E6/E7 pro-
teins, Ki67 and PTEN on formalin xed, paran embedded orthotopic mouse tumors (5 micron sections) was
performed. Briey, the tumor tissues were deparanized, rehydrated, treated with 0.3% hydrogen peroxide and
processed for antigen retrieval using a heat-induced technique. Following blocking with background sniper
(Biocare Medical, Concord, CA), the samples were processed for staining with E6 (Abcam, Cambridge MA),
E7 (Invitrogen), Ki67 and PTEN antibodies (Cell Signaling Technologies). e expression of these proteins was
detected using a MACH 4 Universal HRP Polymer detection kit (Biocare Medical) and 3,39-diaminobenzidine
(DAB substrate kit, Vector Laboratories, Burlingame, CA). The slides were counterstained with hematoxy-
lin, dehydrated, mounted with VectaMount (Vector Laboratories) and visualized using an Olympus BX 41
Microscope (Olympus Corporation, Japan).
Statistical analysis. Values were processed using Microsoft Excel 2007 software and presented as
mean ± standard error of the mean (S.E.M.). Statistical analyses were performed using an unpaired, two-tailed
student t-test. e level of signicance was set at *p < 0.05. All graphs were plotted using Origin 6.1 soware.
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Acknowledgements
is work was supported by the NIH U01CA162106, R01CA142736, K22 CA174841; Department of Defense
ARMY GRANT W81XWH-14-1-0154; and College of Pharmacy/University of Tennessee Health Science Center
Seed Grant.
Author Contributions
M.S.Z designed and performed experiments (proliferation assays, animal experiments, RT-PCR experiments and
analysis, confocal microscopy, ISH and IHC), analyzed data and wrote the paper; N.C. performed experiments
(uptake, clonogenic and proliferation assays, ow cytometry experiments, IHC), data analysis, gure preparation,
participated in method section writing and manuscript editing; R.K.G. performed experiments (Western blots,
IL-6 measurement and migration assay); D.M.M. designed experiments and analyzed data; S.K. performed
experiments (IL-6 measurement and migration assay); M.S. participated in animal experiments; S.K. performed
and analyzed ISH experiments; M.M.Y. synthesized Nano-CUR, analyzed data, participated in animal
experiments, prepared gures, wrote the parts of manuscript, drew Figure 7, and edited the manuscript; N.Z. was
involved as a pathologist and oversee the IHC and ISH studies, M.J. and S.C.C. conceived the idea, analyzed data,
and edited the manuscript.
Additional Information
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Zaman, M. S. et al. Curcumin Nanoformulation for Cervical Cancer Treatment.
Sci. Rep. 6, 20051; doi: 10.1038/srep20051 (2016).
is work is licensed under a Creative Commons Attribution 4.0 International License. e images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/
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