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Endocrinology. 2008 Aug; 149(8): 4158–4167. PMCID: PMC2488238

Published online 2008 May 1. doi: 10.1210/en.2007-1760

Curcumin (Diferuloylmethane) Inhibits Cell Proliferation, Induces

Apoptosis, and Decreases Hormone Levels and Secretion in Pituitary
Tumor Cells
Matthew Miller, Shenglin Chen, Jeffrey Woodliff, and Sanjay Kansra

Departments of Neurosurgery (M.M.), Endocrinology, Metabolism, and Clinical Nutrition (S.C., S.K.), Pediatrics (J.W.), and Pharmacology
(S.K.), Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Address all correspondence and requests for reprints to: Sanjay Kansra, Departments of Endocrinology, Metabolism, and Clinical Nutrition
and Pharmacology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226. E-mail: skansra@mcw.edu.

Received 2007 Dec 19; Accepted 2008 Apr 16.

Copyright © 2008 by The Endocrine Society

Abstract
Prolactinomas are the most prevalent functional pituitary adenomas. Dopamine D2 receptor (D2R)
agonists, such as bromocriptine are the first line of therapy; however, drug intolerance/resistance to D2R
agonists exists. Apart from D2R agonists, there is no established medical therapy for prolactinomas;
therefore, identifying novel therapeutics is warranted. Curcumin, a commonly used food additive in South
Asian cooking, inhibits proliferation of several tumor cell lines; however, its effect on pituitary tumor cell
proliferation has not been determined. Our objectives were to: 1) determine whether curcumin inhibits
proliferation of pituitary tumor cell lines; 2) identify the signaling intermediaries that mediate the effect of
curcumin; 3) examine whether curcumin inhibited pituitary hormone production and release; and 4)
examine whether curcumin could enhance the growth-inhibitory effect of bromocriptine. Using rat
lactotroph cell lines, GH3 and MMQ cells, we report that curcumin had a robust dose and time-dependent
inhibitory effect on GH3 and MMQ cell proliferation. Inhibitory effects of curcumin persisted, even on
removal of curcumin, and curcumin also blocked colony formation ability of pituitary tumor cells. The
growth-inhibitory effect of curcumin was accompanied by decreased expression of cyclin D3 and ser 780
phosphorylation of retinoblastoma protein. In addition, curcumin also induced apoptosis in both GH3 and
MMQ cells. Furthermore, curcumin suppresses intracellular levels and release of both prolactin and GH.
Finally, we show that low concentrations of curcumin enhanced the growth-inhibitory effect of
bromocriptine on MMQ cell proliferation. Taken together we demonstrate that curcumin inhibits pituitary
tumor cell proliferation, induces apoptosis, and decreases hormone production and release, and thus, we
propose developing curcumin as a novel therapeutic tool in the management of prolactinomas.

THE PITUITARY PLAYS a major role in regulating metabolic, developmental, and reproductive functions.
Lactotrophs produce prolactin (PRL) and represent about 20–50% of the cellular population of the anterior
pituitary. PRL production and release is a tightly regulated process, and its disruption leads to several
pathological conditions. Hyperprolactinemia, or excessive serum PRL levels, has severe effects on the
reproductive system (amenorrhea, irregular menses, gynecomastia, galactorrhea, sexual dysfunction,
decreased libido, anovulation, azoospermia, and erectile/ejaculatory disorders) and is also believed to
increase the risk of breast cancer and osteoporosis (1,2,3,4,5,6). Prolactinomas are the most commonly

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diagnosed pituitary tumor comprising 27% of all pituitary tumors (7). Despite their common occurrence,
the molecular events that lead to the development of pituitary tumors are still not fully understood.
Oncogenes (jun, fos, ras, erbB1/2, c-myc, etc.) and tumor suppressor genes (p53) that play a significant role
in the development of other tumor types are not major contributors to the development of pituitary tumors.
Furthermore, inherited genetic defects account for less than 3% of pituitary tumors, and these occur in
multiple endocrine neoplasia type 1 and Carney’s complex (8,9,10). Prolactinoma development is generally
thought to occur as a result of excessive signaling by stimulatory factors (e.g. growth factors, steroid
hormones-estrogen, etc.) or lack of inhibitory mechanisms (e.g. dopamine or its receptor) that couple
genetic defects (11,12,13,14,15,16). Although prolactinomas respond to dopamine D2 receptor
(D2R)-based therapy, intolerance/resistance to D2R agonists does exist (2,17,18,19,20). Apart from D2R
agonists, there are no established medical therapies to treat prolactinomas; therefore, identification of novel
therapeutics is warranted.

Curcumin, the major curcuminoid derivative found in spices commonly used in South Asian cooking, has
several known biological effects, including antiinflammatory, antitumorigenic, and antioxidant (21,22,23).
Although curcumin is primarily used as a food additive in several South Asian countries, it also has a
history of several centuries of use as a medicinal agent in the ancient Indian system of Ayurvedic medicine.
In the last decade, there has been extensive interest in the medicinal value of curcumin, and it is currently
in several clinical trials including colon cancer, pancreatic cancer, Alzheimer’s disease, chemotherapy-
induced mucositis, psoriasis, multiple myeloma, and cystic fibrosis (24). The exact molecular mechanism
by which it exerts its effects in target tissues is still to be determined. Some of the signaling pathways that
curcumin affects include those regulated by nuclear factor-κB, activator protein-1, nitric oxide synthase,
receptor tyrosine kinases, cyclins, and matrix metalloproteinases (25,26,27,28,29,30). Most of these
signaling pathways are in an activated state in several tumors when compared with normal cells, which
might explain the relative low in vivo toxicity of curcumin (31). Indeed, work from other laboratories has
demonstrated that curcumin selectively targets tumor cells when compared with normal cells, e.g. curcumin
induced cell death in rat epithelial cell derived hepatocellular carcinoma without having any apoptotic
effect on normal hepatocytes (32,33).

Although intense investigations have been conducted on the antiproliferative and chemotherapeutic effects
of curcumin in several tumor cell lines, its effect on prolactinomas and other pituitary tumors is unknown.

In this study we examined the effect of curcumin on rat epithelial cell-derived pituitary tumor cell
proliferation, apoptosis, and effects on hormone levels. We report that curcumin inhibits pituitary tumor
cell proliferation, induces apoptosis, and blocks their clonogenic ability. The inhibition of cell proliferation
is accompanied by decreased expression of cyclin D3 and decreased phosphorylation of retinoblastoma
protein (Rb). Furthermore, curcumin has a significant suppressive effect on intracellular levels as well as
secretion of PRL and GH in pituitary tumor cells. Finally, we also demonstrate that curcumin enhances the
growth-inhibitory effect of low concentrations of bromocriptine.

Materials and Methods

Chemicals and reagents

Curcumin was purchased from LTK Laboratories (St. Paul, MN). GH3 and MMQ cells were purchased
from the American Type Culture Collection (Manassas, VA). Rat PRL (rPRL), anti-rPRL antibody, and
antirat GH antibody were purchased from the National Institute of Diabetes and Digestive and Kidney
Diseases (National Hormone and Pituitary Program, Torrance, CA). Anti-cyclin D3 and anti-phospho-Rb
(ser 780) antibodies were purchased from Cell Signaling Technologies (Danvers, MA). Anti-Rb antibody
was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase-conjugated

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secondary antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). 2-Bromo-
α-ergocryptine methanesulfonate salt was purchased from Sigma Chemical Co. (St. Louis, MO). To
measure the PRL and GH content in conditioned medium, enzyme immunoassay (EIA) kits from Alpco
Diagnostics (Salem, NH) were used. Unless otherwise indicated, all buffers, solvents, and chemicals were
of analytical grade and purchased from either Thermo Fisher Scientific Company (Pittsburgh, PA) or
Sigma.

Cell culture

Regular maintenance of GH3 cells was done as described (34) in complete medium [DMEM:F-12 (50:50)
mix (Mediatech, Herndon, VA) containing 10% fetal bovine serum (FBS; Life Technologies,
Inc./Invitrogen, Grand Island, NY) and 5 U/ml penicillin/5 μg/ml streptomycin]. Complete medium was
changed every 2–3 d, and cells were subcultured as required.

MMQ cells were maintained in DMEM containing 15% horse serum, 2.5% FBS, and 5 U/ml penicillin/ 5
μg/ml streptomycin.

Assessment of cell proliferation

GH3 or MMQ cells in log phase were washed three times with phenol red-free DMEM:F12 (50:50) mix
medium and seeded (GH3 cells, 20,000–30,000 cells/well; MMQ cells, 30,000–40,000 cells/well) on
protamine-coated 96-well plates in DMEM: F12 (50:50) medium containing insulin, transferrin, and
selenious acid premix (BD Biosciences, San Jose, CA) and Pen/Strep (plating medium). The next day, cells
were treated with different agonists in plating medium. At the end of incubations cell proliferation was
assessed using the colorimetric 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assay as previously described (35). Briefly, 125 μg/well of MTT (Sigma) was added. Two hours later, 100
μl of developer solution [50% (vol/vol) dimethylformamide; 20% (wt/vol) sodium dodecyl sulfate; 0.24%
(vol/vol) glacial acetic acid; 60 mM sodium acetate] was added, and plates were incubated overnight at 37
C. OD was determined at 570 nm by using a microplate reader (BioTek Instruments Inc., Winooski, VT).
Data are presented as either OD or percent change relative to control. We recently demonstrated that the
MTT assay has an excellent corelationship with 5-bromo-2′-deoxyuridine incorporation assay in
determining cell proliferation or inhibition of cell growth in GH3 cells (35).

Colony formation assays

Colony formation assays were essentially performed as previously described (36). Briefly, GH3 cells
growing in log phase were seeded at a density of 1000–2000 cells/well in a six-well plate in complete
medium. After allowing the cells to adhere for 24 h, medium was replaced with complete medium
containing either vehicle or curcumin (5, 20, 50, 100, or 200 μM). Cells were cultured at 37 C for 14–21 d,
with a medium change containing vehicle or curcumin in complete medium, performed every fourth or fifth
day. Colony formation was detected by crystal violet staining and subsequently photographed. To quantitate
colony formation, the number of colonies (each colony consisting of 50 cells or more) in six random fields
was counted. The average was obtained by deleting the highest and lowest count for each treatment, and
decreases from 0 μM curcumin were calculated and are expressed as percent of control.

Annexin V staining assays

GH3 and MMQ cells were treated with the indicated amount of curcumin for 48 h, washed in PBS buffer,
and stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), and analyzed by
flow cytometry according to the manufacturers instructions (BD Biosciences).

Western blotting

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After treatments, ice-cold PBS solution was used two times to rinse cells. Subsequently lysis buffer [Tris
(pH 7.5), 50 mM; EGTA 5 mM; NaCl 120 mM; α-glycerophosphate 20 mM; Nonidet P-40 1%; Na
pyrophosphate 15 mM; Na fluoride 50 mM; Na orthovanadate 10 mM; phenylmethylsulfonyl fluoride 0.5
mM; aprotinin 10 μg/ml; leupeptin 10 μg/ml; glycerol 20%] was added (100–180 μl) and dishes incubated
for 10–30 min at 4 C. Cells were scraped into lysis buffer, and lysates were clarified by centrifugation
(12,000 rpm, 15 min at 4 C). The protein content in the supernatant was determined by the BCA protein
assay (Pierce Chemical Co., Rockford, IL). For Western blotting equal amounts of proteins were used.
Briefly, 7.5–90 μg of cell lysate were subjected to electrophoresis on 8–12% SDS-PAGE gels, and the
separated proteins were electrophoretically transferred onto polyvinyl difluoride membranes. Membranes
were rinsed twice with PBS and Tween 20 (PBST)/Tris-buffered saline and Tween 20 (TBST) and then
incubated with a blocking buffer (4–5% nonfat milk in PBST/TBST) for 1–2 h at room temperature.
Overnight incubation of membranes with primary antibodies [anti-rPRL antibody (Ab), 1:5,000–12,500;
antirat GH Ab, 1:1,000, and antiactin, 1:5,000, anticyclin D3 Ab, 1:2,000; antiphospho-Rb Ab, 1:1,000;
anti-Rb Ab, 1:200] were done at 4 C, followed by three 10-min washes with PBST/TBST. Membranes were
incubated with secondary antibodies at room temperature for 1 h, washed three times with PBST/TBST,
and antibody bound proteins were detected by enhanced chemiluminescence reagents (Pierce), according to
the manufacturer’s protocol. To calculate fold change, density of protein bands was determined by using
the ImageJ 1.38X software provided by the National Institutes of Health (Bethesda, MD). After
normalization to actin, the control sample was assigned an arbitrary value of 1, and fold change, in
response to curcumin treatment, was calculated.

Determination of PRL and GH content in conditioned medium

GH3 and MMQ cells were treated with the indicated amount of curcumin for 48 h, and 100 μl of
conditioned medium (CM) were collected for each treatment and stored at 4 C. PRL levels in CM from
GH3 and MMQ cells as well as GH levels in CM from GH3 cells were determined by using a rat PRL EIA
kit (Alpco Diagnostics) or rat GH EIA kit (Alpco Diagnostics) as per the manufacturer’s instructions.
PRL/GH content in CM was determined from a standard curve generated by using rat PRL/rat GH supplied
in the kit. Values for both PRL and GH were calculated as nanograms per milliliter and expressed as
percent of control.

Data analysis

For cell proliferation/MTT assays, data were calculated as percent of vehicle control (dimethylsulfoxide)
and expressed as a mean ± SEM of multiple experiments, with each experiment including four to six
determinations, or alternatively data are presented as mean ± SEM of OD. Statistical significance was
determined using Student’s t test.

Results

Curcumin inhibits pituitary tumor cell proliferation in a dose-dependent manner

We first investigated the effect of curcumin on GH3 cell proliferation. GH3 cells were treated with
curcumin in a dose-dependent manner (2.5–200 μM), and cell proliferation after 4 d was assessed by using
the MTT assay. Results (Fig. 1A) show that curcumin, in a dose-dependent manner, inhibited GH3 cell
proliferation with significant inhibition (63.47%; P < 0.005) of cell proliferation being observed with 50
μM, and maximal inhibition (96.6%; P < 0.005) being observed with 100 μM curcumin. To rule out cell
type-specific effects of curcumin on lactotroph proliferation, we examined the effects of curcumin on
proliferation of another rat lactotroph cell line, MMQ cells. Our results (Fig. 1B) show that like GH3 cells,
MMQ cells were also susceptible to the growth-inhibitory effects of curcumin with significant (58.5%; P <

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0.005) inhibition of cell proliferation observed with 20 μM and maximal inhibition (96.4%; P < 0.005)
being observed with 100 μM curcumin. These results demonstrate that MMQ cells were more sensitive to
the growth-inhibitory effect of curcumin at lower concentrations.

Curcumin inhibits pituitary tumor cell proliferation in a time-dependent manner

We next determined the kinetics by which curcumin inhibited pituitary tumor cell proliferation. GH3 and
MMQ cells were treated with the indicated amount of curcumin for either 1, 2, 3, or 4 d. At the end of each
24-h period, cell proliferation was assessed by the MTT assay. Results (Fig. 2A) show that curcumin
inhibited GH3 cell proliferation in a time-dependent manner, with growth-inhibitory effects of 50 μ M
curcumin being apparent as early as 1 d. Likewise, MMQ cell growth was also inhibited in a
time-dependent manner, with growth inhibition being observed with 50 μ M curcumin as early as 1 d (Fig.
2B). Consistent with the results from Fig. 1, in both GH3 and MMQ cells, the suppressive effect of
curcumin on cell proliferation was apparent even on d 4. Taken together, our results clearly establish that
curcumin, in a dose- and time-dependent manner, potently inhibits pituitary tumor cell proliferation.

Inhibition of cell proliferation persists on removal of curcumin

We next questioned whether the growth-inhibitory effect of curcumin on pituitary tumor cell proliferation
persists on removal of curcumin. MMQ cells were treated with either vehicle or 50 μ M curcumin for 4 d,
and proliferation was assessed by MTT assay (d 0). Our data (Fig. 3A) show that, consistent with our
results presented in Figs. 1 and 2, curcumin significantly inhibited MMQ cell proliferation (d 0, OD
control, 0.776; OD curcumin, 0.3272, 57.84% inhibition P < 0.001). Plating medium was removed and
replaced with growth medium containing 10% of FBS, and cell growth was monitored after 1, 2, 3, and 4 d.
Our data show that the suppressive effects of curcumin at d 4 were similar to that observed at d 0, (d 4, OD
control, 2.435; OD curcumin, 0.880, 63.87% inhibition P < 0.001). It is important to note that even in
curcumin treated cells, there is a significant increase in cell proliferation (268.95%, P < 0.005) from d 0
(OD 0.3272) to d 4 (OD 0.880). Furthermore, by using the values obtained from three independent
experiments as performed for Fig. 3A, we evaluated the effect of an initial exposure to curcumin and
subsequent long-term suppression of cell proliferation. We found (Fig. 3B) that suppressive effects of
curcumin detected on d 0 (60.1 ± 6.1%) persisted to 4 d (d 1, 65.6 ± 8.2%; d 2, 62.8 ± 2.9%; d 3, 65.7 ±
1.4%; and d 4, 64.2 ± 4.8%) in complete growth medium. These results clearly demonstrate that the
inhibitory effect of curcumin on cell proliferation persists, even on removal of curcumin.

Curcumin blocks the clonogenic ability of GH3 cells

We next questioned whether curcumin had any effect on the ability of GH3 cells to form colonies. GH3
cells were seeded (1000–2000 cells/well) in complete medium in six-well plates and allowed to adhere for
24 h. The medium was then replaced with complete medium containing curcumin (0–200 μ M), and the
ability of GH3 cells to form colonies was monitored over the next 14–21 d, as described in Materials and
Methods. Our results (Fig. 4A) show that in the absence of any curcumin, GH3 cells have a robust ability to
form colonies and that curcumin inhibits this in a dose-dependent manner. As detailed in Materials and
Methods, the number of colonies was counted, and our results (Fig. 4B) show that significant inhibitory
effect of curcumin on GH3 cell colony formation is apparent at even 10 μ M doses of curcumin (0 μM,
100%, 10 μM, 32.6% ± 15.2; P < 0.05), and with a 50-μM dose, the ability of GH3 cells to form colonies is
completely blocked.

Curcumin-induced inhibition of cell proliferation is accompanied by decreased expression

of cyclin D3 and decreased phosphorylation of Rb

We next questioned whether curcumin-induced inhibition of cell proliferation was accompanied by a

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decrease in cyclin D3 levels. To address this issue, GH3 and MMQ cells were treated with 0, 5, or 50 μ M of
curcumin for 48 h. Equal amounts of cell lysates were subjected to Western blotting with the anti-cyclin D3
Ab. Our results (Fig. 5A) show that curcumin caused a robust dose-dependent decrease in the expression of
cyclin D3 in both GH3 and MMQ cells. After normalization to actin, fold change from control (arbitrary
value of 1) reveals that in GH3 and MMQ cells, 5 μM curcumin had no effect on cyclin D3 expression,
whereas 50 μM curcumin significantly (P < 0.05) inhibited cyclin D3 expression (GH3 cells control 1, 5
μM, 0.96 ± 0.18, 50 μM, 0.29 ± 0.11; MMQ cells control 1, 5 μM, 1.03 ± 0.173, 50 μM, 0.01 ± 0.005). The
cyclin D3 bound form of cyclin-dependent kinase (cdk) 4/6 is active and phosphorylates Rb releasing the
transcription factor E2F, thereby permitting E2F to initiate gene expression. We next questioned whether
the curcumin-induced decrease in cyclin D3 was also accompanied by a decrease in phosphorylation of Rb.
To address this issue, cell lysates from Fig. 5 were subjected to Western blotting with an antiphospho (ser
780) Rb Ab. Our results (Fig. 5B) show that curcumin treatment of GH3 and MMQ cells results in
decreased phosphorylation of Rb. Equal loading was confirmed by probing with total Rb Ab. After
normalization to total Rb, fold change from control (arbitrary value of 1) revealed that in GH3 and MMQ
cells, 5 μM curcumin had no effect on Rb phosphorylation, whereas 50 μM curcumin significantly (P <
0.05) inhibited Rb phosphorylation (GH3 cells control 1, 5 μM, 0.96 ± 0.13, 50 μM, 0.22 ± 0.09; MMQ
cells control 1, 5 μM, 0.86 ± 0.09, 50 μM, 0.17 ± 0.08).

Curcumin induces apoptosis in pituitary tumor cells

We next questioned whether curcumin induces apoptosis in pituitary tumor cells. GH3 and MMQ cells
were treated with the indicated amount of curcumin for 48 h, and after trypsinization, annexin V-FITC
binding and PI staining was analyzed by flow cytometry. Our results (Fig. 6) show that curcumin in a
dose-dependent manner induced apoptosis in GH3 and MMQ cells. In vehicle-treated GH3 cells, 34.73 ±
5.3% were undergoing apoptosis, and this did not significantly change on treatment with 5 μ M curcumin,
44.13 ± 6.7%. However, treatment with 50 μM curcumin resulted in a significant (P < 0.05) increase, 86.4 ±
11.5%, of cells undergoing cell death. The percentage of untreated MMQ cells undergoing apoptosis,
8.74% ± 2.0, did not significantly change on treatment with 5 μM curcumin 8.1% ± 1.56, whereas treatment
with 50 μM curcumin resulted in a significant (P < 0.05) increase, 53.06% ± 6.64 of cells undergoing cell
death.

Curcumin decreases hormone levels and secretion in pituitary tumor cells

We next questioned whether curcumin had any inhibitory effect on the production and release of prolactin
in GH3 and MMQ cells, and GH in GH3 cells. To address this issue, GH3 or MMQ cells were treated with
0, 5, or 50 μM curcumin for 48 h, and prolactin content in equal amount of cell lysates was determined by
Western blotting. Our results show (Fig. 7A) that curcumin in a dose-dependent manner decreased the
expression of PRL in both GH3 and MMQ cells and equal loading was confirmed by probing the filter with
anti-actin Ab. Quantitation of the Western blotting results reveals that in GH3 cells compared with control
(arbitrary value of 1), 5 μM curcumin had no significant effect (0.75 ± 0.14) on intracellular PRL levels,
whereas 50 μM curcumin significantly (0.28 ± 0.08, P < 0.05) inhibited cellular PRL content. Likewise, in
MMQ cells, compared with control (arbitrary value of 1), 5 μM curcumin had no significant effect (0.87 ±
0.10) on intracellular PRL levels, whereas 50 μM curcumin significantly (0.47 ± 0.1, P < 0.05) inhibited
cellular PRL content. Furthermore, in GH3 cells, curcumin also caused a robust decrease in the expression
of GH as determined by Western blotting and equal loading was confirmed by probing the filter with
anti-actin Ab. Quantitation of the western blotting results reveals (Fig. 7B) that in GH3 cells compared with
control (arbitrary value of 1), 5 μM curcumin had no significant effect (0.83 ± 0.08) on intracellular GH
levels, whereas50 μM curcumin significantly (0.37 ± 0.13, P < 0.05) inhibited cellular GH content. These
results clearly demonstrate to us that curcumin inhibits both PRL and GH production in pituitary tumor
cells.

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Next, we questioned whether curcumin had an inhibitory effect on PRL and GH release from pituitary
tumor cells. To address this issue, GH3 or MMQ cells were treated with 0, 5, or 50 μM curcumin for 48 h,
and CM was harvested. Amount of PRL in CM from GH3 and MMQ cells and amount of GH in GH3 cell
CM were determined by using an EIA as described in Materials and Methods. Our data (Fig. 7C) show that
in GH3 cells, compared with control (100%), 5 μM curcumin had no significant inhibitory effect (98.26 ±
7.0) on PRL secretion, whereas 50 μM curcumin significantly (61.24 ± 10.77, P < 0.05) inhibited PRL
secretion. Likewise, in MMQ cells, compared with control (100), 5 μ M curcumin had no significant effect
(88.79 ± 5.38) on PRL secretion into the CM, whereas 50 μM curcumin significantly (9.58 ± 4.22, P <
0.05) inhibited PRL secretion. Finally, in GH3 cells, compared with control (100%), 5 μ M curcumin had no
significant inhibitory effect (100.34 ± 12.83) on GH secretion, whereas 50 μM curcumin significantly
(62.52 ± 7.99, P < 0.05) inhibited GH secretion into the CM.

Curcumin enhances bromocriptine mediated inhibition of MMQ cell proliferation

We next questioned whether curcumin would enhance the growth-inhibitory effect of bromocriptine on
MMQ cell proliferation. To address this issue, MMQ cells were treated with either bromocriptine (0.01,
0.1, 1, and 10 μM) or the indicated doses of bromocriptine in the presence of 5 μM curcumin, and cell
proliferation after 4 d was determined by MTT assay. Our data (Fig. 8) show that bromocriptine had a
dose-dependent inhibitory effect on MMQ cell proliferation with significant (P < 0.05) inhibition being
observed with concentrations of 1 and 10 μM bromocriptine, whereas 0.01 and 0.1 μM were ineffective. The
growth-inhibitory effect of bromocriptine on MMQ cell proliferation is consistent with a previous report
(37). Similar to the results presented in Fig. 1, 5 μM curcumin by itself had no significant growth-inhibitory
effect on MMQ cell proliferation; however, when MMQ cells were exposed to bromocriptine in the
presence of 5 μM curcumin, significant inhibition (P < 0.05) in cell proliferation was detected, even with
0.01 μM of bromocriptine. These results suggest that curcumin enhances the growth-inhibitory effect of
bromocriptine.

Discussion
Pituitary adenomas constitute a significant proportion of intracranial tumors and have several pathological
effects due to excessive hormone secretion as well as visual defects due to compression of the optic chiasm.
Prolactinomas are the most common type of pituitary adenoma, and excessive PRL secretion is a leading
cause of infertility and hypogonadism (1,2,4). Thus, regulating lactotroph proliferation and hormone
secretion is of therapeutic interest. Even though prolactinomas respond well to medical therapy with
dopamine receptor agonists, intolerance/resistance does occur. Alternatives to medical therapy include
surgery and radiation therapy. These treatments do not normalize serum PRL levels in all cases and are
associated with significant morbidity, including pituitary insufficiency. Therefore, development of
alternative medical therapies is warranted. In this study we examined the role of curcumin, a natural
compound with proven antitumor activity and very low toxicity as determined by in vivo studies (32), as a
potential inhibitor of pituitary tumor cell proliferation and hormone production and release.

Our data show that curcumin in a dose- and time-dependent manner inhibited the proliferation of pituitary
tumor cell line GH3 cells as determined with the MTT assay. We have previously shown using GH3 cells
that there is an excellent correlation between cell proliferation as measured by the MTT assay when
compared with 5-bromo-2′-deoxyuridine labeling (35). We also show that this inhibitory effect was not
restricted to a particular cell type because similar inhibitory effects of curcumin were observed with another
rat lactotroph cell line, MMQ cells (Fig. 1, A and B). The growth-inhibitory effects of curcumin (50 μM)
were apparent within 24 h of treatment, in both MMQ and GH3 cells, and persisted up to 4 d. The ability of
curcumin to inhibit cell proliferation persisted even on removal of curcumin, as demonstrated in Fig. 3, A
and B. Although consistent with our results from Figs. 1 and 2, a 4-d treatment with 50 μM curcumin

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resulted in robust growth inhibition of MMQ cells (60.1 ± 6.1%). Subsequent culture of both vehicle-
treated and curcumin-treated cells in medium containing 10% FBS revealed that even on removal of
curcumin, its suppressive effects on cell proliferation persisted (Fig. 3, A and B). These results suggest to
us that curcumin might have an irreversible effect on pituitary tumor cell proliferation. Furthermore,
because even in the 50 μM curcumin-treated cells, there is an increase in the cell proliferation from d 0 to d
4, we conclude that curcumin-induced inhibition of cell proliferation is not due to a toxic effect of
curcumin on pituitary tumor cells. Note the concentration of curcumin used in this study is comparable
with that used in other in vitro studies in which curcumin inhibited cell proliferation and induced apoptosis
in human basal cell carcinomas, bladder cancer cells, human colon cancer cells, hepatoblastomas, and
myeloid leukemic cells (38,39,40,41,42).

In addition, the robust ability of GH3 cells to form colonies (Fig. 4) was blocked in a dose-dependent
manner by curcumin, with 50 μM curcumin completely blocking the colony formation ability of GH3 cells.
The colony formation assays are an excellent readout of the long-term survival of tumor cells. Curcumin
clearly has a potent inhibitory effect on the long-term survival of pituitary tumor cells.

We next wanted to identify the molecular mechanism by which curcumin inhibits pituitary tumor cell
proliferation. We examined the effect of curcumin on cyclin D3 expression. One of the molecular
mechanisms by which curcumin inhibits cell proliferation is by mediating a decrease in cyclin D1 levels.
This decrease in cyclin D1 levels correlates with decreased Cdk4 activity and consequently decreased
phosphorylation of Rb. It has been reported that the decrease in cyclin D1 levels occurs both via
transcriptional and posttranslational mechanisms (43). In lactotrophs cyclin D3 is the major rate-limiting
cyclin mediating G1 to S transition (44,45). Therefore, we questioned whether curcumin had any effect on
cyclin D3 levels in pituitary tumor cells. Our data clearly show that curcumin inhibits the expression of
cyclin D3 in pituitary tumor cells. Consistent with the decrease in cyclin D3 levels, curcumin treatment also
results in decreased phosphorylation of Rb at ser 780 as detected with a phospho-specific anti-Rb Ab.
These results clearly demonstrate that curcumin mediated inhibition of pituitary tumor cell proliferation
involves modulation of Rb. It is of interest to note that mice that bear a single Rb mutant allele develop
aggressive pituitary tumors, suggesting that even in vivo modulation of Rb is important for pituitary
tumorigenesis (46,47). In human pituitary adenomas, both hypermethylation of Rb promoter regions and
decreased Rb expression have been reported, implicating the Rb pathway in pituitary tumorigenesis
(48,49). During the early G1 phase of cell cycle, Rb gets phosphorylated initially at ser 780 by a cyclin
D-cdk4/6 complex, and later during the G1 phase Rb is phosphorylated at ser 795 by both cyclin E-cdk2
and cyclin D-cdk4/6 complexes (50,51,52). Although our data clearly demonstrate that curcumin induced a
decreased phosphorylation of Rb at ser 780, whether this is the exclusive mechanism by which it inhibits
cell proliferation cannot be claimed. It is possible that in addition to inhibiting cyclin D3-cdk4/6 mediated
phosphorylation of Rb at ser 780, curcumin might also inhibit the cyclin E-cdk2-mediated phosphorylation
of Rb.

Curcumin-induced blockade of cell proliferation is often accompanied by apoptosis (53,54,55,56). Our own
results, with the MTT assay (Fig. 1), suggest that curcumin affects pituitary tumor cell viability.
Furthermore, morphological analysis of curcumin (50 μM)-treated GH3 and MMQ cells revealed cell
shrinkage and loss of membrane integrity (data not shown), a phenotype that is commonly seen in cells
undergoing apoptosis. To investigate whether curcumin induced apoptosis in pituitary tumor cells, we used
annexin V-FITC and PI staining to quantitate apoptosis. Our results clearly show (Fig. 6) that lower
concentrations of curcumin (5 μM) that had no significant effect on cell proliferation (Fig. 1) had no
significant effect on apoptosis. However, consistent with the growth-inhibitory effect as well as the
observed morphological changes (data not shown), 50 μM curcumin clearly induced significant apoptosis in
both GH3 and MMQ cells. Together these results clearly suggest that together with inhibiting cell
proliferation, curcumin has an apoptotic effect on pituitary tumor cells. Although there exists a strong

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correlation between the dose of curcumin that inhibits cell proliferation and induces apoptosis, whether
these events are sequentially linked remains to be determined and constitutes the next phase of our study.

Hyperprolactinemia and excessive levels of GH are commonly associated with prolactinomas and
acromegaly, respectively. We questioned whether curcumin had any effect on the levels of PRL and GH in
GH3 and MMQ cells. We clearly demonstrate that curcumin has a potent dose-dependent inhibitory effect
on PRL expression and release in both GH3 and MMQ cells and on GH levels and release in GH3 cells
(Fig. 7, A–C).

Finally, we demonstrate that a low level of curcumin (5 μM) enhances the growth-inhibitory effect of
bromocriptine on pituitary tumor cell proliferation. This result suggests that in bromocriptine-refractory
tumors, simultaneous exposure to curcumin might enhance the inhibitory effect of bromocriptine. As
mentioned in the introduction, D2R resistance/intolerance does occur; therefore, agents that could augment
the growth-inhibitory effect of D2R agonists could be clinically useful in the management of
prolactinomas. Our current studies were conducted by coincubating curcumin with bromocriptine; whether
a short exposure to curcumin would sensitize cells to the growth-inhibitory effect of bromocriptine remains
to be determined. Furthermore, whereas in the current study we examined the effect of curcumin only on
bromocriptine-induced inhibition of cell proliferation, it would be interesting to examine the effect of
curcumin on growth-inhibitory effects of other D2R agonists.

In conclusion, we have shown for the first time that curcumin inhibits proliferation, induces apoptosis, and
abolishes clonogenic ability of pituitary tumor cells. In addition, we show that curcumin-induced decrease
in cell proliferation is associated with decreased expression of cyclin D3 and phosphorylated Rb. We also
demonstrate that curcumin suppresses intracellular hormone levels as well as hormone secretion and can
enhance the growth-inhibitory effects of bromocriptine. Based on this, we propose developing curcumin as
a novel therapeutic tool in the management of PRL- and GH-secreting tumors.

Footnotes
Disclosure Statement: The authors have nothing to disclose.

First Published Online May 1, 2008

Abbreviations: Ab, Antibody; cdk, cyclin-dependent kinase; CM, conditioned medium; D2R, dopamine D2 receptor; EIA,
enzyme immunoassay; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; MTT, 3-[4,5-dimethylthiazole-2-yl]-
2,5-diphenyltetrazolium bromide; PBST, PBS and Tween 20; PI, propidium iodide; PRL, prolactin; Rb, retinoblastoma
protein; rPRL, rat PRL; TBST, Tris-buffered saline and Tween 20.

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Figures and Tables

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Figure 1

Curcumin inhibits GH3 and MMQ cell proliferation in a dose-dependent manner. GH3 cells (A) or MMQ (B) were treated
with the indicated dose of curcumin, and cell proliferation was assessed after 4 d using the MTT assay as described in
Materials and Methods. Data are calculated as percent of vehicle control and expressed as a mean ± SEM of five separate
experiments. *, Significant differences (P < 0.01) from control values.

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Figure 2

Curcumin inhibits GH3 and MMQ cell proliferation in a time-dependent manner. To assess the time-dependent effect of
curcumin on lactotroph proliferation, GH3 cells (A) and MMQ cells (B) were treated with vehicle or 50 μ M curcumin for
the indicated times, and cell proliferation was assessed as above. Data are expressed as OD and they are the mean ± SEM of
four independent determinations. Results are from a single experiment and are representative of three independent
experiments yielding similar results. *, Significant differences (P < 0.05) from control values at each corresponding day.

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Figure 3

Inhibition of cell proliferation persists on removal of curcumin. A, MMQ cells were treated with vehicle or 50 μ M
curcumin for 4 d. Cell proliferation was assessed by the MTT assay as described in Materials and Methods (d 0). Medium
was then replaced with growth medium containing 10% FBS. Cell proliferation was measured using the MTT assay after
1, 2, 3, and 4 d of culture. Data were expressed as OD and is the mean ± SEM of three to six independent determinations.
Results are from a single experiment and are representative of three independent experiments yielding similar results. *,
Significant differences (P < 0.001) from corresponding day control values; **, significant differences (P < 0.005) from
curcumin-treated d 0; ***, significant differences (P < 0.001) from vehicle-treated d 0. B, Mean ± SEM values from the
three experiments in A were expressed as percent inhibition of cell proliferation relative to control at each day (curcumin
treated vs. control).

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Figure 4

Curcumin inhibits clonogenic ability of GH3 cells. A, GH3 cells were seeded at a density of 1000–2000 cells per 60-mm
dish in complete growth medium. After 24 h the medium was replaced with fresh complete growth medium containing (A,
0 μM; B, 5 μM; C, 20 μM; D, 50 μM; E, 100 μM; and F, 200 μM) curcumin, and colony formation ability of GH3 cells was
assessed after 14–21 d, as described in Materials and Methods. Data shown are from a single experiment and are
representative of five similar experiments yielding similar results. B, Colonies were counted as described in Materials and
Methods and are expressed as percent of control (0 μM curcumin) treatment. Data are mean ± SEM of five independent
experiments. *, Significant differences (P < 0.05) from control values.

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Figure 5

Curcumin decreases cyclin D3 and phosphorylated Rb levels in GH3 cells. GH3 cells or MMQ were treated with the
indicated doses of curcumin for 48 h, and equal amount of cell lysates were subjected to Western blotting with cyclin D3
Ab (A, upper panel) or antiactin Ab (A, lower panel) and anti-phospho ser 780 Rb Ab (B, upper panel) or total Rb (B,
lower panel). Western blot data are from a single experiment that is representative of at least three independent
experiments for each cell line. Data from Western blots were quantitated as described in Materials and Methods, and fold
change from control was determined. Data are mean ± SEM of three independent experiments. *, Significant differences (P
< 0.05) from control values.

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Figure 6

Curcumin induces apoptosis in GH3 and MMQ cells. GH3 cells (left panel) and MMQ cells (right panel) were treated with
the indicated amount of curcumin for 48 h. Annexin V-FITC and PI staining was analyzed by flow cytometry. Data are
presented as percent of apoptotic cells and is the mean ± SEM of three independent experiments. *, Significant differences
(P < 0.05) from control values.

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Figure 7

Curcumin decreases intracellular levels and release of PRL and GH. A, GH3 or MMQ cells were treated with the indicated
doses of curcumin (Cur) for 48 h, and equal amounts of cell lysates were subjected to Western blotting with anti-rPRL Ab
or antirat GH Ab (upper panels). Equal loading was confirmed by determining actin levels (lower panels). Data are from a
single experiment that is representative of at least three independent experiments for each cell line. B, Fold change in
intracellular hormone levels, detected in A, were quantitated as described in Materials and Methods. Data are mean ± SEM
of three independent experiments. *, Significant differences (P < 0.05) from control values. C, GH3 or MMQ cells were
treated with the indicated doses of curcumin for 48 h, and PRL and GH released into the CM were determined by EIA as
described in Materials and Methods. PRL/GH release was calculated as nanograms PRL or GH per milliliter of CM and is
expressed as percent of control. Data are mean ± SEM of three to four independent experiments. *, Significant differences
(P < 0.05) from control values.

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Figure 8

Curcumin enhances bromocriptine-induced inhibition of MMQ cell proliferation. MMQ were treated with the indicated
dose of bromocriptine (Bromo) either by itself or in the presence of 5 μ M curcumin, and cell proliferation after 4 d was
assessed using the MTT assay as described in Materials and Methods. Data are expressed as OD and they are the mean ±
SEM of four independent determinations. Results are from a single experiment and are representative of three independent
experiments yielding similar results. *, Significant differences (P < 0.05) from controls; **, significant differences (P <
0.05) from bromocriptine treatment.

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