2Methoxyestradiol overcomes drug resistance in multiple myeloma cells

doi:10.1182/blood-2002-02-0376Prepublished online May 24, 2002;

Kenneth C AndersonMartin Sattler, Paul Richardson, Robert L Schlossman, Klaus Podar, Edie Weller, Nikhil Munshi and Dharminder Chauhan, Laurence Catley, Teru Hideshima, Guilan Li, Richard Leblanc, Deepak Gupta, cells2-Methoxyestradiol (2ME2) overcome drug resistance in multiple myeloma

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1

2-METHOXYESTRADIOL (2ME2) OVERCOME DRUG RESISTANCE IN MULTIPLE

MYELOMA CELLS

Dharminder Chauhan, Laurence Catley, Teru Hideshima, Guilan Li,

Richard Leblanc, Deepak Gupta, Martin Sattler, Paul Richardson,

Robert L. Schlossman, Klaus Podar, Edie Weller, Nikhil Munshi,

Kenneth C. Anderson$

Department of Adult Oncology, Dana-Farber Cancer Institute;

Harvard Medical School, Boston, MA 02115

Running title: 2ME2 overcomes drug resistance in MM

This investigation was supported by National Institutes of Health

Grants R0-1 50947 and PO-1 78378, the Multiple Myeloma Research

Foundation (MMRF) Senior Research Awards (DC, TH), International

Myeloma Foundation Fellow Award (KP), and the Doris Duke

Distinguished Clinical Research Scientist Award (KA)

$Address correspondence and reprints to:

Kenneth C. Anderson, MD

Dana-Farber Cancer Institute

44 Binney Street, Boston, MA 02215

phone: (617)-632-2144 FAX:(617)-632-2569

E-mail: [email protected]

Total text Word count: 4689 Total abstract word count: 220

Scientific Heading: Neoplasia

Copyright 2002 American Society of Hematology

Blood First Edition Paper, prepublished online May 24, 2002; DOI 10.1182/blood-2002-02-0376 For personal use only. by guest on January 15, 2014. bloodjournal.hematologylibrary.orgFrom





2

Abstract

2-Methoxyestradiol (2ME2), an estrogen-derivative, induces

growth arrest and apoptosis in leukemic cells and is also

antiangiogenic. In this study, we demonstrate that 2ME2 inhibits

growth and induces apoptosis in MM cell lines and patient cells.

Importantly, 2ME2 also inhibits growth and induces apoptosis in

MM cells resistant to conventional therapies including melphalan

(LR-5), doxorubicin (Dox-40 and Dox-6), and dexamethasone

(MM.1R). In contrast to its effects on MM cells, 2ME2 does not

reduce survival of normal peripheral blood lymphocytes.

Moreover, 2ME2 enhances Dex-induced apoptosis and its effect is

not blocked by interleukin-6 (IL-6). We next examined the effect

of 2ME2 on MM cells in the bone marrow (BM) milieu. 2ME2

decreases survival of BM stromal cells (BMSCs) and secretion of

vascular endothelial growth factor (VEGF) as well as IL-6

triggered by adhesion of MM cells to BMSCs. We show that

apoptosis induced by 2ME2 is mediated by release of mitochondrial

cytochrome-c (cyto-c) and Smac; followed by activation of

caspases-8, -9, and -3. Finally, 2ME2 inhibits MM cell growth,

prolongs survival, and decreases angiogenesis in a murine model.

These studies therefore demonstrate that 2ME2 mediates anti-MM

activity both directly on MM cells and in the BM

microenvironment. They provide the framework for the use of

2ME2, either alone or in combination with Dex, to overcome drug

resistance and improve outcome in MM.

[email protected]

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3

Introduction

2-Methoxyestradiol (2ME2), a natural metabolite of

estradiol, is a potent anti-tumor and anti-angiogenic agent in

leukemic cells 1-7. Although 2ME2 has potent anti-proliferative

activity in various tumorigenic and non-tumorigenic cell lines,

the mechanisms mediating 2ME2 biologic effects remain unclear.

We and others have demonstrated that multiple myeloma (MM) cells

express estrogen receptors (ERs) and that anti-estrogen compounds

such as, tamoxifen and ICI 182, 780 induce apoptosis in MM cells

8-11. Treatment of MM cells with estrogen (E2) alone did not

alter the growth of MM cells 9. Moreover, despite being a

natural derivative of estradiol (E2), 2ME2 binds poorly to the

ERs and therefore the anti-proliferative effects of 2ME2 are not

mediated by ERs 12,13. These findings, coupled with our studies

suggest that 2ME2-mediated effects on MM cells are independent of

ER.

Previous studies using xenografts and metastatic disease

models in mice have suggested that 2ME2 targets not only the

tumor cell, but also the tumor vasculature 2,14. Recent reports

of increased bone marrow (BM) angiogenesis in multiple myeloma

(MM) 15-17, coupled with the known anti-angiogenic properties of

2ME2 2,14, provide a potential rationale for evaluating 2ME2 as a

novel therapeutic in MM.

In the present study, we characterize the in vitro effects

of 2ME2 against MM cells in the BM microenvironment, as well as

its in vivo anti-MM activity in an animal model. We demonstrate

that 2ME2 induces apoptosis in MM cells resistant to conventional






4

therapies and delineate the signaling cascades mediating its

anti-MM activity. Importantly, we show that 2ME2 directly

decreases cytokine secretion in bone marrow stromal cells

(BMSCs), thereby modulating the tumor BM environment. These

data, along with our demonstration of its anti-MM and anti-

angiogenesis effects in vivo, provide the framework for clinical

trials of 2ME2 to improve patient outcome in MM.






5

Materials and Methods

Cell culture and reagents. Dex-sensitive MM.1S and Dex-

resistant MM.1R human MM cell lines were kindly provided by Dr

Steven Rosen (Northwestern University, Chicago, IL) 18. Dox-6,

Dox-40, and LR-5- drug resistant RPMI 8226 human MM cell lines

were kindly provided by Dr. William Dalton (Moffit Cancer Center,

Tampa, FL) 19. U266 MM cells were obtained from the American

Type Culture Collection (Rockville, MD). All cell lines were

grown in RPMI-1640 media supplemented with 10% heat inactivated

fetal-bovine serum, 100 units/ml penicillin, 100 µg/ml

streptomycin, and 2 mM L-glutamine. Drug resistant cell lines

were cultured with either Dox or Dex to confirm their lack of

drug sensitivity. Mononuclear cells were prepared from MM

patient BM samples by Ficoll-Hypaque density gradient

centrifugation. The MM cells were freshly isolated from patients

relapsing after multiple prior therapies including Dexamethasone,

Melphalan, and thalidomide. An informed consent was obtained from

all patients in accordance with the Helsinki protocol. Tumor

cells (CD138+ 97 + 2.0%) were isolated by CD138 positive

selection method using CD138 (Syndecan-1) Micro Beads and Auto

MACS magnetic cell sorter machine, according to manufacturer's

instructions (Miltenyi Biotec Inc., Auburn, CA). CD138+ myeloma

cells were viable (94-97%) for 2-3 weeks in vitro. Cells were

treated with 0.05 µM Dex (Sigma Chemical Co, St. Louis, MO) or

2ME2 (1-20 µM).






6

Proliferation assays. The BrdUrd incorporation assay was

performed using BrdUrd proliferation assay kit, according to

manufacturer's instructions (Calbiochem, San Diego, CA) with some

modifications 20. Briefly, cells were seeded onto 96-well plates

in RPMI 1640 medium containing 10% FBS. Medium was replaced 24h

later with RPMI-1640 medium containing 2% FBS, with or without

2ME2. Plates were incubated for 48h and 72h, pulsed for 2h-6h

with BrdUrd primary and secondary antibodies, and developed using

a colorimetric reaction. The absorbance at 460 nm was measured

using a Molecular Device plate reader.

MTT assays. Cell viability was assessed by 3-(4,5-

dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT;

Chemicon International Inc., Temecula, CA) assay according to

manufacturer's instructions (Roche Molecular Biochemical’s), with

some modifications. Cells were seeded in 96-well plates in RPMI-

1640 medium containing 5% FBS. 2ME2 or Dex was added 24h later

and incubated for 72h. Cell viability was evaluated as

previously described 21,22.

Enzyme linked Immunosorbent (ELISA) assays. Conditioned

media were generated from 24h cultures of MM patient-derived

BMSCs, MM.1S cells, and BMSCs + MM.1S cells, either untreated or

treated with 3 µM 2ME2; IL-6 and VEGF levels were measured using

ELISAs (R & D Systems, Minneapolis, MN), as previously described

23. BMSC viability at 24h was >80%, as determined by Trypan blue

exclusion assay. Briefly, 96 well plates were coated with either






7

anti-human VEGF or IL-6 antibodies overnight, washed, and then

blocked with 300 µL PBS, 1% bovine serum albumin (BSA), 5%

sucrose and 0.05% NaN3 for 2 h. After washing, 100 µL of sample

or standards diluted in Tris-Cl, 0.1% BSA, and 0.05% Tween 20 (pH

7.3) were added to the wells and incubated for 2 h at room

temperature (RT). The cells were rinsed, and either biotinylated

anti-human VEGF or IL-6 antibodies were added for 2 h at RT.

After a further washing step, the wells were incubated with

streptavidin horseradish peroxidase for 20 min at RT and rinsed.

The reaction was started by the addition of 100 µL H2O2 and

tetramethylbenzidine for 30 min at RT. After stopping the

reaction with 1 µM H2SO4, the optical density of each well was

detected by means of a microtitre plate reader at 450nm, with

correction at 540 nm.

Western blotting. Total cell lysates and cytosolic

extracts were prepared as previously described 24. Briefly,

equal amounts of proteins were resolved by 10% SDS-PAGE and

transferred onto nitrocellulose membranes. Proteins were then

transferred to nitrocellulose filters, blocked by incubation in

5% dry milk in PBST (0.05% Tween-20 in PBS), and probed with

anti-cyto-C, anti-Smac (kindly provided by Dr. Xiaodong Wang,

University of Texas Southwestern Medical Center at Dallas), anti-

Caspase-3 (Santa-Cruz, CA), anti-caspase-8 and 9 (Transduction

Labs, MI), or anti-SHP2 (Santa Cruz, CA) Abs. Blots were then

developed by enhanced chemiluminesence (ECL; Amersham, Arlington

Heights, IL). Preparation of cell lysates for PARP immunoblot






8

analysis was performed as described using C-2-10 anti-PARP

monoclonal antibody 25.

Quantification of apoptosis. Dual fluorescence staining

with DNA-binding fluorochrome Hoechst 33342 (HO) and propidium

iodide (PI) was used to quantitate the percentage of apoptotic

(PI-HO

+) cells using flow cytometry (The Vantage, Becton

Dickinson), as previously described 26. DNA fragmentation assays

were also performed, as in prior studies 27.

Preparation of S-100 cytosolic fractions from MM.1S cells.

MM.1S MM cells were washed twice with PBS, and the pellet was

suspended in 5 ml of ice cold buffer A (20 mM HEPES, pH 7.5, 1.5

mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF,

10 µg/ml leupeptin and aprotinin and pepstatin A) containing 250

nM sucrose. Cells were homogenized by douncing three times in a

Dounce homogenizer with a sandpaper-polished pestle. Cytosolic

fractions were isolated as previously described 24. The

immunoblots were scanned using an LKB produkter (Bromma, Sweden)

Ultrascan XL laser densitometer and analysed with the Gelscan

software package. Signal intensity was determined in a linear

range and normalized to that for SHP2.

Drug preparation and in vivo evaluation in murine tumor

model. For animal studies, 2ME2 was kindly provided by Entremed,

Inc. (Rockville, MD). 2ME2 (100 mg/kg) was suspended in 0.5%

carboxymethycellulose (Sigma). Six-week-old triple immune






9

deficient beige-nude-xid (BNX) mice were obtained from Frederick

Cancer Research and Development Center (Frederick, MD). All

animal studies were conducted according to protocols approved by

the Animal Ethics Committee of the Dana-Farber Cancer Institute.

Mice were observed daily for signs of toxicity. Terminal bleeding

was done under anesthesia using isoflourane inhalation, and

animals were sacrificed by CO2 asphyxiation.

To determine the in vivo anti-MM activity of 2ME2, mice were

inoculated subcutaneously in the flank with 3x107 RPMI 8226 MM

cells in 100 µl of RPMI-1640 media, together with 100 µl matrigel

(Becton Dickinson, Bedford, MA). Drug treatment was started

after the development of measurable tumor. The drug (100 mg/kg)

was given daily using an oro-gastric feeding tube, with

carboxymethylcellulose 0.5% as a control. Serial caliper

measurements of perpendicular diameters were done every other day

to calculate tumor volume, using the following formula: 4/24 x

(shortest diameter)2 x (longest diameter). Animals were

sacrificed if the tumor was > 2 cm or necrotic. Tumor was then

harvested for H/E staining and microvessel density (MVD)

evaluation. For tumor growth studies, 10 mice were used in each

group.

CD31 immunostaining. Tumor tissues were fixed in 10%

neutral buffered formalin and embedded in paraffin, according to

standard histological procedures. After deparaffinization,

tissue sections were pretreated with proteinase K (Roche

Molecular Biochemical’s, Indianapolis, IN) at 37°C for 30 min






10

before staining with rat anti-mouse CD-31 Ab (Pharmingen, San

Diego, CA). Positive staining was detected using secondary

biotinylated rabbit anti-rat Ab, followed by incubation with

streptavidin-horseradish peroxidase (DAKO, Carpenteria, CA). 3-

Amino-9-ethylene carbazol (DAKO) was used as the chromogenic

substrate, and sections were counterstained with Gill’s

Hematoxylin (Fisher Scientific, Fair Lawn, NJ). MVD was

determined by light microscopy, as previously described 28. MVD

assessment was done without knowledge of mouse treatment history.

Areas of most intense neovascularization were counted at high

magnification (400 X), and two investigators using double-headed

light microscopy analyzed at least 5 separate fields. The mean

MVD was compared among the treatment groups and analyzed using

the student's t test.

Statistical Analysis. Non-parametric tests and mixed models

were used to analyze the data. This includes the Wilcoxon's

signed rank test to compare proliferation in untreated and

treated patient cells and the Jonchkeere-Terpstra (J-T) trend

test for assessing viability of MM cell lines resistant to

conventional therapy and of normal donors. The mixed model was

used to adjust for repeated measures in the analysis of the 2ME2

patient MM-derived BMSCs. Analysis of in vivo data included a

Wilcoxon's rank-sum test on the change in the tumor volume and a

log-rank test to compare the overall survival.






11

Results and Discussion

2ME2 induces apoptosis in MM.1S cells. To examine whether

2ME2 induces apoptosis in the MM.1S MM cell line, we performed

both DNA-fragmentation and flow cytometric analyses. MM.1S MM

cells were cultured in the presence or absence of 2ME2 (3 µM) for

24h and 48h. DNA cleavage was analyzed by agarose gel

electrophoresis of the α32P-ATP labeled genomic DNA (Fig 1A, upper panel). Low to undetectable DNA cleavage was observed in

the untreated cells; in contrast, 2ME2 induced significant

cleavage of DNA into oligonucleosomal fragments of approximately

200 base pairs. This pattern was more pronounced following

treatment with 2ME2 at 48h. The finding that 2ME2 induces

fragmentation of DNA into multiples of nucleosome-sized fragments

confirms changes in chromatin structure observed during apoptosis

or program cell death 29. Apoptosis induced by 2ME2 was further

confirmed by flow cytometric analysis using dual PI and Hoechst

staining. Treatment of cells with 2ME2 (3 µM) significantly (P <

0.005) increases the percentage of apoptotic cells (Fig 1A, lower

panel).

Effect of 2ME2 on MM cells resistant to conventional

therapy.

The cytotoxic effects of 2ME2 on various MM cell lines, including

cell lines resistant to conventional therapy (RPMI 8226, Dox-40,

Dox-6, LR5, and MM.1R) was determined using an MTT assay, in the

presence or absence of drug at various concentrations (0.5 µM, 1

µM, 3 µM, 10 µM, and 20 µM.). 2ME2 induced a significant (P <






12

0.0001) decrease in cell viability in a dose dependent manner for

all cell lines (Fig 1B). Fifty-percent decrease in viable cells

was noted at 1.0-3.0 µM of 2ME2 in most of the cell lines. IC50

of 2EM2 for LR-5 cells was 0.5-1.0 µM. We next examined the

effect of 2ME2 on apoptosis in these cells. PI staining

demonstrated a dose-dependent increase in the percentage of

apoptotic (Sub-G1) cells in response to 2ME2 (P < 0.001);

however, estrogen (E2) alone did not induce apoptosis in these

cells (data not shown). This finding is consistent with our

prior study 9, showing that exposure to E2 does not affect growth

kinetics or induce apoptosis of these cells.

Effect of 2ME2 on normal lymphocytes. Normal lymphocytes

from five healthy donors were also treated with various does (0.5

µM, 1.0 µM, 3.0 µM, 10 µM, and 20 µM) of 2ME2 and analyzed for

both inhibition of proliferation and apoptosis by BrdUrd and PI

staining, respectively. In contrast to MM cells, survival of

normal lymphocytes from five healthy donors was not altered

significantly (P = 0.23 from J-T trend test), even at higher

doses (3-20 µM) of 2ME2 (Fig 1C). Moreover, no significant (P =

0.31) apoptosis of normal lymphocytes was observed in response to

2ME2 (data not shown). Taken together, these findings indicate

that 2ME2 has selective anti-MM activity.






13

Fig. 1. Selective cytotoxicity of 2ME2 against human MM cells. (A) MM.1S cells

were treated with 2ME2 (3 µM) for the indicated times and analyzed for apoptosis by






14

both DNA fragmentation (upper panel is a representative of three separate experiments

with similar results) and flow cytometric analysis of PI- and HO+ apoptotic cells

(lower panel is the mean + SD from three independent experiments, P < 0.005). (B) MTT

assay were performed after incubation of MM cell lines (MM.1S --∆--; RPMI-8226 --Ο---;

LR-5 --♦--; Dox-6 --◊--; Dox-40 --�--; MM.1R --�--) with the indicated doses of 2ME2

for 72h. Results are mean + S.D from five independent experiments, P < 0.0001 for all

cell lines. (C) Effect of treatment with 2ME2 (0-20 µM) for 72h on normal lymphocytes

viability, assessed by MTT assay. Results are the mean + SD of five independent

experiments; P = 0.23 from Jonchkeere-Tepstra (J-T) test for trend. (D) MM cells

(CD138+) from five patients (#1-5) were treated with 2ME2 (9 µM) for 72h, followed by

BrdUrd assay. Values are the mean + SD of triplicate samples, P = 0.06; experiments

were repeated three times with similar results. (E) 2ME2 induces proteolytic cleavage

of PARP in patient MM cells. CD138+ cell from 2 MM patients and normal lymphocytes

from 2 healthy donors were treated with 2ME2 (10 µM) for 72h, harvested, and total

protein lysates were subjected to SDS-PAGE analysis. Immunoblot analysis of the

lysates was performed with anti-PARP Ab. 'FL' indicates 'full length' and 'CF' denotes

'cleaved fragment'.

Effect of 2ME2 on patient MM cells. We next determined the

effects of 2ME2 on MM cells freshly isolated from patients

relapsing after multiple prior therapies including Dexamethasone,

melphalan and thalidomide. Tumor cells were purified from bone

marrow aspirates by CD138 positive selection using CD138

(Syndecan-1) Micro Beads and Auto MACS magnetic cell sorting.

Treatment of MM cells from 5 patients (Patients # 1-5), with 2ME2

(9 µM) significantly (P = 0.06) decreased growth, as measured by

BrdUrd incorporation (Fig 1D). In order to further determine






15

whether growth inhibition correlates with apoptosis in patient MM

cells, we next determined the effects of 2ME2 (9 µM) on poly

(ADP-ribose) polymerase (PARP) cleavage, a known signature event

during apoptosis 30,31. 2ME2 (9 µM) induced PARP cleavage in

patient MM cells, consistent with apoptosis, but failed to induce

PARP cleavage in normal lymphocytes (Fig 1E). These findings

correlate with our earlier results (Fig 1C) as well as other

study 32 demonstrating lack of cytotoxicity of 2ME2 (9 µM) on

normal lymphocytes. Taken together, our results suggest that

2ME2 inhibits growth and induces apoptosis in patient MM cells.

Effect of 2ME2 on patient MM-derived Bone Marrow Stromal

Cells (BMSCs). In MM, tumor cells are predominantly localized in

the BM microenvironment due to their adherence both to

extracellular matrix proteins and to BMSCs. This interaction

between MM cells and BMSCs triggers production of cytokines

mediating autocrine and paracrine growth and survival of MM

cells, as well as protection against drug-induced apoptosis 33.

We therefore next examined the effect of 2ME2 on patient MM-

derived BMSCs from 5 MM patients. Treatment of BMSCs with 2ME2

significantly (Slope= -5.5, P = 0.002) decreases the viability

(Fig 2A, 3 Patients A-C are shown). In order to determine

whether 2ME2-induced growth inhibition correlates with apoptosis

in

patient BMSCs, we next examined the effects of 2ME2 (9 µM) on

PARP cleavage. 2ME2 (3 µM) induced PARP cleavage, consistent

with apoptosis (Fig 2B, patient A and B). Taken together, these






16

results suggest that 2ME2 acts not only directly on MM cells, but

also affects the BM microenvironment.

Fig. 2. Effect of 2ME2 on BMSCs and IL-6 secretion (A) Patient MM-derived BMSCs

(Patients A-C) were treated with 2ME2 (3-9 µM) for 72h, and viability was assessed by






17

MTT assay. Results are the mean + SD from triplicate samples, P = 0.002. (B) 2ME2

induce proteolytic cleavage of PARP in patient MM-BMSCs cells. BMSCs from 2MM

patients were treated with 2ME2 (3 µM) for 72h, and harvested; total protein lysates

were subjected to SDS-PAGE analysis. Immunoblot analysis of the lysates was performed

with anti-PARP antibody. 'FL' indicates 'full length' and 'CF' denotes 'cleaved

fragment. (C) Effect of 2ME2 on MM cell adhesion-induced IL-6 secretion in BMSCs. IL-

6 levels were measured using IL-6 specific ELISA in supernatants of 24h cultures of

BMSCs, MM.1S cells, and BMSCs + MM.1S cells, in the presence or absence of 2ME2 (3

µM).

Effect of 2ME2 on Interleukin-6 (IL-6) secretion in BMSCs.

Adhesion of MM cells to BMSCs trigger secretion of factors such

as, IL-6, from BMSCs, which regulate the growth of MM cells in

autocrine or paracrine manner 34. In order to determine whether

2ME2 also affect secretion of IL-6, we next examined the effect

of 2ME2 on IL-6 production in the BM microenvironment. As seen

in Fig 2C, 2ME2 significantly (P < 0.005) inhibits IL-6 secretion

in MM patient BMSCs triggered by MM cell adhesion (Fig 2C).

Reports that high serum levels of IL-6 contribute to clinical

chemo resistance and treatment failure 35, coupled with our

demonstration that 2ME2 decreases the MM adhesion-induced IL-6

secretion from BMSCs, suggests that 2ME2 may overcome drug

resistance in patients with advanced MM.






18

Fig. 3. Effects of Dex and IL-6 on 2ME2-induced cytotoxicity in MM cells. (A) MM.1S

cells were cultured in control media alone and with Dex (0.05 µM), 2ME2 (3 µM) or Dex






19

+ 2ME2. At 48 h, cells were harvested and analyzed by BrdUrd assay. Results are mean

+ SD of three independent experiments (P < 0.005). (B) MM.1S cells were treated with

2ME2 (3 µM) or Dex (0.05 µM), in the presence or absence of IL-6 (10 ng/ml). At 48 h,

cells were harvested and viability analyzed by MTT assay. The median viability was

41% for Dex and 78% for Dex + IL-6 (P = 0.05, as determined by one-sided Wilcoxon

rank-sum test), whereas for 2ME2 the median viability was 45% with 2ME2 alone and 48%

with 2ME2 + IL-6 (P = 0.20, Wilcoxon test, as above). Results are mean + SD of three

independent experiments.

Effect of Dex and IL-6 on response of MM cells to 2ME2. To

determine whether the effects of 2ME2 are additive with

conventional therapies, we next examined whether Dex (0.0005- 0.5

µM) enhanced 2ME2 (3 µM) effects on proliferation of Dex-

sensitive (MM.1S) MM cells. As seen in Fig 3A, the combination

of 2ME2 and Dex induces more growth inhibition as compared to the

treatment of MM.1S cells with 2ME2 alone. Dex (0.05 µM) and 2ME2

(3 µM) increased growth inhibition by 30%, relative to cultures

with 2ME2 alone (P = 0.05, one-sided Wilcoxon rank-sum test).

PI staining after Dex (0.05 µM) and 2ME2 (3 µM) revealed 93 +

6.1% apoptosis versus 66 + 4.3% cell death induced by Dex alone.

We and others have shown that IL-6 protects against Dex-

induced apoptosis 36-39, and we therefore next examined the

effect of IL-6 on 2ME2-induced apoptosis. MM.1S cells were

treated with 2ME2 (3 µM) or Dex (0.05 µM ), in the presence and

absence of IL-6 (10 ng/ml). As seen in Fig 3B, the median

viability was 41% for Dex and 78% for Dex + IL-6 (P = 0.05, as

determined by one-sided Wilcoxon rank-sum test), whereas for 2ME2






20

the median viability was 45% with 2ME2 alone and 48% with 2ME2 +

IL-6 (P = 0.20, Wilcoxon test, as above). These findings suggest

that IL-6 fail to abrogate effects of 2ME2 on MM.1S cells. In

contrast and as in our prior studies 38, IL-6 blocks Dex-induced

decreased in MM.1S cell viability. Taken together, our data

suggest that 2ME2 overcomes the growth and protective effects of

IL-6 on MM cells and also indicate distinct mechanisms of action

for 2ME2 and Dex against MM cells.

2ME2 induces mitochondrial release of cytochrome-c and Smac.

Having shown the biologic sequelae of 2ME2 in MM cells, we next

examined the signaling mechanisms mediating apoptosis triggered

by 2ME2. Our prior studies have characterized Dex-induced

apoptotic mechanisms in MM.1S MM cells, and we therefore studied

2ME2 effects using these cells in order to compare differential

signaling cascades triggered by Dex versus 2ME2. Previous

studies have shown that 2ME2-induced apoptosis involves

mitochondrial damage 32, we first therefore determined whether

treatment of MM.1S cells is associated with the release of

mitochondrial apoptogenic proteins, including cytochrome-c (cyto-

c) and second mitochondrial activator of caspases (Smac). Our

recent studies have demonstrated that Smac, but not cyto-c, is

released from mitochondria into the cytosol during Dex-induced

apoptosis in MM.1S cells 24,40. To determine whether 2ME2-

induced apoptosis in MM.1S cells is associated with the

mitochondrial release of cyto-c and/or Smac, we treated MM.1S

cells with 2ME2 for various times and analyzed cytosolic and






21

mitochondrial extracts for levels of cyto-c and Smac. As seen in

Fig. 4A and 4B (upper panels), 2ME2 increase cyto-c and Smac

levels in cytosol at 48 h. Densitometric analysis of the

immunoblot demonstrated that 2ME2 induces a 4-5 fold increase in

the cytosolic cyto-c and Smac levels, as compared to untreated

cells (Fig 4A and 4B). The increase in cyto-c and Smac levels

was associated with a concomitant decrease in mitochondrial Smac

or cyto-c levels (data not shown). Moreover, the 2ME2-induced

increases in cytosolic cyto-c or Smac levels were specific, since

no changes were observed in levels of cytosolic SHP2 (Fig 4A and

4B, lower panels). These results suggest that 2ME2-induced

apoptosis is accompanied by accumulation of both Smac and cyto-c

in the cytosol. In contrast, and as in our prior studies 24,

Dex-induced apoptosis was associated with the mitochondrial

release of Smac, but not of cyto-c (Fig 4A and 4B).

To determine whether 2ME2 also induces cyto-c or Smac

release in other MM cell lines, we treated Dox-40, MM.1R and LR-5

MM cells with 2ME2 (3 µM) for 48h, followed by analysis of

cytosolic extracts for levels of cyto-c and Smac. As seen in

Fig. 4C and 4D (upper panels), 2ME2 also triggers accumulation of

cyto-c and Smac levels in the cytosol of Dox-40 and LR-5 MM

cells. Similar results were obtained using MM.1R MM cell line

(data not shown). 2ME2-induced increases in cytosolic cyto-c or

Smac levels were specific, since no changes were observed in

levels of cytosolic SHP2 (Fig 4C and 4D, lower panels). Taken

together, these results suggest that 2ME2-induced apoptosis is

accompanied by accumulation of both Smac and cyto-c in the






22

cytosol.






23

Fig. 4. 2ME2 induces mitochondrial release of cytochrome-c (A) and Smac (B).

MM.1S cells were treated with 2ME2 (3 µM) or Dex (0.05••M) and harvested at 48h.

Cytosolic proteins were separated by 12.5% SDS-PAGE and analyzed by immunoblotting

with anti-cyto-c (A, upper panel) or anti-Smac (B, upper panel) Abs. As a control for

equal loading of proteins, filters were also reprobed with anti-SHP2 Ab (A, and B,

lower panels). Blots are representative of three independent experiments. (C) and

(D) Dox-40 and LR-5 MM cells were treated with 2ME2 (3 µM) and harvested at 48h.

Cytosolic proteins were separated by 12.5% SDS-PAGE and analyzed by immunoblotting

with anti-cyto-c (C, upper panels) or anti-Smac (D, upper panel) Abs. As a control

for equal loading of proteins, filters were reprobed with anti-SHP2 Ab (C, and D,

lower panels). Blots are representative of three independent experiments.

Densitometric analysis of the immunoblot demonstrated that 2ME2 induces a 4-5 fold

increase in the cytosolic cyto-c and Smac levels, as compared to untreated cells.

2ME2-induced apoptosis is mediated by activation of caspase

cascade. Smac promotes caspase activation via the cyto-c/Apaf-

1/caspase-9 pathway 41, and our prior study in MM cells

demonstrated that Dex triggers release of Smac from mitochondria

into cytosol, with sequential activation of caspase-9 and

caspase-3 40. To examine whether 2ME2-induced Smac release and

apoptosis in MM cells is associated with activation of caspase-9,

cytosolic extracts from 2ME2-treated MM.1S cells were subjected

to immunoblot analysis using anti-caspase-9 antibody. As seen in

Fig. 5A, treatment of MM.1S cells with 2ME2 (3 µM) induces

proteolytic cleavage of procaspase-9 into 37 and 35 KDa

fragments. We also assayed for catalytic activity of caspase-9,

using LEHD-pNA conjugated substrate in a colorimetric protease






24

assay 40. Incubation of cytosolic extracts from 2ME2-treated

MM.1S cells with LEHD-pNA was associated with cleavage of LEHD-

pNA (data not shown). Taken together, these findings demonstrate

that treatment of MM.1S cells with 2ME2 is associated with

activation of caspase-9.

Multiple studies have shown that upstream events mediating

mitochondrial cyto-c release induce sequential activation of

caspase-8, cleavage of Bid, translocation of cleaved Bid from the

cytoplasm into mitochondria, and release of cyto-c 42,43. Since

2ME2 induces release of cyto-c, we next assayed for activation of

caspase-8 by 2ME2. As seen in Fig. 5B, 2ME2 (3 µM) also triggers

proteolytic cleavage of caspase-8. In contrast, and as in our

prior study 40, Dex-induced apoptosis is not associated with

caspase-8 activation (Fig 5B).

Previous studies have demonstrated that both caspase-8 and -

9 activate downstream apoptotic caspase-3 (CPP32) 44.

Furthermore, in a cell free system, the addition of purified

cyto-C to cyto-c-depleted extracts activates caspase-3 and DNA-

fragmentation 45. Since treatment of MM.1S cells with 2ME2

activates both caspase-8 and -9, we next determined whether

caspase-3 is cleaved and activated in response to 2ME2. MM.1S

cells were treated with 2ME2 (3 µM), and cell lysates were

subjected to immunoblotting with either caspase-3 or its known

substrate PKC δ 24. 2ME2 induces caspase-3 cleavage and

activation, as evidenced by cleavage of caspase-3 (Fig 5C and

data not shown). Dex treatment served as a positive control.






25

Fig 5. Delineation of 2ME2 and Dex-induced caspase cascade. 2ME2 induces

activation of caspase-9 (A) and -8 (B). MM.1S cells were treated with 2ME2 (3 µM) and






26

Dex (0.05•µM) and harvested at 48h. Cytosolic proteins were separated by 12.5% SDS-

PAGE and analyzed by immunoblotting with anti-caspase-8 (cas-8) and -9 (Cas-9) Abs.

Blots are representative of three independent experiments. (C) Cleavage of caspase-3

induced by 2ME2. MM.1S cells were treated with 2ME2 (3 µM) and Dex (0.05•µM), and

harvested at 48h. Total cell lysates were analyzed by immunoblotting with anti-

caspase-3 Ab. 'FL' indicates 'full length' and 'CF' denotes 'cleaved fragment'. (D)

2ME2 and Dex-induced differential signaling cascades in MM.

Collectively, a comparative analyses of 2ME2 and Dex-induced

apoptotic signaling cascades therefore suggests that 2ME2, in

contrast to Dex, triggers multiple signaling pathways (Fig. 5D).

For example, 2ME2 induces release of both cyto-c and Smac,

whereas Dex triggers the release of only Smac; 2ME2 activates

both caspase-8 and -9, whereas Dex triggers activation of only

caspase-9; and finally, IL-6 protects against Dex-, but not 2ME2-

induced apoptosis. Since 2ME2 triggers apoptosis in Dex-

resistant MM cells, elucidating the 2ME2-induced apoptotic

signaling pathways may not only identify additional apoptotic

targets, but also delineate abnormalities in signaling pathways

which confer Dex-resistance.

2ME2 suppresses in vivo tumor growth in Scid-mouse model.

Having shown the signaling mechanisms mediating the anti-MM

effects of 2ME2 in vitro, we next determined whether these in

vitro effects correlate with in vivo activity of 2ME2, using our

Scid-mouse model. Immune deficient beige-nude-xid (BNX) mice

were inoculated subcutaneously in the flank with 3x107 RPMI 8226






27

MM cells in 100 µl of RPMI-1640 media, together with 100 µl

matrigel. Daily oral administration of 2ME2 (100mg/kg) starting

after the development of measurable tumor significantly reduced

MM tumor growth (35-40%, P < 0.05)(Fig 6A) and increased survival

(Fig 6B), compared with the control group treated with the

vehicle (Matrigel) only. No significant toxicity, as evidenced

by lack of weight changes, was observed in any treatment groups.

These findings suggest that 2ME2 at molar concentrations is

effective in controlling growth of human MM in mice. Other in

vivo preclinical studies have shown that 2ME2 is also effective

in reducing tumor burden in Meth-A sarcoma or B16 melanoma; in

non estrogen-dependent human breast carcinoma (MDA-MB-435); and

in human pancreatic (MIA PaCa-2) xenograft mouse models 2,14.

Since 2ME2 is a known anti-angiogenic agent and recent

reports show increased BM angiogenesis in MM 15-17, we next

examined the effect of 2ME2 on intratumoral microvessel density

(MVD), evidenced by CD-31 immunostaining of human MM tumors in

mice. A significant decrease in MVD was noted in 2ME2-treated

animals (Fig 6D) versus the cohort treated with control vehicle

alone (Fig 6C). The number of CD-31+ blood vessels per low power

(400X) field was 10.30 + 1.0 in 2ME2-treated versus 19.10 + 1.9

in control groups (P < 0.05) (Fig 6E). Taken together, these

studies demonstrate that 2ME2 is effective in inhibiting both

tumor cell growth and angiogenesis in MM.






28

Fig. 6. 2ME2 suppresses plasmacytoma growth and increases survival in immune

deficient beige-nude-xid (BNX) mice. Mice were inoculated subcutaneously in the flank

with 3 x 107 RPMI 8226 MM cells in 100 µl of RPMI-1640 media, together with 100 µl

matrigel. 2ME2 (100 mg/kg) was started after the development of measurable tumor and

given daily using an oro-gastric feeding tube; carboxymethylcellulose 0.5% served as a

control. Serial caliper measurements of perpendicular diameters were done every other

day to calculate tumor volume (A). Each time point represents the mean + SD of 10

mice. Survival of 2ME2 versus control animals (B). Frozen tumor samples were stained

by anti-CD-31 antibody, and the number of vessels were counted and averaged in control

(C) versus 2ME2-treated (D) animals. Arrows denote CD31+ positive blood vessels. (E)

Significantly fewer CD31+ blood vessels were observed in 2ME2-treated versus control

animals (p < 0.05).






29

Having shown in vivo effects of 2ME2 on angiogenesis, we

next determined whether 2ME2 altered VEGF secretion in the BM

microenvironment. 2ME2 induces a significant (P < 0.05) decrease

in VEGF secretion in MM.1S cells (34 + 2.1% decrease, n=3) and in

BMSCs (41 + 3.2% decrease, n=3). Our results are consistent with

other study suggesting that VEGF expression, as evaluated by

immunohistochemical analysis, is down regulated concomitant with

tumor angiogenic suppression. 46. Since we and others have

recently demonstrated that VEGF is not only an angiogenic factor,

but also triggers growth and migration in MM cells 16,17, our

present findings suggest an additional mechanism whereby 2ME2

mediates anti-MM activity.

Our study therefore demonstrates that 2ME2 can both directly

induce apoptosis in MM cells and act in the BM microenvironment

to decrease cytokines mediating tumor cell growth and survival,

as well as angiogenesis. These studies provide the framework for

clinical trials of 2ME2 to overcome drug resistance and improve

patient outcome in MM.






30

Acknowledgements

We thank Dr. Xiaodong Wang for providing Smac related

reagents. The authors gratefully acknowledge the technical

assistance of Kamal Chauhan.






31

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2Methoxyestradiol overcomes drug resistance in multiple myeloma cells

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