The Effects of Elecampane on 4T1 Murine Breast Cancer CellsEvan McElwain & Sarah Kelly, Wayland Baptist University

www.cherylgal.com

Background on breast cancer• Why breast cancer?• 1 in 8 women will get breast cancer in

their lifetime (www.cancer.org).• In 2014 alone• 231,840 invasive breast cancer cases• 40,290 deaths from breast cancer

• Treatments (National Cancer Institute)• Surgery• Chemotherapy• Hormone therapy• Medicinal plant extracts (high percentage of the world only has access to

these treatment options)

4T1 Cells

• Murine breast cancer cells that emulate stage 4 cancer cells• Can be injected into balb/c mice and they form a tumor

at the injection site (typically mammary fat pad), then the cells metastasize to the liver, lungs and brain.

Elecampane• We previously found elecampane to have cytotoxic effects

on 4T1 cells when we screened a series of plants commonly used in Native American medicinal treatments (you may have seen that poster presentation last year)

• Elecampane is known to have anti inflammatory effects

• In this study, we sought to purify and isolate the specific molecules that may account for the cytotoxic effects of elecampane

EtOH

Pipsissewa

Poke Root

Horsetai

l

Slippery

Elm

Barberry

St. Jo

hn's Wort

Yellow Dock

Root

Goldseal

Elecampan

e

Comfrey

Witch Haze

l

Lungw

ort

Mugwort

Fringe

Tree

Yarrow Le

af

Solomon's L

eaf

Chaparr

al

Celandine

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

Native American Medicinal Plant Extracts Re

lativ

e Lig

ht U

nits

EtOH

Horsetail 1

00 ug/mL

Horsetail 7

5 ug/mL

Horsetail 5

0 ug/mL

Horsetail 2

5 ug/mL

Horsetail 1

0 ug/mL

Horsetail 5

ug/mL

Yello

w Dock Root 1

00 ug/mL

Yello

w Dock Root 7

5 ug/mL

Yello

w Dock Root 5

0 ug/mL

Yello

w Dock Root 2

5 ug/mL

Yello

w Dock Root 1

0 ug/mL

Yello

w Dock Root 5

ug/mL

Elenca

mpane 100 ug/m

L

Elenca

mpane 7

5 ug/mL

Elenca

mpane 5

0 ug/mL

Elenca

mpane 2

5 ug/mL

Elenca

mpane 10 ug/m

L

Elenca

mpane 5 ug/m

L0

2000

4000

6000

8000

10000

12000

14000

16000

18000Titration of Plant Extracts

Rela

tive

Light

Uni

ts

Yellow Dock presented in a poster by Ashley Rivera

Elecampane story presented here

Methods• Reflux• Distillation• Fraction collection & UV• Cytotoxicity assay of fractions (MTS)• HPLC separation and collection of cytotoxic fractions • Individual peaks from HPLC were collected and assayed for cytotoxicity• Mass Spectrometry was performed to identify cytotoxic molecules

Reflux

25 grams of powdered elecampane was refluxed in 500 mL of dichloromethane.

Distillation

25 ml of ethanol was added and the dichloromethane was distilled off, yielding a concentrated extract in ethanol.

Fraction Collection& UV Analysis

1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 970

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Elecampane @ 280 nm

Fractions (from large to small molecules)Ab

sorb

ance

Cytotoxicity was determined for fractions 26-77

Crude extracts were size fractionated on a sephadex LH20 column. In

general, the larger the molecule, the lower the retention time.

MTS Assay

Fracti

on 26

Fracti

on 28

Fracti

on 30

Fracti

on 32

Fracti

on 34

Fracti

on 36

Fracti

on 38

Fracti

on 40

Fracti

on 42

Fracti

on 44

Fracti

on 46

Fracti

on 48

Fracti

on 50

Fracti

on 52

Fracti

on 54

Fracti

on 56

Fracti

on 58

Fracti

on 60

Fracti

on 62

Fracti

on 64

Fracti

on 66

Fracti

on 68

Fracti

on 70

Fracti

on 72

Fracti

on 74

Fracti

on 760

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Fraction Number

Abso

rban

ce a

t 492

nm

Fractions in brackets were hypothesized to contain cytotoxic molecules and were analyzed and separated with HPLC

A B C

High Performance Liquid Chromatography (HPLC)

A

B

C

Solid phase: C18Mobile phase: 0-75% methanol gradient over 30 minutes

MTS

Cel

l Via

bilit

y As

say

This fraction identified via

mass spec

9.0 167.8 326.6 485.4 644.2 803.0Mass (m/z)

9.9E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tensit

y

4700 Reflector Spec #1 MC[BP = 125.1, 99443]

125.1031

379.1454

172.0545

190.0551

212.0349

228.0072

191.0504

38.9244

294.0913

141.0882

335.1234

192.0597

164.0609

146.0463

189.0399

249.9926

227.0741

147.0378

637.3599

17.9876

337.1362

265.9694

116.0490

279.1028

208.0588

441.0521

568.1853

40.9211

368.4475

312.1510

417.0792

61.0067

401.1024

148.0579

454.3597

659.3532

134.0995

85.0472

622.1078

301.2954

175.1112

326.3906

104.0824

524.1852

550.1758

356.2477

675.3231

468.3640

226.1839

251.9913

382.2947

483.1634

240.2399

194.1002

428.3129

74.0660

606.1305

506.1744

644.0947

344.1421

159.0710

539.1312

290.1364

494.4045

587.1989

29.1495

688.0797

263.1346

710.2012

632.4156

753.4590

783.4502

739.2372

9.0 167.8 326.6 485.4 644.2 803.0Mass (m/z)

1.0E+5

0

10

20

30

40

50

60

70

80

90

100

% In

tensit

y

4700 Reflector Spec #1 MC[BP = 379.1, 100000]

379.1423

172.0485

125.0852

190.0344

212.0092

335.0842

191.0273

192.0366

294.0565

164.0421

146.0289

87.9840

440.9996

306.2652

135.9894

278.1878

233.9871

454.3086

38.9183

209.1735

147.0200

189.0182

442.9978

100.0756

337.0942

227.0464

568.1176

206.1254

426.2885

258.2530

46.0227

107.0127

362.0544

148.0408

179.0352

401.0518

60.0141

280.2400

167.9648

412.2741

468.3101

17.9855

382.2794

122.0275

334.0726

482.3340

296.2312

317.0674

424.2896

445.0124

524.1223

456.2936

231.1075

550.1048

247.1065

89.9885

74.0566

220.1410

503.2545

535.2457

656.0407

346.2424

358.2123

265.1320

40.9161

570.1277

370.2290

203.1190

29.1353

582.3360

145.0558

109.0637

637.2966

672.0117

622.0347

598.3282

133.0546

751.4176

783.3980

767.4052

188.1002

610.3452

53.1121

685.3591

710.3797

735.3952

699.3600

724.3777

control

elecampane

MALDI Mass Spectrometry

440 mw

9.0 167.8 326.6 485.4 644.2 803.0Mass (m/z)

1.0E+5

0

10

20

30

40

50

60

70

80

90

100

% In

tensit

y

4700 Reflector Spec #1 MC[BP = 379.1, 100000]

379.1423

172.0485

125.0852

190.0344

212.0092

335.0842

191.0273

192.0366

294.0565

164.0421

146.0289

87.9840

440.9996

306.2652

135.9894

278.1878

233.9871

454.3086

38.9183

209.1735

147.0200

189.0182

442.9978

100.0756

337.0942

227.0464

568.1176

206.1254

426.2885

258.2530

46.0227

107.0127

362.0544

148.0408

179.0352

401.0518

60.0141

280.2400

167.9648

412.2741

468.3101

17.9855

382.2794

122.0275

334.0726

482.3340

296.2312

317.0674

424.2896

445.0124

524.1223

456.2936

231.1075

550.1048

247.1065

89.9885

74.0566

220.1410

503.2545

535.2457

656.0407

346.2424

358.2123

265.1320

40.9161

570.1277

370.2290

203.1190

29.1353

582.3360

145.0558

109.0637

637.2966

672.0117

622.0347

598.3282

133.0546

751.4176

783.3980

767.4052

188.1002

610.3452

53.1121

685.3591

710.3797

735.3952

699.3600

724.3777

Multiple Charge Variances of 440 mw

440 mw

Conclusion

• We have purified a molecule that appears to have a molecular weight of 440 from elecampane that is cytotoxic to 4T1 breast cancer cells.

Future Studies

• First, we will scale up the purification• This study used a 1cm diameter LH 20 column loading 1mL of

extract, next we will use a 4cm diameter LH 20 column loading 15mL of extract

• Determine the identity of the 440 molecular weight molecule by using NMR and magnetic sector mass spectrometry• Mouse studies • We plan to utilize GFP/luciferase expressing 4T1 cells to perform in vivo mouse studies using 3D fluorescence tomography

Acknowledgements• Welch Foundation (Grant # BW-0044)• Wayland Baptist University• Dr. Robert Moore• Texas Tech University Biotechnology core facility• Susan Sanfrancisco

• Trevor Burrow• Jarrett Ross• Libby Saultz

Questions??