Quanta Confidential QUANTA WBU Study Report 1 昭正 2008/08/01.
Evan WBU spring research day 2015
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Transcript of Evan WBU spring research day 2015
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
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
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