Therapeutic efficacy of Gc protein-derived macrophage ...
Transcript of Therapeutic efficacy of Gc protein-derived macrophage ...
Therapeutic efficacy of Gc protein-derived
macrophage activating factor (GcMAF)
for adenocarcinoma.
Nobuto Yamamoto, Masahiro Urade, and Masumi Ueda.
Socrates Institute for Therapeutic Immunology, Philadelphia, PA
and Hyogo College of Medicine, Hyogo, Japan.
Inflammation in Cancerous and Noncancerous Tissues Inflammation Induced by Administration of BCG
a. b.
a. Intratumor administration of BCG results in eradication of
b. Administration of BCG to noncancerous tissues results in no
Refs: 1. Morton, D. et al. 1970, Surgery 68: 158-164.
2. Zbar, B. and Tanaka, T. 1971, Science 172: 271-273.
local as well as metastasized tumors, indicating development of immunity against the tumors.
significant effect on tumors.
O
phospholipase A 2
I. Ester phospholipids:
O
H2 C OC(CH 2 )n CH 3
H3 C(CH 2 )n CO C H
O
H2 C O P O(CH 2 )2 N(CH 2 )3
O
OH
A. O
H2 C OC(CH 2 )n CH3
HO C H
H2 C O P O(CH 2 )2 N(CH 2 )3
O
OH
B.H2 C OH
HO C H
H2 C O P O(CH 2 )2 N(CH 2 )3
O
OH
C.
acyltransferase
lysophospholipase
II. Ether phospholipids:
H2 C OCH 2 (CH 2 )n CH3
H3 C(CH 2 )n CO C H
O
H2 C O P O(CH 2 )2 N(CH 2 )3
O
OH
D.H2 C OCH 2 (CH 2 )n CH 3
HO C H
H2 C O P O(CH 2 )2 N(CH 2 )3
O
OH
E.H2 C OCH 2 (CH 2 )n CH3
HO C H
H2 C O P OH
OH
F.
phospholipase A 2
acyltransferase
lysophospholipase D
acid phosphatase
Metabolic pathways of ester- and ether-phospholipids
H2 C OCH 2 (CH 2 )n CH3
HO C H
H2 C OH
G.
in inflamed tissues
I. Inflammation in noncancerous tissues
1. Phosphatidylcholine (or other Phospholipids)
Phospholipase A 2
Lysophospholipase
3. Inert compounds
1. Alkylphospholipids
Phospholipase A 2
Lysophospholipase D and acid phosphatase
3. Alkylglycerols: are potent macrophage activating agents
Dodecylglycerol ( DDG : one of alkylglycerols)
For macrophage activation, DDG is 400 times more active than lyso-Pc
Conclusion: These information suggests that highly activated macrophages can kill and eradicate cancerous cells.
2. Lysophosphatidylcholine: ( Lyso-Pc ; one of lysophospho-
lipids) is capable of activation
of macrophages.
II. Inflammation in cancerous tissues (e.g., administration of BCG)
2. Lyso-alkylphospholipids: are potent macrophage activating agents
Publications:
1. Yamamoto, N., et al. 1987. Cancer Immuno. Immunother. 25: 185-192.
2. Yamamoto, N. and Ngwenya, B. Z. 1987. Cancer Res. 47: 2008-2013.
3. Yamamoto, N., St. Claire, D. A., Homma, S., and Ngwenya, B. Z. 1988.
Cancer Res. 48:6044-6049.
4. Homma, S. and Yamamoto, N. 1990. Clin. Exp. Immunol. 79: 307-313.
activates macrophages
II. In vitro: Cultivation of [Macrophages] + lyso -Pc or DDG
lyso-Pc or DDG
macrophages alone
no macrophage activation
III. In vitro: Cultivation of [Macarophages + lymphocytes] + lyso -Pc or DDG
lyso-Pc or DDG
IV. Macrophage activation signaling pathway
macrophages + lymphocytes
I. In vivo: Administration of lyso-Pc (20 µg) or DDG (50ng) into mice
lyso-Pc or DDG
B T Mφ activation
activation
B
BT
T
Lyso-Pc or DDG
Proactivating factorB
Mφ
Activated M φ
Mφ
Inflammation primed macrophage activation requires participation of B and T cells.
TB
To search for macromolecular factor to activate macrophages(macrophage activating factor)
1. In serum free medium
lyso-Pc or DDG
Suggesting macrophage activation requires at least one serum component.
2. Macrophage activation requires serum vitamin D binding proteins
lyso-Pc or DDG
+ Gc protein (1 ng/ml)B T Mφ
activation
(known as Gc protein)
B T Mφ no activation
Publications:
1. Yamamoto, N., Homma, S. and Millman, I. 1991. J. Immunol. 147: 273-280.
2. Yamamoto, N. and Homma, S. 1991. Proc. Natl. Acad. Sci. USA 88: 8539-8543.
3. Yamamoto, N., Kumashiro, R., Yamamoto, M., Willett, N. P. and Lindsay, D. D.
1993. Infect. Imm. 61: 5388-5391.
4. Yamamoto, N. and Kumashiro, R. 1993. J. Immunol. 151: 2794-2902.
5. Yamamoto, N., Willett, N. P. and Lindsay, D. D. 1994. Inflammation 18: 311-322.
6. Yamamoto, N., Naraparaju, V. R. and Asbell, S. O.1996. Cancer Res. 56: 2827-
7. Yamamoto, N. 1996. Molecular Immunol. 33: 1157-1164.
8. Yamamoto, N. and Naraparaju, V. R. 1997. Cancer Res. 57: 2187-2192.
9. Koga, Y., Naraparaju, V. R. and Yamamoto, N. 1999. Proc. Soc. Exp. Biol. Med.
220: 20-26.
2931.
12Val
13Cys
14Lys
15Glu
16Phe
17Ser
18His
19Leu
20Gly
21Lys
22Glu
23Asp
24Phe
25Thr
26Ser
27Leu
28Ser
29Leu
30Val
31Leu
32Tyr
33Ser
34Arg
35Lys
36Phe
37Pro
38Ser
39Gly
40Thr
41Phe
42Glu
43Gln
44Val
45Ser
46Gln
47Leu
48Val
49Lys
50Glu
51Val
52Val
53Leu
54Leu
55Thr
56Glu
57
Ala58Cys
59Cys
60Ala
61Glu
62Glu
63Ala 64
Asp65Pro
66Asp
67Cys
68Tyr
69Asp
70Thr
71Arg
72Thr
73Ser
74Ala
75Leu
76Ser
77Ala
78Lys
79Ser
80Cys
81Glu
82Ser
83Asn
84Ser
85Pro
86Phe
87Pro
88
Val
89His
90Pro
91Gly
92Thr
93Ala
94Glu
95Cys
96Cys
97Thr
98Lys
99Glu100
Gly
101Leu 102
Glu103Arg
104Lys
105Leu
106Cys
107Met 108
Ala
109Ala
110Leu
111Lys
112His
113Gln
114Pro
115Gln
116Glu
117Phe
118Pro
119Thr
120Tyr
121Val
122Glu
123Pro
124Thr
125Asn
126Asp127
Glu
128 Ile 129
Cys130Glu
131A l a
132Phe
133Arg
134Lys
135Asp
136Pro
137Lys
138Glu
139Tyr
140Ala
141Asn
142Gln
143Phe
144Met
145Trp
146Glu
147Tyr
148Ser
149
Thr150Asn 151
Tyr
152Glu153
Gln154
Ala155
Pro156Leu
157Ser
158
Leu159
Leu160
Val161
Ser162
Tyr163
Thr164Lys
165Ser
166
Tyr167Leu
168Ser
169
Met170
Val171
Gly172
Ser173
Cys174
Cys175
Thr176
Ser177
Ala
178Ser 179
Pro180Thr
181Val
182Cys
183Phe
184Leu
185Lys
186Glu
187Arg
188Leu
189Gln
190Leu
191Lys
192His
193
Leu194
Ser195
Leu196Leu
197
Thr198
Thr199Leu
200
Ser201
Asn202
Arg203
Val
204Cys
205Ser
206Gln
207Tyr
208Ala
209Ala
210Tyr
211Gly
212Glu
213Lys
214Lys
215Ser
216Arg
217Leu
218Ser
219Asn
220Leu
221Ile
223Leu
222Lys
224Ala
225Gln
226Lys
227Val
228Pro
229Thr
231
Asp232Leu
233
Glu234
Asp235
Val236Leu
237Pro
238Leu
239
Ala240
Glu241
Asp242
Ile243
Thr244Asn
245
Ile246
Leu247
Ser248Lys
249Cys
250
Cys251
Glu252
Ser230Ala
253Ala
254Ser
255
Glu256Asp
257
Cys258
Met259
Ala260
Lys261
Glu262
Leu263
Pro264
Glu265
His266
Thr267
Val268
Lys269
Leu270
Cys271
Asp272
Asn273
Leu274
Ser275
Thr276Lys
277Asp
278Ser
279Lys
280Phe
281
Glu282
Asp283
Cys284
Cys285
Gln286
Glu287
Lys288
Thr289Ala
290Met
291Asp
292
Val293
Phe294Val
295
Cys
296
Thr297Tyr
298Phe 299
Met
300Pro301
Ala302
Ala303
Gln304Leu
305Pro
306
Glu307Leu
308Pro
309Asp
310
Val311Arg
312Leu
313Pro
314Thr
315Asn
316Lys
317Asp
318Val
319Cys
320Asp
321Pro
322Gly
323Asn
324Thr
325Lys
326Val
327Met
328Asp
329Lys
330Tyr
331Thr
332Phe
333
Glu334
Leu335Ser
336Arg
337Arg 338
Thr
339
His340Leu
341Pro
342Glu
343Val
344Phe
345Leu
346Ser
347Lys
348Val
349Leu
350Glu
351Pro
352Thr
353Leu
354Lys
355Ser
356Leu
357Gly
358Glu
359Cys
360Cys
361Asp
362Val363
Glu
364Asp 365
Ser366Thr
367Thr
368Cys
369Phe
370Asn
371
Ala372
Lys373
Gly374Pro
375Leu
376Leu
377Lys
378Lys
379Glu
380Leu
381Ser
382Ser
383Phe
384 Ile
385Asp
386Lys
387Gly
388Gln
389Glu
390Leu
391Cys
392Ala
393Asp
394Tyr
395Ser
396Glu
397Asn
398Thr
399Phe
400Thr
401Glu
402Tyr
403Lys
404Lys
405Lys
406Leu
407Ala
408Glu
409Arg
410Leu
411Lys
412
Ala413Lys
414Leu
415Pro
416Glu
417Ala
418Thr
419Pro
420Thr
421Glu
422Leu
423Ala
424Lys
425Leu
426
Val427
Asn428Lys
429Arg
430Ser
431Asp
432Phe
433Ala
434Ser
435Asn
436Cys
437Cys
438Ser
439 Ile440
Asn
441Ser 442
Pro443Pro
444Leu
445Tyr
446Cys
447Asp
448Ser
449Glu
450
Ile451
Asp452
Ala453Glu
454Leu
455Lys
456Asn
457
Ile458
Leu
1Leu
2Glu
3Arg
4Gly
11Lys
5Arg
6Asp
7Tyr
8Glu
9Lys
10Asp
COOH
NH2
Domain I
Domain II
Domain III
Vitamin D-binding protein (known as Gc protein)
Gc protein Macrophage activatingfactor (MAF)
....
Gal
SA
GalNAc Thr
....
SA
GalNAc Thr
....
GalNAc Thr
β-galactosidaseof B cells *
▼
sialidaseof T cells
▼
Gc protein Macrophage activatingfactor (GcMAF)
....
Gal
SA
GalNAc Thr
....
SA
GalNAc Thr
....
GalNAc Thr
β-galactosidaseimmobilized immobilized
sialidase
▼
In vitro Tumoricidal Capacity of Macrophages Activated by GcMAFa. Time course observation of tumoricidal process with trypan blue penetration.
b. Tumoricidal capacity of GcMAF-treated human macrophages for prostate cancer cells LNCaP (i) and breast cancer cells MDA-MB-231 (ii)
100
50
4 18
Hrs of cocultivation
100
50
4 18
Hrs of cocultivation
, tumor cells only; , tumor cells with macrophages; , tumor cells with activated macrophages
Approximately 3.5 x 10 5 tumor cells were cultured with 1.1 x 10 6 macrophages/well.
(i) (ii)
Mφ
Cancer
Macrophages were activated by preincubation with GcMAF (100pg/ml) for 3hr.
Characterization of monocytes/macrophages, lymphocytes andSerum Gc protein of individual oral cancer patients.
nmole superoxide produced/min/10 6 phagocytesPatient
Assayed on: phagocytes* lymphocytes/phagocytes**
12
3
4
5
6
78
9
10
Healthy humans
0.210.66
0.71
0.40
0.62
0.56
0.330.72
0.29
0.620.27
6.385.99
6.34
5.96
6.83
5.92
5.845.96
6.70
6.346.68
5.876.03
5.65
5.72
5.64
5.87
5.945.63
6.26
6.086.22
2.611.42
5.19
1.07
3.45
1.11
0.721.19
2.14
0.314.25
* lyso-Pc untreated. ** lyso-Pc-treated lymphocytes + phagocytes.
Precursor activity of serum Gc protein were analyzed using 0.1% patient serum.
none 100pg GcMAF 1ng Gc protein 0.1% serumNo.
Deglycosylated Gc proteinGc protein
Gc protein Macrophage activating
factor (MAF)
....
Gal
SA
GalNAc Thr
....SA
GalNAc Thr
....
GalNAc Thr
....
Thr
....
Gal
SA
GalNAc Thr
sialidaseof T cells
▼
β-galactosidaseof B cells*
▼
α-N-acetylgalacto-
saminidase
▼
....
........
....
....
25
20
15
10
5
00 200 400 600 800 1000 1200
Tumor weight (mg)
Correlation between serum α-N-acetylgalactosaminidase
activity and tumor burden (measured by total weight) in
nude mouse transplanted with human oral squamous cell
carcinoma (KB) cell line.
0 2 4 8 10 12 14 16 18 20 22 246
Weeks
GcMAF therapy for prostate cancer patients
0
1
2
3
4
5
6123456789
10111213141516
Healthy Control
Weekly administration of 100ng GcMAF
0 2 4 8 10 12 14 16 18 20 2260
1
2
3
4
5
6
7
Weeks
GcMAF therapy for breast cancer patients
123456789
10111213141516
Healthy Control
Weekly administration of 100ng GcMAF
Tumoricidal capacity of GcMAF-treated human macrophagescancer cells LNCaP (a) and breast cancer cells for prostate
MDA-MB-231 (b).
, tumor cells only;, tumor cells with macrophages;, tumor cells with activated macrophages;, patient serum coated cancer cells with activated macrophages.
In vitro Tumoricidal Capacity of Macrophages Activated by GcMAF
100
50
4 18
(a)
hrs of cocultivation
100
hrs of cocultivation
(b)
50
4 18
Approximately 3.5 x 10 5 tumor cells were cultured with
Time course observation of 1.1 x 10 6 macrophages/well.
tumoricidal process was performed by trypan blue
exclusion vital assay.
IMMUNE DEVELOPMENTPrincipal Immune Development Cascade
Inflammation
Inflamed lesions release lysophospholipids(chemotactic agents)
LymphocytesMacrophages
(activation)
Activated macrophages(Highly activated
Cell killing
macrophages)phagocytosis
(degradation product/
antigen processing
antigen presentation
B and T lymphocytes
Development of Humoral and Cellular Immunity*
*Macrophage activation is the first mandatory step for immune development. Thus, lack of macrophage
activation leads to immunosuppression.
fragment component)