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Gene expression and functions
specific to acidic cancer nests
Hiroshi Kobayashi, Professor Emeritus
(Graduate School of Pharmaceutical Sciences, Chiba University)
International Conference on Nuclear Medicine & Radiation Therapy,
July 14-15, 2016, Cologne, Germany
Our basic idea
1. The extra-cellular pH decreases below 6.5 in the central
regions of solid tumors.
2. All enzymes have pH-dependent activity, suggesting
that cells have alternative enzymes functioning at
acidic pH to compensate functional decline of enzymes
working at alkaline pH.
3. Genes encoding enzymes functioning at acidic pH may
be potential targets for cancer diagnosis and therapy.
Basic question
Do cells have gene products which function
preferentially under acidic conditions?
We have investigated
1. Genes whose expression increases at acidic pH,
2. Proteins for signal pathways working at acidic pH,
3. Drugs which inhibit cell proliferation at acidic pH.
[1] Genes whose expression increases under acidic conditions
Number of genes whose expression was enhanced at acidic pH in
mesothelioma cells (NCI-H2052)
Group
A
B
C
D
E
F
G
Expression level*
2h
5h
>2
>2
>2
<=2
<=2
>2
<=2
<=2
>2
>2
<=2
>2
>2
<=2
24h
>2
>2
>2
>2
<=2
<=2
<=2
Total
Number of genes
Total
Signal**
15
7
22
3
37
8
305
66
32
6
91
32
191
43
693 (2.9%)a 165 (24%)b
Microarray chips we used contained 24,000 genes.
*Ratio of the expression in cells cultured at pH 6.6 to that at pH 7.5.
**Genes encoding receptors, signal proteins, transcription factors, cytokines, and growth factors.
a 693/24,000,
b 165/693
For original DNA array data, see Fukamachi T, et al. Genes 2013, 4:65-85.
Genes expressed in human cancer nests
Genes
Mesothelioma cells
Increase at
Expression
acidic pH (fold) level (%)*
IL-32
TNFRSF9
ARFG
ATP6V0D2
ErbB3
LOC553158
DMGDH
MnSOD
3.7
5.5
5.7
3.8
6.0
4.3
4.3
1.6
8.91
2.58
0.92
0.69
0.92
0.44
0.39
13.77
Total number of patients tested
% of patients
(expression: cancer > normal)
Colon Stomach Liver Renal
100
100
100
28
91
91
82
73
80
80
70
80
70
90
60
80
60
40
50
100
90
30
60
90
100
90
90
0
40
0
60
100
11
10
10
10
Mesothelioma cells had 64 genes whose expression increased more than 3-fold in acidic medium, and 7 genes were
selected. MnSOD was also selected because MnSOD had been reported to participate in metastasis. The expressions of
selected genes were measured in specimens from cancer patients.
*percent ratio of the mRNA level to the level of GAPDH at pH 6.6
For original data, see Fukamachi T, et al. Molecular and Clinical Oncology, 2014, 2:1160-1166.
[2] Gene expression is pH-independent, but its function is
essential for cell proliferation under acidic conditions
1.
CTIB (C-terminal region protein of IκB-β) in CHO (Chinese hamster
ovary) cells
Similarity is 94%
IκB-β (mouse, 359 amino acids)
CTIB (CHO, 138 amino acids)
Western blotting with anti-IκB-β (C-20)
pHe, medium pH; siCTIB, the CTIB silencing strain with RNAi; siNone, control strain.
Lao Q. et al. J Cell Physiol 2006, 207:238–243.
[3] Anti-cancer drugs whose efficacy increases at acidic pH
Cytotoxicity of approximately 280 compounds were measured using HeLa cells
cultured for 5 days in media of initial pH 7.7 and 6.7.
higher cytotoxicity at pH 6.6
Lovastatin, Cantharidin, Doxorubicin(1),
Manumycin A(2), Ionomycin(3)
lower cytotoxicity at pH 6.6
Vinblastine sulfate, Paclitaxel, Aclarubicin,
Aphidicolin, Trichostatin A, 17-AAG, Cisplatin(4)
no difference in cytotoxicity
between pH 6.6 and 7.5
Bleomycin sulfate, Methotrexate, Mitomycin C,
Daunorubicin, Actinomycin D, Camptothecin,
Etoposide, Cytochalasin D, Kenpaullone,
Cycloheximide, Radicicol, Cucurbitacin I,
Bisindolymaleimide I, MG-132, Hydroxyurea
Medium pH decreased from 7.7 and 6.7 to 7.5 and 6.6, respectively, after 5 days.
Other compounds showed very weak cytotoxicity at 1 μM at both pH values.
Fukamachi T, et al, Recent Patents on Biomedical Engineering, 4, (2011) 141-152.
(1)Cell shape was changed at acidic pH.
(2)mesothelioma cells, Fukamachi T, et al. Cancer Letters 297 (2010) 182–189.
(3)synovial cells, Fukamachi T, et al. International Immunopharmacology 17 (2013) 148–153.
(4)gene manipulated pancreatic cells, unpublished.
Inhibition of cell survival by lovastatin
Mesothelioma cells
NCI-H2052
% Survival
PANC-1
NCI-H2452
150
BxPC-3
pH 7.5
pH 7.5
pH 7.5
100
pH 6.6
pH 6.6
pH 6.6
50
0
150
% Survival
Pancreatic carcinoma cells
HeLa
100
50
0
pH 6.6
pH 7.5
pH 6.6
0 0.01 0.1 1 10 0 0.01 0.1 1
Lovastatin concentration (mM)
pH 6.6
0.01 0.1 1 10 0 0.01 0.1 1
Lovastatin concentration (mM)
SW982
pH 7.5
pH 7.5
10
10
Cells were cultured in modified RPMI-1640 or DMEM
(HeLa, SW982) media of pH 7.7 and 6.7 without a CO2
supply for 4 (HeLa) or 5 (others) days with lovastatin at
indicated concentrations, and cytotoxicity was measured.
After the incubation, the pH values of the media
decreased from 7.7 to 7.5 and 6.7 to 6.6.
Fukamachi T, et al. Cancer Letters 2010, 297:182–189.
Fukamachi T, et al. International Immunopharmacology
2013, 17:148–153.
Statins inhibit any kinds of cells at acidic pH.
Inhibition of cell survival by simvastatin in THP-1 and Jurkat T cells
THP-1
% Survival
150
Jurkat
pH 7.5
pH 7.5
100
0
pH 6.6
pH 6.6
50
0 0.01
0.1
1
0 0.01
0.1
1
10
Simvastatin (open ring form) concentration (μM)
Statins do not inhibit immune cell proliferation
in normal tissues whose pH is around 7.4.
Acidosis-dependent anti-cancer drugs are far superior
because of less effect on normal tissues, especially on
immune systems in the body.
How to use statins for anti-cancer themotherapy?
Clinical investigations of statins as an anti-cancer drug
Kawata S, Yamasaki E, Nagase T, et al. Effect of pravastatin on survival in patients with
advanced hepatocellular carcinoma. A randomized controlled trial. Br J Cancer 2001 84:
886–891.
Huang WY, Li CH, Lin CL, Liang JA. Long-term statin use in patients with lung cancer
and dyslipidemia reduces the risk of death. Oncotarget. 2016 Jun 7 [Epub ahead of print]
Manthravadi S, Shrestha A, Madhusudhana S. Impact of statin use on cancer recurrence
and mortality in breast cancer: A systematic review and meta-analysis. Int J Cancer. 2016
May 13 [Epub ahead of print]
Lee HS, Lee SH, Lee HJ, Chung MJ, Park JY, Park SW, Song SY, Bang S. Statin use and
its impact on survival in pancreatic cancer patients. Medicine (Baltimore). 2016
May;95(19):e3607.
Chen BK, Chiu HF, Yang CY. Statins are associated with a reduced risk of brain cancer:
A population-based case-control study. Medicine (Baltimore). 2016 Apr;95(17):e3392.
Meng Y, Liao YB, Xu P, Wei WR, Wang J. Statin use and mortality of patients with
prostate cancer: a meta-analysis. Onco Targets Ther. 2016 Mar 21;9:1689-96.
Jung YS, Park CH, Eun CS, Park DI, Han DS. Statin use and the risk of colorectal
adenoma: A meta-analysis. J Gastroenterol Hepatol. 2016 Apr 4. [Epub ahead of print]
Zhou YY, Zhu GQ, Wang Y, et al. Systematic review with network meta-analysis: statins
and risk of hepatocellular carcinoma. Oncotarget. 2016 Mar 1. [Epub ahead of print]
Alexandre L, Clark AB, Bhutta HY, et al. Association between statin use after diagnosis
of esophageal cancer and survival: a population-based cohort atudy. Gastroenterology.
2016 Jan 8 [Epub ahead of print]
Lohinai Z, Dome P, Szilagyi Z, et al. From bench to bedside: Attempt to evaluate
repositioning of drugs in the treatment of metastatic small cell lung cancer (SCLC). PLoS
One. 2016 Jan 6;11(1):e0144797.
Lin CJ, Liao WC, Lin HJ, et al. Statins attenuate helicobacter pylori cagA translocation
and reduce incidence of gastric cancer: In vitro and population-based case-control
studies. PLoS One. 2016 Jan 5;11(1):e0146432.
McKay RR, Lin X, Albiges L, et al. Statins and survival outcomes in patients with
metastatic renal cell carcinoma. Eur J Cancer. 2016 Jan;52:155-62.
Clinical reports to show negative effects of statins
Bachy E, Estell JA, Van de Neste E, et al. Statin use is safe and does not impact prognosis
in patient with de novo follicular lymphoma treated with immunochemotherapy: An
exploratory analysis of the PRIMA cohort study. Am J Hematol. 2016 Jan 22. [Epub
ahead of print]
Jeon CY, Goodman MT, Cook-Wiens G, et al. Statin use and survival with early stage
hepatocellular carcinoma. Cancer Epidemiol Biomarkers Prev. 2016 Feb 9. [Epub ahead
of print]
Keskiväli T, Kujala P, Visakorpi T, et al. Statin use and risk of disease recurrence and
death after radical prostatectomy. Prostate. 2016 Apr;76(5):469-78.
The efficacy of statins is low at the early stage of cancer
development and/or in small cancer nests because acidosis is
not enough in these areas.
Statins work under acidic conditions.
Drawn from http://www.medicinenet.com/slideshows/article.htm
0
I
II
III
IV
stage
acidosis
efficacy of statins
Therefore, verification of acidosis of cancer nests is
indispensable for chemotherapy with statins,
and
the methods to measure acidosis may be useful for
efficient use of statins.
Combination therapy would be recommended,
but not in the same stage
Drawn from http://www.medicinenet.com/slideshows/article.htm
acidosis
efficacy of statins
Excision of cancer nests
Radiation therapy
Chemotherapy with pH-independent drugs
Conclusions
1. Mammalian cells have systems working preferentially under
acidic conditions, which may be potential targets for cancer
diagnosis and therapy.
2. Anti-cancer drugs specific to acidic cancer nests do not work at
the early stage of cancer development and/or in small cancer
nests.
3. However, acidosis-dependent anti-cancer drugs are superior
because of less effect on cells in normal tissues, especially on
immune systems in the body.
4. For efficient use of statins, the development of the easy method
to measure acidosis is now being aspired.
Acknowledgements to collaborators
My laboratory:
Dr. Toshihiko Fukamachi
Dr. Hiromi Saito
graduate students
Chiba Cancer Center Research Institute:
Prof. Masatoshi Tagawa
Thank you
and
Welcome to the acidic world