Lauretta Massai Giuseppe Fioritoni Francesco Lo Coco The anticancer properties of Vitamin C in vitro update 10 th World Congress on Healthcare and Technologies July 1718 2017 Lisbon Portugal ID: 931188
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Slide1
Domenico Mastrangelo*, Lauretta Massai*, Giuseppe Fioritoni**, Francesco Lo Coco°
The anticancer properties of Vitamin C: in vitro update
10
th
World Congress on Healthcare and TechnologiesJuly 17-18, 2017 Lisbon, Portugal
*
University
of Siena (Italy)
University
of Roma Tor Vergata (Italy)
**
°
Slide2Physiologic functions
Slide3Helps the metabolism of tyrosine, folic acid and tryptophanIncreases
the elimination of cholesterol
Contributes
to the synthesis of
catecholaminesHelps the body to absorb and breakdown histamine
Enhances
the absorption of non-heme
iron
Promotes the synthesis of collagen (its most widely known physiological
function)Neutralizes free radicals (it is a reducing agent, “scavenger” of free
radicals)
Protects DNA from damage due to free radicals and
mutagens
Reduces
the risk of premature
death
Fights off widespread environmental pollutantsPrevents the development of nitrosaminesRegulates gene expression… and more!
Slide4Rationale for the use ofintravenous Vitamin C (IVC)
in
cancer
Slide5Vitamin C:Is a prodrug
of H2
O2
Neutralizes
free radicalsAs a cofactor of
proly
hydroxilases, inactivates
HIFIs
a booster of the immune system
Promotes collagen
synthesis
Neutralizes
chemical
carcinogens
Modulates gene methylation / expressionSynergizes conventional chemo- and radiotherapy
Vs
Slide6Vitamin C anduveal melanomacell lines (C918 and OCM1)
Slide7UVEAL MELANOMA FACT SHEET
Most common form of eye cancer in adults
Unrelated to sun exposure
Uveal tract: iris, ciliary body, and choroid2000 Americans diagnosed/year
5 – 12% of all melanoma
Diagnosed at around 50-60 years
Bulging eyes, change in color, poor vision, or red, painful eye … or no symptoms at allRadiation for small/medium size
Eye
removal (enucleation) for larger tumors
Metastatizes
in 50% of all cases
Most
commonly
to the
liverThere is no cure for metastatic disease
Slide8Phase-contrast photomicrograph of
OCM1
(A) and
C918
(B) UM cell lines, growing as a monolayer in 25 cm2 flasks (see text). Magnification: 200 X. Details of cell morphology showing a larger mean size with prevalence of spherical elements in C918 and spindle-like elements in OCM1.
OCM1
C918
Slide9Two
cell
lines
, OCM-1 and OCM-2, were established from biopsied specimens of choroidal melanomas
of spindle
B and mixed cell
type morphologies. Both cell
lines were
phenotypically malignant. Karyotypic analyses revealed human
chromosomes with a modal
number of 95 and 85, respectively.
C918
cell lines were also established from
biopsied specimens
of
choroidal melanomas. They are more invasivBoth cell lines were phe than OCM1.Both OCM1 and C918 cell lines were established by Prof. Folberg and Dr. Valyi-Nagy of the Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, USA
OCM1
C918
Slide10Chen MJ et al.:Arsenic Trioxide Induces Apoptosis in Uveal Melanoma Cells Through the Mitochondrial Pathway
The American Journal of Chinese Medicine 2010 38:06, 1131-1142
As
2
O
3
(arsenic
trioxide – ATO) in the treatment of uveal melanoma
Kim KB et al.:
A Phase II Trial of Arsenic Trioxide in Patients with Metastatic Melanoma
Cancer 2005. 104,8: 1687 – 1692
“… Future clinical trials should evaluate arsenic trioxide in combination with other anticancer drugs that may improve its clinical activity in melanoma.”
Slide11Formula =
C
6
H
8O6Synonymous =
Ascorbic AcidCompounds with similar activity =
Sodium, potassium, calcium … Ascorbate … Its deficiency determines the«scurvy»
2-oxo-L-treo-exono-1,4-lacton-2,3-enediol
Vitamin
Slide12ATO =
aresnic
trioxide
2, 4, 6 & 8 = µg/mlASC =
sodium
ascorbate1, 3, 5 & 7
mM
ATO =
aresnic
trioxide
2, 4, 6 & 8 = µg/ml
ASC =
sodium
ascorbate1, 3, 5 & 7 mMFlow cytometry (cell viability)
Slide13OCM1
C918
82%
8.6%
86%
5.3%
82%
58%
86%
58%
- 73.4 %
- 24 %
- 80,7 %
- 24 %
Slide14Ascorbate at 7 millimoles induces a reduction of more than 73% and more than 80% in the number of live cells in vitro for both OCM1 and C918, respectively.The maximum reduction in the percentage of live cells obtained with Arsenic trioxide, is 24%.Ascorbate is almost three times more effective than Arsenic trioxide in killing uveal melanoma cell lines
in vitro.
The concentration of 7 mM
of ascorbate in plasma, can be easily obtained by administering ascorbate in high doses, by intravenous injection.
OCM1C918
ASC
ATO
ASC
ATO
-73,4%
-24%
-80.7%
-24%
Slide15Slide16Combining ASC and ATO
Slide17ATO
2 µg/ml
ASC 1, 3, 5
mM
MIX (
Ato
+ ASC)
ATO
4 µg/ml
ASC 1, 3, 5
mM
MIX (
Ato
+ ASC)
Slide18Vitamin C andretinoblastoma
Slide19NORMAL RETINAL CELLS
Y79
cell
line
Slide20Slide21Y79 human
retinoblastoma
cell
line, grown from a little
patient
of 2.5 years, affected by
bilateral retinoblastoma
Slide22Intra -
arterial
Melphalan
in retinoblastoma
Zanaty M et al:
Update on Intra-Arterial Chemotherapy for Retinoblastoma
The Scientific World JournalVolume 2014, Article ID 869604, 6 pages
Hadjistilianou
T,
Coriolani
G, Bracco S, Gennari P, Caini M, Cerase A,
Galimberti
D, De Francesco S, De Luca M, Domenico Mastrangelo:
Successful Treatment of Macular Retinoblastoma With
Superselective
Ophthalmic Artery Infusion of Melphalan.Journal of Pediatric Ophthalmology & Strabismus • Vol. 51, No. 1, 2014
Slide23Treating retinoblastoma cell line (Y79) with high
doses of
sodium
ascorbate
incomparison to melphalan
Slide24CELL VIABILITY
after
exposure
to sodium ascorbate
CELL VIABILITY
after
exposure
to
melphalan
Flow
cytometry
(
cell
viability)
Slide25% of live cells (%L)
after treatment with ascorbate
(A1 to A7) and
Melphalan (M1 to m7)
Slide26HOECHST/PI
STAINING
Y79
C
M7
A7
Slide27C
M3
M5
M7
M1
M
=
melphalan
1, 3, 5, 7 µM
C = control
C
A3
A5
A
7
A1
A
=
sodium
ascorbate
1, 3, 5, 7
mM
C = control
Slide28CONCLUSIONS
Vitamin
C in high
concentration
is
clearly cytotoxic for
both uveal melanoma (OCM1, C918) and retinoblastoma (Y79) cell lines
in vitro
High concentrations of Vitamin C, are significantly more effective (only 1 – 2 hours of exposure!)
than any other
known chemotheraputic agent for both
uveal
melanoma and
retinoblastoma
Megadoses
of Vitamin C are toxic only for cancer cells and may be particularly indicated for metastatic melanoma, for which there is no cureGiven its low cost, high effectiveness against cancer cells, selectivity of action (it kills only cancer
cells!), lack
of
any
toxicity
and/or side
effects
,
Vitamin
C in high
doses
is
highly
recommended
for the treatment of
both
uveal
melanoma and
retinoblastoma
Slide29High doses of Vitamin C and
Acute Promyelocytic
Leukemia
(APL)
Slide30… a malignancy of the bone marrow in which there is a deficiency of mature blood cells belonging to the myeloid line and an excess of immature cells called
promyelocytes
. APL is due to a translocation (an exchange of chromosome material) between chromosomes 15 and 17 which is symbolized t(15;17). This translocation is not a mere marker of APL. It is the cause of APL.
HL60
Human
promyelocytic
leukemia
cell
line
Treatment
of
choice
All
-trans
retinoic acid(ATRA)Arsenic trioxide(ATO)+
Slide31HL60
cell
line
C = control
ASC =
sodium
ascorbate
1, 3, 5, 7
mM
C
C
ASC 1
ASC 1
ASC 5
ASC 5
ASC 3
ASC 3
ASC 7
ASC 7
ATO (2
μ
g/ml)
ATO (2
μ
g/ml)
ATO (4
μ
g/ml)
ATO (4
μ
g/ml)
ATO (6
μ
g/ml)
ATO
(6
μ
g/ml)
ATO (8
μ
g/ml)
ATO (8
μ
g/ml)
C
C
HL60
cell
line
C = control
ATO =
arsenic
trioxide
2, 4, 6, 8 µg/ml
Flow
cytometry
(
cell
viability
)
Slide32Comparison
of
sodium
ascorbate
(ASC)and
arsenic trixide (ATO)
in the treatmentof APL in vitro
C
ASC 3 mM
ASC 5 mM
ATO 8μg/ml
ATO 6μg/ml
HOECHST/PI
STAINING
HL60
Slide33Slide34Slide35Slide36Automated viability profile of K562 cells exposed to different concentrations of ASC (0.5–7 mM
). As shown in the figure
, the percentage of viable cells
decreases progressively, by increasing
the concentration of ASC in the culture medium
Slide37Automated apoptotic profile of NB4 cells exposed to different concentrations of ASC (0.5–7 mM
). As shown in the figure
, the percentage of both early
and late apoptotic cells increases
progressively, by increasing the concentration of ASC in the culture medium
Slide38Hydrogen PeroxideFluorescent staining
ROS
Fluorescent
staining
Hydrogen ProxideDAB stainingMyeloperoxidase
(O-
tolidine staining
)
Slide39K562 –
Chronic
Myelogenous
Leukemia
(CML)
HL60 – Acute
Promyelocytic
Leukemia
(APL) – M2 FAB
class
.
NB4 – Acute
Promyelocytic
Leukemia (APL) – M3 FAB classNB4-As – Acute Promyelocytic Leukemia-ATO resistantNB4-RI – Acute Promyelocytic Leukemia
-ATRA
resistant
U937 –
Hystiocytic
Lymphoma
Slide40Effect of ASC on the survival, proliferation, and differentiation of normal CB CD34+ cells. CD34+ cells have been purified from CB and then incubated or not for 2 h with 3m MASC
; the cells were then washed and grown in vitro in the presence of a cocktail of myeloid growth
factors suitable
to induce their granulocytic differentiation. At various times of culture
, the number of viable cells (a) or of dead cells (b) or the percentage of CD34+, CD33+, and CD11b+ cells (c) was determined
Slide41Our studies represent a further demonstration of the efficacy of high concentration of ASC in killing, in vitro, cancer cells derived from uveal melanoma, retinoblastoma, and the myeloid lineage. Although further
investigations may be necessary to confirm the data reported herein, we believe that ASC in pharmacologic
doses, administered
by the IV route, may represent an innovative and potentially
effective treatment for uveal melanoma, retinoblastoma, and acute myeloid leukemia (AML). Given its safety profile already established in solid tumors, high-dose ASC could be tested in patients with uveal melanoma, retinoblastoma, and AML in phase II pilot studies.
10
th
World Congress on Healthcare and
Technologies
July 17-18, 2017 Lisbon, Portugal C
onclusions
Thank
you
!