Parkinson disease is the progressive neurodegenerative disorder in which mainly dopaminergic neuron in the substantia nigra pars compacta.
Melanoma is a type of skin cancer.
Melanoma and Parkinson disease & Link between them.
1. PRESENTED BY :-
SAKEEL AHMED (PC01)
ANKAN SARKAR (PC02)
NIRAJ TADASARE (PC03)
SWAPNIL RAUT (PC06)
SATWIK DASTANE (PC05)
Treatment with diphenyl–pyrazole compound
anle138b/c reveals that α-synuclein protects
melanoma cells from autophagic cell death
1
3. Introduction and Hypothesis.
Status of α -Synuclein/SNCA, LRRK2/PARK8, and Parkin/PARK2
Expression.
Level of Expression, Subcellular Localization, and State of α-Synuclein
Protein in VGP and MGP Melanoma Cells.
Action of Anle138b/c on melanoma cell line.
Action of Anle138b on cell membrane, mitochondria and autophagy.
Xenograft study in nude mice.
Discussion and References.
3
1
2
3
4
5
6
7
FLOW OF SEMINAR
4. INTRODUCTION
PARKINSON DISEASE (PD)
• Parkinson disease is the progressive neurodegenerative
disorder that affects predominately dopaminergic neuron
in the substantia nigra pars compacta.
• PD is characterized by the bradykinesia , tremor, rigidity,
postural instability.
• In PD there is mutation in the SNCA, PARK8, PARK2.
4
5. α -SYNUCLEIN:-
• It is made up of 140 amino acids and encoded by SNCA
gene.
• It may help in regulate the release of dopamine from the
synaptic vesicles.
• In PD there is the mutation in the α-synuclein gene .
• α-synuclein aggregates to form insoluble fibrils
characterized as a lewy bodies. ( hallmark for PD
identification)
5
6. MELANOMA
• Melanoma is a type of cancer that develops from the pigment-
containing cells known as melanocytes.
• Melanocytes are located in the stratum basale and produce melanin.
• Most aggressive type of skin cancer that accounts for 75% of all skin-
cancer–related deaths.
• The primary cause of melanoma is ultraviolet light (uv) exposure .
6
7. EPIDEMIOLOGY
• There are 3.1 million people with melanoma which result in 59,800
deaths.
• Australia has the highest incidence of melanoma in the world – 32 new
cases annually per 100,000 of the population.
• In us about 87,110 new melanomas will be diagnosed and 9,730 people
are expected to die.
Worldwide age-standardized annual incidence of melanoma by age.
ASR = Age-standardized rate (world), expressed per 100,000 persons
7
10. STATUS OF α -SYNUCLEIN/SNCA, LRRK2/PARK8,
AND PARKIN/PARK2 EXPRESSION
They took the tissue
sample from the
different organs slides
like Nevus, VGP, MGP.
Then embedded these
tissue on the donor
paraffin block fixed
with the formalin &
stained with H&E.
Transfer these tissue
to the donor paraffin
block.
Sectioning with
microtome & probing
the antibody.
Immunohistochemistry
TMA method
10
11. STATUS OF α -SYNUCLEIN/SNCA
RESULT
SNCA is expressed at
elevated levels in VGP
and MGP melanomas
compared with MIS
and AN.
Abbreviation
NS = normal skin , BN = benign nevi.
AN = atypical nevi.
MIS = melanoma in situ.
VGP = vertical growth phase.
MGP = metastasis growth phase.
11
14. DETERMINING LEVEL OF EXPRESSION OF α-SYNUCLEIN
To determine the level of expression of α-synuclein
immunoblot detection is done on various cell lines is done.
Immunoblot analyses of the VGP melanoma cell line WM983-
A, the three MGP melanoma cell lines WM983-B, SK-MEL-5,
WM852, and the WM1158 melanoma cell line that was
established from a superficial spreading melanoma in the
radial growth phase RGP/VGP is done.
14
15. ANALYSIS
The cell line lysate in done by
probe sonicator and is
separated at 12%SDS/PAGE.
It is transferred to PVDF
membrane, cross-linked by a
30-min treatment at room
temperature with 0.4%
paraformaldehyde (PFA) in
PBS.
Membrane is then blocked by
5% non fat dried powdered
milk along with PBS.
Protein of interest is then
probed with primary antibody
against α-synuclein.
(MJFR1) (Abcam)
It is followed by incubation with
an HRP-conjugated
secondary antibody and
SuperSignal West Pico
chemiluminescent
Substrate for detection.
15
16. RESULT
The cell lines WM983-A and WM983-
B, which were derived from a VGP
and an MGP melanoma of the same
patient, and the MGP melanoma cell
line SK-MEL-5 express high levels of
α-synuclein.
In comparison, the WM852 (MGP)
and the WM1158 (RGP/VGP)
melanoma cell lines contain lower
levels of α-synuclein protein.
16
17. SUBCELLULAR LOCALISATION OF α -SYNUCLEIN IN
VARIOUS CELL LINES OF MELANOMA
(A) Localisation of α-synuclein in cell lines of WM983-A, WM983-B, SK-
MEL-5, WM1158 is detected by immunofluorescence detection.
Procedure
α-synuclein protein expression in the melanoma cell lines WM983-A,
WM983-B, SK-MEL-5, and WM1158 was made by fixation in 3.7% PFA
for 20 min and permeabilization.
Probing is done with a mouse monoclonal anti–α-synuclein
(Syn211) antibody. (EMD-Millipore)
Secondary probing is done by a goat anti-mouse Alexa-546
secondary antibody.
Confocal image stacks were acquired with a 63× N.A. 1.4 oil
immersion objective on a Zeiss LSM 510 Meta confocal microscope
at the same gain and laser powers. 17
18. RESULT
Immunofluorescence analysis of expression of α-synuclein
protein in melanoma cell lines WM983-A, WM983-B, SK-MEL-5,
and WM1158.
On performing immunofluorescence
detection they found the localisation
of α-synuclein in melanoma cell lines
WM983-A, WM983-B, SK-MEL-5,
and WM1158 shows higher
expression in WM983-A, WM983-B,
SK-MEL-5 and distinctively low level
in WM1158.
18
19. (B) Localisation of α-synuclein and ser129 phosphorylated α-synuclein in nucleus
detected by using direct immunofluorescence detection under inverted microscope.
Procedure
WM983-B melanoma cells, fixed with 4% PFA and blocked for
30 min at 37 °C with 5 mg/mL of BSA in 0.05% Triton-X
100/PBS.
α-synuclein were probed with a rabbit polyclonal anti–α
synuclein (C-20) antibody. (Santa Cruz Biotechnology)
ser129 phosphorylate α-synuclein a mouse monoclonal
antiphosphorylated α-synuclein (pSyn#64) antibody. (Wako
Chemicals)
Both are imaged with a 40×/0.60 objective on a Leica
DMI6000B inverted microscope.
19
20. RESULT
By performing
immunofluorescence they
concluded the evidence and
localisation and presence of
both α-synuclein and ser129
phosphorylated α-synuclein in
the nucleus of the WM983-B
melanoma cell line.
Immunofluorescence analysis of α-synuclein expression in WM983-B
melanoma cells probed with antibody to α-synuclein (pseudocolored
green) or phosphorylated α-synuclein (pSer129) (pseudocolored red).
(Scale bars, 30μm.)
20
21. STATE OF α -SYNUCLEIN PROTEIN IN VGP/MGP
MELANOMA CELL LINES
Separation
• WM983-B melanoma cells were lysed in PBS containing 0.5% Triton X-100 and protease
inhibitors.
• The protein lysate (2.2 mg/0.5 mL) filtered through a 0.45-μm centrifuge tube filter before
loading onto a Superose 6 10/300GL column (GE Healthcare Bio-Sciences) connected to an
ÄKTApurifier 10. (GE Healthcare Life Sciences)
Transfer
• The collected fractions were placed for 10 min in a 95 °C water bath, and thereafter the entire
volume of every collected fraction was loaded onto nitrocellulose membranes by way of a dot-
blot vacuum system.
Detection
• After blocking in Tris-buffered saline-Tween 20 containing 5% nonfat dry milk, the membranes
were probed with mouse monoclonal anti–α-synuclein (Syn-1) antibody (BD Biosciences)
followed by incubation with a corresponding HRP-conjugated secondary antibody and
chemiluminescent substrate. (EMD Millipore)
21
22. RESULT
WM983-B melanoma whole-cell lysate,
separated by SEC followed by filter trap–dot
blot analysis of the collected fractions with an
anti–α-synuclein antibody.
It showed monomeric α-synuclein as well as its
distribution in fractions of higher-molecular-
weight species between 17–158 kda and >670
kd indicating that in these cells α-synuclein is
oligomerized.
Presence of monomeric as well as α-synuclein oligomeric
species in WM983-B melanoma cells detected by SEC–
filter trap assay
22
25. Cell Line used : WM983-B, SK-MEL-5
Drug used for treatment : anle138b , anle138c
Solvent to make drug solution : Dimethyl sulfoxide (DMSO)
Dose : 10 μM
Technique used : Phase contrast microscopy
Instrument : Zeiss Axiovert 100
Magnification : 10×
Observed for : Cell morphology (cell shape , cluster of cells)
Cell proliferation
Experimental Design
25
26. Change in the morphology of cells and decrease in cell
number is more in anle138b treated cell lines than in
anle138c treated cell lines. 26
27. Cell Line used : WM983-A , WM1158
Drug used for treatment : anle138b , anle138c
Solvent to make drug solution : Dimethyl sulfoxide (DMSO)
Dose : 10 μM
Technique used : Phase contrast microscopy
Instrument : Zeiss Axiovert 100
Magnification : 10×
Observed for : Cell morphology (cell shape , cluster of cells)
Cell proliferation
Experimental Design
27
28. Change in the cell morphology and decrease in cell
number is significant than observed in WM1158 cell
line treated with anle 138b/c compounds.
DMSO anle138b anle138c
WM983-A
WM983-A
28
29. WM1158 cell line does not show significant change in cell
morphology and cell number after treatment with both of the
anle 138b/c compounds after 48 hrs of treatment.
WM1158
WM1158
DMSO anle138b anle138c
29
30. Both cell lines shows significant decrease in cell number after
24 hrs treated with compound anle 138b
30
31. Anle138 treatment of melanoma cells damages their
plasma membrane, disrupts their mitochondrial
membrane potential, and dysregulates autophagy.
31
32. LDH Cytotoxicity Assay
Replicates of WM983-B as
well as WM852
melanoma cells were
cultured at per well in
tissue culture plates.
24 h and 48 h later, the
cells were rinsed three
times with serum-free
medium.
Addition of increasing
doses of anle138b or
DMSO only
Culture medium was
collected 24 and 48 h
later, was centrifuged to
pellet and remove cellular
debris
Using a microplate reader and
spectrophotometric absorbance
measurement, the release of LDH
enzyme activity into the tissue
culture medium was determined
with an LDH cytotoxicity assay kit.
32
33. At 10μM dose of anle138b showed the maximum killing of
WM983-B cells.
33
34. Mitochondrial Membrane
Potential.
Melanoma cell lines
WM983-B, SK-MEL-5, and
WM1158 were treated
with anle138b or received
DMSO only for 24 h or 48
h.
The cells were
incubated with 200 nM
MitoTracker Red
CMXRos for 30 min at
37 °C
The cells were fixed
with 3.7% PFA
34
35. Mitochondrial membrane potential of WM983-B and SK-MEL-5
was significantly reduced at 24 h after addition of 10μM dose
of anle138b.
35
36. Immunofluorescence
WM983-B and SK-MEL-5 cells
that for 24 h or 48 h were
treated with anle138b or had
received DMSO only were
fixed for 20 min with 3.7%
PFA
probed with an anti-LC3B
(clone 2G6) antibody
followed by a donkey anti-
mouse Alexa-488 secondary
antibody and Draq5 DNA
counterstain
Confocal image stacks were
obtained with a 63× on a
Zeiss LSM 510 Meta confocal
microscope
36
37. LC3 protein was more expressed in WM983-B cell line at
10μM dose of anle138b .
37
38. Immunoblot
WM983-B melanoma
whole-cell lysates
were separated on
12% SDS/PAGE
transferred onto
nitrocellulose
membrane
blocked with 3% BSA
probed with an anti-
p62/SQSTM1
antibody
followed by incubation with a
corresponding HRP conjugated
secondary antibody and
chemiluminescent substrate
38
40. Anle138b Administered Systemically to Nude Mice Bearing
High-Level α-Synuclein–Expressing Human Melanoma
Xenografts Reaches the Tumors and Affects Their Morphology
and Autophagy
40
41. ANLE138B ADMINISTERED SYSTEMICALLY TO NUDE MICE BEARING HIGH-LEVEL Α-SYNUCLEIN–EXPRESSING
HUMAN MELANOMA XENOGRAFTS REACHES THE TUMORS AND AFFECTS THEIR MORPHOLOGY AND
AUTOPHAGY
(7 days)
Without anle138b
With anle138b
Tumors were
resected
4°C
Transfer into tissue
homogenizing tubes and
homogenization in 1 mL
acetonitrile using a Precellys
Evolution Super homogenizer.
Ultrasonicated for
2 min at 25 °C
and centrifuged.C
A 100-μL aliquot of the
supernatant was injected into
an HPLC system.
The effluent was monitored under UV
at 260nm and the samples were
quantified by peak area ratio of
compounds to external standard.
WM-983-B cell
lines
Four-week-old female
nude mice
41
45. • H&E-stained tissue sections, of
the WM983-B human
melanoma xenografts that had
received food pellets not
containing anle138b (A and C)
• From one of the WM983-B
human melanoma xenografts
that had been resected from the
animal that had received food
pellets mixed with anle138b
(B and D).
• (E and F) LC3
immunohistochemical staining
of a tissue section
• The Inset in F shows a tissue
section from the anle138b-
containing WM983-B tumor,
probed with Alexa-488
secondary antibody only and
counterstained with fluorescent
DAPI
Anle138b negative group Anle138b positive group 45
46. DISCUSSION
There are clear evidences that there are genetic factors which plays a
key role in development of these two diseases (SNCA, PARK2, PARK8
genes).
From the TMA core analysis they conclude that unlike PARK8 and Parkin
it is the α-synuclein that is exclusively expressed in the advanced
melanoma cells.
Interestingly, cell lines derived from cancer tissue from nine different
origin types (breast, central nervous system, colon, leukemia,
melanoma, non-small cell lung, ovarian, prostate, and renal), malignant
melanoma cell lines overall have the highest level of SNCA expression.
46
47. DISCUSSION CONTINUED…
High level of expression of α-synuclein in WM983-A, WM983-B and SK-MEL-
5 was found and low level of expression in WM1158 and WM852.
Subcellular localisation of α-synuclein was found high in WM983-A, WM983-
, SK-MEL-5 and low in WM1158.
Significant decrease in cell number and morphology expressing high level of
a-synuclein observed due to removal of toxic oligomer of alpha synuclein.
By performing LDH assay, MM{ assay and autophagy they have concluded
that anle-138-b is cytotoxic, decreases mitochondrial membrane potential
and increases autophagy.
47
48. In the setting of a preclinical human melanoma xenograft study
researchers provide evidence that systemically administered anle138b
not only reaches the tumor, but also is present at high levels in the tumor
cells and that it affects their morphology and autophagy.
In summary, researcher’s findings presented here provide evidence that
α-synuclein, which in PD exerts severe toxic functions, promotes and
thereby is highly beneficial to the survival of melanoma in its advanced
stages. In addition, data suggest that dysregulating autophagy in VGP and
MGP melanoma cells by way of interfering with the aggregation of α-
synuclein might be a powerful approach to a therapy encompassing an
anle138b-like compound.
DISCUSSION CONTINUED…
48
49. REFERENCES:
1. Skibba JL, Pinckley J, Gilbert EF, Johnson RO (1972) Multiple primary melanoma
following administration of levodopa. Arch Pathol 93:556–561
2. Bertoni JM, et al.; North American Parkinson’s and Melanoma Survey Investigators
(2010) Increased melanoma risk in Parkinson disease: A prospective clinicopathological
study. Arch Neurol 67:347–352.
3. Ferreira JJ, et al. (2010) Skin cancer and Parkinson’s disease. Mov Disord 25:139–148.
4. Paisán-Ruiz C, Houlden H (2010) Common pathogenic pathways in melanoma and
Parkinson disease. Neurology 75:1653–1655.
5. Bajaj A, Driver JA, Schernhammer ES (2010) Parkinson’s disease and cancer risk: A
systematic review and meta-analysis. Cancer Causes Control 21:697–707.
6. Olsen JH, Jørgensen TL, Rugbjerg K, Friis S (2011) Parkinson disease and malignant
melanoma in first-degree relatives of patients with early-onset melanoma.
Epidemiology 22:109–112.
49
Four-week-old female nude mice (CAnN.CgFoxn1nu/Crl) (Charles River Laboratories) WM983-B (MGP) human melanoma cells (1 × 107 cells per side).when the tumors had reached a size of 2.3–3.0 mm in any direction, the normal food pellet diet of one of the animals was replaced with food pellets mixed with anle138b (2 g of anle138b/kg of food pellets) These studies were carried out under approved (LAVES) protocol
Analytical HPLC was performed using a Waters HPLC system with a Waters 996 Photodiode Array Detector. All separations involved a mobile phase of 0.1% trifluoroacetic acid (TFA) (vol/vol) in water (solvent A) and 0.1% TFA in acetonitrile (solvent B). HPLC was performed using reversed-phase (RP) column Eurospher RP 18, 100 Å, 5 μm, 250 × 4.6 mm at flow rates of 1 mL/min with a gradient of solvent B from 0 to 100% in 50 min.
H&E staining, prepared from one of the WM983-B, the animal that had received the food pellets mixed with anle138b (B and D), showed that the morphology of these tumor cells, arranged in a scattered pattern with focally pronounced disturbances in the tissue’s architecture, differed from the morphology of the tumor cells in a WM983-B control tumor that didn’t contain anle138b. The anti-LC3B antibody-probed WM983-B human melanoma xenograft tissue sections (pseudocolored green) were counterstained with fluorescent DAPI (pseudocolored blue). Tissuesections,preparedfromtheWM983-Bhumanmelanoma xenografts, were fixed with methanol, treated with blocking solution (goat serum: 0.25% Triton X-100), and probed with an antiLC3(NB100-2220)antibody(Bio-Techne),followedbyadditionof
a goat anti-rabbit Alexa-488 secondary antibody, counterstaining withfluorescentDAPI,andfluorescentmountingmedium(Dako). Imagesoftheanti-LC3antibody-probedandcounterstainedtissue sections were obtained with a 10× N.A. 0.45 objective on a Zeiss AXIO Imager M1 epifluorescence and brightfield microscope. H&E staining was performed according to standard protocols.
