RESPONSE OF A RESISTANT HUMAN MELANOMA CELL LINE TO A THERAPEUTIC PROTON BEAM

1
RESPONSE OF A RESISTANT HUMAN MELANOMA CELL LINE
TO A THERAPEUTIC PROTON BEAM

• A. Ristic-Fira1, I Petrovic1, D. Todorovic1, L.
  Koricanac1, L. Valastro2 and G. Cuttone2
• 1 Vinca Institute of Nuclear Sciences, Belgrade,
  Serbia and Montenegro
• 2 Istituto Nazionale di Fisica Nucleare, LNS,
  Catania, Italy

2

• Malignant melanomas
• highly metastatic tumours,
• poor curing prognosis.
• Phenotypic heterogeneity of melanoma cells
• differences in degree and type of pigmentation,
• different cell morphology,
• different growth rate and metastatic capacity.
• Therapeutic approaches
• surgery,
• chemotherapy,
• radiotherapy,
• but uneven effectiveness.

For certain melanoma (uveal melanoma and other
eye tumours - conjunctival melanoma, iris
melanoma, choroidal melanoma or retinoblastoma)
rather good results obtained with proton beams.
3

• Individual response of malignant melanoma to
  radiotherapy is variable, reflecting different
  radiobilogical characteristics of tumour,
  especially the ability to repair radiation
  damage.
• Radiosensitivity of melanomas is related to
  intrinsic characteristic of the melanocyte -
  specific metabolic activity, i.e., melanogenesis.
• Types of melanin
• eumelanin (black, dark brown, efficient
  radioprotector)
• pheomelanin (red, Cys rich)
• mixed-type pigmentation cells

4
Aim

• To investigate effects of protons at four
  positions within the spread-out Bragg peak
  (SOBP), thus simulating corresponding therapeutic
  effects along the tumour volume. Parameters of
  analyses
• level of cell inactivation,
• quality of cell inactivation.

5
Cell culture conditions

• Irradiation of exponentially growing HTB140 human
  melanoma cells.
• Plating efficiency (PE) for HTB140 cells -
  approximately 60 - 70.
• Doubling time (Td) for HTB140 cells - 242,7 h.

6
Irradiation conditions

• Irradiations at 6.6, 16.3, 25.0 and 26.0 mm in
  Perspex (Polymethyl methacrylate - PMMA) within
  the SOBP of the 62 MeV proton beam (produced by
  the superconducting cyclotron at the CATANA
  treatment facility, INFN, LNS Catania).
• Reference dosimetry - plane-parallel PTW 34045
  Markus ionization chamber calibrated according to
  IAEA code of practice (IAEA-TRS-398 2000).
• Single doses delivered to the cells 2, 4, 8, 12
  and 16 Gy, at dose rate of 15 Gy/min.
• Irradiations with ?-rays, at the same dose
  levels, were performed using 60Co source at the
  Vinca Institute of Nuclear Sciences in Belgrade,
  at average dose rate of 1 Gy/min.
• All cell irradiations were carried out in air at
  room temperature.

7
SOBP
Figure 1. Depth dose distribution of the
spread-out Bragg peak in Perspex of the 62 MeV
proton beam produced at the CATANA treatment
facility in the INFN-LNS, Catania. Arrows
correspond to irradiation positions at 6.6 mm
(A), 16.3 mm (B), 25 mm (C) and 26 mm (D).
8
Table 1. Irradiation position parameters in SOBP
for HTB140 cells
Irradiation Depth in
Dose E position
Perspex (mm) ()
(MeV)
A 6.6
87.242.61 50.904.33
B 16.3
99.420.58
34.882.15 C
25.0 102.213.43
11.741.23 D
26.0
32.124.27 5.991.36
mean energy
9
Biological assays

• Cell viability
• microtetrasolium (MTT) assay,
• sulforhodamine B (SRB) assay,
• clonogenic assay (CA).
• Cell proliferation
• incorporation of 5-bromo-2-deoxyuridine (BrdU)
• during DNA synthesis.
• Cell cycle redistribution
• fluorescence activated cell sorter (FACS) with
  propidium iodide (PI) staining.
• DNA ladder fragmentation on 2 agarose gel
  electrophoresis.

10
Results
11
A
Figure 2. Dose dependent surviving fractions,
estimated by microtetrasolium, sulforhodamine B
and clonogenic assay, of HTB140 melanoma cells
irradiated with ? -rays and protons. Irradiation
position within the proton spread-out Bragg peak
corresponds to 6.6 mm depth in Perspex (A).
12
A
B
C
D
Figure 3.
13
BrdU
Figure 4. Cell proliferation of HTB140 melanoma
cells irradiated at 2, 4, 8, 12 and 16 Gy as a
function of depth, estimated by
5-bromo-2-deoxyuridine assay. Irradiation
position within the proton spread-out Bragg peak
correspond to 6.6 mm (A), 16.3 mm (B), 25 mm (C)
and 26 mm (D) depth in Perspex.
14
FACS
A
B
D
C
Figure 5.
15
A
B
1 2 3 4 5 6 7
1 2 3 4 5 6 7

• Figure 6. DNA gel electrophoresis of HTB140
  cells irradiated with ?-rays 6 h (panel A) and 48
  h (panel B) post-irradiation. Lane 2
  non-irradiated melanoma cells, lanes 3 7 cells
  irradiated with 8, 12, 16, 20 and 24 Gy
  respectively. Lane 1 molecular weight marker, 100
  bp DNA Ladder (Gibco BRL).

16
C
D
1 2 3 4 5 6 7
1 2 3 4 5 6 7

• Figure 7. Proton induced DNA fragmentation in
  HTB140 cells 6 h (panel C) and 48 h (panel D)
  post-irradiation. Lane 2 non-irradiated melanoma
  cells, lanes 3 7 cells irradiated with 8, 12,
  16, 20 and 24 Gy respectively. Lane 1 molecular
  weight marker, 100 bp DNA Ladder (Gibco BRL).

17

• Figure 8. Phase-contrast photomicrographs of
  HTB140 melanoma cells irradiated with ?rays and
  protons at 8, 12, 16, 20 and 24 Gy (original
  magnification, x 100).

18
Conclusions

• The number of viable cells estimated by
  microtetrasolium (MTT), sulforhodamine B (SRB)
  and clonogenic (CA) assays revealed cell
  inactivation, showing an increase when
  approaching the end of the SOBP at lower doses.
• With increase of the doses applied (8 to 16 Gy)
  and position within the SOBP, the level of cell
  elimination, although increasing, had a less
  important descent than for smaller doses (2 and 4
  Gy), suggesting that these cells are very
  radio-resistant.

19
Conclusions

• Cell cycle phase distribution exhibited major
  accumulation of irradiated HTB140 cells in G1/S
  phase, expressing mainly high metabolic activity
  of melanoma cells.
• The level of G2/M cell population was relatively
  low, thus confirming the very pronounced
  radio-resistant nature of these cells.
• With the increase of dose and position within the
  SOBP, this tendency was kept in general,
  indicating that even 7 days after proton
  irradiation cells that survived were still rather
  active.
• BrdU assay has shown considerable proliferative
  activity of irradiated cells with the increase of
  depth within the SOBP. Inside the same
  irradiation position, with the increase of dose
  cell proliferative capacity, although still very
  high, was significantly reduced.

20

• From our previous results, including this
  study, it seems that protons eliminate HTB140
  cells both by apoptosis and irreparable DNA
  damage, including genomic instability generation,
  while ?-rays, almost only by the irreparable DNA
  damage.
• Time course, extent and qualitative features of
  various lesions are reported to be different
  after irradiation with ?-rays and protons,
  indicating higher effectiveness of proton
  irradiation.