The anti-cancer potency of photodynamic therapy of a novel chlorin derivative Amidochlorin p6 (ACP)

Article abstract of DOI:10.1039/c7ra07368b

Photodynamic therapy (PDT) is a minimally invasive method in cancer treatment and has attracted considerable attention recently. In this paper, we have performed a detailed study of photodynamic activity of a chlorophyllous derivative, Amidochlorin p6 (ACP), and evaluated its potential as a photosensitizer in PDT. The singlet oxygen quantum yield (Φ_Δ), the photoreaction mechanisms in PDT, the anti-photobleaching ability in phosphate buffer saline (PBS), the photocytotoxicity and dark toxicity against HeLa cells, cellular uptake and the influence on the expression of survivin and cyclin-dependent kinase (CDK2), were all investigated. The title compound showed significant photocytotoxicity and negligible toxicity in dark, and remarkable photostability. Moreover, ACP could be uptaken by HeLa cells successfully at 20 min leading to damage of cancer cells under light, during which Type I and Type II photodynamic reactions occurred simultaneously on HeLa cells in PDT treatment, and the influence of Type I (the generation of hydroxyl radicals) is slightly larger than Type II (the generation of singlet oxygen). In addition, real-time fluorescent quantitative PCR (RT-qPCR) suggested that ACP could significantly regulate the expression of survivin, which partly explained why ACP could induce the HeLa cell apoptosis and accelerate cell death.

Full text of DOI:10.1039/c7ra07368b

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The anti-cancer potency of photodynamic therapy
of a novel chlorin derivative Amidochlorin p6 (ACP)
Cite this: RSC Adv., 2017, 7, 40873
a
a
b
b
a
Hongyue Zhang, Wenting Li, Guanghui Tan, Guohua Ding,* Zhiqiang Wang*
a
and Yingxue Jin
*
Photodynamic therapy (PDT) is a minimally invasive method in cancer treatment and has attracted
considerable attention recently. In this paper, we have performed a detailed study of photodynamic
activity of
a
chlorophyllous derivative, Amidochlorin p6 (ACP), and evaluated its potential as
), the photoreaction mechanisms in PDT,
a photosensitizer in PDT. The singlet oxygen quantum yield (F
D
the anti-photobleaching ability in phosphate buffer saline (PBS), the photocytotoxicity and dark toxicity
against HeLa cells, cellular uptake and the influence on the expression of survivin and cyclin-dependent
kinase (CDK2), were all investigated. The title compound showed significant photocytotoxicity and
negligible toxicity in dark, and remarkable photostability. Moreover, ACP could be uptaken by HeLa cells
successfully at 20 min leading to damage of cancer cells under light, during which Type I and Type II
photodynamic reactions occurred simultaneously on HeLa cells in PDT treatment, and the influence of
Type I (the generation of hydroxyl radicals) is slightly larger than Type II (the generation of singlet
oxygen). In addition, real-time fluorescent quantitative PCR (RT-qPCR) suggested that ACP could
significantly regulate the expression of survivin, which partly explained why ACP could induce the HeLa
cell apoptosis and accelerate cell death.
Received 4th July 2017
Accepted 16th August 2017
DOI: 10.1039/c7ra07368b
rsc.li/rsc-advances
and low toxic PSs. Among various PSs under development,
chlorophylls have aroused widespread attention. For example,
Introduction
Photodynamic therapy (PDT) is a new cancer treatment chlorin e6 (Ce6), a promising PS in phase II clinical trial, is one
modality. Compared with conventional methods such as of chlorophyll degradation products, which has the advantage
surgery, chemotherapy, radiotherapy, PDT has the advantages of single chemical component, denite chemical structure,
12
of safety, less side effects, reproducibility and relative low cost. long-wavelength absorption at 660 nm; a derivative of Ce6,
It plays an important role in the comprehensive treatment of single aspartyl Ce6 (N-aspartyl chlorin Npe6) also showed
1,2
tumors. PDT involves the administration of light sensitive and promising potential in PDT due to the high singlet oxygen yield,
non-toxic compounds (photosensitizers, PSs), which accumu- short retention time, fast clearance and hardly causing photo-
13
late in tumor cells and are activated by penetrating light of sensitive skin side effects. Moreover, 2-[1-hexyloxyethyl]-2-
appropriate wavelength (within “optical window”). Subse- devinyl pyropheophorbide-a (HPPH), which is a second gener-
quently in the presence of tissue oxygen the activated PS ation PS, has been progressed through phase II clinical trials,
produces reactive oxygen species (ROS) as an intermediate showing excellent safety and efficacy for the treatment of lung,
3
–5
6
14
resulting in cell death. The key to PDT is the photosensitizer,
head and neck cancers.
which determines the tissue penetration depth and photo-
Our group have devoted to synthesizing chlorophyll deriva-
15–19
chemical process including Type I reaction (generating super- tives and preparing the PSs for PDT application.
In our
7
oxide anion, hydroxyl radical and hydrogen peroxide ) and Type previous work, we had reported a novel chlorophyll derivative
5
6
II reaction (generating singlet oxygen).
with two hydroxyl groups at C-13 , 15 named Amidochlorin p6
2
+
20
8
At present, only hematoporphrphyrin derivative (HpD),
(ACP), which showed excellent potential in Cu detection
9
11
Photofrin II, PsD-007 (ref. 10) and 5-Ala were truly applied as (Fig. 1). Considering that ACP has two hydroxyl groups in its
PDT drugs in clinical application, which are unable to meet the structure, it may improve the water solubility. Moreover, the
clinical needs. Therefore, it is urgent to develop high efficient structure of ACP and the absorption of long-wavelength
absorption are similar to those of Ce6, hence it may exhibit
a
promising photodynamic activity. Therefore, in this paper, we
Ministry of Education, Key Laboratory of Photochemical Biomaterials and Energy
Storage Materials, Harbin Normal University, Harbin, 150025, China. E-mail: try to systematically study the photophysical properties of ACP,
jyxprof@163.com; wzq70402@163.com
including singlet oxygen quantum yield (F ) and photostability.
Moreover, we have performed a detailed bioactivity test of ACP
D
b
College of Life Science and Technology, Harbin Normal University, Harbin, 150025,
China. E-mail: dghprof@163.com
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ꢂ1
experiments was 25 mmol L in DMSO, which was redistributed
in DMEM without any nutrient to obtain the required concentra-
tions for MTT assay. For the other bio-experiments (such as RNA
extract, cellular uptake and morphology changes), the samples
ꢂ1
were prepared as above using 8 mmol L stock solutions of ACP.
The nal DMSO concentration in all the samples was <0.1%.
Detection of singlet oxygen quantum yield of ACP
1
To detect O2 generation during the exposure to light for
0–120 s, DPBF was used as trapping reagent. Methylene blue
(
MB) was used as the reference compound, the singlet oxygen
Fig. 1 The structure and atom numbers of Amidochlorin p6 (ACP).
21
quantum yield (F ) of which in DMF is 49.1%. 3 mL stock
D
ꢂ5
ꢂ1
solution of DPBF in DMF (6 ꢃ 10 mol L ) containing 20 mL
PSs solution (the concentration of ACP and MB were 2.8 ꢃ
against HeLa cells, including phototoxicity, dark toxicity, the
mechanism of photodynamic reaction, cellular uptake and the
changes of cell morphology aer PDT. Finally, to further study
the mechanism of PDT, we have successfully extracted the total
cellular RNA from HeLa cells treated with ACP and investigated
the expression of survivin and cyclin-dependent kinase 2
ꢂ6
ꢂ1
1
0
mol L ) was placed in a quartz cuvette and irradiated with
light for different periods of time at room temperature. The
absorption spectra of the solution at 415 nm were monitored
every 10 s for a 120 s period. The singlet oxygen quantum yield
(F
D
) of ACP was calculated using the following formula:
S
(CDK2) in HeLa cells.
F
D
ðRÞk IaT ðRÞ
F
D
ðSÞ ¼
R
k IaT ðSÞ
Materials and methods
General information
ꢂ2.3A
I ¼ I (1 ꢂ e
)
a
0
Dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF)
were obtained from Sinopharm Chemical Reagent (Shanghai, superscript S and R respect the sample and reference
China). Other chemical reagents were obtained from Alfa Aesar compound, respectively. I is dened as the total amount of
Tianjin, China). All solvents and reagents were analytically pure light absorbed by PSs. A is the corresponding absorbance at
without further purication. 4,6-Diamidino-2-phenylindole (DAPI), irradiation wavelength. k is a slope of a plot of the dependence
a
(
sodium azide (SA), D-mannitol (DM), methylene blue (MB), of ln(A
/A
t
) on irradiation time t, with A
and A
being the
0
0
t
1
,3-diphenylisobenzofuran (DPBF) and 6-well or 96-well plates absorbance of the DPBF at 415 nm before irradiation and aer
were bought from Sigma-Aldrich (Beijing, China). Dulbecco's irradiation time t(s), respectively.
Modied Eagle Medium (DMEM), fetal bovine serum (FBS),
22
phosphate buffer saline (PBS), 3-(4,5-dimethy lthiazol-2-yl)-2,5-
dipheny-ltetrazolium bromide (MTT), ethidium bromide (EB),
penicillin and streptomycin were bought from Gibco (Shanghai,
China). UV-vis absorption was recorded using a UV-160A spectro-
photometer (Shimadzu, Kyoto, Japan). All measurements were
Anti-photobleaching ability of ACP
ꢂ5
ꢂ1
The concentration of ACP was 1 ꢃ 10
mol L
in PBS.
Appropriate blanks corresponding to the PBS were subtracted to
correct the background. Before irradiation, ACP solution was
transferred to a 1 ꢃ 1 cm glass cuvette to record the absorbance
of ACP at long-wavelength absorption. Light irradiates vertically
on the solution surface of the glass cuvette every ten minutes.
The absorption spectrum of ACP was recorded with UV-vis
spectrometer at 10 min intervals for a period of 60 min. All
the operations were carried out at room temperature. The
curves of absorbance of ACP at each time point against irradi-
ating time were drawn. The percentage of ACP was calculated by
the following formula:
ꢀ
performed at room temperature (about 25 C), unless otherwise
stated. All spectra were obtained in a quartz cuvette (path length ¼
1
6
cm). Xenon Lamp (HSX-UV300) passed through a band-pass
70 ꢁ 10 nm lter (DT670 quartz lter, NBeT, Beijing, China) to
supply the near infrared light in the range of 660–680 nm. Human
cervical cell lines (HeLa) were purchased from Sigma-Aldrich. The
viability of HeLa cells was measured by MTT assay and recorded on
enzyme-linked immunosorbent assay reader, Synergy II, BioTek,
VT. All the confocal images were obtained utilizing a Leica DM IL
LED uorescence inverted microscope (FIM, Germany). RNA pure
Tissue&Cell Kit, PrimeScript™ 1st Strand cDNA Synthesis Kit and
UltraSYBR One Step RT-qPCR Kit (High ROX) were purchased from
Kang century Biotechnology (Beijing, China).
A
t
Percentage of ACP ð%Þ ¼
ꢃ 100%
A
0
0 t
A , A are the absorbance measured before irradiation and aer
irradiation t seconds, respectively.
Preparation of ACP and stock solutions of ACP
Cell culture
20
ACP was prepared according to our previous reported procedure.
Human cervical carcinoma cell line (HeLa) was routinely
The concentration of stock solutions of ACP applied in bio- maintained in Dulbecco's Modied Eagle Medium (DMEM)
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ꢂ1
ꢂ1
containing 100 mg mL penicillin, 100 mg mL streptomycin cultured another 0 h, 6 h, 12 h, 24 h, the solution was removed
and 10% fetal bovine serum (FBS). HeLa cells were grown at and ACP was rinsed three times with PBS. The cell morpho-
ꢀ
3
7 C under an atmosphere of 5% CO . The cells in logarithmic logical changes were analyzed by FIM.
2
phase were applied into 6-well or 96-well plates for further use
aer dissociation and suspension adherent growth 24 h.
RNA extraction
Collect the cells aer ACP–PDT at different time (0, 6, 12, 24 h).
The total cellular RNA of HeLa cells were extracted using a RNA
pure Tissue&Cell Kit according to the manufacturer's instruc-
tions. The products were separated by 1% agarose gel electro-
phoresis, stained with EB, and visualized using UV light. The
experiment was repeated for three times. All samples were
subjected to DNase treatment to avoid DNA contamination,
followed by reverse transcription (RT) using a PrimeScript™ 1st
Strand cDNA Synthesis Kit. The products above could be sub-
Cytotoxicity and MTT assay
HeLa cells were seeded in 96-well cell culture plates at a density
3
ꢀ
of 4 ꢃ 10 cells per well at 37 C and 5% CO
2
. Aer overnight
incubation, cells were incubated with ACP at different concen-
ꢂ1
trations (0.195, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25 mg mL ) at
ꢀ
ꢂ2
3
1
7 C for 4 h, followed by irradiation with light (5 mW cm
,
ꢂ1
0 min). Then, 20 mL MTT (5 mg mL ) were added in each wells
aer 24 h. Aer 4 h of incubation with MTT, the media were
removed and 150 mL DMSO were added to solubilize the for-
mazan crystals. The cell viability was measured with enzyme-
ꢀ
jected to the next step at once or preserved at ꢂ20 C.
linked immunosorbent assay reader at an absorbance of Detection of real-time uorescent quantitative PCR
5
70 nm. To evaluate dark toxicity, the cells were treated with
Apoptosis-related gene (survivin) and cell cycle-related gene
CDK2) expression levels were quantied by real-time uores-
ACP under the same conditions but without the light exposure.
(
cent quantitative PCR (RT-qPCR). Primer glyceraldehyde
3-phosphate dehydrogenase (GAPDH) was used as reference, in
which the levels of each mRNA were normalized relative to the
quantity of human GAPDH in each cell. The RT-qPCR was
performed by UltraSYBR One Step RT-qPCR Kit (High ROX)
according to the manufacturer's instructions. The reaction
procedure of RT-qPCR is as follows: 40 cycles of denaturation at
Cell uptake
Cellular uptake of ACP was investigated by FIM. HeLa cells were
23
5
seeded into 6-well cell culture plates (1 ꢃ 10 cells per well) and
incubated for 24 h. The cells were incubated with ACP
ꢂ1
ꢀ
(
8 mg mL ) at 37 C for 0, 20, 40, 60, 120, 180 min. Cells were
washed with PBS three times and xed with 4% para-
formaldehyde for 10 min. Then, cells were again washed with
PBS three times and stained with 1 mg mL DAPI for 10 min.
ꢀ
ꢀ
9
5 C for 30 s, primer annealing at 56 C for 30 s, and extension
ꢀ
ꢂ1
at 72 C for 1 min. All experiments were done 4 times. All the
PCR products which had been recovered and sequenced were
contrasted with the design of primers and probe sequences to
analysis effect of primer amplication.
The photoreaction process of ACP
3
HeLa cells were seeded into 96-well plates at a density of 4 ꢃ 10
cells per well. D-mannitol (DM) and sodium azide (SA) are
Statistical analysis
quenchers which could trap the hydroxyl radical (cOH)
quencher and singlet oxygen ( O
during irradiation. The tests were divided into three groups:
ACP–PDT; ACP–DM–PDT and ACP–SA–PDT. The cells of the
ACP–PDT groups were treated with PS. ACP–DM–PDT groups
and ACP–SA–PDT groups were treated with PS and quenching
1
All the results were expressed as the mean ꢁ SD of three inde-
pendent experiments. Statistical analysis was performed using
Student's t-test using the SPSS 19.0, and p < 0.05 was considered
statistically signicant.
2
) generated by photosensitizer
ꢂ1
ꢂ1
Results and discussion
The UV-vis absorption and singlet oxygen detection of ACP
agent DM (20 mL, 40 mmol L ) or SA (20 mL, 20 mmol L ),
respectively. The nal concentrations of ACP were 0.195, 0.39,
ꢂ1
0.78, 1.56, 3.12, 6.25, 12.5, 25 mg mL , respectively. Aer
Fig. 2a displayed the UV-vis absorption spectra of ACP (c ¼ 1 ꢃ
additional 4 h of incubation with samples, all wells were
exposed to light for 10 min. The plates were then kept in the
incubator for additional 24 h. Then, cell viability was estimated
by MTT assay, as described above. Each experiment was paral-
leled three times.
ꢂ5
ꢂ1
10
mol L ) in DMF and PBS. In DMF, ACP absorbed
throughout the UV-vis region between about 300 and 800 nm
with four peaks: a strong Soret absorption peak at 401 nm, three
weak absorption peaks at 500 nm, 529 nm and 608 nm, and
a Q(0,0) band at 663 nm, showing typical absorption charac-
teristics of chlorophylls. Meanwhile, the spectrum of ACP in
PBS also showed a strong absorption Soret band at 400 nm and
Cell morphology observation
To study the changes of cellular morphology aer PDT, cells a Q(0,0) band at 660 nm, displaying a 3 nm blue-shi compared
5
were incubated into six-well plates (1 ꢃ 10 cells per well) with with that in DMF.
1
mL DMEM. Aer the cells adhered to the plate, the DMEM
Before a novel PS is used as a candidate for PDT agent, it is of
1
medium was removed and the cultures were washed three times great important to measure the O quantum yield (F ). It is
with PBS. Then add 1 mL ACP (8 mg mL ) solution in each known that DPBF could be oxidized rapidly into o-dibenzoyl-
wells, which were then incubated for 4 h, followed by the irra- benzene at the present of
diation (10 min, 5 mW cm ). Aer irradiation, cells were meanwhile the intensity of DPBF characteristic peak (415 nm)
2
D
ꢂ1
1
2
O via a ring opening reaction,
ꢂ2
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Fig. 3 The photobleaching plot based on the absorption in PBS.
absorbance and the absorption of PSs would not affect the
absorption of DPBF at 415 nm. As shown in Fig. 2a, the
absorption of DPBF in DMF at 415 nm gradually decreased from
0
s to 120 s, indicating that the target compound could generate
1
2
the O under the light. The slope of ACP and MB was obtained
aer tting with a linear function (R > 0.9983). The singlet
2
oxygen quantum yield (F ) of ACP in DMF is 29.3% (Fig. 2b),
D
exhibiting that ACP has promising ability to produce singlet
oxygen aer irradiation.
Photobleaching test of ACP
Photobleaching can reduce the yield of reactive oxygen species
25
(
ROS) and decrease the ability to destroy tumors. In PDT,
photobleaching refers to the phenomenon of degradation of PS
26
under light. Photobleaching is inevitable in the PDT process
with the formation of singlet oxygen. The therapeutic effect of
27
PDT is related to the amount and the rate of photobleaching.
Therefore, the anti-photobleaching ability of photosensitizer
was an important parameter of PDT. In this paper, the long-
wavelength absorption of ACP was 660 nm in PBS, which was
used to calculate the photobleaching rate. As shown in Fig. 3,
ACP showed a slow trend of degradation over the time. The
photobleaching rate was 1.1% at 10 min, which could be
ignored. At the endpoint of irradiation, only 8.2% of ACP was
photobleached in PBS. Generally, the photosensitizer could
achieve good PDT effects aer being exposed to light for 10 min,
therefore the photobleaching of ACP has little inuence to PDT
effects. All in all, ACP showed a very satisfactory photostability
during 10 min by irradiation, which could be used as a potential
photosensitizer in PDT.
Fig. 2 (a) The UV-vis absorption spectra of ACP (the concentration of
ꢂ5
ꢂ1
ACP is 1 ꢃ 10 mol L ) in DMF and PBS. (b) UV-vis absorption spectra
of decomposition of DPBF after irradiation of ACP in DMF. (The inset is
a locally enlarged drawing of (b); (c) first-order plots for the photo-
decomposition of DPBF monitoring the long-wavelength absorption
of DPBF at 415 nm.
24
was degraded, which corresponds to the ability of PSs to Photodynamic therapy activities of ACP
produce singlet oxygen. To observe the process of dynamic
quenching of ROS and avoid PSs interfering the absorption of
The cytotoxicity of the title compound against HeLa cells was
shown in Fig. 4, in which red columns and black columns stand
for the cell viability of HeLa cells treated with ACP with and
without light, respectively. The control groups treated with
different concentrations of ACP in the dark showed high cell
viability (>90%), indicating that the dark toxicity of ACP was
DPBF at 415 nm, we set a very low concentration of PS (2.8 ꢃ
ꢂ6
ꢂ5
ꢂ1
ꢂ1
1
1
0
0
mol L ) and a relative high concentration of DPBF (6 ꢃ
mol L ), because the PS absorbed very little at the low
concentration, therefore it can be corrected as background of
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Fig. 4 The photodynamic activity of the ACP against HeLa cells after
24 h.
Fig. 5 Cell viability of three groups on the HeLa cells. ACP–PDT stands
for the groups treated with ACP under irradiation. ACP–DM–PDT
stands for the groups treated with ACP and D-mannitol under irradi-
negligible on HeLa cells without light. The cell viability of ation; ACP–SA–PDT stands for the groups treated with ACP and
sodium azide under irradiation.
ACP–PDT declined gradually with increasing concentration of
ACP under light. With a 10 min irradiation, 0.78 mg mL of ACP
ꢂ1
could cause approximately 39% cell mortality. When the
ꢂ1
concentration of ACP was up to 6.25 mg mL , the cell viability the cytotoxicity of target compound was remarkable, but singlet
decreased to 19%, suggesting that ACP had a strong cytotoxicity oxygen quantum yield (F
on HeLa cells. The cytotoxicity against HeLa cells of pyropheo- DMF.
phorbide-a (PPa) and Ce6 were conducted in parallel in our
D
) of ACP (29.3%) was not very high in
laboratory, which suggested that the title compound showed
a stronger cytotoxicity (the cell viability was 21% at the
concentration of 8 mmol L ) than PPa (cell viability, 38%) and
Cell uptake of ACP
Reasonable ability to penetrate the cell membrane would make
ꢂ1
30
the photosensitizer more efficient. To further validates the
potential in clinical application, we have investigated the
uptaking of HeLa cells against ACP in vitro. To identify
the intracellular uptake of ACP, HeLa cells were stained with
Ce6 (cell viability, 61%) at the same conditions (10 min irradi-
ꢂ2
ation, 5 mW cm ). Based on the above analysis, our proposed
compound is an excellent candidate in PDT drug development.
4,6-diamidino-2-phenylindole (DAPI), which is extensively used
nuclear stain agent showing blue uorescence. ACP would emit
red uorescence under FIM. As shown in Fig. 6, the uores-
cence intensity of ACP showed time-dependent increase. Aer
incubated with ACP for 20 min, some HeLa cells with red
The photoreaction process of ACP aer PDT
The excited PS could produce reactive oxygen species (ROS) by
two ways that induced the tumor necrosis or apoptosis, that is,
Type I process involving generation of hydroxyl radicals (HR)
and other reactive oxygen species; Type II process involving

uorescence were observed, suggesting partial ACP has been
uptaken by the cells. Cell treated with ACP for 40 min displayed
the enhanced uorescence signal, implying that more PS
entered the cells. The red signal became brighter and brighter
over the time, suggesting that the photosensitizer (ACP) had
a good ability to entering HeLa cells. Moreover, from the image
of overlay, it had been conrmed that ACP could accumulate
and distribute in cytoplasm of HeLa cells.
1
28
generation of singlet oxygen ( O
2
). To investigate the type of
photochemical reaction of ACP, sodium azide (SA, a quencher
1
2
of O ) and D-mannitol (DM, a quencher of hydroxyl radical)
29
were introduced. Fig. 5 showed the cell viability of the three
experimental groups: ACP–PDT, ACP–DM–PDT and ACP–SA–
PDT. With the quenching agent's adding, the cell viability of
ACP–DM–PDT and ACP–SA–PDT increased typically, demon-
strating that corresponding reactive oxygen species were
quenched and also indicating that the ROS were generated in
Cell morphology observation
HeLa cells treated with ACP during the light. Meanwhile, the The changes of cell morphology in bright eld were analyzed by
mechanism of generating ROS against HeLa cells in PDT uorescence inverted microscope (FIM) aer irradiation for 0,
treatment may be performed via two ways, Type I and Type II 6, 12, 24 h, respectively. As shown in Fig. 7a (0 h), the HeLa cells
photodynamic reaction. Moreover, the cell viability of the morphology were spindle-shaped and closely linked to each
ACP–DM–PDT groups were slightly higher than the cell viability other. All the cells maintained normal cell morphology and
of ACP–SA–PDT groups, from which we can conclude that the grew adherent to the plate with steady cytoplasm and high
generation of hydroxyl radicals play a more important role than refractive index. As the passage of time, cells became rounder,
that of singlet oxygen in PDT. Herein, this partly explained why the cell gap became larger and the membrane became fuzzy
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Fig. 6 In vitro uptake of ACP was observed by fluorescence inverted microscopic after incubating HeLa cells with ACP for 0, 20, 40, 60 and
20 min, respectively. The blue fluorescence was attributed to cell nucleus (stained by DAPI), the red fluorescence was due to ACP and the
1
overlay image in DAPI and ACP channels on the same cell.
with signicantly decreased shading degree. Aer treated with proteins, such as survivin, which is a member of the inhibitor of
PDT and cultured for additional 6 h (Fig. 7b), a majority of cells apoptosis proteins (IAP) family. Survivin could inhibit
gradually began to rounder and some cells maintain normal cell apoptosis, promote cell proliferation and regulate cell mitosis.
morphology. The rounder cells had change to an irregular Its anti-apoptotic effect may be through apoptosis inhibition of
31
shape and the shading degree slightly decreased, indicating caspase-3 to achieve. If the expression of survivin is inhibited,
that the efficient PDT activities of ACP to induce cell damage. the expression of caspase-3 would be promoted directly or
32
Aer prolonged incubation for 12 h (Fig. 7c), the obviously indirectly, thereby leading to apoptosis. Moreover, it is widely
injury of the cell membrane structure, remarkably pathological acknowledged that cell cycle arrest is an important reason of
changes and signicantly decreased shading degree could be cell apoptosis. The family of cyclin-dependent kinases (CDKs)
observed, showing that HeLa cells undergone apoptosis and are well known in the cell division cycle. Among them, cyclin-
damage aer treated with PDT. Finally, in Fig. 7d (24 h), the dependent kinase 2 (CDK2) is an important member of the
membrane was destroyed completely and the cells were dead. CDKs family, which is believed to be essential to complete G1
All in all, the cell morphological changes aer ACP–PDT treat- and to initiate S phase. If the expression of CDK2 declined, S
ment implying that ACP had a efficiently PDT activity to induce phase would fail to start normally, and the incomplete S phase
3
3
apoptosis and damage cell death against HeLa cells.
events would lead to cell death. Fig. 8a showed the gel elec-
trophoresis of total RNA from HeLa cells aer treated with ACP
in different time. It can be observed that both 18s and 28s RNA
were clearly visible and 28s band is two times brighter than 18s
band, demonstrating that RNA was successfully extracted.
Expression of survivin and CDK-2 in HeLa cells
The mechanism of apoptosis induced by PDT may be related to
the inhibition of the expression of apoptosis regulating
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ACP could inhibited the expression of survivin, result in cells
apoptosis and cell death, while the change of expression of
CDK2 is negligible aer treatment of ACP on HeLa cells.
Conclusion
In this paper, we have systematically investigated the photody-
namic therapy activity of a chlorin derivative Amidochlorin p6
(
(
ACP), which showed excellent phototoxicity against HeLa cells
1.17 ꢁ 0.029 mg mL , 10 min, dosage 5 mW cm ), negligible
ꢂ1
ꢂ2
dark toxicity, remarkable photostability, and being easily
uptaken by cancer cells. Moreover, Type I and Type II photo-
dynamic reactions occurred simultaneously on HeLa cells in
PDT treatment, and the inuence of Type I was slightly larger
than that of Type II. Finally, the RT-qPCR experiment showing
that ACP could regulate the expression of survivin, which partly
explained why ACP could effectively lead to the cell apoptosis
and accelerating cell death. To sum up, ACP is potential as
photosensitizer in PDT cancer treatment, and deserves a further
investigation.
Fig. 7 The cell morphology observation of HeLa cells incubated with
ꢂ1
8
mg mL ACP after PDT for: (a) 0 h; (b) 6 h; (c)12 h; (d) 24 h.
Fig. 8b showed the relative expression of survivin and CDK2
under ACP treatment in HeLa cells at 0 h, 6 h, 12 h and 24 h,
respectively. The expression of survivin in HeLa cells treated
with ACP dropped suddenly at 6 h, then increased close to half
of the control group at 12 h. Finally it decreased to 0.06 times
the size of control group (Fig. 8b). The result indicated that the
Conflicts of interest
There are no conicts to declare.
title compound (ACP) could inhibit the expression of survivin Acknowledgements
effectively at 6 h and then accelerate apoptosis. The higher
Financial support of this research was provided by National
expression of survivin at 12 h might be due to the cellular stress
response to external stimulus. For CDK2 (Fig. 8c), the expres-
sion in HeLa cells treated with ACP at 6 h and 24 h are 0.8 times
and 0.9 times the size of control group, respectively, revealing
that the expressions of CDK2 were inhibited aer 6 h and 24 h.
The expression of CDK2 is 3.1 times as high as of control group
at 12 h, which could be attributed to be cellular stress response,
leading a recovery of expression. Based on the above analysis,
Natural Science Foundation of China (No. 21272048), the
Natural Science Foundation of Heilongjiang Province (No.
B20913), the Program for Scientic Technological Innovation
Team Construction in the Universities of Heilongjiang Province
(No. 211TD010), the Scientic Research Fund of Heilongjiang
Provincial Education Department (No. 12531194), the Natural
Science Youth Foundation of Heilongjiang Province (No.
QC2016011) and the Innovation Fund for Graduate Students of
Harbin Normal University.
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