Photodynamic therapy using zinc phthalocyanine with low dose of diode laser combined with doxorubicin is a synergistic combination therapy for human SK-MEL-3 melanoma cells

doi:10.1016/j.pdpdt.2019.08.027

Photodiagnosis and Photodynamic Therapy

Volume 28, December 2019, Pages 88-97

https://doi.org/10.1016/j.pdpdt.2019.08.027 Get rights and content

Highlights

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Photodynamic therapy with ZnPc (ZnPc-PDT) inhibits SK-MEL-3 cancer cells proliferation.
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Pre-treatment with ZnPc-PDT, in low dose of laser, sensitizes SK-MEL-3 cells towards very lower concentration of Doxorubicin.
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Combined ZnPc-PDT with DOX (chemo-photodynamic therapy) decreases cell viability synergistically.
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Chemo-PDT decreases cell migration and increases rate of apoptosis, autophagy, cell cycle arrest at subG1 and G2-M phase.

Abstract

Chemotherapy is a generally used anticancer strategy for melanoma and it may have improved outcomes in combination with other approaches. One such strategy is photodynamic therapy (PDT), where a photosensitizer (PS) generates reactive oxygen species (ROS) after illumination of target cells. Interestingly, in low doses and high doses of light sources, special cellular responses can be induced. Regarding this fact, in this study, the combination of zinc phthalocyanine (ZnPc)-PDT and Doxorubicin (DOX) was applied at low and high dose of diode laser to treat SK-MEL-3 cells. Cytotoxic effects were determined by MTT assay for assessment synergistic effects were estimated by calculation of Combination Index (CI); that synergistic effects were observed in most groups. In low dose of laser irradiation higher synergism effects were observed. Significant changes of ROS were not observed with combinations, but autophagy, subG1 and G2/M phase cell cycle arrest, decreased cell migration ability and apoptosis induction were significantly increased compared to either treatment alone. The expression of caspase-8, -9, -3 and Bcl-2 genes revealed caspase-dependent apoptosis in all groups. Moreover, ZnPc-PDT and chemo-PDT down-regulated the expression of MMP-9 and Vimentin genes that impaired cell migration. In conclusion, it can be suggested that pre-treatment with ZnPc-PDT has high effects to sensitize SK-MEL-3 cells to DOX, in particular with low dose of diode laser.

Graphical abstract

Introduction

Malignant Melanoma is the most common aggressive skin cancer worldwide. As reported by the American Cancer Society, about 91,270 new cases of melanoma were approximated to be detected in 2018. About 9,320 Americans are estimated to die from melanoma in 2018 [1]. Heavy prices are spent every year on research in order to treat melanoma or improve the quality of life in patients. The most common strategies of melanoma treatment are surgery, radiation therapy and chemotherapy [2]. Chemotherapy is a commonly used anticancer approach and has been significantly developed to date [3]. Nevertheless, serious side effects and drug-resistance can lead to limited therapeutic outcomes of chemotherapy in most kinds of cancers such as melanoma [[4], [5], [6]]. Owing to the heterogeneity and rapid proliferation of melanoma cells along with the high probability of melanoma invasion and metastasis [5], any single treatment approach is generally not sufficient to destroy the entire tumor [7]. Combination therapy using two or more therapeutic agents either simultaneously or sequentially has been explored since the 1960s, in order to improve the effectiveness of cancer therapy [[8], [9], [10]]. Combination therapy shows better therapeutic results when compared to single therapies, especially in reducing drug-resistance and preventing cancer recurrence. Combination therapy can also decrease side effects and improve the survival rate in cancer patients [7].

The combination of chemotherapy with photodynamic therapy (PDT) has received significant attention as a promising approach to treat cancer [[11], [12], [13]]. PDT is an effective minimally-invasive approach to the treatment of cancers of different types and sites as well as non-cancerous diseases. Excellent therapeutic results and capability of the parallel application of PDT with other treatments make it attractive [14]. PDT is based on local or systemic administration of a photoactive agent (photosensitizer), which is selectively accumulated in the tumor. The photosensitizer molecules absorb light of the specific wavelength and produce reactive oxygen species (ROS) inducing a biological cascade leading to selective death of the targeted cells. In the treatment of melanoma with PDT immediately suggests the question of the interaction of light with endogenous molecules of skin (such as melanin and hemoglobin) which absorb light. The most appropriate wavelength for PDT is 600–800 nm, which is known as the “therapeutic window”. Melanin and hemoglobin exhibit absorption across the visible region of the spectrum, but both fall to a minimum level in the therapeutic window [15,16].

Research over the last decades has shown that the use of two or more therapeutic approach in cancer treatment has more effective results than the single therapy approach. Notwithstanding the success of combined chemotherapy with DOX and ZnPc-PDT (chemo-PDT,) identify the most important factors and innovative methods are yet being researched and needed to improve the effective use of chemo-PDT in clinical oncology. Although PDT and chemotherapy have shown high potential for cancer therapy, both strategies have complex limitations, such as high side effects and drug-resistance (for chemotherapy) and difficulty in the light delivery of some internal tumors (for PDT). Biological mechanisms and effective factors of chemo-PDT effectiveness are extremely intricate and not completely identified to date. In this regard, the purpose of this work are (I) the comparative study of effects of low and high dose of light source in combination ZnPc-PDT with DOX to investigate the role of light source in chemo-PDT synergistic outcomes; (II) the comparative study of cellular and molecular mechanisms after chemo-PDT to highlighting significant results. So, we characterizing these mechanisms aimed at maximizing therapeutic outcomes with minimum invasive effects by employing the advantages of both strategies.

Section snippets

Cell line and cell culture

Human melanoma cell line (SK-MEL-3) was obtained from the Pasteur Institute of Iran. The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (Gibco, USA) and 1% antibiotics (penicillin /streptomycin, Gibco, USA). Subsequently, cells were incubated in the incubator at 37 °C under an atmosphere of 5% CO2 and 95% humidity.

In vitro cellular uptake of ZnPc

According to our previously reported article [17], DMSO was used to make ZnPc dissolve in the culture medium. The

Results and discussion

The role of the light source used in PDT is highly complex and remarkable. So, characterizing roles of the light source in synergistic effects of combination therapy leads to design better PDT and chemo-PDT protocols that extensively improve these efficiencies. Although the use of chemotherapy and PDT are clinically extended, the mechanisms underlying cancer cells death after chemo-PDT and effective factors are not completely characterized. In fact, one of the main areas of studies in the

Conclusion

Chemotherapy has always played and still play vital roles in melanoma treatment. Chemo-PDT is a powerful combination therapeutic approach with two anticancer strategies (PDT and chemotherapy) that can decrease the rate of metastasis and initiate melanoma cells death.

In 2016, studies by Tahmasebi et al. on melanoma cell line demonstrated that the melanoma cells incubated with 5-Aminolevulinic acid (5-ALA) and 5-fluorouracil(5-FU), enhanced the efficiency of 5-ALA-PDT [41].

In 2018, Gao et al.

Funding

Michael R Hamblin was supported by USNIH grant R01AI050875.

References (41)

Z. Chen
Zinc phthalocyanine conjugated with the amino-terminal fragment of urokinase for tumor-targeting photodynamic therapy
Acta Biomater.
(2014)
Y. Huang et al.
Membrane transporters and channels in chemoresistance and-sensitivity of tumor cells
Cancer Lett.
(2006)
U. Gatzemeier
Randomized phase II trial of gemcitabine–cisplatin with or without trastuzumab in HER2-positive non-small-cell lung cancer
Ann. Oncol.
(2004)
C.-M.J. Hu et al.
Nanoparticle-based combination therapy toward overcoming drug resistance in cancer
Biochem. Pharmacol.
(2012)
A. Khdair
Nanoparticle-mediated combination chemotherapy and photodynamic therapy overcomes tumor drug resistance in vitro
Eur. J. Pharm. Biopharm.
(2009)
M. Wainwright et al.
The use of photosensitisers in acne treatment
J. Photochem. Photobiol. B, Biol.
(2011)
M.A. Doustvandi
The interaction between the light source dose and caspase-dependent and-independent apoptosis in human SK-MEL-3 skin cancer cells following photodynamic therapy with zinc phthalocyanine: a comparative study
J. Photochem. Photobiol. B, Biol.
(2017)
E. Buytaert et al.
Molecular effectors of multiple cell death pathways initiated by photodynamic therapy
Biochimica et Biophysica Acta (BBA)-Reviews on Cancer
(2007)
T. Yan
pH-Sensitive mesoporous silica nanoparticles for chemo-photodynamic combination therapy
Colloids Surf. B Biointerfaces
(2018)
H.-L. Xu
Anti-proliferative effect of Juglone from Juglans mandshurica Maxim on human leukemia cell HL-60 by inducing apoptosis through the mitochondria-dependent pathway
Eur. J. Pharmacol.
(2010)

J. Nackiewicz et al.

A novel pro-apoptotic role of zinc octacarboxyphthalocyanine in melanoma me45 cancer cell’s photodynamic therapy (PDT)

J. Photochem. Photobiol. B, Biol.

(2019)

S. Li

Autophagy activation alleviates nonylphenol-induced apoptosis in cultured cortical neurons

Neurochem. Int.

(2019)

M. Russo et al.

Autophagy inducers in cancer

Biochem. Pharmacol.

(2018)

H.L. Best

Characterisation of early changes in ovine CLN5 and CLN6 batten disease neural cultures for the rapid screening of therapeutics

Neurobiol. Dis.

(2017)

C.A. Robertson et al.

Photodynamic therapy (PDT): a short review on cellular mechanisms and cancer research applications for PDT

J. Photochem. Photobiol. B, Biol.

(2009)

N.B.R. Vittar

Caspase-independent apoptosis, in human MCF-7c3 breast cancer cells, following photodynamic therapy, with a novel water-soluble phthalocyanine

Int. J. Biochem. Cell Biol.

(2010)

S. Yang

Hinokiflavone induces apoptosis in melanoma cells through the ROS-mitochondrial apoptotic pathway and impairs cell migration and invasion

Biomed. Pharmacother.

(2018)

H. Tahmasebi

Enhancing the efficiency of 5-aminolevulinic acid-mediated photodynamic therapy using 5-fluorouracil on human melanoma cells

Photodiagnosis Photodyn. Ther.

(2016)

American Cancer Society

Cancer Facts & Figures 2018

(2018)

H. Lee

Combination of chemotherapy and photodynamic therapy for cancer treatment with sonoporation effects

J. Control. Release

(2018)

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Photodynamic therapy using zinc phthalocyanine with low dose of diode laser combined with doxorubicin is a synergistic combination therapy for human SK-MEL-3 melanoma cells

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Cell line and cell culture

In vitro cellular uptake of ZnPc

Results and discussion

Conclusion

Funding

Acta Biomater.

Cancer Lett.

Ann. Oncol.

Biochem. Pharmacol.

Eur. J. Pharm. Biopharm.

J. Photochem. Photobiol. B, Biol.

J. Photochem. Photobiol. B, Biol.

Biochimica et Biophysica Acta (BBA)-Reviews on Cancer

Colloids Surf. B Biointerfaces

Eur. J. Pharmacol.

J. Photochem. Photobiol. B, Biol.

Neurochem. Int.

Biochem. Pharmacol.

Neurobiol. Dis.

J. Photochem. Photobiol. B, Biol.

Int. J. Biochem. Cell Biol.

Biomed. Pharmacother.

Photodiagnosis Photodyn. Ther.

Cancer Facts & Figures 2018

Combination of chemotherapy and photodynamic therapy for cancer treatment with sonoporation effects

J. Control. Release