Hypericin-mediated photodynamic therapy for the treatment of cancer: A review

Abstract

Objectives
Hypericin is a polycyclic aromatic naphthodianthrone that occurs naturally. It is also an active ingredient in some species of the genus Hypericum. Emerging evidence suggests that hypericin has attracted great attention as a potential anticancer drug and exhibits remarkable antiproliferative effect upon irradiation on various tumour cells. This paper aims to summarise the anticancer effect and molecular mechanisms modulated by hypericin-medicated photodynamic therapy and its potential role in the cancer treatment.

Key findings
Hypericin-medicated photodynamic therapy could inhibit the proliferation of various tumour cells including bladder, colon, breast, cervical, glioma, leukaemia, hepatic, melanoma, lymphoma and lung cancers. The effect is primarily mediated by p38 mitogen-activated protein kinase (MAPK), JNK, PI3K, CCAAT-enhancer-binding protein homologous protein (CHOP)/TRIB3/Akt/mTOR, TRAIL/TRAIL-receptor, c-Met and Ephrin-Eph, the mitochondria and extrinsic signalling pathways. Furthermore, hypericin-medicated photodynamic therapy in conjunction with chemotherapeutic agents or targeted therapies is more effective in inhibiting the growth of tumour cells.

Summary
During the past few decades, the anticancer properties of photoactivated hypericin have been extensively investigated. Hypericin-medicated photodynamic therapy can modulate a variety of proteins and genes and exhibit a great potential to be used as a therapeutic agent for various types of cancer.

Full text

425
Journal of Pharmacy and Pharmacology, 2021, Vol 73, 425–436
doi:10.1093/jpp/rgaa018
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Review
Hypericin-mediated photodynamic therapy for
the treatment of cancer: areview
XiaoxvDong1,2,, YawenZeng1, ZhiqinZhang1, JingFu3, LongtaiYou1,
YuanyuanHe2, YangHao2, ZiliGu2, ZhenfengYu2, ChanghaiQu1,
XingbinYin1, JianNi1,4 and LuisJ.Cruz2,*
1School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China
2Department of Radiology, Division of Translational Nanobiomaterials and Imaging, Leiden University Medical Center,
Leiden, The Netherlands
3Beijing Institute of Traditional Chinese Medicine, Beijing Hospital of Traditional Chinese Medicine, Capital Medical
University, Beijing, China
4Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China
*Correspondence: Luis J. Cruz, Department of Radiology, Division of Translational Nanobiomaterials and Imaging, Room
C2-187h, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands. Email: l.j.cruz_ricondo@lumc.nl
Received April 4, 2020; Accepted October 5, 2020.
Abstract
Objectives Hypericin is a polycyclic aromatic naphthodianthrone that occurs naturally. It is also
an active ingredient in some species of the genus Hypericum. Emerging evidence suggests that
hypericin has attracted great attention as a potential anticancer drug and exhibits remarkable
antiproliferative effect upon irradiation on various tumour cells. This paper aims to summarise the
anticancer effect and molecular mechanisms modulated by hypericin-medicated photodynamic
therapy and its potential role in the cancer treatment.
Key findings Hypericin-medicated photodynamic therapy could inhibit the proliferation of various
tumour cells including bladder, colon, breast, cervical, glioma, leukaemia, hepatic, melanoma,
lymphoma and lung cancers. The effect is primarily mediated by p38 mitogen-activated protein kinase
(MAPK), JNK, PI3K, CCAAT-enhancer-binding protein homologous protein (CHOP)/TRIB3/Akt/mTOR,
TRAIL/TRAIL-receptor, c-Met and Ephrin-Eph, the mitochondria and extrinsic signalling pathways.
Furthermore, hypericin-medicated photodynamic therapy in conjunction with chemotherapeutic
agents or targeted therapies is more effective in inhibiting the growth of tumour cells.
Summary During the past few decades, the anticancer properties of photoactivated hypericin have
been extensively investigated. Hypericin-medicated photodynamic therapy can modulate a variety
of proteins and genes and exhibit a great potential to be used as a therapeutic agent for various
types of cancer.
Keywords: Hypericin; photosensitiser; photodynamic therapy; molecular targets
Introduction
Photodynamic therapy (PDT) is an emerging noninvasive therapy
that possesses many advantages over traditional cancer therapies
(surgery, chemotherapy and radiation therapy), such as minimal
invasiveness and limited side effects, and low mutation rate. [1, 2] In
addition, PDT could be used to treat a variety of diseases, including
bacterial infections, psoriasis and age-related macular degener-
ation.[3, 4] PDT consists of three essential elements: photosensitiser,
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426 Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 4
light and oxygen.[5, 6] The photosensitiser is irradiated under the
appropriate wavelength of light, and the singlet basic energy state
is converted to the excited state due to photon absorption, which
might spontaneously undergo intersystem crossing to form a rela-
tively stable triplet state (Figure1). The triplet state photosensitiser
reacts with molecule oxygen and then generates reactive oxygen
species (ROS), which can direct cell killing, devastate the vascular
system and stimulate immune response. [7] Hence, the photosensitiser
plays a critical role in the PDT process.
Hypericin (4,5,7,4′,5′,7′-hexahydroxy-2,2′-
dimethylnaphthodianthrone) (HYP; Figure 2) is a hydroxyl-
ated phenanthroperylenequinone in some species of the genus
Hypericum. The photoactivation behaviour of HYP is induced by
the delocalised π-electron system in its aromatic rings.[8, 9] A lot of
researches have conrmed that HYP selectively accumulates in tu-
mour tissue via diffusion, pinocytosis or endocytosis in PDT of tu-
mours.[10–13] Hypericin mainly accumulates in the membranes of the
endoplasmic reticulum (ER), lysosomes, Golgi apparatus and mito-
chondria due to its hydrophobic character.[14, 15] Furthermore, HYP
has a maximum absorption at 590nm and generates high singlet
oxygen. It also has minimal dark toxicity, excellent photosensitivity
and high clearance rate. [16, 17] In all, these advantages make HYP to
be a promising excellent photosensitiser, as well as its photochem-
ical properties in PDT. Hence, the purpose of this review is intended
to summarise the anticancer activity and molecular mechanisms of
HYP-medicated PDT (HYP-PDT) and highlight its potential thera-
peutic value in cancer treatment.
Anticancer Activity
Bladdercancer
De Witte’s group for the rst time reported that HYP-PDT could
induce apoptosis or necrosis in AY-27 urinary bladder carcinoma
in a concentration-dependent manner. This indicated that HYP
might play an important role for the PDT of supercial bladder car-
cinoma.[18] The mechanism of HYP-PDT in AY-27 cells was through
vascular effects rather than mediated by cellular destruction in these
tumours.[19] Moreover, it was shown for the rst time that HYP-
PDT could be used to induce selective AY-27 cells damage without
damaging the detrusor musculature.[20] In another experiment, their
group also evaluated the biodistribution of HYP in AY-27 cells line
and normal bladder wall. The data demonstrated that there was
much more HYP uorescence intensity in tumour cells without
causing damage to the underlying muscle layers. [21] Huygens etal.[22]
showed that HYP combined with hyperoxygenation resulted in a
virtually complete bladder RT-112 cancer cells killing through apop-
tosis. Similarly, peruorocarbons were also applied in increasing
the photodynamic activity of HYP in RT-112 carcinoma spheroids.
Since HYP was specically concentrated in bladder carcinoma cells,
indicating that HYP combined with peruorocarbons could be util-
ised for effective bladder wall photodynamic treatment.[23] Kocanova
etal.[24] studied the signalling pathways leading to the upregulation
of heme oxygenase (HO-1) enzyme in T24 tumour cells treated
with HYP-PDT. This mechanism might involve the stimulation of
HO-1 expression controlled by the PI3K and p38 mitogen-activated
protein kinase (MAPK) pathways. Buytaert etal.[25] investigated the
molecular targets of HYP-induced cell apoptosis in T24 cells. The
results indicated that inhibition of p38 MAPK prevented the autono-
mous regeneration and migration. It has been shown that HYP-PDT
combined with angiogenesis inhibitor (bevacizumab) signicantly
downregulated the expression of vascular endothelial growth factor
(VEGF) proteins in the MGH cells.[26] Moreover, HYP-PDT induced
the upregulation of angiogenic proteins including VEGF, interferon-α
(IFN-α), tumor necrosis growth factor-α (TNF-α) and basic bro-
blast growth factor (bFGF) in MGH xenograft model. Hepatocyte
growth factor (HGF) and Ephrin-A3 (EFNA3) proteins were also
increased by the activation of c-Met and Ephrin-Eph pathways.[27]
Coloncancer
Blank et al.[28] evaluated the wavelength effects of HYP-PDT in a
C26 colon carcinoma model. In-vitro irradiation of HYP-sensitised
cells resulted in a loss of cell viability in a light dose-dependent
manner. In-vivo irradiation of C26-derived tumours, HYP caused
extensive vascular damage and tumour necrosis. It was shown for
the rst time that pre-treatment with proadifen affected the function
Figure 1 Mechanism of photoactivation of photosensitizer.
Figure 2 Chemical structure of hypericin.
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Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 4 427
of multidrug resistance-associated protein (MRP1) and breast cancer
resistance protein (BCRP), leading to an increase of HYP content
and the improvement of HY-PDT.[29] 5-Lipoxygenase (5-LOX) in-
hibitor (MK-886) and HYP-PDT also induced apoptosis and the
accumulation in the S phase of HT-29 cells, along with the repres-
sion of DNA synthesis.[30] Moreover, the mechanisms associated
with the cell cycle progression of HT-29 cells were accompanied by
increase in cyclin E level and decrease of cyclin A, cdk-2 and pRb
expression. The stimulation of apoptosis was accomplished prefer-
entially via the mitochondrial pathway.[31] Asimilar study showed
that HYP-PDT combined with hyperforin stimulated the onset of
apoptosis in HT-29 cells through inhibiting cell cycle arrest and sup-
pressing expression of matrix metalloproteinase-2 (MMP-2)/-9.[32]
Furthermore, the expression of some pro-angiogenic factors, such as
VEGF-A, PD-ECGF, PDGF-A (V1) and PDGF-A (V2) in HT-29 cells,
was increased.[33, 34] The nonspecic cyclooxygenase (COX) inhibi-
tors or polyunsaturated fatty acids potentiated the cytotoxic effect
of HYP-PDT.[35, 36] Treatment with HYP and the inhibitor of survivin
expression (YM155) caused more severe dissipation of mitochon-
drial membrane potential and induced the activation of caspase-3
and poly(ADP-ribose)polymerase (PARP) proteins in HT-29 cells.[37]
Similarly, a synergistic combination of photoactive HYP and
Manumycin Aexhibited anticancer effects on oxaliplatin-resistant
HT-29-OxR cells. This mechanism might involve the inhibition
of cell viability, colony formation and induction of apoptosis.[38]
Meanwhile, HYP-PDT decreased the volume of the primary tu-
mour and signicantly prolonged the survival of the CT26 tumour-
bearing BALB/c mice by inducing antitumoral immune response.[39]
Lin etal.[40] reported that HYP-PDT caused severe ER stress and led
to the induction of CCAAT-enhancer-binding protein homologous
protein (CHOP), which thereby activated CHOP/TRIB3/Akt/mTOR
pathway and triggered autophagic cell death of HCT116 and HCT8
cells. Their group also found that treatment with HYP-PDT en-
hanced the antitumour activity of oxaliplatin in HCT8 and HCT116
cells. This was mediated by ROS, and its mechanism involved af-
fecting drug efux and GSH-related detoxication.[41] Furthermore,
the inhibition of ABCG2 potentially enhanced the efcacy of HYP-
PDT in 3D spheroid models of HT-29 cells.[42] In another study,
F-actin and dysadherin could be the target proteins for HYP-PDT
in HT-29 cells.[43] Montanha etal.[44] investigated the photodynamic
activity of HYP in Pluronic P123 against HT-29 cells. The data dem-
onstrated that the nanocarrier played an important role in the pene-
tration of HYP through cell membranes, suggesting that HYP was
a promising photosensitiser that might be suitable for the treatment
of colon diseases. Similarly, the phototoxic effects of HYP loaded on
superparamagnetic iron oxide nanoparticles were also studied. The
results indicated that nanoparticle-loaded HYP resulted in a com-
plete stop of HT-29 cells’ proliferation and induction of cell death.[45]
In another study, HYP-PDT exhibited immunomodulatory activities
resulting from an increase of interleukin-8 (IL-8) secretion in SW620
cells and a signicant decrease in IL-8 in the SW480 cells. [46]
Breastcancer
Ferenc et al.[47] found that combination of HYP-PDT and genistein
inhibited the proliferation of MCF-7 and MDA-MB-231 cells and
induced their apoptosis. The mechanisms were related to alterations
in the protein levels of Bcl-2, Bax, Akt and cell cycle perturbations
in G2/M-phase. It has been shown that HYP-PDT combined with
17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-
DMAG) obviously decreased the metabolic activity and viability
of SKBR-3 cells through decreasing the levels of human epidermal
growth factor receptor-2 (HER2), Akt, survivin and P-Erk1/2.[48]
Similarly, photoactivated HYP signicantly decreased the mRNA
and protein expression of HER2 in SKBR-3 and MCF-7 cells.[49]
HY-PDT and MK-886 also synergistically induced the MCF-7 cell
death.[50] Asimilar study showed that combination of HYP-PDT and
carmustine inhibited the reduction of GSH, resulting in an increase in
the sensitivity of MCF-7 cells.[51] Photoactivated HYP increased the
production of ROS in MCF-7 and MDA-MB-231 cells. Additionally,
the superoxid dismutase-2 (SOD-2) inhibitor (methoxyestradiol)
signicantly enhanced the sensitivity of MCF-7 cells to HYP.[52]
In-vitro assays have shown that HYP and hypericinates permeated
the membrane of MCF-7 cells and accumulated in organelles near
the nucleus. Furthermore, the photodynamic studies showed that
HYP inhibited the formation of cellular colonies, indicating a pos-
sible ability to prevent the recurrence of tumours.[53] Gao et al.[54]
demonstrated that single-walled carbon nanohorns (SWNH)-HYP
effectively ablated the 4T1 mouse breast cells upon light irradiations.
Moreover, the remarkable tumour growth inhibition as well as tu-
mour cell death was proved by PDT of SWNH-HYP. In another ex-
periment, the hybrid therapy of HYP-PDT and tamoxifen exhibited
90% cytotoxicity in MDA-MB-231 and MCF-7 cells. The cytotox-
icity was in the form of necrosis and autophagy accompanied by
high levels of lipid peroxidation.[55]
Cervicalcancer
Delaey et al.[56] demonstrated for the rst time that HYP-induced
photocytotoxicity in HeLa cells depends on the cells’ number, as low
cell density cultures were more responsive to PDT than conuent
cell cultures. The inhibition of JNK and p38 MAPK upregulated the
HYP-PDT-induced apoptosis of HeLa cells, which might provide
molecular basis for developing new therapeutic strategies.[57] The
apoptosis induced by HYP-PDT in HeLa cells was mediated by the
mitochondrial pathway, which involved the release of cytochrome c
and the further activation of caspase-3.[58] HYP-PDT and pyridinyl
imidazole might improve the therapeutic effect by preventing the
upregulation of COX-2, which contributed to the growth of HeLa
cells through the release of pro-angiogenic factors, as well as by
sensitising the cells to apoptosis.[59] Similarly, Bianchini etal.[60] re-
ported that the complexation of HYP with apomyoglobin (apoMb)
improved its efcacy as a photosensitiser on HeLa cells. Application
of the HYP-apoMb to HeLa tumour spheroids followed by illumin-
ation obviously decreased the growth rate of the structure. It has
been shown that PDT with ER-associated HYP induced the imme-
diate loss of SERCA2 protein levels, leading to HeLa cells death and
disruption of Ca2+ homeostasis.[61] Moreover, Barras investigated the
photodynamic effect of HYP-loaded lipid nanocapsules on HeLa
cells. The results demonstrated that the nanocapsules held promise
for potential application for cervical cancer due to their extended
circulation and accumulation in tumour-bearing mice.[62]
Nasopharyngealcancer
Du et al.[63] evaluated the expression of IL-6 in two different
nasopharyngeal cancer (NPC) cells after HYP-PDT. The results
showed the transcription of IL-6 was obviously elevated in PDT-
treated poorly differentiated CNE-2 cells but not in well differ-
entiated HK-1 cells. In vivo, the mRNA expression of IL-6 was
also upregulated in CNE-2 cells. However, HYP-PDT did not alter
the expression level of IL-8 in HK-1 and CNE-2 cells.[64] HYP-
PDT induced the phototoxicity in HK1 cells, and led to tumour
shrinkage and necrosis with accompanying lipid peroxidation
in the HK-1 murine tumour model.[65] Moreover, there was
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428 Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 4
signicant reduction of glutathione S-transferase (GST) activity
in HK-1 and CNE-2 cells as well as in the HK-1 tumour tis-
sues.[66] Photoactivation of HYP elicited an increase in the protein
and mRNA expression of MMP-1 in HK-1 and CNE-2 cells.
Similarly, the mRNA expression of MMP1 was also upregulated
in HYP-PDT-treated HK-l tumours.[67] In addition, HYP-PDT
downregulated the expression of MMP-9 by inhibiting the pro-
duction of granulocyte-macrophage colony-stimulating factor
(GM-CSF), thereby regulating the transcriptional effect of AP1/
NF-kB.[68] HYP-PDT could activate cysteine-type endopeptidase,
which inhibited the growth and induced the apoptosis of CNE-2
cells through the mitochondrial-dependent intrinsic and extrinsic
pathways.[69] Furthermore, HYP-PDT activated the p38 MAPKs
by generating singlet oxygen. The inhibition of p38 MAPKs
pathway with chemical inhibitors could enhance the apoptosis
of HK-1 cells induced by HYP-PDT.[70] HYP-PDT had been re-
ported to exhibit the desirable characteristics of inducing more
apoptosis and lowering serum VEGF levels in the HK-1 tumour
murine model.[71] Olivo etal. conducted a study to improve the
tumour growth by using HYP-PDT and celebrex together. The
data showed that the mRNA level of COX-2 was decreased in
CNE-2 cells in vitro. Meanwhile, the downregulation of COX-2
and hypoxia inducible factor-lα (HIF-lα) genes was observed in
vivo.[72] Additionally, their group reported that HYP-PDT along
with celebrex decreased the protein level of VEGF, indicating that
angiogenesis inhibitors might enhance the therapeutic efcacy of
HYP-PDT in HK-1 cells.[73]
Glioma
PDT can be utilised to regulate the proliferation of glioma cells,
which belong to the most inltrative types of cancers. Miccoli
etal.[74] were the rst to show that photoactivation of HYP affected
the energy metabolism of the SNB-19 cells through suppressing
hexokinase binding to mitochondria. These data demonstrated
that HYP could be a promising phototoxic drug for the treat-
ment of glioma tumours. The intracellular localisation of HYP in
D54Mg cells was investigated by Uzdensky etal.[75] The results
indicated that HYP was concentrated in the perinucleolar cyto-
plasmic area mainly on one side of the nucleus.[75] Furthermore,
HYP-PDT effectively inactivated three glioblastoma cells (U373
MG, LN229 and T98G) after a short incubation and exposure
to low light.[76] Balogová studied the distribution patterns of Bax
and Bak after HYP-PDT treatment in U-87 MG cells. The data
demonstrated that Bak was located predominantly at the plasma
membrane, while Bax was translocated from cytosol to mitochon-
dria.[77] In another experiment, photoactivated HYP exhibited an
inhibitory activity on U-87 MG and U-373 MG cells. However,
the efcacy of the inhibitory effect was involved in key steps of
angiogenesis, such as the cell proliferation, migration and degrad-
ation of MMP.[78] HYP is one of the protein kinase C (PKC) regu-
lators, which are considered as promising drugs that can mediate
glioma cells apoptosis in PDT. HYP-PDT induced the activation
and translocation of PKC along the plasma membrane and par-
tially in the nucleus of U87 MG cells. The prolonged effect of
HYP-PDT resulted in PKC being located mainly in the cytoplasm
and nucleus.[79] In addition, HYP and PKCδ inhibitor (rottlerin)
after light irradiation led to a signicant increase in apoptosis of
U87 MG cells accompanied by the downregulation of intracel-
lular oxidative stress and upregulation of phosphorylated Bcl-2
in the cytoplasm.[80]
Leukaemia
Lavie et al.[81] reported that photoactivation HYP and dimethyl
tetrahydroxyhelianthrone (DTHe) induced apoptosis and necrosis
with nucleolar condensation of chromatin in HL-60 and K-562 cells.
HYP-PDT also inhibited the proliferation of Jurkat cells by induction
of apoptosis. The mechanism might be mediated by the activation
of the TRAIL/TRAIL-receptor system and caspase-8.[82] Moreover,
HYP loaded on superparamagnetic iron oxide nanoparticles
(SPIONs) following laser irradiation induced the death of Jurkat
cells in a dose and illumination time-dependent manner due to the
generation of ROS.[83] Xu etal.[84] investigated the cytotoxic effect
of HYP-PDT on the growth of K562 cells as well as its underlying
mechanism. The results demonstrated that HYP-PDT reduced the
viability and induced mitochondria-dependent apoptosis by regu-
lating the JNK signalling pathway. The overexpression of BCRP
can confer a multidrug-resistant (MDR) phenotype on cancer cells.
Pretreatment with BCRP inhibitor (Ko143) signicantly increased
HYP accumulation and sensitised HL-60 cells to HYP.[85] Treatment
with HYP-PDT was highly effective against adult T-cell leukemia
(ATL) cells through mitochondrial-dependent apoptotic signalling
and suppression of viral transcription.[86]
Livercancer
HYP-PDT resulted in the decrease of cell viability, proliferative ac-
tivity and apoptosis in hepatoblastoma (HUH6 and HepT1) and
HepG2 cells.[87] Barathan etal.[88] also showed that treatment with
HY-PDT induced apoptosis in HepG2 cells by promoting the gen-
eration of ROS, and recruited apoptosis mediators and IL-6. Fadel
et al.[89] found that the binding of anti-hepatocyte-specic antigen
(anti-HAS) to HYP played an important part in increasing the mor-
tality of HepG2 cells compared with free HYP. This suggested that
the conjugation of HYP with anti-HAS might be a potential agent
for liver cancer. Moreover, HYP-loaded on polydopamine-coated
cerium oxide nanorods under light illumination had specic targeting
ability and good biocompatibility. It also specically recognised the
asialo-glycoprotein (ASGP) receptors, which were overexpressed on
the membrane of HepG2 cells.[90]
Melanoma
Melanoma is the main cause of death in skin cancers. Hadjur etal.[91]
for the rst time demonstrated that the activity of total SOD was
increased in G361, M18 and M6 cells, while the glutathione perox-
idase and catalase activities were decreased. Davids etal.[92] showed
that treatment with HYP-PDT induced different cell death modes
in melanoma cells associated with the absence or presence of a pig-
mented phenotype. In addition, UCT Mel-1 pigmented cells died
due to necrosis, whereas UCT Mel-3 nonpigmented cells died via
apoptosis.[92] However, both melanoma cell types exhibited an ini-
tial cytoprotective response to HYP-PDT cytotoxicity by inducing
autophagy.[93] In another study, their group studied that HYP-PDT
signicantly induced UCT Mel-1 cells death after depigmentation
through a likely mechanism involving a necrotic or autophagic mode
of cell death.[94] HYP-PDT was effective in inhibiting the growth of
A375 and UCT Mel-1 cells through participating in the loss of cell
membrane integrity and the externalisation of phosphatidylserine.
In addition, this treatment led to extrinsic and intrinsic caspase-
dependent apoptotic modes of cell death.[95] Under the illumination
effect, HYP in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)
liposomes had a higher selectivity index in B16-F10 cells than Hyp
solubilised in ethanol.[96]
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Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 4 429
Lymphoma
Chen etal.[97] evaluated the PDT efcacy of HYP in a P388 murine
lymphoma model. The results indicated that HYP-PDT was ef-
fective in prolonging the survival time. Moreover, the damage of
tumour vasculature might be the main mechanism of HYP-PDT
action. Treatment with HYP-PDT decreased the viability of U937
cells through altering the levels of apoptosis-related proteins, such
as Bcl-2, Bax and PARP.[98] Asimilar study showed that HYP-PDT
signicantly restrained the proliferation and induced apoptosis of
U937 cells. However, intracellular ATP levels increased markedly.[99]
Compared with the placebo vehicle, HYP combined with visible
light led to a signicant improvement in skin lesions in most patients
with cutaneous T-cell lymphoma.[100]
Squamouscancer
Due to the limitations of surgery and recurrence of the disease, the
clinical treatment of patients with squamous cancer is still poor.
Blank et al.[101] evaluated the tumoricidal properties of HYP-PDT
in a highly metastatic adenocarcinoma (DA3Hi) and anaplastic SQ2
tumours in vivo. The data demonstrated that HYP-PDT reduced
the growth of primary tumours and obviously prolonged the sur-
vival of tumour-bearing mice. Additionally, HY-PDT signicantly
upregulated the mRNA levels of inammatory cytokines (IL-1β,
IL-6, GM-CSF and TNF-α). In-vitro testing revealed that HYP-
PDT exhibited an optimal tumoricidal response in SNU-1 cells.
Moreover, in-vivo tumour retention of injected HYP following laser
led to regression of SNU-1 tumour transplants.[102] Moreover, HYP
combined with pulsed laser light resulted in the membrane damage
of the SNU-1 cells. The phototoxic response increased linearly with
HYP dose of 0.1–2µm.[103] Sanovic etal.[104] investigated gene ex-
pression proles of A-431 cells to analyse the molecular background
for skin treatment and apoptosis induced by HYP-PDT. The results
reported that 168 genes were signicantly upregulated and 45 genes
were downregulated. These functions were related to the antioxidant
response, adaptation mechanism and cell damage protection func-
tion against ROS. HYP-PDT also decreased the cell viability of SCC
cells in a concentration-dependent manner via increasing the ROS
production. Moreover, the cell death mode was a necrotic caspase-
independent mechanism.[105] The FaDu cells treated with HYP-PDT
exhibited a large amount of metabolic reduction and excessive apop-
tosis, indicating that HYP might be suitable for clinical application
of head neck squamous cancers.[106] In another study, the mixtures
[HYP and meso-tetra-hydroxyphenyl-chlorin (mTHPC)] decreased
the cell viability and increased the level of ROS in UMB-SCC 745
and 969 cells. Examination of death signalling pathways exhibited
that the mixtures-mediated cell death was apoptotic and necrotic.[107]
Othercancer
Liu was the rst to show that HYP-PDT signicantly decreased
the growth of MiaPaCa-2 and PANC-1 cells in vivo and in vitro,
indicating that HYP and laser therapy might be effective in pancre-
atic cancer.[108] Chen etal.[109,110] reported that HYP-PDT induced vas-
cular injury and apoptosis in a tumour model of radiation-induced
brosarcoma-1 (RIF-1) mice. Their studies also showed that the
Figure 3 Anticancer effect of hypericin.
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430 Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 4
Table 1 Anticancer activities of HYP-PDT
Cell lines/model Drug Dose Laser Energy Mechanism(s) of action Application Reference
AY-27 cells 0.05–10μm 0.45–3.6 J/cm2Induces apoptosis or necrosis In vitro [18]
30μm 6–48 J/cm2Induces selective the cells damage In vitro [20]
RT-112 cells 5,30,100μm 7.2 J/cm2Induces apoptosis In vitro [22]
T24 cells 125, 150 nm 4 J/cm2Involves the stimulation of HO-1 expression
governed by the p38 MAPK and PI3K
pathways
In vitro [24]
150 nm 4 J/cm2Inhibits p38 MAPK pathway In vitro [25]
Mice, MGH cells 5mg/kg 120 J/cm2Downregulates the VEGF expression In vivo [26]
5mg/kg 120 J/cm2Activates c-Met and Ephrin-Eph signalling
pathways
In vivo [27]
C26 cells 0.5–4μm
5mg/kg
60–120 J/cm2Induces vascular damage and tumour necrosis In vitro and
vivo
[28]
HT-29 cells 0.1μm 3.15 J/cm2Affects the function of MRP1 and BCRP In vitro [29]
0.1μm 4.4 J/cm2Induces S phase arrest and increases apoptosis In vitro [30]
0.04, 0.1μm 4.4 J/cm2The mitochondrial pathway In vitro [31]
30, 50 nm 3.15 J/cm2Suppresses expression of MMP-2 In vitro [32]
50, 500 nm 3.15 J/cm2Increases expression of VEGF-A, PD-ECGF In vitro [33]
50–200 nm 3.15 J/cm2Induces an increase in caspase-3 activation and
subsequent PARP cleavage
In vitro [37]
0–100 nm 4.4 J/cm2Inhibits cell viability and Induces apoptosis In vitro [38]
0–200 nm 1 J/cm2Upregulates the ABCG2 In vitro [42]
Mice, CT26 cells 1mg/ml 14, 60 J/cm2Induces immune response In vivo [39]
HCT116 and
HCT8 cells
0.0625–1μm 7.2 J/cm2Activates CHOP/TRIB3/Akt cascade and
triggered autophagic cell death
In vitro [40]
0–2.5μm 7.2 J/cm2Affects drug efux and GSH detoxication In vitro [41]
MCF-7 cells 0.021μm 4.4 J/cm2Regulates Bcl-2, Bax, Akt In vitro [47]
0.04, 0.1μm 3.15 J/cm2Induces cell death In vitro [50]
2μm 200 mJ/cm2Inhibits the GSH reduction In vitro [51]
0.1–50μm 0.96 J/cm2Inhibits the formation of cellular colonies In vitro [53]
2μm
12.5mg/kg
40 J/cm2Induces necrosis and autophagy In vitro and
vivo
[55]
SKBR-3 cells 0.84–210 nm 4.4 J/cm2Decreases HER2, Akt, P-Erk1/2 and In vitro [48]
50, 250 nm 3.15 J/cm2survivin [49]
Mice, 4T1 cells 10μg/ml
25.6μg/ml
150 J/cm2Inhibits tumour growth In vitro and
vivo
[54]
HeLa cells 125 nm 4 J/cm2Inhibits JNK and p38 MAPK In vitro [57]
80 nM-10μm 4 J/cm2The mitochondrial pathway In vitro [58]
150 nm 4 J/cm2Blocks COX-2 upregulation In vitro [59]
200 nm 4 J/cm2Induces loss of SERCA2 protein levels In vitro [61]
Mice, CNE-2 cells 0.5μm
2mg/kg
0.5 J/cm2Upregulates IL-6 mRNA expression In vitro and
vivo
[63]
0.5–4.0μg/ml 5.67 J/cm2The mitochondria pathway and the extrinsic
pathway
In vitro [69]
2mg/kg 42.4 J/cm2Decreases the COX-2 mRNA expression In vivo [72]
Mice, HK-1 cells 2mg/kg 120 J/cm2 Induces tumour shrinkage and necrosis In vivo [65]
2mg/kg 120 J/cm2Reduces GST activity In vivo [66]
0.125–2μm 0.25 J/cm2Upregulates MMP1 mRNA expression In vitro [67]
0.25, 0.5μm
2mg/kg
0.5 J/cm2Downregulates MMP-9 expression; Induces
apoptosis and lowers VEGF
In vitro and
vivo
[68]
2, 5mg/kg 30 J/cm2levels In vivo [71,73]
SNB-19 cells 0.5, 2.5μg/ml 1.5 J/cm2Inhibits hexokinase bound to mitochondria In vitro [72]
U-87 MG cells 500 nm 4 J/cm2Induces Bax translocated from cytosol to
mitochondria
In vitro [77]
5×10–7 M 5 J/cm2Inhibits angiogenesis In vitro [78]
500 nm 4 J/cm2Activates PKC In vitro [79]
0.5μm 4 J/cm2Induces apoptosis In vitro [80]
HL-60 cells 0–65μm 7.2 J/cm2Induces apoptosis and necrosis In vitro [81]
Jurkat cells 0.25–1μg/ml 5 J/cm2The TRAIL/TRAIL-receptor pathway and
activates the caspase-8
In vitro [82]
0–0.3μg/ml 12 J/cm2Upregulates reactive oxygen species In vitro [83]
K562 cells 0.4μg/ml 90 mJ/cm2Regulates the JNK pathway In vitro [84]
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Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 4 431
combination of HYP-PDT with hyperthermia or mitomycin C ef-
fectively enhanced tumour response with minimal side effects.[111,112]
HYP might also be an effective tool for treating and detecting
ovarian cancer. HYP-loaded polylactic acid (PLA) nanoparticles
exhibited higher photoactivity on NuTu-19 ovarian cells than free
drugs. In addition, the antitumour activity was improved with the
increasing incubation time and laser dose.[113] The combination of
HYP and illumination resulted in an obvious decrease in the meta-
bolic activity and induced apoptosis of renal cancer cells (A498 and
ACHN) in vitro.[114] In a GH4C1 rat pituitary cell line, treatment
of HYP-PDT in vitro signicantly slowed tumour growth and in-
duced apoptosis in a dose-dependent manner.[115] Penjweini etal.[116]
compared the phototoxicity of HYP on a mixture of human lung
broblast (HLF) cells and A549 lung cancer cells. Compared with
HLF cells, A549 cells were more responsive by polyvinylpyr-
rolidone (PVP)-HYP treatment after 4h PDT. HYP-PDT also in-
duced damage-associated molecular patterns (DAMPs) and surface
integrin-related protein (CD47) dysregulation on the surface of
Lewis cancer cells, leading to the destruction of immune homeo-
stasis.[117] Furthermore, HYP-PDT was effective in inhibiting the
growth of FRO anaplastic thyroid cancer cells and tumours in mice.
The mechanisms were mediated by the intracellular ROS generation
and mitochondrial damage.[118] HYP tetraether liposomes irradiated
with a LED-device were suitable for antivascular targeting of tu-
mours, while HYP encapsulated in hydroxypropyl-β-cyclodextrin
(HPβCD) inclusion complex could be more safely delivered to tu-
mour sites.[119]
Perspectives and Conclusions
PDT is a rapidly developing targeted cancer therapy with less toxic
adverse effect due to its excellent photosensitising and tumoritropic
properties. Hypericin, a powerful candidate photosensitiser, is a
natural naphthodianthrone isolated from the genus Hypericum, es-
pecially Hypericum perforatum L.[120] In recent decades, the photo-
dynamic compound has attracted great attention as a potential
anticancer drug. Many researchers have conrmed its powerful
antitumour effects on different tumour cells in vivo and in vitro
studies, such as bladder, colon, breast, cervical, glioma, leukaemia,
hepatic, melanoma, lymphoma, etc. (Figure3). The photosensitising
effects of HYP are generally described as oxygen-dependent where
singlet oxygen is the main contributor for HYP-PDT.
The underlying antitumour mechanisms of HYP-PDT are associ-
ated with inhibition of various proteins and genes including VEGF,
MMP-2, MMP-9, HER2, Akt, P-Erk1/2, SERCA2, COX-2, GST,
glutathione peroxidase and catalase. However, the levels of MRP-1,
HO-1, VEGF-A, PD-ECGF, ABCG2, Bax, IL-6, IL-1β, MMP-1, PKC,
caspase-3, caspase-8, PARP, GM-CSF and TNF-α are upregulated.
Furthermore, HYP-PDT may affect the p38 MAPK, JNK, PI3K,
CHOP/TRIB3/Akt/mTOR, TRAIL/TRAIL-receptor, c-Met and
Ephrin-Eph, mitochondria and the extrinsic signalling pathways
(Table1; Figure4). Meanwhile, these results strongly establish that
HYP-PDT possesses the ability to inhibit various events related to
cell growth, apoptosis, necrosis, autophagy, angiogenesis, cell cycle
arrest and the formation of cellular colonies, and clearly supports its
great promising in cancer treatment.
Cell lines/model Drug Dose Laser Energy Mechanism(s) of action Application Reference
ATL cells 0–200ng/ml 11.28 J/cm2The mitochondrial pathway and suppresses the
viral transcription
In vitro [86]
HUH6 and
HepT1 cells
0–12.5μm 1000 Lux Decreases the cell viability and Induces apoptosis In vitro [87]
HepG2 cells 0–1μg/ml 21 J/cm2Facilitates cytotoxic ROS and recruits IL-6 In vitro [88]
0.025–0.5μm 15.48 J/cm2Recognises ASGP-R In vitro [90]
G361, M18 and
M6 cells
5×10–8 M 12 J/cm2Decreases glutathione peroxidase and catalase
activities
In vitro [91]
UCT Mel-1 and
UCT Mel-3 cells
0.2–10μm 1 J/cm2Induces necrosis or apoptosis In vitro [92]
0.2–10μm 1 J/cm2Induces autophagy In vitro [93]
A375 cells 3μm 1 J/cm2The extrinsic and intrinsic caspase-dependent
apoptotic pathway
In vitro [95]
Mice, P388 cells 1, 5, 20mg/kg 120 J/cm2Induces tumour vasculature damage In vivo [97]
SQ2 cells 10mg/kg 7.2 J/cm2Upregulates the mRNA expression levels of
IL-1β, IL-6, TNF-α and GM-CSF
In vivo [101]
SNU-1 cells 0.1–2μm 3 J/cm2Induces the membrane damage In vitro [103]
A-431 cells 200ng/ml 0.6 J/cm2Induces apoptosis In vitro [104]
human SCC cells 3μm 1 J/cm2The necrotic caspase-independent pathway In vitro [105]
FaDu cells 5–50μm 50 000 Lux Reduces metabolism and induces apoptosis In vitro [106]
UMB-SCC 745
and 969 cells
2.5μg/ml 1.92 J/cm2Induces apoptosis and necrosis In vitro [107]
RIF-1 mouse
tumour
5mg/kg 120 J/cm2Induces vascular damage and apoptosis In vivo [109,110]
A498 and ACHN
cells
50μm 50 000 Lux Decreases in metabolic activity and causes
apoptosis
In vitro [114]
Rats, GH4C1 cells 1mg/kg 130 J/cm2Inhibits cellular growth In vivo [115]
Mice, Lewis cells 0.25μm 1.85 J/cm2Induces dysregulation of DAMPs In vitro and
vivo
[117]
FRO cells 1–20μg/ml 46.8 J/cm2Induces mitochondrial damage In vivo [118]
SK-OV-3 cells 0.2–1μm 2.1–12.4 J/cm2Induces antivascular targeting of the tumor In vitro [119]
Table 1 Continued
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432 Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 4
Nevertheless, hypericin has a very hydrophobic behav-
iour and easily forms aggregates in water, resulting in the loss
of its spectroscopic and tumour-selective properties as well as
low bioavailability.[121] To overcome these limitations, previous
studies have shown that HYP-PDT in conjunction with tar-
geted therapies, such as nanoencapsulation, superparamagnetic
iron oxide nanoparticles, single-walled carbon nanohorns, lipid
nanocapsules, cerium oxide nanorods, PLA nanoparticles, lipo-
somes and HPβCD inclusion complex, is a more potential ap-
proach in cancer treatment.
Moreover, previous researches have demonstrated that HYP-
PDT combined with various chemotherapeutic agents, such as
peruorocarbons bevacizumab, MK-886, hyperforin, diclofenac,
urbiprofen, ibuprofen, YM155, 17-DMAG, carmustine,
methoxyestradiol, tamoxifen, pyridinyl imidazole, apoMb, celebrex,
rottlerin, DTHe and Ko143, is more effective in killing cancer cells.
Hence, it is essential to investigate the activity of HYP-PDT on
the metabolism of other agents in the future. What’s more, further
studies are still needed to improve its oral bioavailability and en-
hance its efciency before HYP-PDT can be safely used in clinical
application for the treatment ofcancer.
Acknowledgements
This work was nancially supported by the China Scholarship Council
(201906557012) and Beijing Natural Science Foundation (7194289,
7202121, 7202123).
Conflict of Interest
The authors have declared that there is no conict of interest.
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