Peter de Witte

Hypericin in cancer treatment: more light on the way.

Photodynamic therapy (PDT) has been described as a promising new modality for the treatment of cancer. PDT involves the combination of a photosensitizing agent (photosensitizer), which is preferentially taken up and retained by tumor cells, and visible light of a wavelength matching the absorption spectrum of the drug. Each of these factors is harmless by itself, but when combined they ultimately produce, in the presence of oxygen, cytotoxic products that cause irreversible cellular damage and tumor destruction. Hypericin, a powerful naturally occurring photosensitizer, is found in Hypericum perforatum plants, commonly known as St. Johns wort. In recent years increased interest in hypericin as a potential clinical anticancer agent has arisen since several studies established its powerful in vivo and in vitro antineoplastic activity upon irradiation. Investigations of the molecular mechanisms underlying hypericin photocytotoxicity in cancer cells have revealed that this photosensitizer can induce both apoptosis and necrosis in a concentration and light dose-dependent fashion. Moreover, PDT with hypericin results in the activation of multiple pathways that can either promote or counteract the cell death program. This review focuses on the more recent advances in the use of hypericin as a photodynamic agent and discusses the current knowledge on the signaling pathways underlying its photocytotoxic action.

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

Surface‐exposed calreticulin (ecto‐CRT) and secreted ATP are crucial damage‐associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)‐based (reactive oxygen species (ROS)‐regulated) pathway for ecto‐CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS‐mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80high, CD83high, CD86high, MHC‐IIhigh) and functional stimulation (NOhigh, IL‐10absent, IL‐1βhigh) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto‐CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK‐orchestrated pathways that require a functional secretory pathway and phosphoinositide 3‐kinase (PI3K)‐mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase‐8 signalling are dispensable for this ecto‐CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto‐CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase‐8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK‐dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS‐mediated ER stress.

The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin.

In this study, we elucidate signaling pathways induced by photodynamic therapy (PDT) with hypericin. We show that PDT rapidly activates JNK1 while irreversibly inhibiting ERK2 in several cancer cell lines. In HeLa cells, sustained PDT-induced JNK1 and p38 mitogen-activated protein kinase (MAPK) activations overlap the activation of a DEVD-directed caspase activity, poly(ADP-ribose) polymerase (PARP) cleavage, and the onset of apoptosis. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-fmk) protect cells against apoptosis and inhibit DEVD-specific caspase activity and PARP cleavage without affecting JNK1 and p38 MAPK activations. Conversely, stable overexpression of CrmA, the serpin-like inhibitor of caspase-1 and caspase-8, has no effect on PDT-induced PARP cleavage, apoptosis, or JNK1/p38 activations. Cell transfection with the dominant negative inhibitors of the c-Jun N-terminal kinase (JNK) pathway, SEK-AL and TAM-67, or pretreatment with the p38 MAPK inhibitor PD169316 enhances PDT-induced apoptosis. A similar increase in PDT-induced apoptosis was observed by expression of the dual specificity phosphatase MKP-1. The simultaneous inhibition of both stress kinases by pretreating cells with PD169316 after transfection with either TAM-67 or SEK-AL produces a more pronounced sensitizing effect. Cell pretreatment with the p38 inhibitor PD169316 causes faster kinetics of DEVD-caspase activation and PARP cleavage and strongly oversensitizes the cells to apoptosis following PDT. These observations indicate that the JNK1 and p38 MAPK pathways play an important role in cellular resistance against PDT-induced apoptosis with hypericin.

Hypericin-induced photosensitization of HeLa cells leads to apoptosis or necrosis Involvement of cytochrome c and procaspase-3 activation in the mechanism of apoptosis

Here we report that photoactivated hypericin can induce either apoptosis or necrosis in HeLa cells. Under apoptotic conditions the cleavage of poly(ADP‐ribose) polymerase (PARP) into the 85‐kDa product is blocked by the caspase inhibitors benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone (z‐VAD‐fmk) and benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp‐fluoromethylketone (z‐DEVD‐fmk). Both inhibitors protect cells from apoptosis but cannot prevent hypericin‐induced necrosis. Conversely, HeLa cells overexpressing the viral cytokine response modifier A (CrmA), which inhibits caspase‐1 and ‐8, still undergo hypericin‐induced apoptosis and necrosis. Evidence is provided for the release of mitochondrial cytochrome c in the cytosol and for procaspase‐3 activation in the hypericin‐induced cell killing.

Up-regulation of Cyclooxygenase-2 and Apoptosis Resistance by p38 MAPK in Hypericin-mediated Photodynamic Therapy of Human Cancer Cells*

Photodynamic Therapy (PDT) is an approved anticancer therapy that kills cancer cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which selectively accumulates in tumors. We report that hypericin-mediated PDT of human cancer cells leads to up-regulation of the inducible cyclooxygenase-2 (COX-2) enzyme and the subsequent release of PGE2. Dissection of the signaling pathways involved revealed that the selective activation of p38 MAPK α and β mediate COX-2 up-regulation at the protein and messenger levels. The p38 MAPK inhibitor, PD169316, abrogated COX-2 expression in PDT-treated cells, whereas overexpression of the drug-resistant PD169316-insensitive p38 MAPK α and β isoforms restored COX-2 levels in the presence of the kinase inhibitor. Transcriptional regulation by nuclear factor-κB was not involved in COX-2 up-regulation by PDT. The half-life of the COX-2 messenger was drastically shortened by p38 MAPK inhibition in transcriptionally arrested cells, suggesting that p38 MAPK mainly acts by stabilizing the COX-2 transcript. Overexpression of WT-p38 MAPK increased cellular resistance to PDT-induced apoptosis, and inhibiting this pathway exacerbated cell death and prevented PGE2 secretion. Hence, the combination of PDT with pyridinyl imidazole inhibitors of p38 MAPK may improve the therapeutic efficacy of PDT by blocking COX-2 up-regulation, which contributes to tumor growth by the release of growth- and pro-angiogenic factors, as well as by sensitizing cancer cells to apoptosis.

Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death

The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called “damage-associated molecular patterns” (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation.

Phosphorylation of Bcl-2 in G2/M Phase-arrested Cells following Photodynamic Therapy with Hypericin Involves a CDK1-mediated Signal and Delays the Onset of Apoptosis

The role of Bcl-2 in photodynamic therapy (PDT) is controversial, and some photosensitizers have been shown to induce Bcl-2 degradation with loss of its protective function. Hypericin is a naturally occurring photosensitizer with promising properties for the PDT of cancer. Here we show that, in HeLa cells, photoactivated hypericin does not cause Bcl-2 degradation but induces Bcl-2 phosphorylation in a dose- and time-dependent manner. Bcl-2 phosphorylation is induced by sublethal PDT doses; increasing the photodynamic stress promptly leads to apoptosis, during which Bcl-2 is neither phosphorylated nor degraded. Bcl-2 phosphorylation involves mitochondrial Bcl-2 and correlates with the kinetics of a G2/M cell cycle arrest, preceding apoptosis. The co-localization of hypericin with α-tubulin and the aberrant mitotic spindles observed following sublethal PDT doses suggest that photodamage to the microtubule network provokes the G2/M phase arrest. PDT-induced Bcl-2 phosphorylation is not altered by either the overexpression or inhibition of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH2-terminal protein kinase 1 (JNK1) nor by inhibiting the extracellular signal-regulated kinases (ERKs) or protein kinase C. By contrast, Bcl-2 phosphorylation is selectively suppressed by the cyclin-dependent protein kinase (CDK)-inhibitor roscovitine, completely blocked by the protein synthesis inhibitor cycloheximide and enhanced by the overexpression of CDK1, suggesting a role for this pathway. However, in an in vitro kinase assay, active CDK1/cyclin B1 complex failed to phosphorylate immunoprecipitated Bcl-2, suggesting that this protein kinase may not directly modify Bcl-2. Mutation of serine-70 to alanine in Bcl-2 abolishes PDT-induced phosphorylation and restores the caspase-3 activation to the same levels of the vector-transfected cells, indicating that Bcl-2 phosphorylation may be a signal to delay apoptosis in G2/M phase-arrested cells.

Cytotoxicity and antiproliferative effect of hypericin and derivatives after photosensitization.

The toxicity on three human tumor cell lines (A431, HeLa and MCF7) of five phenanthroperylenequinones (hypericin and derivatives) and two perylenequinones (cercosporin and calphostin C) was investigated after photosensitization (4 J/cm2). Furthermore, the antiproliferative effect on HeLa cells was studied for the phenanthroperylenequinones. Hypericin, 2,5‐dibromohypericin, 2,5,9,12‐tetrabromohypericin and perylenequinones displayed a potent cytotoxic and antiproliferative effect in the nanomolar range. Hypericin dicarboxylic acid exhibited no photoactivity. In general, the antiproliferative activity correlated well with the photocytotoxicity. However, the nonphotocytotoxic compound hexamethylhypericin showed potent antiproliferative activity in the nanomolar range, probably exerting its action by protein kinase C inhibition. Without light irradiation, no cytotoxic and antiproliferative effect was observed for any photocytotoxic phenanthroperylenequinone compound. Furthermore, confocal laser microscopy revealed that the subcellular localization in A431 cells was similar for the photoactive compounds; the photosensitizers were mainly concentrated in the perinuclear region, probably corresponding with the Golgi apparatus and the endoplasmic reticulum. In addition, the accumulation of the photosensitizers in HeLa cells was investigated. All compounds except hypericin dicarboxylic acid were found to concentrate to a large extent in the cells. The compound 2,5,9,12‐tetrabromohypericin seemed intrinsically more effective than hypericin since the intracellular concentration of the bromoderivative was a magnitude of order lower than that of hypericin although both compounds showed similar photobiological activity.

Targeting of the photocytotoxic compound AlPcS4 to hela cells by transferrin conjugated peg-liposomes

Photodynamic therapy has attracted increasing interest over the last few years, whereby the activation of photosensitizers by light causes the production of reactive oxygen species (ROS), such as singlet oxygen, which are cytotoxic. The goal of our study was to enhance the photodynamic activity of the photosensitizer aluminum phthalocyanine tetrasulfonate (AlPcS4) through its specific delivery to tumor cells. Since many tumor cells, among which are HeLa cells, overexpress the transferrin receptor, we synthesized transferrin conjugated PEG‐liposomes that contained AlPcS4 that could be internalized by receptor mediated endocytosis. The antiproliferative activity of the targeted liposomes was evaluated and compared to the native AlPcS4 and the non‐targeted liposome. These findings were supplemented with data on intracellular concentration of the photo‐active compounds. The accumulation together with ROS production after irradiation was visualized by using confocal microscopy to confirm the data found in the antiproliferative and accumulation assay. Tf‐Lip‐AlPcS4 was 10 times more photocytotoxic (IC50, 0.63 μM) than free AlPcS4 at a light dose of 45 kJ/m whereas Lip‐AlPcS4 displayed no photocytotoxicity at all. The high photocytotoxicity of Tf‐Lip‐AlPcS4 was shown to be the result of a high intracellular concentration (136.5 μM) in HeLa cells, which could be lowered dramatically by incubating the conjugate with a competing transferrin concentration. The images of intracellular accumulation and ROS production matched the accumulation and photocytotoxicity profile of the different photo‐active compounds. The photodynamic activity of the Tf‐Lip‐AlPcS4 conjugate on HeLa cells is much more potent than free AlPcS4 as a result of selective transferrin receptor mediated uptake.

Induction of heme-oxygenase 1 requires the p38MAPK and PI3K pathways and suppresses apoptotic cell death following hypericin-mediated photodynamic therapy

Photodynamic therapy (PDT) is an established anticancer modality utilizing the photogeneration of reactive oxygen species (ROS) to kill the cancer cells and hypericin is a promising photosensitizer for the treatment of bladder tumors. In this paper we characterize the signaling pathways and the mechanisms leading to the up-regulation of the antioxidant enzyme heme oxygenase (HO-1) in PDT treated cancer cells. We show that PDT engages the p38MAPK and PI3K signaling cascades for HO-1 induction. p38MAPK inhibitors or small interfering RNA (siRNA) for p38MAPK suppress HO-1 induction after PDT and complete repression is attained when p38 and PI3K antagonists are combined. Blocking these signaling pathways increases additively the propensity of the cells to undergo PDT-induced apoptosis, mirroring the effect of HO-1 silencing. Conversely, increasing HO-1 protein level by hemin prior to irradiation is cytoprotective. HO-1 stimulation by PDT is dependent on transcription and de novo protein synthesis and it is preceded by the nuclear accumulation of the Nrf2 transcription factor, which is reduced by inhibitors of p38MAPK and PI3K. Altogether these results indicate that stimulation of HO-1 expression by hypericin-PDT is a cytoprotective mechanism governed by the p38MAPK and PI3K pathways, likely through the control of the nuclear availability of the Nrf2 pool.