Annelies Vantieghem

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.

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.

Cell death and growth arrest in response to photodynamic therapy with membrane-bound photosensitizers

Photodynamic therapy (PDT) is a treatment for cancer and for certain benign conditions that is based on the use of a photosensitizer and light to produce reactive oxygen species in cells. Many of the photosensitizers currently used in PDT localize in different cell compartments such as mitochondria, lysosomes, endoplasmic reticulum and generate cell death by triggering necrosis and/or apoptosis. Efficient cell death is observed when light, oxygen and the photosensitizer are not limiting (high dose PDT). When one of these components is limiting (low dose PDT), most of the cells do not immediately undergo apoptosis or necrosis but are growth arrested with several transduction pathways activated. This commentary will review the mechanism of apoptosis and growth arrest mediated by two important PDT agents, i.e. pyropheophorbide and hypericin.

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.

Ultraviolet B radiation-induced apoptosis in human keratinocytes: cytosolic activation of procaspase-8 and the role of Bcl-2

In this study, we show that ultraviolet B radiation (UVB)‐induced apoptosis of human keratinocytes involves mainly cytosolic signals with mitochondria playing a central role. Overexpression of Bcl‐2 inhibited UVB‐induced apoptosis by blocking the early generation of reactive oxygen species, mitochondrial cardiolipin degradation and cytochrome c release, without affecting Fas ligand (FasL)‐induced cell death. It also prevented the subsequent activation of procaspase‐3 and ‐8 as well as Bid cleavage in UVB‐treated cells. Comparative analysis of UVB and FasL death pathways revealed a differential role and mechanism of caspase activation, with the UVB‐induced activation of procaspase‐8 only being a bystander cytosolic event rather than a major initiator mechanism, as is the case for the FasL‐induced cell death. Our results suggest that Bcl‐2 overexpression, by preventing reactive oxygen species production, helps indirectly to maintain the integrity of lysosomal membranes, and therefore inhibits the release of cathepsins, which contribute to the cytosolic activation of procaspase‐8 in UVB‐irradiated keratinocytes.

Different Pathways Mediate Cytochrome c Release After Photodynamic Therapy with Hypericin

In this study we show that overexpression of Bcl‐2 in PC60R1R2 cells reveals a caspase‐dependent mechanism of cytochrome c release following photodynamic therapy (PDT) with hypericin. Bcl‐2 overexpression remarkably delayed cytochrome c release, procaspase‐3 activation and poly(adenosine diphosphate‐ribose)polymerase cleavage during PDT‐induced apoptosis while it did not protect against PDT‐induced necrosis. PDT‐treated cells showed a reduction in the mitochondrial membrane potential which occurred with similar kinetics in PC60R1R2 and PC60R1R2/Bcl‐2 cells, and was affected neither by the permeability transition pore inhibitor cyclosporin A nor by the caspase inhibitor N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone (zVAD‐fmk). Hypericin‐induced mitochondrial depolarization coincided with cytochrome c release in PC60R1R2 cells while it precedes massive cytochrome c efflux in PC60R1R2/Bcl‐2 cells. Preincubation of PC60R1R2 cells with zVAD‐fmk or cyclosporin A did not prevent the mitochondrial efflux of cytochrome c, and caspase inhibition only partially protected the cells from PDT‐induced apoptosis. In contrast, in PC60R1R2/Bcl‐2 cells cytochrome c release and apoptosis were suppressed by addition of zVAD‐fmk or cyclosporin A. These observations suggest that the progression of the PDT‐induced apoptotic process in Bcl‐2–overexpressing cells involves a caspase‐dependent feed‐forward amplification loop for the release of cytochrome c.

In Vitro Photobiological Evaluation of Rhodac, A New Rhodacyanine Photosensitizer¶

We have previously shown that the rhodacyanine dye, Rhodac, exhibits a potent photocytotoxic activity in HeLa cells. In this study several aspects of the photobiological activity of Rhodac were further examined. Rhodac displayed no selective cytotoxicity toward several malignant cell lines after photosensitization (3.6 J/cm2), although HeLa cells were found to be the most sensitive. Interestingly, MCF‐7/Adr cells, a multidrug‐resistant subline, were less sensitive to the antiproliferative effect of photoactivated Rhodac. The subcellular localization, as revealed by confocal laser microscopy, demonstrated that the dye was mainly concentrated in the cytosolic membranes of the perinuclear region. The Rhodac‐induced inhibition of HeLa cell proliferation after light exposure was found to be strictly oxygen dependent. In addition, photoactivated Rhodac induced poly(adenosine 5′ diphosphate‐ribose)polymerase cleavage, caspase‐3 activation and apoptosis in HeLa cells. In the current work it was further demonstrated that Rhodac binds specifically to high‐density lipoproteins and low‐density lipoproteins, while no binding was observed to very low‐density and heavy proteins. To sum up, our results show that Rhodac is an interesting and potent photosensitizer. Further in vivo experiments are required to elucidate whether the lipoprotein binding leads to a selective uptake of Rhodac in tumor cells and to address its efficacy in photodynamic therapy.