St John's Wort (Hypericum perforatum L.) photomedicine: hypericin-photodynamic therapy induces metastatic melanoma cell death

Hypericin, an extract from St John's Wort ( Hypericum perforatum L. ), is a promising photosensitizer in the context of clinical photodynamic therapy due to its excellent photosensitizing properties and tumoritropic characteristics. Hypericin-PDT induced cytotoxicity elicits tumor cell death by various mechanisms including apoptosis, necrosis and autophagy-related cell death. However, limited reports on the efficacy of this photomedicine for the treatment of melanoma have been published. Melanoma is a highly aggressive tumor due to its metastasizing potential and resistance to conventional cancer therapies. The aim of this study was to investigate the response mechanisms of melanoma cells to hypericin-PDT in an in vitro tissue culture model. Hypericin was taken up by all melanoma cells and partially co-localized to the endoplasmic reticulum, mitochondria, lysosomes and melanosomes, but not the nucleus. Light activation of hypericin induced a rapid, extensive modification of the tubular mitochondrial network into a beaded appearance, loss of structural details of the endoplasmic reticulum and concomitant loss of hypericin co-localization. Surprisingly the opposite was found for lysosomal-related organelles, suggesting that the melanoma cells may be using these intracellular organelles for hypericin-PDT resistance. In line with this speculation we found an increase in cellular granularity, suggesting an increase in pigmentation levels in response to hypericin-PDT. Pigmentation in melanoma is related to a melanocyte-specific organelle, the melanosome, which has recently been implicated in drug trapping, chemotherapy and hypericin-PDT resistance. However, hypericin-PDT was effective in killing both unpigmented (A375 and 501mel) and pigmented (UCT Mel-1) melanoma cells by specific mechanisms involving the externalization of phosphatidylserines, cell shrinkage and loss of cell membrane integrity. In addition, this treatment resulted in extrinsic (A375) and intrinsic (UCT Mel-1) caspase-dependent apoptotic modes of cell death, as well as a caspase-independent apoptotic mode that did not involve apoptosis-inducing factor (501 mel). Further research is needed to shed more light on these mechanisms

St John's Wort photomedicine for Melonoma

The use of photomedicine in ancient civilizations dates back 4000 years ago but it wasn't until the beginning of the 20th century that photodynamic therapy was discovered by man. The “trinity” of photodynamic therapy (PDT) comprises a photosensitizer, light and molecular oxygen. Following cellular uptake of the photosensitizer, its activation by light produces reactive oxygen species in the presence of oxygen. The resulting cytotoxic oxidative stress elicits cancer cell death by various mechanisms including apoptosis, necrosis and autophagy. Hypericin, an extract from St John's Wort, is a promising photosensitizer in the context of clinical photodynamic therapy due to its excellent photosensitizing properties and tumoritropic characteristics. However, limited reports on the efficacy of this photomedicine for the treatment of melanoma have been published. South Africa has the second highest incidence of malignant melanoma skin cancer in the world; a highly aggressive tumor due to its metastasizing potential and resistance to conventional cancer therapies. The aim of this study was to investigate the response mechanisms of melanoma cells to hypericinPDT in an in vitro tissue culture model. This investigation was three-fold. Firstly, the susceptibility of melanoma cells to the treatment was determined using cell viability assays. We found a dose of 3 µM light-activated hypericin was effective in reducing cell viability to 50 % or less than the control, for all melanoma cells employed in this study. We therefore used this killing-dose for further experiments. Next, hypericin uptake and its specific association with intracellular organelles was characterized using organelle-specific fluorescent-fusion proteins and dyes, in conjunction with the red fluorescent nature of hypericin and visualization by live confocal fluorescent microscopy. The intracellular localization of a photosensitizer directly influences its cytotoxic action and is thus crucial for effective cell death induction. Hypericin was taken up by all melanoma cells and co-localized with lysosomes and variably with melanosomes, the pigment producing organelles. No co-localization with the cell membrane, mitochondria, endoplasmic reticulum or nucleus was found. Investigating intracellular hypericin after treatment revealed a time-dependent decrease in all melanoma cells. Finally, melanoma cell death mechanisms were elucidated in response to the killing-dose of lightactivated hypericin. Ultrastructural examination of the cells with transmission electron microscopy 2 revealed extensive cytoplasmic vacuolisation, at 4 hours after treatment. In pigmented melanoma cells, the treatment furthermore induced the formation of glycogen aggregations. Fluorescent activated cell sorting analyses revealed a time-dependent increase in phosphatidylserine exposure, indicating apoptosis, in conjunction with a loss of cell membrane integrity, indicating necrosis, in all melanoma cells. An initial early necrotic population was found which decreased with time after treatment, whereas the late apoptotic/necrotic population increased. Minimal early apoptotic populations were found in all cell lines. In addition, melanoma cells showed a decrease in cellular size accompanied by an increase in granularity/pigmentation after treatment. Western blot analyses of proteins involved in specific cell death cascades furthermore verified the induction of apoptosis in melanoma cells by hypericin-PDT. The extrinsic apoptotic cascade was initiated in unpigmented A375 melanoma cells at 24 hours after treatment, mediated by activation of the suicidal proteases caspase 8 and caspase 3. Intrinsic apoptosis was found in pigmented UCT Mel-1 cells at 4 and 7 hours, mediated by activation of caspase 3 and cleavage of poly(ADP-ribose)polymerase 1 (PARP1). Induction of apoptosis by cleavage of PARP1 was furthermore evident in 501mel cells at 7 hours after treatment; however this cleavage was not mediated by caspase 3. Apoptosis inducing factor was found in its vital form in all melanoma cells, indicating that caspase-independent apoptosis or regulated necrosis by parthanatos were not induced by hypericin-PDT. In summary, this study demonstrated the effectiveness of hypericin-PDT in killing both unpigmented and pigmented melanoma cells by the induction of apoptosis. Further investigations into the exact mechanisms of the cell death response, including the observed loss of cell membrane integrity and the involvement of lysosomes and melanosomes are interesting avenues to explore in future studies. Translation of hypericin-PDT into a three-dimensional skin model with melanoma invasion is of particular interest, to further simulate the natural environment of this aggressive cancer and thereby enable the identification of enhanced treatment options

Hypericin-PDT induced loss of structural details of calreticulin positive structures (endoplasmic reticulum).

<p>Cells expressing calreticulin-YFP (ER-YFP) were exposed to 3 µM hypericin (red) for 4 h, followed by light-activation and imaging using Super-resolution structured illumination microscopy (SR-SIM). (A) Control (hypericin-treated, sham-irradiated). (B) 30 min post PDT. (C) 60 min post PDT. Images are shown at lower magnification (top panel, scale bars: 5 µm) and higher magnification (zoom, lower panel, scale bars: 1/2 µm.) Co-localization plots indicate co-localization of the fluorophores. A representative result is shown (n = 2).</p

Organelle-specific G/YFP-fusion plasmids and dyes used to visualize intracellular organelles.

<p>A summary of the experimental conditions, functions and sources of the organelle-specific G/YFP-fusion plasmids and dyes used in this study.</p

Hypericin uptake and intracellular localization.

<p>Cells were exposed to 3 µM hypericin for 4 h without light activation. (A) Hypericin uptake assay. Data is shown as mean±SEM relative fluorescent units per microgram of protein (RFU/µg of protein, n = 3, *p<0.05). (B) Live confocal fluorescent microscopy images of melanoma cells indicate the intracellular localization of hypericin (red) in relation to the endoplasmic reticulum (ER-YFP), mitochondria (OTC-GFP), lysosomes (Lysotracker yellow) and mature melanosomes (MyosinVa-GFP). Nuclei were counterstained with Hoechst (blue). Profiles taken at different locations through the cell indicate co-localization of the fluorophores. A representative result is shown (n = 3, scale bars: 10/20 µm).</p

Cell death protein expression in response to hypericin-PDT.

<p>Melanoma cells were treated with 3 µM light-activated hypericin and analyzed for protein expression of caspase 3 (CASP3), caspase 8 (CASP8), apoptosis inducing factor (AIF) and poly(ADP-ribose)polymerase 1 (PARP1) at 1, 4, 7 and 24 hours after treatment by Western blot analyses (−: uncleaved, +: cleaved).</p

Phenotypic heterogeneity of melanoma cells.

<p>(A) Phase contrast images. A representative result is shown (n≥3, scale bars: 20 µm, inset: higher magnification). (B) Cell pellets. A representative result is shown (n≥3). (C) Mean±SEM tyrosinase activity in counts per minute (cpm)/120 µg protein (n = 3, ***p<0.0001, *p<0.05). MCF7 breast cancer cells were included as a negative control.</p

Hypericin-PDT induced expression of apoptotic proteins.

<p>(A) Caspase 3 (CASP3), (B) caspase 8 (CASP8), (C) poly(ADP-ribose)polymerase 1 (PARP1) and (D) apoptosis inducing factor (AIF) Western blot analyses of whole cell lysates detected at 1, 4, 7 and 24 h after treatment. Data is shown as mean±SEM normalized OD ratio (n≥3, ***p<0.0001, **p<0.01, *p<0.05, CTRL: vehicle-treated control, HYP: hypericin and L: light).</p

Melanoma response mechanisms to hypericin-PDT.

<p>Hypericin (HYP) was taken up by melanoma cells and localized to various intracellular organelles, including the endoplasmic reticulum, mitochondria, lysosomes and melanosomes. Light activation (yellow arrow) of hypericin, in the presence of oxygen (O₂), resulted in loss of structural details of various intracellular organelles, phosphatidylserine (PS) exposure, loss of melanoma cell membrane integrity, cell shrinkage and an increase in granularity/pigmentation. Hypericin-PDT furthermore initiated caspase-dependent apoptotic modes of cell death of both extrinsic (caspase 8 (CASP8)) and intrinsic (caspase 3 (CASP3)) nature, as well as a caspase-independent apoptotic mode that did not involve apoptosis inducing factor (AIF). Both caspase-dependent and caspase-independent apoptotic modes of cell death resulted in the cleavage of poly(ADP-ribose)polymerase 1 (PARP1).</p

Hypericin-PDT induced phosphatidylserine exposure and loss of cell membrane integrity.

<p>(A) Annexin V (phosphatidyl serine exposure) and VIVID (loss of cell membrane integrity) median fluorescent intensities (MFI) normalized to the vehicle-treated, sham-irradiated control (Control −Light) at 30 min, 1, 4, 7 and 24 h after treatment. Flow fluorocytometric data is shown as the median±SEM (n≥3, ***p<0.0001, **p<0.01, *p<0.05, L: light). (B) Percentage gated cells of 4 different populations labeled with Annexin V and VIVD: live (AV− VIVD−), early apoptotic (AV+ VIVID−), necrotic (AV− VIVID+) and late apoptotic/necrotic (AV+ VIVID+) at 30 min, 1, 4, 7 and 24 h after treatment. Data is shown as mean±SEM of gated cells (n≥3).</p