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

Kleemann B, Loos B, Scriba TJ, Lang D, Davids LM - PLoS ONE (2014)

Bottom Line: In line with this speculation we found an increase in cellular granularity, suggesting an increase in pigmentation levels in response to hypericin-PDT.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.

View Article: PubMed Central - PubMed

Affiliation: Redox Laboratory and Confocal and Light Microscope Imaging Facility, Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.

ABSTRACT
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.

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Hypericin-PDT induced loss of structural details of calreticulin positive structures (endoplasmic reticulum).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).
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pone-0103762-g004: Hypericin-PDT induced loss of structural details of calreticulin positive structures (endoplasmic reticulum).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).

Mentions: Upon visualization of hypericin in GFP-labeled mitochondria with live confocal fluorescent microscopy, we noticed distinct changes in the mitochondrial structure of melanoma cells. To investigate this in more detail we used the unpigmented 501mel melanoma cells and activated hypericin in real time on the confocal fluorescent microscope using time-lapse technology. The 501mel cells displayed a rapid and extensive modification from its normal tubular mitochondrial network into a beaded appearance upon light-activation of hypericin (Vid. S1). This was not observed in the untreated control cells expressing the mitochondrial-specific GFP fusion protein (Vid. S2). Despite hypericin partially co-localizing with the endoplasmic reticulum and lysosomes in the melanoma cells (Fig. 2A, Fig. S1), rapid photo-destruction was only observed in mitochondria. Whether this is related to an organelle-specific, concentration-dependent effect or simply an energetic, metabolic effect remains to be determined. However, to further investigate the light-activation effect on the structural details of intracellular organelles, we transiently expressed calreticulin and lysosomal-associated membrane protein (LAMP1) in 501mel cells to visualize the endoplasmic reticulum and lysosomes respectively, before and after PDT using super-resolution structured illumination microscopy (SR-SIM). A partial co-localization of hypericin with LAMP1 positive structures (lysosomes, endosomes and melanosomes, Fig. 3A) and calreticulin-positive structures (endoplasmic reticulum, Fig. 4A) was observed before light-activation, confirming the confocal fluorescent microscopy data (Fig. 2, Fig. S1). Surprisingly, an increased co-localization of hypericin with LAMP1 positive structures was found at 30 and 60 minutes post-PDT, whilst the structure of these organelles remained intact (Fig. 3B, C). In contrast, a loss in structural detail for calreticulin-positive structures was found post-PDT, suggestive of organelle disruption, which was associated with a loss in co-localization of hypericin and calreticulin-positive structures (Fig. 4B, C).


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

Kleemann B, Loos B, Scriba TJ, Lang D, Davids LM - PLoS ONE (2014)

Hypericin-PDT induced loss of structural details of calreticulin positive structures (endoplasmic reticulum).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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4116257&req=5

pone-0103762-g004: Hypericin-PDT induced loss of structural details of calreticulin positive structures (endoplasmic reticulum).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).
Mentions: Upon visualization of hypericin in GFP-labeled mitochondria with live confocal fluorescent microscopy, we noticed distinct changes in the mitochondrial structure of melanoma cells. To investigate this in more detail we used the unpigmented 501mel melanoma cells and activated hypericin in real time on the confocal fluorescent microscope using time-lapse technology. The 501mel cells displayed a rapid and extensive modification from its normal tubular mitochondrial network into a beaded appearance upon light-activation of hypericin (Vid. S1). This was not observed in the untreated control cells expressing the mitochondrial-specific GFP fusion protein (Vid. S2). Despite hypericin partially co-localizing with the endoplasmic reticulum and lysosomes in the melanoma cells (Fig. 2A, Fig. S1), rapid photo-destruction was only observed in mitochondria. Whether this is related to an organelle-specific, concentration-dependent effect or simply an energetic, metabolic effect remains to be determined. However, to further investigate the light-activation effect on the structural details of intracellular organelles, we transiently expressed calreticulin and lysosomal-associated membrane protein (LAMP1) in 501mel cells to visualize the endoplasmic reticulum and lysosomes respectively, before and after PDT using super-resolution structured illumination microscopy (SR-SIM). A partial co-localization of hypericin with LAMP1 positive structures (lysosomes, endosomes and melanosomes, Fig. 3A) and calreticulin-positive structures (endoplasmic reticulum, Fig. 4A) was observed before light-activation, confirming the confocal fluorescent microscopy data (Fig. 2, Fig. S1). Surprisingly, an increased co-localization of hypericin with LAMP1 positive structures was found at 30 and 60 minutes post-PDT, whilst the structure of these organelles remained intact (Fig. 3B, C). In contrast, a loss in structural detail for calreticulin-positive structures was found post-PDT, suggestive of organelle disruption, which was associated with a loss in co-localization of hypericin and calreticulin-positive structures (Fig. 4B, C).

Bottom Line: In line with this speculation we found an increase in cellular granularity, suggesting an increase in pigmentation levels in response to hypericin-PDT.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.

View Article: PubMed Central - PubMed

Affiliation: Redox Laboratory and Confocal and Light Microscope Imaging Facility, Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus