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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 phosphatidylserine exposure and loss of cell membrane integrity.(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).
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pone-0103762-g005: Hypericin-PDT induced phosphatidylserine exposure and loss of cell membrane integrity.(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).

Mentions: Melanoma cells were treated with 3 µM hypericin and analysed for PS exposure (Annexin V) and loss of cell membrane integrity (VIVID), 30 minutes, 1, 4, 7 and 24 hours after treatment using fluorescent activated cell sorting (FACS). Cells treated with vehicle with sham-irradiation (Control –light) displayed intact cell membranes with no evidence of PS externalization (Fig. 5A, left panel). The Annexin V median fluorescent intensity (MFI) analyses revealed a time-dependent increase in PS exposure after light-activated hypericin treatment in all melanoma cells, significantly different to controls (Fig. 5A, left panel). The highest Annexin V MFI was observed at 24 hours after treatment, most pronounced in A375 (14.1±1.5 fold) and similar in 501mel (7.2±1.0 fold) and UCT Mel-1 (7.5±1.3 fold), compared to that of the vehicle-treated, sham-irradiated control (Control –light, Fig. 5A, left panel).


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 phosphatidylserine exposure and loss of cell membrane integrity.(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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103762-g005: Hypericin-PDT induced phosphatidylserine exposure and loss of cell membrane integrity.(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).
Mentions: Melanoma cells were treated with 3 µM hypericin and analysed for PS exposure (Annexin V) and loss of cell membrane integrity (VIVID), 30 minutes, 1, 4, 7 and 24 hours after treatment using fluorescent activated cell sorting (FACS). Cells treated with vehicle with sham-irradiation (Control –light) displayed intact cell membranes with no evidence of PS externalization (Fig. 5A, left panel). The Annexin V median fluorescent intensity (MFI) analyses revealed a time-dependent increase in PS exposure after light-activated hypericin treatment in all melanoma cells, significantly different to controls (Fig. 5A, left panel). The highest Annexin V MFI was observed at 24 hours after treatment, most pronounced in A375 (14.1±1.5 fold) and similar in 501mel (7.2±1.0 fold) and UCT Mel-1 (7.5±1.3 fold), compared to that of the vehicle-treated, sham-irradiated control (Control –light, Fig. 5A, left panel).

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