**4.2 Subcellular distribution of hypericin**

188 Advances in the Biology, Imaging and Therapies for Glioblastoma

used, the survival time was enlarged in newly and also in patients with tumor recurrence. Side effects of PDT were modest, including skin sensitivity against sunlight and sometimes

Due to the small penetration depth of 5-ALA derived PpXI of 2-3mm, Eljamel et al. combined fluorescence guided resection and PDT.(Eljamel 2008) They used 5-ALA PpXI for resection and HpD for PDT. In respect to the limitation of these PSs we were encouraged to

Hypericin, a Naphtodianthron is a naturally occurring compound of the plant *Hypericum perforatum*, better known as St. John's wort. St. John's wort is a plant that has been used since the Middle Ages to treat wounds and depression. It grows bushy and has its peak season between May and August, see figure 2. St. John's wort contains the following active ingredients: essential oils, flavonoids (Biapigenin, hyperoside, Isoquercitin, rutin), tannins, glycosides, resins, Naphtodianthrone (hypericin, Pseudohyericin) and phloroglucinol (hyperforin). In 1942 by Pace a pronounced sensitization of the skin by light for grazing animals taken St. John's wort containing feed was described. The changes in the skin were reversible after the animals were protected from sun exposure. The phenomenon was described as hypericism.(Pace 1942) Hypericin is a lipophilic molecule that is incorporated into the phospholipid bilayer of cell membranes and has already in the dark versatile pharmacological activities. These include antiviral, anticancer and antiangiogenic properties. Takahashi et al. could show an inhibitory effect on proteinkinase C, which is involved in cell proliferation.(Takahashi et al., 1989) Malignant gliomas have, compared to glial cells, a high proteinkinase C activity.(Couldwell et al. 1991) Hypericin has excellent properties as a PS. It has a high triplet quantum yield and a high efficiency in the formation of ROS.(Diwu & Lown

The excessive production of the so called ROS leads to oxidative stress to many biomolecules, e.g. proteins, causing cell death by induction of apoptosis, necrosis or

increased intracranial pressure.

investigate a new PS combining both positive properties.

1993;Ehrenberg, Anderson, & Foote 1998;Hadjur et al. 1996)

Fig. 2. *St. John's wort, also known as Hypericum perforatum*;

[www.awl.ch/heilpflanzen/hypericum-perforatum/index.htm].

autophagy associated cell death.(Buytaert, Dewaele, & Agostinis 2007)

**4. Hypericin – high potential for PD and PDT** 

Predominantly perinuclear localization of hypericinis is in common concordance.(Uzdensky et al. 2001) The more detailed description of subcellular distribution of hypericin is not uniform. Due to the very short life span of the singlet-oxygen for cytotoxic reactions, the subcellular distribution of hypericin is off interest to understand PDT mechanisms in more detail. Many investigators studied in different cultured cell lines with different methods the subcellular hypericin localisation. According to our studies hypericin enriches particularly in the endoplasmatic reticulum (ER) and the Golgi apparatus (GA) in U37MG glioblastoma cells after incubation with noncytotoxic hypericin (1µM, 2h incubation time). ER is predominantly found in the perinuclear region while the GA is more distant to the nucleus, see figure 3.(Ritz et al. 2007b;Ritz et al. 2008)

Fig. 3a. Long-term glioblastoma, incubated with 20µM Hypericin. Note the perinuclear granular enrichment.

Visualization and Photodynamic Therapy in Malignant Glioma - An Overview and Perspectives 191

**Fluorescence intensity [a.u.]**

**Fluorescence intensity [a.u.]**

**0**

**25**

**50**

**75**

**100**

20

**10**

found at higher incubation concentrations.

40 60 80

200

**100**

400 600 800

2000

**1000**

4000

**Incubation concentration [µM] 0 5 10 15 20**

**Incubation time [h] 01234**

Fig. 4b. Cellular accumulation of hypericin in U373MG glioblastoma cells (incubation concentration 2.5µM) dependent on incubation temperature [(4°C for 2h and subsequently

37°C for further 2 h (●) vs. 37°C for 4h (o)] measured by flow cytometry.

Fig. 4a. Incubation concentration dependent hypericin uptake in U373 MG cells. Cells were incubated for 2 hours; up to 5 µM cellular fluorescence increased. No further increase was

**A**

Fig. 3b. Fluorescence microscopic images of U373 MG glioblastoma cells, co stained with ER-Tracker (2 M/20 min) and Hypericin (1µM/2h). (A) staining for ER, (B) hypericin fluorescence. (C) demonstrates the costaining image. C-I original image, C-II after image processing.

#### **4.3 Kinetic of intracellular accumulation of hypericin**

The cellular accumulation of hypericin in glioma cell culture is time and concentration dependent. Short incubation times of 2 h lead to saturation up to 5µM hypericin, higher concentrations do not increase hypericin accumulation. Cellular hypericin uptake is subjected by active temperature dependent transport mechanism, although details are not clear at all (Fig. 4).(Ritz, Wein, Dietz, Schenk, Roser, Tatagiba, & Strauss 2007b)

**A** 20 µm **B** 20 µm

**C-I**

Fig. 3b. Fluorescence microscopic images of U373 MG glioblastoma cells, co stained with ER-Tracker (2 M/20 min) and Hypericin (1µM/2h). (A) staining for ER, (B) hypericin fluorescence. (C) demonstrates the costaining image. C-I original image, C-II after image

**C** 20 µm **C-II**

The cellular accumulation of hypericin in glioma cell culture is time and concentration dependent. Short incubation times of 2 h lead to saturation up to 5µM hypericin, higher concentrations do not increase hypericin accumulation. Cellular hypericin uptake is subjected by active temperature dependent transport mechanism, although details are not

clear at all (Fig. 4).(Ritz, Wein, Dietz, Schenk, Roser, Tatagiba, & Strauss 2007b)

**4.3 Kinetic of intracellular accumulation of hypericin** 

processing.

Fig. 4a. Incubation concentration dependent hypericin uptake in U373 MG cells. Cells were incubated for 2 hours; up to 5 µM cellular fluorescence increased. No further increase was found at higher incubation concentrations.

Fig. 4b. Cellular accumulation of hypericin in U373MG glioblastoma cells (incubation concentration 2.5µM) dependent on incubation temperature [(4°C for 2h and subsequently 37°C for further 2 h (●) vs. 37°C for 4h (o)] measured by flow cytometry.

Visualization and Photodynamic Therapy in Malignant Glioma - An Overview and Perspectives 193

Fig. 6. Cryosections of the C6 glioma in rat brain. Contralateral hemisphere without tumor

demonstrated in the upper row. Corresponding sections stained by hematotoxylin and eosin

**5. Photodynamic therapy and anti-tumor immunity – A chance for PDT to be** 

In malignant brain tumors standard therapy is based on surgery, radiation and chemotherapy. The prognosis of patients is still dismal. There is no doubt about the necessitiy of other treatment options. As mentioned in this chapter PDT represents an interesting therapy option in addition to the modern therapeutical strategies described in this book. At the first moment PDT seems only as one additional tool of local tumor treatment, with all advantages, e.g. low costs compared to modern biotechnical products, selective and repetitive application. Therapy resistancy has been seldom observed. PDT is able to occluse tumor associated vessels, mainly PDT induces apoptosis and necrosis, also autophagy plays a role. Great hope lies in the modulation of immune system by PDT. Fluorescence guided tumor resection is able to eradicate tumor locally. In combination with

(left), BAT zone (middle) and tumor (right). Selective hypericin accumulation (red fluorescence) in the tumor and tumor infiltration zone co-stained with DAPI (blue) is

(lower row).

**more than a local cancer therapy?** 

#### **4.4 Photodynamic therapy with hypericin in glioma**

Hypericin exhibits high phototoxicity combined with weak to negligible dark cytotoxicity, as reported previously.(Ritz et al. 2007a) Optimal illumination wavelength for PDT is at 595nm. In our *in vitro* studies on glioma cells, a dosis of 0.15-0.2 J/cm2 resulted in cell survival to 50% (ID50-value); after exposure to 0.4 J/cm2 cell survival was reduced to about 10% as compared to non-illuminated controls. In comparison other investigators applied light doses between 2 J/cm2 (ID50 in U373 MG) up to 15J/cm2 for 5-ALA PDT.(Blake & Curnow 2010)

Fig. 5. Phototoxicity of hypericin in T98G cell line. Phototoxicity depends on incubation concentration (incubation time of 2h) and light dose. Cells were incubated with 0.5 µM (●) 1.5 µM (♦) and 2.5 µM (▲). Illumination was performed at 595 nm, light was delivered at 5- 10 mW/cm2. Cell viability is given on the ordinate.

#### **4.5 Tumor selectivity of hypericin** *in vivo*

Basical for a successful clinical application of hypericin for fluorescence guided resection and PDT is a selective enrichment in tumor tissue without enrichment in normal brain tissue. For this we evaluated in a C6 rat glioma model selective hypericin accumulation in tumor tissue compared to BAT zone and normal brain tissue. By these experiments it could be demonstrated that ratios of hypericin in rat glioma compared to BAT and normal brain tissue were 19.8:2.5:1 (Fig. 6).(Noell et al. 2011) Hypericin was found in a high concentration in tumor tissue, BAT zone was also enriched by hypericin in contrast to normal brain tissue were no hypericin was found.

Our first clinical results demonstrated also a high potential of hypericin for fluorescence guided surgery in malignant glioma, data are submitted for publication.

Hypericin exhibits high phototoxicity combined with weak to negligible dark cytotoxicity, as reported previously.(Ritz et al. 2007a) Optimal illumination wavelength for PDT is at 595nm. In our *in vitro* studies on glioma cells, a dosis of 0.15-0.2 J/cm2 resulted in cell survival to 50% (ID50-value); after exposure to 0.4 J/cm2 cell survival was reduced to about 10% as compared to non-illuminated controls. In comparison other investigators applied light doses between 2 J/cm2 (ID50 in U373 MG) up to 15J/cm2 for 5-ALA PDT.(Blake &

**<sup>100</sup> A**

**Light dose [J/cm2**

Fig. 5. Phototoxicity of hypericin in T98G cell line. Phototoxicity depends on incubation concentration (incubation time of 2h) and light dose. Cells were incubated with 0.5 µM (●) 1.5 µM (♦) and 2.5 µM (▲). Illumination was performed at 595 nm, light was delivered at 5-

Basical for a successful clinical application of hypericin for fluorescence guided resection and PDT is a selective enrichment in tumor tissue without enrichment in normal brain tissue. For this we evaluated in a C6 rat glioma model selective hypericin accumulation in tumor tissue compared to BAT zone and normal brain tissue. By these experiments it could be demonstrated that ratios of hypericin in rat glioma compared to BAT and normal brain tissue were 19.8:2.5:1 (Fig. 6).(Noell et al. 2011) Hypericin was found in a high concentration in tumor tissue, BAT zone was also enriched by hypericin in contrast to normal brain tissue

Our first clinical results demonstrated also a high potential of hypericin for fluorescence

guided surgery in malignant glioma, data are submitted for publication.

**0,0 0,3 0,6 0,9 1,2 1,5**

**]**

**4.4 Photodynamic therapy with hypericin in glioma** 

Curnow 2010)

**Cell survival [% of control]**

**0**

were no hypericin was found.

10 mW/cm2. Cell viability is given on the ordinate.

**4.5 Tumor selectivity of hypericin** *in vivo*

**25**

**50**

**75**

Fig. 6. Cryosections of the C6 glioma in rat brain. Contralateral hemisphere without tumor (left), BAT zone (middle) and tumor (right). Selective hypericin accumulation (red fluorescence) in the tumor and tumor infiltration zone co-stained with DAPI (blue) is demonstrated in the upper row. Corresponding sections stained by hematotoxylin and eosin (lower row).
