Photodynamic therapy and tumor imaging of hypericin-treated squamous cell carcinoma
© Head et al; licensee BioMed Central Ltd. 2006
Received: 11 May 2006
Accepted: 05 December 2006
Published: 05 December 2006
Conventional cancer therapy including surgery, radiation, and chemotherapy often are physically debilitating and largely ineffective in previously treated patients with recurrent head and neck squamous cell carcinoma (SCC). A natural photochemical, hypericin, could be a less invasive method for laser photodynamic therapy (PDT) of these recurrent head and neck malignancies. Hypericin has powerful photo-oxidizing ability, tumor localization properties, and fluorescent imaging capabilities as well as minimal dark toxicity. The current study defined hypericin PDT in vitro with human SCC cells before the cells were grown as tumor transplants in nude mice and tested as a model for hypericin induced tumor fluorescence and PDT via laser fiberoptics.
SNU squamous carcinoma cells were grown in tissue culture, detached from monolayers with trypsin, and incubated with 0.1 μg to 10 μg/ml of hypericin before exposure to laser light at 514, 550, or 593 nm to define optimal dose, time, and wavelength for PDT of tumor cells. The SCC cells also were injected subcutaneously in nude mice and grown for 6–8 weeks to form tumors before hypericin injection and insertion of fiberoptics from a KTP532 surgical laser to assess the feasibility of this operating room instrument in stimulating fluorescence and PDT of tumors.
In vitro testing revealed a hypericin dose of 0.2–0.5 μg/ml was needed for PDT of the SCC cells with an optimal tumoricidal response seen at the 593 nm light absorption maximum. In vivo tumor retention of injected hypericin was seen for 7 to10 days using KTP532 laser induced fluorescence and biweekly PDT via laser fiberoptics led to regression of SCC tumor transplants under 0.4 cm2 diameter, but resulted in progression of larger size tumors in the nude mice.
In this preclinical study, hypericin was tested for 514–593 nm dye laser PDT of human SCC cells in vitro and for KTP532 surgical laser targeting of SCC tumors in mice. The results suggest hypericin is a potent tumor imaging agent using this surgical laser that may prove useful in defining tumor margins and possibly in sterilizing post-resection margins. Deeply penetrating pulsed infrared laser emissions will be needed for PDT of larger and more inaccessible tumors.
Current estimates indicate that 30,000 people will be diagnosed with squamous cell carcinoma (SCC) of the head and neck region each year in the USA and greater than 8,000 of these patients will die of their disease. Despite recent advances in surgical reconstruction permitting larger resections in addition to new radiation and chemotherapy regimes, the morbidity and mortality of SCC has not decreased significantly in the last 20 years. The median age of death remains 68 years of age and over 50% of head and neck SCC patients recur within 5 years of treatment. For recurrent SCC, the five-year survival rate is less than 10% . Conventional cancer therapies including surgery, radiation, and chemotherapy often are physically debilitating and largely ineffective in previously treated recurrent head and neck tumors. Recent use of laser thermal therapy to palliate these unresectable cancers has not proved effective in reducing the morbidity and mortality of recurrent head and neck cancer patients.
The natural photosensitizing agent, hypericin, may provide a novel approach for less invasive treatment of locally recurrent head and neck tumors [2–4]. Hypericin has powerful photo-oxidizing ability, tumor-seeking characteristics, and minimal dark toxicity, so it is potentially an ideal therapeutic agent for treatment of recurrent SCC. Previous preclinical studies have shown that use of hypericin in photodynamic therapy (PDT) for cancer treatment is effective and may prove to be a clinically valuable procedure in targeting unresectable tumors via this minimally invasive therapy [2–6]. Recently, we found hypericin PDT of cultured human SCC cells in vitro can be activated by visible laser light or infrared pulsed laser excitation . In the current study a low power variable wavelength dye laser was used for hypericin-induced PDT of human SCC cells in vitro and a KTP532 laser tested for effects on SCC cells grown as tumors in mice.
Materials and methods
The human squamous carcinoma cell line SNU1 from Seoul National University was grown in tissue culture. SNU cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 50 ug/ml gentamicin as described previously . Subconfluent monolayers of the cells were detached with 0.25% trypsin in PBS, counted in trypan blue with a hemocytometer, and resuspended in microfuge tubes at 500,000 cells/0.5 ml in RPMI 1640 media without phenol red. These in vitro experiments included hypericin concentrations ranging from 0.1 to 10 μg/mL, dye laser wavelengths of 514, 550, and 593 nm, laser power at the fiberoptic tip of 50, 100, and 150 mW with illumination times of 0–120 seconds for a total light fluence of 0–60 J/cm2 measured at the sample tube liquid interface. After PDT each sample was added to microplate wells at 50,000 cells/0.2 ml in triplicate and incubated for 48 hours at 37°C before cell viability was measured by adding 0.5 mg/ml MTT tetrazolium bromide for 3 hours followed by media aspiration, addition of dimethyl sulfoxide (DMSO) to solubilize the dye product, and optical density of each well recorded at 450 nm with a Molecular Devices microplate reader .
This preclinical evaluation of hypericin was carried out to assess its potential use as a new agent for sterilizing postoperative resection margins in adjuvant treatment of recurrent head and neck SCC where specific tumor targeting by photo-activation is needed to avoid normal tissue damage to adjacent vital structures. Hypericin was tested in the cultured SCC cells to define drug and light dosage as well as light wavelength, exposure time, and laser power delivery needed for tumoricidal effects. Both the in vitro and in vivo experiments described in this study shows that the photosensitizing effects of hypericin induced significant killing of human SCC when excited by visible light from a laser delivered via fiberoptics.
Initial treatment of head and neck squamous cell carcinoma is directed by several considerations with the single most important factor being stage of disease . However, with the advent of newer chemoradiation protocols, an increase in the number of recurrent squamous cell carcinomas has been seen. These recurrent patients usually have no surgical options and need newer modalities of treatment in hopes of palliation after receiving maximum dose radiation or appearance of drug resistant tumors . Tumor imaging and PDT with hypericin have been tested in vivo for therapy of pancreas and skin cancer as well as in nasopharyngeal carcinomas [5, 8, 11]. Experimental studies have shown that hypericin photodynamic therapy is tumoricidal for human cancer cells both in vitro and in vivo with potential application in targeting recurrent or unresectable malignancies through this minimally invasive new adjuvant procedure [2–7].
The current study supports previously reported data and provides new evidence that hypericin may prove to be clinically useful for both tumor imaging and in PDT of locally recurrent head and neck squamous cell carcinoma. As seen in Figs 5 and 6, hypericin is effective as a fluorescent tumor imaging agent when activated by a conventional operating room KTP532 surgical laser. In an earlier study, we reported that intravenous hypericin injection resulted in significant uptake by SCC tumors, but higher levels of this fluorescent dye were deposited in the lungs and other vascular organs . Although 532 nm laser light was sufficient for hypericin excitation and in fluorescent tumor imaging, the in vitro SCC experiments of the current study show clearly that the higher 593 nm laser wavelength is superior in phototherapy. This conclusion also was indicated by a decreased PDT response observed for large SCC tumors after hypericin and KTP532 laser treatment even when fiberoptics were inserted directly into the tumor center to maximize the light delivery. Recent reports show hypericin phototherapy can be enhanced by multi-fraction treatment, by the addition of synthetic oxygen carriers, or by co-administration of a cyclo-oxygenase inhibitor to block prostaglandin synthesis and prevent new angiogenesis in tumor sites [9–11]. Further improvement of hypericin PDT also may be possible using 2-photon infrared pulsed laser emissions to increase tissue penetration of light during tumor imaging and treatment . These advances will make hypericin and lasers a more effective cancer therapy.
The photodynamic response of hypericin dye was tested in vitro with cultured human squamous carcinoma cells after exposure to laser emissions at green, yellow, and orange visible light wavelengths (514, 550, and 593 nm). This study revealed that the greatest PDT responses occurred at hypericin's absorption and fluorescence maximum of 593 nm. A range of hypericin concentrations was tested to determine the minimum dosage for photosensitization of the tumor cells, which was found to be from 0.2 to 0.5 μg/mL or 1 μM. Laser power delivery at 150 mW produced the highest level of tumor cell killing at 593 nm. In vivo studies with nude mice bearing human squamous cell carcinoma tumors showed that injected hypericin remained within the tumor site for 10 days allowing repeated laser illumination.
This study also confirms that placement of the fiberoptic within the tumor provides the greatest photo-oxidation and is the optimal route of laser light delivery. During these PDT tests of subcutaneous human SCC tumors using a surgical KTP532 laser emitting visible green light, the limiting factor appears to be tumor size. Hypericin PDT also should be enhanced via deeper penetration of 593 nm light as in our in vitro studies above or by using longer wavelengths of pulsed infrared light for 2-photon excitation . This study provides new evidence hypericin is an ideal photosensitizer for PDT that may prove to be clinically useful in tumor imaging and minimally invasive treatment of locally recurrent head and neck squamous cell carcinoma.
We thank N. Jongewaard, T. Day, J. Kim, T. Kim, and Dr. P. Felker for help with this research.
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