Yukari Kuroiwa

Increased cytotoxic effects of photodynamic therapy in IL-6 gene transfected cells via enhanced apoptosis

PDT has been reported to induce cancer cell expression of cytokines, such as IL‐6 and TNF‐α, but it has been unclear whether cytokine expression by cancer cells is directly related to the antitumor effect of PDT. We treated Lewis lung carcinoma (LLC) cells with a new photosensitizer, mono‐L‐aspartyl chlorin e6 (NPe6) and light from a diode laser and found that expression of the mRNA of IL‐2, IL‐6, and TNF‐α was increased by NPe6‐mediated‐PDT 6 hr later. To elucidate the mechanism of the direct anti‐tumor effect of cytokine expression, we examined the photosensitivity of cytokine‐gene‐transfected cells, namely LLC‐IL‐2, LLC‐IL‐6, and LLC‐TNF‐α cells, by MTT assay. The IL‐6 gene transfected, LLC‐IL‐6 cells were significantly more sensitive to cytotoxic effects than the parent LLC cells and other cytokine gene‐transfected cells. This finding indicates that IL‐6 expression modulates cellular sensitivity to PDT and that IL‐2 and TNF‐α expressions does not. In addition, the apoptosis of LLC‐IL‐6 cells induced by NPe6‐PDT was greater than in the other cells as determined by DNA fragmentation and staining of apoptotic nuclei. Because IL‐6 has been reported to induce apoptosis by downregulating expression of Bcl‐2, we analyzed the expression of apoptosis‐related Bcl‐2, Bax, and cytochrome C by Western blot analysis. Decreased expression of Bcl‐2 and cytochrome C was observed in both LLC cells and LLC‐IL‐6 cells. Bax protein increased in a time‐dependent manner, and the ratio of Bax to Bcl‐2 rose markedly after PDT in LLC‐IL‐6 cells. These results suggest that the increased sensitivity of LLC‐IL‐6 cells to PDT‐induced cytotoxicity results from the high ratio of Bax to Bcl‐2 in the IL‐6‐dependent apoptotic pathway. In conclusion, IL‐6 expression plays a role in cellular sensitivity to PDT, and combination of IL‐6 and PDT may provide a new strategy for cancer treatment.

Photodynamic Therapy with a Diode Laser for Implanted Fibrosarcoma in Mice Employing Mono-L-Aspartyl Chlorin E6

Abstract— The authors performed photodynamic therapy (PDT), avoiding any hyperthermic effects, using a newly developed diode laser and photosensitizer, mono‐L‐aspar‐tyl chlorin e6 (NPe6), of Meth‐A fibrosarcoma implanted in mice and achieved tumor therapeutic benefit. The photodynamic light treatment was performed 5 h following the photosensitizer administration. With 5.0 mg/kg NPe6 and light doses of 50, 100, 150 and 200 J/cm2, the tumor cure rates were 20, 50, 70 and 90%, respectively. With 100 J/cm2 laser exposure and NPe6 doses of 1.25, 2.5, 5.0, 7.5 and 10.0 mg/kg, the tumor cure rates were 0, 20, 50, 70 and 90%, respectively. A charge‐coupled device (CCD) camera system was employed to measure the NPe6 fluorescence intensity correlating with the residual amount of the photosensitizer at deferent depth from the tumor surface. The ratios of the NPe6 fluorescence intensity at 3 mm from the tumor surface following 50, 100, 150 and 200 J/cm2 laser exposure to no laser exposure were 0.73, 0.36, 0.22 and 0.16, respectively. With samples sectioned at 1 mm depth, after 50 J/cm2 and the same photosensitizer dose (5 mg/kg) this ratio was 0.19. These results suggest that a certain increase in the tumor tissue level of NPe6 and a certain increase of laser light dose reaching deeper layers of tumor caused an increase in percent cure. In addition, the effectiveness of PDT depends on the total laser dose reaching deeper layers of tumors. Furthermore, the effectiveness of PDT tends to correlate with the amount of NPe6 photobleaching by PDT.

Photodynamic Therapy Using a Diode Laser with Mono-l-aspartyl Chlorin e6 for Implanted Fibrosarcoma in Mice

We have developed a new high‐power red (664 nm) laser diode system for photodynamic therapy (PDT) with mono‐L‐aspartyl chlorin e6 (NPe6). Meth‐A fibrosarcoma cells (1X106) were implanted subcutaneously in the right hind leg of 4‐week‐old BALB/c female mice. One week later, diode laser irradiation was applied 5 h after the intravenous administration of NPe6 to each tumor‐bearing mouse. In the first study, the time course of intratumor temperature increase during PDT was measured by using a 23‐guage thermocouple hypodermic needle at a depth of 2 mm from the tumor surface. In the second study, 6 groups of 10 to 17 tumor‐bearing mice were treated with the diode laser 5 h after intravenous administration of NPe6 at the dose of 1.25, 2.5, 5.0 or 7.5 mg/kg i.v. per mouse. Total photoirradiation ranged from 0 to 150 J/cm2 and the dose rate was adjusted to 100 mW/cm2. Percentages of cures were determined from numbers of mice apparently disease‐free 50 days after treatment. The results showed that this diode laser is effective in PDT of implanted fibrosarcoma after NPe6 administration. It also confirmed that the therapeutic effects of PDT were not due to hyperthermia. Moreover, the diode laser beam was demonstrated by CCD technology to be uniform in intensity throughout the photoirradiated field.

Direct visualization of atherosclerosis in small coronary arteries using the epifluorescence stereoscope

OBJECTIVE To visualize specifically at the beating heart surface atherosclerosis in small coronary arteries using the photosensitiser, mono-L-aspartyl chlorin e6 (NPe6). METHODS Cholesterol-fed atherosclerotic rabbits were injected intravenously with 2.0 mg/kg of NPe6. Atherosclerosis was visualized by allowing NPe6 to accumulate in atheromatous plaques, and then used as a potent fluoroprobe to illuminate atherosclerotic coronary arteries upon excitation by light. An epifluorescence stereoscope system was used to visualize atherosclerosis in small coronary arteries. RESULTS Although it was unable to specify the parts of the coronary arteries which had atherosclerotic changes under room light with the naked eye, several brightly illuminated branching small coronary arteries were observed clearly against the dark heart surface through the epifluorescence stereoscope, as an exciting mercury blue light beam was used to irradiate the beating heart. A fluorescence micrograph of the coronary artery, at which orange-red fluorescence was seen through the epifluorescence stereoscope, showed that the atheromatous plaques emitted orange-red fluorescence. CONCLUSIONS The presence and extent of small coronary atherosclerosis were demonstrated in the beating heart. Such information may help assess the clinical significance of atherosclerosis in small coronary arteries.

Photodynamic therapy using a diode laser with mono-L-aspartyl chlorin e6 (NPe6)

The authors developed a new high-power red laser diode system (Matsushita Industrial Equipment Co., Ltd., Osaka) for photodynamic therapy (PDT) with mono-L-aspartyl chlorin e6 (NPe6, Meiji Seika Kaisha LTD.). The laser wavelength was adjusted to 664 nm and the power output could be varied in the range of 50-500 mW at the fiber tip in a continuous wave (cw) mode. The delivered energy could be adjusted from 50 - 1000 J. The system has a size of 49 by 20 by 40 cm, weighs 20 kg, and is readily portable. It runs on 100 V current. The laser power is easily controlled and the wavelength is stable (less than plus or minus 0.2 nm). The output beam is delivered via a quartz fiber. Furthermore, the full width at half-maximum power is less than 2 nm, which enables uniform, high-density photoirradiation. The power density distribution of the laser, analyzed by a CCD camera, was uniform throughout the photoirradiated field. In an animal study, tumor-bearing Balb/c mice were treated with the diode laser 5 hours after intravenous administration of NPe6 at a dose of 1.25 to 12.5 mg/kg i.v.. Total photoirradiation ranged from 3.13 to 250 J/cm2 which the energy density was adjusted to 100 mW/cm2. Percentages of cures were determined histopathologically from numbers of mice apparently disease-free 1 week after treatment. The results show that a laser energy of more than 12.5 J/cm2 was necessary to obtain 90% tumor cure rate at a maximum dose of NPe6 (12.5 mg/kg) and NPe6 dose of more than 2.5 mg/kg was necessary to obtain 90% cure rate at high laser energy of 200 J/cm2.

Effectiveness of photodynamic therapy with a diode laser using mono-L-aspartyl chlorin e6 for implanted fibrosarcoma in mice

The authors developed a new high power red laser diode system for photodynamic therapy (PDT) with Mono-L-aspartyl Chlorin e6 (NPe6). The laser beam has a low wavelength (664 nm) and relatively high power. Our results suggest that a certain increase in the tumor tissue level of NPe6 and a certain increase of laser light dose reaching a deeper layer of tumor caused an increase in percent cure. In addition, the effectiveness of PDT and also tumor recurrence following PDT depend to some extent on the total laser dose reaching deeper layers of tumors. Furthermore, the effectiveness of PDT tends to correlate with the amount of photobleaching by PDT.

Percutaneous transluminal photodynamic therapy of atheroma using mono-L-aspartyl chlorin e6

Structural changes after photodynamic therapy of atherosclerotic lesions of the thoracic aorta were analyzed by scanning electron microscopy. Cholesterol fed atherosclerotic rabbits were injected intravenously with 5 mg/kg of NPe6. At 6 hours after NPe6 loading, a diode laser irradiated angioscopically on the surface of atheroma with the total energy of 200 mJ/cm2. Scanning electron microscopy showed degeneration of atherosclerotic plaques of the thoracic aorta examined at one week after photodynamic therapy. NPe6 could be a potent photosensitizer for photodynamic therapy of atheroma.