Krzysztof Symonowicz

In vitro and in vivo matrix metalloproteinase expression after photodynamic therapy with a liposomal formulation of aminolevulinic acid and its methyl ester

Photodynamic therapy (PDT) is a well-known method for the treatment of malignant tumors, and its principles have been well established over the past 30 years. This therapy involves the application of a chemical called a photosensitizer and its subsequent excitation with light at the appropriate wavelength and energy. Topical photodynamic therapy with aminolevulinic acid (5-ALA) is an alternative therapy for many malignant processes, including nonmelanoma skin cancers such as basal-cell carcinoma (BCC). Our novel approach for this study was to use a liposomal formulation of 5-ALA and its methyl ester (commercially available as metvix) both in vitro and in vivo, and to check whether the liposome-entrapped precursors of photosensitizers can induce the expression of metalloproteinases (MMPs) in animal tumor cells and in other tissues from tumor-bearing rats and in selected cell lines in vitro. We also checked whether the application of tissue inhibitors of matrix metalloproteinases (TIMPs) has any effect on MMPs in the above-mentioned experimental models, and if they can cause complete inhibition of MMP expression. Immunohistochemical studies revealed that after the PDT, the intensity of expression of MMPs in healthy animals was very low and seen in single cells only. After the PDT in tumor-bearing rats, MMP-3 was expressed in the tumor cells with the highest intensity of staining in the tissues directly adjacent to the tumors, while MMP-2 and -9 were not found. In the control groups, there was no observed expression of MMPs. In vitro studies showed that MMP-3 was expressed in MCF-7 cells after PDT, but MMP-9 was not observed and MMP-2 was only seen in single cases. Our studies confirmed that the application of an MMP-3 inhibitor may block an induction of MMP-3 expression which had previously been initiated by PDT. The preliminary data obtained from cancer patients revealed that new precursors are effective in terms of PDT, and that using MMP inhibitors should be considered as a potential enhancing factor in clinical PDT.

New potent sensitizers for photodynamic therapy : 21-oxaporphyrin, 21-thiaporphyrin and 21,23-dithiaporphyrin induce extensive tumor necrosis

Abstract New sensitizers for photodynamic therapy (PDT) are reported. These compounds, namely 21-thiaporphyrin, 21,23-dithiaporphyrin and 21-oxaporphyrin, reveal some of the properties required for such therapy. Their physicochemical, chemical and pharmacological features meant that we could use them in the treatment of transplantable BFS1 fibrosarcoma in Balb/c mice. New sensitizers and the well-known chlorin e6 (Ce6) were used in doses of 2.5, 5.0, 7.5 and 10.0 mg/kg body weight, given intraperitoneally and followed by light irradiation, the total light doses being 50, 100 and 150 J/cm2 within 24 h after injection. The effectiveness of new sensitizers in PDT was evaluated with in terms of tumor necrosis intensity, the survival time of treated animals, the rate of tumor response (complete/partial/no response), and skin photosensitivity. These results were compared to results obtained in analogous conditions after Ce6-PDT. Distribution studies revealed that the highest concentration of new compounds occurred within 24 h after injection. The results of these experiments confirmed that 21-thiaporphyrin, 21,23-dithiaporphyrin and 21-oxaporphyrin can be considered as potent tumor photosensitizers that do not exert any unwanted effects, primarily skin photosensitization. We suggest that these porphyrins are possible sensitizers to be applied in clinical PDT.

5,20-bis(4-sulphophenyl)-10,15-bis (2-methoxy-4-sulphophenyl)-21-thiaporphyrin as a new potent sensitizer in photodynamic therapy.

The main purpose of the study was to investigate the effectiveness of a new photosensitizer for photodynamic therapy. 5,20-bis(4-sulphophenyl)-10,15-bis(2-methoxy-4-sulphophenyl)-21-thiaporphyrin (21-thiaporphyrin) was compared to chlorin e6 and tetra(m-hydroxyphenyl)porphyrin (m-THPP) for its ability to sensitize tumors and skin to light. Chlorin e6 and m-THPP induced a strong tumor and skin photosensitization. In contrast, the same doses of 21-thiaporphyrin produced no skin sensitization and gave approximately 10 mm tumor necrosis after light exposure, in comparison to the 5-6 mm necrosis induced by chlorin e6 or m-THPP under identical conditions. 21-Thiaporphyrin, tested as a potential photosensitizer, induced no skin sensitization even at doses as high as 7.5 mg/kg body weight. 21-Thiaporphyrin presents a high potency in tumor sensitizing, i.e. a feature required for an efficient photosensitizer in photodynamic therapy applications.

Photodynamic Treatment of Epithelial Tissue Derived from Patients with Endometrial Cancer: A Contribution to the Role of Laminin and Epidermal Growth Factor Receptor in Photodynamic Therapy

Photodynamic therapy (PDT) was used to treat endometrial G1 cancer tissue derived from patients who had undergone a total hysterectomy and bilateral salpingo-oophorectomy. After surgical treatment the cancerous tissue was kept in a medium containing Dulbecco solution, fetal calf serum, and antibiotics. The tissue was then exposed to hematoporphyrin derivative (0.1 mg/ℓ) and 24 h later exposed to light (total light dose-18 J/sq cm). Necrosis depth was evaluated 24 h later using a light microscope. In order to assess the possible role of the basal membrane component laminin, as well as epidermal growth factor receptor susceptibility to PDT, immunohistochemical studies were carried out. Additionally, nucleolar organizer regions evaluation was performed. Our experiment confirmed that PDT results in the necrosis in the treated endometrial cancer, while not affecting the laminin in the cancerous tissue. In contrast, PDT strongly affects the epidermal growth factor receptor and nucleolar organizer regions in cancer cells. We suggest that laminin may contribute to the prevention of cancer dissemination in the cases where PDT has to be repeated, and that after PDT the cells become less susceptible to a mitogen, like, e.g., epidermal growth factor.

Immunohistochemical study of nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 in invasive breast carcinoma of no special type

The aim of the present study was to investigate the immunohistochemical expression of nuclear ubiquitous casein and cyclin-dependent kinases substrate 1 (NUCKS1) in invasive breast carcinoma of no special type, in association with clinicopathological characteristics, including the tumor grade, frequency of lymph node involvement and distant metastasis. In addition, associations between NUCKS1 and other tumor subtype markers, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), Ki-67 and cytokeratin 5/6 (CK 5/6), were investigated. NUCKS1 expression was shown to be associated with the formation of distant metastases and lymph node involvement. Furthermore, an association between the presence of NUCKS1 and histological grading was observed. The results confirmed that the expression of NUCKS1 in low grade invasive breast carcinoma of no special type was significantly less common compared with cases of high grade carcinoma. With regard to the additional tumor subtype markers, NUCKS1 expression was demonstrated to be significantly associated with Ki-67 and CK 5/6; however, no association was identified with ER, PR and HER2. Therefore, NUCKS1 may be a novel prognostic marker in the histopathological evaluation of invasive breast carcinoma of no special type.

Expression of metallothioneins I and II in kidney of doxorubicin-treated rats

Metallothioneins I/II (MT) are commonly expressed in mammalian tissues and are highly inducible in the response to stress conditions. Doxorubicin (DOX) intoxication promotes oxidative stress and subsequent apoptosis leading to kidney damage. The present study investigates a correlation between endogenous MT expression and DOX-induced apoptosis in renal tubular cells. Experiments were conducted on Buffalo rats receiving DOX (8 mg/kg b.w. for 3 weeks) versus control rats injected with saline. The histopathological alterations and apoptosis (TUNEL) were evaluated in tissue sections. MT expression and tissue localization was examined using immunohistochemical method (IHC). Western blot (WB) was used to evaluate pro-caspase-3, active caspase-3 and MT expression level in tissue homogenates. Examination of renal tissue revealed severe nephrotoxicity in DOX-treated animals. Apoptosis was observed in distal convoluted tubular cells, whereas MT was detected in proximal tubular cells. A significant increase in pro-caspase-3, active caspase-3 and MT expression levels (WB) were seen in DOX group. Positive correlations between histopathological lesions, apoptosis and MT expression were observed. The results obtained in this study could suggest the protective and antiapoptotic effect of MT expression in renal proximal tubular cells under DOX intoxication.

In-vivo interleukin-1-alpha induction following chlorin-e6 photodynamic therapy in Balb/c mice

Photodynamic therapy may induce in in-vivo conditions the cytokine Interleukin-1-alpha in Balb/c mice. The sensitizer, i.e. chlorin e6, in the doses 2.5 and 5.0 mg/kg of body weight followed by light treatment with doses 50 and 100 J/sq.cm resulted in the increase in serum levels of the cytokine. The levels of Interleukin-1-alpha have been determined at different time points using enzyme-linked immunosorbent assay (ELISA). These levels in control animals did not exceed the mean value of 15 pg/ml, whereas in photodynamically treated mice the levels were almost 3 - 4 times higher. The entire experiment has been carried out on healthy animals.

A brief history of photodynamic therapy in Wrocław

A brief history of photodynamic therapy in Wroclaw was presented in this paper.

Assessment of in vivo experiments: The newly synthesized porphyrin with proper light source enhanced effectiveness of PDT comparing to 5-ALA-mediated PDT

BACKGROUND The search for new photosensitizers for application in photodynamic therapy has quite a long history. In the past, a large number of potent photosensitizers were used in both basic and clinical studies; however, only a few turned out to be effective and safe. METHODS In the present study, two compounds were used: 5-aminolevulinic acid in two formulations (free and liposomal), and the newly synthesized porphyrin, 5,10,15,20-tetra-p-tolyl-22,24-dithiadibenzocarbaporphyrin, termed DTDB. Two different light sources, a halogen lamp (wavelength 450+/-20nm) and a diode laser (wavelength 450nm), were used to sensitize the compounds. The entire experiment was performed on mice bearing mouse mammary carcinoma, 4T1. RESULTS The results showed that the DTDB-PDT applied by means of a laser proved to be most effective and caused the 83.3% necrosis of treated tumors. The overall effect of laser PDT was more potent than that of the halogen lamp-mediated PDT. CONCLUSIONS In the present study, we would like to show that modifications of porphyrins lead to an increase in the effectiveness of PDT and that this effect could also be potentiated by using a proper light source.

Particle position measuring with optical tweezers using video processing

The stiffness of a single optical trap is a basic parameter of optical manipulators and detecting the displacement of trapped object belongs to fundamental measurements [1]. The trap stiffness can be determined by position measurements of oscillating (with high frequency) dielectric microsphere. Usually the signal is captured by simple detector like quadrant photodiode [2]. However, this method is hard to apply when working in multitrap mode, especially in case of holographic optical tweezers. In multitrap mode, widely used method is video image processing focused on determining the center of mass of an object [3]. In this contribution we present the method for position measurment based on video sequence analysis from fast camera. The position is obtained by correlating the video frame with templates of known positions of that object. This algorithm can work on several objects, finding their positions independently. It has also scalable accuracy up to sub-pixel level.