The role of interleukin-8 (CXCL8) and CXCR2 in acquired chemoresistance of human colorectal carcinoma cells HCT116.

Colorectal cancer is one of the most common malignant diseases and is a leading cause of cancer mortality in the Western world. Primary or acquired resistance to chemotherapeutic drugs is a common phenomenon which causes a failure in cancer treatment. A diverse range of molecular mechanisms has been implicated in drug resistance: DNA damage repair, alterations in drug metabolism, mutation of drug targets, increased rates of drug efflux, and activation of survival signaling pathways. The aim of this study was to investigate the expression of CXCL8-CXCR1/2 pathway, its impact on cell proliferation and cytokine expression in human colorectal carcinoma HCT116 cells, and their chemotherapy-resistant subline. We found that IL-1 alpha stimulates the production of CXCL8 through IL-1 receptor signaling. Our data indicate that CXCL8 is upregulated in chemoresistant subline of colorectal cancer cells HCT116, and modulation of CXCR2 pathway can be a target for proliferation inhibition of chemoresistant colorectal cancer cells.

Differential expression of VEGF and IL-1alpha after photodynamic treatment in combination with doxorubicin or taxotere.

AIM: To show the impact of chemotherapeutic drugs doxorubicin and taxotere on the molecular pattern of cell response to photodynamic treatment (PDT). MATERIALS AND METHODS: Human squamous cell carcinoma cells A-431 were studied. Apoptosis was investigated by recording caspase-3 activity. Expression of IL-1alpha and VEGF on mRNA and protein levels was measured by qPCR and ELISA. RESULTS: PDT in combination with either doxorubicin or taxotere was found to be more cytotoxic in comparison to either single-treatment. The expression of IL-1alpha and VEGF was up-regulated in PDT-treated cells, either alone or in combination with doxorubicin or taxotere. Addition of doxorubicin to the cytokine induction after PDT was not detected, however, taxotere promoted significant over-expression of IL-1alpha and VEGF on the protein level. CONCLUSION: Contribution of chemotherapeutic drugs to IL-1 alpha and VEGF release from cells which received dual treatment involving PDT could be significantly different, despite the same level of cytotoxicity.

mTHPC-mediated photodynamic treatment up-regulates the cytokines VEGF and IL-1alpha.

Photodynamic therapy (PDT) of cancer induces oxidative stress, which intervenes in the expression of cytokines by tumor cells. The cytokines might have either a positive or a negative impact on tumor eradication. Here, we studied the expression of cytokines vascular endothelial growth factor (VEGF) and interleukin-1alpha (IL-1alpha) in the human epidermoid carcinoma A-431 cells following m-tetra(3-hydroxyphenyl)-chlorin (mTHPC)-mediated PDT in vitro and assessed the IL-1alpha effect on VEGF expression. Quantitative polymerase chain reaction and enzyme-linked immunosorbent assay revealed the enhanced production of VEGF and IL-1alpha both on mRNA and protein levels by mTHPC-loaded cells after light exposure. The silencing of IL1A by small interfering RNA resulted in decreased production of IL-1alpha and a reduced amount of VEGF. Furthermore, exogenous recombinant IL-1alpha stimulated the VEGF expression after PDT. Thus, in addition to the cytotoxic action on the A-431 cells, mTHPC-mediated PDT stimulated the production of VEGF and IL-1alpha, and IL-1alpha contributed to the VEGF overexpression. These data establish IL-1alpha as a possible target of combined cancer treatment.

mTHPC-mediated photodynamic treatment of Lewis lung carcinoma in vitro and in vivo.

BACKGROUND AND OBJECTIVE: The ongoing search for the enhancement of efficacy of photodynamic therapy stimulates the interest in molecular mechanisms of the response to the treatment. Looking for the cell line suitable for investigation of cellular response both in vivo and in vitro, we evaluated phototoxicity of m-tetrakis-(3-hydroxyphenyl)-chlorin (mTHPC) on viability of Lewis lung carcinoma (LLC1) cells in vitro, growth of murine transplantable tumor, and mice survival. MATERIAL AND METHODS: LLC1 cell culture and male C57BL/6 mice bearing Lewis lung carcinoma were used for the experiments. Photodynamic treatment was mediated by m-tetrakis-(3-hydroxyphenyl)-chlorin as a photosensitizer. Light emitting diode array was used for illumination. The effect of the photodynamic treatment was evaluated by comparison of viability of control and treated cells, growth of tumors, and survival of the control and treated mice. RESULTS: In vitro, a cytotoxic dose inducing a reduction in viability of LLC1 cells by 50% was achieved at 60 mJ/cm(2) and approximately 400 ng/mL of the photosensitizer, or 30 mJ/cm(2) and 600 ng/mL of mTHPC. Both the concentration of the photosensitizer and duration of light exposure were significant determinants of cytotoxic effect. In vivo, an injection of 0.25 mg/kg of mTHPC to mice bearing Lewis lung tumor and illumination at 120 J/cm(2) taking place after 24 h significantly inhibited tumor growth and prolonged mice survival. However, the tumors regained their growth potential after 9 days. CONCLUSIONS: Photodynamic treatment mediated by m-tetrakis-(3-hydroxyphenyl)-chlorin had a significant effect on LLC1 cells in vitro and growth of Lewis lung carcinoma in vivo.

Damage targeted to the mitochondrial interior induces autophagy, cell cycle arrest and, only at high doses, apoptosis.

It is generally accepted that permeabilization of the outer mitochondrial membrane is a crucial event in the induction of apoptosis. In order to know how the cell would respond to the damage targeted not to the outer mitochondrial membrane, but to the mitochondrial interior, we followed the changes in cell morphology and some biochemical parameters triggered by ROS in the mitochondrial inner space. The experiments were carried out in epidermoid carcinoma A431 cells. For stimulated production of singlet oxygen in the inner space of mitochondria, we employed photodynamic treatment (PDT) mediated by a cationic photosensitizer 7-diamino-2,8-dimethyl-5-phenylphenazinium chloride (Safranin O, Safr), accumulating in the inner space of mitochondria. At low to intermediate cytotoxic doses (up to CD50 reducing cellular viability by 50%), Safr-PDT did not reveal hallmarks of dead cells, and the decrease of cellular viability could be attributed to cell cycle inhibition, and enhanced autophagy. High Safr-PDT doses (beyond CD70) did induce cell death by apoptosis involving release of cytochrome c and caspase-3 activation, in addition to enhanced autophagy.

Enhancement of photodynamic tumor therapy effectiveness by electroporation in vitro.

The aim of our study was to determine if electroporation could improve the efficacy of photodynamic tumor therapy. A disadvantage of photodynamic therapy is a slow and in some cases insufficient accumulation of photosensitizer in tumor tissue, which could restrict the achievement of an efficient dose. Under the action of electric pulses, cells undergo membrane electroporation, which results in an increased permeability to various exogenous molecules. In this study, murine hepatoma MH22A cells were exposed to light in vitro in the presence of a photosensitizer, either chlorin e6 or aluminum phthalocyanine tetrasulfonate, following electroporation. Accumulation of the photosensitizers was registered by fluorescence microscopy. Cell viability was determined by the MTT assay. Our results demonstrate that electroporation improves an access of chlorin e6 and aluminum phthalocyanine tetrasulfonate to MH22A cells. Electroporation in combination with photosensitization significantly reduces viability of the treated cells even at low doses of photosensitizers.

Apoptosis, autophagy and cell cycle arrest following photodamage to mitochondrial interior.

Cell death induced by oxidative insult targeted to mitochondrial interior of A431 cells was investigated. For stimulated production of ROS in the inner space of mitochondria, safranin-mediated photodynamic treatment (PDT) was employed. Another photosensitizer, mTHPC, which diffusely localizes to cellular membranes, was used for comparison. Cell response to the oxidative insult in mitochondrial interior was different from the response to the photodamage produced in cellular membranes. Autophagy and apoptotic features of cell death in response to mTHPC-PDT was observed in a wide range of PDT doses. Cell response to the oxidative stress in mitochondrial interior was dose-dependent. Damage up to CD50 did not reveal hallmarks of dead cells. At intermediate damage (CD50), cells manifested enhanced autophagy and reduced population of S-phase, but not apoptosis. Severe damage (beyond CD70) induced apoptosis following release of cytochrome c and caspase activation, in addition to autophagy and cell cycle arrest.

Experimental survey of non-clonogenic viability assays for adherent cells in vitro.

Results of rapid cell viability assays were experimentally compared in order to reveal the most suitable test for in vitro investigations of the combination of photodynamic therapy (PDT) with chemotherapeutic drugs. meso-Tetra(3-hydroxyphenyl)-chlorin (m-THPC) accumulating in cell membranes and meso-tetra(4-sulfonatophenyl)-porphin (TPPS4) accumulating in lysosomes were used as photosensitisers. Doxorubicin that localises, mainly, to nucleus and vincristine that binds to microtubules were used as cytostatic drugs. Two adherent rodent cell lines, baby hamster kidney (BHK-21) and murine hepatoma (MH-22A), were used to examine the contribution of a cell. We tested cytotoxicity assays of the main groups of fast (non-clonogenic) methods of cell viability measuring. Plasma membrane integrity was estimated by trypan blue exclusion and LDH leakage, metabolic activity was tested by [3H]-thymidine incorporation and MTT assay, loss of monolayer adherence was measured by staining with crystal violet and CyQUANT. The most sensitive test in each case was the assay related to the site of the direct damage, and measurement of the loss of monolayer adherence proved to be as sensitive assay as the damage-specific one. All the assays applied, except for the LDH release, revealed a higher effect of combination of m-THPC-mediated phototreatment and doxorubicin compared to either of the single treatments.

Correlation of death modes of photosensitized cells with intracellular ATP concentration.

The impact of intensity of glycolysis and oxidative phosphorylation on death of photosensitized murine hepatoma MH22 cells in vitro has been investigated. Cells photosensitized with meso-tetra(4-sulfonatophenyl)-porphine localized to lysosomes died mostly by necrosis, and the mode of cell death did not depend on the energy metabolism. Photosensitization with 5-aminolevulinic acid-stimulated endogenous porphyrins localized mainly in mitochondria or 5,10,15,20-tetrakis(m-hydroxyphenyl)-chlorine localized to cell membranes, including mitochondria, led to cell death mostly by apoptosis. In this case, the mode of cell death depended on the medium: under conditions unfavorable to glycolysis the ratio apoptosis/necrosis decreased significantly.