Photodynamic Effects with 5-Aminolevulinic Acid on Cytokines and Exosomes in Human Peripheral Blood Mononuclear Cells from Patients with Crohn's Disease.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) which is the precursor of the photosensitizer protoporphyrin IX (PpIX) is an available treatment for several diseases. ALA-PDT induces the apoptosis and necrosis of target lesions. We have recently reported the effects of ALA-PDT on cytokines and exosomes of human healthy peripheral blood mononuclear cells (PBMCs). This study has investigated the ALA-PDT-mediated effects on PBMC subsets from patients with active Crohn's disease (CD). No effects on lymphocyte survival after ALA-PDT were observed, although the survival of CD3-/CD19+ B-cells seemed slightly reduced in some samples. Interestingly, ALA-PDT clearly killed monocytes. The subcellular levels of cytokines and exosomes associated with inflammation were widely downregulated, which is consistent with our previous findings in PBMCs from healthy human subjects. These results suggest that ALA-PDT may be a potential treatment candidate for CD and other immune-mediated diseases.

Evaluation of the PSMA-Binding Ligand 212Pb-NG001 in Multicellular Tumour Spheroid and Mouse Models of Prostate Cancer.

Radioligand therapy targeting the prostate-specific membrane antigen (PSMA) is rapidly evolving as a promising treatment for metastatic castration-resistant prostate cancer. The PSMA-targeting ligand p-SCN-Bn-TCMC-PSMA (NG001) labelled with 212Pb efficiently targets PSMA-positive cells in vitro and in vivo. The aim of this preclinical study was to evaluate the therapeutic potential of 212Pb-NG001 in multicellular tumour spheroid and mouse models of prostate cancer. The cytotoxic effect of 212Pb-NG001 was tested in human prostate C4-2 spheroids. Biodistribution at various time points and therapeutic effects of different activities of the radioligand were investigated in male athymic nude mice bearing C4-2 tumours, while long-term toxicity was studied in immunocompetent BALB/c mice. The radioligand induced a selective cytotoxic effect in spheroids at activity concentrations of 3-10 kBq/mL. In mice, the radioligand accumulated rapidly in tumours and was retained over 24 h, while it rapidly cleared from nontargeted tissues. Treatment with 0.25, 0.30 or 0.40 MBq of 212Pb-NG001 significantly inhibited tumour growth and improved median survival with therapeutic indexes of 1.5, 2.3 and 2.7, respectively. In BALB/c mice, no signs of long-term radiation toxicity were observed at activities of 0.05 and 0.33 MBq. The obtained results warrant clinical studies to evaluate the biodistribution, therapeutic efficacy and toxicity of 212Pb-NG001.

Application of Photodynamic Therapy with 5-Aminolevulinic Acid to Extracorporeal Photopheresis in the Treatment of Cutaneous T-Cell Lymphoma: A First-in-Human Phase I/II Study.

Extracorporeal photopheresis (ECP) is a therapeutic modality used for T-cell-mediated disorders. This approach involves exposing isolated white blood cells to photoactivatable 8-methoxypsoralen (8-MOP) and UVA light, aiming to induce apoptosis in T-cells and thereby modulate immune responses. However, conventional 8-MOP-ECP lacks cell selectivity, killing both healthy and diseased cells, and has shown limited treatment efficacy. An alternative approach under investigation involves the use of 5-aminolevulinic acid (ALA) in conjunction with light, referred to as ALA-based photodynamic therapy. Our previous ex vivo studies suggest that ALA-ECP exhibits greater selectivity and efficiency in killing T-cells derived from patients with T-cell-mediated disorders compared to those treated with 8-MOP-ECP. We have conducted a clinical phase I-(II) study evaluating ALA-ECP safety and tolerability in cutaneous T-cell lymphoma (CTCL). Here, 20 ALA-ECP treatments were administered to one CTCL patient, revealing no significant changes in vital signs. Two adverse events were reported; both evaluated by the Internal Safety Review Committee as non-serious. In addition, five conceivable events with mainly mild symptoms took place. During the study period, a 53% reduction in skin involvement and a 50% reduction in pruritus was observed. In conclusion, the results indicate that ALA-ECP treatment is safe and well tolerated.

Blebbistatin, a myosin inhibitor, is phototoxic to human cancer cells under exposure to blue light.

BACKGROUND: Blebbistatin is a new inhibitor of cell motility. It is used to study dynamics of cytokinesis machinery in cells. However, the potential of this inhibitor as an anticancer agent has not been studied so far. METHODS: Cytotoxicity of blebbistatin was evaluated in five human cell lines, FEMX-I melanoma, U87 glioma, androgen independent Du145 and androgen sensitive LNCaP prostate adenocarcinoma, and F11-hTERT immortalized fibroblasts. Phototoxicity of blebbistatin was assessed in these cell lines after their exposure to a blue light (390-470 nm). Photostability of blebbistatin and its reactive oxygen species (ROS) generating properties were measured during irradiation with the blue light. RESULTS: Blebbistatin at a concentration range of 10-200 µmol/L was toxic to all studied cells. Toxic concentrations (TC) were about 10-25 µmol/L corresponding to TC10, 50-100 µmol/L to TC50 and 140-190 µmol/L to TC90. Only for the U87 glioma cells TC90 could not be measured as the highest studied concentration of 200 µmol/L gave around 70% toxicity. However, after exposure to the blue light blebbistatin exhibited phototoxicity on the cells, with a cytotoxicity enhancement ratio that was greatest for the FEMX-I cells (about 9) followed by LNCaP (5), Du145 (3), U87 (2) and F11-hTERT (1.7) cells. CONCLUSIONS: Blebbistatin inhibits cell motility and viability. Under exposure to the blue light blebbistatin exhibits photodynamic action on human cancer cells. During the irradiation blebbistatin oxidizes dihydrorhodamine 123 but not Singlet Oxygen Sensor Green. GENERAL SIGNIFICANCE: Our findings offer new possibilities for blebbistatin as a potential anticancer and photodynamic agent.

Carbon-core silver-shell nanodots as sensitizers for phototherapy and radiotherapy.

Spherical carbon nanoparticles (carbon nanodots) with a silver shell were investigated as potential sensitizing agents. The cytotoxicity of the combination of ultraviolet radiation or x-rays with the nanodots was examined in cancer cells in vitro. The cell viability decreased following the exposure to the radiation. The carbon nanodots enhanced the radiation effects by significantly reducing the amount of surviving cells compared to that of the cells exposed only to the radiation. Carbon-core silver-shell nanodots can be proposed as a bimodal sensitization platform for biological and medicinal applications employing non-ionizing or ionizing radiation.

Photodynamic Effects with 5-Aminolevulinic Acid on Cytokines and Exosomes in Human Peripheral Blood Mononuclear Cells.

Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA), a precursor to the potent photosensitizer, protoporphyrin IX (PpIX), is an established modality for several malignant and premalignant diseases. This treatment is based on the light-activated PpIX in targeted lesions. Although numerous studies have confirmed the necrosis and apoptosis involved in the mechanism of action of this modality, little information is available for the change of exosome levels after treatment. We report from the first study on the effects of ALA-PDT on cytokines and exosomes of human healthy peripheral blood mononuclear cells (PBMCs). The treatment reduced the cytokines and exosomes studied, although there was variation among individual PBMC samples. This reduction is consistent with PDT-mediated survivals of subsets of PBMCs. More specifically, the ALA-PDT treatment apparently decreased all pro-inflammatory cytokines included, suggesting that this treatment may provide a strong anti-inflammatory effect. In addition, the treatment has decreased the levels of different types of exosomes, the HLA-DRDPDQ exosome in particular, which plays an important role in the rejection of organ transplantation as well as autoimmune diseases. These results may suggest future therapeutic strategies of ALA-PDT.

Amphiphilic Protoporphyrin IX Derivatives as New Photosensitizing Agents for the Improvement of Photodynamic Therapy.

Photodynamic therapy (PDT) is a non-invasive therapeutic modality based on the interaction between a photosensitive molecule called photosensitizer (PS) and visible light irradiation in the presence of oxygen molecule. Protoporphyrin IX (PpIX), an efficient and widely used PS, is hampered in clinical PDT by its poor water-solubility and tendency to self-aggregate. These features are strongly related to the PS hydrophilic-lipophilic balance. In order to improve the chemical properties of PpIX, a series of amphiphilic PpIX derivatives endowed with PEG550 headgroups and hydrogenated or fluorinated tails was synthetized. Hydrophilic-lipophilic balance (HLB) and log p-values were computed for all of the prepared compounds. Their photochemical properties (spectroscopic characterization, photobleaching, and singlet oxygen quantum yield) were also evaluated followed by the in vitro studies of their cellular uptake, subcellular localization, and photocytotoxicity on three tumor cell lines (4T1, scc-U8, and WiDr cell lines). The results confirm the therapeutic potency of these new PpIX derivatives. Indeed, while all of the derivatives were perfectly water soluble, some of them exhibited an improved photodynamic effect compared to the parent PpIX.

Predictive biomarkers for 5-ALA-PDT can lead to personalized treatments and overcome tumor-specific resistances.

BACKGROUND: Photodynamic therapy (PDT) is a minimally invasive, clinically approved therapy with numerous advantages over other mainstream cancer therapies. 5-aminolevulinic acid (5-ALA)-PDT is of particular interest, as it uses the photosensitiser PpIX, naturally produced in the heme pathway, following 5-ALA administration. Even though 5-ALA-PDT shows high specificity to cancers, differences in treatment outcomes call for predictive biomarkers to better stratify patients and to also diversify 5-ALA-PDT based on each cancer's phenotypic and genotypic individualities. AIMS: The present study seeks to highlight key biomarkers that may predict treatment outcome and simultaneously be exploited to overcome cancer-specific resistances to 5-ALA-PDT. METHODS AND RESULTS: We submitted two glioblastoma (T98G and U87) and three breast cancer (MCF7, MDA-MB-231, and T47D) cell lines to 5-ALA-PDT. Glioblastoma cells were the most resilient to 5-ALA-PDT, while intracellular production of 5-ALA-derived protoporphyrin IX (PpIX) could not account for the recorded PDT responses. We identified the levels of expression of ABCG2 transporters, ferrochelatase (FECH), and heme oxygenase (HO-1) as predictive biomarkers for 5-ALA-PDT. GPX4 and GSTP1 expression vs intracellular glutathione (GSH) levels also showed potential as PDT biomarkers. For T98G cells, inhibition of ABCG2, FECH, HO-1, and/or intracellular GSH depletion led to profound PDT enhancement. Inhibition of ABCG2 in U87 cells was the only synergistic adjuvant to 5-ALA-PDT, rendering the otherwise resistant cell line fully responsive to 5-ALA-PDT. ABCG2 or FECH inhibition significantly enhanced 5-ALA-PDT-induced MCF7 cytotoxicity, while for MDA-MB-231, ABCG2 inhibition and intracellular GSH depletion conferred profound synergies. FECH inhibition was the only synergism to ALA-PDT for the most susceptible among the cell lines, T47D cells. CONCLUSION: This study demonstrates the heterogeneity in the cellular response to 5-ALA-PDT and identifies biomarkers that may be used to predict treatment outcome. The study also provides preliminary findings on the potential of inhibiting specific molecular targets to overcome inherent resistances to 5-ALA-PDT.

Evaluation of In Vitro Phototoxicity of a Minibody-IR700 Conjugate Using Cell Monolayer and Multicellular Tumor Spheroid Models.

Photodynamic therapy (PDT) is a treatment strategy that utilizes photosensitizers (PSs) and light of a specific wavelength to kill cancer cells. However, limited tumor specificity is still a drawback for the clinical application of PDT. To increase the therapeutic efficacy and specificity of PDT, a novel human minibody (MS5) that recognizes a cell surface receptor expressed on various cancer cells was labeled with the hydrophilic phthalocyanine PS IR700 to generate an MS5-IR700 conjugate that is activated by near-infrared (NIR) light. The phototoxicity of the conjugate was mainly tested against the PC3 prostate cancer cell line. The MS5-IR700 conjugate killed PC3 cells after NIR light irradiation as compared to untreated cells or cells treated with IR700 alone. Time-course analysis of cell viability revealed a high percentage of cell death during the first hour in PC3 cells exposed to the MS5-IR700 conjugate and NIR light irradiation. After irradiation, the MS5-IR700 conjugate-treated PC3 cells displayed cellular swelling, round shape, and rupture of the cell and nuclear membranes. In a co-culture model, the MS5-IR700 conjugate killed MS5-positive Ramos lymphoma cells specifically, while leaving MS5-negative cells unaffected. In line with the data obtained with the monolayer cultures, the MS5-IR700 conjugate also killed PC3 cancer cell spheroids. The treatment induced relocation of heat shock protein 70 and calreticulin to the cell surface, implying the induction of immunogenic cell death. Overall, the data suggest that the developed MS5-IR700 conjugate is a promising therapeutic agent that warrants further preclinical studies.

Correction: Sioud et al. Evaluation of In Vitro Phototoxicity of a Minibody-IR700 Conjugate Using Monolayer and Multicellular Tumor Spheroid Models. Cancers 2021, 13, 3356.

The authors wish to make the following corrections to this paper [...].

Application of Photodynamic Therapy with 5-Aminolevulinic Acid to Extracorporeal Photopheresis in the Treatment of Patients with Chronic Graft-versus-Host Disease: A First-in-Human Study.

Extracorporeal photopheresis (ECP), an immunomodulatory therapy for the treatment of chronic graft-versus-host disease (cGvHD), exposes isolated white blood cells to photoactivatable 8-methoxypsoralen (8-MOP) and UVA light to induce the apoptosis of T-cells and, hence, to modulate immune responses. However, 8-MOP-ECP kills diseased and healthy cells with no selectivity and has limited efficacy in many cases. The use of 5-aminolevulinic acid (ALA) and light (ALA-based photodynamic therapy) may be an alternative, as ex vivo investigations show that ALA-ECP kills T-cells from cGvHD patients more selectively and efficiently than those treated with 8-MOP-ECP. The purpose of this phase I-(II) study was to evaluate the safety and tolerability of ALA-ECP in cGvHD patients. The study included 82 treatments in five patients. One patient was discharged due to the progression of the haematological disease. No significant persistent changes in vital signs or laboratory values were detected. In total, 62 adverse events were reported. Two events were severe, 17 were moderate, and 43 were mild symptoms. None of the adverse events evaluated by the internal safety review committee were considered to be likely related to the study medication. The results indicate that ALA-ECP is safe and is mainly tolerated well by cGvHD patients.

Selective Killing of Activated T Cells by 5-Aminolevulinic Acid Mediated Photodynamic Effect: Potential Improvement of Extracorporeal Photopheresis.

Extracorporeal photopheresis (ECP), a modality that exposes isolated leukocytes to the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light, is used to treat conditions such as cutaneous T-cell lymphoma and graft-versus-host disease. However, the current procedure of ECP has limited selectivity and efficiency; and produces only partial response in the majority of treated patients. Additionally, the treatment is expensive and time-consuming, so the improvement for this modality is needed. In this study, we used the concept of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA), a precursor of an endogenously synthesized photosensitizer protoporphyrin IX (PpIX) in combination with blue light to explore the possibility of targeting activated human blood T cells ex vivo. With various T-cell activation protocols, a high ALA-induced PpIX production took place in activated CD3+, CD4+CD25+, and CD8+ T cell populations with their subsequent killing after blue light exposure. By contrast, resting T cells were much less damaged by the treatment. The selective and effective killing effect on the activated cells was also seen after co-cultivating activated and resting T cells. Under our clinically relevant experimental conditions, ALA-PDT killed activated T cells more selectively and efficiently than 8-MOP/UV-A. Monocyte-derived dendritic cells (DCs) were not affected by the treatment. Incubation of ALA-PDT damaged T cells with autologous DCs induced a downregulation of the co-stimulatory molecules CD80/CD86 and also upregulation of interleukin 10 (IL-10) and indoleamine 2,3-dioxygenase expression, two immunosuppressive factors that may account for the generation of tolerogenic DCs. Overall, the data support the potential use of ALA-PDT strategy for improving ECP by selective and effective killing of activated T cells and induction of immune tolerance.

Depth profile of protoporphyrin IX fluorescence in an amelanotic mouse melanoma model.

Protoporphyrin IX (PpIX) fluorescence was measured at different depths in a subcutaneous amelanotic melanoma model (LOX) in mice. PpIX was induced by topical application of 5-aminolevulinic acid (ALA) and two of its derivatives, the methylester (MAL) and hexylester (HAL) onto the normal skin covering the tumor. The PpIX fluorescence intensity on the surface of the tumors was the highest for HAL, followed by ALA and MAL. Using equimolar concentrations (0.5 mmol g(-1)), HAL induced nearly twice as much fluorescence as ALA did. The depth profile of PpIX fluorescence was measured at different layers of the tumor, which was carefully sliced and controlled in situ ex vivo. The PpIX fluorescence was mainly localized within the upper 2 mm of the tissue for ALA and within 1 mm for MAL and HAL. There were no significant differences in the shape of the fluorescence excitation spectra, but the long wavelength excitation peak (633 nm) was so weak that these results are unreliable for depth estimation. When considering the low fluorescence intensity (around 5% of the intensity at the tumor surface), the actual penetration depth of HAL was comparable to that of ALA. The fluorescence after topical application of ALA and HAL was significantly above the background level down to a depth of around 6 mm, and there were traces of PpIX fluorescence even at the tumor base (10 mm). The fluorescence after topical application of MAL was detectable down to 1 mm. In the depth of 2-6 mm, the fluorescence was slightly higher for HAL than for ALA. Using the estimated diffusion coefficients for topically applied ALA (0.16 +/- 0.03 mm(2) h(-1)), MAL (0.045 +/- 0.005 mm(2) h(-1)) and HAL (0.037 +/- 0.003 mm(2) h(-1)), the behavior of the drugs after different application times could be estimated in this tumor model.

Microneedle arrays permit enhanced intradermal delivery of a preformed photosensitizer.

Silicon microneedle (MN) arrays were used to puncture excised murine and porcine skin in vitro and transdermal and intradermal delivery of meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP) investigated using topical application of a bioadhesive patch containing 19 mg TMP cm(-2). Animal studies, using nude mice, were then conducted to investigate the in vivo performance of the bioadhesive patch following MN puncture of skin. MN puncture significantly enhanced both intradermal and transdermal delivery of TMP in vitro, though the total amounts of drug delivered (25.22% into porcine skin and 0.07% across murine skin) were still quite small in each case. Notwithstanding this, in vivo experiments showed that MN puncture was capable of permitting a prolonged increase in TMP fluorescence at the site of application. Importantly, fluorescence was negligible at distant sites, meaning systemic delivery of the drug was not sufficient to induce TMP accumulation other than at the application site. In this study we have conclusively demonstrated proof of principle; MN puncture allows true intradermal delivery of a preformed photosensitizer in animal skin models in vitro and in vivo. Importantly, transdermal delivery was much reduced in each case. Increasing MN density would allow increased amounts of photosensitizer to be delivered. However, as MNs create aqueous pores in the stratum corneum, a preformed photosensitizer must possess at least some degree of water solubility in order to permit enhanced intradermal delivery in this way. We believe that use of MN array technology in this way has the potential to significantly improve topical photodynamic therapy of skin tumors.

Influence of formulation factors on PpIX production and photodynamic action of novel ALA-loaded microparticles.

A novel 5-aminolevulinic acid (ALA)-containing microparticulate system was produced recently, based on incorporation of ALA into particles prepared from a suppository base that maintains drug stability during storage and melts at skin temperature to release its drug payload. The novel particulate system was applied to the skin of living animals, followed by study of protoporphyrin IX (PpIX) production. The effect of formulating the microparticles in different vehicles was investigated and also the phototoxicity of the PpIX produced using a model tumour.Particles formulated in propylene glycol gels (10% w/w ALA loading) generated the highest peak PpIX fluorescence levels in normal mouse skin. Peak PpIX levels induced in skin overlying subcutaneously implanted WiDr tumours were significantly lower than in normal skin for both the 10% w/w ALA microparticles alone and the 10% w/w ALA microparticles in propylene glycol gels during continuous 12 h applications. Tumours not treated with photodynamic therapy continued to grow over the 17 days of the anti-tumour study. However, those treated with 12 h applications of either the 10% w/w ALA microparticles alone or the 10% w/w ALA microparticles in propylene glycol gel followed by a single laser irradiation showed no growth. The gel formulation performed slightly better once again, reducing the tumour growth rate by approximately 105%, compared with the 89% reduction achieved using particles alone. Following the promising results obtained in this study, work is now going on to prepare particle-loaded gels under GMP conditions with the aim of initiating an exploratory clinical trial.

The Akt pathway in oncology therapy and beyond (Review).

Protein kinase B (Akt), similar to many other protein kinases, is at the crossroads of cell death and survival, playing a pivotal role in multiple interconnected cell signaling mechanisms implicated in cell metabolism, growth and division, apoptosis suppression and angiogenesis. Akt protein kinase displays important metabolic effects, among which are glucose uptake in muscle and fat cells or the suppression of neuronal cell death. Disruptions in the Akt­regulated pathways are associated with cancer, diabetes, cardiovascular and neurological diseases. The regulation of the Akt signaling pathway renders Akt a valuable therapeutic target. The discovery process of Akt inhibitors using various strategies has led to the identification of inhibitors with great selectivity, low side­effects and toxicity. The usefulness of Akt emerges beyond cancer therapy and extends to other major diseases, such as diabetes, heart diseases, or neurodegeneration. This review presents key features of Akt structure and functions, and presents the progress of Akt inhibitors in regards to drug development, and their preclinical and clinical activity in regards to therapeutic efficacy and safety for patients.

Photodynamic therapy with di-l-arginine protoporphyrinate on WiDr human colon adenocarcinoma xenografts in athymic nude mice.

The fluorescence kinetics of a new photosensitizer for photodynamic therapy, di-l-arginine protoporphyrinate (PP(Arg)2), was studied in the skin of healthy mice. Furthermore, induction of necrosis in WiDr human colon adenocarcinoma xenografts in athymic nude mice was studied after photodynamic therapy (PDT) with PP(Arg)2. After intravenous administration of PP(Arg)2 maximal fluorescence was reached after 72h in normal mouse skin. Complete elimination of the drug from the mouse skin was not found even after 32 days. Exposure of WiDr tumours in mice to red light (λ=632nm, fluence 150J/cm(2), fluence rate 250mW/cm(2)) 24 and 72h after intravenous administration of 10mg/kg of PP(Arg)2 caused extensive tumour necrosis. Epidermal damage and infiltration of inflammatory cells was seen 24h after light exposure but not after 72h.

Biological activity of 5-aminolevulinic acid and its methyl ester after storage under different conditions.

5-Aminolevulinic acid (ALA) is a natural precursor of protoporphyrin IX (PpIX) and heme in cells. Photodynamic therapy (PDT) utilizes a metabolic imbalance in cancer cells, leading to increased PpIX generation from exogenous ALA. Due to chemical instability of ALA in therapeutic concentrations at pH values larger than 5.0 and at high temperatures, it looses its activity by spontaneous dimerization to 2,5-dicarboxyethyl-3,6-dihydropyrazine (DHPY). ALA esters are now supplementing ALA in PDT, but little is known about their stability. We have studied the stability of ALA and its methyl ester (MAL) stored under different conditions (temperatures, pH values) by measuring their ability to generate PpIX. 100mM solutions of both compounds were found to be stable at pH 4 and at 4 degrees C. However, at pH 5.5 they lost almost 10% of the initial activity during 5days of storage at 4 degrees C. The fastest decay of ALA and MAL was seen at pH 7.4 and at 37 degrees C, and followed first order kinetics. At pH 7.4 and at 4 degrees C MAL lost its PpIX producing ability more slowly than at 37 degrees C. Our work shows that solutions should be prepared immediately before use and stored at low temperatures. The pH of stock solutions should not exceed 5.

Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading.

Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor MK1775. Strikingly, the Chk1-inhibitors AZD7762 and LY2603618 were among the top candidate hits of 1664 tested compounds, suggesting that the synergistic cytotoxic effects are due to increased S-phase DNA damage. Combined Wee1- and Chk1-inhibition caused a strong synergy in induction of S-phase DNA damage and reduction of clonogenic survival. To address the underlying mechanisms, we developed a novel assay measuring CDK-dependent phosphorylations in single S-phase cells. Surprisingly, while Wee1-inhibition alone induced less DNA damage compared to Chk1-inhibition, Wee1-inhibition caused a bigger increase in S-phase CDK-activity. However, the loading of replication initiation factor CDC45 was more increased after Chk1- than Wee1-inhibition and further increased by the combined treatment, and thus correlated well with DNA damage. Therefore, when Wee1 alone is inhibited, Chk1 suppresses CDC45 loading and thereby limits the extent of unscheduled replication initiation and subsequent S-phase DNA damage, despite very high CDK-activity. These results can explain why combined treatment with Wee1- and Chk1-inhibitors gives synergistic anti-cancer effects.

The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of cultured human cancer cells.

5-Aminolevulinic acid (ALA), a precursor of the endogenous photosensitizer protoporphyrin IX, is used in the photodynamic therapy (PDT) of cancer. Sub-lethal ALA-PDT (1-min irradiation with 370-450 nm blue light, 0.6 mW/cm(2) after 2-h incubation with 1 mM ALA) has been earlier shown to change cell morphology and to inhibit both trypsin-induced detachment of cultured cancer cells from the plastic substrata and cell attachment to the bottom of the plastic well plates. In the present study, we found that such treatment of human adenocarcinoma WiDr cells grown in dense colonies stimulated the formation of actin cortex between cells in the colonies and increased the number of actin stress fibres in some, but not in all, cells. However, ALA-PDT did not change the microtubular cytoskeleton in these cells. A similar treatment of glioblastoma D54Mg cells, which grow separately and communicate by protrusions, caused loss of fibrillar actin structures in growth cones, retraction of protrusions, and surface blebbing in some cells. The application of the cytoskeleton inhibitors cytochalasin D, colchicine or taxol showed that the inhibition of trypsin-induced detachment of photosensitized WiDr cells was related to ALA-PDT-induced changes in actin and microtubular cytoskeleton. Some signal transduction processes are suggested to be involved in ALA-PDT-induced changes in cytoskeleton, cell shape, and adhesion.