Abstract In the present study, some of the difficulties in the treatment of the most common malignant lymphoma, diffuse large B-cell lymphoma (DLBCL), were evaluated. Some patients develop local or central nervous system (CNS) relapse after first-line treatment. The treatment of relapsed disease is challenging and despite all efforts, some patients die of the disease. Chemoresistant disease also remains challenging because some patients suffer from refractory disease of a progressive nature. The antioxidant enzymes peroxiredoxins (Prxs) and thioredoxin-1 (Trx) were evaluated as prognostic and predictive markers of DLBCL. High cytoplasmic expression of Prx VI was found to correlate with poor prognosis in patients with DLBCL. Trx knockdown in lymphoma cell culture revealed a possible predictive role of Trx. Trx knockdown sensitized cells to doxorubicin, a widely used chemotherapeutic agent in treatment schemas of DLBCL. Etoposide, another widely used chemotherapeutic agent, on the other hand, killed more native DLBCL cells than did doxorubicin. Patients with high Trx expression at the diagnostic stage of the disease benefitted from etoposide-containing high-dose chemotherapy and autologous stem cell transplantation and did not develop post-transplantation relapses which Trx-negative patients did. Antithrombin III (AT III) in cerebrospinal fluid has been suggested to be a biomarker in previous studies of CNS lymphoma. In the present study, AT III was evaluated in patients with CNS lymphoma and with neurological diseases. High concentrations of AT III in cerebrospinal fluid reflected the magnitude of blood-brain barrier leakage and because of this, AT III should not be used as a biomarker in clinical practice. / Tiivistelmä Tutkimuksessa arvioitiin osaa yleisimmän pahanlaatuisen imukudossyövän eli lymfooman, diffuusin suurisoluisen B-solulymfooman, hoidon haasteista. Osa potilaista saa ensilinjan hoidon jälkeen joko paikallisen tai aivoston alueen taudin uusiutuman. Uusiutuneen taudin hoito on haasteellista, ja hoitoyrityksistä huolimatta osa potilaista kuolee tautiinsa. Solunsalpaajille resistentti tauti on myös yksi haastavista hoitotilanteista, ja osa potilaista kärsiikin hoitojen läpi etenevästä taudista. Antioksidatiivisia entsyymejä, kuten peroksiredoksiineja ja tioredoksiinia, arvioitiin ennusteellisina ja ennakoivina merkkiaineina diffuusissa suurisoluisessa B-solulymfoomassa. Peroksiredoksiini VI:n korkea sytosolinen ilmaantuvuus korreloi tavallista huonompaan diffuusin suurisoluisen B-solulymfooman ennusteeseen. Tioredoksiinin hiljentäminen lymfoomasoluviljelyssä paljasti sen mahdollisen ennakoivan merkityksen hoitoon liittyvässä päätöksenteossa. Solut herkistyivät tiodredoksiinin hiljentämisen vuoksi doksorubisiinille, jota käytetään laajalti diffuusin suurisoluisen B-solulymfooman solunsalpaajahoidoissa. Etoposidi, joka on huomattavasti myrkyllisempi solunsalpaaja, päinvastoin tappoi enemmän tavallisia diffuusia suurisoluisia B-solulymfoomaa edustavia soluja kuin doksorubisiini. Potilaat, joilla oli korkea tioredoksiinin määrä taudin diagnostisessa vaiheessa, hyötyivät etoposidia sisältävästä korkea-annoshoidosta sekä autologisesta kantasolusiirrosta. Näille potilaille ei kehittynyt kantasolusiirron jälkeisiä taudin uusiutumia kuin taas niitä kehittyi potilaille, joilla oli tioredoksiini negatiivinen. Antitrombiini III:a on ehdotettu soveltuvaksi aikaisempien tutkimusten perusteella aivoston lymfooman merkkiaineeksi aivo-selkäydinnesteestä. Tässä tutkimuksessa antitrombiini III:n määrää mitattiin potilailta, joilla oli aivoston lymfooma tai neurologinen sairaus. Korkeat konsentraatiot antitrombiini III:a aivo-selkäydinnesteessä kuitenkin vain heijastivat veri-aivoesteen vuotamisen määrää, ja näin ollen antitrombiini III:a ei tulisi käyttää kliinisessä käytössä.

No description available.

<p>We treated 16 patients with somatostatin analogs combined with α-interferon and achieved a biochemical and/or radiological response in 56% (median duration 22 months). We consider this treatment a good alternative for patients who fail during chemotherapy or who do not want to/cannot receive cytotoxic drugs.</p><p>Thirty-six patients with neuroendocrine tumors were treated with cisplatin combined with etoposide. Of 14 patients with evaluable EPTs, 50% responded radiologically and/or biochemically (median duration 9 months). We consider this treatment useful as first-line medical treatment in aggressive EPTs or in patients failing prior chemotherapy.</p><p>Twenty-eight tumor tissues from EPTs were examined with immunohistochemistry regarding expression of somatostatin receptors (ssts) 1 to 5 on tumor cells and in intratumoral vessels. We found that sst₂ and sst₄ were highly expressed on tumor cells and in vessels. However, sst₃ and sst₅ were lacking in half of the tumor tissues and in most of the vessels. Because of the variability in sst expression, we recommend analysis of each individual’s receptor expression before starting treatment.</p><p>Endocrine pancreatic tumors (EPTs) are rare with an incidence of 4 per million inhabitants. In the majority of cases they grow slowly, but there are exceptions with very rapidly progressing malignant carcinomas. First-line medical treatment is streptozotocin combined with 5-fluorouracil.</p><p>We examined 38 tumor samples regarding expression of tyrosine kinase receptors platelet-derived growth factor receptors (PDGFRs), c-kit and epidermal growth factor receptor (EGFR). We found that the receptors were expressed in more than half of the tumor tissues. Further studies will reveal if tyrosin kinase antagonists can be part of the future treatment arsenal.</p>

We treated 16 patients with somatostatin analogs combined with α-interferon and achieved a biochemical and/or radiological response in 56% (median duration 22 months). We consider this treatment a good alternative for patients who fail during chemotherapy or who do not want to/cannot receive cytotoxic drugs. Thirty-six patients with neuroendocrine tumors were treated with cisplatin combined with etoposide. Of 14 patients with evaluable EPTs, 50% responded radiologically and/or biochemically (median duration 9 months). We consider this treatment useful as first-line medical treatment in aggressive EPTs or in patients failing prior chemotherapy. Twenty-eight tumor tissues from EPTs were examined with immunohistochemistry regarding expression of somatostatin receptors (ssts) 1 to 5 on tumor cells and in intratumoral vessels. We found that sst2 and sst4 were highly expressed on tumor cells and in vessels. However, sst3 and sst5 were lacking in half of the tumor tissues and in most of the vessels. Because of the variability in sst expression, we recommend analysis of each individual’s receptor expression before starting treatment. Endocrine pancreatic tumors (EPTs) are rare with an incidence of 4 per million inhabitants. In the majority of cases they grow slowly, but there are exceptions with very rapidly progressing malignant carcinomas. First-line medical treatment is streptozotocin combined with 5-fluorouracil. We examined 38 tumor samples regarding expression of tyrosine kinase receptors platelet-derived growth factor receptors (PDGFRs), c-kit and epidermal growth factor receptor (EGFR). We found that the receptors were expressed in more than half of the tumor tissues. Further studies will reveal if tyrosin kinase antagonists can be part of the future treatment arsenal.

No description available.

The aim of this thesis is the characterization of a protein involved in apoptosis. Our group has identified an early step common to different forms of intrinsic apoptosis stimuli. This step requires de novo synthesis of a novel Cyclin, Cyclin O, that upon apoptosis induction in lymphoid cells forms active complexes, primarily with Cdk2. Cyclin O expression precedes glucocorticoid and gamma radiation-induced apoptosis in vivo in mouse thymus and its overexpression induces apoptosis in cultured cells. Knocking down the endogenous expression of Cyclin O by shRNA leads to the inhibition of glucocorticoid and DNA damage-induced apoptosis while leaving CD95 death receptor mediated apoptosis intact. This data demonstrates that apoptosis induction in lymphoid cells is one of the physiological roles of Cyclin O and it does not act by perturbing a normal cellular process such as the cell cycle. In addition we have identified c-Myb a substrate of Cdk2-Cyclin O complexes and we show that c-Myb is downregulated during apoptosis of lymphoid cells. / L'objectiu d'aquesta tesi és la caracterització d'una proteïna involucrada en l'apoptosi. El nostre grup ha identificat un pas primerenc comú en diversos estímuls apoptòtics de la ruta intrínseca. Aquest pas requereix la síntesi de novo d'una nova Ciclina, Ciclina O, que quan s'indueix apoptosi en cèl·lules limfoides forma complexes actius majoritàriament amb Cdk2. L'expressió de la Ciclina O és prèvia a l'apoptosi induïda per glucocorticoids i radiació gamma i la seva sobreexpressió indueix apoptosi en cultius cel·lulars. La baixada dels nivells d'expressió de la Ciclina O endògena amb shRNA provoca una inhibició de l'apoptosi induïda per glucocorticoids o agents que danyen el DNA, mentre que l'apoptosi mediada pel receptor CD95 es manté intacta. Aquests resultats demostren que la inducció d'apoptosi en cèl·lules limfoides és una de les funcions fisiològiques de la Ciclina O i que no es deu a una pertorbació de processos cel·lulars normals com ara el cicle cel·lular. A més a més, hem identificat c-Myb com a substrat dels complexes Cdk2-Ciclina O i demostrem que els nivells de c-Myb baixen durant l'apoptosis de cèl·lules limfoides.

Cancer is a leading cause of death that affects millions of people across the globe each year. Photodynamic therapy (PDT) is a relatively new treatment approach for cancer in which anticancer drugs are activated by light at an appropriate wavelength to generate highly cytotoxic reactive oxygen species (ROS) and achieve tumor destruction. Compared with conventional chemo- and radiotherapy, PDT can be performed with minimal invasiveness, local targeting and reduced side effects. However, most of the currently available PDT drugs mainly absorb in the visible part of the spectrum, where light penetration depth into human tissues is very limited. Therefore, increasing the treatment depth of PDT has been considered to be an important approach to improve the effectiveness of PDT for treating larger and thicker tumor masses. In this thesis, we present our investigation into the potential of two-photon activated PDT (2-γ PDT), combination therapy of PDT and chemotherapy, and bioluminescence-activated PDT as a means to increase the treatment depth of this modality. In 2-γ PDT, the photosensitizing agents are activated through simultaneous absorption of two photons. This approach allows the use of near-infrared (NIR) light that can penetrate deeper into tissues and thus, has the potential of treating deep-seated tumors and reducing side effects, while the non-linear nature of two-photon excitation (TPE) may improve tumor targeting. We have evaluated the PDT efficacy of a second-generation photosensitizer derived from chlorophyll a, pyropheophorbide a methyl ester (MPPa), through both one- and two-photon activation. We observed that MPPa had high one-photon (1-γ PDT efficacy against both cisplatin-sensitive human cervical (HeLa) and cisplatin-resistant human lung (A549) and ovarian (NIH:OVCAR-3) cancer cells when activated by femtosecond (fs) laser pulses at 674 nm. At a low light dose of 0.06 J cm-2, the MPPa concentration required to produce a 50% cell killing effect (IC50) was determined to be 5.3 ± 0.3, 3.4 ± 0.3 and 3.6 ± 0.4 μM in HeLa, A549 and NIH:OVCAR-3 cells, respectively. More significantly, we also found that MPPa could be effectively activated at the optimal tissue-penetrating wavelength of 800 nm through TPE. At a light dose of 886 J cm-2, where no measurable photodamage was observed in the absence of MPPa, the IC50 values were measured to be 4.1 ± 0.3, 9.6 ± 1.0 and 1.6 ± 0.3 μM in HeLa, A549 and NIH:OVCAR-3 cells, respectively. We obtained corresponding LD50 (the light dose required to produce a 50% killing effect) values of 576 ± 13, 478 ± 18 and 360 ± 16 J cm-2 for 10 μM MPPa, which were approximately 3-5 times lower than the published 2-γ LD50 of Visudyne® and 20-30 times lower than that of Photofrin®. These results indicate that MPPa may serve as a photosensitizer for both 1- and 2-γ activated PDT treatment of difficult-to-treat tumors by conventional therapies. Indocyanine green (ICG), a dye having an absorption maximum near 800 nm, has been considered to be a potential NIR PDT agent. However, the PDT efficacy of ICG has been found to be very limited probably due to the low yield of cytotoxic ROS. In the present work, we have evaluated the combination effects of ICG-mediated PDT with conventional chemotherapy mediated by two types of chemotherapeutic drugs, namely the type II topoisomerase (TOPII) poisons etoposide (VP-16)/teniposide (VM-26) and the platinum-based drugs cisplatin (CDDP)/oxaliplatin (OXP). Synergistic enhancement of cytotoxicity and increased yields of DNA double strand breaks (DSBs) were observed in HeLa, A549 and NIH:OVCAR-3 cancer cells treated with the combination of ICG-PDT and VP-16. The presence of VP-16 during the laser irradiation process was found to be critical for producing a synergistic effect. An electron-transfer-based mechanism, in which ICG could increase the yield of highly cytotoxic VP-16 metabolites, was proposed for the observed synergistic effects, although direct spectroscopic detection of the reaction products was found to be very challenging. Moreover, we observed a much lower degree of synergy in the human normal fibroblast GM05757 cells than that in the three cancer cell lines investigated. Synergistic effects were also observed in A549 cells treated with the combination of ICG-PDT and VM-26 (i.e. an analog of VP-16). Furthermore, the combination of low-dose CDDP/OXP and ICG-PDT was demonstrated to produce an additive or synergistic effect in selected cancer cell lines. These preliminary results suggest that the combination of ICG-PDT with VP-16/VM-26 or CDDP/OXP chemotherapy may offer the advantages of enhancing the therapeutic effectiveness of ICG-PDT and lowering the side effects associated with the chemotherapeutic drugs. Bioluminescence, the generation of light in living organisms through chemical reactions, has been explored as an internal light source for PDT in recent years. This approach, in principle, does not suffer from the limited tissue penetration depth of light. In the present project, we have evaluated the effectiveness of luminol bioluminescence in activating the porphyrin photosensitizers meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TPPS4) and Fe(III) meso-tetra(4-sulfonatophenyl)porphine chloride (FeTPPS). The combination treatment induced significant killing of HeLa cells, while additive effects were observed in two normal human fibroblast cell lines (GM05757 and MRC-5). Our observations indicate that bioluminescence of luminol may generate sufficient light for intracellular activation of PDT sensitizers. Furthermore, the combination treatment may have intrinsic selectivity towards cancerous tissues. In summary, we have demonstrated effective killing of cancer cells by MPPa-mediated 1- and 2-γ PDT, combination of ICG-PDT and VP-16/VM-26 or CDDP/OXP chemotherapy, and bioluminescence of luminol activated PDT mediated by TPPS4/FeTPPS. These positive preliminary results indicate that all these three approaches have the potential of increasing the treatment depth of PDT and facilitating the development of more effective PDT treatment strategies.