Tadeusz Issat

Statins impair antitumor effects of rituximab by inducing conformational changes of CD20.

Background Rituximab is used in the treatment of CD20+ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas. Methods and Findings Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a 51Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-β-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells. Conclusions Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.

Proteasome Inhibition Potentiates Antitumor Effects of Photodynamic Therapy in Mice through Induction of Endoplasmic Reticulum Stress and Unfolded Protein Response

Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by inducing production of reactive oxygen species such as singlet oxygen. PDT leads to oxidative damage of cellular macromolecules, including proteins that undergo multiple modifications such as fragmentation, cross-linking, and carbonylation that result in protein unfolding and aggregation. Because the major mechanism for elimination of carbonylated proteins is their degradation by proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to accumulation of carbonylated proteins in endoplasmic reticulum (ER), aggravated ER stress, and potentiated cytotoxicity toward tumor cells. We observed that Photofrin-mediated PDT leads to robust carbonylation of cellular proteins and induction of unfolded protein response. Pretreatment of tumor cells with three different proteasome inhibitors, including bortezomib, MG132, and PSI, gave increased accumulation of carbonylated and ubiquitinated proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells of murine (EMT6 and C-26) as well as human (HeLa) origin to PDT-mediated cytotoxicity. Significant retardation of tumor growth with 60% to 100% complete responses was observed in vivo in two different murine tumor models (EMT6 and C-26) when PDT was combined with either bortezomib or PSI. Altogether, these observations indicate that combination of PDT with proteasome inhibitors leads to potentiated antitumor effects. The results of these studies are of immediate clinical application because bortezomib is a clinically approved drug that undergoes extensive clinical evaluations for the treatment of solid tumors.

The influence of photodynamic therapy on the immune response

Photodynamic therapy (PDT) is a clinically approved therapeutic modality used for the management of several types of tumors as well as non-malignant diseases. Most of the effects of this treatment regimen result from direct action of singlet oxygen and reactive oxygen species. However, accumulating evidence indicates that antitumor effects are also mediated by indirect stimulation of inflammatory and immune responses. These responses include rapid local infiltration of tumors by neutrophils and macrophages accompanied by systemic release of inflammatory mediators. This early response can initiate and translate into a more precise immune reaction that involves activation of specific T lymphocytes that seem to be necessary for the ultimate control of residual tumor cells. Although still incompletely understood, PDT can not only activate but also suppress the immune response depending on several variables. This review summarizes the influence of PDT on the immune response and discusses its importance in the management of human diseases.

Zinc protoporphyrin IX, a heme oxygenase-1 inhibitor, demonstrates potent antitumor effects but is unable to potentiate antitumor effects of chemotherapeutics in mice

BackgroundHO-1 participates in the degradation of heme. Its products can exert unique cytoprotective effects. Numerous tumors express high levels of HO-1 indicating that this enzyme might be a potential therapeutic target. In this study we decided to evaluate potential cytostatic/cytotoxic effects of zinc protoporphyrin IX (Zn(II)PPIX), a selective HO-1 inhibitor and to evaluate its antitumor activity in combination with chemotherapeutics.MethodsCytostatic/cytotoxic effects of Zn(II)PPIX were evaluated with crystal violet staining and clonogenic assay. Western blotting was used for the evaluation of protein expression. Flow cytometry was used to evaluate the influence of Zn(II)PPIX on the induction of apoptosis and generation of reactive oxygen species. Knock-down of HO-1 expression was achieved with siRNA. Antitumor effects of Zn(II)PPIX alone or in combination with chemotherapeutics were measured in transplantation tumor models.ResultsZn(II)PPIX induced significant accumulation of reactive oxygen species in tumor cells. This effect was partly reversed by administration of exogenous bilirubin. Moreover, Zn(II)PPIX exerted potent cytostatic/cytotoxic effects against human and murine tumor cell lines. Despite a significant time and dose-dependent decrease in cyclin D expression in Zn(II)PPIX-treated cells no accumulation of tumor cells in G1 phase of the cell cycle was observed. However, incubation of C-26 cells with Zn(II)PPIX increased the percentage of cells in sub-G1 phase of the cells cycle. Flow cytometry studies with propidium iodide and annexin V staining as well as detection of cleaved caspase 3 by Western blotting revealed that Zn(II)PPIX can induce apoptosis of tumor cells. B16F10 melanoma cells overexpressing HO-1 and transplanted into syngeneic mice were resistant to either Zn(II)PPIX or antitumor effects of cisplatin. Zn(II)PPIX was unable to potentiate antitumor effects of 5-fluorouracil, cisplatin or doxorubicin in three different tumor models, but significantly potentiated toxicity of 5-FU and cisplatin.ConclusionInhibition of HO-1 exerts antitumor effects but should not be used to potentiate antitumor effects of cancer chemotherapeutics unless procedures of selective tumor targeting of HO-1 inhibitors are developed.

Synthesis of novel, peptidic kinase inhibitors with cytostatic/cytotoxic activity

The utility of a novel, chemoenzymatic procedure for the stereocontrolled synthesis of small peptides is presented in the preparation and structure optimisation of dipeptides with cytostatic/cytotoxic activity. The method uses Passerini multicomponent reaction for the preparation of racemic scaffold which is then enantioselectively hydrolysed by hydrolytic enzymes. Products of these transformations are further functionalised towards title compounds. Both activity and selectivity towards tumor cells is optimised. Final compound is shown to be an inhibitor of the protein kinase signaling pathway.

Bilateral ectopic tubal pregnancy, following in vitro fertilisation (IVF).

This is a case report of a 33 year-old-woman who underwent in vitro fertilization, because of primary infertility caused by fallopian tube factor. The patient underwent three trials of frozen embryo transfers (ET-CRYO). On the 26th day after the third probe of ET-CRYO she suffered from pelvic pain and vaginal bleeding. Transvaginal ultrasonographic scan revealed bilateral tubal ectopic pregnancy without fluid in the Douglas pouch and no gestational sac in the uterus. Laparoscopic bilateral salpingectomy was performed on the next day after admission. The postoperative course was uneventful.

PCOS and cancer risk.

Polycystic ovary syndrome (PCOS) affects approximately 5 to 10% of women of reproductive age. It is the most common reason of anovulation in infertile women. PCOS is accompanied by such conditions as oligo- or anovulation, hipertestosteronism, lower cell sensitivity to insulin, type II diabetes, hyperlipidemia and obesity. Each of the above-mentioned conditions is an approved risk factor proved to predispose towards cancer. However, PCOS is also a disease entity which differs in its clinical manifestation. For example not all patients suffer from obesity or hipertestosteronism related symptoms. From the analysis of literature it is possible to draw conclusions, that there is a possible correlation between PCOS and endometrial cancer, which emerges from clinical trials or research focused on molecular changes in endometrium patients with PCOS. On the other hand, correlation between PCOS and breast or ovary cancer is not so strong, in spite of single papers which are showing the link. The main problem in researching the correlation between PCOS and any cancer risk, is there is a very small group of women or the trial is imperfect (e.g. no control group). There is no meta-analysis focused on this correlation in literature. The change of criteria of PCOS in the past is also a big problem, because there was a number of definitions of PCOS, which results in inconsistent PCOS diagnoses over time. In this paper we would like to provide a description of studies that aimed at showing correlation between PCOS and cancer risk and underlying theoretical assumptions.

Photodynamic therapy-driven induction of suicide cytosine deaminase gene

Photodynamic therapy (PDT) of tumors is associated with induction of hypoxia that results in activation of hypoxia-inducible factors (HIFs). Several observations indicate that increased HIFs transcriptional activity in tumor cells is associated with cytoprotective responses that limit cytotoxic effectiveness of PDT. Therefore, we decided to examine whether this cytoprotective mechanism could be intentionally used for designing more efficient tumor cell cytotoxicity. To this end we transfected tumor cells with a plasmid vector carrying a suicide cytosine deaminase gene driven by a promoter containing hypoxia response elements (HRE). The presence of such a genetic molecular beacon rendered tumor cells sensitive to cytotoxic effects of a non-toxic prodrug 5-fluorocytosine (5-FC). The results of this study provides a proof of concept that inducible cytoprotective mechanisms can be exploited to render tumor cells more susceptible to cytotoxic effects of prodrugs activated by products of suicide genes.

In vitro fertilization and age. When old is too old

This case report refers to a 53-year-old female patient who deliberately misinformed the staff of the in vitro fertilization (IVF) clinic using counterfeit documents concerning her real age and underwent the IVF procedure. During the pregnancy it was discovered accidentally that the patient was not telling the whole truth about her real age. In the 34th week of pregnancy two healthy twins have been delivered by a cesarean section. Somehow the trust between the patient and the doctor was compromised, however we can acknowledge this case of postmenopause pregnancy as a therapeutical success for the clinic and for the patient herself. The mother invested her time, emotions and financial resources, and risked her health undergoing IVF procedures and carrying the pregnancy to fulfill her wish and deliver a child. Regardless of her intentions, we can argue about her knowledge concerning this procedure and awareness of threats to pregnancy resulting from her age. The age of childbearing in Western societies has been considerably delayed nowadays, mainly because of social aspects. Many institutions nowadays try to face these aspects, which produces certain medical issues. In this case report authors also present literature review on age and pregnancy after IVF.